BIOLOGICAL SCAFFOLD AND METHOD FOR FABRICATING THE SAME

Abstract

A biological scaffold in the present invention comprises a main body, a biological material layer, and an optional tissue adhesive layer. The main body at least has a non-constituted collagen matrix. The biological material layer is coated at least on a surface of the main body, and the tissue adhesive layer is disposed at least on another surface of the main body. When the biological scaffold is adhered to a tissue through the tissue adhesive layer, a plurality of cells move from the tissue to either the adhesive layer or the biological material layer for tissue repairing or regeneration.

Claims

1. A biological scaffold, comprising: a main body including a de-antigenic, non-reconstituted collagen matrix, wherein the non-reconstituted collagen matrix contains a plurality of bundled collagen fibrils that are interwoven and overlapped and a plurality of pores formed by the bundled collagen fibrils; and a biological material coating a first outer surface and inside the plurality of pores of the main body, optionally, the biological material substantially coats the entire surface and inside of the main body.

2. The biological scaffold of claim 1, wherein the biological material is selected from the group consisting of a cell attachment material, a tissue repair material, a cell induction material, a growth factor material, an antibacterial material, and a combination thereof.

3. The biological scaffold of claim 2, wherein: the antibacterial material is an antibiotic, an antimicrobial protein, an antimicrobial peptide, or a combination thereof; the cell attachment material is a saccharide, a peptide, a protein, a phospholipid, or a combination thereof, optionally, the saccharide material is a glycosaminoglycan material; the growth factor material is epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), hepatocyte growth factor (HGF), colony-stimulating factor (CSF), stem cell factor (SCF), keratinocyte growth factor (KGF), granulocyte colony-stimulating factor (GCSF), macrophage colony-stimulating factor (MCSF), granulocyte macrophage colony-stimulating factor (GMCSF), glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial-monocyte activating polypeptide, epithelial neutrophil activating peptide, erythropoietin, bone morphogenetic protein, brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factor, interleukin, tumor necrosis factor (TNF), or a combination thereof; the cell induction material is a vitamin, mineral, chemical, medicine, herbal medicine, metabolite, intermediate metabolite, saccharide, peptide, protein, phospholipid, or a combination thereof; or the tissue repair material is a biomaterial, an extracellular matrix, a nutrient, or a combination thereof; optionally, the biomaterial is collagen, gelatin, hyaluronan, elastin, glycosaminoglycan, chitosan, alginate, polyglutamic acid (γ-PGA), polylysine, poly(lactic-co-glycolic acid) (PLGA), silk fibroin, polyamino acid, cellulose and its derivatives, or a combination thereof; optionally, the extracellular matrix is collagen, gelatin, elastin, glycosaminoglycan, proteoglycan, glycoprotein, fibronectin, laminin, aggrecan, metalloproteinase, or a combination thereof.

4-12. (canceled)

13. The biological scaffold of claim 1, further comprising cells in the plurality of the pores in the main body, optionally, the cells are stem cells, satellite cells, progenitor cells, precursor cells, or tissue cells.

14-15. (canceled)

16. The biological scaffold of claim 1, further comprising a tissue adhesive layer coated on a second surface of the main body.

17. The biological scaffold of claim 16, wherein the tissue adhesive layer includes: a cross-linking agent having at least a functional group or a bifunctional group; and a quenching agent for reacting with any excess functional group of the crosslinking agent that has not reacted.

18. The biological scaffold of claim 17, wherein the cross-linking agent is transglutaminase or has a functional group selected from the group consisting of amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidoester, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyldithiol, hydrazide, photo-reacting, carbodiimide, diazirine, aziridine, acryloyl, arylate, thiol, genipin, riboflavin, flavonoid and its derivatives, hydroxymethyl phosphine, isocyanate, maleimide, 6-maleimidohexanoic acid active ester, disuccinimidyl suberate, bis(sulfosuccinimidyl)suberate, N-hydroxy-succinimide ester (NHS-ester), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, pentafluorophenyl ester (PFP-ester), ethylene glycol diglycidyl ether, glutaraldehyde, 2,3-dibromopropionyl N-hydroxysuccinimide ester, sulfo-N-hydroxysuccinimide ester, chlorambucil-N-hydroxysuccinimide ester, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, psoralen, vinyl sulfone, and a combination thereof.

