Cartilage-derived implants and methods of making and using same
Abstract
Cartilage fibers and implants made therefrom are disclosed, with and without cartilage particles. Methods for making the cartilage fibers and the implants containing them are also disclosed. The implants may be pre-shaped and may be reshapable and provide good shape retention and little swelling when placed into a cartilage defect.
Term
9.9 yearsleft in the term
Expires 18 August 2036.
- Priority and filed
- Granted
- Today
- Expires
12 claims: 1 independent, 11 dependent
- 1CLAIMS:1. A cartilage-derived implant for treating a cartilage defect of cartilage, said implant being shapeable and comprising: a plurality of milled lyophilized cartilage particles having a size of 1 millimeter or less and a reduced water content of from 0.1 to 8.0%, by weight based on the weight of the plurality of lyophilized cartilage particles;and a plurality of cartilage fibers which are elongated curled pieces of cartilage tissue, each having three or more laterally oriented striations, wherein the striations of the cartilage fibers enable the cartilage fibers to entangle with one another to a greater degree than cartilage fibers without such striations and form the implant which retains its shape until reshaped and conforms to and completely fills the cartilage defect when implanted in the cartilage defect.
343 paragraphs in 31 sections, as filed
CIPO
CARTILAGE-DERIVED IMPLANTS AND METHODS OF MAKING AND USING SAME
Technical Field of the Invention
The present invention relates to compositions for repairing damaged or defective tissues, to methods for making such compositions, and to surgical methods for making such repairs.
Background of the Invention
Articular cartilage in a joint acts as a lubricating smooth surface and shock absorber between joint members. Articular cartilage ranges from 1-4 mm in thickness, and is located on the mating surfaces of joints. Articular cartilage is an anatomical tissue that wears out with age, or tears due to excess loading such as in sport or other trauma related activities.
Damaged or worn articular cartilage does not heal without surgical intervention. It can be repaired in several ways using both autograft and allograft techniques. These repairs fall into two categories: Osteochondral (OC) repair that addresses both the damaged cartilage and damaged subchondral bone and chondral repair that address only damaged cartilage.
Current autograft surgical repair techniques include microfracture, osteoarticular transfer system (OATS) surgery, and autologous chondrocyte implantation (ACI). Of these only OATS addresses damage to the bone. Microfracture consists of drilling holes within the
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CA 02939280 2016-08-18 subchondral bone to provide blood flow to the damaged areas of cartilage. OATS utilizes small plugs of cartilage harvested from non-load bearing portions of the knee joint to replace damaged areas of load-bearing cartilage. These plugs can also be used in the ankle. ACI utilizes lab-grown chondrocytes generated from harvested cells within the patient’s non-loadbearing cartilage. The developed cells are placed within the damaged cartilage area and held so that they can produce new cartilage-like tissue within the defect.
Current allograft surgical repair techniques include OC transplant utilizing donor cartilage and subchondral bone, and, cartilage-only grafts made from minced or milled cartilage. OC allograft tissues are provided as a whole or portion of the OC region of a long bone from which the surgeon selects and prepares an appropriately sized transplant specimen. The tissue is selected from available donated tissue to match the patients defect.
When utilizing donor cartilage and subchondral bone, worn or damaged articular cartilage and subchondral bone is removed from the patient. The damaged site is then prepared to receive the transplant, while the allograft specimen is sized and shaped. Finally the allograft specimen is press-fit in the prepared site.
Minced or milled donor cartilage is provided ready to use. The damaged cartilage area is prepared to receive the transplant and the minced or milled cartilage is placed in the prepared area either by itself or mixed with/covered by saline or biologic liquid (blood, platelet rich plasma (PRP), fibrin glue, etc.).
While current autologous and allograft cartilage therapies are, on the whole, beneficial, each suffers from its own limitations. Microfracture techniques damage the subchondral bone. Other autograft procedures generate donor sites which experience morbidity and subsequent osteoarthritis. ACI is only successful if the transplanted cells develop into
CA 02939280 2016-08-18 cartilage-like tissue. ACI is also a two-step procedure that requires two surgeries, thus posing greater risk to the patient. OC allograft procedures require on-site preparation of the donor specimen. In addition, OC allografts are limited by the availability of suitably-sized tissues from tissue banks. Current minced and milled allograft products are limited by challenges posed by the available surgical techniques, such as tedious application or limited adhesion of the allograft product to the prepared surgical site.
Summary of the Invention
In an embodiment, a cartilage-derived implant includes cartilage fibers. In an embodiment, the cartilage fibers include viable native chondrocytes. In an embodiment, the cartilage fibers include non-viable native chondrocytes. In an embodiment, the cartilage fibers include viable non-native chondrocytes. In an embodiment, the cartilage fibers include nonnative non-viable chondrocytes. In an embodiment, the cartilage fibers are substantially free of chondrocytes and their components. In an embodiment, the cartilage fibers are freeze-dried. In an embodiment, the cartilage-derived implant consists of cartilage fibers. In an embodiment, the cartilage-derived implant consists of cartilage fibers and cartilage particles. In an embodiment, the cartilage particles are freeze-dried. In an embodiment, the cartilage particles are from about 0.1 millimeter (mm) to about 1 mm. In an embodiment, the cartilage-derived implant includes a biologically-compatible material other than cartilage. In an embodiment, the cartilage-derived implant is a putty-like material. In an embodiment, the cartilage-derived implant is a pre-shaped form. In an embodiment, the cartilage-derived implant is a pre-shaped form that may be reshaped by a surgeon to fit an articular cartilage defect.
In an embodiment, the cartilage fibers are prepared by a method including the step of separating the cartilage fibers, by mechanical techniques such as, without limitation, grating, scraping, slicing, cutting, or combinations thereof, from recovered cartilage, then recovering cartilage fibers having desired dimensions, and storing the recovered fibers in a growth media. In an embodiment, the selected cartilage fibers are lyophilized instead of being stored in a growth media. In an embodiment, unwanted material such as, without limitation, bone fragments and tissue fragments, are separated from the cartilage fibers after they are separated from recovered cartilage. In an embodiment, measured portions of the cartilage fibers are eventually placed in containers for storage and shipping.
In an embodiment, cartilage particles are prepared by a method including the steps of freezer-milling strips of cartilage, sieving the cartilage particles to separate cartilage particles of a desired size range, then lyophilizing the separated cartilage particles. Various suitable methods for preparing the cartilage particles are described for example, without limitation, in U.S. Patent Application Publication No. US2006/0210643, published September 21,2006.
In an embodiment, cartilage fibers and cartilage particles are packaged separately from each other and provided to a surgeon as part of a kit. In such an embodiment, the surgeon mixes the cartilage fibers and cartilage particles to form the cartilage-derived implant. The surgeon may further mix the cartilage fibers and cartilage particles with a biologically-oompatible carrier. In an embodiment, the cartilage-derived implant is provided to the surgeon in a pre-mixed form, as a putty-like material, as a pre-shaped form, or as a preshaped form that may be reshaped by the surgeon.
Brief Description of the Figures
Date Reçue/Date Received 2022-01-10
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For a more complete understanding of the present invention, reference is made to the following detailed description of exemplary embodiments considered in conjunction with the accompanying figures, in which:
FIG. 1 is a block diagram of a process for preparing cartilage fibers having viable native chondrocytes, according to an embodiment of the present invention;
FIG. 2 is a block diagram of a process for preparing a cartilage-derived implant having viable native chondrocytes;
FIG. 3 is a block diagram of a process for preparing cartilage particles, according to an embodiment of the present invention;
FIG. 4 is a top view of a planar grater suitable for use in an embodiment of the present invention;
FIG. 5 is a perspective view of a rotary grater suitable for use in an embodiment of the present invention;
FIG: 6A is a perspective view of an exemplary embodiment of a cutting feature of a grater suitable for use in an embodiment of the present invention;
FIG. 6B is a cross-sectional view of the cutting feature of FIG. 6A;
FIG. 6C is a perspective view of a cutting feature similar to that of FIG. 6A but which has been modified;
6D is a cross-sectional view of the modified cutting feature of FIG. 6C;
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FIG. 7 is a series of cross-sectional views of a cutting feature similar to that of FIG. 6A, but having differently sized cutting angles;
FIG. 8A is a top view of another exemplary embodiment of a cutting feature of a grater suitable for use in an embodiment of the present invention
FIG. 8B is a cross-sectional view of the cutting feature of FIG. 8A;
FIG. 9A is a schematic diagram showing the formation of a cartilage fiber having striations by a cutting edge;
FIG. 9B is a schematic diagram showing the formation of discontinuous pieces of cartilage by a cutting edge;
FIGS. 10A, 10B and 10C are a series of diagrams showing how cutting edges having smaller cutting angles will produce less curved cartilage fibers;
FIGS. 11A-11B are schematic diagrams showing an exemplary embodiment of a cartilage-derived implant in accordance with the present invention, with and without protruding cartilage fibers, respectively; and
FIGS. 12A-12B are schematic diagrams showing the use of the cartilage-derived implant of FIG. 11 A, where FIG. 12B shows swelling of the cartilage-derived implant upon rehydration.
Detailed Description of the Invention
An embodiment of the present invention comprises a cartilage-derived implant including cartilage fibers. In an embodiment, the cartilage fibers include viable native chondrocytes. In an embodiment, the cartilage fibers are freeze-dried, and do not contain
CA 02939280 2016-08-18 measureable amounts of viable chondrocytes. In an embodiment, the cartilage-derived implant includes cartilage fibers and cartilage particles. Embodiments of the present invention include methods for producing cartilage fibers from recovered cartilage, including without limitation one or more condyles. Embodiments of the present invention also include methods of making the aforementioned cartilage-derived implants. Embodiments of the present invention also include methods of using the aforesaid cartilage-derived implants for the repair of cartilage defects. Non-limiting exemplary embodiments of the cartilage-derived implants of the present invention, and methods of making and using same, are discussed herein.
The cartilage-derived implants of the present invention have uses in the repair of defects in articular cartilage, costal cartilage, or other types of cartilage in a patient. The cartilage-derived implants of the present invention have uses in meniscal repair, sternotomy repair, the repair of subchondral bone, the repair of articulating joints, and the repair or replacement of cartilaginous anatomical features, including, but not limited to, nasal and auricular cartilage.
In an embodiment of the present invention, the cartilage-derived implant is a scaffold for the delivery of growth-inductive factors. In an embodiment, the cartilage-derived implant is growth-inductive. In an embodiment, the cartilage-derived implant is a scaffold for the delivery of cells. In an embodiment, the cartilage-derived implant is a scaffold for the migration of cells. In an embodiment, the cartilage-derived implant is a growth-conductive medium for the in-growth of tissue. In an embodiment, the cartilage-derived implant is a tissuegenic matrix.
I. Cartilage Fiber Implante
Cartilage fibers according to some embodiments of the present invention are derived from cartilage which is allogeneic to the patient. In an embodiment, cartilage fibers are derived from cartilage which is autologous to the patient. In an embodiment, cartilage fibers are derived from cartilage which is xenogeneic to the patient. In an embodiment, the cartilage includes articular cartilage. In an embodiment, the cartilage includes costal cartilage. In an embodiment of a method of making such cartilage fibers, cartilage is recovered from deceased human donors, and the tissue is treated to reduce bioburden according to methods known in the art. In an embodiment, the donor is a mature adult human donor. In an embodiment, the donor is a mature adult human donor having an age in the range of 20 to 55 years. In an embodiment, the donor is a human donor having an age of less than 20 years. In an embodiment, the donor is a juvenile human donor. Methods of selecting, recovering, and treating cartilage are known in the art, and exemplary methods are disclosed in co-owned U.S. Patent Nos. RE 42,208, RE 43,258, 8,292,968, 8,834,928, and 8,883,210.
In embodiments of the present invention, cartilage fibers are obtained by a dissection of the recovered cartilage so as to obtain fibers therefrom (e.g., by grating, scraping, slicing, or cutting) and collecting the fibers, as described in further detail hereinafter. The cartilage fibers are then stored in media. The cartilage fibers are cleaned by means of buffered saline rinses (e.g., phosphate buffered saline (PBS) solution) or other rinses that would remove or reduce the potential for antigenic reactions in a patient receiving the cartilage fibers. In an embodiment of the present invention, the cartilage fibers are cleaned by means that conserve the viability of chondrocytes within the cartilage fibers. The cartilage fibers may then be sieved or filtered to collect fibers having desirable dimensions for use in making the cartilage-derived implant.
In embodiments of the present invention, the cartilage fibers have lengths in the range of about 0.2 mm to about 50 mm. In an embodiment of the present invention, at least one 8
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CA 02939280 2016-08-18 of the cartilage fibers has a length in the range of about 0.2 mm to about 0.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 0.5 mm to about 1.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 1.0 mm to about 1.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 1.5 mm to about 2.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 2.0 mm to about 2.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 2.5 mm to about 3,0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 3.0 mm to about 3.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 3.5 mm to about 4.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 4.0 mm to about 4.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 4.5 mm to about 5.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 5.0 mm to about 5.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 5.5 mm to about 6.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 6.0 mm to about 6.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 6.5 mm to about 7.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 7.0 mm to about 7.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 7.5 mm to about 8.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 8.0 mm to about 8.5 mm. In an
CA 02939280 2016-08-18 embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 8.5 mm to about 9.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 9.0 mm to about 9.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 9.5 mm to about 10.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 10 mm to about 11 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 11 mm to about 12 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 12 mm to about 13 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 13 mm to about 14 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 14 mm to about 15 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 15 mm to about 16 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 16 mm to about 17 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 17 mm to about 18 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 18 mm to about 19 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 19 mm to about 20 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 20 mm to about 25 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 25 mm to about 30 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 30 mm to about 35 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the
CA 02939280 2016-08-18 range of about 35 mm to about 40 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 40 mm to about 45 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a length in the range of about 45 mm to about 50 mm.
