Method for preparing a solid-fibrin web

<u style="single"> Cross-reference of related applications </u> This application claims priority under 35 U.SC § 119 (e) under US Provisional Application No. 60 / 392,669 filed June 27, 2002.

The present invention relates to systems, kits and methods for producing solid fibrin nets or self-derived fibrin roux. Fibrin loops are primarily known as blood inducers used as topical surgical adhesives or hemostatic agents. Several kits containing enriched fibrinogen from donors are available on the market in connection with protein activators of human or animal origin such as thrombin or batroxobin to obtain fibrin roux of heterologous origin. ..

Such known kits involve the use of materials of human or animal origin, which can pose a viral contamination and serious risk to the recipient of fibrin roux. In the past, competent authorities have even been forced to suspend or even ban the trading of blood inducers obtained using materials of human or animal origin. In addition, cases of rejection in patients due to reimplantation of fibrin produced using human or animal proteins are known from the literature. Such cases are indeed due to the fact that some of the re-implanted sealant proteins or components used in their production are of heterologous origin for the recipient organism.

Self-derived fibrin roux, i.e., self-derived fibrin roux from the patient's own blood, is more reliable against the risk of rejection and / or infection. Although some methods for obtaining instant self-derived fibrin roux have already been described, "ready-to-use" kits, despite the fact that some relevant references can be found in the patent literature. Not available on the market.

U.S. Pat. No. 5,733,545 discloses the combination of plasma-pial concentrate and fibrinogen activator to form a wound sealant with platelet glue. While the method disclosed in this patent allows the treatment of a patient's blood to obtain a self-derived fibrin roux, the method uses thrombin or batroxobin as the fibrinogen activator. These activators are of human or animal origin and therefore still carry the risk of rejection and / or viral infection to the patient.

U.S. Pat. No. 5,555,007 discloses methods and instruments for making concentrated plasma for use as a tissue sealant. The method comprises separating the plasma from whole blood and removing water from the plasma by contacting it with a concentrating agent to provide concentrated plasma, which is then subjected to thrombin and calcium. Can be coagulated with a solution containing. The instrument includes a first centrifuge in a first chamber, a concentrate contained in a second chamber communicating with the first chamber (eg, dextranomer or polyacrylamide), and a second. Includes a separator. The method disclosed in this reference requires a long time to obtain the plasma concentrate necessary for the subsequent production of self-derived fibrin roux, and the instruments are expensive and not disposable. This method does not disclose the use of calcium coagulation activators and requires a prior concentration step.

Many methods and systems require the transfer of fluid from one container to another. For example, many chemical and medical devices require the transfer of various reagents and an essential volume of liquid that sequentially reacts with a particular volume of aliquots. A common method is to remove the lids of the two containers and pipette the liquid in one container to another. However, this method exposes the sample to environmental pollution. For example, this technique is used to transfer plasma separated from red blood cells in a blood sample. However, special techniques are required to remove plasma with the interfacial meniscus. Often, high density, unwanted lower partial red blood cell cells contaminate the aspirated sample. To avoid this problem, the pipette is often kept at a safe distance from the meniscus (ie, the isolate between plasma and red blood cells), which results in incomplete sample transfer. Incomplete transfer of the desired fraction results in lower volume yields than optimal, and the ratio of sample reagent to reagent in the second container is no stoichiometric. This second condition can be a significant cause of fluctuations in product performance. This is the case in many enzymatic reactions where the reaction rate is maximal at a particular stoichiometric ratio and sharply decreases at a larger or smaller ratio.

In medical care, especially in the case of chronic ulcers, fistulas, etc., wound care is one of the most important matters. This is important not only because of the high management costs, but also because of the low success rate. Other issues related to wound care and burn care include the potential for fluid loss and infection. Synthetic membranes or animal-derived membranes are used to separate bone cavities from soft tissue during the process of reossification.

One treatment for treating a wound may include applying an animal-derived biological tissue or sponge (generally protein-based) to the wound site, for example collagen, fibrin, albumin. However, such applications are generally associated with allergic and immunological responses. 50% of these cases do not resolve with a single application. More than 20% may not resolve after two applications.

Other procedures include skin grafts performed on the most difficult cases. However, skin grafts are expensive and can cost about $ 600-700 per application. A modified horse collagen mesh is used to support new autologous tissue. This application is a difficult process that can take up to 20 days to culture skin tissue, depending on the area, as well as the potential to contaminate the sample.

Overall, methods and systems for producing self-derived fibrin roux or solid fibrin capable of regenerating tissue in living organisms are desired.

Prior to elaborating on one embodiment of the invention, the invention is not limited in its application to the details of the structure and arrangement of the components described in the following description or shown in the drawings. Should be understood. Other embodiments are possible, and the invention can be embodied or implemented in a variety of ways. It is also understood that the expressions and terminology used herein are for illustration purposes only and should not be considered limiting.

<u style="single">Detailed description of the invention</u> This application is incorporated by citing the subject matter of US Pat. No. 6,368,298, issued April 9, 2002. This application is also filed on January 15, 2002, US Patent Application No. 10 / 053,247, and on June 27, 2002, US Patent Application No. 60 / 392,669. Incorporate by quoting the subject of.

The present invention provides a ready-to-use kit that, when used surgically, alleviates at least partial alleviation of viral infections and / or rejections and allows rapid acquisition of self-derived fibrin roux. Can be done.

This is an inorganic compound, not of human or animal origin, and can therefore be achieved by using a coagulation activator that is non-infectious and does not cause rejection. The "ready-to-use" kit according to the invention may include a sealed container containing calcium chloride as a coagulation activator. Calcium chloride activates fibrinogen present in the patient's plasma when the patient's plasma is introduced into a sealed container.

The systems and kits according to the invention have the very advantageous effect of allowing the production of self-derived fibrin roux that can be used without the risk of viral infection or rejection cases. The kit according to the invention may also allow the production of self-derived fibrin roux from patient plasma in a very short time and during the formation of clots or membranes or during spraying. Ready-to-use kits according to the invention may also allow self-derived fibrin loops to be obtained at a lower cost compared to known systems. Also, this ready-to-use kit may provide platelets and their associated growth factors for rapid tissue regeneration.

Further favorable effects of the kit according to the present invention will become apparent to those skilled in the art from the following detailed description of some embodiments thereof. Containers suitable for kits according to the invention include glass containers for antibiotics, as described below in Example 1. Glass or plastic test tubes can also be used. The preferred volume of the container is 5-15 ml. The test tube is preferably in the range of 12 to 16 mm in diameter and 75 to 100 mm in height. The container must be of appropriate thickness to withstand the pressure due to the pressure difference between its inner space and the atmosphere when the container is evacuated. Preferably, a test tube having a hemispherical or conical bottom has a thickness of 0.7 mm, and a test tube having a flat bottom has a thickness of 1 mm. The plastic container is preferably made of a clear polyester resin with a thickness of 0.2-0.8 mm to ensure that the vacuum pressure is maintained for at least 12 months after production. Manufacturing Later, the plastic test tube is preferably placed in a tin foil vacuum airtight container with a heat-sealed inner polyethylene layer to ensure complete airtightness until the date of use.

It is advisable to exhaust the container or test tube, but it should be understood that it is not essential to embody the present invention. The container or test tube is a rubber suitable to ensure that the container is completely airtight and to allow vacuum plugging after the introduction of chemical components and before the steam or radiation sterilization step. Alternatively, it can be sealed with a silicone piercing cap.

After sealing, the container can be sterilized with steam at 121 ° C for 30 minutes. The sterilization process can also be performed by irradiation with a gamma ray or an electron beam. Although the fibrin stabilizer tranexamic acid can be used, pure crystalline ε-aminocaproic acid is also suitable. When using a 25 ml container suitable for a volume of 20 ml plasma, the amount is about 1 g. It may not be necessary to use fibrin stabilizers. Other healing agents that improve performance may be added to the second vessel for inclusion in the network of fibrin and platelets. Examples include, but are not limited to, bone and soft tissue implants, scaffolding materials, antibiotics, analgesics, stem cells, chemotoxicants for cancer cure, immunosuppressants, cells designed to express the desired molecule, and Including those combinations.

Solid CaCl as a coagulation activator₂ 2H₂A liquid solution containing O or calcium can be used in the kit according to the invention, but other coagulation activators (listed below) can also be used. For example, using a precision dosage meter (maximum error: 1-2 mg), 11.76 mg of CaCl₂ 2H₂O can be introduced into a 5 ml container to prevent the introduction of contaminated external components. Alternatively, other cation species such as magnesium, manganese or zinc ions, which have a greater affinity for anticoagulants than endogenous calcium, are used in place of divalent calcium cations. can do. When anticoagulant platelet-rich plasma (PRP) is added, the endogenous calcium ions are replaced from the anticoagulant by a higher affinity cation species, and the original endogenous calcium ions are available for blood clot activation. become.

For a 15 ml container for a plasma volume of 12 ml, the amount of solid anhydrous calcium chloride to be introduced would be 35.28 mg, while the amount of tranexamic acid would be as much as 300 mg crystals.

For a 25 ml container for 20 ml of plasma, the amount of anhydrous calcium chloride to be introduced would be 58.8 mg, while the amount of tranexamic acid would be as much as 500 mg of crystals.

Calcium chloride can be in the anhydrous form used in the examples, as well as, for example, CaCl.₂ 2H₂It can be any other suitable form available on the market such as O. A solution of this salt can also be used, as described in Example 1 below.

The present invention also provides a system and method for forming a solid fibrin network or self-derived glue capable of regenerating tissue in a living organism. In these methods and systems, anticoagulant plasma is obtained by centrifuging blood samples. The transfer device described herein allows plasma to be transferred to a second container containing a calcium clotting agent and then immediately centrifuged for the purpose of obtaining a stable and dense autologous fibrin and platelet network. To do. The transfer device described herein can also be used to transfer other liquids in other applications. In other words, the methods, transfer devices and systems described herein allow for simultaneous centrifugation and coagulation. By using these systems and methods, the following: 1) Manipulate the sample in such a way that it remains sterile; 2) Transfer the total amount of plasma to maximize the total yield of clots. 3) Keep the stoichiometric ratio of anticoagulant to calcium coagulant within a narrow range to minimize clotting time; 4) Transfer completes quickly and platelet-derived growth What can be done while effective within the half-life of the factor; 5) Health care providers (eg, dentists) who normally do not do this can easily perform these methods and activate the system. 6) The device can achieve at least one of being single-use to prevent reuse and possible contamination by pathogens contained in the blood.

Generally speaking, the present invention provides an integrated system and method for producing a solid fibrin network or self-derived glue that can be used to regenerate tissue in a living organism. In one embodiment (shown in FIG. 1), the system comprises a primary container 10, a secondary container 14, and a transfer device 18. Preferably, the primary container 10 and the secondary container 14 are tubes, more specifically test tubes, but any container capable of holding a fluid or liquid and centrifuging is the invention of the invention. Suitable for use. Preferably, the containers 10 and 14 are made of glass or plastic.

The primary container 10 must be able to aspirate blood into it using standard venipuncture techniques. Preferably, the primary container 10 is sealed with a seal 22 to prevent contamination while blood is aspirated, although the container 10 may be sealed immediately thereafter. The primary container 10 can be sealed using a variety of seals 22, such as rubber stoppers, caps, foams, elastomers or other composites. The seal 22 must be piercable or piercable, and for this reason rubber and silicone are the preferred materials for making the seal, but any material that provides the seal and is pierceable is used. Can be done. The primary container 10 may contain the anticoagulant solution 25. The anticoagulant 25 in solution preferably comprises a calcium binder. More specifically, the anticoagulant 25 may include sodium citrate, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dipotassium salt and tripotassium, and combinations thereof. Preferably, the primary container 10 contains a sodium citrate solution. The anticoagulant 25 tends to dilute the blood collected in the primary container 10 for the purpose of placing the blood in a centrifuge state. In addition, the primary vessel includes a density gradient separation medium 26, air 27, and a high viscosity low density fluid 28 (see Figure 10, which shows a kit further described below).

The density gradient separation medium 26 must be capable of separating different fractions of a particular liquid or fluid within the primary vessel 10 having different densities. Separation medium 26 allows a dense, undesired fraction of the liquid to be separated by centrifugation and then removed. For example, the separation medium 26 can separate red blood cell 30 from platelet-rich plasma 34 while centrifuging the blood sample. In one example, the separation medium 26 can be found at the bottom of the primary container 10. In another example, the separation medium 26 may be mounted as a ring around the interior of the primary vessel 10 or in any other suitable inner position. Any density gradient separation medium 26 capable of separating liquids of different densities during centrifugation is suitable for use with the present invention, but preferably the medium 26 is a gel, more preferably thixotropy. It is a tropic gel. FIG. 2 shows the primary container 10 after centrifugation of the blood sample, and also shows the gel separation medium 26. Preferably, the thixotropic gel does not flow in or around the primary vessel 10 under normal ambient conditions, but flows at a greater centrifugation force encountered during centrifugation. Has a sufficient yield point. Most preferably, the gel has a density less than the high density of the unwanted red blood cell fraction 30 but greater than the density of the desired plasma fraction 34. In other words, a gel or other medium capable of separating the red blood cell 30 from the plasma 34 after centrifuging the blood sample is most preferred. Such medium 26 moves or flows in the vessel during centrifugation, but does not flow thereafter, thereby forming a semi-permanent barrier between the separated fractions when centrifugation is complete.

As shown in FIG. 3, another suitable density gradient separation medium 26 that can be employed in the primary vessel 10 is a plurality of plastic beads 26 that retain the desired density for separating the fractions. The beads can then be suspended in the high viscosity, low density fluid required to seal the transfer device 38. During centrifugation, the beads 26 migrate to the boundary between the two fractions 30, 34, compact in a state very similar to sintering, and fractions with different densities (ie, red blood cells). It forms a stable barrier between 30 and plasma 34). The residual high-viscosity, low-density fluid that coats the pellets contributes to the stabilization of the dense layers.

Other suitable density gradient separation media are such as those disclosed in US Pat. No. 5,560,830 and US Pat. No. 5,736,033 issued to Coleman, which are incorporated herein by reference. Includes system float device. FIG. 4 shows the polymerization system float device 26.

