Concentrated aqueous silk fibroin solution and use thereof

This application claims the benefits of US Patent Provisional Application No. 60 / 461,716 filed on April 10, 2003 and US Patent Provisional Application No. 60 / 551,186 filed on March 8, 2004. is there.

<u style="single">Government assistance</u> The present invention relates to fund numbers RO1EB003210, RO1DE13405-O1A1, DMR-0090384, F49620-01-C of the United States National Institutes of Health (NIH), the United States National Scientific Fund (NSF) and the United States Air Force (outsourced contract from Foster Miller). -Supported by 0064, the United States Government reserves certain rights to the invention.

<u style="single">Technical field of invention</u> The present invention generally relates to methods of preparing concentrated aqueous silk fibroin solutions and the use of these solutions in the production of silk fibroin materials such as fibers, films, spongy porous foams, three-dimensional scaffolds, and hydrogels. In particular, all aqueous means for the preparation of silk fibroin solutions are described.

<u style="single">Background of the invention</u> Silk is a well-described natural fiber produced by Bombyx mori, which has traditionally been used in the form of woven yarns for thousands of years. This silk is a fibroin protein called fibroin (both heavy and light chains) that forms the thread core, and a glue- called sericin that surrounds and holds the fibroin fibers together. like) Contains protein. Fibroin is a highly insoluble protein containing up to 90% of the amino acids glycine, alanine and serine that lead to the formation of β-pleated sheets in fibers (Asakura, et al., Encylopedia of Agricultural Science, Arntzen, CJ, Ritter). , EM Eds .; Academic Press: New York, NY, 1994; Vol.4, pp1-11).

The unique mechanical properties of reprocessed silk, such as fibroin, and its biocompatibility make this silk fiber particularly attractive for use and medical applications in biotechnology materials. Silk offers an important set of material options for biomaterials and tissue engineering due to its excellent mechanical properties, biocompatibility and biodegradability.<img file="JP4698596B2_D0001.tif" />For example, the use of 3D porous silk scaffolds in tissue engineering has been described (Meinel et al., Ann Biomed Eng, 2004 Jan; 32 (1): 112-22; Nazarov, R., et al., Biomacromolecules, printing). Further regenerated silk fibroin films have been studied as oxygen permeable and drug permeable membranes, supports for enzyme fixation, and substrates for cell culture.<img file="JP4698596B2_D0002.tif" />In addition, silk hydrogels have many uses in drug delivery in addition to tissue engineering (Megeed et al., Pharm Res., 2002 Jul; 19 (7): 954-9; Dinerman et al., J. Control. Release., 2002 Aug 21; 82 (2-3): 277-87).

However, chemicals or organic solvents such as hexafluoroisopropanol (HFIP) are used for cross-linking or processing to prepare the silk-based materials described above (Li, M., et. al., J. Appl. Poly. Sci., 2001, 79, 2192-2199; Min, S., et al., Sen'i Gakkaishi, 1997, 54, 85-92; Nazarov, R., et al. , Biomacromolecules, printing). For example, HFIP is used to optimize the solubility of silk, and methanol is used to induce a transition to amorphous β-sheet conformation in fibroin to create a stable silk structure in water. used.

The use of organic solvents in the preparation of silk fibroin materials presents significant drawbacks, as organic solvents pose biocompatibility problems when the processed material is exposed to cells in vitro or in vivo. The organic solvent can also change the properties of the fibroin material. For example, impregnation of silk fibroin film with an organic solvent such as methanol causes dehydration of the hydrated or swollen structure, which leads to crystallization, resulting in loss of solubility in water. Moreover, with respect to scaffolding for tissue engineering, the use of organic solvents makes the silk material more resistant to decomposition. Therefore, it is necessary to develop silk-based materials that can be formed in the absence of chemical cross-linking and / or organic solvents.

<u style="single">Outline of the invention</u> The present invention provides an all-aqueous mode for the preparation of concentrated aqueous silk fibroin solutions and concentrated aqueous fibroin solutions that avoid the use of organic solvents or harsh chemicals. The present invention further provides the use of these solutions in the manufacture of materials such as fibers, films, foams, meshes, scaffolds, and hydrogels.

In one embodiment, an aqueous silk fibroin solution having a fibroin concentration of at least 10 wt% is provided, where the solution is free of organic solvents. Aqueous silk fibroin solutions having a fibroin concentration of at least 15 wt%, at least 20 wt%, at least 25 wt%, or at least 30 wt% are also provided. If desired, this solution can be combined with a biocompatible polymer prior to processing.

Fibroin in an aqueous silk fibroin solution can be obtained, for example, from a solution containing dissolved silk moth silk from Bombix moth, such as dissolved spider silk from Nephila clavipes, or from a solution containing genetically modified silk. Obtainable.

In one aspect of the invention, the aqueous silk fibroin solution described herein further contains a therapeutic substance. Therapeutic substances include, for example, proteins, peptides, nucleic acids, and small molecule drugs.

In another embodiment, a method for producing a concentrated aqueous fibroin solution is provided. The method comprises preparing an aqueous silk fibroin solution and dialyzing the solution against the hygroscopic polymer for a time sufficient to produce at least 10 wt% aqueous fibroin solution.

Hygroscopic polymers useful in the methods of the invention include, for example, polyethylene glycol, amylase, or sericin. A preferred hygroscopic polymer is polyethylene glycol (PEG) with a molecular weight of 8,000 to 10,000 g / mol. Most preferably, PEG has a concentration of 25-50%.

In one embodiment, a method of producing fibers is provided. This method involves processing a concentrated aqueous silk fibroin solution to form fibers. The treatment includes, for example, electrospinning or wet spinning. Alternatively, the fibers can be stretched directly from this solution. If desired, the fibers are processed and then treated with methanol, preferably impregnated. The fibers are then preferably washed with water.

Compositions containing fibers and therapeutic substances produced by the methods of the invention are also provided.

In another embodiment, a method for producing silk foam is provided. The method comprises processing the concentrated aqueous silk solution of the present invention to make a foam. Processing methods include, for example, salt leaching, gas foaming, micropatterning, or contact of the solution with salt particles. The salt is preferably monovalent, such as NaCl, KCl, KFl, or NaBr. Or a divalent salt, eg CaCl₂, DDL₄, Or MgCl₂Can also be used.

Compositions containing foams and therapeutic substances produced by the methods of the invention are also provided.

In another embodiment, a method of producing a film is provided. This method casts a concentrated aqueous salt solution to form a film. In some embodiments, it is useful to bring the film into contact with water vapor. In addition, the film can be stretched in the uniaxial and biaxial directions.

Also provided are films and compositions containing therapeutic substances produced by the methods of the invention.

In another embodiment, a method for producing silk hydrogel is provided. The method comprises inducing a sol-gel transition in the concentrated aqueous silk solution of the present invention.

Sol-gel transitions include increased silk fibroin concentration, increased temperature, decreased pH, salts (eg, KCl, NaCl, or CaCl).₂) Can be induced by increasing the concentration or by adding a polymer (eg, polyethylene oxide (PEO)).

Also provided are compositions containing silk hydrogels and therapeutic substances produced by the methods of the invention.

<u style="single">Detailed description of the invention</u> A method of preparing a concentrated aqueous silk fibroin solution in the absence of organic solvents or harsh chemicals is described. This method involves forming a solution containing silk fibroin. Preferably, the solution is an aqueous salt such as lithium bromide. The solution is then dialyzed against the hygroscopic polymer for a time sufficient to yield an aqueous silk fibroin solution of 10-30 wt% or greater. A preferred hygroscopic polymer is polyethylene glycol (PEG).

We have increased the viscosity of aqueous silk fibroin solutions to at least 10 wt% for other applications such as fiber formation by electrospinning, scaffolding for porous 3D tissue engineering, and foam and film formation. While enabling, it has been found to avoid the use of organic solvents that can cause problems when the processed material is exposed to cells in vitro or in vivo. Dialysis of this solution against the hygroscopic polymer is also sufficient to control the water content in the formation of silk hydrogels.

The term "fibroin" as used herein includes silk moth fibroin and silk proteins of insects or spiders (Lucas et al., Adv. Protein Chem, 13: 107-242 (1958)). Fibroin is preferably obtained from a solution containing dissolved silk moth silk or spider silk. The silk moth silk protein is obtained from, for example, Bombix moth, and the spider silk is obtained from Nephila clavata. Another silk protein suitable for use in the present invention can be obtained from a solution containing genetically modified silk derived from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants and the like. See, for example, International Publication No. 97/08315 and US Pat. No. 5,245,012.

The concentrated silk fibroin solution can be prepared by any conventional method known to those skilled in the art. For example, Bombix Mori cocoons are boiled in an aqueous solution for about 30 minutes. This aqueous solution is about 0.02M Na₂CO₃Is preferable. The cocoon is rinsed with, for example, water to extract the sericin protein, and the extracted silk is dissolved in an aqueous salt solution. Salts useful for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate or other chemicals capable of solubilizing silk. Preferably, the extracted silk is dissolved in a solution of about 9-12 M LiBr. This salt is finally removed, for example using dialysis.

The solution is then concentrated using dialysis against hygroscopic polymers such as PEG, polyethylene oxide, amylose, or sericin.

Preferably, PEG has a molecular weight of 8,000 to 10,000 g / mol and a concentration of 25 to 50%. A Slide-A-Lyzer dialysis cassette (Pierce, MWCO 3500) is preferably used. However, any dialysis system can be used. This dialysis is performed for a time sufficient to produce a final concentration of 10-30% aqueous silk solution. In most cases, 2-12 hours of dialysis will suffice.

The concentrated aqueous solution of the present invention can be processed into hydrogels, foams, films, threads, fibers, meshes, and scaffolds using methods known in the art. See, for example, Altman, et al., Biomaterials, 24: 401, 2003.

A biocompatible polymer can be added to this silk solution to make a composite matrix in the method of the invention.

Biocompatible polymers useful in the present invention include, for example, polyethylene oxide (PEO) (US 6,302,848), polyethylene glycol (PEG) (US 6,395,734), collagen (US 6,127,143), fibronectin (US 5,263,992), keratin (US 6,379,690), Polyaspartic acid (US 5,015,476), polylysine (US 4,806,355), alginate (US 6,372,244), chitosan (US 6,310,188), chitin (US 5,093,489), hyaluronic acid (US 387,413), pectin (US 6,325,810), polycaprolactone (US) Includes 6,337,198), polylactic acid (US 6,267,776), polyglycolic acid (US 5,576,881), polyhydroxyalkanoate (US 6,245,537), dextran (US 5,902,800), and polyacid anhydride (US 5,270,419). Two or more biocompatible polymers can be used.

Silk films can be produced by preparing a concentrated aqueous silk fibroin solution and casting this solution. In one embodiment, the film is contacted with water or water vapor in the absence of alcohol. The film is then uniaxially or biaxially stretched or stretched. See, for example, Figures 5a and 5b. Stretching the silk-blended film induces molecular alignment of the film, thereby improving the mechanical properties of the film.

In one embodiment, the film is composed of about 50 to about 99.99 parts by volume of an aqueous silk protein solution and about 0.01 to about 50 parts by volume of a biocompatible polymer such as polyethylene oxide (PEO). Preferably, the resulting silk-blended film is about 60 to about 240 μm thick, but thicker samples are easily formed by the use of larger volumes or adhesion of multiple layers.