In embodiments of the present invention, the cartilage fibers have an average length in the range of about 1 mm to about 20 mm. In an embodiment, the cartilage fibers have an average length of about 1 mm. In an embodiment, the cartilage fibers have an average length of about 2 mm. In an embodiment, the cartilage fibers have an average length of about 3 mm. In an embodiment, the cartilage fibers have an average length of about 4 mm. In an embodiment, the cartilage fibers have an average length of about 5 mm. In an embodiment, the cartilage fibers have an average length of about 6 mm. In an embodiment, the cartilage fibers have an average length of about 7 mm. In an embodiment, the cartilage fibers have an average length of about 8 mm. In an embodiment, the cartilage fibers have an average length of about 9 mm. In an embodiment, the cartilage fibers have an average length of about 10 mm. In an embodiment, the cartilage fibers have an average length of about 12 mm. In an embodiment, the cartilage fibers have an average length of about 14 mm. In an embodiment, the cartilage fibers have an average length of about 16 mm. In an embodiment, the cartilage fibers have an average length of about 18 mm. In an embodiment, the cartilage fibers have an average length of about 20 mm.
In an embodiment, the cartilage fibers have an average length of at least 1 mm. In an embodiment, the cartilage fibers have an average length of at least 2 mm. In an embodiment, the cartilage fibers have an average length of at least 3 mm. In an embodiment, the cartilage fibers have an average length of at least 4 mm. In an embodiment, the cartilage fibers have an average length of at least 5 mm. In an embodiment, the cartilage fibers have an
CA 02939280 2016-08-18 average length of at least 6 mm. In an embodiment, the cartilage fibers have an average length of at least 7 mm. In an embodiment, the cartilage fibers have an average length of at least 8 mm. In an embodiment, the cartilage fibers have an average length of about at least 9 mm. In an embodiment, the cartilage fibers have an average length of at least 10 mm. In an embodiment, the cartilage fibers have an average length of at least 12 mm. In an embodiment, the cartilage fibers have an average length of at least 14 mm. In an embodiment, the cartilage fibers have an average length of at least 16 mm. In an embodiment, the cartilage fibers have an average length of at least 18 mm. In an embodiment, the cartilage fibers have an average length of at least 20 mm.
In an embodiment, the cartilage fibers have an average length in the range of about 1 mm to about 3 mm. In an embodiment, the cartilage fibers have an average length in the range of about 2 mm to about 4 mm. In an embodiment, the cartilage fibers have an average length in the range of about 3 mm to about 5 mm. In an embodiment, the cartilage fibers have an average length in the range of about 4 mm to about 6 mm. In an embodiment, the cartilage fibers have an average length in the range of about 5 mm to about 7 mm. In an embodiment, the cartilage fibers have an average length in the range of about 6 mm to about 8 mm. In an embodiment, the cartilage fibers have an average length in the range of about 7 mm to about 9 mm. in an embodiment, the cartilage fibers have an average length in the range of about 8 mm to about 10 mm. In an embodiment, the cartilage fibers have an average length in the range of about 9 mm to about 11 mm. In an embodiment, the cartilage fibers have an average length in the range of about 10 mm to about 12 mm. In an embodiment, the cartilage fibers have an average length in the range of about 12 mm to about 14 mm. In an embodiment, the cartilage fibers have an average length in the range of about 14 mm to about 16 mm. In an embodiment, the cartilage fibers have an average length in the range of about 16 mm to about
CA 02939280 2016-08-18 mm. In an embodiment, the cartilage fibers have an average length in the range of about 18 mm to about 20 mm.
In embodiments of the present invention, the cartilage fibers have widths or thicknesses in the range of about 0.01 mm to about 5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.01 mm to about 0.05 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.05 mm to about 0.10 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.10 mm to about 0.15 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.15 mm to about 0.20 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.20 mm to about 0.25 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.25 mm to about 0.30 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.30 mm to about 0.35 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.35 mm to about 0.40 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.40 mm to about 0.45 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.45 mm to about 0.50 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.50 mm to about 0.55 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.55 mm to about 0.60 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.60 mm to about 0.65 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a
CA 02939280 2016-08-18 width in the range of about 0.65 mm to about 0.70 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.70 mm to about 0.75 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.75 mm to about 0.80 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.80 mm to about 0.85 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.85 mm to about 0.90 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.90 mm to about 0.95 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 0.95 mm to about 1.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.0 mm to about 1.1 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.1 mm to about 1.2 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.2 mm to about 1.3 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.3 mm to about 1.4 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.4 mm to about 1.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.5 mm to about 1.6 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.6 mm to about 1.7 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.7 mm to about 1.8 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.8 mm to about 1.9 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 1.9 mm to about 2.0 mm. In an embodiment of the present
CA 02939280 2016-08-18 invention, at least one of the cartilage fibers has a width in the range of about 2.0 mm to about 2.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 2.5 mm to about 3.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 3.0 mm to about 3.5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 3.5 mm to about 4.0 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 4.0 mm to about 4,5 mm. In an embodiment of the present invention, at least one of the cartilage fibers has a width in the range of about 4.5 mm to about 5.0 mm.
In embodiments of the present invention, the cartilage fibers have an average width in the range of about 0.1 mm to about 2 mm. In an embodiment, the cartilage fibers have an average width of about 0.1 mm. In an embodiment, the cartilage fibers have an average width of about 0.2 mm. In an embodiment, the cartilage fibers have an average width of about 0.3 mm. In an embodiment, the cartilage fibers have an average width of about 0.4 mm. In an embodiment, the cartilage fibers have an average width of about 0.5 mm. In an embodiment, the cartilage fibers have an average width of about 0.6 mm. In an embodiment, the cartilage fibers have an average width of about 0.7 mm. In an embodiment, the cartilage fibers have an average width of about 0.8 mm. In an embodiment, the cartilage fibers have an average width of about 0.9 mm. In an embodiment, the cartilage fibers have an average width of about 1.0 mm. In an embodiment, the cartilage fibers have an average width of about 1.2 mm. In an embodiment, the cartilage fibers have an average width of about 1.4 mm. In an embodiment, the cartilage fibers have an average width of about 1.6 mm. In an embodiment, the cartilage fibers have an average width of about 1.8 mm. In an embodiment, the cartilage fibers have an average width of about 2 mm.
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In an embodiment, the cartilage fibers have an average width of at least 0.1 mm.
In an embodiment, the cartilage fibers have an average width of at least 0.2 mm. In an embodiment, the cartilage fibers have an average width of at least 0.3 mm. In an embodiment, the cartilage fibers have an average width of at least 0.4 mm. In an embodiment, the cartilage fibers have an average width of at least 0.5 mm In an embodiment, the cartilage fibers have an average width of at least 0.6 mm. In an embodiment, the cartilage fibers have an average width of at least 0.7 mm. In an embodiment, the cartilage fibers have an average width of at least 0.8 mm. In an embodiment, the cartilage fibers have an average width of at least 0.9 mm. In an embodiment, the cartilage fibers have an average width of at least 1.0 mm. In an embodiment, the cartilage fibers have an average width of at least 1.2 mm. In an embodiment, the cartilage fibers have an average width of at least 1A mm. In an embodiment, the cartilage fibers have an average width of at least 1.6 mm. In an embodiment, the cartilage fibers have an average width of at least 1.8 mm. In an embodiment, the cartilage fibers have an average width of at least 2.0 mm.
In an embodiment, the cartilage fibers have an average width in the range of about 0.1 mm to about 0.3 mm. In an embodiment, the cartilage fibers have an average width in the range of about 0.2 mm to about 0.4 mm. In an embodiment, the cartilage fibers have an average width in the range of about 0.3 mm to about 0.5 mm. In an embodiment, the cartilage fibers have an average width in the range of about 0.4 mm to about 0.6 mm. In an embodiment, the cartilage fibers have an average width in the range of about 0.5 mm to about 0.7 mm. In an embodiment, the cartilage fibers have an average width in the range of about 0.6 mm to about 0.8 mm. In an embodiment, the cartilage fibers have an average width in the range of about 0.7 mm to about 0.9 mm. In an embodiment, the cartilage fibers have an average width in the range of about 0.8 mm to about 1.0 mm. In an embodiment, the cartilage fibers have an
CA 02939280 2016-08-18 average width in the range of about 0.9 mm to about 1.1 mm. In an embodiment, the cartilage fibers have an average width in the range of about 1.0 mm to about 1.2 mm. In an embodiment, the cartilage fibers have an average width in the range of about 1.2 mm to about 1.4 mm. In an embodiment, the cartilage fibers have an average width in the range of about 1.4 mm to about 1.6 mm. In an embodiment, the cartilage fibers have an average width in the range of about 1.6 mm to about 1.8 mm. In an embodiment, the cartilage fibers have an average width in the range of about 1.8 mm to about 2.0 mm.
In embodiments of the present invention, the cartilage fibers have individual volumes in the range of about 0.1 mm<sup>3</sup> to about 200 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 0.1 mm<sup>3</sup> to about 0.5 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 0.5 mm<sup>3</sup> to about 1.0 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 1.0 mm<sup>3</sup> to about 1.2 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 1.2 mm<sup>3</sup> to about 1.4 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 1.4 mm<sup>3</sup> to about 1.6 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 1.6 mm<sup>3</sup> to about 1.8 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 1.8 mm<sup>3</sup> to about 2.0 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 2.0 mm<sup>3</sup> to about 2.2 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 2.2 mm<sup>3</sup> to about 2.4 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 2.4 mm<sup>3</sup> to about 2.6 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 2.6 mm<sup>3</sup> to about 2.8 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 2.8 mm<sup>3</sup> to about 3.0 mm<sup>3</sup>. In an embodiment, at least one of the
CA 02939280 2016-08-18 cartilage fibers has a volume in the range of about 3.0 mm<sup>3</sup> to about 3.2 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 3.2 mm<sup>3</sup> to about 3.4 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 3.4 mm<sup>3</sup> to about 3.6 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 3.6 mm<sup>3</sup> to about 3.8 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 3.8 mm<sup>3</sup> to about 4.0 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 4.0 mm<sup>3</sup> to about 4.2 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 4.2 mm<sup>3</sup> to about 4.4 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 4.4 mm<sup>3</sup> to about 4.6 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 4.6 mm<sup>3</sup> to about 4.8 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 4.8 mm to about 5.0 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 5.0 mm<sup>3</sup> to about 5.5 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 5.5 mm<sup>3</sup> to about 6.0 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 6.0 mm<sup>3</sup> to about 6.5 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 6.5 mm<sup>3</sup> to about 7.0 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 7.0 mm<sup>3</sup> to about 7.5 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 7.5 mm<sup>3</sup> to about 8.0 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 8.0 mm<sup>3</sup> to about 8.5 mm<sup>3</sup>. In an
Q embodiment, at least one of the cartilage fibers has a volume in the range of about 8.5 mm to about 9.0 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 9.5 mm<sup>3</sup> to about 10 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 10 mm<sup>3</sup> to about 12 mm<sup>3</sup>. In an embodiment, at least one of the
CA 02939280 2016-08-18 cartilage fibers has a volume in the range of about 12 mm<sup>3</sup> to about 14 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 14 mm<sup>3</sup> to about 16 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 16 mm<sup>3</sup> to about 18 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 18 mm<sup>3</sup> to about 20 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 20 mm<sup>3</sup> to about 25 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 25 mm<sup>3</sup> to about 30 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 30 mm<sup>3</sup> to about 35 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 35 mm<sup>3</sup> to about 40 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 40 mm<sup>3</sup> to about 45 mm<sup>3</sup>. In an
A embodiment, at least one of the cartilage fibers has a volume in the range of about 45 mm to about 50 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 50 mm<sup>3</sup> to about 60 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 60 mm<sup>3</sup> to about 70 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 70 mm<sup>3</sup> to about 80 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 80 mm<sup>3</sup> to about 90 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 90 mm<sup>3</sup> to about 100 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 100 mm<sup>3</sup> to about 120 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 120 mm<sup>3</sup> to about 140 mm<sup>3</sup>. In an
A embodiment, at least one of the cartilage fibers has a volume in the range of about 140 mm to about 160 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 160 mm<sup>3</sup> to about 180 mm<sup>3</sup>. In an embodiment, at least one of the cartilage fibers has a volume in the range of about 180 mm<sup>3</sup> to about 200 mm<sup>3</sup>.
CA 02939280 2016-08-18
In embodiments of the present invention, the cartilage fibers have an average volume in the range of about 1 mm<sup>3</sup> to about 20 mm<sup>?</sup>. In an embodiment, the cartilage fibers have an average volume of about 1 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 1.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 1.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 1.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 1.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 2.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 2.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 2.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 2.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 2.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 3.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 3.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 3.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 3.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 3.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 4.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 4.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 5 0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 5.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 6.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 6.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 7.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 7.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 8.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 8.5 mm<sup>3</sup> In an embodiment, the cartilage fibers have an average volume of about 9.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average
CA 02939280 2016-08-18 volume of about 9.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 10 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 12 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 14 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 16 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 18 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of about 20 mm<sup>3</sup>.
In an embodiment, the cartilage fibers have an average volume of at least 1 mm<sup>3 </sup>In an embodiment, the cartilage fibers have an average volume of at least 1.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 1.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 1.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 1.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 2.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 2.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 2.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 2.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 2.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 3.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 3.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 3.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 3.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 3.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 4.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 4.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume of at least 5.0 mm<sup>3</sup>. In an
CA 02939280 2016-08-18 embodiment, the cartilage fibers have an average volume of at least
5.5 mm<sup>3</sup>.
In an embodiment, the cartilage fibers have an average volume of at least
6.0 mm.
In an embodiment, the cartilage fibers have an average volume of at least
6.5 » mm
In an embodiment, the cartilage fibers have an average volume of at least
7.0 mm
In an embodiment, the cartilage fibers have an average volume of at least
7.5 mm.
In an embodiment, the cartilage fibers have an average volume of at least
8.0 mm<sup>3</sup>.
In an embodiment, the cartilage fibers have an average volume of at least
8.5 mm<sup>3</sup>.
In an embodiment, the cartilage fibers have an average volume of at least
9.0 mm .
In an embodiment, the cartilage fibers have an average volume of at least
9.5 mm .
In an embodiment, the cartilage fibers have an average volume of at least mm .
In an embodiment, the cartilage fibers have an average volume of at least mm .
In an embodiment, the cartilage fibers have an average volume of at least mm.
In an embodiment, the cartilage fibers have an average volume of at least ^3 mm .
