WO2004028584A1 - Substitut dermique compose d'amnios et d'un polymere biodegradable, procede de preparation et utilisation de ce substitut - Google Patents
Substitut dermique compose d'amnios et d'un polymere biodegradable, procede de preparation et utilisation de ce substitut Download PDFInfo
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- WO2004028584A1 WO2004028584A1 PCT/KR2003/002012 KR0302012W WO2004028584A1 WO 2004028584 A1 WO2004028584 A1 WO 2004028584A1 KR 0302012 W KR0302012 W KR 0302012W WO 2004028584 A1 WO2004028584 A1 WO 2004028584A1
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- amnion
- biodegradable polymer
- collagen
- dermal substitute
- sponge
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
Definitions
- the present invention relates to the dermal substitute comprising the biodegradable polymer such as collagen and the like and the biomaterial such as amnion obtained from placenta, the preparation method and the use thereof.
- wound-covering materials should have the properties such as suitable water-permeability and protective activity from harmful environment such as microbes.
- synthetic polymer membranes for instance, urethane polymer, poly-L-leucine and the like, have been used as wound-covering materials with a limit to use, since they do not have any biological functions.
- Biobrane one of successful products in the early 1980's, has the bilayer structure consisting of collagen-covered nylon textile and micro-porous silicone membrane and shows many advantages, for example, the adhesiveness to skin, the expandability, the easy delivery of antibiotics to the burned area through silicone membrane to be commonly used for superficial burns, deep burns, after necrectomy on extensive granulating wound surface, on autografts, in donor regions, and after dermoabrasion
- collagen in the form of sponge, gel or biodegradable polymers is used as a dermal substitute.
- collagen sponge When the collagen sponge is applied to full thickness skin defected wound, fibroblasts and capillary blood vessels are infiltrated and proliferated in the sponge and collagen fibers are formed by self-collagen synthesis to build the dermis like-tissue. And then the collagen sponge is degraded and absorbed. Finally, the dermis like-tissue becomes complete self-dermal tissue.
- This collagen sponge is called as dermal substitute because the material itself turns to dermal tissue in the body. It is already commercially available and lots of studies and clinical reports have been published till now.
- Korea patent publication No. 2000-0013701 discloses the porous, double structure wound protection membrane for inducing dermis-regeneration, which consists of porous layer and rigid layer comprising collagen, laminin and hyaluronic acid.
- Korea patent publication No. 2000-0007983 discloses the biological tissue containing solidified collagen layer of complex structure comprising collagen sponge and mesh, and the use thereof.
- Korean patent No. 94-1379 discloses tissue equivalent that is dermal substitute comprising collagen-grid form dermal layer wherein collagen layer was made by constructing fibroblastic cell Additionally IntegraTM (USA) and TerudermisTM (Japan) allowed by FDA and KFDA as medical device are commercially available dermal substitute now. Those are consisting of silicone membrane synthetic polymer and collagen sponge layer. Upper layer is silicone membrane, preventing microbial infection and the evaporation of body fluid and the lower layer is collagen sponge layer, which induces the regeneration of the new blood vessel and the connective tissue, and is degraded and absorbed by itself in the body.
- the treatment for severe burns using such commercialized dermal substitute usually requires 2 steps of grafting procedure; the first step is the formation of new dermis in 2- 3 weeks by grafting dermal substitute on the wound; and the second step is the skin grafting for epidermis formation on the newly formed dermis.
- An above-mentioned conventional grafting procedure accompanies the long-term hospitalization and the pain of the patient, hi order to solve the problems, dermal substitute grafting and autologous split-thickness skin graft/cultured epithelial autografts have been tried simultaneously, however, engraftment(take) rates of these grafts are not good since the present commericial product ; dermal substitute does not have the basement membrane. Therefore, a certain material instead of basement membrane of skin has been need for increasing the rate of skin graftance till now.
- biocompatible dermal substitute which allows one-step grafting procedure by autologous split-thickness skin graft(autograft) or cultured epithelial autografts simultaneous with dermal substitute grafting synchronized with promoting wound healing effect through increasing the rate of the skin graftance and anti-inflammation.