The low viscosity, high density, immiscible fluid 28 (LDHV fluid) in the primary vessel generally contains an inert oil. Most preferably, the LDHV fluid comprises polyester, silicone or other inert fluid and is fed by a replacement or pressure pump to a position above the gel in the primary vessel. The LDHV fluid must be able to block or eliminate the flow through the cannula 38 of the transfer device 18 as it enters its interior, as further described below.

Secondary vessel 14 (particularly illustrated in FIGS. 1 and 10) contains the chemical reagents required for a particular reaction. The secondary container 14 is sealed with the seal 24 in the same manner as for the primary container 10, ie, with a rubber stopper, cap, foam, elastomer or other composite material. In one application of the invention described below, the secondary tube may contain a calcium coagulation activator 36. Examples of suitable calcium coagulation activators include, but are not limited to, calcium chloride, calcium fluoride, calcium carbonate and combinations thereof, but any salt containing calcium is sufficient as a calcium coagulation activator. Let's go. In addition, other activators include calcium gluconate, calcium fumarate, calcium pyruvate and other organic calcium salts that are soluble in water and compatible with humans. The coagulation activator coagulates the plasma when the activator comes into contact with the plasma. The secondary container 14 can be completely evacuated so that the internal pressure becomes substantially zero. Exhausting the secondary container 14 facilitates the transfer of fluid from the primary container 10 to the secondary container 14 through the device 18. When the secondary container 14 is filled during transfer, there are no gas molecules at all, so that the residual gas is compressed and the pressure does not rise. As a result, the flow rate is maximized, complete transfer is facilitated, sterilization is maintained by eliminating the need for exhaust, and the ratio of the desired stoichiometric value to the desired reaction is maintained.

In another embodiment, the secondary vessel is also an antibiotic, an analgesic, a cancer healing agent, a platelet growth factor, a bone morphogenetic protein, a stem cell, a bone graft material, a soft tissue graft and a cell culture material, an immunosuppressant and It may contain one or more healing enhancers such as combinations thereof. Other therapeutic agents that can be administered topically may also be included. Examples of antibiotics include, but are not limited to, ampicillin, erythromycin, tobramycin and combinations thereof. Analgesics include, but are not limited to, aspirin, codeine and combinations thereof. Cancer cures include, but are not limited to, 5-fluorouracil. Bone graft materials include, but are not limited to, autologous bone, allograft from corpse (allograft) or allogeneic graft (homograft), animal-derived bone (xenografts) or xenograft. ); For example, sheep, cows, pigs, horses), synthetic bone grafts (tricalcium phosphate, hydroxyapatite, calcium sulfate ceramics), orthobiologic compounds (platelet-derived growth factor (PDGF)), bone morphogenetic factors ( Includes BMP), recombinant human bone morphogenetic factor (rhBMP), and combinations thereof. Soft tissue grafts and cell culture materials are not limited to skin grafts, skin graft materials (Apligraf sold by Organogenesis), gingival grafts (eg, from flexible palettes). , Collagen, bioabsorbable graft, vascular graft, PDGF, thrombocytopenic factor 4 (PF4), thromboglobulin, thrombosponton, DuPont's TEFLON (registered brand name) and Dacron (registered brand name) Includes DACRON) (registered trade name) and combinations thereof. Immunosuppressants include, but are not limited to, immunosuppressants for organ transplantation (eg, corticosteroids, calcin, neuroin blockers (cyclosporin, tacrolimus, SK506), mycophenolate mofetil, rapamycin) and skin. Includes immunosuppressants (serolimus, spingosine 1-phosphate receptor agonist (STY720)). Living cells for expression of desired molecules and gene therapy may also be included.

The transfer device 18 may include, for example, two parts as shown in FIG. 1, or may be alternative to, for example, a single part as shown in FIGS. 17 and 18. As best shown in FIGS. 5 and 6 and 17 and 18, the transfer device 18 has a first end 42 having a first opening 46 and a second having a second opening 54. Includes a cannula 38 with an end 50. The ends 42, 50 of the cannula 38 are sharpened or pointed (or at these ends) so that the seals 22, 24 of the primary container 10 and the secondary container 14 can be perforated or penetrated. It also has an acute angle surface). The cannula 38 is retracted into the housing 58 and coaxially attached to prevent accidental finger sticking during operation of the container. The housing 58 has two cylindrical opposed guides 62, 64 oriented centrally and axially with respect to the cannula 38. The guides 62 and 64 serve to guide the primary container 10 and the secondary container 14 to the first end 42 and the second end 50 of the transfer device 18. 5 and 6 show guides 62 and 64 that guide the containers 10 and 14 to the first end 42 and the second end 50.

The ends 42, 50 of the cannula 38 may be surrounded or covered by safety valves, sheaths or elastomeric sleeves 68, 72 forming a sealing seal. Safety sheaths 68, 72 also cover the first and second openings 46, 54. When the first and second ends 42, 50 pierce the elastomeric sleeves 68, 72, the sleeves 68, 72 retract accordingly. In FIG. 5, the first end 42 begins to puncture the seal 22 of the primary vessel 10, and the sleeve 68 retracts accordingly, while the sleeve 72 still completely covers the second end 50. Is shown. The ends 42, 50 extend sufficiently to fully pierce the seals 22, 24, but do not extend further into the containers 10, 14 (as shown in Figure 6). This allows the maximum amount of liquid volume of the upside-down primary container 10 to be transferred to the secondary container 14. Figure 6 also shows that the first and second ends 42, 50 completely pierce the seals 22, 24 of the first and second containers 10, 14, and both the sleeves 68, 72 are fully pierced. Indicates a retracted state. Elastomer sleeves 68, 72 block the flow of gas or liquid when not punctured. Suitable materials for sleeves 68, 72 include, but are not limited to, a variety of rubbers and thermoplastic elastomers.

To describe the operation of the first embodiment, if blood is aspirated into the primary vessel 10 using standard venipuncture techniques, the blood is anticoagulated by the anticoagulant 25 inside it. .. Typically, the primary container 10 is sealed while the blood is aspirated, after which the primary container may be sealed. Sealing the primary container 10 prevents contamination of its contents. The primary container and its contents 10 (eg, blood, anticoagulant 25, separation medium 26 and LDHV fluid 28) are then centrifuged. Allowable centrifugation can be performed for 5-15 minutes under a gravity of 900-3,500 xG. In a preferred embodiment, the primary vessel is centrifuged for about 10 minutes under a gravity of about 1,000 xG. This first centrifugation separates the contents or fractions of the primary vessel into multiple layers, eg, as shown in FIG. The layers (in the order from the bottom of the primary vessel 10 to the top of the vessel after centrifugation) are the red blood cell layer 30, the separation medium 26, the platelet-rich plasma layer 34, the LDHV fluid layer 28 and finally the residual gas. Contains 27 volumes at pressure equal to that of the atmosphere. The proportions of these layers can vary from application to application and are therefore shown only for illustration purposes. Following centrifugation, the sealed primary holder 10 is turned upside down before using the transfer device 18 to puncture the seal 22. In other words, as shown in FIG. 7, the primary container 10 is turned upside down so that the sealed opening is in the lowest vertical position. Inverting the primary vessel changes the order in which the layers are placed. Above the seal 22, in the order from bottom to top, is the next layer: platelet-rich plasma 34, high viscosity, low density immiscible fluid 28, residual gas 27, separation medium 26 and red blood cell 30.

Next, as best shown in FIG. 8, the secondary container 14 is placed in a vertical position with its sealed opening 24 in the uppermost position. As a result, the secondary container 14 is arranged so that the contents of the primary holder can be transferred. FIG. 8 shows the centrifuge primary container 10 in the upside-down position above the transfer device 18 above the secondary container 14, which is the proper position for transfer. Next, the guide portion 64 of the transfer device is placed above the secondary container 14 to guide the secondary container 14, while the inverted primary container 10 is placed in the other guide portion 62 (or its). Reverse). In other words, either the ends 42 or 50 of the cannula 38 can be used to drill holes in either the seals 22 or 24. Since the transfer device 18 is symmetrical at both ends, the user can perform operations without making any mistakes to some extent. The user then presses the containers against each other to puncture both the seals 22 and 24 at the respective cannula ends 42 and 50, respectively. The two valve sleeves 68, 72 that cover the ends 42, 50 make it even more confusing. First, if the first end 42 punctures the primary seal 22 (again, either end can be used to puncture either seal), then there is a hole covering the other end 50. The sleeve 72 holds the fluid and thereby prevents the fluid from flowing out. On the other hand, if the other end 50 first punctures the other seal 24 (and thus the sleeve 72), the vacuum is maintained by the sleeve 68 covering the first end 42.

As shown in FIGS. 6 and 9, when the ends 42, 50 pierce both sleeves 68, 72 and seals 22, 24, the desired fluid is transferred from the primary vessel 10 to the secondary vessel 14 by differential pressure. Is done. In other words, since the pressure in the secondary container 14 is exhausted, the contents of the primary container 10 (more specifically, plasma 34) flow into the secondary container 14. Initially, the pressure in the primary vessel 10, which is equal to the ambient pressure, drops as the amount of liquid decreases and the volume of gas expands. However, there is no time when the pressure becomes zero. Since the secondary vessel 14 is completely exhausted to zero or a pressure slightly higher than zero, when the tube is filled, there is little or no gas to compress and the pressure inside it does not rise. Therefore, instrument 18 can be used to transfer a variety of liquids and solutions from one tube to another and should not be construed to be confined to the transfer of blood alone.

Platelet-rich plasma 34 is readily transferred by the specific ordering of the layers within the primary vessel 10. Further, since the primary container 10 is preset to the exhaust level, the container is only partially filled after blood is collected. This allows the gas in the "upper space" to remain significantly above zero when the volume of gas expands during transfer, thereby allowing rapid and complete transfer to the secondary vessel 14. .. This is due to the law of ideal gases and the Poiseuille-Hagen equation.

Transfer of the contents or portion of the primary vessel (ie, platelet-rich plasma) continues until LDHV fluid 28 enters cannula 38. The high viscosity of the LDHV fluid clogs the narrow lumen of the cannula 38, which interrupts the flow. This prevents the reuse of the transfer device 18, which is especially important when attempting to remove the contaminated blood transfer device, and also causes accidental contamination by pathogens in the blood associated with previous use or use in another patient. Also prevent.

The transfer of the plasma fraction 34 to the secondary vessel 14 is complete, which allows maximum yield and to maintain the proper stoichiometric ratio of the reagents. Plasma 34 then contacts the coagulation activator 36 in the secondary vessel 14 to form a mixture 60, which can be immediately centrifuged to form a solid fibrin network. The differential pressure between the primary vessel 10 and the secondary vessel 14 is substantially maintained throughout the transfer, allowing rapid transfer. The transfer device 18 is not affected by the order in which the tubes engage and makes the device virtually error-proof. Finally, the transfer is performed without exhaust to maintain the sterilization and contamination-free condition of the sample.

Overall, the transfer device 18 is capable of contacting the plasma 34 with the calcium coagulation activator 36 for the purpose of forming a solid fibrin network, immediately followed by simultaneous coagulation and centrifugation of the plasma, quickly and efficiently. Providing a method. Solid fibrin nets are suitable for regenerating body tissues in living organisms. Such a method alleviates the need to first pre-concentrate the plasma by removing its water before it comes into contact with the calcium coagulation activator 36. In addition, the transfer device 18 can be used to transfer blood or other fluids in a variety of applications.

The present invention also provides a ready-to-use kit as shown in FIG. The kit includes a primary container 10, a secondary container 14, and a transfer device 18. In one embodiment of the kit, the kit may have two trays 70, 74 to be placed on the packaging. The first tray 70 has all the components required for step 1 and the second tray 74 has all the components required for step 2. Of course, the components can be arranged according to various specifications.

Step 1 involves collecting blood in a primary vessel 10 and then centrifuging to obtain platelet-rich plasma. The components of the first tray 70 are an alcohol cotton stick 78 that cleans the puncture site, a large number of sample blood sampling needles 82 (21 gauge x 25.4 mm (1 inch)), a safety holder 86, and an anticoagulant (eg, citrate). Includes a primary container 10 containing (acetate), gel, LDHV fluid, and bandage 90 covering the venipuncture site. Clean the venipuncture site with a sterile alcohol swab 78. Open the needle cartridge 84 and screw it into the safety holder 86. The needle 82 is then inserted into the patient's vein and the container 10 is connected to the holder 86. The container is then filled with blood and the needle 82 is withdrawn and retracted into the holder 86. The ends of the holder are closed by hinged flaps. Close the vein with bandage 90. Centrifuge vessel 10 at about 1000xG for about 10 minutes to separate plasma from erythrocyte cells.

The components of the secondary tray are the components used in step 2 and include the AF tube (self-derived fibrin tube) or secondary container 14, and the transfer device 18. Step 2 involves placing the primary container 10 in an inverted position and within the transfer device 18. The secondary container 14 contains a coagulant and is punctured by the other end of the transfer device. Containers 10 and 14 are connected, and platelet-rich plasma flows from the primary container 10 to the secondary container 14. The secondary vessel is then immediately centrifuged at 2300xG for about 30 minutes to give a dense fibrin or solid fibrin network with platelets.

A second embodiment of the present invention provides another integrated system for producing a solid fibrin network, as shown in FIGS. 11-14. The system includes a primary sampling device 10 that is very similar to the primary container 10 of the first embodiment. The collection device 10 may contain a density gradient cell separation medium 26 (as described above) and an anticoagulant (not shown), as well as a reservoir 94 that can be connected to or integrated with the primary collection device 10. More specifically with respect to the first embodiment of the present invention, the above with respect to the separation medium 26 also applies to the second embodiment of the present invention. In other words, the same material can be used for the separation medium 26, and the same material is preferable. For example, most preferably, the separation medium 26 comprises a thixotropic gel, where the yield point of the gel prevents the gel from flowing under normal ambient conditions, such that the gel is encountered during centrifugation. Allows flow with greater centrifugal force. The separation medium 26 may be located at the bottom of the primary sampling device (ie, the other end of the opening), as shown in FIG. Alternatively, the separation medium may form a ring around the interior of the primary sampling device. The primary sampling device 10 is essentially the same as the primary container 10 described above, except that the primary sampling device does not have to hold a high-density, low-viscosity fluid. Preferably, the primary sampling device 10 has a seal 22 (as described above) such as a rubber stopper or cap.