Foams can be produced by methods known in the art, including lyophilization and gas foaming, where water is the solvent or nitrogen or other gas is the foaming agent, respectively. Alternatively, the foam is produced by contacting the silk fibroin solution with a granular salt. The pore size of the foam can be controlled, for example, by adjusting the concentration of silk fibroin and the particle size of the granular salt (eg, the preferred diameter of the salt particles is from about 50 microns to about 1000 microns). These salts can be monovalent or divalent. Preferred salts are monovalent, such as NaCl and KCl. CaCl₂Divalent salts such as are also available. Contact with the salt of the concentrated silk fibroin solution is sufficient to induce a conformational change to the β-sheet structure that is insoluble in the solution of amorphous silk. After foam formation, excess salt is extracted, for example by impregnation with water. The resulting porous foam is then dried and the foam is used, for example, as a cell scaffold in biomedical applications. See Figure 2.

In one embodiment, the foam is a micropatterned foam. Micropatterned foams can be prepared, for example, using the methods disclosed in US Pat. No. 6,423,252, which disclosure is incorporated herein by reference. In this method, the concentrated silk solution of the present invention is brought into contact with the surface of a mold, which mold is placed on the foam and integrated with at least one surface of the foam on at least one surface of the mold. This solution is lyophilized while in contact with the micropatterned surface of the mold, which comprises a three-dimensional shade shape of a predetermined μ pattern to be lyophilized and micropatterned foam. And involves removing the lyophilized and micro-patterned foam from the mold. Foams prepared according to this method have a pre-determined and designed micropattern on at least one surface, which pattern is effective in promoting tissue repair, endoculture, or regeneration. ..

The fibers are produced, for example, using wet spinning or electrospinning. Alternatively, the concentrated solution has a gel-like consistency, so that the fibers are stretched directly from this solution.

Electrospinning can be performed by any means known in the art (see, eg, US 6,110,590). Preferably, a steel capillary tube with a tip inner diameter of 1.0 mm is mounted on an adjustable insulated stand. Preferably, the capillary tube is maintained at a high potential and mounted in a parallel plate arrangement. The capillary tube is preferably connected to a syringe filled with a silk solution. Preferably, a constant volume flow rate is maintained using a syringe pump and is set to hold the solution at the tip of the tube without dripping. The potential, solution flow rate, and distance between the capillary tip and the collection screen are adjusted for stable injection. Dry or moist fibers are collected by varying the distance between the capillary tip and the collection screen.

A suitable collection screen for collecting silk fibers can be a wire mesh, a polymer mesh, or a water bath. Another preferred collection screen is aluminum foil. The aluminum foil can be coated with a Teflon solution to facilitate the exfoliation of the silk fibers. Those skilled in the art will be able to readily select other means of collecting the fibrous solution as the fibrous solution travels through the electric field. As described in more detail in the Examples section below, the potential difference between the tip of the capillary and the pair of aluminum foil electrodes is preferably gradually increased to about 12 kV, but one of ordinary skill in the art will provide a suitable jet. To achieve this, the potential can be adjusted.

The present invention also provides a non-woven network of fibers containing the fibers of the present invention. The fibers can form knitting yarns and, for example, woven or knitted fabrics.

The fibroin silk solution of the present invention can also be coated on products of various shapes, including medical devices (eg, stents), as well as other fibers, including silk or fragments of such fibers.

Silk hydrogels can be prepared by methods known in the art and as exemplified herein. The sol-gel transition of the concentrated silk fibroin solution is the silk fibroin concentration, temperature, salt concentration (eg CaCl).₂, NaCl, and KCl), pH, hydrophilic polymer, etc. can be changed. Prior to the sol-gel transition, the concentrated aqueous silk solution can be placed in a mold or form. The resulting hydrogel can then be cut into any shape using, for example, a laser.

Materials made using the present invention, such as hydrogels, fibers, films, foams, or meshes, include drug (eg, small molecule, protein, or nucleic acid) delivery devices, vascular wound repair devices, including controlled release systems. Various medical applications such as wound closure systems, hemostatic bandages, patches and adhesives, sutures, as well as products for long-term or biodegradable transplantation into living organisms, such as tissue regeneration scaffolds, ligament prostheses. Can be used for organizational engineering applications. Films can also be used in a wide range of materials science and engineering needs, such as controlled drug release systems, coatings, composites or stand-alone materials.

In addition, these biomaterials include, but are limited to, spinal disc, cranial tissue, dural, nervous tissue, liver, pancreas, kidney, bladder, spleen, myocardium, skeletal muscle, tendons, ligaments, and breast tissue. It can be used for organ repair / replacement or regeneration strategies that can benefit from these unique scaffolds, which are not.

In another aspect of the invention, the silk biomaterial can contain a therapeutic substance. To form these materials, the silk solution is either mixed with the therapeutic material before forming the material or loaded into the material after it has been formed. The variety of therapeutic substances that can be used in combination with the biomaterials of the present invention is plentiful and includes small molecules, proteins, peptides, and nucleic acids. In general, therapeutic substances that can be administered according to the invention include, but are not limited to: anti-infective agents such as antibiotics and antiviral agents; chemotherapeutic agents (ie, anticancer agents); anti-rejection. Reactive agents; A combination of analgesics and analgesics; Anti-inflammatory agents; Hormones such as steroids; Growth factors (bone morphogenetic proteins (ie, BMP1-7), bone morphogenetic proteins (ie, GFD-5, GFD-7 and GFD) -8), epithelial growth factor (EGF), fibroblast growth factor (ie, FGF1-9), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I and IGF-II), transforming growth factor (Ie, TGF-β-III), vascular endothelial growth factor (VEGF)); anti-angiogenic proteins such as endostatin, and other naturally occurring or genetically modified proteins, polysaccharides, glycoproteins, or lipoproteins. Growth factors are "The Cellular and Molecular Basis of Bone Formation and Repair" by Vicki Rosen and R. Scott Thies (RG) Landes Company), which is incorporated herein by reference. In addition, the silk biomaterials of the invention deliver any type of molecular compound, including pharmaceuticals, vitamins, sedatives, steroids, hypnotics, antibiotics, chemotherapeutic agents, prostaglandins, and radiopharmaceuticals. Can be used for. The delivery system of the present invention comprises the above substances, as well as proteins, peptides, nucleotides, carbohydrates, monosaccharides, cells, genes, antithrombotic agents, metabolic antagonists, growth factor inhibitors, growth promoters, anticoagulants, antimitotic substances. Suitable for delivery of others, including, but not limited to, fibrinolytics, anti-inflammatory steroids, and monoclonal antibodies.

Silk biomaterials, including bioactive substances, can be formulated by mixing one or more therapeutic substances with the polymers used to make this material. Alternatively, the therapeutic material can be coated on this material, preferably with a pharmaceutically acceptable carrier. Any pharmaceutical carrier that does not dissolve in this silk material can be used. The therapeutic substance can be present as a liquid, a fine powder solid, or any other suitable physical form.

The biomaterials described herein can be further modified after secondary processing. For example, these scaffolds can be coated with additives such as bioactive substances that act as receptors or chemical attractants for the desired cell population. This coating can be applied via absorption or chemical bonding.

Additives suitable for use in the present invention include biologically or pharmaceutically active compounds. Examples of biologically active compounds include, but are not limited to: cell adhesion mediators such as collagen, elastin, fibronectin, vitronectin, laminin, proteoglycans, or known integrin binding domains. Contains peptides such as "RGD" integrin binding sequences known to affect cell adhesion or variants thereof (Schaffner P and Dard, 2003, Cell Mol Life Sci., Jan; 60 (1): 119-32). Hersel U. et al., 2003, Biomaterials., Nov; 24 (24): 4385-415); Biologically active ligands; as well as substances that enhance or eliminate certain variants of cell or tissue endoculture. For example, the step of cell rearrangement of a three-dimensional scaffold matrix is preferably carried out in the presence of growth factors effective in promoting the growth of cultured cells used to reassemble the matrix. The substance that promotes proliferation will depend on the type of cell used. For example, when fibroblasts are used, the growth factors for use here are fibroblast growth factor (FGF), most preferably basic fibroblast growth factor (bFGF) (human recombinant bFGF, UPSTATE Biotechnology). ,, Inc.) can be. Other examples of additives that enhance growth or differentiation are bone inducers such as bone morphogenetic proteins (BMP); cytokines such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF). It includes, but is not limited to, growth factors such as -I and II) and TGF-β. The term additive as used herein also includes antibodies, DNA, RNA, modified RNA / protein complexes, glycogen or other sugars, and alcohols.

These biomaterials can be shaped into commodities for tissue engineering and tissue-guided regeneration applications, including reconstructive surgery. The structure of this scaffold allows the inoculation of abundant cells and eliminates the need for pre-seed cells. These scaffolds can also be molded to form an external scaffold for the support of in vitro cell culture for the production of external support organs.

The scaffold functions to mimic the body's extracellular matrix (ECM). This scaffold serves as both a physical support and an adhesive substrate for the isolated cells during in vitro culture and subsequent transplantation. The transplanted cell population proliferates, and as the cells function normally, they begin to secrete their own extracellular matrix (ECM) support.

In the reconstruction of structural tissues such as cartilage and bone, the shape of the tissue is essential for its function and it is necessary to shape this scaffold into commodities of various thicknesses and shapes. Any desired gap, opening, or improvement in the three-dimensional structure can be made by removing part of the matrix with scissors, a scalpel, a laser beam, or other excision instrument. Application of the scaffold includes regeneration of tissues such as nerves, musculoskeletal, cartilage, tendons, liver, pancreas, eye, integument, arteries and veins, urinary tissue or any other tissue that form parenchymal or hollow organs.

Scaffolds can also be used in transplantation as a matrix of dissociated cells, such as chondrocytes or hepatocytes, to create three-dimensional tissues or organs. Tissues or organs can be produced by the methods of the invention for any species.

Many different cell types or combinations thereof can be employed in the present invention, depending on the intended function of the tissue engineering constructs produced. These cell types include, but are not limited to: smooth muscle cells, skeletal muscle cells, myocardial cells, epithelial cells, endothelial cells, urinary tract epithelial cells, fibroblasts, myoblasts, Cartilage cells, chondroblasts, osteoblasts, osteoclasts, keratinocytes, hepatocytes, bile duct cells, pancreatic islet cells, thyroid gland, parathyroid gland, adrenal gland, hypothalamus, pituitary gland, ovary, testis, salivary gland cells, fat cells, And progenitor cells. For example, smooth muscle cells and endothelial cells can be employed for muscle, tubular constructs, such as those intended as vascular, esophageal, intestinal, rectal, or urinary tract constructs; chondrocytes are chondrocytes. Can be used in constructs; chondrocytes can be used in cardiac constructs; hepatocytes and bile duct cells can be used in liver constructs; epithelial cells, endothelial cells, fibroblasts and Hepatocytes can be used in constructs intended to function to replace or enhance any of the wide variety of histological types, including these cells. In general, any cell found in the natural tissue to which the construct is intended to correspond can be used. In addition, progenitor cells, such as myoblasts or stem cells, can be used to produce their corresponding differentiated cell types. In some cases, it may be preferable to use neonatal cells or tumor cells.

Cells can be obtained from donors (alogenic) or recipients (autologous). The cell can be an established cell culture or a genetically modified cell. Tissue pieces can also be used to provide many different cell shapes in the same structure.