19. The biological scaffold of claim 17, wherein the tissue adhesive layer further includes a matrix molecule crosslinked with the cross-linking agent, and wherein the matrix molecule is selected from the group consisting of collagen, hyaluronan, gelatin, silk protein, fibroin, fibronectin, elastin, tenascin, laminin, vitronectin, heparan sulfate, chondroitin, chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, alginate, aggrecan, agarose, agar, cellulose, methyl cellulose, carboxyl methyl cellulose, glycogen, fibrin, fibrinogen, thrombin, polyglutamic acid, polylysine, polyamino acid, synthetic polymers, a derivative thereof, and a combination thereof.

20. The biological scaffold of claim 17, wherein the matrix molecule is collagen in the main body.

21. The biological scaffold of claim 17, wherein the quenching agent is selected from the group consisting of amino acid, oligopeptide, polypeptide, protein, amine, diamine, oligoamine, polyamine, carbonyl compound, glycol compound, carboxyl compound, dicarboxylate, oligo-carboxylate, polycarboxylate, sulfhydryl compound, oligosulfhydryl compound, polysulfhydryl compound, hydroxyl compound, oligohydroxyl compound, polyhydroxyl compound, saccharide, oligosaccharide, polysaccharides, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), oligonucleotide, azide, photo-crosslinking compound a monofunctional or heterobifunctional group, and a combination thereof.

22. The biological scaffold of claim 1, wherein the non-reconstituted collagen matrix is prepared by a procedure including: obtaining a layer sheet of connective tissue; and carrying out a swelling step that includes immersing the layer sheet in a first acidic solution for a sufficient period of time to let the layer sheet to form a swelled layer sheet, whereby forming a non-reconstituted collagen matrix.

23. The biological scaffold of claim 22, wherein the layer sheet is a dermal layer sheet obtain from an animal skin tissue, and wherein the procedure further includes, before the swelling step, carrying out a depilating step that includes immersing the dermal layer sheet in a high ionic strength salt solution and then adding a proteolytic enzyme to the salt solution, whereby a depilated layer sheet is produced.

24. A method of preparing a non-constituted collagen matrix, comprising: obtaining a layer sheet of connective tissue; and carrying out a swelling step that includes immersing the layer sheet in a first acidic solution for a sufficient period of time to let the layer sheet to form a swelled sheet layer, whereby forming a non-reconstituted collagen matrix, the non-reconstituted collagen matrix having a plurality of bundled collagen fibrils that are interwoven and overlapped and a plurality of pores formed by the bundled collagen fibrils; optionally, the layer sheet is a dermal layer sheet obtain from an animal skin tissue, and the method further includes, before the swelling step, carrying out a depilating step that includes immersing the dermal layer sheet in a high ionic strength salt solution and then adding a proteolytic enzyme to the salt solution, whereby a depilated layer sheet is produced.

25. The method of claim 24, wherein the ionic strength of the salt solution is greater than 0.15 N, optionally, the ionic strength is between 0.5 N and 10 N.

26. (canceled)

27. The method of claim 24, wherein the salt solution is a phosphate solution containing sodium chloride, ammonium sulfate, or a mixture thereof.

28. The method of claim 24, wherein the proteolytic enzyme is selected from the group consisting of a serine protease, threonine protease, cysteine protease, aspartate protease, glutamate protease, metalloprotease, and a mixture thereof.

29. The method of claim 24, further comprising a disinfection step or a degreasing step before or after the depilation step.

30. (canceled)

31. The method of claim 24, further comprising, after the swelling step, a de-antigen step that includes immersing the non-reconstituted collagen matrix in a second acidic solution containing pepsin.

32. The method of any of claim 24, further comprising, after the swelling step, a strengthening step that includes immersing the swelled dermal sheet layer in an additive.

33. A method for preparing a biological scaffold, comprising: providing a non-reconstituted collagen matrix prepared by a procedure including: obtaining a layer sheet of connective tissue; and carrying out a swelling step that includes immersing the layer sheet in a first acidic solution for a sufficient period of time to let the layer sheet to form a swelled dermal sheet layer, whereby forming a non-reconstituted collagen matrix, the non-reconstituted collagen matrix having a plurality of bundled collagen fibrils that are interwoven and overlapped and a plurality of pores formed by the bundled collagen fibrils; optionally, the procedure further includes a disinfection step, a degreasing step, a de-antigen step, or a strengthening step; optionally, the layer sheet is a dermal layer sheet obtain from an animal skin tissue, and the procedure further includes, before the swelling step, carrying out a depilating step that includes immersing the dermal layer sheet in a high ionic strength salt solution and then adding a proteolytic enzyme to the salt solution, whereby a depilated layer sheet is produced; contacting the non-reconstituted collagen matrix with a solution containing a biological material under a decompression environment, whereby the biological material is coated on a first outer surface and inside the plurality of pores of the non-reconstituted collagen matrix.