The reservoir 94 includes a chamber 96 and a cannula 100 that is in fluid communication with the chamber. Chamber 96 contains liquid reagent 104, most preferably a calcium coagulation activator. Preferably, the calcium coagulation activator is calcium chloride, calcium fluoride, calcium carbonate, calcium gluconate, calcium fumarate, calcium pyruvate or a combination thereof. The cannula 96 must be capable of drilling holes in the seal 22 of the primary sampling device 10. In certain preferred embodiments, the cannula contains a blocking medium 108, such as a gel at a yield point that prevents reagent 104 in chamber 96 from flowing out of cannula 100 under ambient conditions. Other suitable blocking media are, but are not limited to, spring-loaded balls, valves, spring-loaded valves, puncture membranes, and ampoules (ie, hollow membranes filled with fluid or powder). Includes force-operated mechanical systems. The yield point of gel 108 is such that when chamber 96 and primary collector 10 are engaged, gel 108 moves, especially when centrifuged under high gravity to allow them to communicate. Shall be. The reservoir 94 may also have a guide housing 110 used to guide the reservoir to the sampling device 10. The cannula 100 may be surrounded or covered by an elastomeric sleeve 112 to maintain its sterility. The sleeve 112 is as described above with respect to the first embodiment.

In other embodiments, the chamber 96 is also among antibiotics, analgesics, cancer healing agents, platelet growth factor, bone morphogenetic proteins for gene therapy, stem cells for additional use, and other hormonal agents. May contain one or more. Other administrable healing agents may also be included. Examples of antibiotics include, but are not limited to, ampicillin, erythromycin and tobramycin. Analgesics include, but are not limited to, aspirin and codeine. Cancer cures include, but are not limited to, 5-fluorouracil.

At the time of implementation, the patient's blood 116 is collected in the primary collection device 10 by the conventional venipuncture technique described above. The anticoagulant in the primary sampling device 10 dilutes the blood prior to centrifugation. The reservoir 94 is then tied to the primary sampling device 10 by puncturing the cannula 100 of the reservoir 94 through the seal 22 of the primary sampling device 10, as shown in FIGS. 13 and 14. When the cannula 100 punctures the seal 22, the sleeve 112 retracts. Although the length of the cannula 100 is sufficient to puncture the seal 22, it is preferred that the cannula does not extend further into the sampling device 10, if possible.

Next, the sampling device 10 and the reservoir 94 are centrifuged. The centrifugal force applied to the tube is the equation F = mω²Expressed by r, where; F = force, m = system mass, r = radial distance from the center of the rotor, ω = rotation angle factor. Since the reservoir has a smaller r than the gel in the primary tube, the shear stress generated is not sufficient and the gel in the cannula of the reservoir cannot move. The primary tube 10 is swirled in low gravity until the cells separate and the gel 26 moves to the cell / plasma boundary as shown in FIG. In other words, as in the first embodiment, after the first centrifugation at about 1000xG for about 10 minutes, the separation medium 26 separates the red blood cells 30 from the platelet-rich plasma 34. Centrifugal force of about 900-1500xG is also acceptable for the first centrifugation for about 5-15 minutes.

The centrifugation rate is then increased and the reservoir receives a sufficiently large centrifugal force so that the blocking medium 108 in the cannula 100 is discharged into the primary sampling device 10 and the liquid reagent 108 (eg, calcium coagulant activator). Is drained from the reservoir, as shown in FIG. The contents can then be centrifuged for about 15-40 minutes at about 2300-6000xG. When the calcium coagulation activator comes into contact with the plasma in the primary sampling device, the sample is still centrifuged, resulting in immediate and simultaneous coagulation and centrifugation. As a result, a solid fibrin network suitable for tissue regeneration is formed. Performing cell separation in the primary tube and subsequent addition of the liquid coagulant at the proper stoichiometric ratio are performed in one tube without transfer. By programming the centrifuge in terms of speed and time, the present invention provides a simple and unmistakable method.

In one alternative embodiment, the single sampling device 10 has an inner compartment 119 and a reservoir 94, as shown in FIGS. 15 and 16. The reservoir 94 integrates or connects with the primary sampling device 10 and fluidly communicates with the compartment. The pipe, conduit or opening 120 provides a fluid communication state between the compartment 119 and the reservoir 94 and is sealed with a blocking medium 108. Again, as described above, the blocking medium 108 has a yield point that activates and moves to allow communication between the reservoir 94 and the primary sampling device 10 when exposed to particularly high gravity. The yield point of the gel or medium is such that it does not move during the first centrifugation to separate the blood cells from the plasma. In a third embodiment, each end of the device has an opening, and each end is removable or non-removable, such as a rubber stopper, cap, foam, elastomer or other composite. It is sealed by seals 22 and 122. The reservoir 94 with the stopper 122 is located at the seal 22 of the sampling device and at the other end of the opening.

In another embodiment, the reservoir 94 may contain one or more of antibiotics, analgesics, cancer cures, platelet growth factor and bone morphogenetic proteins. Other administrable healing agents may also be included. Examples of antibiotics include, but are not limited to, ampicillin, erythromycin and tobramycin. Analgesics include, but are not limited to, aspirin and codeine. Cancer cures include, but are not limited to, 5-fluorouracil.

Alternative embodiments are used in the same manner as described above for the second embodiment. That is, the centrifuge is controlled by two different centrifugal forces: 1) the first is enough force to separate the plasma from the erythrocyte cells; 2) the second is with the reservoir Enough force to move the blocking medium 108 in the tube, conduit or opening 120 to and from the device into the main body. As a result, the calcium coagulation activator is allowed to enter the inside of the device. This, on the other hand, allows for simultaneous centrifugation and coagulation of plasma for the purpose of forming a solid fibrin network as the centrifugation proceeds at a second higher gravity. The seal 122 can be removed to obtain a solid fibrin net or self-derived glue. In a preferred embodiment, the seal 122 is pierced and fitted and can be unscrewed from device 10 as shown in FIG.

In one aspect, the invention provides a system for producing a self-derived solid fibrin network suitable for tissue regeneration in living organisms. The system includes a sealed primary container containing a separation medium and a low density, high viscosity liquid. The separation medium is capable of separating erythrocyte cells from plasma when the vessel contains blood, is centrifuged and the primary vessel has a primary pressure. The system further includes a sealed second container containing a calcium coagulation activator. The second container has a second pressure that is lower than the first pressure. The system also includes a transfer device that includes a cannula with a first end and a second end. The first and second ends can be pierced in sealed primary and secondary vessels for the purpose of providing fluid communication between the first and second vessels. The low-density, high-viscosity liquid in the primary vessel can block the flow through the cannula when it enters the cannula.

In another aspect, the invention provides another system for producing a solid fibrin network capable of regenerating tissue in a living organism. The system includes a sealed primary container that has a primary pressure into which blood can be aspirated. The system further includes a sealed second container that has a second pressure and contains a calcium coagulation activator. The second pressure is lower than the first pressure. The system also includes a transfer device that includes a cannula with a first end and a second end. The first and second ends can be punctured in a sealed container and the transfer device can transfer the portion of blood sucked into the primary container to the second container by differential pressure. The system is also the blood transferred from the primary vessel to the secondary vessel through a device and is contacted with a calcium coagulation activator for the purpose of forming a solid fibrin network capable of regenerating the tissues of living organisms. Includes a centrifuge device for simultaneously centrifuging and coagulating parts of blood.

In another aspect, the invention provides a method of making a solid fibrin network for regenerating body tissue in a living organism. The method comprises aspirateing blood from the patient into a primary vessel and separating plasma from the blood in the primary vessel. Plasma from the primary container contains the calcium coagulant, using a transfer device containing a cannula with a first end and a second end for the purpose of contacting the plasma with the calcium coagulant. Transfer to a secondary container. Plasma and calcium coagulation activators are simultaneously coagulated and centrifuged in a secondary vessel for the purpose of forming a solid-fibrin network. Solid fibrin nets are suitable for regenerating body tissues in living organisms.

In another aspect, the invention provides another system for producing a solid fibrin network suitable for regenerating tissue in a living organism. The system includes a sealed primary sampling device that has an interior and contains a separation medium. The primary collection device can have the blood sucked inside, and the separation medium can separate plasma from the red blood cells when the primary collection device contains blood and is centrifuged. The system further includes a chamber and a reservoir having a conduit that communicates fluidly with the chamber. The chamber has a calcium coagulation activator inside and the conduit is at least partially filled with a blocking medium to prevent the activator from flowing out of the chamber under ambient conditions.

In another aspect, the invention provides another method of producing a solid fibrin network capable of regenerating tissue in a living organism. The method comprises aspirateing blood from a patient into a primary sampling device having a seal and providing a reservoir having a chamber and a fluidly communicating conduit with the chamber. The chamber is at least partially filled with a calcium coagulation activator and the conduit is at least partially filled with a blocking medium to prevent the activator from flowing out of the chamber under ambient conditions. The reservoir is connected to the primary sampling device so that the chamber, conduit and sampling device are fluidly accessible in the absence of blocking media. The primary sampling device is then centrifuged at the first rate. The first rate is sufficient to separate the plasma from the blood, but not enough to move the blocking medium in the conduit into the primary collection device. The primary sampling device is then centrifuged at a second rate. The second rate is sufficient to allow at least a portion of the blocking medium to move from the conduit into the primary sampling device, which allows the calcium coagulation activator to flow into the sampling device and come into contact with the plasma. This forms a solid fibrin network suitable for regenerating the tissues of living organisms.

Most of the systems described above employ radial centrifugation (ie, the arrangement of the axis of the tube perpendicular to the axis of the centrifuge) during the second centrifugation for the purpose of compressing the clot. However, when these systems and devices are used, the centrifugation step may not be possible in the operating room due to sterilization concerns. Therefore, in another aspect, the present invention provides an outer portion of a sterile tube that extends into the operating room, collects a specimen under sterile conditions, carries the specimen out of the operating room, and centrifuges the specimen in a centrifuge. It is treated and thus ensured that the outside of the tube is sterile when reintroduced into the operating room.

Thus, along with any of the two tube systems described above, both the primary and secondary tubes can be packaged in an easily removable film or, in place of this, a molded carrier. FIG. 52 shows the design of a film that may include a shrink wrap with stripped pieces or a shrink wrap with a jagged end with an easy stripped bottom. FIG. 52 shows a sterile tube carrier that allows the primary or secondary container to be introduced into a sterile space such as an operating room. The carrier can be shrink-wrapped to meet further standards of handling, as shown in FIG. The assembly can be sterilized by radiation. The assembly is then opened in the operating room and the inside and outside of the tube are kept sterile. FIG. 53 shows the design of the carrier. The packaging is generally of the smallest thickness during production. Both the tube and the wrap can be sterilized by radiation, providing sterilization of the inner and outer surfaces. Immediately before entering the operating room, the film or carrier is removed from the primary tube so that a sterile outer tube enters the operating room. In addition, the outer surface of the film, the wrapped tube or carrier can be decontaminated or sterilized using suitable chemicals known in the art before introduction into the operating room. This allows the film, wrap or carrier to be opened in the operating room, which guarantees absolute sterilization of the product. Blood is collected, the tube is removed from the operating room and centrifuged. The sterile plasma is transferred to a second tube containing the activator and centrifuged while in the film or container. Immediately before re-entering the operating room, the outer wrap is removed and the sterile product enters the operating room. An improvement in this design is the addition of a removable adhesive film to the stopper of the primary tube, which allows a sterile surface during the transfer operation.

Further, when using any of the one-tube systems described above, the tube may have a film or carrier pre-assembled during manufacturing. Place the assembly in a sealed bag and sterilize the assembly. Open the bag just before entering the operating room. The needle then punctures the tube stopper and film to collect blood. An adhesive film can also be added to the stopper of the tube. The tube exits the operating room and adds a liquid reservoir with activator or other substance to the assembly. Two-step centrifugation is performed and the carrier is removed immediately before reintroduction into the operating room.

Overall, both the primary and secondary tubing can be maintained in the sterile film or carrier during processing and can be reintroduced into the operating room with the outer portion of the sterile condition. The film or carrier is pre-assembled into a tube and its use is visible when collecting and centrifuging a normal blood collection tube. The film or carrier can be made of a material that improves the shelf life and reliability of the tubing by providing a permeation barrier to gas and water vapor, especially materials that are useful for plastic tubing.

The above description establishes a variety of methods and devices used to form dense fibrin and platelet networks, and solid fibrin networks by simultaneous centrifugation and coagulation. Many of these devices and methods employ radial centrifugation, where the axis of the tube can be located substantially perpendicular to the axis of the centrifuge. For example, the tube may be located at the radius of the centrifuge and may have a stopper near the center and the bottom of the tube towards the outer edge of the centrifuge. In most of the applications described above, the clots compress during the second centrifugation cycle. As a result, the centrifugal force changes linearly along the length of the tube. Differences in centrifugal force can be beneficially used by using different speeds of the centrifuge to activate additional reagents. Radial centrifuges are the most common type found in commercial applications, and their adaptable use is beneficial to product designers, especially when focusing on their wide range of availability. Is.

A solid fibrin network or fibrin platelet network can mean plasma, or, more specifically, a substance formed by simultaneous centrifugation and coagulation of platelet-rich plasma. As mentioned above, solid fibrin nets can be used for unrestricted tissue regeneration applications. Fibrinogen in plasma is converted to fibrin strands, which deposit at the same time as platelets deposit. In the final step of the solidification cascade, the fibrin strands crosslink in random directions, resulting in a gel-like consistency. When a very large centrifugal force is applied, fibrin is compressed into a high-strength membrane. Thus, the membrane can mean a solid fibrin network that is further compressed by greater centrifugal force or gravity (as described above).

However, one alternative to radial centrifugation is axial centrifugation. When using axial centrifugation, the container holding the liquid rotates about its central axis. In other words, the container acts like a rotor, in short. As a result, heavy rotors in the centrifuge are no longer needed. Axial centrifuges can generally have smaller radii than radial rotors and therefore require larger rpm to achieve equivalent g-force. For example, instead of swirling the centrifuge up to 10,000 rpm, the centrifuge can swirl up to 200,000 rpm. The rpm required is higher, but a significant reduction in weight minimizes the safety risk and disproportionately reduces the cost of the motor. In other words, removing the rotor can significantly reduce the weight of the centrifuge, resulting in the centrifuge being swiveled at higher rpm.