- the present inventors had prepared a reinforced collagen sponge by inserting mesh type of collagen thread into the collagen sponge, disclosed in Korea patent publication No. 2000-7983. The disclosures of which are incorporated herein by references.
- the present inventors have studied on the biocompatible amnion and have found the wound healing effect of amnion that prevents the infection, protects the loss of water, protein, etc. in the wound and facilitates epithelial cell migration to the wound by various kinds of growth and differentiation factors. And also the inventive dermal substitute prepared by attaching the amnion to collagen scaffold, makes it possible that one-step grafting procedure is enough to complete grafting, not conventional two steps required grafting.
- the present inventors have endeavored to develop the bioartif ⁇ cial skin using amnion as a basement membrane and completed the present invention by deveolping inventive dermal substitute comprising amnion and biodegradable polymer.
- the present invention provides the dermal substitute comprising the biodegradable polymer such as collagen and the like and the biomaterial such as amnion obtained from placenta, the preparation method and the use thereof.
- a dermal substitute comprising complex of the biodegradable polymer structure and the biocompatible amnion which have a major role of basement membrane, the preparation method and the use thereof.
- complex herein comprises the attached form, incorporated form or inserted form of biodegradable polymer and amnion.
- biodegradable polymer herein comprises at least one selected from the group consisting of natural materials such as collagen, gelatin, hyaluronic acid and its derivatives, chitin, chitosan, alginate, fibronectin and dextran; synthetic materials such as PLGA (poly(D,L-lactic-co-glycolic acid)), PGA (polyglycolic acid), PLA (poly(lactic acid)) and copolymer analog thereof, poly ⁇ -caprolactone, polyanhydride, polyorthoesters, polyurethane and the like.
- natural materials such as collagen, gelatin, hyaluronic acid and its derivatives, chitin, chitosan, alginate, fibronectin and dextran
- synthetic materials such as PLGA (poly(D,L-lactic-co-glycolic acid)), PGA (polyglycolic acid), PLA (poly(lactic acid)) and copolymer analog thereof, poly ⁇ -cap
- biodegradable polymer herein is preferably in the form of sponge, film, fiber and the like.
- amnion herein can be prepared by procedure comprising the steps: preparing sheet structure using double ring, insert or silicone ring, mesh structure or an amnion extract and subsequent attaching, incorporating or inserting said amnion onto/into biodegradable polymer.
- attached and inserted means that amnion and biodegradable polymer is in physically and closely contacted condition each other or any of them is entered into between layered structures to bind each other.
- incorporated means that one or both of amnion and biodegradable polymers are bound each other by mixing, for example, immersing in other structure.
- the inventive dermal substitute can be prepared by various methods as follows; 1) by attaching the amnion to the biodegradable polymer sponge in the process of polymer structure manufacturing step; 2) by attaching the amnion to the biodegradable polymer sponge after polymer structure manufacturing process comprising cross-linking; or 3) by immersing the biodegradable polymer sponge into amnion extract.
- the present invention provides improved treating and healing effect and the increased convenience to use the product by introducing amnion into inventive dermal substitute, which is attached, incorporated or inserted to/in/into biodegradable polymer substrate, a conventional and artificial substrate.
- the biodegradable polymer of the present invention should be as a backbone to which cells can be attached easily for three-dimensional structure; be immunologically inactive in order not to provoke an inflammatory response or a foreign substance response; be able to react actively with surrounding tissues and induce the neighboring cells to grow/proliferate into the structure of itself; have proper degradation rate since it has to act as a supporting layer not to be degraded quickly by foreign body response after grafting; and be completely degraded and disappeared by itself in a certain period.
- the polymer of the present invention comprises all the materials which can be degraded in the body.
- collagen a kind of structural protein in the body, can be used as a biodegradable polymer.
- the inventive dermal substitute can be prepared by various methods as follows; 1) by attaching the amnion to the biodegradable polymer sponge in the process of polymer structure manufacturing step; 2) by attaching the amnion to the biodegradable polymer sponge after polymer structure manufacturing process comprising cross-linking; or 3) by immersing the biodegradable polymer sponge into amnion extract.