34-38. (canceled)

Description

BRIEF DESCRIPTION OF SCHEMA/FIGURE

[0055] FIG. 1 is a schematic cross-sectional view showing a non-reconstituted biological scaffold in an embodiment of the present invention.

[0056] FIG. 2 is a schematic cross-sectional view showing the main body of a non-reconstituted biological scaffold in an embodiment of the present invention.

[0057] FIG. 3 shows a 100× magnified electron microscope image of a conventional reconstituted porous collagen matrix.

[0058] FIG. 4 shows a flow chart for preparation of a non-reconstituted collagen matrix of the present invention.

[0059] FIG. 5 shows another flow chart for preparation of a non-reconstituted collagen matrix of the present invention.

[0060] FIGS. 6A to 6C show electron microscope images of a non-reconstituted collagen matrix prepared by the method of the present invention.

[0061] FIGS. 7A to 7B show electron microscope images of a biological scaffold from an embodiment of the present invention.

[0062] FIG. 7C demonstrates alcian blue staining indicating well-distribution of hyaluronan inside the biological scaffold.

[0063] FIG. 8 shows a comparison diagram of a shear stress test between biological scaffolds from an embodiment of the present invention and from a conventional technique.

[0064] FIGS. 9A and 9B show the storage and loss modulus of a non-reconstituted porous collagen matrix of the present invention and a reconstituted porous collagen matrix from a conventional technique.

[0065] FIG. 10A is a comparison diagram showing the healing of wounds of a biological scaffold in present invention and a reconstituted porous collagen-chondroitin sulfate matrix prepared by a conventional method.

[0066] FIG. 10B is a graph showing healing of wounds under different treatments.

[0067] FIG. 11 is a set of microscopy images showing comparison of the wound healing status at day 28 post-operation. (A) normal skin control, (B) healed matrix of PCHM group, (C) healed matrix of Integra group.

[0068] FIG. 12 is a flowchart showing a method for preparing a biological scaffold in an embodiment of the present invention.

[0069] FIGS. 13A to 13B are schematic cross-sectional view and partial enlargement showing a biological scaffold according to another embodiment of the present invention.

[0070] FIGS. 14A to 14C are comparison diagrams of water absorption capacity of biological scaffolds having different extent of biological material coating in an embodiment of the present invention.

[0071] FIGS. 15A to 15D show the antibacterial effect of the biological scaffolds covered with different antibacterial materials in an embodiment of the present invention.

DETAILED DESCRIPTION

[0072] Referring to FIG. 1, the present invention provides a non-reconstituted material biological scaffold 1 which includes: a main body 10, a biological material layer 20, and a tissue adhesive layer 30. The main body 10 includes at least one non-reconstituted collagen matrix 10a. The biological material layer 20 and the tissue adhesive layer 30 are coated on one surface of the body and the adjacent or opposite surface of the body respectively. When the above-mentioned biological scaffold is adhered to a tissue 2 by the tissue adhesive layer, a plurality of cells are chemoattracted by the biological material and moved from the tissue adhesive layer to the biological material layer, and reconstruction of the tissue 2 is performed.

[0073] Referring to FIG. 2, which shows a schematic cross-sectional view of a main body 10 of a biological scaffold in an embodiment of the present invention. As shown in the figure, main body 10 is preferably a non-reconstituted collagen matrix 10a, and this collagen matrix 10a is composed of a plurality of columnar collagen fibrils and includes a plurality of pores 10b structured by collagen fibrils. A biological material 10c is coated on the inner surface of pores 10b.

[0074] Referring to FIG. 4, a wound dressing prepared from a biological scaffold with a non-reconstituted collagen matrix of the present invention comprises: firstly, as shown in step S100, providing an animal tissue or an animal skin tissue, such as pig skin, cowhide, or sheepskin tissue, in an environment below 15° C., removing the subcutaneous fat of the animal skin tissue by a sharpener, and cutting the tissue to a size of 28±1 cm×70±1 cm. Next, the animal skin tissue was depilated and the oil and moisture remaining thereon were scraped off, and each skin tissue was cut into 4 pieces with each size at 13±1 cm×34±1 cm. Then, the dermal layer of the pig skin is peeled by a machine to obtain at least one dermal layer sheet, and then rinsed with pure water. Most preferably, the dermal layer is taken from pig skin.