In an embodiment, the cartilage fibers have an average volume of at least * mm.
In an embodiment, the cartilage fibers have an average volume of at least 20 mm<sup>3</sup>.
In an embodiment, the cartilage fibers have an average volume in the range of about 1 mm<sup>3</sup> to about 1.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 1.2 mm<sup>3</sup> to about 1.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 1.4 mm<sup>3</sup> to about 1.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 1.6 mm<sup>3</sup> to about 2.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 1.8 mm<sup>3</sup> to about 2.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 2.0 mm<sup>3</sup> to about 2.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 2.2 mm<sup>3</sup> to about 2.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers
CA 02939280 2016-08-18 have an average volume in the range of about 2.4 mm<sup>3</sup> to about 2.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 2.6 mm<sup>3</sup> to about 3.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 2.8 mm<sup>3</sup> to about 3.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 3.0 mm<sup>3</sup> to about 3.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 3.2 mm<sup>3</sup> to about 3.6 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 3.4 mm<sup>3</sup> to about 3.8 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 3.6 mm<sup>3</sup> to about 4.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 3.8 mm<sup>3</sup> to about 4.2 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 4.0 mm<sup>3</sup> to about 4.4 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 4.2 mm<sup>3</sup> to about 5.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 4.5 mm<sup>3</sup> to about 5.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 5.0 mm<sup>3</sup> to about 6.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 5.5 mm<sup>3</sup> to about 6.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 6.0 mm<sup>3</sup> to about 7.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 6.5 mm<sup>3</sup> to about 7.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 7.0 mm<sup>3</sup> to about 8.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 7.5 mm<sup>3</sup> to about 9.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 8.0 mm<sup>3</sup> to about 10.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 8.5 mm<sup>3</sup> to about 10.5 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 9.0 mm<sup>3</sup> to about 11.0 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 9.5 mm<sup>3</sup> to about 11.5 mm<sup>3</sup>. In an
CA 02939280 2016-08-18 embodiment, the cartilage fibers have an average volume in the range of about 10 mm<sup>3</sup> to about 14 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 12 mm<sup>3</sup> to about 16 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 14 mm<sup>3</sup> to about 18 mm<sup>3</sup>. In an embodiment, the cartilage fibers have an average volume in the range of about 16 mm<sup>3</sup> to about 20 mm<sup>3</sup>.
In embodiments of the present invention, at least some of the cartilage fibers have an aspect ratio (length/width) in the range of about 1:1 to about 50:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 1:1 to about 2:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 2:1 to about 3:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 3:1 to about 4:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 4:1 to about 5:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 5:1 to about 6:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 6:1 to about 7:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 7:1 to about 8:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 8:1 to about 9:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 9:1 to about 10:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 10:1 to about 12:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 12:1 to about 14:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 14:1 to about 16:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 16:1 to about 18:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 18:1
CA 02939280 2016-08-18 to about 20:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 20:1 to about 25:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 25:1 to about 30:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 30:1 to about 35:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 35:1 to about 40:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 40:1 to about 45:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio in the range of about 45.Ί to about 50:1.
In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 1:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 2:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 3:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 4:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 5:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 6:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 7:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 8:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 9:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 10: 1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 12:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 14:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 16:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 18:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 20:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at
CA 02939280 2016-08-18 least 30:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 35:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 40:1. In an embodiment, at least some of the cartilage fibers have an aspect ratio of at least 45:1.
In an embodiment of the present invention, at least some of the cartilage fibers are ribbon-like, having, for example, a width/thickness ratio in the range of about 10:1 to about 500:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 10:1 to about 20:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 20:1 to about 40:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 40:1 to about 60:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 60:1 to about 80:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 80:1 to about 100:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 100:1 to about 150:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 150:1 to about 200:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 200:1 to about 250:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 250:1 to about 300:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 300:1 to about 350:1. In an embodiment, at least some of the ribbon-like 26
CA 02939280 2016-08-18 cartilage fibers have a width/thickness ratio in the range of about 350:1 to about 400:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio in the range of about 450:1 to about 500:1.
In an embodiment of the present invention, at least some of the cartilage fibers are ribbon-like, having, for example, a width/thickness ratio in the range of about 10:1 to about 500:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 10:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 20:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 40:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 60:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 80:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 100:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 150:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 200:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 250:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 300:1, In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 350:1. In an embodiment, at least some of the ribbon-like cartilage fibers have a width/thickness ratio of at least 450:1.
CA 02939280 2016-08-18
In an embodiment of the present invention, at least some of the cartilage fibers are thread-like, having, for example, a width/thickness ratio in the range of about 1:1 to about 10:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 1:1 to about 2:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 2:1 to about 3:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 3:1 to about 4:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 4:1 to about 5:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 5:1 to about 6:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 6:1 to about 7:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 7:1 to about 8:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 8:1 to about 9:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio in the range of about 9:1 to about 10:1.
In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 1:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 2:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 3:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness
CA 02939280 2016-08-18 ratio of at least 4:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 5:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 6:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 7:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 8:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 9:1. In an embodiment, at least some of the thread-like cartilage fibers have a width/thickness ratio of at least 10:1.
In embodiments of the present invention, the cartilage fibers may have crosssections of virtually any shape. In an embodiment, at least some of the cartilage fibers have cross-sectional shapes that are similar to each other. In an embodiment, at least some of the cartilage fibers have a cross-sectional shape having at least one straight edge. In an embodiment, at least some of the cartilage fibers have a cross-sectional shape having at least two straight edges. In an embodiment, at least some of the cartilage fibers have a crosssectional shape having at least two straight edges that intersect each other at an angle. In an embodiment, at least some of the cartilage fibers have a polygonal cross-sectional shape. In an embodiment, at least some of the cartilage fibers have a cross-sectional shape with at least one curved edge In an embodiment, at least some of the cartilage fibers have a semi-circular cross-sectional shape. In an embodiment, at least some of the cartilage fibers have a semielliptical cross-sectional shape. In an embodiment, at least some of the cartilage fibers have a circular cross-sectional shape. In an embodiment, at least some of the cartilage fibers have a crescent cross-sectional shape. In an embodiment, at least some of the cartilage fibers have an irregular cross-sectional shape.
CA 02939280 2016-08-18
In an embodiment, at least some of the cartilage fibers are similar to each other in their respective dimensions. In an embodiment, at least some of the cartilage fibers are a mixture of ribbon-like and thread-like cartilage fibers. In an embodiment, at least some of the cartilage fibers have selected dimensions and/or cross-sectional shapes. In an embodiment, the cartilage fibers have various lengths. In an embodiment, the cartilage fibers have various widths and thicknesses. In an embodiment, some of the cartilage fibers have long lengths and some of the cartilage fibers have short lengths. In an embodiment, at least some of the cartilage fibers have one cross-sectional shape and at least some of the cartilage fibers have a different cross-sectional shape. In an embodiment, the dimensions and/or cross-sectional shape of a cartilage fiber are generated by the tool selected to perform the blunt dissection of the recovered cartilage.
In an embodiment, the cartilage fibers are substantially free of cells and their components. In an embodiment, the cartilage fibers include viable native cells. In an embodiment, the cartilage fibers include non-viable native cells. Non-viable native cells may, for example without limitation, be present in the cartilage fibers when the cartilage fibers are produced from recovered cartilage that has been frozen. In an embodiment, the cartilage fibers include viable non-native cells. In an embodiment, the cartilage fibers include non-native nonviable cells. Cartilage fibers may, for example without limitation, include non-native cells (viable or non-viable) when they have been added or cultured on the cartilage fibers after production of the cartilage fibers. Native and non-native cells include, without limitation, chondrocytes and other types of cells.
In an embodiment, the cartilage fibers are in the form of a mass of cartilage fibers, which are then used to prepare implants that may be used to fill a cartilage defect in a patient. Such implants may be used as a standalone treatment device or be applied in
CA 02939280 2016-08-18 combination with one or more of a variety of bioactive materials or cells that facilitate the. reconstruction and healing of cartilage. The cartilage fibers serve as a matrix or scaffold for the remodeling of chondral tissue, resulting in the growth of new cartilage or of collagen fibrous material that fills spaces in the defect. Remodeling occurs along the edges of the fibers, which provide a greater number of remodeling sites relative to the number provided by other types of cartilage pieces.
In an embodiment, the cartilage fibers are manipulated to form a putty without adding a carrier or other biologically-compatible material. In an embodiment, the cartilage fibers are combined with a carrier or other biologically-compatible material to form a paste, putty or gel. In an embodiment, the cartilage fibers are combined with a carrier or other biologicallycompatible material such that the resulting mixture may be injected into a cartilage defect site in a patient. In an embodiment, the paste, putty, gel, or injectable mixture is used to fill a defect site, or are otherwise applied to a defect site or to a site of a diseased or defective cartilage, or to other tissues in a patient in need of such a paste, putty, gel, or injectable mixture.
In an embodiment, suitable carriers for forming a putty or an injectable mixture include, but are not necessarily limited to, the following: an aqueous buffer, an isotonic solution; a sodium chloride solution at a concentration of about 0.1% to about 1%, more particularly, about 0.9%; a lactated Ringer’s solution, with or without D5LR (dextrose); phosphate buffered saline (“PBS’’); platelet rich plasma (PRP); glycerin; lecithin; alginate; hyaluronic acid (HA); a derivative of HA; or sodium hyaluronate; or other suitable carriers known in the art. The carrier may also comprise biological components” added to the carrier, such as, but not limited to, DNA, RNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), micro RNA (mRNA), polysaccharides, peptides, matrix proteins, glycosaminoglycans (e.g, hyaluronic acid), viral vectors, and liposomes. The carrier further should maintain the stability and bioavailability of an
CA 02939280 2016-08-18 active agent, if such an active agent is added to the carrier. Biologically-compatible materials that are suitable for combination with the cartilage fibers include, but are not necessarily limited to, bone marrow aspirate (BMA), platelet rich plasma (PRP), growth factors, blood, synovial fluid, fibrin glue, and acellular bulking agents derived from soft tissues such as fascia, adipose tissue, placental tissues, or other soft tissues. In an embodiment, exogenous viable cells are added to the cartilage fibers, or to the putty, paste, gel, or injectable mixture. Such exogenous viable cells may include, but are not necessarily limited to, autologous or allogeneic chondrocytes, autologous cells such as those obtained from the patient’s bone marrow or stromal vascular fraction (SVF), allogeneic cells such as those obtained from a cell bank (e.g., stem cells, progenitor cells or other cell types available from cell banks), or bone marrow and bone marrow components including bone marrow cells (both autologous and allogeneic).
II. Producing Cartilage Fibers
As already mentioned, methods for preparing cartilage fibers suitable for use in producing cartilage-derived implants as described and contemplated herein include separating cartilage fibers by mechanical techniques selected from grating, scraping, slicing, cutting, or combinations thereof, from recovered cartilage, such as without limitation one or more condyles. The grating, scraping, slicing or cutting of the recovered cartilage may be performed using any suitable tools or devices, known now or in the future, that are capable of separating cartilage fibers from the recovered cartilage that have the desired dimensions and shapes. In some embodiments, for example without limitation, a grater is used to prepare the cartilage fibers from recovered cartilage held in a vice. As grating proceeds, the position of the recovered cartilage in the vice may be changed so as to allow the grater better access to the cartilage without bumping against the vice. Such graters are commercially available from various sources for performing the desired grating operation, including: Kitchen Aid of Benton Harbor, Michigan,
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U.S.A.; Oxo located Chambersburg, Pennsylvania, U.S.A.; Microplane of McAllen, Texas, U.S.A.; Chefn, a division of Taylor Precision Products located in Las Cruces, New Mexico, U.S.A.; and Cuisinait of East Windsor, New Jersey, U.S.A. In some embodiments, commercially available graters are modified to perform the desired grating operation. In some embodiments, the graters are custom designed and fabricated to perform the desired grating operation. The characteristics (e.g., size, shape, degree of pliability or stiffness, degree of curling, quantity of viable chondrocytes, etc.) of cartilage fibers appear to be related to the type of grater used, as well as the characteristics of the cutting features of the grater, as will be discussed in further detail hereinafter.
Different techniques may be employed to form the cutting features of graters such as, without limitation, die stamping, acid etching, microplaning, laser etching, and electroforming, regardless of the type of grater. Applicants have surprisingly discovered that differences in cutting feature forming techniques affect certain characteristics of the resulting cartilage fibers. For example, Applicants have surprisingly discovered that cartilage fibers prepared using a grater having cutting features formed by die stamping techniques are rougher and have more striations, are more curled, more pliable, and swell less upon hydration. Similarly, Applicants have surprisingly found that cartilage fibers prepared using a grater having cutting features formed by acid etching techniques are smoother (fewer or no striations), less curled, more stiff (less pliable), and swell more upon hydration. Additionally, Applicants have surprisingly found that cartilage fibers prepared using a grater having cutting features formed by acid etching techniques have a greater quantity of viable chondrocytes, while cartilage fibers prepared using a grater having cutting features formed by die stamping techniques have a fewer viable chondrocytes. While cartilage fibers produced with acid etched cutting features are easier to cut and contain more viable chondrocytes, they also swell more upon rehydration. It is
CA 02939280 2016-08-18 preferred, when the fibers are formed into an implant and implanted in a cartilage defect, that the implant remains substantially flush with the native surface of the cartilage having the defect. “Substantially flush” as used herein means that the implant swells less than 2 millimeters beyond or above the native surface of the cartilage having the defect after the implant is implanted therein. Cartilage fibers produced with die stamping techniques have better handling characteristics, but fewer viable chondrocytes. Thus, persons of ordinary skill in the art will recognize that selection and balancing among the various possible characteristics of cartilage fibers is possible and may be advantageous when selecting the type of cutting tool to use.