In general, the centrifugal force is the radius squared rpm (rpm).²) Is multiplied by. In fact, a significantly larger g-force can be obtained by this method. A significantly large cylindrical area can be obtained with a very uniform centrifuge field strength. Since the vessel is generally a rotor, axial centrifugation usually employs the operation of a single vessel rather than a batch. For this reason, the membrane covers most of the outside of the tube, even when a thin tube is used and swivels around its axis. More specifically, the surface area of a covered cylinder can be defined as about 2πrl, where r is the radius of the cylinder and l is the length of the cylinder. On the contrary, when using radial centrifugation, the surface area will be substantially the diameter of the tube. Therefore, radial centrifugation produces a membrane equal to 2πrl, while axial centrifugation produces πr.²A film equal to is produced.

The form of axial centrifugation can be realized in a variety of ways. For example, different cartridges can be placed in a modified existing rotor. Alternatively, the rotor may be removed and a disposable cartridge or container may be inserted therein. Typically, the system used with axial centrifugation is two chambers, a cell separation chamber where blood is separated into red blood cells and platelet-rich plasma, and the platelet-rich plasma is in contact with the coagulation activator and simultaneously. A densification chamber will be adopted in which the membrane is formed by various centrifugation and coagulation. Again, the membrane can be used for a variety of tissue regeneration and wound sealant applications.

In one embodiment, a sterile drum rotor disposable cartridge 200 is provided, for example as shown in FIGS. 19 and 20. The cartridge 200 can be formed from a variety of materials, such as a variety of ceramics, glass and plastics. Unless otherwise specified, the devices and systems formed in the present invention may be made from a variety of ceramics, plastics, glasses or other suitable materials. The cartridge 200 can generally have a circular crown cross-sectional shape that can be mounted within the drum rotor of the centrifuge, but as described below, the two chambers separated by the filtration device. The shape is not as important as having. The shape of the cross section shall be such that the fibrin platelet-rich plasma membrane can be subsequently removed for recovery. In one embodiment, the cartridge 200 has an inner chamber 204 contoured by a central wall 208, side walls 212, top and bottom walls 216, 220 and filtration device 224. The inner chamber 204 acts as the cell separation chamber described above. The apical wall 216 can have a punctureable loading port 228 through which blood from the patient can be introduced or injected into the inner chamber 204. The filtration device 224 may be made of a selective centrifugable (mechanically supported) filter that receives a separate amount of whole blood. Filtration device 224 may be formed of a variety of materials, including but not limited to polycarbonate, cellulose, polyethylene, polypropylene, nylon or Teflon®. The filter can have a pore size of about 4-9 microns. The inner chamber 204 may contain, for example, one or more anticoagulants 232, such as the anticoagulants described above for radial centrifugation methods and equipment. The anticoagulant 232 prevents blood entering the inner chamber 204 from clotting.

The cartridge 200 also has an outer chamber 236 that is generally smaller in volume than the inner chamber 204. The outer chamber 236 or peripheral tank is contoured by filtration device 224 and peripheral wall 240, as well as top and bottom walls 216, 220, as shown in FIG. The outer chamber 236 may include one or more coagulation activators 244, such as the calcium coagulation activators described above. The outer chamber 236 acts as a densification chamber, where the platelet-rich plasma is activated by the coagulation activator 244. These materials are simultaneously centrifuged and coagulated to form a membrane. The densification chamber 236 may also contain one or more secondary activators 248 or healing enhancers. Secondary activator 248 is, but is not limited to, one or more antibiotics, analgesics, cancer healing agents, platelet growth factor, bone morphogenetic proteins, cells for gene therapy, stem cells for further use, Includes other hormones and combinations thereof. Other administrable healing agents may also be included. Examples of antibiotics include, but are not limited to, ampicillin, erythromycin and tobramycin. Analgesics include, but are not limited to, aspirin and codeine. Cancer cures include, but are not limited to, 5-fluorouracil. Secondary agents may be included herein in any densification chamber, which is further described below, more specifically. Secondary activators or healing enhancers are described in more detail above.

During operation, the cartridge 200 is inserted into a drum rotor (not shown) for centrifugation. The inner chamber 204 may already contain blood or may be inserted into the drum rotor and then injected with blood through port 228. Injection of blood into the inner chamber 204 is performed using standard venipuncture. In other words, the inner chamber 204 can be kept in a vacuum state. An anticoagulant 232 may be used in the inner chamber 204 to prevent blood clots. Port 228 maintains the sterile condition of the inner chamber 204 and provides a closed system. The drum rotor can accommodate several different cartridges. Generally, each of the cartridges 200 needs to be balanced in the rotor by arranging similar cartridges or counterweights on opposite sides in the rotor. When the blood sample is centrifuged, the filter 224 retains red blood cells and white blood cells in the inner chamber 204, but plasma and platelets penetrate into the outer chamber 236 for a given period of time under proper centrifugation force. Allow to flow. The proper centrifugal force is in the range of 1000 to 15,000xg, and the predetermined time can be 5 minutes or more, more specifically in the range of 5 to 60 minutes or 5 to 30 minutes. possible. Once the platelet-rich plasma enters the secondary chamber 236, it contacts the coagulation activator 244 in the cartridge.

As described for this embodiment and the following embodiments, any of the coagulation activators 244 described above are suitable for use. Upon contact with activator 224, plasma is simultaneously centrifuged and coagulated, thereby forming a solid fibrin network or membrane. Providing a mixing operation of the rotor helps to completely mix the plasma and activator 244 to initiate the coagulation process. After mixing, the rotor is swirled at about 3000-15000xg for 10 minutes or more, more specifically about 20 minutes or more, to white blood cell resistance (white) to the peripheral wall 240 of the second chamber 236. A resistant) fibrin platelet-rich plasma membrane can be obtained. To remove the membrane from the cartridge 200 for use, the device can be broken or opened in two parts. Alternatively, one of the walls may have a removable portion or other access area so that the membrane can be obtained from it. Other methods of removing the membrane from the cartridge include winding and folding the membrane. For hygienic purposes, the cartridge 200 may be disposable. Membranes have a variety of uses, including, but not limited to, wound care and burn care. More generally, membranes can be used in an unlimited number of tissue regeneration applications.

In another embodiment of the invention known as large axial swirl, for example, but not limited to, membranes such as membranes up to 1000 mm in diameter can be obtained. 21 to 25 show this embodiment. The size of the membrane depends on the size of the rotor. Therefore, the size of the membrane depends on which rotors are commercially available. In this embodiment, both the radial centrifuge and the operation of the primary and secondary centrifuges described above with respect to the device are performed in one axial swivel vessel. The secondary chamber can be partitioned so as to provide a large area, a large number of separate area membranes. This partition will be described in more detail below.

This system includes a centrifuge (not shown) and the device 252 shown in FIGS. 21-25 that can be inserted therein. The device 252 has two chambers, namely a primary or upper chamber 256 and a secondary or lower chamber 260 that are fluidly communicated with each other. The primary or upper chamber 256 functions as a cell separation chamber, while the secondary or lower chamber 260 functions as a densification chamber. Device 252 also has a diaphragm 264 with at least one opening, opening or vent outlined in the figure, as shown in FIGS. 21-25. The separator 264 separates the two chambers 256, 260. The opening, opening or vent 268 provides a fluid communication state between the primary chamber 256 and the secondary chamber 260. The separator plate 264 may be made of, for example, plastic, ceramic or glass.

The primary chamber 256 may contain a separation medium 272. Although any of the separation media 272 described above can be used with the system, specific examples of the separation medium 272 may include at least one of silicone gel, polyester gel, thixotropic gel and combinations thereof. More specifically, sufficient to block one or more vents 268 of the diaphragm 264, block one or more vents 268, and separate erythrocyte cells from plasma after the first centrifugation. It can be closed with an amount of separation medium 272 (eg, gel). The primary chamber 256 typically receives whole blood from the patient through a punctureable stopper 276 or other suitable device such as a threaded cap lined with a bottle cap. In FIGS. 21-25, the system is shown as having a punctureable stopper 276 used to introduce blood into the upper chamber 256. The primary chamber 256 may also contain an anticoagulant 232. Chamber 256 may also be evacuated to allow vacuum collection of specimens by standard venipuncture. The secondary chamber 260 can include the coagulation activator 244 and can include one or more of the secondary activators 248 described above.

After blood 280 is collected in the upper chamber 256, as shown in FIG. 22, the device 252 is axially centrifuged with an appropriate g-force to separate the cells, i.e., platelet-rich plasma of erythrocyte cells 288. Separation from 284. A typical g-force used to separate cells can include 500 to 15,000 xg for a given time, for example 5 minutes or more. Preferably, the first centrifugation is performed at about 1000-1500xg for about 5-15 minutes. This applies to all membrane-related embodiments described herein. The first centrifuge moves the separation medium 272 from the position where the vent 268 is blocked to the interface. For example, thixotropic gel 272 can maintain the separation of the two chambers 256, 260 while filling the primary chamber 256 with blood 280, but moves during the first centrifugation to separate the cells. Open the connecting fluid path that separates the two chambers 256 and 260. The gel 272 flows radially, outwardly and upwards, and the gel 272 does not fall into the bottom chamber 260. The result of the first centrifugation is shown in FIG. Due to the relative density of platelet-rich plasma 284, separation medium 272 and erythrocyte cells 288, by centrifugation, these three substances are in the order described above from the inside of the primary chamber 256 to the outside of the primary chamber 256, as shown in FIG. Place at. As used in the drawings and herein, PRP represents platelet-rich plasma and RBC represents erythrocyte cells.

The first centrifugation was then stopped and the results are shown in FIG. When centrifugation is stopped, the platelet-rich plasma 284 is expelled by gravity into the lower chamber 260 through the vent 268, in which the platelet-rich plasma is present as a coagulation activator 244 and a secondary activator 248 (present). If) and mix. However, the separation medium 272 stays in place, thereby preventing the red blood cell 288 from entering the second chamber 260 through the vent 268. Vent 268 can be funnel-shaped to ensure that all of the platelet-rich plasma 284 flows into the secondary densification chamber 260 due to the applied g-force.

As shown in FIG. 25, centrifugation is resumed with a proper g-force, for example 500-15,000xg, and a large membrane 292 is formed on the outer circumference of the lower chamber 260. Preferably, the centrifugation is carried out at about 2500-10,000 xg for about 20 minutes to 1 hour, depending on the density of the membrane to be achieved. This applies to all embodiments used for film formation described herein. It should be recognized that the separation medium 272 and erythrocyte cells 288 tend to remain in the same position during the second centrifugation. This system allows simultaneous centrifugation and coagulation, resulting in a large platelet / fibrin membrane 292. The device 252 can also be removable and has a bottom 294 that allows the membrane to be easily withdrawn from the device.

The following systems and devices are modifications of the basic system shown in FIGS. 21 to 25. For example, the cell separation chamber or primary chamber 256 may have a different radius than the densification chamber or secondary chamber 260. As shown in more detail in FIG. 26, the radii of the upper and lower chambers can be different, which allows different g-forces to be applied at the perimeter wall. As a result, one speed rpm produces two different g-forces, which simplifies motor and programming. More specifically, providing different radii for the chambers results in different g-forces at the same rpm, eliminating the need for multi-velocity programming.

27 to 30 show one modification of the system shown in FIGS. 21 to 25, in which concentric cylinders are used. System 296 includes a primary tube 300 having an upper portion 304 separated from a lower portion 308 by a separator or other separator 309. The primary tube 300 functions as a cell separation chamber. At least one vent, hole or opening 312 provides fluid communication between the upper portion 304 and the lower portion 308 of the primary tube 300. Again, blood can be introduced into the primary tube 300 through one or more punctureable stoppers 316 or other suitable device described above. The primary tube 300 may retain the anticoagulant 232 to prevent premature blood clotting. The separation medium 272 prevents blood from flowing from the upper portion 304 to the lower portion 308 of the primary tube 300 through at least one vent 312. The lower portion 308 may also have voids, holes or openings 310 through which the liquid can flow. The densification of platelet-rich plasma 384 takes place in the secondary concentric tube 320. The secondary tube 320 may contain one or more and / or one or more secondary activators of the coagulation activator 244 described above.

First, blood is separated into plasma and erythrocyte cells by centrifugation of the system, which plasma and erythrocyte cells are separated by the separation medium described above and shown in FIG. 28. The first centrifugation is generally performed for about 10 minutes or longer, at about 1000xg or higher. As shown in FIG. 29, if centrifugation is stopped, the platelet-rich plasma 284 falls through one or more vents 312 into the lower portion 308 of the primary tube 300 and the erythrocyte cells 288 , Will be trapped in the upper portion 304 of the primary tube 300 by the separation medium 272. The wall of the lower portion 308 of the primary tube 300 is provided with at least one vent 310. The system is then centrifuged as shown in FIG. 30, whereby at least a portion of the platelet-rich plasma 284 leaves the lower portion 308 through the void 310 of the primary tube 300 and enters the secondary tube 320. Again, the erythrocyte cells 288 would remain trapped in the upper portion 304 of the primary tube 300 by the separation medium 272. As shown in FIGS. 27-30, the secondary tube contains at least one coagulation activator 244, where the platelet-rich plasma 284 contacts the coagulation activator. This modification then provides simultaneous coagulation and centrifugation to form membrane 292. This modification allows smaller units and reduces plastic usage. Device 296 may also have a removable bottom 324 to facilitate removal of the membrane.

As another alternative, the hydrophobic membrane 325 may be used instead of the separation medium. Hydrophobic membrane 325 can be used in place of any system that uses a separation medium. The hydrophobic membrane 325 only allows the platelet-rich plasma to flow at a predetermined g-force, eliminating the need for a separation medium. In other words, instead of using a diaphragm with holes blocked by the gel, a hydrophobic membrane can be used as shown in FIGS. 31 and 32. When using membranes, the lower and upper chambers may have the same radius as shown in FIG. 21, or the two chambers may have different radii as shown in FIG. 26, an example thereof. In addition, the hydrophobic membrane can be used for the concentric design shown in FIG.