[0075] Furthermore, as shown in step S102, a depilation treatment step is performed by immersing the dermal layer sheet in a salt solution containing high ionic strength, and after adding a proteolytic enzyme, heating the salt solution to 25-40° C. An ultrasonic oscillating treatment is then carried out for two hours. A phosphate buffered solution is used as a washing solution to clean the dermal layer sheet after the depilation treatment. A salt solution containing high ionic strength is a salt solution in which anion and cation are ionically bonded. The ionic strength of the salt solution containing high ionic strength is greater than 0.15 N. Most preferably, the ionic strength of the salt solution containing high ionic strength is between 0.5 N and 10 N. Most preferably, the salt solution containing high ionic strength is a solution containing a sodium chloride, ammonium sulfate, or a mixture of the above compounds.

[0076] In one embodiment, the salt solution containing high ionic strength is a phosphate buffered solution containing sodium chloride. This high salt condition stabilizes the structure of the non-reconstituted collagen matrix, does not cause the tissue to swell and is extruded into the hair follicle, so that the enzyme can smoothly enter the hair follicle for reaction.

[0077] In one embodiment, the proteolytic enzyme acts on the salt solution containing high ionic strength. Most preferably, the proteolytic enzyme is selected from the group consisting of serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamate proteases, metalloproteases, and a combination of the above enzymes. Most preferably, the cysteine protease is selected from the group consisting of papaya enzymes, pineapple enzymes, and mixture of the above enzymes. Most preferably, the metalloprotease is dispase.

[0078] Referring to Table 1 below, when the dermal layer sheet was immersed with papain (a papaya enzyme) in a phosphate buffered solution containing ˜5 mM or 0.16% (w/w) L-cysteine, and the concentration of papain was higher than 0.6 mg/mL (groups B, C, and D), the pig hairs were completely removed. When the dermal layer sheet was immersed with bromelain (a pineapple enzyme) in a phosphate buffered solution, and when the bromelain concentration was 0.2 mg/mL and 0.6 mg/mL (groups A and B), there was still pig hair that had not fallen off. When the concentration of bromelain was 1.2 mg/mL, the pig hairs were completely removed. The pig follicles could also be removed completely with more than 0.2 U/mL of dispase. As for the pigs in the control group (phosphate buffer only), all pig hair did not fall off.

TABLE-US-00001 TABLE 1 The dermal layer sheet (groups A-D) immersed in a phosphate buffered solution containing papain, bromelain, or dispase. Groups Concentration A B C D Papain (mg/mL) 0.2 0.6 1.2 2.4 Bromelain (mg/mL) 0.2 0.6 1.2 2.4 Dispase (U/mL) 0.2 0.6 1.2 2.4

[0079] Referring to FIG. 4, as shown in step S104, a swelling treatment step is performed, in which the dermal layer sheet after depilation treatment is immersed in a first acidic solution, and the first acidic solution is heated to 33-37° C. with an ultrasonic oscillating treatment. The ultrasonic oscillating treatment time is continuous for 24 hours, so that the volume of the dermal layer sheet is expanded by at least fifty percent. Preferably, the first acidic solution is a weak acid solution selected from the group consisting of formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid, hydrochloric acid, and a mixture thereof. Most preferably, the first acidic solution is an acetic acid solution.

[0080] As shown in step S105, a washing treatment step is performed to remove non-collagenous material from the dermal layer sheet and a purified collagen matrix is obtained. This follows with freezing the purified collagen matrix at −20° C. and −80° C., and a freeze-drying or lyophilization process to obtain a purified non-reconstituted porous collagen matrix.

[0081] Referring to FIG. 5, which shows another embodiment of a preparation method of the non-reconstituted collagen matrix of the present invention, comprising: as shown in step S101a, a disinfection treatment before or after performing any step, in which the sheet or the dermal layer sheet is washed with a disinfecting solution. Most preferably, the disinfecting solution is a peracetic acid solution.