Appropriate growth conditions for mammalian cells are well known in the art (Freshney, RI, (2000), Culture of Animal Cells, a Manual of Basic Technique. Hoboken NJ, John Wiley &amp; Sons; Lanza et al. Principles of Tissue Engineering, Academic Press; 2nd edition May 15, 2000; and Lanza &amp; Atala, Methods of Tissue Engineering Academic Press; 1st edition October 2001). Cell culture media generally contain essential nutrients and additional elements such as growth factors, salts, minerals, vitamins, etc. that can be optionally selected depending on the cell type to be cultured. Specific components may be selected to enhance cell proliferation, differentiation, secretion of specific proteins, and the like. Generally, standard growth medium is Dalbeco's modified Eagle's medium low glucose (DMEM) containing 10-20% fetal bovine serum (FBS) or fetal bovine serum and 110 mg / L pyruvate and glutamic acid supplemented with 100 U / ml penicillin. ), As well as various other standard media known to those skilled in the art. Proliferation conditions will vary depending on the mammalian cell type used and the desired tissue.

In one embodiment, there is provided a method of making bone or cartilage tissue in vitro, comprising culturing pluripotent cells on a porous silk fibroin scaffold under conditions suitable for inducing bone or cartilage formation. Will be done. Suitable conditions for bone and cartilage formation are well known to those of skill in the art. For example, conditions for cartilage tissue growth often include non-essential amino acids, ascorbic acid-2-phosphate, dexamethasone, insulin, and TGF-β1. In one preferred embodiment, the non-essential amino acids are present at a concentration of 0.1 mM, ascorbic acid-2-phosphate is present at a concentration of 50 ug / ml, dexamethasone is present at a concentration of 10 nM, and insulin is present at a concentration of 5 ug / ml. However, TGF-β1 exists at a concentration of 5 ng / ml. Suitable conditions for bone growth often include ascorbic acid-2-phosphate, dexamethasone, β-glycerophosphate and BMP-2. In a preferred embodiment, ascorbic acid-2-phosphate is present at a concentration of 50 ug / ml, dexamethasone is present at a concentration of 10 nM, β-glycerophosphate is present at a concentration of 7 mM, and BMP-2 is present at a concentration of 1 ug / ml. To do.

In general, the length of the growth period will depend on the particular tissue engineering construct being manufactured. The growth period can be continued until the construct reaches the desired properties, eg, until the construct reaches a particular thickness, size, strength, composition of protein components, and / or a particular cell density. Methods of evaluating these parameters are known to those of skill in the art.

These constructs can be seeded with a second cell population following the first proliferation period, which comprises the same or different types of cells used in the first seeding. be able to. The construct can then be maintained for a second growth period, which is different in length from the first growth period and can employ different growth conditions. Multiple rounds of cell seeding with an intervening growth period can be employed.

In one preferred embodiment, tissues and organs are made for humans. In another embodiment, tissues and organs are made for animals such as dogs, cats, horses, monkeys, or any other mammal.

These cells are obtained from any suitable human or animal donor, or from the subject to which it should be transplanted. As used herein, the term "host" or "subject" includes, but is limited to, humans, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. Includes mammalian species that are not.

The cells used in the methods of the invention must be derived from a source that is compatible with the intended recipient. The cells are dissected using standard techniques and seeded on and into the scaffold. In vitro culture can optionally be performed prior to transplantation. Alternatively, the scaffold is implanted in the subject, angioplasted, and then cells are injected into the scaffold. Methods and reagents for in vitro culture of cells and transplantation of tissue scaffolds are known to those of skill in the art.

Cells can be seeded within the matrix either before or after matrix formation, depending on the method of matrix formation. Uniform seeding is preferred. Theoretically, the number of cells seeded does not limit the final tissue produced, but optimal seeding may increase its production rate. The number of cells seeded can be optimized using dynamic seeding (Vunjak-Novakovic et al., Biotechnology Progress, 1998; Radisic et al., Biotechnoloy and Bioengineering, 2003).

Another aspect of the invention is that the three-dimensional porous silk scaffolds described herein can themselves be implanted in vivo and utilized as tissue substitutes (eg, bone or cartilage substitutes). Is. Such implants do not require cell dissemination, but include, for example, the addition of RGD to attract cells.

In one embodiment, the silk matrix scaffold is seeded with pluripotent cells in the presence of medium that induces the formation of either bone or cartilage. Suitable media for the production of cartilage and bone are well known to those of skill in the art.

As used herein, "pluripotent" cells have the ability to differentiate into more than one cell type in response to individual differentiation signals. Examples of pluripotent cells include, but are not limited to, bone marrow stroma cells (BMSCs) and adult or embryonic stem cells. In a preferred embodiment, BMSC is used. BMSCs are pluripotent cells of the bone marrow that can proliferate undifferentiated and differentiate into mesenchymal cells such as cartilage, bone, or fat by appropriate exogenous signals (Friedenstein, AJ, 1976, Int Rev Cytol, 47: 327-359; Friedenstein et al., 1987, Cell Tissue Kinet, 20: 263-272; Caplan, AI, 1994, Clin Plast Surg, 21: 429-435; Mackay et al., 1998, Tissue Eng, 4: 415-428; Herzog et al., Blood, 2003 Nov 15; 102 (10): 3483-93, Epub 2003 Jul 31).

Cartilage tissue or bone formation can be monitored by assay methods well known to those of skill in the art, including, but not limited to, histological, immunohistochemical, and confocal or scanning electron microscopy. Yes (Holy et al., J. Biomed. Mater. Res (2003) 65A: 447-453).

Silk-based scaffolds can be used to create organized tissue with predetermined shapes and structures, either in vitro or in vivo. For example, tissues produced in vivo function from the beginning and can be used as in vivo implants. Alternatively, this silk-based structure can be seeded with cells capable of forming either bone or cartilage and then transplanted to promote proliferation in vivo. Thus, these scaffolds can be designed to form tissue with a "customized fit" designed specifically for transplantation into a particular patient. For example, the cartilage or bone tissue produced by the method of the present invention can be used to replace the huge cartilage or bone defects found in musculoskeletal and degenerative diseases such as osteoarthritis or rheumatism. it can. The engineered bone and cartilage are suitable for the replacement of joints such as the spinal column and elbow, knee, hip, or finger joints, or can also be used in osteochondral implants.

All biomaterials of the invention may be sterilized using conventional sterilization methods such as radiation sterilization (ie gamma rays), chemical sterilization (ethylene oxide), autoclaves, or other suitable techniques. Preferably, it is a sterilization method using ethylene oxide at a temperature of 52 to 55 ° C. for less than 8 hours. After sterilization, the biomaterial can be packaged in a suitable sterile, moisture-proof package for shipping and use in hospitals and other health care facilities.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, but preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference. In addition, the materials, methods and examples are merely exemplary and are not intended to be limiting. In case of conflict, this specification, including the definition, governs.

The invention is further characterized by the following examples intended to be exemplary of the invention.

<u style="single">Example</u><u style="single">(Example 1) Preparation of pure silk fiber from water derived from regenerated silk solution by electrospinning</u><u style="single">Method</u><u style="single">Preparation of regenerated B. morisilk fibroin solution</u> Bombix molysilk fibroin was prepared as follows as a modification of our early method (Sofia, et al., Journal of Biomedical Materials Research, 2001, 54: 139-148). The cocoon is 0.02M Na₂CO₃It was boiled in aqueous solution for 30 minutes and then rinsed thoroughly with water to extract the paste-like sericin protein. The extracted silk was then dissolved in 9.3M LiBr solution at room temperature to give a 20% (w / v) solution. This solution was dialyzed in water for 48 hours using a Slide-A-Lyzer dialysis cassette (Pierce, MWCO 2000). The final concentration of the aqueous silk solution was 8.0 wt%, which was determined by weighing the residual solids after drying.

This solution is further osmoticly placed in an aqueous polyethylene glycol (PEG) (MW 8,000-10,000) solution (25-50 wt%) outside the Slide-A-Lyzer dialysis cassette (Pierce, MWCO 3500) for 2-12 hours. Concentrated by exposure (Fig. 1). The final concentration of the aqueous silk solution formed 10-30 wt% or more.

<u style="single">Electric spinning</u> This solution was concentrated using the PEG solution method described above to increase the viscosity of the aqueous silk solution for spinning above 8 wt%. Concentration was needed because the viscosity and surface tension of the pure silk solution (8 wt%) was not sufficient to maintain stable droplets at the tip of the capillary. The increase in silk solution resulted in viscosity and surface tension suitable for electrospinning. A new, more concentrated pure silk solution (10-30%) makes direct spinning feasible. The distance between the tip and the collector was 10-15 cm and the liquid flow rate was 0.01-0.05 ml / min. Since the potential difference between the tip of the capillary and the pair of aluminum foil electrodes gradually increased to 30 kV (E = 2-3 kV / cm), the droplet at the end of the tip of the capillary extended from a hemisphere to a cone. The morphology and diameter of electrospun fibers were tested using SEM. The silk / PEO combination solution produced microsized fibers having a diameter of 1.5 μm to 25 μm. The morphology of the fiber surface and the crushed surface in liquid nitrogen matched well with the natural silk fibers.

<u style="single">(Example 2) Preparation of silk fibroin scaffolding</u> Porous three-dimensional scaffolds were prepared by salt leaching from an aqueous silk fibroin solution. Adjustment of silk fibroin concentration and granular NaCl particle size controls the morphological and functional properties of the scaffold. These scaffolds were highly uniform and had interconnected pores, exhibiting pore diameters in the range of 470-940um, depending on the mode of preparation. These scaffolds had a porosity of&gt; 90%. The compressive strength and compressive modulus of the scaffold were up to 320 ± 10 KPa and 3330 ± 500 KPa, respectively. The scaffold was completely degraded by protease in 21 days. These novel silk-based 3D matrices are all useful as biomaterial matrices for tissue engineering due to their aqueous mode preparation, pore size control, pore connectivity, degradability and useful mechanical properties. Provides properties.

<u style="single">Method</u><u style="single">Preparation of aqueous silk fibroin solution</u> B. Mori's cocoon, 0.02M Na₂CO₃It was boiled in aqueous solution for 20 minutes and then rinsed thoroughly with distilled water to extract the paste-like sericin protein and wax. The extracted silk was then dissolved in 9.3M LiBr solution at 60 ° C. for 4 hours to give a 20% (w / v) solution. This solution was dialyzed in distilled water for 2 days using a Slide-A-Lyzer dialysis cassette (MWCO 3500, Pierce). The final concentration of the aqueous silk fibroin solution was about 8 w / v%, which was determined by weighing the residual solids after drying. To prepare a concentrated silk fibroin solution, use a Slide-A-Lyzer dialysis cassette (MWCO 3500) with 10 ml of 8w / v% silk fibroin solution and 1 liter of 25 wt% polyethylene glycol (PEG, The 10,000 g / mol) solution was dialyzed against room temperature. After the required time, the concentrated silk fibroin solution was slowly collected with a syringe to avoid excessive shearing and the concentration was determined. An aqueous silk fibroin solution having a concentration of less than 8 wt% was prepared by dilution with distilled water. All solutions were stored at 7 ° C before use to avoid premature precipitation. Evaluate silk fibroin film prepared from 8w / v% solution and Li by XPS⁺Proved the removal of ions. Remaining Li⁺No ions were detected.