The hydrophobic membrane 325 substantially blocks the flow of aqueous solutions such as platelet-rich plasma through its pores until a predetermined hydrostatic pressure is reached. Examples of the hydrophobic membrane 325 may include, but are not limited to, polypropylene, polycarbonate, cellulose, polyethylene, DuPont Teflon® and combinations thereof. Other examples include Millipore® membranes and screens manufactured by Millipore, or Nucleopore® membranes and screens manufactured by Nucleopore. Alternatively, a plastic diaphragm perforated with precision holes by a laser may be used. Hydrophobic membranes allow blood to be introduced into the cell separation chamber but not to fall into the densification chamber. First, proper hydrostatic pressure can be achieved by separating erythrocyte cells from plasma at low rpm. The centrifuge rate is then increased to achieve the desired pressure and overcome the surface energy / surface tension constraints that define the flow pressure. In other words, gravity increases with the rate of centrifugation, resulting in platelet-rich plasma flowing through the membrane but not red blood cells. The membrane substantially blocks red blood cells.

Another mode modification to the above system includes changing the form of the secondary or densification chamber of any of the embodiments described herein. These modified densification chambers can be used within the system, where the primary and secondary chambers have the same or different radii, the chambers are concentric and / or the separation medium or Hydrophobic membranes are used. The densification chamber may have different inner walls that facilitate membrane removal and ensure maximum degree of membrane recovery. For example, a densification chamber improves the peeling strength of the membrane for first placement in the body and then decomposes, woven biodegradable fabrics (eg Goretex manufactured by Goretex). (Registered trademark name)) may be contained. The outer wall of the chamber can also contain molded overhangs or grooves that support the woven fabric at a uniform length away from the wall, with desired dimensions of fibrin and platelet thickness on both sides of the woven fabric. Realize.

More specifically, as shown in FIG. 39, the inner wall or side wall of the densification chamber 318 is one or more to allow removal of the membrane in the form of a flat sheet rather than a cylinder. It may include solid or serrated ribs 319. The perforated ribs promote membrane aeration. The inner wall of the chamber may be in a form that provides holes in the film to be formed and facilitates peeling. FIG. 39 shows a bottom view of a solid rib on one or more of the inner walls.

FIG. 33 shows a woven biocompatible woven fabric 328 that can be seen inside the densification chamber 326. Such a fabric keeps the membrane 292 away from the wall itself. Woven 328 facilitates the separation of the membrane from the cylinder in order to obtain a flat membrane and increases the peel strength of the membrane for specific applications. The woven fabric 328 is embedded in the membrane 292. Further, as shown in FIGS. 34 and 35, an overhang 332 or groove 340 can be formed on the wall 336 of chamber 326 to control the thickness of the fibrin layer on either side of the woven fabric. These serve as small support ribs that keep the woven fabric away from the wall. In short, FIG. 33 shows that the woven fabric 328 itself prevents the film from adhering to the wall 336; FIG. 34 shows the overhang 332 of the wall 336 that facilitates the removal of the film 324; FIG. 35. Shown are grooves or molded support ribs 340 that keep the membrane 292 away from the wall. A wall having an overhang 332 or a groove 340 may be adopted independently of the woven fabric 328.

Alternatively, as shown in FIG. 36, the densification chamber can be lined with removable film 344 to facilitate removal of film 292. The film 344 may include plastics such as polyolefins, including polyethylene, polypropylene, polycarbonate, or Teflon®. The film 344 can have tabs 348 for ease of operation and can also help color the film 292 to distinguish it from the film 348. In addition, film 348 can be treated to obtain desirable properties such as glass-like contact activity. By manipulating the tab 348, the entire film 344 with the film 292 on top can be removed. For example, the densification chamber 326 may be lined with a treated film that provides both activation of platelets for coagulation and release of growth factors and facilitation of membrane manipulation. Alternatively, PRP or PPP may be flowed through a high surface energy capillary to activate PRP or PPP for blood clots, allowing rapid blood clot adhesion for use as a fibrin sealing layer or adhesive layer. Good.

With respect to other surfaces in the chamber, the plastic surface may be effective, but may not be ideal for clot activation and release of platelet growth factor. As a result, alternative examples of plastic are outlined in FIGS. 50 and 51. For example, platelet-rich plasma can come into contact with the glass in the lower chamber. In other words, the glass can be attached to the bottom cap as shown in FIG. 50, or the glass spheres can be glued to the bottom or heat connected as shown in FIG. The glass may also be heated and dropped onto the plastic. Alternatively, the surface can be plasma treated using a glow discharge process that employs an activated gas such as oxygen or nitrous oxide. More specifically, the surface can be treated using plasma chemical vapor deposition. Alternatively, the surface can be modified using a variety of chemical coatings, such as silicone surfactants or PVPyr. Another way to modify the plastic is to add small size silica beads or particles to the citrate solution in the upper chamber. Due to the high density of silica compared to gels and red blood cells, most of the silica stays in the upper chamber below or embedded in the gel. Thus, these plastic modifications can be used to cover a portion of the system and, more specifically, a portion of any densification chamber of the embodiments described herein.

In a different aspect, the present invention has made it possible to produce a square platelet-rich fibrin membrane used in the context of wound care, which takes advantage of platelet-derived cell division-promoting characteristics and is derived from platelets. It provides the protective effect of growth factor (PDGF) and betathromboglobulin (BTG), and solid fibrin film. Growth factors, BTG, platelet factor 4 (PT4) and thrombospondin are all factors that can promote cell proliferation in the solid fibrin network. More specifically, the protective action includes a microaerobic environment, a disinfecting action, and a separating action. Devices that can be used to perform simultaneous centrifugation and coagulation include the rotor medical device shown in FIG. 38. The device includes a disposable cartridge 352, which may be made of plastic or some other suitable material. Again, the plastic modification techniques described above apply to any of the embodiments described herein. The cylindrical cartridge has two concentric chambers, namely the inner chamber 356 and the outer chamber 360.

The inner chamber 356 is cylindrical and is contoured by an inner filtration wall 364 as shown in FIGS. 38 and 39. Any filter or filtration device described herein is suitable for use with this embodiment. The inner chamber 356 has a top end 368 and a bottom end 372, each of which has a rotor shaft 376 attached to that end. The rotor shaft 376 allows the cartridge 352 to be inserted into the centrifuge and used (not shown). At least one of these ends 368, 372 of the inner chamber 356 may have a port or a suitable opening 380 through which blood from the patient is introduced or injected. As mentioned above, in one embodiment, the inner chamber 356 can be kept in vacuum to facilitate standard venipuncture. The inner chamber 356 contains the anticoagulant 232 and can prevent the coagulation of blood entering the inner chamber 356. The inner chamber 356 functions as a cell separation chamber. The inner filtration wall 364 is a selectively centrifugable (mechanically supported) filter that accepts individual volumes of whole blood. The filtration function of the filtration wall substantially prevents red blood cells and white blood cells from penetrating and flowing. However, the filter allows plasma and platelets to flow to the second chamber 360, for example, for a predetermined time of 10 minutes or longer, eg, when a predetermined centrifugal force of 1000 xg or greater is applied.

The second chamber 360 is contoured by an outer wall 384, an inner filtration wall 364, and a top and bottom wall. The second outer chamber 384 may contain one or more coagulation activators 244 as described above and one or more secondary activators 248. The second chamber 360 functions as a densification chamber. As shown in FIGS. 38 and 39, the inner chamber 356 and the outer chamber 360 are concentric.

During operation, the device 352 is centrifuged after blood has been introduced into the inner chamber 356. As described above, centrifugation is performed for a predetermined time and with a predetermined force so that blood separates into plasma and erythrocyte cells. Again, the filtration wall 364 allows the platelet-rich plasma to penetrate, while the red blood cells clog the filter. Once passed through the filter 364, the plasma contacts the coagulation activator 244 and / or the secondary activator 248, resulting in simultaneous coagulation and centrifugation to form a membrane. It may be helpful to provide a mixing action to promote coagulation. Centrifugation performed after the plasma has entered the second chamber is usually performed at about 1500-15,000 xg for 10 minutes or longer to obtain a leukocyte-resistant fibrin platelet-rich plasma membrane. The membrane can be used for any of the tissue regeneration applications described herein, but is particularly useful in the context of wound or burn care.

One or more pins 388 may be present in the inner portion of the outer chamber 360 to allow the membrane to be pulled vertically from the top of the device. Everything described with respect to the surface of the densification chamber (eg, using woven fabrics, overhangs, grooves, etc.) applies in this case to the outer chamber 360. Further, the description of modification of the plastic surface also applies in this case. The membrane can be pulled out by breaking the device or opening it in two parts. Typically, for hygienic reasons, the device is disposable. The device is easy to operate and provides a safe and sterile condition.

Another aspect of the invention relates to devices and methods that can be used to form formed dense fibrin and platelet networks by radial and axial centrifugation, as well as modifications to the devices and methods. This aspect also relates to a method of dividing the measured liquid into a large number of aliquots in order to form a large number of networks at the same time. The clinical efficiency and ease of use of autologous fibrin and platelet networks have been described above. There are several clinical applications for regenerating soft tissue, such as the formation of the networks or membranes described above in a particular shape prior to transplantation (eg, knee half-moon repair). In the case of crescent cartilage, the ideal shape would be a semi-circular wedge shape, similar to the part that can be used to replace a significantly damaged crescent. The platelets present provide the vascular tissue formation needed to regenerate the tissue, and fibrin provides an absorption cushion to withstand the load.

Current practice of repairing soft tissues such as cartilage can treat only 20% of cases. Often, in the remaining cases, the soft tissue is permanently removed and the patient's movements are impaired. This syndrome is prominent in professional athletes and is of great concern in sports medicine. Although synthetic materials are available to form a scaffold for new tissue growth, they have the disadvantage of producing unfavorable immune responsiveness and low success rates due to lack of angiogenic function. There is. A successful method would be to separate the platelet-rich plasma into controlled volumes in order to simultaneously form the multiple forms and shapes used in a given method.

The molding system may include a formed cavity in any of the above-mentioned centrifugation vessels, which is contoured by the shape of the desired portion at the time when the centrifugal force is maximized. The cavity may form at the bottom of the vessel when centrifugation is performed within a radial centrifuge. Alternatively, the cavity can be contoured within the cylindrical wall of the vessel that is axially centrifuged. FIG. 40 shows a mold oriented for use in radial centrifugation as shown in FIG. 40 (a) and oriented for use in axial centrifugation as shown in FIG. 40 (b). An example of a determined template is shown. The mold can be integrated with the container or separate parts can be connected, combined, stretched or added to the container, as shown in FIGS. 43-46. If the cavity is not integral, the split design may allow the formation of complex geometries and allow for easy removal. The cavity may also contain a funnel-shaped feature to direct the flow of the fibrin / platelet mixture into the cavity, as shown in FIG. More specifically, FIG. 41 shows a mold 392 with a funnel 396 such that it reaches a runner 400 that allows material to flow into the cavity 404. The cross-sectional area of funnel opening 396 determines the relative amount of fibrin / platelet monofilament enrichment. The runner 400 can also be connected to the funnel 396 and the cavity 404, as shown in FIG. 41, allowing direct flow into the cavity 404 and minimizing trimming of the molded part. As shown in FIG. 42, a vent or passage 408 may be included within the mold frame 392 to allow gas and / or liquid to be pushed out of the cavity 404 by replacement with invading fibrin. In other words, the ventilation holes 408 allow the release of gases and liquids.

In methods that require a large number of implants, especially those that require different volumes and densities, the axial centrifuge described above can be provided by including a vertical vane at the bottom, as shown in FIGS. 43-47. It can be divided into controlled volumes. In other words, one of the devices described above, eg, concentric chamber embodiments, can be employed, but multiple cavities are used for each of the centrifuge vessels to simultaneously provide multiple shaped objects. Can be done. The relative amount of platelet-rich plasma to be fed to each of the axial swirl design templates can be obtained by including the vertical vane 412 as shown in FIGS. 43-47. The blades 412 may extend to the height of the device, but this is not always necessary, as they project towards the central axis but do not contact the central axis, thereby increasing the volume between the volumes contoured by the blades. Allows platelet-rich plasma to flow freely.

FIG. 44 is a plan view showing the blades B1 and B2. Vents 416 allow fluid connection between chambers W1 and W2 when blades B1 and B2 do not touch and fluid is first added and the centrifuge is dormant. The cross-sectional areas of chambers W1 and W2 can be proportional to the volume of fluid to be fed to each of the molds. The liquid level in the initial dormant state is equal in all compartments, so the relative volume is proportional to the cross-sectional area contoured by the positioning of the blades. Therefore, the position of the blade will determine the volume in each compartment. As a result, larger "pie pieces" can be employed for deeper molds.

Once centrifuged, the volume in each compartment moves radially to the target mold. FIG. 45 shows a three-chamber device with unequal "pie pieces". FIG. 47 shows a three-chamber device having each mold 420 set to a different radius, thereby applying a g-force proportional to its radius to the contents of each mold 420. The number of chambers depends on the particular application. The formed material has different densities depending on the radius of the mold 420. FIG. 46 shows molds in three different positions: one-piece position, one-to-one position, and one extending from the device. The positioning of the mold affects the density of the film formed. Since the relative volume of each aliquot and the position of the cavity are set in advance, it is possible to add a molded balance weight to obtain an appropriate balance. A useful application of this feature would be for molding films at high densities and pastes at low densities.

During activation, the platelet-rich plasma may be added to a container as described above, or whole blood may be added to the pretreatment chamber to transfer the platelet-rich plasma to a second vessel containing the appropriate blood coagulant activator. Produces platelet-rich plasma. The container is quickly placed in the centrifuge and swirled with the desired g-force required for the application. This provides simultaneous centrifugation and coagulation. Fibrin strands and platelets deposit rapidly towards the cavity and fill the cavity. The fibrin strands are then crosslinked to form a stable network. When removed from the centrifuge, the molded portion can be removed and all excess portions can be trimmed. For more complex shapes, split cavity molds may be employed. As described above and shown in FIG. 41, a funnel-shaped pretreatment device can be employed in this design to minimize the required blood volume and increase efficiency. As shown in FIGS. 41 and 42, it is very similar to the runner system used in hobbyist plastic model kits to fully fill the cavity and facilitate the handling of complex geometries. Runners and vents can be included.

47 to 49 show cross-sectional views of the operating device. Figure 47 shows a dormant device; a mixture of platelet-rich plasma 284 and coagulation activator 244 until a single horizontal fluid surface is achieved and the fluid is properly balanced between the chambers. , Flowing between the chambers. Figure 48 shows the device when centrifugation begins; the liquid is formed in the shape of a swirling stream by axial rotation. During centrifugation, the blades 412 prevent communication between channels, thereby maintaining proper supply to each of the molds. The wall 428 gradually thins towards the mold and can function as a concentrated funnel. As the speed of the centrifuge and the associated g-force increase, the parabolic swirling flow increases until all fluid is delivered to the mold. FIG. 49 shows the device in complete centrifugation, at which point the mold is filled.