FIGS. 4 and 5 show two exemplary types of graters suitable for performing the methods for preparing cartilage fibers described and contemplated herein. FIG. 4 shows the top view of a planar grater 10 having a handle 12, a convex planar body 14, and a plurality of cutting features 16 on the planar body 14. Another type of grater, i.e., a rotary grater 110, is shown in FIG 5 having a handle 112 adapted for connection with a drill device (not shown) and a cylindrical body 114 with a plurality of cutting features 116 on the cylindrical body 114. As easily determinable by persons of ordinary skill in the art, other types of graters are also suitable for preparing cartilage fibers from recovered cartilage according to the methods described and contemplated herein. The cutting features 16,116 shown on the graters 10,110 of FIGS. 4 and 5 were formed by a die stamping technique, which is common and well-known in the industry of grater manufacture. However, the cutting features could have been made by any technique known now or in the future, such as those mentioned above. Applicants have surprisingly found that using a rotary type of grater produces cartilage fibers having a more consistent fiber length compared to cartilage fibers produced using a planar grater.
With reference now to FIGS. 6A and 6B, FIG. 6A shows a perspective view of an individual cutting feature 16 and FIG. 6B shows a front cross-sectional view of that cutting
CA 02939280 2016-08-18 feature 16 taken along line I-I and looking in the direction of the arrows. Each individual cutting feature 16 has a configuration which includes an opening 20 and a raised cutting surface 22 with a cutting edge 24. As shown, the cutting surface 22 and cutting edge 24 of this cutting feature 16 have a curved or arced shape, which produces cartilage fibers having a commensurately sized fiber thickness.
Applicants have also explored modifications to the cutting features 216 to create modified cutting surfaces 222 and cutting edges 224, as shown for example in FIGS. 6C and 6D, in which the cutting surface 222 has a flattened portion 226. FIG. 6D Shows a front crosssectional view of the cutting feature 216 of FIG. 6C taken along line ΙΠ-ΠΙ and looking in the direction of the arrows. As shown in FIG. 6D, the cutting edge 224 of such modified cutting features 216 also has a flattened portion 228 which produces cartilage fibers having a commensurately smaller sized fiber thickness than those produced with the unmodified cutting features 16. Although not shown per se, other modifications may be made to the cutting features 16 such as making the cutting surface 22 smaller, which in turn makes the opening 20 larger and the cutting edge 24 shorter and less curved, which produced cartilage fibers having a commensurately smaller sized fiber thickness than those produced with the unmodified cutting features 16.
With reference to FIG. 7, Applicants also explored modifications to the cutting edge 324 of cutting features 316 whereby cutting angles (a1, a2, a3, a4, a5, a6) of different sizes are created on the cutting edge 324. Various cutting angle sizes were created and investigated. FIG. 7 shows a series of lateral cross-sectional views (#1, #2, #3, #4, #5, #6) of a cutting feature 316, of the type shown in FIG. 6A, taken along line II-II and looking in the direction of the arrows. As shown, the cutting edges 324 of various cutting features 316 were modified to have cutting angles (α1, a2, a3, a4, a5. a6) of various sizes. More particularly, in
CA 02939280 2016-08-18 one embodiment (#1), the cutting edge 24 has a cutting angle a1 of -20°. In another embodiment (#2), the cutting edge 24 has a cutting angle a2 of -5<sup>e</sup>. In another embodiment (#3), the cutting edge 24 has a cutting angle a3 of -35°. In another embodiment (#4), the cutting edge 24 has a cutting angle a4 of 0<sup>e</sup>. In another embodiment (#5), the cutting edge 24 has a cutting angle a5 of 10°. In another embodiment (#6), the cutting edge 24 has a cutting angle αδ of 20°. Of course, as will be recognized by persons of ordinary skill in the art, the exemplary embodiments shown in FIG. 7 are not the only possible configurations, and cutting angles (a1, a2, a3, a4, a5, a6) of other sizes are possible and suitable for use in the methods described and contemplated herein.
With reference now to FIGS. 8A · 8B, other configurations of cutting features 416 are possible. FIG 8A shows a top plan view of a cutting feature 416 having an alternative configuration and FIG. 8B shows a front cross-sectional view of the cutting feature 216 of FIG. 6C taken along line 1V-IV and looking in the direction of the arrows. More particularly, in some embodiments, individual cutting features 416 each comprise an opening 420 with a planar cutting surface, such as a blade 422, and a linear cutting edge 424 on the blade 422. For cutting features such as these 416, the size of the blade angle β may be varied, which will commensurately change the size of the cartilage fibers produced. For example, a cutting feature 416 having a smaller blade angle β is expected to produce cartilage fibers having a smaller fiber thickness than a cutting feature 416 having a larger blade angle β. Additionally, the cutting angle a7 of these cutting features 416 may be modified to be any one of various sizes, in the manner of the modifications to the cutting angle (a1, a2, a3, a4, a5, a6) of the cutting features 316 described above in connection with FIG. 7.
It has been discovered that aspects of the cutting process have an effect on how curled the resulting cartilage fibers become during production. It is important, for example, to
CA 02939280 2016-08-18 keep the recovered cartilage moist during production of cartilage fibers using cutting tools. With reference to FIG. 9A, when the recovered cartilage 530 is kept moist during the fiber production process by periodic or continuous addition of water or another biocompatible solvent or solution, including but not limited to saline or buffered saline, the cartilage fibers 532 produced using a cutting tool (not shown) with a cutting edge 534 will be continuous or semi-continuous, often with striations 536 and curled, as shown in FIG. 9A. During the cutting of the cartilage fiber 532 from the recovered cartilage 530 by the action of the cutting edge 534 of the cutting tool, a high shear strain zone HS is created in the fiber 532 first, followed by a low shear strain zone LS. As shown in FIG. 9B, if the recovered cartilage 630 is too dry, discontinuous cartilage pieces 638a, 638b, 638c, 638d will likely be created by the action of the cutting edge 634. Also, as shown in FIGS. 10A, 10B and 10C, the smaller the cutting angle a8, a9, alO of the cutting edge 734a, 734b, 734c, respectively, of the cutting tool (not shown), the less curled the cartilage fibers 732a, 732b, 732c will be. Continuous or semi-continuous cartilage fibers 532 having striations 536 and being curled are generally preferred over discontinuous pieces and cartilage fibers that are more curled are generally preferred over less curled ones, for reasons discussed below in connection with formation of implants comprising the cartilage fibers.
As mentioned previously, the cartilage fibers are generally in the form of a mass of cartilage fibers which is then manipulated, with or without carrier or other biologicallycompatible material, into a paste, putty, or gel that may then be used to fill a cartilage defect in a patient. For example, a mass of cartilage fibers may be manipulated into a more cohesive cartilage-derived implant 840, such as shown in FIG. 11A Such a cartilage-derived implant 840 behaves like a paste, putty, or gel in that it is deformable to different shapes, but the fibers remain intertwined with one another and cohesive, so the cartilage-derived implant can be applied to a defect site or to a site of diseased or defective cartilage without coming apart or
CA 02939280 2016-08-18 falling out of the defect. It is preferred that after manipulation to form the cartilage-derived implant, no cartilage fibers protrude from the surface of the implant. For example, FIG. 11B shows a cartilage-derived implant 940 having cartilage fibers 932 undesirably protruding from its surface. A smooth and substantially homogenous surface is most beneficial for the cartilagederived implants (as shown in FIG. 11 A). It has been surprisingly found by Applicants that cartilage fibers that have striations and are curled perform better in this respect to form a cartilage-derived implant without fibers protruding from its surface.
FIG. 12A provides a schematic side cross-sectional view of cartilage 1050 having a defect 1052 which has been filled with a cartilage-derived implant 1040 according to the methods described and contemplated herein. Generally the implant 1040 is pushed into the defect until its outward facing surface 1042 is flush or level with the native surface 1054 of the healthy or undamaged portion cartilage 1050. If the implant is thereafter hydrated either by exposure to biological fluids of the patient or purposeful addition of fluids by a surgeon or other user of the implant 1040, the implant 1040 may swell, as shown in FIG. 12B, and the outward facing surface 1042 of the implant 1040 may undesireably protrude from the native surface 1054 of the cartilage 1050. Thus, the swelling of the implant 1040 after rehydration is generally not preferred. As mentioned above, Applicants have determined that cartilage fibers produced using a grater having cutting features formed by die stamping techniques have better handling characteristics, including more curling and having striations, which features are believed to minimize the protrusion of individual cartilage fibers from the surface of a cartilage-derived implant comprising such fibers.
III. Cartilage Fibers Having Viable Cells
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In an embodiment of the present invention, the aforementioned dissection of the recovered cartilage to produce cartilage fibers is performed so as to preserve viable cells within the cartilage fibers (hereinafter referred to as “fresh cartilage fibers”). For example, in an embodiment of the present invention, a cutting tool having cutting features formed by acid etching techniques is used to produce cartilage fibers having viable cells. In an embodiment, about 90-100% of the cells in the fresh cartilage fibers are viable. Fresh cartilage fibers are those which include viable cells and have not been subjected to lyophilizing or freezing. The fresh cartilage fibers may then be used in the operating room to prepare a cartilage-derived implant, or packaged in such a way that the viability of the cells in the cartilage fibers is maintained. The packaged fibers may then be recovered for use in a cartilage-derived implant, as described elsewhere herein. In an embodiment, more than 50% of the cells in the recovered fresh cartilage fibers are viable. In an embodiment, about 50% to about 70% of the cells in the recovered fresh cartilage fibers are viable. In an embodiment, more than 85% of the cells in the recovered fresh cartilage fibers are viable. In an embodiment, about 85% to about 95% of the cells in the recovered fresh cartilage fibers are viable. FIG. 1 is a block diagram of steps in a process for preparing fresh cartilage fibers, which may be understood with reference to Sections I and II of the present disclosure.
Turning to methods of packaging and storing the fresh cartilage fibers, various conditions are possible. For example, in an embodiment, fresh cartilage fibers may be stored in a growth media or other media that is suitable for maintaining the viability of the cells within the fresh cartilage fibers. In an embodiment, the fresh cartilage fibers are stored with a serum. In an embodiment, the fresh cartilage fibers are stored without a serum. In an embodiment, the fresh cartilage fibers are stored at room temperature, such as from about 15°C to about 30’C. In an embodiment, the fresh cartilage fibers are stored at a refrigeration temperature, such as
CA 02939280 2016-08-18 from about 2’C to less than 15°C, such as, without limitation, from about 4°C to about 10'C. In an embodiment, the fresh cartilage fibers are stored for a period of time from about 1 hour to less than about 7 days. In an embodiment, the fresh cartilage fibers are stored for a period of time from about 7 days to about 3 months. In an embodiment, the fresh cartilage fibers are stored for a period of time from greater than about 3 months to about 6 months. In an embodiment, the fresh cartilage fibers are stored for a period of time from greater than about 6 months to about 9 months. In an embodiment, the fresh cartilage fibers are stored for a period of time from greater than about 9 months to about 12 months. In an embodiment, the fresh cartilage fibers are stored for a period of time greater than about 12 months.
In an embodiment, the fresh cartilage fibers are stored under cryogenic conditions. In an embodiment, cryogenic conditions are achieved using controlled-rate freezing. In an embodiment, cryogenic conditions are achieved using freezing at an un-controlled rate. In an embodiment, the fresh cartilage fibers are stored under cryogenic conditions in at least one cryopreservative. In an embodiment, the at least one cryopreservative is a solution. According to an embodiment, the cryopreservative is dimethylsulfoxide (DMSO). According to an embodiment, the cryopreservative is basal media solution comprising about 5% DMSO. According to an embodiment, the cryopreservative is basal media solution comprising about 10% DMSO. According to an embodiment, the cryopreservative is basal media solution comprising about 15% DMSO. According to an embodiment, the cryopreservative is fetal bovine serum comprising about 5% DMSO. According to an embodiment, the cryopreservative is fetal bovine serum comprising about 10% DMSO. According to an embodiment, the cryopreservative is a human serum comprising about 15% DMSO. According to an embodiment, the cryopreservative is human serum comprising about 5% DMSO. According to an embodiment, the cryopreservative is human serum comprising about 10% DMSO.
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According to an embodiment, the cryopreservative includes ethylene glycol. According to an embodiment, the cryopreservative includes propylene glycol. According to an embodiment, the cryopreservative includes glycerol. In an embodiment, the cryopreservative is another cryopreservative known in the art for maintaining the viability of cells at cryogenic or other subfreezing temperatures (i.e., a temperature below 0°C or below). In an embodiment, the fresh cartilage fibers are cooled to a subfreezing temperature. In an embodiment, the fresh cartilage fibers are stored cryogenically at the temperature of liquid nitrogen (e.g., at a temperature of about -196 C). In an embodiment, the fresh cartilage fibers are stored cryogenically at a temperature in the range of about -20°C to about -90*0.
The packaged fresh cartilage fibers may be recovered according to methods that preserve the viability of the cells in the fresh cartilage fibers and are appropriate to the media in which they are stored. In an embodiment, the packaged fresh cartilage fibers are brought to room temperature in the media. In an embodiment, the frozen or cryogenically-preserved fresh cartilage fibers and the media are thawed. In an embodiment, the fresh cartilage fibers are separated from the media by sedimentation. In an embodiment, the fresh cartilage fibers are separated from the media by centrifugation. In an embodiment, the fresh cartilage fibers are separated from the media by decantation. In an embodiment, the fresh cartilage fibers are separated from the media by filtration. In an embodiment, the fresh cartilage fibers are separated from the media by gravity filtration. In an embodiment, the fresh cartilage fibers are separated from the media by vacuum filtration. In an embodiment, the fresh cartilage fibers are separated from the media by capturing the fresh cartilage fibers in a sieve.
In an embodiment, the separated fresh cartilage fibers are rinsed to remove media from the fibers. In an embodiment, the separated fresh cartilage fibers are rinsed with a liquid. In an embodiment, the liquid comprises a physiological saline solution. In an
CA 02939280 2016-08-18 embodiment, the liquid comprises a buffered isotonic solution. In an embodiment, the physiological saline solution is a phosphate-buffered saline (PBS) solution. In an embodiment, the fresh cartilage fibers are rinsed at a temperature in the range from about 4°C to about 10°C. In an embodiment, the fresh cartilage fibers are rinsed at a temperature in the range from about 10°C to about 20°C. In an embodiment, the fresh cartilage fibers are rinsed at a temperature in the range from about 20’C to about 30’C. In an embodiment, the fresh cartilage fibers are rinsed at a temperature in the range from about 30’C to about 45°C. In an embodiment, the fresh cartilage fibers are rinsed at a temperature of about 25’C. In an embodiment, the fresh cartilage fibers are rinsed at an ambient temperature. In an embodiment, the fresh cartilage fibers are rinsed at body temperature (i.e., from about 35’C to about 45’C).