<u style="single">Preparation of silk fibroin scaffolding</u> 4 g of granular NaCl (particle size; 300 to 1180 um) was added to 2 ml of an aqueous silk fibroin solution (4 to 10 wt%) in a disc-type Teflon container (Fig. 2a). The container was covered and left at room temperature. After 24 hours, the vessel was impregnated with water and NaCl was extracted for 2 days. Porous silk fibroin scaffolds formed in this way were stored in water at 7 ° C before use.

<u style="single">X-ray diffraction</u> X-ray diffraction of lyophilized samples of scaffolds was obtained by Cu-Kα irradiation (λ = 0.15418 nm) through a Ni filter from a Rigaku RU-200BH rotary anti-cathode X-ray generator operating at 40 kV and 40 mA. It was. X-ray diffraction patterns were recorded by a point collimated beam and imaging plate (Fuji Film BAS-IP SR 127) in a vacuum camera. The camera length was calibrated with NaF (d = 0.23166 nm).

<u style="single">FTIR spectroscopy</u> Approximately 1 mg of lyophilized sample was compressed into pellets with 200 mg of potassium bromide, and a Fourier transform infrared (FTIR) spectrum was obtained by accumulating 64 scans by Nicolet Magna 860 and 4 cm.^-1Recorded with the resolution of.

<u style="single">Scanning electron microscope (SEM)</u> The silk scaffold was cut into sections in distilled water using a razor blade and then lyophilized. The sample was sputter coated with gold. The morphology of the scaffold was observed with the LEO Gemini 982 field emission gun SEM. Pore diameters were obtained using ImageJ software developed by the National Institutes of Health.

<u style="single">Porousness</u> Density and porosity of silk scaffolds were measured by liquid exchange (Zhang, RY, et al., J. Biomed. Mater. Res., 1999, 44: 446-455). Hexane penetrated through the silk scaffold without swelling or shrinking the matrix and was used as a replacement. Silk scaffolds (dry mass, W) were impregnated with a known volume (V1) of hexane in a graduated cylinder for 5 minutes. The total volume of hexane and hexane-impregnated scaffolds was recorded as V2. The hexane-impregnated scaffold was then removed from the cylinder and the remaining hexane volume was recorded as V3. The total volume of the scaffold is as follows: V = (V2-V1) + (V1-V3) = V2-V3 V2-V1 is the volume of the polymer scaffold and V1-V3 is the volume of hexane in the scaffold. The porosity (ε) of the scaffold was obtained from the following equation: ε (%) = (V1-V3) / (V2-V3) x100

<u style="single">Swelling characteristics</u> Silk fibroin scaffolds were impregnated in distilled water at room temperature for 24 hours. After removing excess water, the water content mass (Ws) of the scaffold was determined. The sample was then dried overnight in an oven at 65 ° C. under vacuum to determine the dry mass (Wd) of the scaffold. The scaffold swelling ratio and scaffold water content were calculated as follows: Swelling ratio = (Ws-Wd) / Wd Water intake (%) = [(Ws-Wd) / Ws] x100

<u style="single">Mechanical properties</u> Resistance to mechanical compression of the scaffold (12 mm diameter, 10 mm height, disc) was performed at room temperature on an Instron 8511 loaded with 0.1 kN-loaded cells. The crosshead speed was 10 mm / min. The compression test was performed by the open-sided / confined method for convenience. Four samples were evaluated for each composition. Cylindrical samples measured 12 mm in diameter and 10 mm in height were used according to a modification method based on ASTM method F451-95. The compressive stress and strain were graphed and the compressive modulus and standard deviation were determined in addition to the average compressive strength. The modulus of elasticity was defined by the slope of the first straight section of the stress-strain curve. The compressive strength was determined by drawing a straight line parallel to it, starting from 1% strain. The intersection of this straight line with the stress-strain curve was defined as the compressive strength of the foam (Thomson RC et al., Biomaterials, 1998, 19; 1935-1943).

<u style="single">In vitro enzymatic degradation</u> Degradation of silk fibroin scaffolds was evaluated using protease XIV (EC 3.4.24. 31, Sigma-Aldrich) with an activity of 5.6 U / mg. The sample (12 mm in diameter, 5 mm in height) was impregnated with 5 ml (pH 7.4) of phosphate buffered saline containing protease (1U) at 37 ° C. After a specified time, the samples were washed with phosphate buffered saline and distilled water and lyophilized. The enzyme solution was replaced with a freshly prepared solution every 24 hours. As a control, the sample was impregnated with enzyme-free phosphate buffered saline.

<u style="single">Results and Discussion</u><u style="single">Preparation of water-based scaffolding</u> Porous silk fibroin scaffolds were prepared using the salt leaching methods previously used in the preparation of porous scaffolds from other polymers such as collagen and polylactic acid. The pore size and porosity of the scaffold were adjusted by adding granular NaCl having a diameter of 300 to 1180 μm to the aqueous silk fibroin solution. In this method, part of the surface of the NaCl particles was dissolved in an aqueous silk fibroin solution, but most of this salt remained as solid particles due to the saturation of the solution. This aqueous silk fibroin solution formed a hydrogel in the mixture after ~ 24 hours, which resulted in the formation of a stable porous matrix in water. Table 1 shows the silk fibroin concentration and the particle size of NaCl used in this test. With increasing silk fibroin concentration, the matrix was formed homogeneously by the use of larger particle size NaCl. When NaCl having a particle size of 500 to 600 μm was added to the 8 wt% silk fibroin solution, the surface of the silk fibroin aqueous solution rapidly formed a hydrogel.

(Table 1) Scaffold preparation from various silk fibroin concentrations and NaCl particle size<img file="JP4698596B2_D0003.tif" />Degree of homogeneity: &gt; &gt; ×

In a concentrated salt solution, the solvation power is significantly different from that of a dilute electrolyte because the salt ions change the structure of the intervening water (Curtis RA et al., Biophys Chem, 2002, 98: 249). -265). NaCl, KC1, CaCl₂And MgCl₂The action of the concentrated salt solution containing chlorine ions such as on silk fibroin was determined at room temperature with a maximum salt concentration of 3M. When droplets of silk fibroin solution (8 wt%) were added to a 3M concentrated salt solution, silk hydrogels formed rapidly in NaCl and KCl solutions, but CaCl.₂And MgCl₂It was not formed in solution. Ions are classified as cosmotropic or chaotropic based on their size and charge (Grigs by JJ et al., Biophys Chem., 2001, 91: 231-243). Ca²⁺And Mg²⁺Ions with high charge density such as are extremely cosmotropic and K⁺Ions with low charge density such as are chaotropic. Na⁺Is a weak kosmotropic, Cl^-Is a weak chaotropic. Kosmotropic ions bind more strongly to adjacent water molecules than chaotropic ions. In addition, kosmotropic ions strongly interact with oppositely charged residues on the protein surface due to their high charge density. At low salt concentrations, this solution contains a sufficient number of water molecules to hydrate both the protein surface and the ions. At higher salt concentrations, more water molecules are needed to hydrate the increased number of ions. Therefore, water molecules are easily removed from the protein as the concentration of the salt solution increases.

From the primary sequence of the silk moth silk fibroin heavy chain, there are 7 internal hydrophobic blocks with 2 large hydrophilic blocks at the end of the chain and 7 much smaller internal hydrophilic blocks (Zhou, CZ, et). al., Nucleic Acids Res., 2000, 28: 2413-2419). The proportion of hydrophobic residues in silk fibroin is 79% (Braun, FN, et al., Int. J. Biol. Macromol., 2003, 32: 59-65) and repeats in these hydrophobic blocks. The sequence is dominant in the β-sheet structure that forms the crystalline region in silk fibroin fibers and films GAGAGS<u style="single">(SEQ ID NO: 4)</u>It consists of peptides (Mita, K., et al., J. Mol. Evol., 1994, 38: 583-592).

Protein solubility typically decreases with increasing salt concentration, favoring protein-protein interactions (Curtis, RA, et al., Biophys. Chem .. 2002, 98: 249-265). It is well known that the hydrophobic interaction between non-polar residues is increased by the addition of salts, which leads to salting out (Robinson, DR, et al., J. Am. Chem. Soc., 1965, 1965, 87: 2470-2479). The behavior of fibroin in the described salt system would otherwise cover the hydrophobic fibroin domain, promote chain-chain interactions, and lead to new, more stable structures of salt ions in water extraction. Is related to the role of. These hydrophobic interactions induce protein folding that results in the formation of β-sheets (Li, GY, et al., Biochem., 2001, 268: 6600-6606).

Alginate beads or glass beads were tested to further demonstrate the ionic effect of silk fibroin (8 wt%) on hydrogelation. The gelation time of silk fibroin with glass beads was similar to that observed in silk fibroin over 30 days in the previous test (Kim UJ et al., Biomacromolecules, in printing), but of the silk fibroin solution. The gelation time with alginate beads was ~ 2 times faster, probably due to the removal of water molecules from the proteins associated with the swollen alginate beads. Compared to the gelation time (24 hours) of silk fibroin in saturated NaCl solution, salt ions strongly induced protein-protein interaction.

<u style="single">Structural analysis</u> Changes in the structure of silk fibroin were determined by X-ray diffraction and FTIR (Fig. 3). X-ray diffraction of the silk fibroin scaffold showed a clear peak of 20.8 ° and a small peak of 24.6 °. These peaks were similar to those of the β-sheet crystal structure (Silk II) of natural silk fibroin (Asakura, T., et al., Macromolecules, 1985, 18: 1841-1845). These results show β crystals with spacings of 4.3 and 3.6 Å, respectively, following reflections at 20.8 ° and 24.6 °. The FTIR spectrum of the silk fibroin scaffold is 1701 cm^-1And 1623 cm^-1(Amid I) showed a characteristic peak of Silk II (Asakura, T., et al., Macromolecules, 1985, 18: 1841-1845). Silk fibroin in aqueous solution showed a random coil structure at neutral pH. From the results of X-ray diffraction and FTIR analysis, the formation of silk fibroin scaffolds from these solutions induced a structural transition from the random coil to the β sheet.

<u style="single">form</u> SEM images of lyophilized scaffolds prepared from NaCl particles of various silk fibroin concentrations and sizes show highly interconnected porous structures, and the distribution of pores is the air-water interface on the outermost surface of the scaffold. It was homogeneous throughout the scaffold, except for the thin layer formed in. The scaffold showed a coarse pore surface that was highly interconnected by many smaller pores. A spherical structure with a diameter of 1 to 3 μm was observed on the surface of the pores. As the silk fibroin concentration increased, the pore walls became thicker. Table 2 shows the actual pore size in the scaffold, which ranges from 350 to 920 μm.

(Table 2) Measured pore size of silk fibroin scaffold (μm)<img file="JP4698596B2_D0004.tif" />The value is mean ± standard deviation (N = 20).

The actual pore size in the scaffold was 80-90% smaller than the particle size of NaCl used in this method. The pore size of scaffolds prepared with the same particle size of NaCl produced pores of similar size, regardless of the silk fibroin concentration used.

<u style="single">Porousness and swelling properties</u> A silk fibroin scaffold with a porosity of&gt; 90% was formed, and the porosity increased as the pore size and silk fibroin concentration decreased (Table 3). These values were similar to those of HFIP-derived silk scaffolds prepared by salt leaching or gas foaming (84-98%) (Nazarov R, et al., Biomacromolecules, in print). Scaffold swelling ratios and water uptake are shown in Tables 4 and 5.

(Table 3) Porousness of silk fibroin scaffolding (%)<img file="JP4698596B2_D0005.tif" />The value is mean ± standard deviation (N = 3).