[0082] As shown in step S102a, the preparation method of the present invention further comprises a degreasing step before or after the depilation treatment step S102. The degreasing step is a saponification treatment, an organic solvent treatment, or a combination thereof. The saponification step is to contact the sheet or the dermal layer sheet with an alkaline substance, and one specific surface can be contacted if only one surface is greasing. Most preferably, the alkaline substance is in the form of granules of strong base particle. Most preferably, the strong base particles are sodium hydroxide particles. The organic solvent treatment is an alcohol treatment, a hexane treatment, or a chloroform treatment. Nevertheless, optimal amount and time period of treatment are required to prevent denaturation of collagen in the matrix.

[0083] As shown in step S104a, the preparation method of the present invention further comprises a de-antigen treatment step after the swelling treatment step S104. Most preferably, the de-antigen treatment step is to immerse the sheet or dermal layer sheet in a second acidic solution containing pepsin. The second acidic solution containing pepsin is heated to between 33° C. and 37° C. An ultrasonic oscillation treatment is then carried out for 4 hours. The solution remaining on the sheet or the dermal layer sheet is washed away with pure water. The sheet or dermal layer sheet is treated again with a potassium dihydrogen phosphate solution at 25° C. Preferably, the second acidic solution is a weak acid solution selected from the group consisting of formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid, hydrochloric acid, and mixtures thereof. Most preferably, the second acidic solution is an acetic acid solution.

[0084] As shown in step S104b, the preparation method of the present invention further comprises a strengthening treatment step after the swelling treatment step, wherein a plurality of sheets or dermal layer sheets are laminated and immersed in an additive, preferably a crosslinking agent solution. The crosslinking agent solution is heated to between 33° C. and 37° C., and an ultrasonic oscillation treatment is performed for 24 hours to complete the crosslinking reaction. The remaining solution on the sheets or dermal layer sheets is rinsed off with pure water. Most preferably, one functional group of the cross-linking agent is selected from the group consisting of amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidoester, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyldithiol, hydrazide, photo-reacting, carbodiimide, diazirine, aziridine, acryloyl, arylate, thiol, genipin, riboflavin, flavonoid and its derivatives, hydroxymethyl phosphine, isocyanate, maleimide, 6-maleimidohexanoic acid active ester, disuccinimidyl suberate, bis(sulfosuccinimidyl)suberate, N-hydroxy-succinimide ester (NHS-ester), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, pentafluorophenyl ester (PFP-ester), ethylene glycol diglycidyl ether, glutaraldehyde, 2,3-dibromopropionyl N-hydroxysuccinimide ester, sulfo-N-hydroxysuccinimide ester, chlorambucil-N-hydroxysuccinimide ester, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, psoralen, vinyl sulfone, and a combination thereof.

[0085] Referring to FIGS. 6A to 6C, which show the appearance of the non-reconstituted collagen matrix prepared according to the method described above. FIG. 6A is a 100× magnification image of a scanning electron microscope of a non-reconstituted collagen matrix in an embodiment of the present invention. FIG. 6B is a 400× magnification image of a scanning electron microscope of a non-reconstituted collagen matrix in an embodiment of the present invention. FIG. 6C is a 10,000× magnification of a scanning electron microscope of a non-reconstituted collagen matrix in an embodiment of the present invention.

[0086] Compared with the porous collagen matrix prepared by the prior art (see FIG. 3), the non-reconstituted porous collagen matrix prepared by the present invention is composed of a plurality of collagen fibril bundles interlaced and aggregated, and has a plurality of pores having a depth and having a groove wall. The above-mentioned collagen fibril bundles are composed of procollagen molecules and has a diameter of less than 100 nm, but the present invention is not intended to be limited thereto. The appearance of the pores is a random radius opening, and the aperture size is controlled to a narrower range. Thus, a collagen matrix prepared by the method described herein has a relatively uniform aperture.

[0087] Please refer to FIGS. 7A to 7C, which show the appearance of a biological scaffold made from a non-reconstituted collagen matrix prepared according to the method described above. FIG. 7A is a 100× magnification image of a scanning electron microscope of a biological scaffold in an embodiment of the present invention. FIG. 7B is a 400× magnification image of a scanning electron microscope of a biological scaffold in an embodiment of the present invention. FIG. 7C is an image of a light micrograph of a biological scaffold with hyaluronan pre-activated by CNBr and crosslinked to the non-reconstituted collagen matrix in an embodiment of the present invention. Alcian blue was used to stain for the presence of hyaluronan derivatives inside the scaffold after sectioning of the histological sample. The image indicates a well-distribution of hyaluronan derivatives inside the biological scaffold.