IV. Dehydrated or Lyophilized Cartilage Fibers
In an embodiment of the present invention, the fresh cartilage fibers are dehydrated or lyophilized before being packaged or used. In an embodiment, dehydration or lyophilization devitalizes the cartilage fibers (i.e., viable cells are removed or killed). In an embodiment, the dehydrated or lyophilized cartilage fibers have an amount of residual moisture that maintains the integrity of the tissue, but does not permit biological activity.
In an embodiment, the fresh cartilage fibers are dehydrated without being frozen. In an embodiment, the fresh cartilage fibers are air-dried. In an embodiment, the fresh cartilage fibers are dehydrated under vacuum. In an embodiment, the fresh cartilage fibers are dehydrated at a temperature in the range from about 4’0 to about 10°C. In an embodiment, the fresh cartilage fibers are dehydrated at a temperature in the range from about 10°C to about 20’C. In an embodiment, the fresh cartilage fibers are dehydrated at a temperature in the range from about of about 20°C to about 30’C. In an embodiment, the fresh cartilage fibers are
CA 02939280 2016-08-18 dehydrated at a temperature of about 25°C. In an embodiment, the fresh cartilage fibers are dehydrated at a temperature that is no greater than 35°C. In an embodiment, the fresh cartilage fibers are dehydrated at a temperature that is no greater than 40°C. In an embodiment, the fresh cartilage fibers are dehydrated at a temperature that is no greater than 50°C. In an embodiment, the fresh cartilage fibers are dehydrated at an ambient temperature. In an embodiment, the fresh cartilage fibers are dehydrated at body temperature (i.e., from about 35°C to about 45°C). In an embodiment, the dehydrated cartilage fibers have residual moisture in the range of about 0% to about 20%. In an embodiment, the dehydrated cartilage fibers have residual moisture in the range of about 6% to about 15%.
In an embodiment, the fresh cartilage fibers are dried in a frozen state (i.e., “freeze-dried or “lyophilized”). Lyophilization methods suitable for use in the present invention are those known now or in the future to persons of ordinary skill in the relevant art. In an embodiment, the lyophilized cartilage fibers have residual moisture of about 6% or less, such as about 5% or less, or about 4% or less, or about 3% or less, by weight based on the total weight of the lyophilized cartilage fibers.
In an embodiment, the dehydrated or lyophilized cartilage fibers are packaged in a container without the addition of other materials. In an embodiment, the dehydrated or lyophilized cartilage fibers are stored at a temperature in the range from about 2°C to about 15°C, such as, without limitation, from about 4’C to about 10<sup>û</sup>C. In an embodiment, the dehydrated or lyophilized cartilage fibers are stored at an ambient temperature.
In an embodiment, the dehydrated or lyophilized cartilage fibers are rehydrated, then manipulated to form a putty without adding a carrier or other biologically-compatible material. In an embodiment, the dehydrated or lyophilized cartilage fibers are rehydrated using
CA 02939280 2016-08-18 a rehydration solution known in the art. In an embodiment, the dehydrated or lyophilized cartilage fibers are rehydrated with a biologically compatible buffer solution. In an embodiment, the dehydrated or lyophilized cartilage fibers are rehydrated with PBS. In an embodiment, the dehydrated or lyophilized cartilage fibers are rehydrated with Ringer solution. In an embodiment, the rehydration solution includes a preservative, which may include, but is not necessarily limited to, mannitol, ascorbic acid, and glucose.
V. Implants Comprising Cartilage Fibers and Cartilage Particles
In an embodiment of the present invention, a cartilage-derived implant includes both cartilage fibers and cartilage particles. In an embodiment, the cartilage fibers are fresh cartilage fibers, as described in Section III above. In an embodiment, the cartilage fibers are dehydrated, lyophilized, or rehydrated cartilage fibers, as described in Section IV above. FIG. 2 is a block diagram of steps in a method of preparing a cartilage-derived implant from fresh cartilage fibers and lyophilized cartilage particles, which may be understood in conjunction with
FIGS. 1 and 3 and Sections I, II, III and IV of the present disclosure. In an embodiment, the cartilage fibers and the cartilage particles are from the same donor. In an embodiment, the cartilage fibers and the cartilage particles are from different donors. In an embodiment, the cartilage fibers and the cartilage particles are from the same species. In an embodiment, the cartilage fibers and the cartilage particles are from different species. In an embodiment, the cartilage fibers and the cartilage particles are from the same species but the recipient is a different species.
In an embodiment of the present invention, the cartilage particles are derived from articular cartilage recovered from a human donor. In an embodiment, the cartilage particles are derived from meniscal cartilage recovered from a human donor. In an embodiment, the cartilage particles are derived from costal cartilage recovered from a human donor. In an embodiment, the cartilage particles are derived from hyaline cartilage recovered from a human donor. In an embodiment, the cartilage particles are derived from other cartilage recovered from a human donor. In an embodiment, the donor is a mature adult human donor having an age in the range of 20 to 55 years. In an embodiment, the donor is a human donor having an age of less than 20 years. In an embodiment, the donor is a juvenile human donor.
FIG. 3 is a block diagram of steps in a method of preparing cartilage particles useful in the present invention. Additional methods of selecting, recovering, and treating cartilage are known in the art, and exemplary methods are disclosed in co-owned U.S. Patent Nos. RE 42,208, RE 43,258, 8,292,968, 8,834,928, and 8,883,210.
Methods of reducing cartilage to particulate form are also known in the art, and exemplary methods are disclosed in the aforesaid patents. Additionally, methods for making and using cartilage particles are also disclosed in U.S. Patent Application Publication Nos. 2006/0210643, 2008/0133008, 2011/0070271 and 2011/0196508.
In an embodiment of the present invention, the cartilage particles include viable cells (hereinafter, “fresh cartilage particles). In an embodiment, the cartilage particles are dehydrated cartilage particles having residual moisture in the range of about 6% to about 15%. In an embodiment, the cartilage particles are lyophilized cartilage particles having residual moisture of about 6% or less.
In an embodiment of the present invention, the cartilage particles have sizes in the range of 212 pm and less. In an embodiment, the cartilage particles have sizes in the range
Date Reçue/Date Received 2022-01-10
CA 02939280 2016-08-18 of 500 pm and less. In an embodiment, the cartilage particles have sizes in the range of about 50 pm to about 212 pm.
In an embodiment of the present invention, the cartilage-derived implant comprises only cartilage fibers and no cartilage particles (i.e., a fiberparticle ratio of about 100/0 (w/w)). In an embodiment, the cartilage-derived implant comprises cartilage fibers and cartilage particles in a ratio of about 95/5 (w/w), a ratio of about 90/10 (w/w), a ratio of about 85/15 (w/w), a ratio of about 80/20 (w/w), a ratio of about 75/25 (w/w), a ratio of about 70/30 (w/w), a ratio of about 65/35 (w/w), a ratio of about 60/40 (w/w), a ratio of about 55/45 (w/w), a ratio of about 50/50 (w/w), a ratio of about 45/55 (w/w), a ratio of about 40/60 (w/w), a ratio of about 35/65 (w/w), a ratio of about 30/70 (w/w), a ratio of about 25/75 (w/w), a ratio of about 20/80 (w/w), a ratio of about 15/85 (w/w), a ratio of about 10/90 (w/w), a ratio of about 5/95 (w/w), or at some other ratio between 100/0 (w/w) and less 0/100 (w/w), where the w/w ratio is the ratio of the weight of the cartilage fibers to the weight of the cartilage particles.
In an embodiment of the present invention, the cartilage-derived implant consists of cartilage fibers and cartilage particles. In an embodiment of the present invention, the cartilage-derived implant comprises cartilage fibers, cartilage particles, and at least one additional component. In an embodiment, the at least one additional component is a carrier or another biologically-compatible material. In an embodiment, suitable carriers and biologicallycompatible materials include, but are not necessarily limited to, the following: an aqueous buffer, an isotonic solution; a sodium chloride solution at a concentration of about 0.1% to about 1%, more particularly, about 0.9%; a lactated Ringer’s solution, with or without D5LR (dextrose); phosphate buffered saline (“PBS”); platelet rich plasma (PRP); glycerin; lecithin; alginate; hyaluronic acid (HA); a derivative of HA; or sodium hyaluronate; or other suitable carriers known in the art. The carrier may also comprise “biological components” added to the carrier, such as,
CA 02939280 2016-08-18 but not limited to, DNA, RNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), micro RNA (mRNA), polysaccharides, peptides, matrix proteins, glycosaminoglycans (e.g, hyaluronic acid), viral vectors, and liposomes. Biologically-compatible materials that are not necessarily inert, but are also suitable for combination with the cartilage fibers and cartilage particles include, but are not necessarily limited to, bone marrow aspirate (BMA), platelet rich plasma (PRP), growth factors, blood, synovial fluid, fibrin glue, and acellular bulking agents derived from soft tissues such as fascia, adipose tissue, placental tissues, or other soft tissues. In an embodiment, exogenous viable cells are added to the cartilage fibers and cartilage particles. Such exogenous viable cells may include, but are not necessarily limited to, autologous or allogeneic chondrocytes, autologous cells such as those obtained from the patient's bone marrow or stromal vascular fraction (SVF) allogeneic cells such as those obtained from a cell bank (e.g., stem cells, progenitor cells or other cell types available from cell banks), or bone marrow and bone marrow components including bone marrow cells (both autologous and allogeneic).
In an embodiment of the present invention, the cartilage fibers are provided separately from the cartilage particles (e.g., in a kit). In an embodiment, the cartilage fibers and cartilage particles are provided separately from each other, and are combined to make the cartilage-derived implant in the operating room prior to being administered to a patient in need thereof. In an embodiment, the cartilage fibers and cartilage particles are combined with a carrier or other biologically-compatible material to make a paste. In an embodiment, the cartilage fibers and cartilage particles are combined with a carrier or other biologicallycompatible material to make a putty. In an embodiment, the cartilage fibers are combined with a carrier or other biologically-compatible material to make a paste. In an embodiment, the cartilage fibers are combined with a carrier or other biologically-compatible material to make a
CA 02939280 2016-08-18 strip. In an embodiment, the cartilage fibers are combined with a carrier or other biologicallycompatible material to make a gel. In an embodiment, the cartilage fibers and cartilage particles are combined with a earner or other biologically-compatible material to make a mixture that is injectable into a cartilage defect.
In an embodiment of the present invention, the cartilage-derived implant is prepared, and then stored under conditions that preserve the viability of native or added cells. The cartilage-derived implant is then thawed and warmed to ambient or physiological temperatures before being administered to a patient in need thereof. Suitable storage conditions include those discussed in Section III of the present disclosure with respect to fresh cartilage fibers.
VI. Shaped Cartilage-Derived Implants
In an embodiment of the present invention, shaped cartilage-derived implants include three-dimensional shaped structures formed by a process in which articular cartilage is broken into fibers, then reformed into a three-dimensional structure. Suitable cartilage fibers may be prepared using methods disclosed in Sections I and II of the present application or by other methods that may be known in the art. Suitable cartilage fibers may be fresh cartilage fibers, dehydrated cartilage fibers, frozen cartilage fibers, cartilage fibers including non-viable cells, lyophilized cartilage fibers, or rehydrated cartilage fibers.
In an embodiment of the present invention, cartilage fibers are suspended in a liquid (e.g., water or a buffer solution) to form a flowable mass, such as a slurry. The flowable mass may be poured into a mold of a desired shape, in which it may form a porous or spongelike shaped acellular tissue upon dehydration or lyophilization. In an embodiment, a mass of cartilage fibers is manipulated to form a putty, which can then be molded into a desired shape.
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In an embodiment, the cartilage fibers are manipulated with a liquid to form a paste. In an embodiment, the cartilage fibers are mixed with a polymer to form a paste or a putty. In an embodiment, a shaped cartilage-derived implant is formed by dehydrating the slurry, paste or gel In an embodiment, a shaped cartilage-derived implant is formed by lyophilizing the slurry, paste or gel. In an embodiment, the density or porosity of a shaped cartilage-derived implant is controlled by selecting the amount of carrier or other liquid relative to the amount of cartilage fiber. In an embodiment, the shaped cartilage-derived implant is a solid piece that conforms to the shape of a mold after being dehydrated or lyophilized. In an embodiment, a shaped cartilage-derived implant may be provided in a lyophilized, dehydrated, cryopreserved, or frozen form.
In embodiments of the present invention, the liquids added to tissue before and/or during the molding process could optionally contain therapeutic factors, cytokines, growth factors, pharmaceuticals, antibiotics, free-radical scavengers, sugars, vitamins including, but not limited to, riboflavin and ascorbic acid, surfactants, DMEM medium, human or animal serum, or other additives. The addition or removal of liquid from the tissue also allows the density of the final implant to be controlled.
In an embodiment of the present invention, the shaped cartilage-derived implant includes cartilage fibers that have been aligned to form a sheet. In an embodiment, the shaped cartilage-derived implant includes cartilage fibers that have been interwoven to form a sheet. In an embodiment, the shaped cartilage-derived implant includes a mass of cartilage fibers that have been pressed to form a sheet. In an embodiment, the shaped cartilage-derived implant includes cartilage fibers that have been placed into a mold to form a pre-shaped implant. In an embodiment, the pre-shaped implant is a sheet. In an embodiment, the pre-shaped implant is a block. In an embodiment, the pre-shaped implant is a disk. In an embodiment, the pre-shaped
CA 02939280 2016-08-18 implant is a dome. In an embodiment, the pre-shaped implant is sphere. In an embodiment, the pre-shaped implant is portion of a sphere.