(Table 4) Swelling ratio of silk fibroin scaffolding<img file="JP4698596B2_D0006.tif" />The value is mean ± standard deviation (N = 3).

(Table 5) Water intake of silk fibroin scaffolding (%)<img file="JP4698596B2_D0007.tif" />The value is mean ± standard deviation (N = 3).

The swelling ratio gradually decreased as the pore size decreased. However, the swelling ratio decreased significantly as the silk fibroin concentration increased due to the decrease in porosity. The swelling ratio of scaffolds prepared from 8 wt% silk fibroin was ~ 8 times lower than that of collagen scaffolds due to differences in protein hydrophilicity (Ma L. et al., Biomaterials, 2003, 24: 4833). -4841). This value is similar to that of polylactic acid scaffolds (Maquet V. et al., Biomaterials, 2004, 25: 4185-4194). Scaffold water uptake in distilled water was&gt; 93% over a 24-hour period. The high water binding capacity of the scaffold is due to the highly porous structure of the protein network.

<u style="single">Mechanical properties</u> The scaffold exhibited ductile sponge-like behavior with varying hardness depending on the concentration of silk fibroin used in this method. The elastic region was observed at the initial strain, followed by peak stress. Table 6 shows the mechanical properties of the silk fibroin scaffold. The compressive strength and compressive modulus of the scaffold increased with increasing silk fibroin concentration.

(Table 6) Mechanical properties of silk fibroin scaffolding<img file="JP4698596B2_D0008.tif" />The value is mean ± standard deviation (N = 4).

The improvement in mechanical properties was due to an increase in polymer concentration with an increase in the thickness of the pore walls. Scaffolds prepared with smaller particle size NaCl at the same silk fibroin concentration showed higher compressive strength and compressive modulus due to the reduced pore size. The increased pore wall site induced by the reduced pore size is believed to have provided a larger pathway for distributing the applied stress. The increased pore site acted as a barrier, such as crack disipation, to reduce the spread of cracks. In addition, it has been reported that a more uniform pore distribution improved the mechanical properties of the polymer matrix. Therefore, the stress applied to the porous material is concentrated at the interface of the pores, and if the pore distribution is not uniform, the polymer matrix is typically deformed with lower stress (Harris LD. Et al., J. . Biomed Mater Res., 1998, 42: 396-402). For example, a recent study by the inventors (Nazarov R. In et al., Biomacromolecules, in print), a three-dimensional silk fibroin scaffold was developed using HFIP salt leaching. These scaffolds had smaller pore diameters and utilized higher concentrations of silk fibroin during processing, but the compressive strength (30 ~) of HFIP-derived silk scaffolds (17 wt% silk in HFIP) prepared by salt leaching. 250 kPa) was similar to that found for the aqueous (8-10 wt% silk in water) silk scaffolds of the present invention. However, the compressive modulus of the water-based silk scaffold was 3 to 4 times higher than that of the HFIP-derived silk scaffold (100 to 790 kPa).

<u style="single">Enzymatic decomposition</u> FIG. 4a shows the mass of the scaffold prepared from 4-8 wt% silk fibroin with NaCl having a particle size of 850 to 1000 μm in diameter for a decomposition period of 21 days over time. Scaffolds in protease-free phosphate buffer showed no degradation within 21 days. Scaffolds prepared with 4 wt% fibroin were rapidly degraded, with only 2% remaining mass after 10 days. Scaffolds prepared from 6 and 8 wt% fibroin gradually degraded over time, and after 21 days the mass decreased to 30 and 20%, respectively. Figure 4b shows the mass of scaffolding remaining when prepared from 6 wt% silk fibroin with NaCl of various particle sizes. These degradation patterns showed that pore size did not correlate with degradation rate in relation to the nature of the initial concentration of fibroin.

<u style="single">Conclusion</u> Porous silk fibroin scaffolds were prepared directly by salt leaching from aqueous silk fibroin in the complete absence of organic solvents or chemical cross-linking. Scaffold formation involved a structural transition from a random coil to a β-sheet. This transfer provides a mechanical basis for the transfer, as the salt promotes the loss of water from the hydrophobic domain, which enhances chain-chain interactions, which in turn leads to the formation of β-sheets. The functional and morphological properties of the scaffold were controlled by the concentration of silk fibroin solution used in this method and the particle size of NaCl.

<u style="single">(Example 3) Preparation of silk hydrogel</u> Control of silk fibroin concentration in aqueous solution via osmotic stress was tested to evaluate the relationship between gel formation and structural, morphological, and functional (mechanical) changes associated with this method. Environmental factors that may be important in the in vivo processing of aqueous silk fibroin were also tested and their contribution to this method was determined. Gelation of silk fibroin aqueous solution is temperature, Ca²⁺, PH, and polyethylene oxide (PEO). Gelation time is increased protein concentration, decreased pH, increased temperature, Ca²⁺Decreased with the addition of and PEO. K⁺No change was observed in the gelation time with the addition of. Upon gelation, the random coil structure of silk fibroin was transferred to the β-sheet structure. Hydrogels with a fibroin concentration of&gt; 4% by weight showed a network and sponge-like structure based on a scanning electron microscope. The pore size of the lyophilized hydrogel became smaller as the silk fibroin concentration or gelation temperature increased. Ca²⁺The lyophilized hydrogel formed in the presence of was showing larger pores as this ion concentration increased. The mechanical compressive strength and compressive modulus of hydrogels increased with increasing protein concentration and gelation temperature.

<u style="single">Method</u><u style="single">Preparation of aqueous silk fibroin solution</u> Bombix Mori cocoons were obtained with the kindness of M. Tsukada (Institute of Sericulture, Tsukuba, Japan) and M. Goldsmith (U. Rhode Island), 0.02M Na.₂CO₃Boiled in aqueous solution for 20 minutes, then rinsed thoroughly with distilled water to extract paste-like sericin protein and wax. The extracted silk fibroin was then dissolved in a 9.3M LiBr solution at 60 ° C. for 4 hours to give a 20% (w / v) solution. This solution was dialyzed in distilled water for 2 days using a Slide-A-Lyzer dialysis cassette (MWCO 3500 Pierce). The final concentration of the aqueous silk fibroin solution was about 8 w / v%, which was determined by weighing the residual solids after drying. Evaluate silk film prepared from 8w / v% solution and Li by XPS⁺Proved the removal of ions. Remaining Li⁺No ions were detected.

<u style="single">Preparation of concentrated silk fibroin solution by osmotic stress</u> Aqueous silk fibroin solution (8 wt%, 10 ml) was dialyzed against a 10-25 wt% polyethylene glycol (PEG, 10,000 g / mol) solution at room temperature using a Slide-A-Lyzer dialysis cassette (MWCO 3500). The volume ratio of PEG to the silk fibroin solution was 100: 1. Due to osmotic stress, water molecules in the silk fibroin solution migrated through the dialysis membrane to the PEG solution (Parsegian, VA, et al., Methods in Enzymology, Packer, L., Ed .; Academic Press: 1986; Vol. .127, p400). After the required time, the concentrated silk fibroin solution was slowly collected with a syringe to avoid excessive shearing and the concentration was determined. Aqueous silk fibroin solutions with a concentration of less than 8 wt% were prepared by diluting the 8 wt% solution with distilled water. All solutions were stored at 7 ° C before use.

<u style="single">Sol-gel transition</u> 0.5 ml of an aqueous silk fibroin solution was placed in a 2.5 ml flat bottom vial (diameter: 10 mm). These vials were sealed and maintained at room temperature, 37 ° C and 60 ° C. The gelation time was determined when the sample showed an opaque white color and did not fall from the inverted vial within 30 seconds. To investigate the effects of ions and ion concentrations on this method, CaCl₂Alternatively, a KCl solution was added to the silk fibroin aqueous solution to give a final salt concentration of 2.5-30 mM. The pH of the silk fibroin solution was adjusted with HCl or NaOH solution. For the preparation of a silk fibroin-poly (ethylene) oxide (PEO, 900,000 g / mol) solution, the required amount of PEO solution (5 wt%) was added to the silk fibroin solution over 5 minutes with gentle stirring. The blending ratios of silk fibroin / PEO were 100/0, 95/5, 90/10, 80/20 and 70/30 (w / w).

<u style="single">Wide-angle X-ray scattering (WAXS)</u> X-ray profiles were recorded for lyophilized silk fibroin solutions and hydrogels by Ni-filter Cu-Kα irradiation at 40 kV and 20 mA using a Bruker D8 X-ray diffractometer.

<u style="single">Scanning electron microscope (SEM)</u> The silk fibroin solution and hydrogel were frozen at -80 ° C and then lyophilized. These samples were destroyed in liquid nitrogen and tested using a LEO Gemini 982 field emission gun SEM. To check for anthropogenic morphological changes due to lyophilization, an alternative preparation method used a Karnovsky fixative at room temperature for 4 hours. With or without fixatives, hydrogels showed little morphological change upon lyophilization. Pore diameters were obtained using ImageJ software developed by the US NIH.

<u style="single">Mechanical properties</u> Hydrogel compression tests were performed at room temperature on Instron 8511 loaded with 2.5 kN-loaded cells. The crosshead speed was 10 mm / min. The cross section of the sample was 12 mm in diameter and 5 mm in height. The compression test was performed by the open side method for convenience. The compression limit was 98% strain to protect the loaded cells. Five samples were evaluated for each composition.

<u style="single">result</u><u style="single">Concentrated silk fibroin solution</u> An aqueous solution of silk fibroin having an initial concentration of 8 wt% was dialyzed against a 10 to 25 wt% PEG solution at room temperature. The aqueous silk fibroin solution was concentrated over time due to osmotic stress, and after 9 hours of dialysis against a 25 wt% PEG solution, a concentration of about 21 wt% was obtained (Fig. 6). When using lower concentrations of PEG solution, longer dialysis times were required to make higher concentrations of aqueous silk fibroin. 23-33 wt% silk fibroin gel was spontaneously produced in the dialysis cassette during the concentration method. These gels were clear even after drying at room temperature and 60 ° C.

<u style="single">Gelation of aqueous silk fibroin solution</u> Temperature for gelation of aqueous silk fibroin solution, Ca²⁺And K⁺The effects of concentration, pH, and PEO concentration were investigated. FIG. 7 shows the gelation time of an aqueous silk fibroin solution (pH 6.5 to 6.8) at various temperatures. The gelation time of the aqueous silk fibroin solution decreased with increasing fibroin content and temperature. At the same time, a structural change from a random coil to a β-sheet structure was observed, and the formation of the β-sheet structure in the hydrogel was confirmed by X-ray diffraction as described below. Figure 8 shows various Ca²⁺And K⁺It shows the gelation time of the aqueous silk fibroin solution at the concentration. Ca²⁺And K⁺The pH of the silk fibroin solution containing ions was 5.6 to 5.9 and 6.2 to 6.4, respectively. Ca²⁺Produced a relatively short gelation time, whereas K⁺There was no change in gelation time at any temperature with the addition of. These results with regenerated silk moth fibroin show that K was added to the spider silk solution.⁺Unlike previous tests where ions affected protein aggregation and precipitation, Ca²⁺There was no change in rheology after the addition of ions. FIG. 9 shows the gelation time of an aqueous silk fibroin solution (4 wt%) at various pH values. The gelation time was significantly shortened as the pH decreased. This behavior is similar to that observed for silk from the European garden spider (Araneus diadematus), which gels at pH 5.5 but behaves as a viscous liquid at pH 7.4 (Vollrath, F., et al. , Proc. R. Soc. London B, 1998, 265: 817-820). FIG. 10 shows the gelation time of an aqueous silk fibroin solution (4 wt%) containing various polyethylene oxide (PEO) contents. With the addition of the PEO solution, the pH dropped slightly to the range of 6.1-6.4. The gelation time was significantly shortened by the addition of 5% PEO alone, whereas there was no difference in the gelation time when the concentration exceeded 5%.