[0088] Referring to FIG. 8, which shows a shear stress test of a non-reconstituted porous collagen matrix (line B) according to an embodiment of the present invention and a conventional porous collagen matrix (line A) obtained by the prior art. It can be seen from the results that the shear stress of the porous collagen matrix obtained by the prior art was 63.56e-03 MPa at a temperature of 20° C., and the shear stress of the non-reconstituted porous collagen matrix of the present invention was 189.07 MPa. Thus, it was demonstrated that, under the same test conditions, the shear strength of a non-reconstituted porous collagen matrix of the present invention is about 3 orders of magnitude higher than the porous collagen matrix of the prior art. Since the hair follicles can be completely removed at the beginning, the non-reconstituted porous collagen matrix is indeed superior in structure strength and tension than conventional porous collagen matrix. In summary, the present invention provides a process for preparing a non-reconstituted porous collagen matrix, which can destroy the hair follicle tissue without destroying the natural crosslinks of collagen in the dermis layer, thereby a porous matrix having a structural strength higher than that of the prior art is prepared.

[0089] Referring to FIGS. 9A and 9B, which shows the changes of storage modulus and loss modulus along with variation of oscillation strain monitored by a reometer. The storage modulus is an indication of a matrix's ability to store deformation energy in an elastic manner. This is directly related to the extent of cross-linking, the higher the degree of cross-linking the greater the storage modulus. FIG. 9A is the data of a non-reconstituted porous collagen matrix of the present invention, whereas FIG. 9B shows the data of a conventional reconstituted porous collagen matrix. A microstructure means that there are forces between the molecules or particles in the material. To break the microstructure, a force larger than the ones holding it is needed. When the applied force is smaller than the molecular or inter particle forces, then G′ is larger than G″; the material has some capacity to store energy and should be able to return, to some extent, to its initial configuration before a mechanical force is applied. The material behaves as an elastic solid, although not an ideal one because some of the mechanical energy is dissipated. The results in FIG. 9A and 9B indicate that the storage modulus of the non-reconstituted porous collagen matrix is higher than that of the reconstituted porous collagen matrix prepared by the prior art. Most native crosslinks in the non-reconstituted porous collagen matrix are preserved, whereas most native crosslinks are lost in the conventional reconstituted porous collagen matrix.

[0090] Furthermore, since the non-reconstituted collagen matrix 10a itself is not a hydrophilic material, the main body 10 may further include a hydrophilic biomaterial 10c coated on the outer surfaces of the collagen fibrils. That is, the inner walls of these pores 10b are also coated with a hydrophilic biological material 10c (as shown in FIG. 2) to facilitate the infiltration of cells into the non-reconstituted porous collagen matrix 10a and subsequently move and adhere/attach within it. Preferably, the above-mentioned biological material may be a growth factor or a glycosaminoglycan such as hyaluronan (HA), chondroitin sulfate (CS) or heparan (Hep). More preferably, the hydrophilic biological material 10c is hyaluronan, but the present invention is not intended to be limited thereto.

[0091] Referring to FIG. 1 again, in a preferred embodiment of the present invention, the growth factor material layer 20 coated on one surface of main body 10 is selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), hepatocyte growth factor (HGF), colony-stimulating factor (CSF), stem cell factor (SCF), keratinocyte growth factor (KGF), granulocyte colony-stimulating factor (GCSF), macrophage colony-stimulating factor (MCSF), granulocyte macrophage colony-stimulating factor (GMCSF), glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial-monocyte activating polypeptide, epithelial neutrophil activating peptide, erythropoietin, bone morphogenetic protein (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factor (TGF-α, TGF-β), interleukin (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF), and a combination thereof.

[0092] In addition, although tissue adhesives used in clinical medicine and tissue engineering, by virtue of their adhesive properties, they can achieve the effects of controlling drug release, regulating the breakdown of biocompatible materials, or promoting joint of blood vessels. However, because the tissue adhesives of the conventional techniques are made of cross-linking agents such as glutaraldehyde (GA), glyoxal, formaldehyde, or sodium tripolyphosphate (TPP) and cross-linked with carrier materials such as gelatin, chitosan or collagen, the cross-linking agent is cytotoxic and easily leads to a strong and sustained immune response, making tissue adhesives cytotoxic.