This system can also be used for platelet-rich plasma (PPP) to form substances containing fibrin. In other words, the system can be used in applications that do not require platelets. Platelet-rich plasma can be formed by centrifuging the first tube with a larger g-force, for example, 5,000xg or more instead of 1,000xg. This design can also be used to form the desired non-self-derived fibrin or fibrin / platelet network once donor and recipient compatibility has been established.

Overall, the template provides complete, self-derived patient compatibility. As a result, the fibrin-platelet network can be formed with a precise molding shape and density. Multiple shapes can be formed simultaneously, such as the left and right half moons of the knee. In addition, molded tubules, kinuta and stapedius bones for the inner ear, and circumflex tendons for the shoulder can be formed using these templates. In addition, cartilage of the elbow cartilage, the etepicondyle part, the finger part, the ankle and the wrist can be formed. The formed membrane or network is absorbable, stable, has growth factors and promotes healing. For multi-shape applications, the density of the portions can be varied by setting the radius of the mold.

Another aspect of the invention relates to an apparatus and method for controlling the distribution of platelets in a fibrin / platelet network by utilizing the deposition of different centrifuges. The clinical efficiency and ease of use of autologous fibrin and platelet networks are as described above. Fibrin stabilizes the wound and provides a medium for cells to grow and move. Platelets initially contribute to wound stabilization while containing a variety of anti-inflammatory, growth and vasculature agents. Therefore, in many healing methods it is beneficial to concentrate the location of platelets in the fibrin continuum. For example, in the case of a chronic wound, concentrating platelets on the side of the membrane in contact with the wound will increase the adhesion of the membrane to the wound and increase vascular tissue formation in the subcutaneous layer. For half-moon repair, it may be beneficial to concentrate platelets in the outermost region of the formed half-moon, the "red zone," and increase vascular tissue formation in this region. In the case of bone cement, it is preferable that platelets are evenly distributed throughout the continuum. Therefore, this aspect of the invention provides a method for preferably placing platelets within a fibrin substrate using centrifugal force.

Fibrin formation proceeds at a rate independent of g-force, whereas platelet deposition is a function of g-force. More specifically, platelets deposit at a constant rate, and as a result, platelets deposit at a constant rate until all of them deposit. Platelets are evenly distributed throughout the platelet-rich plasma. When gravity is applied to plasma, platelets deposit at a constant rate that increases with increasing gravity. The time to complete the deposition is proportional to the height of the platelet-rich plasma that the top platelet must traverse. Therefore, in the case of platelets with a height of 100 mm, the time to complete the deposition is about 5 minutes at 6000xg or 15 minutes at 2000xg.

On the other hand, fibrin monomers are formed at a rate independent of gravity. For normal patients, this process is completed in about 30 minutes. Thus, the method presented here solves the problem of achieving a centrifugal profile that accommodates two different deposition rates, thereby achieving a preferred position of platelets in the network. The preferred position of platelets optimizes tissue regeneration to suit each particular application, providing faster healing and higher success rates for that method. The method of placing platelets in a preferred manner involves adjusting the g-force during the deposition process, taking into account differences in platelet deposition and formation and subsequent fibrin deposition rates.

In one example, platelet-rich plasma can be exposed to a coagulation activator and then immediately centrifuged at about 4000-6000 xg. This causes platelets to deposit rapidly in about 5-10 minutes, followed by fibrin, which is formed in the subsequent 25-35 minutes, as the layer above it. The formed structure concentrates the first formed platelets on its surface, and then the number of platelets decreases in the formed layer. This application is particularly useful for half-moon repair and chronic wounds.

In another example, platelet-rich plasma can be exposed to a coagulation activator and then immediately centrifuged at 2000 xg or greater. Platelet deposition and fibrin formation can proceed at equivalent rates. The network thus formed has platelets evenly distributed throughout the network. This application is particularly beneficial for bone cement and soft tissue growth (of periodontology).

In yet another embodiment, the platelet-rich plasma can be exposed to a coagulation activator and immediately centrifuged. However, the centrifugation rate repeats between a rate of about 4000-6000xg for about 1-2 minutes and then a rate of 1000-2000xg for 5-10 minutes. By repeating about 5 to 10 times, a sandwich structure having 10 to 20 separate layers of high-concentration platelets and low-concentration platelets is obtained. This application is particularly beneficial for cartilage repair in joints, which prevents bones from rubbing against each other.

Therefore, as a result of controlling the rate of centrifugation of the platelet-rich plasma and the coagulation activator and the centrifugation time, the platelets are placed in a preferable position. Controlling the position of platelets optimizes tissue regeneration for a particular application, thereby providing faster healing and higher success rates for that method.

FIG. 56 shows another embodiment of mold design. The molded insert 424 is generally made of a plastic or rubber material. The insert is introduced into and removable from the container 426, as shown in FIG. Once the platelet-rich plasma has been placed in the vessel 426, the coagulation activator can be added to the vessel. Alternatively, the coagulation activator may already be present in the plasma upon introduction. The insert has blades 428 similar to those of FIGS. 42-49. The vanes 428 project to contour the chamber 430 and may form or form a membrane within the chamber upon centrifugation. In other words, the vanes may leave a space between the insert core 432 and the vessel when the insert is inserted and use a radial centrifuge to form a cylindrical membrane. Inserts are shown as having three blades, but inserts with one or more blades can also be manufactured. Alternatively, the insert may be split and a rectangular membrane may be formed between the two inserts. The advantage of using the molded insert 424 is that it eliminates the need for a flat bottom container with a swivel head centrifuge.

In another embodiment of the invention, methods and devices used to treat a person suffering from cartilage disease are provided. The fibrous cartilage tissue has a complex structure of a multi-layered structure of chondrocytes enclosed in the non-crystalline fibrous tissue, and the main component of the non-crystalline fibrous tissue is collagen to which hyaluronic acid and polysaccharide are added. Is. The inner layer is the densest layer (ie, the inner layer can be up to 25 times harder than the outer layer), while the other two flexible layers are visible towards the surface. Pathological symptoms associated with cartilage tissue of joints due to infections, autoimmune diseases (such as arthritis), age-related degeneration, and trauma are common in humans and animals. Today's treatments focus on the pharmacological treatment of patients to stop infection, reduce inflammation, or stimulate the natural regeneration of autologous cartilage tissue. In painful cases, such as the treatment of meniscus in the knee, surgical treatment is performed to remove unreplaced cartilage, leaving the patient's bone unprotected. This embodiment provides a method of treating cartilage disorders.

Membranes and fibrin can be used as scaffolds for culturing chondrocytes. More specifically, these methods can be applied to human and animal cells to form biologically active hard solid fibrin cushions (including autologous chondrocytes) and replace damaged living body cartilage. It can support mechanical stress and initiate biological recovery of tissue. In one particular embodiment, the chondrocytes, starting with the biopsies of cartilage tissue digested by enzymes, as known to those skilled in the art, CO.₂Incubate in a monolayer according to conventional protocols in an incubator. Chondrocytes carefully isolated from the support swirl the vessel at approximately 4,000-10,000 xg with the aim of obtaining a "oriented" strong membrane that can be used to replace damaged parts of the cartilage in the body. It can be mixed with PRP just before it is made to. The centrifugal force applied can make a difference in chondrocytes within different types of cartilage.

Fibrin scaffolds with chondrocytes can be cultured under sterile conditions in specialized bioreactors for several days (as described by R. Portner, University of Dortmund, Animal Cell Culture Group). In this device, DMEM (Invitrogen) medium supplemented with serum, TGF (Transforming Growth Factor-Cell Concept), and IGF (Insulin-like Growth Factor-Cell Concept) is continuously replenished on the scaffold in the flow chamber. To do. This method can be done for 19 days. Its purpose is to produce true cartilage in vitro on top of the original fibrin base and shape. This new cartilage can be used in vitro to replace damaged cartilage.

In one method, the simultaneous coagulation and centrifugation methods described above can be used to form extremely tough, self-derived membranes. More specifically, a thick film (eg, 3 mm thick and 24 mm in diameter) can be produced according to Example 5 below. Of course, any of the devices or methods described above can be used to form films of various sizes. One particular membrane is in a sterile vessel (eg, a flat bottom 25 ml glass flask filled with about 20 ml of autologous platelet-rich plasma (PRP) and swirled at about 4500-5000 xg for 30 minutes). Can be formed with. Platelet rich plasma (PPP) may be used in this step. It is also possible to adopt any of the other film forming techniques described above.

After this membrane or any other membrane of the invention is formed, the membrane is thoroughly washed with sterile saline and placed in a larger sterile flask containing the activator and platelet-rich fibrin. A second layer of (PRF) can be manufactured. In this step, a new amount of platelet-rich plasma is introduced into a new flask containing a strong membrane in a completely sterile condition. A second centrifugation step can be performed on the second flask for the purpose of obtaining a three-layer membrane. In one particular embodiment, this centrifugation can be performed for 20 minutes at a rate of 1000xg to form one with a diameter of 30 mm. Centrifugation can be performed at any of the speeds described above (ie, for 10 minutes or longer, at 500-15,000 xg). The membrane formed can be used to transplant the cartilage where it should be replaced. In this case as well, the thickness and dimensions of the film are determined by the above-mentioned conditions. The amount of blood and the type of flask will change accordingly. Importantly, the sterile membrane (formed by any of the methods described above) is exposed to an additional coagulation activator and then its contents to form a second layer of polyplasma fibrin. Centrifuge. Alternatively, additional coagulation and centrifugation can form a third layer of membrane, and so on.

In one relevant method, cartilage tissue (autologous) is placed in an IN VITRO culture in a gel according to the alginate regenerated chondrocyte (ARC) method well known to those skilled in the art. The gel in the ARC method may be replaced with self-derived fibrin prepared according to the method and apparatus described above. More specifically, during the second step in the production of polyplasma fibrin, the selected chondrocyte cell line is added to the secondary vessel with autologous fibrin and the mixture is centrifuged at a low centrifugation rate or by centrifugation. It may be jelly-like without any force.

The form and size of the container in which the jelly is made can be selected according to the subsequent use of the "artificial cartilage" (ie, the form of the cartilage to be replaced). The jelly formation can be carried out in such a way that a gel is formed around a strong film prepared according to the above clause. This can be achieved by placing the strong membrane in a container where the second blood clot is occurring so that the new gel substantially surrounds the original strong membrane and the chondrocytes are contained in the gel. it can. Sterilized vessels with autologous chondrocytes, jelly-like autologous platelet-rich fibrin and finally a strong inner membrane are suitable as known to those with knowledge of growing chondrocytes in vitro. It can be arranged for culturing in an atmosphere (temperature, O2, CO2 and RH levels). This allows new tissue to grow on the fibrin gel scaffold in vitro. If the tissue culture has the correct density of chondrocytes and fibrous tissue, the three-layer tissue will be a membrane that is extremely tough, flexible and ready to replace the host's diseased tissue. Appropriate additives can be added to the medium to optimize the yield of the method. The use of stem cells is also expected because stem cells are the origin of all cells in the body and new chondrocytes can be expressed in vitro if properly treated as known to those of skill in the art. To.

Overall, this embodiment is intended to produce implants for treating the diseases described above, while reducing the risks associated with the use of synthetic or heterologous materials. Autologous chondrocytes are found in platelet-enriched membranes, which provide a suitable foothold for growth in vitro and in vivo, and are the basic chondrocyte matrix for the production of new cartilage. To produce. The membranes formed can be easily produced in sterile cabinets, and the membranes reduce postoperative regeneration time and promote the migration of chondrocytes that build new cartilage. It has physical properties that allow direct transplantation to the required location for the purpose of.

The embodiments and methods described herein can also be used in connection with harvesting PRP from a plasma separation and exchange machine. During surgery, cell savers or separation and exchange machines are used multiple times to save blood by aspirating blood that collects at the surgical site, separating cells, and reinjecting cells into the patient's body. Will be done. This technique, sometimes referred to as "non-invasive surgery," minimizes or eliminates blood transfusions to replace lost blood, making the surgery safer and more economical. Such equipment is manufactured by Haemonetics (Braintree, Mass.) And Cobe (Colorado). These separation and exchange machines are sometimes used to separate platelets and plasma from red blood cells. PRP can be collected by accessing the platelet and plasma ports of these machines. If the PRP is added to the second tube, the PRP is remineralized and can undergo simultaneous centrifugation and coagulation by the methods described above. This makes it possible to obtain higher amounts of PRP while eliminating the need for a first centrifugation step and sampling device. A variety of solid fibrin networks and membranes can also be obtained from this method and can be used in the applications herein.

The majority of centrifuges are designed to be capable of processing collected blood or a second fibrin / platelet network tube approximately 16 mm x 125 mm in diameter. Tubes of these dimensions tend to hold a maximum capacity of 15 mls. These tubes are nested in a removable centrifuge cup for cleaning purposes. The cup is tubular and can support the tube and cup during high speed centrifugation with a collar (Fig. 52). A collar can be integrally formed on the tube by processing a metal or injection molding a polymer-based material. The collar may also be formed separately and bonded to the tube by adhesive, ultrasonic welding, spin welding, induction welding or other material bonding methods. In these methods, the materials of the collar and the tube do not have to be the same, allowing a wide range of material choices. Alternatively, the tube may have a tapered outer diameter that narrows towards the closed lower end, and the collar may have an inside-out taper that matches its inner diameter, with the tube in the collar. When inserted into, the outer diameter of the tube and the inner diameter of the collar can only reach where they engage, and the distance from the open end of the tube during the molding process may be preset. The collar has an inner diameter that allows sufficient contact with the tube and needs to support the tube while the large shear forces generated during centrifugation are applied. The thickness of the collar, i.e. the height of the low-height cylinder, is determined by the nature of the material of the collar and the force applied to the collar during centrifugation. The outer diameter of the collar must be sufficient to prevent the tube from moving radially outward during centrifugation. Color dimensions can be easily calculated by engineering computer manipulation or computerized finite element analysis.