<u style="single">Structural analysis of hydrogel</u> Structural changes in silk fibroin were determined by X-ray diffraction. FIG. 11 shows the X-ray profile of the lyophilized silk fibroin solution and hydrogel prepared from the silk fibroin aqueous solution. When the silk fibroin solution was frozen at a low temperature (-34 to -20 ° C) below the glass transition point, its structure did not change significantly (Li, M., et al., J Appl. Polym. Sci). ., 2001, 79: 2185-2191). The lyophilized silk fibroin sample showed a wide peak near 20 °, regardless of the silk fibroin concentration, indicating an amorphous structure. Silk fibroin in a neutral pH aqueous solution showed a random coil structure (Magoshi, J., et al., Polymeric Materials Encyclopedia; Salamone, JC, Ed .; CRC Press: New York, 1996; Vol.1, p.667 Magoshi, J., et al., Polymeric Materials Encyclopedia; Salamone, J. C., Ed .; CRC Press: New York, 1996; Vol.1, p.667). All hydrogels prepared from silk fibroin solution showed a clear peak at 20.6 ° and two small peaks near 9 ° and 24 °. These peaks were similar to those of the β-sheet crystal structure of silk fibroin (Ayub, ZH, et al., Biosci. Biotech. Biochem., 1993, 57: 1910-1912; Asakura, T., et. al., Macromolecules, 1985, 18: 1841-1845). These peaks show β crystal spacing distances of 9.7, 4.3, and 3.7 Å according to 9 °, 20.6 °, and 24 °, respectively. From the results of X-ray diffraction, gelation of the silk fibroin solution induced a conformational transition from the random coil to the β sheet as previously reported (Ayub, ZH, et al., Biosci. Biotech. Biochem., 1993, 57: 1910-1212; Hanawa, T., et al., Chem. Pharm. Bull., 1995, 43: 284-288; Kang, GD, et al., Marcromol. Rapid Commun., 2000 , 21: 788-791).

<u style="single">Freeze-dried hydrogel morphology</u> The morphological characteristics of the silk fibroin solution and hydrogel were lyophilized at -80 ° C and then observed by SEM. The lyophilized 4-12 wt% silk fibroin solution showed a leaf-like morphology. The lyophilized 16 wt% and 20 wt% silk fibroin solutions showed networks and spongy structures with pore diameters of 5.0 ± 4.2 μm and 4.7 ± 4.0 μm, respectively. SEM images show that lyophilized hydrogels prepared from a 4 wt% silk fibroin solution show foliar morphology and interconnected pores regardless of temperature, and spongy structures are observed at fibroin concentrations above 4 wt%. It was decided. The pore size (&lt;1.1 ± 0.8 μm) of the lyophilized hydrogel was smaller than that observed for the lyophilized silk fibroin solution sample. The pore size of the lyophilized hydrogel decreased with increasing silk fibroin concentration, and the pore size decreased with increasing temperature at the same silk fibroin concentration. Ca²⁺The 4 wt% lyophilized hydrogel containing ions showed a network and spongy structure, but K⁺The 4 wt% lyophilized hydrogel containing ions had a foliar morphology. Ca in lyophilized hydrogels with fibroin concentration&gt; 4 wt%²⁺The pore size of the lyophilized hydrogel containing Ca²⁺It was larger than that of the lyophilized hydrogel prepared from an aqueous solution of silk fibroin containing no ions. Interestingly, the pore size is Ca at the same silk fibroin concentration.²⁺As the concentration increased, it became larger in the lyophilized hydrogel. Ca²⁺In contrast to lyophilized hydrogels containing K⁺The pore size of the lyophilized hydrogel containing was similar in size to that of the lyophilized hydrogel prepared from an aqueous silk fibroin solution. These results are Ca²⁺Is K in inducing interactions between silk fibroin chains⁺It implies that it is more effective than. This result is Ca²⁺Is K⁺It is also consistent with previous data that resulted in shorter gelation times.

<u style="single">Mechanical properties of hydrogel</u> The compressive strength and compressive modulus of hydrogels prepared from an aqueous solution of silk fibroin increased with increasing silk fibroin concentration (Figs. 12a and 12b). The improvement in mechanical properties is due to an increase in polymer concentration with a decrease in pore size. Hydrogels prepared at the same silk fibroin concentration and at higher temperatures showed higher compressive strength and modulus due to the reduced pore size. Hydrogels with 4-8 wt% fibroin showed less than 55% strain, whereas hydrogels with 12-16 wt% fibroin showed higher strains in the 75% -96% range (Fig. 12c). The effect of pore size was taken into account because the smaller pore size distributes the stress in the hydrogel more evenly and resists stress concentration. The smaller pore size and increased number of pores also serve as a barrier to the spread of cracks.

<u style="single">Consideration</u> Gelation results from the formation of intramolecular and intermolecular interactions between protein chains, including hydrophobic and hydrogen bonds (Ayub, ZH, et al., Biosci. Biotech. Biochem., 1993, 57: 1910). -1912; Hanawa, T., et al., Chem. Pharm. Bull., 1995, 43: 284-288; Kang, GD, et al., Marcromol. Rapid Commun., 2000, 21: 788-791). Interactions between fibroin chains increase with increasing fibroin content and temperature. This allows the silk fibroin molecule to interact more rapidly, which leads to physical cross-linking.

Caiko (Bombix Mori) Ca²⁺The ion concentration increases from 5 mM to 15 mM as the silk moves toward the spit tube, whereas K⁺Ion is 5 ~ 8mM³Exists in. Several calcium salts are known to dissolve silk fibroin due to its strong interaction with fibroin (Ajisawa, A., J. Seric. Sci. Jpn., 1998, 67: 91- 94; Ha, SW, et al., Biomacromolecules, 2003, 4: 488-496). The rheological measurements of the diluted solution of silk fibroin derived from Bombix moly show that the protein chain is the COO of the amino acid side chain of fibroin.^-Ion and Ca²+ Or Mg²⁺It was revealed that there is a tendency to form clusters by ionic interactions with divalent ions such as (Ochi, A., et al., Biomacromolecules, 2002, 3: 1187-1196). Through these interactions, Ca²⁺The pH of the ion-containing silk fibroin solution was significantly lower than that of the ion-free silk fibroin solution, whereas K⁺Addition of monovalent ions such as, showed only a slight decrease in pH. Lower pH reduced the repulsive force between silk fibroin molecules, facilitated interchain interactions, and increased the likelihood of forming β-sheet structures via hydrophobic interactions. PH near the isoelectric point (pI = 3.8 ~ 3.9) of silk fibroin (Ayub, ZH, et al., Biosci. Biotech. Biochem., 1993, 57: 1910-1912; Kang, GD, et al., Marcromol. Rapid Commun., 2000, 21: 788-791) accelerated the sol-gel transition of aqueous silk fibroin in a manner similar to other proteins that aggregate near the isoelectric point.

These results reflect subtle differences in how different silk proteins from different organisms utilize physiologically related ions to promote sol-gel transitions. Divalent ions can induce aggregation of silk fibroin molecules, especially by ion interaction with negatively charged amino acids present near the ends of the heavy chain fibroin chain. Different concentrations of Ca²⁺The lack of response to is suggesting a broad window for the role of the combination of ions to fully control the physiological response or perhaps this method in vivo or in vitro. Additional studies may be needed to elucidate these relationships, especially if considered in response to findings on silk domain mapping related to the processing environment (Bini, E., et al., J). . Mol. Biol., 2004, 335: 27-40).

The transfer of water from silk fibroin molecules to hydrophilic PEO promotes intramolecular and intermolecular interactions between protein molecules, as well as the subsequent formation of β-sheet structures. This transition is evident in silk, based on our latest mechanical understanding of this method (Jin, HJ, et al., Nature, 2003, 424: 1057-1061). These transitions can be induced by direct addition of PEO to aqueous fibroin or via separation (by PEG) from aqueous solution across the dialysis membrane. Therefore, no direct contact between the protein and PEO is required, and only facilitating water transport from the protein to PEO / PEG drives the sol-gel transition.

<u style="single">Conclusion</u> From the primary sequence, the silk moth silk fibroin heavy chain is composed of 7 internal hydrophobic blocks with 2 large hydrophilic blocks at the ends of the chain, and 7 much smaller internal hydrophilic blocks (Zhou,). CZ, et al., Nucleic Acids Res., 2000, 28: 2413-2419; Jin, HJ, et al., Nature, 2003, 424: 1057-1061). The proportion of hydrophobic residues in silk fibroin is 79% (Braun, FN, et al., Int. J. Biol. Macromol., 2003, 32: 59-65) and repeat sequences in hydrophobic residues. Consists of silk fibroin fibers and GAGAGS peptides that govern the β-sheet structure that forms the crystalline regions within the film (Mita, K., et al., J. Mol. Evol., 1994, 38: 583-592). The formation of these β-sheets results in insolubility in water (Valluzzi, R., et al., J. Phys. Chem. B, 1999, 103: 11382-11392). The hydrophobic regions of silk fibroin in aqueous solution physically assemble by hydrophobic interactions and are eventually organized into hydrogels (Jin, HJ, et al., Nature, 2003, 424: 1057-1061). ). Silk fibroin concentration, temperature, Ca²⁺, PH and PEO affect the gelation of aqueous silk fibroin. With increasing fibroin content and temperature, physical cross-linking between silk fibroin molecules is more easily formed. Ca²⁺Ions promote these interactions, presumably via a hydrophilic block at the end of the chain. Lowering the pH and adding hydrophilic polymers reduces the repulsive force between silk fibroin molecules, promotes the elimination of water from the protein, resulting in shorter gelation times. Upon gelation, the structural transition from the random coil to the β-sheet structure is induced, and the insolubility and stability of the silk fibroin hydrogel in water is promoted. Silk fibroin hydrogel has a network and sponge-like structure. The pore size became smaller as the silk fibroin concentration and gelation temperature increased. The lyophilized hydrogel is Ca²⁺As the concentration increased, it showed a larger pore size than the lyophilized hydrogel prepared from an aqueous solution of silk fibroin with the same fibroin content. The compressive strength and compressive modulus of hydrogels prepared from an aqueous solution of silk fibroin containing no ions increased with increasing protein concentration and gelation temperature.

Hydrogels derived from natural polymers such as collagen, hyaluronic acid, fibrin, alginate, and chitosan have been recognized for many uses in tissue engineering in addition to drug delivery. However, they generally provide a limited range of mechanical properties (Lee, KY, et al., Chem. Rev., 2001, 101: 1869-1879). In contrast, silk fibroin is important in the field of biomaterials and scaffolds for controlled release, tissue engineering due to its combination of excellent mechanical properties, biocompatibility, biodegradability, and cell interactions. Offers a range of material options.<img file="JP4698596B2_D0009.tif" />

<u style="single">(Example 4) Bone regeneration using a silk scaffold derived from three-dimensional water</u> We tested bone regeneration of human bone marrow stem cells on a three-dimensional silk scaffold from the aqueous silk solution of the present invention. To test the ability of silk scaffolds to support the proliferation and differentiation of bone marrow stem cells, we used silk scaffolds without any modification.