In an embodiment, shaped cartilage-derived implants may be used to surgically repair cartilage defects in a patient or worn or damaged cartilage in a patient. In an embodiment, shaped cartilage-derived implants are used alone or after being seeded or cultured with appropriate exogenous cells, such as the exogenous cells identified with respect to the cartilage-derived implants of Sections I, II and V of the present disclosure. In an embodiment, the shaped cartilage-derived implants are provided with bioactive components such as growth factors, proteins, angiogenic factors, and other bioactive components such as those discussed with respect to the cartilage-derived fiber implants of Sections I, II and V of the present disclosure. In an embodiment, one or more biologically-compatible substances are added to the shaped cartilage-derived implant, such as carriers or other biologically-compatible substances that are discussed with respect to the cartilage-derived implants of Sections I, II and V of the present disclosure. In an embodiment, the exogenous cells, bioactive components, or biologically-compatible substances are added to the slurry, putty, paste, gel, or cartilage fiber mass or sheet before the shaped cartilage-derived implant is formed. In an embodiment, the exogenous cells, bioactive components, or biologically-compatible substances are added to the shaped cartilage-derived implant after it is shaped, but before it is lyophilized, dehydrated, cryopreserved, or frozen. In an embodiment, the degradation profile of the shaped cartilagederived implant and a substance added thereto cause the substance to be released at an appropriate time for growth or healing of tissues to occur.
In an embodiment of the present invention, the shaped cartilage-derived implant has a simple shape. In an embodiment, the shaped cartilage-derived implant has a complex
CA 02939280 2016-08-18 shape. In an embodiment, the shaped cartilage-derived implant has a symmetrical shape. In an embodiment, the shaped cartilage-derived implant has an asymmetrical shape.
In an embodiment of the present invention, the shaped cartilage-derived implant has a porosity customized for its intended use. In an embodiment, the shaped cartilage-derived implant has a pH customized for its intended use. In an embodiment, the shaped cartilagederived implant includes cross-linked collagen. In an embodiment, the shaped cartilage-derived implant includes cross-linked non-coIlagen components. In an embodiment, the shaped cartilage-derived implant has biological polymers that are cross-linked with non-biological (i.e., synthetic) polymers.
In an embodiment of the present invention, a method of forming a shaped cartilage-derived implant includes a step of scanning or imaging a cartilaginous portion of a patient’s body (e g., a nose or an ear), then making shaped cartilage-derived implants to replace those anatomical structures. In an embodiment, a shaped cartilage-derived implant is made to restore the shape of a cartilaginous anatomical structure. In an embodiment, a shaped cartilage-derived implant is made to provide a substitute for a cartilaginous anatomical structure. Methods of making such a shaped cartilage-derived implant include any method of forming a putty, a paste, a gel, a slurry, or a mass of cartilage fibers into a three-dimensional object, and include, but are not necessarily limited to, casting, molding, and three-dimensional printing.
In an embodiment of the present invention, the shaped cartilage-derived implant is formed, then cultured in vitro with exogenous cells. When the cells reach a sufficient number, the shaped cartilage-derived implant is implanted for orthopedic, plastic, reconstructive or regenerative surgery or used in cartilage repair procedures.
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In an embodiment of the present invention, the shaped cartilage-derived implant is formed, then cultured in vitro with cells. When the cells reach a sufficient number, the shaped cartilage-derived implant is cryopreserved, and then reconstituted when needed for use.
In an embodiment of the present invention, mesenchymal stem cells are harvested from a patient in need of a nasal graft, cultured onto a shaped cartilage-derived implant resembling the patient's own nasal structure. In an embodiment of the present invention, mesenchymal stem cells are harvested from a patient in need of an ear graft, cultured onto a shaped cartilage-derived implant resembling the patient's own ear. The shaped cartilage-derived implant could optionally resemble other structures natural to patient's own body, for example but not limited to nasal septum, sternum, trachea, bronchea, articular cartilage, osteroarticular cartilage, costal cartilage. After the cells have differentiated into a sufficient number of chondrocytes, the shaped cartilage-derived implant can be provided to the patient as a viable graft.
In an embodiment of the present invention, a shaped cartilage-derived implant is provided in a lyophilized form. The lyophilized shaped cartilage-derived implant is rehydrated in the operating room, where it may be combined with such substances as the patient’s plateletrich plasma (PRP), autologous cells such as those obtained from the patient’s bone marrow or stromal vascular fraction (SVF) (eg,. SVF from adipose tissue obtained by liposuction), allogeneic cells such as those obtained from a cell bank (e g., stem cells, progenitor cells or other cell types available from cell banks), or bone marrow and bone marrow components including bone marrow cells (both autologous and allogeneic).
In an embodiment of the present invention, a shaped cartilage-derived implant is provided in a dehydrated form. The dehydrated shaped cartilage-derived implant is rehydrated
CA 02939280 2016-08-18 in the operating room, where it may be combined with such substances as the patient's plateletrich plasma (PRP), autologous cells such as those obtained from the patient’s bone marrow or stromal vascular fraction (SVF) (e.g,. SVF from adipose tissue obtained by liposuction), allogeneic cells such as those obtained from a cell bank (e.g., stem cells, progenitor cells or other cell types available from cell banks), or bone marrow and bone marrow components including bone marrow cells (both autologous and allogeneic).
VII. Other Forms of Cartilage
In embodiments of the present invention, the cartilage-derived implant may include cartilage tissue forms other than cartilage fibers and cartilage particles. For example, minced cartilage or sheets of cartilage cut from recovered intact body parts may be used in place of, or in combination with, cartilage fibers and cartilage particles in the implants discussed in the present disclosure. Such minced cartilage or cartilage sheets may be in fresh, dehydrated, or lyophilized form, and may be processed or used in similar fashion to the cartilage fibers and cartilage particles.
VIII. EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a disclosure and description of exemplary embodiments of the described invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.) but some experimental errors and deviations should be accounted for.
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Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade (’C), and pressure is at or near atmospheric.
Example 1 : Viability of Fresh Cartilage Fibers
Fresh cartilage fibers were assayed for viable cells. Viability was determined by total cell counts and live/dead cell counts. Alternative means can be used to assess cell viability of intact fibers, such as standard methods utilizing the adenosine triphosphate (ATP) assays or routine cell viability reagents (e.g., PrestoBlue®; ThermoFisher Scientific, Waltham, MA) as described in the literature, although such methods were not used in the present example.
Femoral condyles were recovered from an adult human donor, cut into hemicondyles, and stored in growth media. The recovered hemi-condyles were submerged in growth media, and separated into two groups: Group A and Group B. Fresh cartilage fibers from Group A were obtained by grating the cartilage of the hemi-condyles while the hemicondyles were totally submerged in growth media. Fresh cartilage fibers from Group B were obtained by grating the cartilage of the hemi-condyles outside of the growth media, and depositing the fibers into the growth media. Fresh cartilage fibers were then separated from the growth media, placed into separate vials, and covered with growth media, after which the vials were sealed and stored in sealed Kapak® pouches until the fibers were tested for viability.
Fresh cartilage fibers from Group A and Group B were tested separately for viability. Prior to testing, the fresh cartilage fibers were separated from the growth media by settling. Cells were recovered from the fresh cartilage fibers by digestion with Pronase® and collagenase II, following protocols known in the art. Cell counts were performed on the resulting cell suspensions using Trypan Blue, following protocols known in the art. Fluorescent live/dead
CA 02939280 2016-08-18 counts were performed on cells plated from the suspensions, following protocols known in the art. Cartilage digestion and cell recovery for Group A and Group B were completed on the same day that the condyles were recovered from the human donor. All viability tests were performed promptly thereafter.
The total number of cells counted for fresh cartilage fibers from Group A and Group B were 2.44x10<sup>6</sup>/mL suspension and 3.09x1 O<sup>e</sup>/mL suspension, respectively. The live/dead counts indicated that 94.5% of the cells of Group A and 93.0% of the cells from Group B were viable.
Example 2: Viability of Fresh and Cryopreserved Cartilage Fibers
Fresh and cryopreserved cartilage fibers were assayed for viable cells. Cryopreserved fibers were assayed at 0 hours and 48 hours after cryopreservation. Viability was determined by total cell counts and live/dead cell counts.
Femoral condyles were recovered from an adult human donor, cut into hemicondyles, and stored in growth media. Each recovered hemi-condyle was removed from the growth media, and securely mounted onto a vise. Fresh cartilage fibers were harvested from the hemi-condyle using a grater, and periodically transferred from the grater to a container of growth media.
When a sufficient amount of fresh cartilage fibers had been collected, the cartilage fibers were separated from the growth media, and divided among vials with growth media and jars with a cryopreservation solution (100 mL DMSO/1 L Hyclone™ media). The vials and jars were immediately sealed in foil pouches, and transferred to a laboratory for viability testing.
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Digestion of the fresh cartilage fibers from the vials (Group A) was started immediately upon receipt at the laboratory. Fresh cartilage fibers from one group of jars (Group B) were subjected to a cryopreservation cycle, and the cartilage digestion was started immediately after the cryopreservation cycle. Fresh cartilage fibers from another group of jars (Group C) were cryopreserved, and the cartilage digestion was started at about 48 hours after the cryopreservation cycle. All viability tests were performed promptly after the cells were recovered.
Prior to testing, the fresh cartilage fibers were separated from the growth media and cryopreserved cartilage fibers were separated from both growth media and cryopreservation solution. Cells were recovered from the cartilage fibers by digestion with Pronase® and collagenase II, following protocols known in the art. Cell counts were performed on the resulting cell suspensions using Trypan Blue, following protocols known in the art. Fluorescent live/dead counts were performed on cells plated from the suspensions, following protocols known in the art.
The total number of cells counted for cartilage fibers from Groups A, B, and C were 3.85x10<sup>6</sup>/mL suspension, 0.8x10®/mL suspension, and 0.91x10<sup>6</sup>/mL suspension, respectively. The live/dead counts indicated that the percentages of viable cells for Groups A, B, and C were 92.0%, 66.6%, and 70.0%, respectively.
Example 3: Viability of Fresh and Cryopreserved Cartilage Fibers
Fresh and cryopreserved cartilage fibers were assayed for viable cells. Cryopreserved fibers were assayed at 0 hours and 48 hours after cryopreservation. Viability was determined by total cell counts, and live/dead cell counts.
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Femoral condyles were recovered from an adult human donor, cut into hemicondyles, and stored in growth media. Each recovered hemi-condyle was removed from the growth media, and securely mounted onto a vise. Fresh cartilage fibers were harvested from the hemi-condyle using a grater, and periodically transferred from the grater to a container of growth media.
When a sufficient amount of fresh cartilage particles had been collected, the cartilage fibers were separated from the growth media, and divided among vials with growth media and jars with a cryopreservation solution (100 mL DMSO/1 L Hyclone™ media). The vials and jars were immediately sealed in foil pouches, and transferred to a laboratory for viability testing.
Digestion of the fresh cartilage fibers from the vials (Group A) was started immediately upon receipt at the laboratory. Fresh cartilage fibers from one group of jars (Group B) were subjected to a cryopreservation cycle, and the cartilage digestion was started immediately after the cryopreservation process. Fresh cartilage fibers from another group of jars (Group C) were cryopreserved, and the cartilage digestion was started at about 48 hours after the cryopreservation cycle. All viability tests were performed promptly after the cells were recovered.
Prior to testing, the fresh cartilage fibers were separated from the growth media and cryopreserved cartilage fibers were separated from both growth media and cryopreservation solution. Cells were recovered from the cartilage fibers by digestion with Pronase® and collagenase II, following protocols known in the art. Cell counts were performed on the resulting cell suspensions using Trypan Blue, following protocols known in the art.
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Fluorescent live/dead counts were performed on cells plated from the suspensions, following protocols known in the art.
The total number of cells counted for cartilage fibers from Groups A, B, and C were 3.38x10<sup>6</sup>/mL suspension, 1.52x10<sup>6</sup>/mL suspension, and 1.05x10<sup>6</sup>/mL suspension, respectively. The live/dead counts indicated that the percentages of viable cells for Groups A, B, and C were 88.1%, 62.0%, and 66.4%, respectively.
Example 4: Viability of Fresh and Cryopreserved Cartilage Fibers
Fresh and cryopreserved cartilage fibers were assayed for viable cells. Cryopreserved fibers were assayed at 48 hours after cryopreservation. Viability was determined by total cell counts and live/dead cell counts.
Femoral condyles were recovered from an adult human donor, cut into hemicondyles, and stored in growth media. Each recovered hemi-condyle was removed from the growth media, and securely mounted onto a vise, Fresh cartilage fibers were harvested from the hemi-condyle using a grater, and periodically transferred from the grater to a container of growth media.
When a sufficient amount of fresh cartilage particles had been collected, the cartilage fibers were separated from the growth media, and divided among vials with growth media and jars with a cryopreservation solution (100 mL DMSO/1 L Hydone™ media). The vials and jars were immediately sealed in foil pouches, and transferred to a laboratory for viability testing
Digestion of the fresh cartilage fibers from the vials (Group A) was started immediately upon receipt at the laboratory. Fresh cartilage fibers from the group of jars (Group
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B) were cryopreserved, and the cartilage digestion was started at about 48 hours after the cryopreservation cycle. All viability tests were performed promptly after the cells were recovered.
Prior to testing, the fresh cartilage fibers were separated from the growth media and cryopreserved cartilage fibers were separated from both growth media and cryopreservation solution. Cells were recovered from the cartilage fibers by digestion with Pronase® and collagenase II, following protocols known in the art. Cell counts were performed on the resulting cell suspensions using Trypan Blue, following protocols known in the art. Fluorescent live/dead counts were performed on cells plated from the suspensions, following protocols known in the art.
The total number of cells counted for cartilage fibers from Group A and Group B were 2.86x10<sup>6</sup>/mL suspension and 0.71x10<sup>6</sup>/mL suspension, respectively. The live/dead counts indicated that the percentages of viable cells for Group A and Group B were 91.1% and 69.2%, respectively.
Example 5: Viability of Fresh and Cryopreserved Cartilage Fibers
Fresh and cryopreserved cartilage fibers were assayed for viable cells. Cryopreserved fibers were assayed at 48 hours and 14 days after cryopreservation. Viability was determined by total cell counts and live/dead cell counts.