The structural material is typically high strength steel or engineering plastic. For many applications of fibrin and platelet networks, such as spinal fusion and plastic surgery, more PRP and / or fibrin platelet networks than those obtained with a 16 x 125 mm tube are desired. A method of obtaining a significantly large amount of collected blood or fibrin / platelets comprises forming a large collection or receiving tube by attaching a support collar or forming the collar integrally. The tube can be placed directly in the rotor of the centrifuge after removing the support cup. The structural material of the tube may be strong enough to hold vacuum pressure, accept stoppers, be compatible with blood, and withstand centrifugation. Suitable materials include, but are not limited to, metals, glass with a support collar attached by adhesive, or high-strength barrier plastics such as polyethylene terephthalate (PET) or polyethylene naphthalate (PEN). Such tubes have a diameter of about 20-30 mm (eg, 25 mm) and a length of 110-140 mm (eg, 125 mm) and can hold 20-30 ml or more. By modifying the shape of the rotor, a thicker tube can be formed and a larger diameter tube can be accepted. This is also useful for diagnostic tests and other methods where larger specimens than those obtained with standard sized tubes are desired.

Delaying centrifugation and / or calcium re-deposition after transfer of PRP may improve the incorporation of fibrin, platelets and growth factors into the implant. Delaying centrifugation and / or calcification does not mean that simultaneous coagulation and centrifugation will not occur. After transfer to the second tube, a small particle size implant material can be added to the PRP or at least one of the secondary tubes prefilled during the manufacturing process. These graft materials may include autologous bone, donor bone, animal bone, synthetic bone, tricalcium phosphate, carbonates, sulfates and combinations thereof. Due to the density of the implant material and its small particle size, it is difficult to evenly integrate the implant into a fibrin platelet network or a solid fibrin network. This can result in the density of the implant being much higher than the density of the PRP and the implant material being rapidly packed into the bottom of the tube during centrifugation. The small particle size of the packed implant material does not always allow the descending fibrin and platelets to easily enter the gaps between the packed implant materials during the subsequent centrifugation cycle. One alternative method for rapid centrifugation is to delay the centrifugation for a period of time before the subsequent cross-linking occurs, with fibrin monomers, platelets and growth factors surrounding and invading the perforated surface of the graft material. To allow that. The mixture can be mixed periodically or continuously during the delay period to improve the dispersion and coverage of each implant particle. After an appropriate amount of time, as determined by the particle size of the graft material, the graft is packed by compressing the network during the cross-linking step of fibrin formation and centrifugation is initiated to stabilize the network of fibrin platelets. can do.

Depending on the particle size of the bone graft material, the delay can be widespread. The larger the particles, the less beneficial the delay will be. For autologous and human and animal grafts of 3 mm and above, no delay may be required to integrate the graft into the fibrin platelet network. For implants between 0.5 mm and 3 mm, 1-20 minutes (eg, a delay of 3 minutes) allows sufficient incorporation of the implant material while allowing centrifugation during the cross-linking process. .. For implant materials of 0.5 mm or less, a delay of 3-25 minutes (eg, a delay of 5 minutes) allows sufficient incorporation and compression during cross-linking. Again, delaying the recalculation of PRP allows PRP to be absorbed into the graft before the onset of coagulation. In one embodiment, the calcium is not prefilled in the second tube and is added after a soaking time of 1-30 minutes, ideally 5-15 minutes.

In other cases, large volume graft materials such as bone rods for spinal fusion are employed. In these cases, prior to coagulation, the graft material is immersed in the PRP to allow plasma, platelets and / or growth factors to penetrate deeper into the porous surface, thereby into the subsequent fibrin-platelet network graft material. It may be desirable to improve the incorporation of. This is achieved by slowing the addition of calcium or other cationic species that will replace the endogenous calcium bound to the anticoagulant chelate to PRP after transfer to the second tube. can do. The calcium coagulation activator can be added directly to the tube after an appropriate time delay, depending on the nature of the implant material and subsequent centrifugation during coagulation. Alternatively, using a reservoir that connects to a second tube and holds a calcium solution that operates by increasing the rate of centrifugation, in a manner similar to that of the single tube system embodiment. , Calcium coagulation activator may be added.

It is also desirable to spray PRP onto the surface of the wound to form a fibrin-platelet network with insitu. This method improves the healing effect. Fibrin acts as an adhesive, while platelets improve healing by adding their growth factors. Examples of methods that employ this technique are: gluing a skin graft to a burn or chronic wound; gluing the skin to the subcutaneous layer during plastic surgery such as facelift; after resection of the burn wound. It may include sealing the wound where the liquid seeps out; and applying a topical hemostatic agent. One way to achieve the desired effect is to transfer the PRP to a remineralized tube or secondary tube, then apply a pump-type aerosol spray or air-assisted spray to the tube and apply the remineralized PRP to the wound area. That is. The PRP will then form a fibrin platelet network in situ.

One alternative would be to pump directly from the first tube after segregation of erythrocyte cells, or transfer the PRP into a tube that does not retain calcium coagulation activators or other cationic species. Calcium can be added by adding the solution to the fluid pathway during pumping, either by utilizing a separate fluid diversified tube or by flowing PRP through a chamber containing calcium crystals.

One alternative method is to concentrate the platelets into the bottom of the second tube without a calcium solution by centrifuging the PRP after transfer. The suction part for the pumping system sucks from the bottom of the tube and causes platelets to act at higher concentrations on large amounts of growth factors. The suction stem can have a sliding diaphragm that traps the platelet concentrate below the diaphragm. The suction stem can have a stopper that limits the initial position of the diaphragm, thereby setting the concentration of platelets in the volume to be distributed to the desired value (see Figure 53). The stopper is located at a preset distance from the bottom of the suction port that will give the platelets in the volume of plasma below the diaphragm the desired concentration. The higher the stopper is placed on the suction stem, the lower the platelet concentration will be. Another embodiment places a high energy coagulation activated surface in the fluid path of the spray system. The surface will activate plasma to form a clot with a higher cross-linking density, which will increase mechanical strength and reduce clotting time.

Other devices can be employed to facilitate the removal of the fibrin-platelet network from the secondary tube with and without the graft material. After completion of the second centrifugation step, the fibrin-platelet network can be packed into the bottom of the second tube. Normally, the tube can gently pour the supernatant into the sterile cup and the network flows freely into the cup. At the higher speeds of centrifugation required for denser networks, the clots can be tightly packed so that the supernatant can be easily poured gently. This stuffing forms a closed seal against the wall of the tube, which creates a vacuum pressure as the clot moves when turned upside down, blocking further flow. This condition is exacerbated by the addition of graft material that is compressed during centrifugation, forming a packed, dense substrate, as in the case of sintering metal powders. For this reason, it is desirable to remove the network from the bottom of the tube, preferably as part of the supply system so that the network can be added to the wound site, such as a bone cavity.

One way to facilitate network removal is to include a cup with a perforated bottom in the second tube during manufacture (see Figure 54). A hole is made in the bottom to allow the drainage of serum generated during centrifugation while removing the cup from the tube (Fig. 54a). The walls of the cup can vary in height from those with a shallow appearance (Fig. 54a) to the overall length of the tube (Fig. 54b). The lip of the cup may contain means to connect to the feeder. The connecting means may be a screw, a plug-in screw lock, a clip or a similar mechanism. The wall of the cup may have a groove in the wall to prevent vacuum pressure during removal so that excessive removal force is avoided (Fig. 54d). The wall of the cup may have a hole portion through which the serum above the clot can flow along the groove into the lower volume formed during the removal of the cup from the tube. The top of the cup can also be connected to a supply system, which operates by a reliable replacement of the piston through the cylindrical portion of the cup; this action is a syringe (Figure 54e) or a pawl-type "caulking gun" mechanism (Figure 54e). It can imitate 54f). The material of the cylindrical portion of the cup or feed cylinder may be radiation opaque to allow accurate feed using fluorescence measurement techniques. In FIG. 55, the second tube is cylindrical and has a stopper at either end of the cylinder. After centrifugation, the stopper is removed and the network on which the piston is formed is replaced. This constitutes a cartridge system. Alternatively, the cup may be attached to the stopper by a fiber so that the cup is pulled out when the stopper is removed.

Membranes and fibrin produced by the methods disclosed herein can serve as scaffolds to be used for culturing cells, as described above. The fibrin substrate can be dense enough to function as a scaffold. The substrate can be formed of a slowly absorbed scaffolding material such as collagen sponge, a biodegradable polymer or a biodegradable polymer such as an abdominal aortic aneurysm implant. These combined scaffolds can provide additional mechanical strength and more uniform tissue regeneration compared to current scaffold materials. In certain embodiments, the membrane obtained by applying a large centrifugal force, eg, about 4,000-10,000 xg, can serve as a scaffold for in vitro adhesive culture of epidermal cells. These cultures are particularly useful for repairing severe skin damage caused by burns or mechanical exfoliation of the original tissue. These fibrin scaffolds typically donate growth factors by platelets and adhesion factors by solid fibrin networks to cells in culture. In order to obtain a good culture in vitro, it is desirable to start with epithelial cells of sufficient density in the medium.

The membranes and solid fibrin networks described herein can also be used to fuse stem cells on it. More specifically, membranes and solid fibrin networks can be used to culture pancreatic cells with monolayer or several layers of cells or stem cells within the membrane. For example, Medvinsky of the University of Edinburgh, UK and X. Wang of the University of Portland, Oregon, recently injected stem cells into the pancreas of diabetic mice to fuse their genomes with lesioned pancreatic cells. And again, it has been demonstrated to produce some degree of multiples with insulin-producing activity. The active growth factors present in the scaffolds and supports described herein allow cells to grow using conventional media and also study fusion, biochemical and cytological properties. It also makes it possible to do.

In different applications, chondrocytes obtained from monolayer cultures are immobilized within alginate beads, as known to those skilled in the art. These solid beads are compressed to become larger "tissue-like" aggregates using centrifugation, and these aggregates are intended to regulate air pressure and stimulate the "tissue" according to physiological stress in vivo. Objects are "compressed" (Giessen, Germany, University of Applied Sciences, Czermak P.)). During centrifugation, PRP can be added to alginate beads and ready to be implanted in place of damaged cartilage or cultured in a biological reactor for several days to induce cartilage growth. As such, dense bioactive aggregates can be produced.

<u style="single">Example</u>

100 mg of tranexamic acid, which functions as a fibrin stabilizer, was introduced into a 5 ml glass container for antibiotics, which can be sealed in a vacuum and is made of transparent white glass, and which is inert and has a thickness of 1 mm. Synthetic tranexamic acid with a purity of 98% or higher is commercially available from the American company Sigma Incorporated. Separately from this, CaCl from the same American company Sigmain Corporation₂ 2H₂By weighing 147.0 g of O (purity&gt; 99%) with a precision scale, 1 M CaCl₂A solution was prepared.

The salt was dissolved in exactly 1 liter of ultra-high purity non-exothermic distilled water at room temperature for several minutes with frequent stirring. An 80 μL activator solution was introduced into a glass container using a precision piston dispenser with a supply accuracy of ± 5% (such as Eppendorf). In this step, at the same time as the feed, filtration was performed using a 0.22 μm Millipore sterilization filter, carefully preventing the possibility of contamination by any powder or filament. Finally, punctureable and under vacuum pressure, taking care not to completely plug the container so that subsequent vacuum plugging and the possibility of further sterilization by using gas can be tolerated. The glass container was plugged with a stoptable rubber stopper. The container was then placed in a suitable device for vacuum closure while preventing the possibility of contamination by solid particles in the atmosphere (ULPA or HEPA filtration in a sterile chamber). By using a membrane vacuum pump and a micrometer control device, 4 ml of vacuum pressure was applied to the internal atmosphere of the device. A precision vacuum instrument was used to control the vacuum level in the internal atmosphere (accuracy # 1 m bar). Finally, the container was evacuated under vacuum so that the device could be recovered without draining and subsequently for use as described in the following examples.

For example, using a Becton-Dickinson VACUTAINER® sterile test tube supplemented with 0.106 M sodium citrate solution, 10 ml from the patient according to quantitative standards for clinical analysis. Intravenous blood was aspirated. For this purpose, test tubes supplemented with disodium or dipotassium ethylenediamine tetraacetate can also be used. The sample was carefully kept sterile while aspirating the blood. Finally, the ingredients were thoroughly mixed and the sample was gently shaken to ensure the anticoagulant effect of sodium citrate. The test tube was then introduced into a suitable centrifuge, carefully balancing the weight of the rotor to prevent damage to the centrifuge. Once the lid was sealed, the sample was centrifuged for 15 minutes at 3500 rpm, which separated erythrocyte cells (high concentration) from citrate-added plasma (supernatant). In this case, the plasma yield, which was largely dependent on the donor's blood characteristics, was as high as 55%. The test tubes containing the separated plasma were kept capped under sterilization and placed vertically in the stand to collect the plasma itself, but to prevent mixing of the two phases separated during centrifugation. Care was taken not to shake the test tube during this step. The outer portion of the test tube cap was then sterilized with denatured alcohol and then a sterile needle connected to a sterile syringe was introduced into the test tube cap. The needle was separated from the two-phase separation meniscus by 3-4 mm and 4 ml of plasma was aspirated. The container cap according to the invention, adjusted as described in Example 1, was sterilized in advance with alcohol and punctured by using the same needle. Immediately after the needle punctured the cap, the citrated plasma retained in the syringe was completely aspirated into the container. The container was gently shaken, and after about 2 minutes at 37 ° C., a blood clot of sterile self-derived fibrin roux that could be used immediately was obtained.

Using a 5 ml sodium citrate vacutina® test tube (Becton-Dickinson), aspirate approximately 18 ml of venous blood from a 49-year-old patient and be careful to shake gently immediately after aspirating the sample. .. The blood thus collected was immediately centrifuged (at 2500 rpm for 15 minutes) to separate the plasma. Prepared as described in Example 1 (but without tranexamic acid) 120 μL each of CaCl₂Plasma (12 ml) was carefully transferred to two 10 ml tubes containing (10 g / 100 ml). After mixing the plasma with the activator, the test tube was centrifuged at 3000 rpm for 30 minutes, resulting in two large fibrin samples, which were prepared with all care for sterilization. Within 2-3 hours, it was inserted into a large small sac-like mandibular cavity. The cavity was formed by extraction of the impacted left canine and the second incisor on the right side, and by excising the cysts present in the central region of the incisors. Finally, the gingival margin was sewn eight times and closed. A radiograph check after 15 days showed that the fibrin remained in its required position, apparently intact. Tissue structure after 7 months revealed that fibrin had been completely replaced in bone tissue, a better postoperative course than traditional methods that required more than 12 months to achieve the same result. It was. Since no anti-fibrin solubilizer is used for the production of self-derived fibrin, it can be explained in this case that the above additives are useful for a particular purpose.