<u style="single">Method</u><u style="single">material</u> Bovine serum, Dalbeco's modified Eagle's medium (DMEM), minimum essential medium α modified (αMEM), basic fibroblast growth factor (bFGF), penicillin-streptomycin (Pen-Strep), fungizone, non-essential amino acids, trypsin , Obtained from Gibco (Carlsbad, CA). Ascorbic acid phosphate, Histopaque-1077, dexamethasone, and β-glycerophosphate were obtained from Sigma (St. Lois, MO). All other substances are analytical or pharmaceutical grade and were obtained from Sigma. The silk moth cocoons were obtained with the kindness of M. Tsukada (Institute of Sericulture, Tsukuba, Japan) and Marion Goldsmith (University of Rhode Island, Cranston, RI).

<u style="single">Scaffolding preparation</u> Aqueous-derived silk scaffolds were prepared by adding 4 g of granular NaCl (particle size; 1000 to 1180 μm) to 2 ml of an 8 wt% silk fibroin solution in a disc-type Teflon container. The container was covered and left at room temperature. After 24 hours, the container was impregnated with water and NaCl was extracted for 2 days. Silk scaffolds derived from HFIP were prepared by adding 4 g of granular NaCl (particle size; 850 to 100 μm) to 2 ml of 8 wt% silk fibroin in HFIP. The container was capped to reduce HFIP, providing sufficient time for a more uniform distribution of this solution. The solvent was allowed to volatilize at room temperature for 3 days. The silk / porogen complex was treated in methanol for 30 minutes to induce insolubility in β-sheet structure and aqueous solution, after which the complex was impregnated with water for 2 days to remove NaCl. This porous silk scaffold was air dried.

<u style="single">Isolation and proliferation of human bone marrow stem cells</u> Whole bone marrow (25 cm)³, Clonetics, Santa Rosa, CA.) Was diluted in 100 ml of isolation medium (5% FBS in RPMI 1640 medium). Cells were separated by density gradient centrifugation. Briefly, a 20 ml aliquot of bone marrow suspension with a polysucrose gradient (1,077 g / cm).³, Histopaque, Sigma, St. Louis, MO) and centrifuged at 800xg for 30 minutes at room temperature. The cell layer was carefully removed, washed with 10 ml of separation medium, pelleted in 5 ml of Pure-Gene lysate, and contaminated erythrocytes were lysed. Cells are pelleted and suspended in growth medium (DMEM, 10% FBS, 1 ng / ml bFGF, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml fungizone, non-essential amino acids), 75 cm.²In the flask, density 5x10⁴Individual cells / cm²Was sown in. Adherent cells reached about 80% confluence (primary passage 12-17 days). The cells were trypsinized, reseeded and passage 2 (P2) cells (80% confluent after 6-8 days) were used in the experiment.

<u style="single">In vitro culture</u> BMSC (5x10) to test cell proliferation and differentiation in vitro on silk scaffolds⁵Individual cells / scaffolds, passage 2) were seeded on pre-moistened (α-MEM, overnight) silk scaffolds. After 24 hours, the medium was removed and the culture was maintained in individual wells on a 6-well plate. The osteogenic medium was αMEM supplemented with 10% FBS, non-essential amino acids, 50 μg / ml ascorbic acid-2-phosphate, 10 nM dexamethasone, and 7 mM β glycerophosphate in the presence of penicillin and streptomycin and fungizone. .. Culture is 5% CO₂Was maintained at 37 ° C in a replenished humidified incubator. Half of the medium was replaced every 2-3 days.

<u style="single">Biochemical analysis and histology</u> Scaffolds were cultured in osteogenic medium for 2 and 4 weeks and treated for biochemical analysis and histology. For DNA analysis, 3-4 scaffolds per group per time point were degraded. DNA content (n = 3-4) was measured using the PicoGreen assay (Molecular Probes, Eugene, OR) according to the manufacturer's protocol. The sample was fluorescently measured at an excitation wavelength of 480 nm and an emission wavelength of 528 nm. For total calcium content, the sample (n = 4) was extracted twice with 0.5 ml of 5% trichloroacetic acid. Calcium content was determined by colorimetric assay using o-cresolphthalein complexone (Sigma, St. Louis, MO). The calcium complex was measured at 575 nm by spectrophotometry. Alkaline phosphatase activity was measured using a Sigma (St. Louis, MO) biochemical assay based on the conversion of p-nitrophenyl phosphate to p-nitrophenol as measured spectrophotometrically at 405 nm. ..

<u style="single">RNA isolation, real-time reverse transcription-polymerase chain reaction (real-time RT-PCR)</u> The new scaffold (n = 3-4 / group) was transferred to a 2 ml plastic tube and 1.0 ml trizol was added. The scaffold was disassembled using a steel ball and a Microbeater. The tube was centrifuged at 12000 g for 10 minutes and the supernatant was transferred to a new tube. Chloroform (200 μl) was added to this solution and incubated for 5 minutes at room temperature. The tube was centrifuged again at 12000 g for 15 minutes and the upper aqueous layer was transferred to a new tube. One volume of 70% ethanol (v / v) was added and added to an RNeasy minispin column (Quiagen, Hilden, Germany). RNA was washed and eluted according to the manufacturer's protocol. RNA samples were reverse transcribed into cDNA using oligo (dT) selection according to the manufacturer's protocol (Superscript Preamplification System, Life Technologies, Gaithersburg, MD). Gene expression of type I collagen, type II collagen, alkaline phosphatase, bone sialoprotein and osteopontin was quantified using the ABI Prism 7000 Real Time PCR System (Applied Biosystems, Foster City, CA). The PCR reaction conditions were 50 ° C for 2 minutes, 95 ° C for 10 minutes, then 95 ° C for 15 seconds for 50 cycles, and 60 ° C for 1 minute. Expression data were standardized for the expression of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). The GAPDH probe was labeled with the fluorescent dye VIC at the 5'end and with the quencher dye TAMRA at the 3'end. The primer sequence for the human GAPDH gene was as follows: forward primer<img file="JP4698596B2_D0010.tif" />, Reverse primer<img file="JP4698596B2_D0011.tif" />,probe<img file="JP4698596B2_D0012.tif" />.. Alkaline phosphatase, bone sialoprotein (BSP), osteopontin primers and probes were purchased from Applied Biosciences (Assay on Demand #, Hs 00240993 ml (ALP), Hs 00173720 ml (BSP), Hs 00167093 ml (osteopontin)).

<u style="single">Western blot analysis</u> For total protein extraction, cells are subjected to RIPA buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.2% SDS, containing protease inhibitors and phosphatase inhibitors. , 5 mM NaF]. The protein content was measured by the Bradfod method. The protein was degraded by 3-8% SDS-PAGE and transferred to a membrane. The blots were probed with the primary antibody at 4 ° C. for 12 hours, washed and incubated with the appropriate peroxidase-labeled secondary antibody for 1 hour at room temperature. The protein band was manifested by ECL (Armersham-Pharmacia).

<u style="single">Scanning electron microscope (SEM) analysis</u> The polymer surface was tested by scanning electron microscopy (SEM) before and after cell adhesion. The matrix was fixed with Karnovsky fixative for 24 hours and washed 3 times in CMPBS to remove residual fixative. These samples were then dried using a series of stepwise ethanol (50-100%) at 15 minute intervals. After drying, the sample was sputter coated with gold and examined with a LEO Gemini 982 field emission gun SEM.

<u style="single">Histological evaluation</u> After fixing with 4% phosphate buffered formaldehyde for at least 24 hours, the specimens were embedded in paraffin and sectioned (4 μm). Serial sections were stained with hematoxylin eosin and alcian blue using standard histochemical techniques.

<u style="single">result</u><u style="single">SEM analysis</u> The characteristics of the 3D silk scaffold were determined by structural evaluation by SEM for analysis of pore size distribution and surface topography. SEM analysis showed that the water-silk scaffold had an interconnected porous network with an average pore size of 920 ± 50 μm. The pore surface had the appearance of a coarse structure containing non-homogeneous micropores. However, the HFIP-silk scaffold had a porous network with an average pore diameter of 900 ± 40 μm and poor interconnection, and showed a smooth surface structure. BMSC (passage 2) was seeded on water-silk sponge and HFIP-silk sponge. More cells adhered to the water-silk sponge than to the HFIP-silk sponge. Water-silk sponge promoted cell seeding. And BMSC was evenly distributed throughout the water-silk sponge. In contrast, the distribution of BMSC on the HFIP-silk sponge was not uniform. BMSC proliferation was observed on water-silk sponges. SEM confirmed widespread growth of BMSC on water-silk sponge, followed by up to 4 weeks of growth.

<u style="single">Exam with the naked eye</u> Cell scaffold construct is 5% CO₂In the atmosphere, the cells were cultured in osteogenic medium at 37 ° C. Constructs were cultured in 6-well plates for up to 28 days. The BMSC water-silk construct began to round after culturing, but the BMSCHFIP-silk construct was initially flat and did not change after culturing. The tissue formed in the water-silk scaffold was whitish and hard to the touch with hand and surgical forceps. However, BMSC seeded on the HFIP-silk scaffold did not form a whitish tissue. The 2-week and 4-week specimens showed no significant difference in the macroscopic study. The uniform cell distribution of BMSC in the water-silk scaffold was qualitatively apparent by uniform matrix staining (an indicator of MTT conversion by viable cells) on the surface and throughout the center of the scaffold. However, the HFIP-silk scaffold showed strong staining along the surface of the construct and weakly stained areas inside the construct.

<u style="single">Biochemical analysis</u> The porosity of the 3D matrix was about 92% for both water-silk and HFIP-silk. The compressive strength and compressive modulus of the water-silk scaffold were 100 ± 10 kPa and 1300 ± 40 kPa. These values for HFIP scaffolding were 50 ± 5 kPa and 210 ± 60 kPa.

The total number of cells cultured on these scaffolds was quantified using a DNA assay over time in this study. For water-silk scaffolds seeded with cells suspended in medium, the number increased from 51,000 ± 12,000 cells after initial seeding to 150,000 ± 12,000 cells after 28 days of culture. HFIP-silk scaffolds seeded with cells suspended in medium showed no significant proliferation from 8,000 ± 3,400 cells after initial cell seeding to 32,000 ± 11,000 cells after 28 days of culture. It was.

Alkaline phosphatase (ALPase) activity, an indicator of the commitment of osteoprogenitor cells to the osteoblast phenotype, was measured per scaffold. For water-silk scaffolds, ALPase activity (9.7 ± 0.3 mmol / scaffold) after 28-day culture was significantly increased compared to day 1 (0.4 ± 0.01 mmol / scaffold). For HFIP-silk scaffolds, 2.9 ± 0.12 mmol / scaffold was detected 28 days after culturing.

The total calcium content of each sample was measured for each scaffold. For water-silk scaffolds, significant calcium deposition (10.5 ± 0.65 μg / scaffold) was observed after 28 days of culture in osteogenic medium. For HFIP-silk scaffolds, after 28 days of culture, Ca²⁺There was 1.4 ± 0.1 μg / scaffolding.