[0093] Therefore, in the present invention, a tissue adhesive layer 30 with better bonding strength and low cytotoxicity is used, which comprises at least one cross-linking agent, at least one matrix component and a quenching agent. The cross-linking agent is transglutaminase or has a functional group, and the matrix component is used for cross-linking with the cross-linking agent. The quenching agent is used to react with the functional group of the crosslinking agent to reduce the cytotoxicity.

[0094] In a preferred embodiment, the above-mentioned cross-linking agent is transglutaminase or one of its functional group is selected from a group consisting of reactive compounds containing amine, sulfhydryl, carbonyl, glycol, hydroxyl, carboxyl, azide, imidoester, epoxide, aldehyde, haloacetyl, pyridyl disulfide, pyridyldithiol, hydrazide, photo-reacting, carbodiimide, diazirine, aziridine, acryloyl, arylate, thiol, genipin, riboflavin, flavonoid and its derivatives, hydroxymethyl phosphine, isocyanate, maleimide, 6-maleimidohexanoic acid active ester, disuccinimidyl suberate, bis(sulfosuccinimidyl)suberate, N-hydroxy-succinimide ester (NHS-ester), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, pentafluorophenyl ester (PFP-ester), ethylene glycol diglycidyl ether, glutaraldehyde, 2,3-dibromopropionyl N-hydroxysuccinimide ester, sulfo-N-hydroxysuccinimide ester, chlorambucil-N-hydroxysuccinimide ester, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, psoralen, vinyl sulfone, and a combination thereof.

[0095] In a preferred embodiment, the matrix component is selected from the group consisting of collagen, hyaluronan, gelatin, silk fibroin, fibronectin, elastin, tenascin, laminin, vitronectin, heparan sulfate, chondroitin, chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, alginate, aggrecan, agarose, agar, cellulose, methyl cellulose, carboxyl methyl cellulose, glycogen, fibrin, fibrinogen, thrombin, polyglutamic acid, polylysine, polyamino acid, synthetic polymers (e.g., acrylate, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid), a derivative thereof, and a combination thereof.

[0096] More preferably, the matrix components can be prepared from any natively produced collagen or functional variants thereof and/or hyaluronan. Moreover, it has been shown that hyaluronan, especially high molecular weight hyaluronan (greater than 5 kDa), can effectively promote angiogenesis and further promote wound healing. For example, the molecular weight of hyaluronan can be 50 kDa to 5,000 kDa, 70 kDa to 1,500 kDa, 200 kDa to 1,500 kDa, 500 kDa to 1,500 kDa, or 700 kDa to 1,500 kDa. When cells grow in the matrix of collagen or hyaluronan, they have good viability. Therefore, in the matrix component of the tissue adhesive layer 30 used in the present invention, the concentration of hyaluronan is 0.001 mg/mL to 100 mg/mL and the concentration of collagen is 0.001 mg/mL to 100 mg/mL. Preferably, the concentration of collagen is 0.1 mg/mL to 100 mg/mL, and the concentration of hyaluronan is 0.01 mg/mL to 35 mg/mL. More preferably, the concentration of collagen is 3 mg/mL to 75 mg/mL (such as 6 mg/mL or 9 mg/mL), and the concentration of hyaluronan is 0.2 mg/mL to 20 mg/mL.

[0097] In a preferred embodiment, the quenching agent is selected from the group consisting of amino acid, oligopeptide, polypeptide, protein, amine, diamine, oligoamine, polyamine, carbonyl compound, glycol compound, carboxyl compound, dicarboxylate, oligo-carboxylate, polycarboxylate, sulfhydryl compound, oligosulfhydryl compound, polysulfhydryl compound, hydroxyl compound, oligohydroxyl compound, polyhydroxyl compound, saccharide, oligosaccharide, polysaccharides, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), oligonucleotide, azide, photo-crosslinking compound, a monofunctional or heterobifunctional group, and a combination thereof. More preferably, the quenching agents used in the present invention include spermine, protamine, 1-6 hexanediamine, and polylysine, which is of different groups of varied molecular weights. The average molecular weights of polylysine are 3.4 kDa, 20 kDa, 99 kDa, 212 kDa, and 225 kDa, respectively.