- the present invention provides the method of preparing dermal substitute having complex structure comprising biodegradable polymer and amnion, which is characterized in comprising steps consisting of (step 1) dissolving collagen fiber in acidic solution, preferably acetic acid or pepsin, at the concentration ranging from 0.3 to 1% and pH ranging from 3 to 4; (step 2) spreading the solution prepared in step (1) on the amnion-attached mold (i.e. 12-well plate); (step 3) freezing the mold in the refrigerator at the temperature ranging from -196°C to 0°C and then freeze-drying over 36 hours.
- steps consisting of (step 1) dissolving collagen fiber in acidic solution, preferably acetic acid or pepsin, at the concentration ranging from 0.3 to 1% and pH ranging from 3 to 4; (step 2) spreading the solution prepared in step (1) on the amnion-attached mold (i.e. 12-well plate); (step 3) freezing the mold in the refrigerator at the temperature ranging from -196°C to
- the present invention provides the dermal substitute manufactured by above- described method, having complex structure attaching the amnion to the biodegradable polymer sponge in the process of polymer structure manufacturing step.
- the present invention provides the method of preparing dermal substitute having amnion-collagen sponge complex structure prepared by comprising crosslinking collagen and amnion in conventional crosslinking manner well known in the art.
- conventional crosslinking method comprises 0.25% glutaraldehyde treatment, 33mM 1,3-carbodiimide and 6mM hydroxysuccinimide (dissolved in 90% acetone) treatment, 33mM 1,3-carbodiimide and 6mM hydroxysuccinimide (dissolved in 40% alcohol) treatment, UN and gamma irradiation, other chemical crosslinking methods and so on.
- the present invention provides the method of preparing dermal substitute having complex structure comprising biodegradable polymer and amnion, which is characterized in comprising the steps consisting of (step 1) dissolving collagen fiber in acid solution, preferably acetic acid or pepsin, at the concentration ranging from 0.3 to 1% about pH ranging from 3 to 4; (step 2) spreading the solution prepared in (step 1) on the mold (i.e.
- step 3) freezing the mold in the refrigerator at the temperature ranging from -196°C to 0°C and then freeze-drying over 36 hours;
- step 4) performing DHT(dehydrothermal) crosslinking that comprises the steps consisting of putting collagen sponge into the vaccum oven, maintaining in vacuo at the room temperature for 2 hours and at 110°C for 24 hours and removing vacuum at 30 °C;
- step 5) performing crosslinking collagen according to above conventional crosslinking method repeatedly;
- step 6) coating the crosslinked-collagen sponge or amnion with 0.01% to 0.05% of collagen solution;
- step 7) attaching collagen sponge to amnion;
- step 8) freezing the complex structure in the refrigerator at the temperature ranging from -196°C to 0°C and then freeze-drying over 36 hours.
- the pore size of which aforementioned collagen sponge ranges from 40 to 150 ⁇ m, preferably 60 to 120 ⁇ m.
- the present invention provides the dermal substitute manufactured by above- described method, having complex structure attaching the amnion to the biodegradable polymer sponge after its manufacture process.
- the present invention provides the method of preparing dermal substitute having complex structure comprising biodegradable polymer and amnion, which is characterized in comprising the steps consisting of ;(step 1) dissolving collagen fiber in acid solution, preferably acetic acid or pepsin, at the concentration ranging from 0.3 to 1% about pH ranging from 3 to 4; (step 2) spreading the solution prepared in (step 1) on the mold (i.e.
- step 3) freezing the mold in the refrigerator at the temperature ranging from -196°C to 0°C and then freeze-drying over 36 hours;
- step 4) performing DHT(dehydrothermal) crosslinking that comprises the steps of putting collagen sponge into the vaccum oven, maintaining in vacuo at the room temperature for 2 hours and at 110°C for 24 hours and removing vacuum at 30°C;
- step 5) performing again crosslinking collagen according to above conventional crosslinking method;
- step 6) preparing the amnion extract;
- step 7) immersing the collagen sponge into amnion extract over 12 hours;
- step 8) freezing the complex structure in the refrigerator at the temperature ranging from -196°C to 0°C and then freeze-drying over 36 hours.
- amnion extract of step 6 can be prepared by pulverizing and homogenizing a freeze-dried amnion, centrifuging and collecting the supernatant and then filtrating them.