<u style="single">Expression of genes related to osteogenic differentiation</u> To characterize the bone-like tissue produced by BMSC, the expression of several marker genes for osteogenic and chondrogenic differentiation was quantified using a real-time RT-PCR assay. The analyzed genes were bone-forming differentiation marker type I collagen (Col I), alkaline phosphatase (ALP), osteopontin (OP), bone sialoprotein (BSP), and chondrogenic differentiation marker type II collagen (Col II). Including. Differences in transcription levels (standardized for GAPDH within a linear range of amplification) between scaffold types were significant. Col I, ALP, and OP transcription levels were increased in water-silk scaffolds compared to HFIP-silk scaffolds. After 28 days of culture on water-silk scaffolds, Col I, ALP and BSP gene expression was significantly increased by 190%, 1100% and 10500%, respectively, compared to after 1 day of culture. However, the expression of OP and Col II was significantly reduced. BSP expression was similarly regulated in water-silk scaffolds and HFIP-silk scaffolds. Differences between scaffold types were not statistically significant.

<u style="single">Expression of proteins associated with osteogenic differentiation</u> Under 3D water-silk scaffold culture conditions, human bone marrow stem cells expressed osteoblast markers. Compared to the HFIP-silk construct, Col I expression showed a significant increase in protein levels after 2 weeks of culture under water-silk culture conditions. However, Col I expression decreased after 4 weeks of culture under both conditions. After 28 days of culture, OP expression was increased in the water-silk construct. The protein showed two bands, the highest of which was estimated to be highly glycosylated, sulfated, or phosphorylated (Singh et al., J. Biol. Chem., 1990, 65). : 18696-18701). BSP, another bone protein, was expressed in cells cultured on both water-silk scaffolds and HFIP-silk scaffolds. However, its expression increased in HFIP-silk constructs after 28 days of culture.

We also analyzed the expression of matrix metalloproteinase 13 (MMP13) and Col II. MMP13 was expressed only in the water-silk construct. Col II was also down-regulated after 4 weeks under both culture conditions.

<u style="single">Histological examination</u> Histological studies using hematoxylin and eosin staining of these specimens revealed that the proportion of osteoblast-like cells in their cubic or columnar morphology increased with extended culture periods in water-silk constructs. I made it. After 14 days of culture, almost all pores were filled with connective tissue, fibroblasts, and cubic osteoblast-like cells. After 28 days, these pores were filled with extracellular matrix, osteoblast-like cells, and slight fibroblast-like morphological cells. However, histological sections of the HFIP-silk scaffold revealed that there was a slight distribution of cells, most of which formed a cell layer on the surface of the scaffold. After 28 days of culture, the majority of cells in the HFIP-silk construct showed flat fibroblast morphology.

After culturing in osteogenic medium, the extracellular matrix of proteoglycans stained with alcian blue revealed that proteoglycans were detected in the water-silk construct. Proteoglycans were not histologically detected in the HFIP-silk construct.

<u style="single">Consideration</u> Silk protein-based matrix scaffolds are of current interest in bone tissue engineering. These scaffolds exhibited higher mechanical properties than other common biodegradable synthetic and natural polymers such as PGA-PLA copolymers and collagen. Porous silk fibroin material has been prepared using HFIP. HFIP-silk scaffolds are known for their unique mechanical properties, but these natural polymers result in inadequate cell adhesion due to the lack of cell recognition signals. To overcome this problem, many approaches have been developed, including surface modification with arginine-glycine-aspartic acid (RGD) and hybrids with natural biodegradable polymers.

Cell adhesion is known as an important cellular process because it directly affects cell proliferation and differentiation. In this example, the silk scaffold was used unmodified. We found sufficient cell adhesion in water-silk scaffolds. More cells adhered to the water-silk scaffold than the HFIP-silk scaffold. Surface tissue, or microtopography diversity, can affect cellular responses. It was found that cells adhered to the rougher surface at a higher rate. SEM and histological analyzes have shown that our water-silk scaffolds have a coarse structure with uniform pores. However, the HFIP-silk scaffold had a smooth surface structure.

For the microstructure of porous scaffolds, high porosity (&gt; 90%) and interconnected pore networks are desirable. In addition, preferred pore sizes are generally in the range of 50-500 μm to allow cell endoculture and tissue regeneration (Katoh K. et al., Biomaterials, 2004, 25: 4255-4262; Thomson). R, et al., In Principles of Tissue Engineering; Lanza R, Langer R and Vacanti J, eds. Academic Press: San Diego, pp.251-262, 2000). Relaxation of vegetative transport restrictions outside the 3D cell / polymer construct affects the proliferation, differentiation, and expression of MSC osteoblast markers seeded on 3D scaffolds (Sikavitsas VI. Et al. , J. Biomed Mater Res, 62: 136-148). Structural characterization showed that the pore size and porosity of the water-silk sponge was controlled by the size of the NaCl particles. We prepared a water-silk sponge with a regulated pore size (920 ± 50 μm) with a porosity greater than 90%. Furthermore, these pores were open to the outside, connected to each other, and evenly distributed throughout the sponge.

Human BMSCs were seeded on porous silk scaffolds and the BMSC silk constructs were cultured in two model silk scaffolds (water-silk scaffold and HFIP-silk scaffold) for an extended period of 28 days. BMSCs seeded on water-silk scaffolds showed accelerated growth during the first 2 weeks of culture, as well as strongest ALP activity and highest calcium attachment at the end of the culture period.

During both fetal development and adult repair, the onset of skeletal formation begins with the condensation of mesenchymal stem cells. Immediately after the condensation stage, cells in the central region of aggregation take the cartilage phenotype (Ferguson C. et al., Mech. Dev., 1999, 87: 57-66). Expression of Col II demonstrated this event in our silk scaffolds. Our study of Col II found that, despite early Col II gene expression, differentiated BMSCs cultured on water-silk scaffolds maintained a differentiated phenotype until the end of the culture period. Indicated. Initially, we also observed Col II expression in HFIP-silk constructs. However, Col II gene expression and protein expression were significantly reduced.

Chondrocytes progress from proliferation to a hypertrophic state. The majority of hypertrophied chondrocytes are ultimately destined to undergo programmed cell death, which is accompanied by extracellular matrix (ECM) remodeling followed by new bone attachment ( Gerber H. et al., Nat. Med. 1999, 5: 623-628). MMP13 regulates the remodeling of the hypertrophic cartilage matrix. Expression of MMP13 was observed in the water-silk construct but not in the HFIP-silk construct, indicating that the cells of the water-silk scaffold are mature and hypertrophied chondrocytes. ..

Switching from a cartilage template to bone during endochondral bone formation was not just a cell phenotypic switch. The cartilage ECM is then replaced by the bone ECM. Proteoglycan synthesis, ALP expression, and type I collagen expression were detected in the water-silk construct, but type II collagen was scarcely detected. Expression of ALP and type I collagen (a marker of osteoblast differentiation) was significantly increased in the water-silk construct, but was thought to contain 8-12% of total non-collagen protein in other bone proteins. The BSP produced was similarly expressed in water-silk scaffolds and HFIP-silk scaffolds. Co-expression of type I and type II collagen has been demonstrated in studies (Nakagawa T. et al., Oral Diseases, 2003, 9: 255-263), where chondrocytes display their phenotypes. It shows that a bone-like matrix is created and modified to remain in the intrachondrial bone.

OP, one of the osteoblast markers, is clearly highly expressed in the following two stages of bone formation: an early proliferative stage, followed by an early formation of a calcified bone matrix. Late stage (Yae, KL. Et al., J. Bone Miner. Res., 1994, 9: 231-240). In early culture, OP expression was upregulated in the water-silk construct. These studies point to the usefulness of water-silk scaffolds in the early formation of bone tissue. Type I collagen makes up the largest part (90%) of the organic matrix of bone, but it is not unique to this tissue. Proteoglycans, or at least their constituent glycosaminoglycan chains, have long been recognized as a small but important component of the calcified bone matrix (Fisher LW. Et al., J. Biol. Chem., 1982, 258). : 6588-6594; Fedarko NS. Et al., J. Biol. Chem., 1990, 265: 12200-12209). Staining of sections with Alcian blue stain after culturing on water-silk scaffolds clearly demonstrated the presence of proteoglycans in the ECM. Proteoglycans can be found in cartilage. The origin and tissue specificity of these proteoglycans have not been determined. In our study, proteoglycans were detected 14 days after culturing in water-silk constructs, but not in HFIP-silk constructs.

References cited throughout this application are incorporated herein by reference.

The accompanying drawings incorporated in and in part thereof are used to illustrate and explain aspects of the invention and to explain the objectives, advantages and principles of the invention.<figref num="1">FIG. 1 illustrates one embodiment of the method of the invention for producing a highly concentrated regenerated silk fibroin solution.</figref><figref num="2">FIG. 2 illustrates one embodiment of the method of the invention for the preparation of porous silk fibroin scaffolds.</figref><figref num="3">Figures 3a and 3b show the (Fig. 3a) X-ray diffraction and (Fig. 3b) FTIR spectra of the silk fibroin scaffold prepared by the water-based method described in Example II.</figref><figref num="4">Figures 4a and 4b show the mass of scaffolding remaining over time when prepared with (Figure 4a) 4 or 8 wt% silk fibroin and NaCl with a particle size of 850-1000 μm, and (Figure 4b) NaCl with various particle sizes. Shows the mass of the scaffold prepared at 6 wt%.</figref><figref num="5">5a and 5b illustrate one aspect of the invention relating to silk film preparation, including (FIG. 5a) water treatment and (FIG. 5b) stretching.</figref><figref num="6">Figure 6 shows the concentrations of silk fibroin solution (black) and gel (white) prepared by dialysis against PEG solution (circle; 25 wt%, square; 15 wt%, triangle; 10 wt%) at room temperature. There is. The value is the mean ± standard deviation of the three samples.</figref><figref num="7">FIG. 7 shows the gelation time of the silk fibroin aqueous solution at various temperatures (pH 6.5 to 6.8, no ions). The value is the mean ± standard deviation of the 7 samples.</figref><figref num="8">Figures 8a, 8b, and 8c show different Ca at room temperature (Figure 8a), 37 ° C (Figure 8b) and 37 ° C (Figure 8c).²⁺(pH 5.6 ~ 5.9) and K⁺The gelation time of the silk fibroin aqueous solution at a concentration of (pH 6.2 to 6.4) is shown.</figref><figref num="9">FIG. 9 shows the gelation time of an aqueous silk fibroin solution at various pH (4 wt% silk fibroin; no ions; room temperature). The value is the mean ± standard deviation of the 7 samples.</figref><figref num="10">FIG. 10 shows the gelation time of an aqueous silk fibroin solution at various PEO contents (4 wt% silk fibroin; pH 6.1 to 6.4; no ions; room temperature). The value is the mean ± standard deviation of the 7 samples.</figref><figref num="11">Figures 11a and 11b show X-ray diffraction of hydrogels prepared from (Fig. 11a) lyophilized silk fibroin solution and (Fig. 11b) aqueous silk fibroin solution at 60 ° C.</figref><figref num="12">Figures 12a, 12b, and 12c show the compressive strength (FIG. 12a), compressive modulus (FIG. 12b), and fracture strain (FIG. 12c) of hydrogels prepared from an aqueous silk fibroin solution at various temperatures.^**Hydrogels prepared at 60 ° C. with a silk fibroin concentration of 16 wt% were not ground under the conditions used in this study. The value is the mean ± standard deviation of the 5 samples.</figref>