Femoral condyles were recovered from an adult human donor, cut into hemicondyles, and stored in growth media. Each recovered hemi-condyle was removed from the growth media, and securely mounted onto a vise. Fresh cartilage fibers were harvested from
CA 02939280 2016-08-18 the hemi-condyle using a grater, and periodically transferred from the grater to a container of growth media.
When a sufficient amount of fresh cartilage particles had been collected, the cartilage fibers were separated from the growth media, and divided among vials with growth media and jars with a cryopreservation solution (100 mL DMSO/1 L Hyclone™ media). The vials and jars were immediately sealed in foil pouches, and transferred to a laboratory for viability testing.
Digestion of the fresh cartilage fibers from the vials (Group A) was started immediately upon receipt at the laboratory. Fresh cartilage fibers from one group of jars (Group B) were cryopreserved, and the cartilage digestion was started at about 48 hours after the cryopreservation cycle. Fresh cartilage fibers from another group of jars (Group C) were cryopreserved, and the cartilage digestion was started on day 14 after the cryopreservation cycle.AII viability tests were performed promptly after the cells were recovered.
Prior to testing, the fresh cartilage fibers were separated from the growth media and cryopreserved cartilage fibers were separated from both growth media and cryopreservation solution. Cells were recovered from the cartilage fibers by digestion with Pronase® and collagenase II, following protocols known in the art. Cell counts were performed on the resulting cell suspensions using Trypan Blue, following protocols known in the art. Fluorescent live/dead counts were performed on cells plated from the suspensions, following protocols known in the art.
The total number of cells counted for cartilage fibers from Groups A, B, and C were 1.61x10<sup>e</sup>/mL suspension, 0.625x10<sup>6</sup>/mL suspension, and 0.29x10<sup>6</sup>/mL suspension,
CA 02939280 2016-08-18 respectively. The live/dead counts indicated that the percentages of viable cells for Groups A, B, and C were 87.6%, 66.1%, and 68.1%, respectively.
Example 6: Viability of Fresh and Cryopreserved Cartilage Fibers
Fresh and cryopreserved cartilage fibers were assayed for viable cells. Cryopreserved fibers were assayed at 0 hours and 35 days after cryopreservation. Viability was determined by total cell counts and live/dead cell counts.
Femoral condyles were recovered from an adult human donor, cut into hemicondyles, and stored in growth media. Each recovered hemi-condyle was removed from the growth media, and securely mounted onto a vise. Fresh cartilage fibers were harvested from the hemi-condyle using a grater, and periodically transferred from the grater to a container of growth media.
When a sufficient amount of fresh cartilage particles had been collected, the cartilage fibers were separated from the growth media, and divided among vials with growth media and jars with a cryopreservation solution (100 mL DMSO/1 L Hyclone™ media). The vials and jars were immediately sealed in foil pouches, and transferred to a laboratory for viability testing.
Digestion of the fresh cartilage fibers from the vials (Group A) was started immediately upon receipt at the laboratory. Fresh cartilage fibers from one group of jars (Group B) were subjected to a cryopreservation cycle, and the cartilage digestion was started at about two hours after receipt at the laboratory. Fresh cartilage fibers from another group of jars (Group C) were cryopreserved, and the cartilage digestion was started at about 35 days after
CA 02939280 2016-08-18 the cryopreservation cycle. All viability tests were performed promptly after the cells were recovered.
Prior to testing, the fresh cartilage fibers were separated from the growth media and cryopreserved cartilage fibers were separated from both growth media and cryopreservation solution. Cells were recovered from the cartilage fibers by digestion with Pronase® and collagenase II, following protocols known in the art. Cell counts were performed on the resulting cell suspensions using Trypan Blue, following protocols known in the art. Fluorescent live/dead counts were performed on cells plated from the suspensions, following protocols known in the art.
The total number of cells counted for cartilage fibers from Groups A, B, and C were 3.42x10<sup>6</sup>/mL suspension, 0.735x10<sup>6</sup>/mL suspension, and 0.615x10<sup>6</sup>/mL suspension, respectively. The live/dead counts indicated that the percentages of viable cells for Groups A, B, and C were 88.1%, 66.8%, and 71.4%, respectively.
Example 7: Handling Characteristics of Mixtures of Fresh Cartilage Fibers with Lyophilized Cartilage Particles
Fresh articular cartilage fibers were mixed at various ratios with lyophilized cartilage particles having sizes of less than 212 microns to assess the handling characteristics of the mixture. It was found that the fresh fibers provided sufficient moisture to bind with the lyophilized particles and form a cohesive putty that was easily handled. Mixtures that included more than about 35% to about 45% particles by weight were not as cohesive as mixtures having particle contents of less than about 35% by weight.
Example 8: Rehydration of Lyophilized Cartilage Fibers
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Fresh cartilage fibers were collected by grating articular cartilage from intact hemi-condyles. The following fluids were added to samples of the fresh cartilage fibers: synovial fluid, blood, platelet rich plasma (PRP), growth media. Sorenson’s buffer, and standard saline. One sample of fresh cartilage fibers received no fluids. Each sample of the fresh cartilage fibers was then lyophilized. All of the lyophilized samples were found to be stiff when handled.
The lyophilized samples of cartilage fibers treated with saline, growth media, and no liquid were then rehydrated. Each sample formed a cohesive putty.
Lyophilized cartilage fibers which had received no fluids were soaked overnight in growth media. The rehydrated fibers looked and felt like fresh cartilage fibers, and had the same handling properties as fresh cartilage fibers.
Example 9: Formulations of Cartilage Fibers and Cartilage Particles
Fresh cartilage fibers were collected by grating articular cartilage from thawed hemi-condyles. The fresh cartilage fibers were soaked for up to 4 hours in growth media, then drained in a 212 pm sieve. Lyophilized cartilage fibers, prepared without adding a fluid, and lyophilized cartilage particles were previously prepared according to methods discussed in the present disclosure.
In a first formulation, lyophilized cartilage fibers (0.12 g) were mixed with lyophilized cartilage particles (0.2 g) and defibrinated sheep blood (0.7 mL). The formulation mixed readily into a cohesive mass. A portion of the mixture was placed in a cartilage defect in a knee-en-bloc, where it stuck well. The mixture remained firm in the defect after the knee-enbloc had been articulated 20 times with a saline drip
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In a second formulation, lyophilized cartilage fibers (0.3 g) were mixed with defibrinated sheep blood (0.7 mL). The formulation was difficult to mix, but gradually improved. A portion of the mixture was placed in a cartilage defect in a knee-en-bloc, where it stuck well. The mixture remained in the defect after the knee-en-bloc had been articulated 20 times without a saline drip. The mixture began to swell after the knee-en-bloc was articulated 20 times with a saline drip.
In a third formulation, fresh cartilage fibers (0.5 g) were mixed with lyophilized cartilage fibers (0.5 g). The formulation did not mix well, and was dry and crumbly.
In a fourth formulation, fresh cartilage fibers (0.6 g) were mixed with lyophilized cartilage fibers (0.4 g). The formulation was dry and did not mix well, but formed a cohesive mass upon further manipulation. A portion of the mixture was placed in a cartilage defect in a knee-en-bloc, where it stuck well. The mixture remained in the defect after the knee-en-bloc had been articulated 20 times with a saline drip.
In a fifth formulation, fresh cartilage fibers (0.8 g) were mixed with lyophilized cartilage particles (0.2 g). The formulation mixed easily into a cohesive putty-like mass. A portion of the mixture was placed in a cartilage defect in a knee-en-bloc, where it stuck well. The mixture remained firm in the defect after approximately 20 articulations with a saline drip.
In a sixth formulation, fresh cartilage fibers (0.85 g) were mixed with lyophilized cartilage particles (0.15 g). The formulation mixed easily into a cohesive putty-like mass. A portion of the mixture was placed in a cartilage defect in a knee-en-bloc, where it stuck well. The mixture remained firm in the defect after approximately 20 articulations with a saline drip.
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In a seventh formulation, fresh cartilage fibers (0.75 g) were mixed with lyophilized cartilage particles (0.25 g). The formulation mixed easily into a cohesive putty-like mass. A portion of the mixture was placed in a cartilage defect in a knee-en-bloc, where it stuck well. The mixture remained firm in the defect after approximately 20 articulations with a saline drip.
Example 10: Viability of Cryopreserved Cartilage Fibers
Cryopreserved cartilage fibers were assayed for viable cells. Cryopreserved fibers were assayed approximately 6 months after cryopreservation. Viability was determined by total cell counts and live/dead cell counts.
Femoral condyles were recovered from an adult human donor, cut into hemicondyles, and stored in growth media. Each recovered hemi-condyle was removed from the growth media, and securely mounted onto a vise. Fresh cartilage fibers were harvested from the hemi-condyle using a grater, and periodically transferred from the grater to a container of growth media.
When a sufficient amount of fresh cartilage particles had been collected, the cartilage fibers were separated from the growth media, and divided among vials with a cryopreservation solution (100 mL DMSO/1 L HycloneTM media) to form Group A. The vials were immediately sealed in foil pouches and cryopreserved using controlled rate freezing. Following cryopreservation, samples were stored in a Liquid Nitrogen (LN) tank until time of testing.
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Digestion of the cryopreserved cartilage fibers from the vials (Group A) was started about 6 months after cryopreservation and storage in LN tanks. All viability tests were performed promptly after the cells were recovered.
Prior to testing, the cryopreserved cartilage fibers were separated from the cryopreservation solution. Cells were recovered from the cryopreserved cartilage fibers by digestion with Pronase® and collagenase II, following protocols known in the art. Cell counts were performed on the resulting cell suspensions using Trypan Blue, following protocols known in the art. Fluorescent live/dead counts were performed on cells plated from the suspensions, following protocols known in the art.
The total number of cells counted for cryopreserved cartilage fibers from Group A was 0.220x10<sup>6</sup>/mL suspension. The live/dead counts indicated that the percentage of viable cells for Groups A was 71,1%.
Example 11: Viability of Cryopreserved Cartilage Fibers and Storage Conditions
Cryopreserved cartilage fibers were assayed for viable cells. Cryopreserved fibers were assayed approximately 6 months after cryopreservation. Viability was determined by total cell counts and live/dead cell counts.
Femoral condyles were recovered from an adult human donor, cut into hemicondyles, and stored in growth media. Each recovered hemi-condyle was removed from the growth media, and securely mounted onto a vise. Fresh cartilage fibers were harvested from the semi-condyle using a grater, and periodically transferred from the grater to a container of growth media.
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When a sufficient amount of fresh cartilage particles had been collected, the cartilage fibers were separated from the growth media, and divided among vials with a cryopreservation solution (100 mL DMSO/1 L HycloneTM media). The vials were immediately sealed in foil pouches and cryopreserved using controlled rate freezing. Following cryopreservation, samples were split into two groups. Group A samples were stored in the LN tank and Group B samples were stored in a -70C freezer until time of testing.
Digestion of the cryopreserved cartilage fibers from the vials of Group A and Group B were started about 6 months after cryopreservation and storage in either an LN tank (Group A) or -70C freezer (Group B). All viability tests were performed promptly after the cells were recovered.
Prior to testing, the cryopreserved cartilage fibers were separated from the cryopreservation solution. Cells were recovered from the cryopreserved cartilage fibers by digestion with Pronase® and collagenase II, following protocols known in the art. Cell counts were performed on the resulting cell suspensions using Trypan Blue, following protocols known in the art. Fluorescent live/dead counts were performed on cells plated from the suspensions, following protocols known in the art.
The total number of cells counted for cryopreserved cartilage fibers from Group A and Group B were 0.205x10<sup>e</sup>/mL suspension and 0.145x10<sup>6</sup>/mL suspension, respectively. The live/dead counts indicated that the percentage of viable cells for Groups A and Group B were 65.5% and 68.1%, respectively.
IX. GLOSSARY
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The term “acellular” as used herein means substantially free of cells and their components (including DNA).
The term “ambient temperature” as used herein refers to the temperature of the immediate, unaltered surroundings. Ambient temperature is between about 15°C and about 30°C. According to some embodiments, ambient temperature is room temperature.
The term “adherent in all of its grammatical forms, as used herein refers to the act of sticking to, clinging, or staying attached.
The term administer as used herein means to give or to apply.
The term “allogeneic” as used herein refers to being genetically different although belonging to or obtained from the same species. The term “allogeneic” may be used interchangeably herein with the term “allogenic”.
The term ‘allogenic graft”, “allogeneic graft” or “allograft” as used herein refers to a tissue that is recovered from one individual and implanted into different individual of the same species.
The term “attached” as used herein refers to being fastened, fixed, joined, connected, bound, adhered to or assembled with.
The term “autologous” as used herein means derived from the same organism.
The term “autologous graft or “autograft” as used herein refers to a tissue that is grafted into a new position in or on the body of the same individual.
The term biocompatible as used herein refers to causing no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to living tissue.
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The term buffer or “buffer solution as used herein refers to a compound, usually a salt, which, when dissolved in an aqueous medium, serves to maintain the free hydrogen ion concentration of the solution within a certain pH range when hydrogen ions are added or removed from the solution. A salt or solution is said to have a buffering capacity or to buffer the solution over such a range, when it provides this function. Generally a buffer will have adequate buffering capacity over a range that is within .±.1 pH unit of its pK.
The term “buffered isotonic solution” as used herein refers to any buffer that is commonly used in biological research or the commercial biotechnology field. Exemplary buffered isotonic solutions include but are not limited to balanced salt solution (BSS), Hank’s Balanced Salt Solution, Grey’s Balanced Salt Solution, Hank’s Buffered Salt Solution, Phosphate Buffered Saline, Tris-Buffered Saline, etc. The term “isotonic solution” as used herein refers to a solution whose osmolarity and ion concentrations closely match those within normal cells of the body and the blood.
The term “carrier as used herein refer to a pharmaceutically acceptable inert agent or vehicle for delivering one or more biological components or active agents to a subject, and often is referred to as an “excipient.” The carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the subject being treated. The carrier further should maintain the stability and bioavailability of the biological component or active agent
The term cell is used herein to refer to the structural and functional unit of living organisms and is the smallest unit of an organism classified as living.