- inventive amnion-collagen sponge dermal substitute was grafted to intra- stroma of rabbit cornea, it showed more potent anti-inflammatory activity by inhibiting the infiltration of inflammatory cell than in case of the grafting with collagen sponge only. Also in the transplant of the inventive collagen sponge immersed into the amnion extract into the subcutaneous tissue of the back of a nude mouse, it causes less inflammatory response and delayed collagen degradation to provide endurance than control.
- amnion-collagen sponge dermal substitute after ⁇ -heptanol treatment at the limbus of rabbit, it showed anti-inflammatory effect by inhibiting the infiltration of inflammatory cells, differently from the grafting collagen sponge.
- the amnion in the present dermal substitute can be prepared by freezing or freeze- drying treatment, however the EGF (epidermal growth factor) therein are not so much decreased that the inventive dermal substitute can express its wound-healing facilitating activity.
- EGF epidermal growth factor
- the dermal substitute of the present invention using amnion instead of silicone membrane has the good biocompatibility, the high engraftment(take) rates of autologous split-thickness skin graft because of providing basement membrane, the anti- inflammatory activity, and wound-healing facilitating activity so it would be useful for as wound dressing for healing.
- the present invention provides the dermal substitute prepared by culturing cell in amnion-biodegradable polymer complex structure manufactured in accordance to the above-described method.
- cell which can be cultured in the polymer structure comprises at least one selected from the group consisting of fibroblast, keratinocytes, chondrocyte, osteocyte, muscle cell, oral mucosal cell, cornea stem cell and so on.
- the present invention provides the bio-artificial skin, and the preparation thereof, which is characterized in culturing epithelial or epidermal, cells repeatedly on the amnion of bio-artificial dermis obtained from culturing stromal or dermal cells in amnion-biodegradable polymer sponge complex.
- cell which can be cultured in the polymer sponge comprises at least one selected from the group consisting of fibroblast, keratinocytes, chondrocyte, osteocyte, muscle cell, oral mucosal cell, cornea stem cell and so on.
- the inventive dermal substitute as an alternative method can provide one-step grafting procedure to substitute with conventional dermal substitute requiring more than one grafting operation, and thereby it can give the pain relief and cost reduction of hospitalization to the patients.
- the inventive artificial dermal substitute culturing human fibroblast can be used as supporter of bio-artificial skin required in the autologous split-thickness skin grafting or in the grafting of cultured epithelial cell from autologous/allogenic organism.
- the dermal substitute of the present invention can be used as substrate for the preparation of various artificial organs such as artificial skin, artificial cornea, artificial cartilage, artificial bone and artificial muscle etc.
- Fig. 1 shows the scanning electron microscopy of porous collagen sponge
- Fig. la is the sectional view thereof
- Fig. lb is the bottom view thereof;
- Fig. 2 shows the scanning electron microscopy of inventive amnion-collagen sponge complex structure
- Fig. 2a is the attached part between amnion and collagen sponge of inventive dermal substitute prepared in Example 1
- Fig. 2b is that of inventive dermal substitute prepared in Example 2;
- Fig. 3 a is the picture of H&E stained tissue section observed at 1 month after transplanting only collagen sponge in the intra-stroma of rabbit cornea
- Fig. 3b is the picture of infiltrated inflammatory cells around the implant
- Fig. 3 c is the picture of H&E stained tissue section observed at 1 month after transplanting inventive amnion- collagen sponge complex in the intra-stroma of rabbit cornea
- Fig. 4a is the picture of H&E stained tissue section observed at 1 week after transplanting only collage sponge in the back of nude mouse
- Fig. 4b is the picture of H&E stained tissue section observed at 1 week after transplanting inventive dermal substitute, prepared by immersing collagen sponge into amnion extract, in the back of nude mouse;
- Fig. 5 a is the picture of H&E stained tissue section observed on 10 days after transplanting TerudermisTM in the full-thickness defected skin wound on the back of guinea pig
- Fig. 5b is the picture of H&E stained tissue section observed on 10 days after transplanting inventive amnion-collagen complex in the full-thickness defected skin wound on the back of guinea pig;
- Fig. 6 shows the pictures on the 3, 7 and 30 days after transplant to the limbus of rabbit cornea, induced alkali-burns injury by N-heptanol (Fig. ⁇ a, 6b and 6c; collagen sponge, Fig. 6d, 6e, 6f; inventive amnion-collagen sponge complex);
- Fig. 7 shows the H&E stained tissue sections at 7 days after transplanting in the full- thickness wound by surgical excision on the back of New Zealand white rabbit;
- Fig. 7a is for only collagen sponge
- Fig. 7b is for collagen sponge and autologous split- thickness skin graft simultaneously
- Fig. 7c is for inventive amnion-collagen sponge complex and autologous split-thickness skin graft simultaneously;
- Fig. 8 is the standard curve of EGF presenting the relation of EGF amount and absorbance
- Fig. 9 is the immunohistochemical staining picture of basement membrane component of amnion; Fig. 9a is for EGF-receptor in amnion; and Fig. 9b is for collagen type IV in amnion; Fig. 10 is the immunohistochemical analysis of bio-artificial skin for cytokeratin.