According to the present invention, in order to produce an adhesive fibrin loop, 12 ml of plasma obtained as in Example 3 was prepared as described in Example 1 by taking all measures to maintain sterilization. Transferred to a 20 ml container.

After careful stirring, the mixed plasma is poured into a sterile glass slide of the type used in the chemistry laboratory, where the plasma is sterilized and very pure coral-derived calcium carbonate (Notebs, France). NOTE BS) SA was mixed with BIOCORAL (registered trade name)) or calcium fluoride (&gt; 98%, Sigmain Corporation). Both of these calcium salts are well known to those skilled in the art as fibroblast stimulants.

Mixing a portion of plasma with a portion of calcium carbonate (eg, 2 ml vs. 500 mg) results in a flexible, sterile adhesive paste for the subgingival space or different cavities after excision of the infected mucous sac. Used as a filler. The paste is placed to fill the empty space, forming a solid fibrin network that acts as a thrombus in minutes, and also supports the mucous edge at the required location, followed by the migration of binding cells. At the starting point, a self-derived biological base layer was formed.

To obtain a fibrin loop membrane, 20 ml of plasma obtained as in Example 3 was placed in a 25 ml flat bottom container according to the invention prepared as in Example 1. After normal careful stirring, the vessel was centrifuged in a swirling rotor for 40 minutes at 4000 rpm. At the end of the centrifugation step, a white, extremely dense and high tensile strength film was recovered from the bottom of the test tube, measuring the same size as the bottom of the test tube (24 mm in diameter) and 3 mm thick. Due to its denseness and strength, this self-derived membrane was used as a replacement for porous synthetic membranes, as a retaining and separating membrane in dentistry and general surgery. The resulting membrane can be stored sterile at 4 ° C for several days.

Approximately 200 ml of citrate-added plasma was aspirated from the patient and collected and separated in a double transfusion bag to obtain a large size fibrin loop membrane. A cold precipitation reaction was carried out by freezing the plasma at -80 ° C for 12 hours, and thawing was carried out at 4 ° C for 24 hours (this method is well known to those skilled in the art). On the morning of the same day, the plasma obtained by this method was centrifuged at 5000 rpm at 4 ° C for 15 minutes to obtain about 20 ml of cold sediment. After careful removal of the supernatant by using a pressurizer (eg XP100 from the French company Jouan SA), the cold sediment was removed with 20 ml of total plasma from the same patient. The resulting 40 ml was placed in a flat bottom sterile polypropylene container with a diameter of 35 mm according to the invention containing an appropriate amount of activator as in Example 1. After careful shaking, the vessel was centrifuged for 40 minutes at 5000 rpm, similar to Example 5, but with a higher fibrin content, a denser, higher tensile strength membrane was obtained. Was done. The membrane can also be stored in sterile form at 4 ° C for several days.

The membrane obtained by the method described in Example 5 can be used in the same patient in order to obtain a graft to be transplanted in a case of extremely severe hot water disease in addition to the use described in Example 4. It can be used as a substrate for in vitro culture of epidermal cells.

Good quality membranes useful for the purposes described above can also be obtained from total isolated plasma transferred directly into the vessel according to the invention. The resulting membrane is thinner than those described above, but remains useful as a substrate for surgery and cell growth.

To obtain sprayed fibrin starting from the cold precipitate in Example 5, 20 ml of cold precipitate was removed with 10 ml of total plasma at room temperature and gently shaken to dissolve completely. The resulting plasma was carefully transferred into a 50 ml container according to the invention prepared as in Example 1 and gently shaken to completely mix the ingredients. After 120 seconds, at room temperature, the test tube is connected to a venturi-type sterile air compressor known to those skilled in the art to perform surgery on the surface of the bleeding organ (lung, heart, spleen, arterial anastomosis). Was evenly distributed. Distributing concentrated plasma containing concentrated fibrinogen, thrombin, calcium ions and other coagulant enzymes onto the organ causes coagulation in seconds due to the presence of tissue coagulation-activating enzymes in the patient's endothelium, providing protective hemostatic activity. A fibrin film having was formed. This reduced internal bleeding during surgery and thus avoided further transfusions or complications.

The membrane obtained by the method described in Example 5 will also incorporate autologous platelets if platelet-rich plasma (PRP) is used as the starting blood component. To obtain PRP from whole blood, blood samples can be centrifuged at 1000xg for 10-15 minutes. Subsequent steps are similar to those described in the examples described above.

Membranes obtained by this application method are used as active substrates for in vitro study of stem cell fusion phenomena described by Wang and Vassillopoulos (Nature, Vol. 422-2003, April 24). be able to. This study discloses the use of stem cells that grow in the presence of hepatocytes in the liver of mice and of them to form new types of cells capable of regenerating damaged or defective tissue. The genome is fused.

The membrane obtained as described above can be used as a support for in vitro studies of this vital phenomenon due to the presence of platelets, which are a donor of growth factors and other stimulants. Also, the finally obtained new cell generation can be used for introduction into vivo (similar to that performed for chondrocytes or epithelial cells).

All of the following methods were operated under a laminar flow cabinet in a sterile condition. Starting with a solid biopsy sample, the epidermal tissue was homogenized and small cell aggregates were separated while preserving the perfect state of the single cell. The homogenate was washed with 40 mM sterile PBS (phosphate buffered solution Ph7.3-Gibco), and the cells were centrifuged in a PP tube for 7 to 15 minutes at 500 to 1000 xg, and the pellet was collected. .. Washing was repeated twice. Next, the obtained cells were digested with a solution of 0.05% trypsin / 0.02% EDTA for 20 minutes at 37 ° C. to remove the collagen structures supporting the cells.

Then, the cells were thoroughly washed with sterile PBS to remove collagenase. The cells were then resuspended in medium (eg, M199, HAM-F12, or others known to those skilled in the art) and uniformly distributed over the surface of the fibrin membrane. Cell density on the surface of the fibrin scaffold is important for good epithelial cell culture results. For example, the best result is 1-3x10⁴Cell / cm²Can be obtained with a membrane of the same density, in which case the cells usually adhere 15-60 minutes after distribution and after 2-5 hours these cells are completely flattened. Keep the culture in a controlled atmosphere of 5% CO2 and RH98% at 37 ° C to regularly control the development of the culture and, as a result, medium every 2-3 days, if necessary. Can be replaced and kept for 5 days. In the case of cylindrical vessels, a suitable roller system was used on a 24-hour basis in the CO2 incubator so that the medium constantly washed the membrane surface to nourish the cells in culture.

Culturing should be carried out in a suitable sterile container with a ventilation system that allows gas exchange but maintains internal sterilization. The fibrin membrane should be formed inside the vessel and adhere completely to the inner surface of the flask throughout the culture time.

After confirming the development of the culture in vitro (eg, using a phase-contrast microscope), the membrane with cells on the upper surface is carefully removed and the membrane is implanted in the patient's wound or burn under sterile conditions. It is ready and is finally held in the required position by sewing the membrane.

<figref num="1">It is a perspective view which shows the 1st Embodiment of this invention.</figref><figref num="2">It is sectional drawing which shows the primary container of 1st Embodiment shown in FIG.</figref><figref num="3">It is sectional drawing which shows the different embodiment of the primary container of FIG.</figref><figref num="4">It is sectional drawing which shows the different embodiment of the primary container of FIG.</figref><figref num="5">FIG. 5 is an enlarged partial cross-sectional view of a portion according to the first embodiment of FIG. 1, showing the first end of a transfer device that begins to drill a hole in a sealed primary container.</figref><figref num="6">Shown in FIG. 5, showing the first end of a transfer device that is fully perforated in a sealed primary container and the second end of a transfer device that is fully perforated in a sealed second primary container. It is the same figure as.</figref><figref num="7">It is the same figure as FIG. 2 which shows the primary tube turned upside down and its contents.</figref><figref num="8">It is a top view of the 1st Embodiment shown in FIG.</figref><figref num="9">FIG. 8 is a partial cross-sectional view of FIG. 8 showing the interlocking primary container, secondary container and transfer device, and the contents of the first container being transferred to the second container.</figref><figref num="10">It is a top view of the kit which embodies the present invention.</figref><figref num="11">It is a perspective view of the 2nd Embodiment of this invention.</figref><figref num="12">It is sectional drawing of the 2nd Embodiment of this invention shown in FIG.</figref><figref num="13">It is a cross-sectional view similar to FIG. 12 which shows the primary sampling device which punctures the reservoir and the primary sampling device.</figref><figref num="14">It is the same cross-sectional view as FIG.</figref><figref num="15">It is a perspective view which shows the 3rd Embodiment of this invention.</figref><figref num="16">It is sectional drawing which shows the 3rd Embodiment of this invention shown in FIG.</figref><figref num="17">It is a perspective view of the transfer device which embodies the present invention.</figref><figref num="18">It is sectional drawing along the line 18-18 in FIG.</figref><figref num="19">It is a perspective view of the cartridge which embodies one aspect of this invention.</figref><figref num="20">It is sectional drawing side view of the cartridge of FIG.</figref><figref num="21">It is a perspective view of the apparatus which can be adopted in the axial centrifugation system which embodies another aspect of this invention.</figref><figref num="22">It is sectional drawing of the apparatus and contents shown in FIG.</figref><figref num="23">FIG. 2 is a cross-sectional view of the apparatus and contents shown in FIG. 21 during the first centrifugation.</figref><figref num="24">FIG. 2 is a cross-sectional view of the apparatus and contents shown in FIG. 21 after the first centrifugation is stopped.</figref><figref num="25">FIG. 2 is a cross-sectional view of the apparatus and contents shown in FIG. 21 during the second centrifugation.</figref><figref num="26">FIG. 2 is a perspective view of one modification of the device shown in FIG. 21 in which the radius of the secondary densification chamber is greater than the radius of the primary cell separation chamber.</figref><figref num="27">FIG. 2 is a cross-sectional view of one modification of the system shown in FIG. 21 in which a concentric chamber is adopted.</figref><figref num="28">FIG. 2 is a cross-sectional view of the apparatus and contents shown in FIG. 27 during the first centrifugation.</figref><figref num="29">FIG. 2 is a cross-sectional view of the apparatus and contents shown in FIG. 27 after the first centrifugation is stopped.</figref><figref num="30">FIG. 2 is a cross-sectional view of the apparatus and contents shown in FIG. 27 during the second centrifugation.</figref><figref num="31">It is sectional drawing of the system which adopts a hydrophobic membrane.</figref><figref num="32">It is the figure of the enlarged part of FIG.</figref><figref num="33">FIG. 5 is a cross-sectional view of a portion of the wall of a densification chamber with woven reinforcement.</figref><figref num="34">FIG. 3 is a cross-sectional view of one modification of the wall of FIG. 33 with an overhang on the wall.</figref><figref num="35">FIG. 3 is a cross-sectional view of one modification of the wall of FIG. 33 with a groove in the wall.</figref><figref num="36">FIG. 5 shows a densification chamber lined with a removable film with tabs that facilitates membrane removal.</figref><figref num="37">It is a figure which shows the membrane which has a hole so that it can be easily peeled off.</figref><figref num="38">FIG. 3 is a partial cross-sectional perspective view of a rotor medical device embodying another aspect of the present invention.</figref><figref num="39">It is a bottom view of a densification chamber having one or more solid ribs on the inner wall.</figref><figref num="40">Molds oriented for use in radial centrifugation systems (shown in Figure 40 (a)) and molds oriented for use in axial centrifugation systems (shown in Figure 40 (b)). ) Is shown in the figure.</figref><figref num="41">FIG. 5 is a diagram of a portion of a device having a mold in which a funnel and a runner are employed to facilitate filling of the cavity.</figref><figref num="42">It is a figure which shows the part of the apparatus which has a mold which the ventilation hole is adopted to allow the gas and liquid to leak properly.</figref><figref num="43">It is a perspective view which showed the apparatus which has the mold, the blade which divides two chambers, and a vent by partial sectional view.</figref><figref num="44">It is a top view of the apparatus of FIG. 43.</figref><figref num="45">FIG. 5 is a plan view of a device having a mold, blades and vents that divide three disparate chambers.</figref><figref num="46">FIG. 5 is a modified plan of the device of FIG. 45, showing a mold that is integrally connected and extends from the device, and the blades are divided into three equal chambers.</figref><figref num="47">FIG. 3 is a cross-sectional side view of a portion of any of the devices shown in FIGS. 43-46, after the platelet-rich plasma has been introduced into at least one chamber, but before the device has been centrifuged.</figref><figref num="48">FIG. 4 is a cross-sectional side view of a portion of the device shown in FIG. 47 immediately after the device was centrifuged.</figref><figref num="49">FIG. 4 is a cross-sectional side view of a portion of the device shown in FIG. 47, in which the platelet-rich plasma is in at least one template and in complete centrifugation.</figref><figref num="50">FIG. 5 is a cross-sectional view showing a plastic alternative, eg glass fixed to the bottom.</figref><figref num="51">FIG. 5 is a cross-sectional view showing a plastic alternative, eg, a glass ball glued or heat-bonded to the bottom.</figref><figref num="52">52a is a perspective view of a primary container wrapped in a sterile film and housed by a carrier. 52b is an exploded view of FIG. 54a showing the color in the primary tube.</figref><figref num="53">FIG. 5 is a partial cross-sectional view of a supply / pumping system that can be used with certain embodiments of the present invention.</figref><figref num="54">54a is a cross-sectional view of a cup having a perforated bottom and housed in a secondary container. 54b is a cross-sectional view showing an alternative embodiment of the cup shown in FIG. 54a. 54c is a cross-sectional view taken along line 54c-54c of FIG. 54b. 54d is a cross-sectional view showing an alternative embodiment of the cup shown in FIG. 54b, having holes along its length. 54e is a partial cross-sectional view showing a supply system operating with reliable replacement with any cup of FIGS. 54a-54d. 54f is a partial cross-sectional view showing a supply system similar to FIG. 54e in which a caulking gun mechanism is used.</figref><figref num="55">FIG. 6 is a series of partial cross-sectional views showing a supply system in which the secondary tube has two stoppers.</figref><figref num="56">It is a perspective view of the moldable insert body which embodies the present invention.</figref>