The term “chondrocytes” as used herein refers to cells found in cartilage that produce and maintain the cartilaginous matrix for, for example, joints, ear canals, trachea,
CA 02939280 2016-08-18 epiglottis, larynx, the discs between vertebrae and the ends of ribs. From least to terminally differentiated, the chondrocytic lineage is (i) Colony-forming unit-fibroblast (CFU-F); (ii) mesenchymal stem cell I marrow stromal cell (MSC); (iii) chondrocyte.
The term chondrogenesis as used herein refers to the formation of new cartilage from cartilage forming or chondrocompetent cells.
The term ’’chondrogenic’ as used herein refers to a potential of precursor cells to differentiate into cartilage forming or chondrocompetent cells.
The term compatible” as used herein means that the components of a composition are capable of being combined with each other in a manner such that there is no interaction that would substantially reduce the efficacy of the composition under ordinary use conditions.
The term component as used herein refers to a constituent part, element or ingredient.
The term “condition, as used herein, refers to a variety of health states and is meant to include disorders or diseases caused by any underlying mechanism or disorder, injury, and the promotion of healthy tissues and organs.
The term “condyle” as used herein means the rounded protuberance or projection at the end of a bone forming an articulation with another bone.
The term “contact” and its various grammatical forms as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination may occur by any means of administration known to the skilled artisan.
CA 02939280 2016-08-18 “Decellularization, as used herein in all of its grammatical forms, is any process by which at least some of the endogenous cells and cellular components (including DNA) are removed from a tissue, thereby leaving the extracellular matrix (ECM) which may be essentially free of such cells and cellular components.
The term “dehydrate, as used herein in all of its grammatical forms, means removal of at least a portion of water that is initially present.
The term “devitalization”, as used herein in all of its grammatical forms, is any process which renders a tissue substantially or essentially free from reproductively or metabolically viable cells, without necessarily leaving the tissue essentially free of such cells and cellular components. In other words, a devitalized tissue is a tissue in which less than 5%, by weight, of its native cells remain metabolically viable, compared to the total weight of the metabolically viable cells originally present in the tissue.
The term “endogenous” as used herein refers to that which is naturally occurring, incorporated within, housed within, adherent to, attached to or resident in.
The term extracellular matrix” as used herein refers to a scaffold in a cell’s external environment with which the cell interacts via specific cell surface receptors. The extracellular matrix serves many functions, including, but not limited to, providing support and anchorage for cells, segregating one tissue from another tissue, and regulating intracellular communication. The extracellular matrix is composed of an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs). Examples of fibrous proteins found in the extracellular matrix include collagen, elastin, fibronectin, and laminin. Examples of GAGs found in the extracellular matrix include proteoglycans (e.g., heparin sulfate), chondroitin sulfate, keratin sulfate, and non-proteoglycan polysaccharide (e.g., hyaluronic acid). The term
CA 02939280 2016-08-18 “proteoglycan” refers to a group of glycoproteins that contain a core protein to which is attached one or more glycosaminoglycans.
The term “factors” as used herein refers to nonliving components that have a chemical or physical effect. For example, a paracrine factor” is a diffusible signaling molecule that is secreted from one cell type that acts on another cell type in a tissue. A “transcription factor” is a protein that binds to specific DNA sequences and thereby controls the transfer of genetic information from DNA to mRNA.
The term “fiber” as used herein refers to an elongated piece of tissue having a thread-like or ribbon-like shape.
The term fluorescence as used herein refers to the result of a three-state process that occurs in certain molecules, generally referred to as fluorophores or fluorescent dyes, when a molecule or nanostructure relaxes to its ground state after being electrically excited. Stage 1 involves the excitation of a fluorophore through the absorption of light energy; Stage 2 involves a transient excited lifetime with some loss of energy; and Stage 3 involves the return of the fluorophore to its ground state accompanied by the emission of light.
The term “graft as used herein refers to a tissue or organ transplanted from a donor to a recipient. It includes, but is not limited to, a tissue transferred from one body site to another in the same individual (“autologous graft), a tissue transferred between genetically identical individuals or sufficiently immunologically compatible to allow tissue transplant (“syngeneic graft”), a tissue transferred between genetically different members of the same species (“allogeneic graft or “allograft”), and a tissue transferred between different species (“xenograft”).
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The term growth as used herein refers to a process of becoming larger, longer or more numerous, or an increase in size, number, or volume.
The term growth conduction as used herein refers to a process by which a tissue is directed to regenerate or grow so as to conform to a material’s surface. A growthconductive surface is one that permits tissue growth on its surface or down into pores, channels or pipes. Growth-conductive material facilitates the spontaneous formation of a tissue by furnishing a microenvironment that supports deposition or adhesion of tissuegenic cells and optionally, vascularization. Examples of growth-conductive materials, include, but are not limited to, processed human bone (e g., allograft bone, which may be an osteoconductive material), purified collagen, calcium phosphate ceramics, synthetic polymers, tissue-derived matrices, BMP-2 and 4, VEGF, bFGF, TGF-β, and PDGF.
The term “growth-conductive matrix as used herein refers to a matrix that may be inert in and of itself but which supports three-dimensional tissue formation. For example, allograft bone tissue may be an osteoconductive matrix.
The term “growth factor as used herein refers to extracellular polypeptide molecules that bind to a cell-surface receptor triggering an intracellular signaling pathway, leading to proliferation, differentiation, or other cellular response. Growth factors include, but are not limited to, cytokines and hormones.
The term “growth induction as used herein refers to a process by which primitive, undifferentiated and tissuegenic cells are stimulated to develop into an ensemble of cells, not necessarily identical, that together carry out a specific function. This ensemble of cells is termed a tissue.
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The term “growth-inductive matrix” as used herein refers to a matrix containing a substance or substances capable of recruiting or stimulating local tissuegenic cells so that the cells are induced (meaning to cause, bring about, bring about, or trigger) to differentiate and/or produce a tissue.
The terms “growth-inductive components or “growth-inductive factors or “tissuegenic factors” are used interchangeably to refer to the plethora of mediators associated with tissue development and repair.
The terms “growth medium”, “growth media, “culture medium”, and “culture media” refer to liquids or gels designed to support the growth of microorganisms or cells. There are different types of media for growing different types of cells, and the selection of appropriate media for certain cell types will be understood by those having ordinary skill in the art.
The term “implant” refers to any device or material inserted or placed, permanently or temporarily, into or onto a subject as well as those used for the administration or delivery of a therapeutic agent(s) or substance.
The term “improve” (or improving) as used herein refers to bring into a more desirable or excellent condition.
The terms in the body, void volume, resection pocket, excavation, injection site, deposition site or implant site as used herein are meant to include all tissues of the body without limit, and may refer to spaces formed therein from injections, surgical incisions, tumor or tissue removal, tissue injuries, abscess formation, or any other similar cavity, space, or pocket formed thus by action of clinical assessment, treatment or physiologic response to disease or pathology as non-limiting examples thereof.
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The term “injury, as used herein, refers to damage or harm to a structure or function of the body caused by an outside agent or force, which may be physical or chemical.
The terms “length”, “width, and “thickness” refer to dimensions of an object measured along three axes which respectively extend in different, non-parallel directions (e.g., in the x-y-z system of three orthogonal axes). As used herein, the thickness of an object (e.g., a fiber or a particle) is equal to or less than the width of the object, which is, in turn, equal to or less than the length of the object.
The term “lyophilize”, as used herein in all its grammatical forms, means tissue dehydrated for storage by conversion of the water content of frozen tissue to a gaseous state under vacuum that extracts moisture. Lyphilization is also known as freeze-drying and these terms are used interchangeable herein.
The term “matrix refers to a surrounding substance within which something is contained or embedded.
The term “mill, and its various grammatical forms, as used herein refers to operations performed to grind, to cut, to shred, to chip, or to pulverize a substance, or equipment for performing such operations on a substance. The terms freezer-mill, “freezemill”, and their various grammatical forms, as used herein refer to milling a substance in a frozen state, or equipment for performing such operations.
The term particle as used herein refers to a chip, fragment, slice, fiber or other small constituent of a larger body (e.g., picoparticles, nanoparticles, microparticles, milliparticle, centiparticle, deciparticle; fractions thereof, or, in some instances, a larger segment or piece).
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The term “piece” as used herein refers to a particle, section, strip, chip, fragment, slice, fiber or other part, derived from, cut off, or broken from a larger unit.
The term “recovered cartilage” as used herein means cartilage which is derived from a donor, whether the donor is the same as the patient, another human, a cadaver human, or another species (alive or not) and recovered cartilage may, for example, be any one or more of articular, costal, hyaline, elastic and fibrocartilage types of cartilage.
The term “reduced or “to reduce, as used herein in all of its grammatical forms, refers to a diminishing, a decrease in, an attenuation or abatement of the degree, intensity, extent, size, amount, density or number of.
The term “regeneration or “regenerate as used herein refers to a process of recreation, reconstitution, renewal, revival, restoration, differentiation and growth to form a tissue with characteristics that conform with a natural counterpart of the tissue.
The term “rehydration as used herein refers to the restoration of water or fluid content to a body, tissue, or substance that has had water or fluid removed via drying processes, including but not limited to dehydration, lyophilization, freeze-drying or any other drying process.
The term “relative” as used herein refers to something having, or standing in, some significant association to something else.
The term repair as used herein as a noun refers to any correction, reinforcement, reconditioning, remedy, making up for, making sound, renewal, mending, patching, or the like that restores function. When used as a verb, it means to correct, to reinforce, to recondition, to remedy, to make up for, to make sound, to renew, to mend, to patch
CA 02939280 2016-08-18 or to otherwise restore function. According to some embodiments repair includes full repair and partial repair.
The term “resident, and its various grammatical forms, as used herein refers to being present habitually, existing in or intrinsic to or incorporated therein.
The term “rinse, and its various grammatical forms, as used herein refers to wash, to douse with a liquid or liquids or to flow a liquid or liquids over the material being rinsed.
The term “scaffold as used herein refers to a structure capable of supporting a three-dimensional tissue formation. A three-dimensional scaffold is believed to be critical to replicate the in vivo milieu and to allow the cells to influence their own microenvironment. Scaffolds may serve to promote cell attachment and migration, to deliver and retain cells and biochemical factors, to enable diffusion of vital cell nutrients and expressed products, and to exert certain mechanical and biological influences to modify the behavior of the cell phase. A scaffold utilized for tissue reconstruction has several requisites. Such a scaffold should have a high porosity and an adequate pore size to facilitate cell seeding and diffusion of both cells and nutrients throughout the whole structure. Biodegradability of the scaffold is also an essential requisite. The scaffold should be absorbed by the surrounding tissues without the necessity of a surgical removal, such that the rate at which degradation occurs coincides as closely as possible with the rate of tissue formation. As cells are fabricating their own natural matrix structure around themselves, the scaffold provides structural integrity within the body and eventually degrades leaving the neotissue (newly formed tissue) to assume the mechanical load.
The term “serum” as used herein refers to the clear portion of any physiological liquid separated from its more solid elements. With regard to sera derived from blood, “serum
CA 02939280 2016-08-18 refers to the clear liquid that separates from blood when it is allowed to clot completely (e.g., blood plasma from which fibrinogen is removed during clotting.
The term similar is used interchangeably with the terms analogous, comparable, or resembling, meaning having traits or characteristics in common.
The term “size reduction, as used herein in all of its grammatical forms, refers to a process by which an object, such as a tissue, is divided or reduced in size. Such processes include, without limitation, cutting, slicing, chopping, grinding, milling, freezer-milling, blending, homogenizing, tearing, shredding, fracturing, breaking, crushing, and morselizing.
A solution generally is considered as a homogeneous mixture of two or more substances. It is frequently, though not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent. The term “solvent” as used herein refers to a substance capable of dissolving another substance (termed a “solute) to form a uniformly dispersed mixture (solution).
The term “stain” as used herein refers to a composition of a dye(s) or pigment(s) used to make a structure, a material, a cell, a cell component, a membrane, a granule, a nucleus, a cell surface receptor, a peptide, a microorganism, a nucleic acid, a protein or a tissue differentiable.
The phrase “subject in need thereof as used herein refers to a patient that (i) will be administered at least one graft, (ii) is receiving at least one graft; or (iii) has received at least one graft, unless the context and usage of the phrase indicates otherwise.
The term “substantially free as used herein means a subject material or composition includes less than 5% of the component or substance of which it is being said to be
CA 02939280 2016-08-18 substantially free, based on the total original native component or substance that was present. For example, cartilage fibers that are substantially free of chondrocytes or their components are cartilage fibers which include less than 5%, by weight, of chondrocytes and their components, based on the original total weight of the native chondrocytes and their components originally present in the cartilage fibers.
The term “substantially similar as used herein means that a first value, aspect, trait, feature, number, or amount is of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of a second value, aspect, trait, feature, number, or amount.
The term therapeutic effect as used herein refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect may include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect also may include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.
The term “tissuegenic as used herein refers to a potential of a precursor cell to differentiate into a mature cell type and to regenerate a tissue. Exemplary tissuegenic cells include but are not limited to a stem cell, a progenitor cell, a precursor cell, a non-terminally differentiated cell, an immature cell anywhere along its maturation pathway up to the point of terminal differentiation, any cell type with differentiation potential or any combination thereof. The term “osteogenic” refers more specifically to cell differentiation and tissue regeneration with regard to bone. The term “adipogénie” refers more specifically to cell differentiation and tissue regeneration with regard to the adipose compartment.
The term “treat or treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating
CA 02939280 2016-08-18 clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorderfs) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).
The term “viable” as used herein refers to having the ability to grow, expand, or develop; capable of living.
The term “xenogenic graft, xenogeneic graft or “xenograft as used herein refers to a tissue that is grafted into an individual of one species from an individual of a different species.
While the disclosed invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the described invention.
Contents31
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Numbers
- Publication
- 2939280
- Application
- 2939280
Titles2
- English
- CARTILAGE-DERIVED IMPLANTS AND METHODS OF MAKING AND USING SAME
- French
- IMPLANTS DERIVES DE CARTILAGE ET METHODE DE FABRICATION ET D'UTILISATION
Classification
- CPC, 3
- A61L27/3612
- A61L27/3654
- A61L2430/06
- IPC, 1
- A61F2 08