- the placenta was serologically screened for guaranteeing its safety according to generally applicable rules of tissue bank.
- the amnion separated from normal placenta was stored in 400 mi of sterile saline solution (0.9% NaCl) at 4°C.
- the amnion was washed 4 times for 10 mins with gentle shaking and was transferred to the fresh sterile saline solution to store at 4 °C overnight.
- the sponge layer of amnion hydrated was removed. Remaining amnion was washed 4 times with sterile saline for 10 mins and was prepared in the form of sheet or mesh to attach to collagen sponge. Mesh form thereof was manufactured by mesher or by perforating artificially.
- the amnion extract was prepared by following procedure. The washed amnion of
- Reference Example 1 was fast-frozen in liquid nitrogen and then was crushed with a mortar and pestle. The crushed substance was homogenized and centrifuged at 6000 rpm for 30 mins. The supernatant thereof was filtered with ultrafiltration membrane (Centrikon Co.) to obtain the amnion extract used in the following experiment.
- 0.5% collagen (Matrixen-ASP, Bioland Ltd.) was prepared by dissolving in acetic acid or pepsin and was adjusted to pH 3.0. Collagen solution was vortexed by a homogenizer (Bead Beater, BioSpec Co.) at 1500rpm for 5mins. The 1.5ml of cream type collagen solution was spread on the amnion-attached mold, i.e., 12-well plate and the complex structure was frozen in the refrigerator at the temperature ranging from - 196°C to 0°C and then freeze-dried over 36 hours.
- a homogenizer Bead Beater, BioSpec Co.
- the above-prepared amnion- collagen sponge complex structure was subjected to the conventional crosslinking procedure treated with 0.25% glutaraldehyde.
- Example 2 Amnion-Collagen sponge complex preparation 2 0.5% collagen (Matrixen-ASP, Bioland Ltd.) was prepared by dissolving in acetic acid or pepsin and adjusted to pH 3.0. Collagen solution was vortexed by a homogenizer at 1500rpm for 5mins. The 1.5ml of cream type collagen solution was spread on the amnion-attached mold, i.e., 12-well plate and the complex structure was frozen in the refrigerator at the temperature ranging from -196°C to 0°C and then freeze-dried over 36 hours.
- Amnion-Collagen sponge complex preparation 2 0.5% collagen (Matrixen-ASP, Bioland Ltd.) was prepared by dissolving in acetic acid or pepsin and adjusted to pH 3.0. Collagen solution was vortexed by a homogenizer at 1500rpm for 5mins. The 1.5ml of cream type collagen solution was spread on the amnion-attached mold, i.e., 12-well plate and
- Freeze-dried porous collagen sponge was put into the vaccum oven maintaining in vacuo at the room temperature for 2 hours to remove a trace amount of water and subsequently vacuum was sustained at the temperature up to 110°C for 24 hours. After 24 hours, it was cooled down at 30 °C and the vacuum was removed.
- DHT dehydrothermal treatment
- the collagen was crosslinked by conventional crosslinking method using 0.25% glutaraldehyde or 1,3- carbodiimide and then surface of collagen sponge was coated with 0.05% collagen solution.
- Reference Example 1 The amnion of Reference Example 1 was attached thereon, at least one side of the collagen sponge and the complex structure was frozen in the refrigerator at the temperature ranging from -196°C to 0°C and then freeze-dried over 36 hours.
- Fig. la and Fig. lb represents the bottom and sectional view of conventionally manufactured-collagen sponge, respectively, observed by SEM (scanning electron microscope).
- the pore size thereof ranges from about 60 ⁇ m to 120jt_n.
- Fig. 2a and Fig. 2b shows the attached forms between amnion and collagen sponge in complex structures prepared in Example 1 and Example 2 of the present invention, respectively.
- the inventors carried out implantation of collagen sponge and inventive amnion-collagen sponge complex structure in the intrastroma of rabbit cornea and at 1 month after grafting, the tissue including graft was biopsied and subjected to the general procedures for histological observation; i.e.,fixed in formalin, washed ⁇ embedded in paraffin, sectioned with a 5/-H1 thickness, stained with H&E and examined under a light microscope.
- Fig. 3a depicts the picture of collagen sponge implanted in the intrastroma of rabbit cornea;
- Fig. 3b shows the ongoing infiltration of inflammatory cells around the implant at day 30 post-grafting and
- Fig. 3c shows that the inventive amnion-collagen sponge complex structure has the anti-inflammatory activity due to inhibiting activity from the infiltration of inflammatory cell.
- the collagen sponge was immersed in the amnion extract prepared in above Reference Example 2 for 24 hours and then used.
- Fig. 4a presents the picture of collagen sponge-transplanted tissue
- Fig. 4b depicts the picture of collagen sponge immersed in amnion extract, transplanted tissue, which leads less inflammatory response and maintains its original form without degradation after a week.
- guinea pig (Samtaco Co.) was injected intramuscularly with ketamine hydrochlonde at a dose of 100 mg/kg bodyweight for anesthesia. The hair of the back was removed with electronic shaver. The back of pigs was washed and 10% povidone-iodine was applied thereon followed by swabbing with 70% ethanol.
- Fig. 5a and Fig. 5b showed the pictures of histological observation of Terudermis- transplanted one and inventive dermal substitute-transplanted one, respectively, 10 days after transplant. In both of them, the new blood vessel formation and fibroblast proliferation was observed and the collagen synthesis was augmented.
- keratinocyte migration under silicone membrane was not observed in Fig. 5 a for control, but the cell migration was detected in Fig. 5b for experimental group using inventive dermal substitute with amnion, which brought fast re- epithalization of epidermis.
- Fig. 6a, 6b and 6c are the pictures of collagen sponge-transplanted tissue at day 3, day 7 and day 30, respectively.
- Fig. 6d, 6e and 6f are the pictures of inventive amnion-collagen sponge complex- transplanted tissue at day 3, day 7 and day 30, respectively. Comparing with collagen sponge-transplanted tissue, amnion-collagen sponge complex suppressed over- growth of blood vessel as corneal injury at 30 th day after transplant and was less degraded.
- Collagen sponge was transplanted on the one side of wound, followed by transplanting 0.25mm thickness of autograft using dermatome and inventive amnion- collagen sponge complex of Example 2 was transplanted on another side of wound, followed by same autografting procedure as described above.
- the criteria evaluating wound healing includes skin graftance, inflammatory cell number, new vascularization and fibroblast proliferation under light microscope.
- the evaluation results were classified into 4 groups, i.e., +, ++, +++ and ++++.
- Fig. 7a shows the control transplanted with only collagen sponge
- Fig. 7b shows the group transplanted with collagen sponge and autograft simultaneously
- Fig. 7c shows the group transplanted with inventive amnion-collagen sponge complex and autograft simultaneously.
- the inventive amnion-collagen sponge complex showed the excellent engraftment of transplated epidermis, however, epidermis in the group transplanted with collagen sponge and autograft was partially sloughed off.
- the fibroblast and blood vessel endothelial cell was actively infiltrated into complex structure in case that the inventive dermal substitute and autograft were simultaneously transplanted.
- the amnion was prepared from the placenta obtained by Cesarean section in 12 hours.
- the amnion was incubated in TSA medium (Tryptic soybean casein digest medium) for at least 7 days at 37 °C in order to determine whether microorganism remains or not.
- the guaranteed amnion was washed 4 times with about 400ml sterile saline(0.9% NaCl) for 10 mins with shaking.
- the washed amnion was stored in fresh sterile saline overnight at 4°C. Thereby, the hydrated amnion sponge layer was eliminated and the amnion was washed again 4 times with about 400ml sterile saline(0.9% NaCl) for 10 mins with shaking.
- the amnion prepared by the procedure disclosed in above was frozen and freeze- dried prior to the extraction with PBS to obtain the amnion extract.
- the amnion extract was centrifuged for 5 mins at 15,000 rpm and the supernatant thereof was used to measure the amount of epidermal growth factor (EGF) using EGF detection kit (Asan pharmaceuticals Co.).
- EGF epidermal growth factor
- EGF-R EGF receptor
- collagen type IN a major component of basal lamina
- immunohistochemical staining was performed with EGF-R antibody (DAKO Co., K0675) and collagen type IN antibody ( ⁇ eoMarkers Co., MS- 747-S1) according to immuno-peroxidase method well known in the art.
- EGF-R antibody DAKO Co., K0675
- collagen type IN antibody ⁇ eoMarkers Co., MS- 747-S1
- Example 2 was laid on the 3.5 cm -diameter cell culture dish. ⁇ 3xl0 6 dermal cells/well were seeded thereon with 10% FBS DMEM media and incubated in a 5% CO 2 incubator at 37°C. 5 hours after seeding, 2ml of medium was added thereto and the medium was replaced with fresh one. After then, the cells were incubated for 7 days with changing medium every 2 days.
- Keratinocytes were multi-layer cultured on basement membrane of amnion of artificial dermis prepared in Example 3-1 to manufacture an artificial skin.
- Fig. 10 shows the picture of immunohistochemical analysis of inventive full thickness bio-artificial skin.
- the cytokeratin expressed in keratinocyte differentiation was determined according to the immuno-peroxidase method using cytokeratin antibody (Biomedical Technologies).
- the present invention using amnion has several advantages, i.e., better biocompatibility, anti-inflammatory activity, durability due to delayed rate of biodegradability and wound healing facilitating activity functioned by various growth factors and basal lamina components, compared with conventional dermal substitute comprising silicone membrane and collagen sponge.
- inventive dermal substitute can allows the one-step skin graft procedure by providing with the basement membrane like role of amnion attached collagen sponge as an alternative method of conventional dermal substitute requiring two separate grafting procedure, which can give the pain relief and cost reduction of hospitalization to the patients.
- the present invention provides with a supporter of bio-artificial skin comprising basement membrane for the split-thickness graft or for the grafting of cultured epithelial cell originated from autologous/allogenic organism.
- the dermal substitute of the present invention comprising the amnion and biodegradable polymer can be useful as a basic matrix for the preparation of various artificial organs such as artificial skin, artificial cornea, artificial cartilage, artificial bone, artificial muscle etc.
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Abstract
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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AU2003265103A AU2003265103A1 (en) | 2002-09-30 | 2003-09-30 | Dermal substitute consisting of amnion and biodegradable polymer, the preparation method and the use thereof |
US11/019,441 US20050107876A1 (en) | 2002-09-30 | 2004-12-21 | Dermal substitute consisting of amnion and biodegradable polymer, the preparation method and the use thereof |
Applications Claiming Priority (4)
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KR10-2002-0059308 | 2002-09-30 | ||
KR20020059308 | 2002-09-30 | ||
KR10-2003-0064761 | 2003-09-18 | ||
KR1020030064761A KR100644078B1 (ko) | 2002-09-30 | 2003-09-18 | 양막과 생분해성 고분자로 구성된 이식용 진피대체물,이의 제조방법 및 용도 |
Related Child Applications (1)
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US11/019,441 Continuation US20050107876A1 (en) | 2002-09-30 | 2004-12-21 | Dermal substitute consisting of amnion and biodegradable polymer, the preparation method and the use thereof |
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WO2004028584A1 true WO2004028584A1 (fr) | 2004-04-08 |
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PCT/KR2003/002012 WO2004028584A1 (fr) | 2002-09-30 | 2003-09-30 | Substitut dermique compose d'amnios et d'un polymere biodegradable, procede de preparation et utilisation de ce substitut |
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Cited By (23)
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WO2006002128A1 (fr) * | 2004-06-23 | 2006-01-05 | Minu, L.L.C. | Utilisation de membranes amniotiques en tant que dispositifs biocompatibles |
FR2892311A1 (fr) * | 2005-10-24 | 2007-04-27 | Sang Etablissement Public A Ca | Pansement pour la cicatrisation des plaies, ulceres ou brulures des epitheliums, endotheliums ou muqueuses. |
US7824711B2 (en) | 2003-12-11 | 2010-11-02 | Isto Technologies, Inc. | Particulate cartilage system |
US20110293691A1 (en) * | 2010-04-28 | 2011-12-01 | Weber Kurt R | Multimodal adhesion barrier |
US8480757B2 (en) | 2005-08-26 | 2013-07-09 | Zimmer, Inc. | Implants and methods for repair, replacement and treatment of disease |
US8497121B2 (en) | 2006-12-20 | 2013-07-30 | Zimmer Orthobiologics, Inc. | Method of obtaining viable small tissue particles and use for tissue repair |
US8580289B2 (en) | 2004-07-12 | 2013-11-12 | Isto Technologies Inc. | Tissue matrix system |
CN103893831A (zh) * | 2012-12-28 | 2014-07-02 | 广州暨南大学医药生物技术研究开发中心 | 一种器官型人工皮肤、其制备方法及应用 |
US9138318B2 (en) | 2007-04-12 | 2015-09-22 | Zimmer, Inc. | Apparatus for forming an implant |
WO2015175752A1 (fr) * | 2014-05-14 | 2015-11-19 | NuTech Spine, Inc. | Traitement de soin de plaie et ses procédés de fabrication et d'utilisation |
US10167447B2 (en) | 2012-12-21 | 2019-01-01 | Zimmer, Inc. | Supports and methods for promoting integration of cartilage tissue explants |
US10179191B2 (en) | 2014-10-09 | 2019-01-15 | Isto Technologies Ii, Llc | Flexible tissue matrix and methods for joint repair |
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US10245306B2 (en) | 2012-11-16 | 2019-04-02 | Isto Technologies Ii, Llc | Flexible tissue matrix and methods for joint repair |
WO2019125962A1 (fr) * | 2017-12-20 | 2019-06-27 | Kci Licensing, Inc. | Pansement pour plaies pour la récolte de greffes épidermiques superficielles |
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US10912861B2 (en) | 2015-04-09 | 2021-02-09 | Kci Licensing, Inc. | Soft-tack, porous substrates for harvesting skin grafts |
US11006974B2 (en) | 2015-11-03 | 2021-05-18 | Kci Licensing, Inc. | Devices for creating an epidermal graft sheet |
US11083487B2 (en) | 2010-08-06 | 2021-08-10 | Kci Licensing, Inc. | Methods for preparing a skin graft |
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US11413372B2 (en) | 2015-09-17 | 2022-08-16 | Stimlabs Llc | Compositions derived from placenta and methods of producing the same |
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US8580289B2 (en) | 2004-07-12 | 2013-11-12 | Isto Technologies Inc. | Tissue matrix system |
US8480757B2 (en) | 2005-08-26 | 2013-07-09 | Zimmer, Inc. | Implants and methods for repair, replacement and treatment of disease |
FR2892311A1 (fr) * | 2005-10-24 | 2007-04-27 | Sang Etablissement Public A Ca | Pansement pour la cicatrisation des plaies, ulceres ou brulures des epitheliums, endotheliums ou muqueuses. |
US8497121B2 (en) | 2006-12-20 | 2013-07-30 | Zimmer Orthobiologics, Inc. | Method of obtaining viable small tissue particles and use for tissue repair |
US9138318B2 (en) | 2007-04-12 | 2015-09-22 | Zimmer, Inc. | Apparatus for forming an implant |
US20110293691A1 (en) * | 2010-04-28 | 2011-12-01 | Weber Kurt R | Multimodal adhesion barrier |
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US10167447B2 (en) | 2012-12-21 | 2019-01-01 | Zimmer, Inc. | Supports and methods for promoting integration of cartilage tissue explants |
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