[0098] FIG. 10A is a comparison diagram showing the healing of full-thickness wounds in the dorsal skin of guinea pigs implanted with Integral® artificial skin or PCMH. Integra® artificial skin, served as a control, is a reconstituted porous collagen-chondroitin sulfate matrix prepared by a conventional method with dehydrothermal crosslinking. PCHM is a biological scaffold of the present invention composed of a non-reconstituted porous collagen matrix and hyaluronan coated on the outer and inner surface of the matrix. A stable porous structure of PCHM allows a better infiltration of dermal cells up to 4 folds than the control group. Thus, the healing speed of the wounds filled with PCHM was demonstrated to be much faster (by about 80%) than that in the Integra group. The healing quality as demonstrated in the histology of regenerative matrix of the PCHM group was also much better than the control group at month 4 post-operation.

[0099] Referring to FIG. 11, at day 28 post-operation, in comparison with normal skin, the biological scaffold of PCHM has degraded within two weeks and the healed matrix of PCHM group was basically replaced with neomatrix, as histology indicated in FIG. 11B. As shown in FIG. 11C, some debris of Integra artificial skin was observed, which interfered with the regeneration process of wound healing.

[0100] Referring to FIG. 12, as shown in steps S106 to S109, the non-reconstituted collagen matrix is contacted with a solution containing a biological material, and the non-reconstituted collagen matrix is provided in a reduced pressure environment. In one embodiment, one end of the non-reconstituted collagen matrix is placed in a solution containing at least one biological material. A reduced pressure environment is created at the other end of the non-reconstituted collagen matrix so that the biological material can be absorbed into the structure of the non-reconstituted collagen matrix to allow the biological material to be evenly coated on the outer and inner surfaces of the bundled collagen fibrils (can also be considered as being evenly coated on the inner walls of the pores), as shown in step S108. It must be noted that, because the porous diameter of the non-reconstituted collagen matrix is extremely small, it is impossible to avoid air residue by merely immersing the non-reconstituted collagen matrix in the solution. Therefore, in the present invention, the principle of siphon is used to indirectly remove air, so that the biological material can completely cover the outer surfaces of the bundled collagen fibrils (also can be regarded as the inner walls of the pores). In addition, the above-mentioned reduced pressure environment is preferably performed by a vacuuming action.

[0101] The above-mentioned biological material is selected from the group consisting of a cell attachment material, a tissue repair material, a cell induction material, a growth factor material, an antibacterial material, and a combination thereof. Preferably, the above-mentioned cell attachment material is a saccharide, a peptide, a protein, a phospholipid, or a combination thereof. Preferably, the saccharide material is a glycosaminoglycan material. Preferably, the glycosaminoglycan material is selected from the group consisting of chondroitin, chondroitin sulfate, heparin, heparan sulfate, heparan sulfate proteoglycan, keratan, keratan sulfate, dermatan sulfate, carrageenan, hyaluronan, and a combination thereof. Preferably, the biomaterial used in the present invention is selected from the group consisting of collagen, elastin, glycosaminoglycan, chitosan, alginate, polyglutamic acid (γ-PGA), polylysine, poly(lactic-co-glycolic acid) PLGA, silk protein, and fibroin, but the present invention is not intended to be limited thereto.

[0102] Preferably, the cell induction material is selected from the group consisting of molecules such as vitamins, minerals, growth factors, chemicals, medicine, herbal medicine, metabolites, intermediate metabolites, a saccharide, a peptide, a protein, a phospholipid, or a combination thereof.

[0103] Preferably, the growth factor material is selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), hepatocyte growth factor (HGF), colony-stimulating factor (CSF), stem cell factor (SCF), keratinocyte growth factor (KGF), granulocyte colony-stimulating factor (GCSF), macrophage colony-stimulating factor (MCSF), granulocyte macrophage colony-stimulating factor (GMCSF), glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNF), endothelial-monocyte activating polypeptide, epithelial neutrophil activating peptide, erythropoietin, bone morphogenetic protein (BMP-1, BMP-2, BMP-3, etc.), brain-derived neurotrophic factor (BDNF), BRAK, serotonin, von Willebrand factor (vWF), transforming growth factor (TGF-α, TGF-β), interleukin (IL-1, IL-2, IL-3, etc.), tumor necrosis factor (TNF), and a combination thereof.

[0104] Preferably, the antibacterial material is an antibiotic, an antimicrobial protein, an antimicrobial peptide, or a combination thereof.

[0105] For the detailed structure of the non-reconstituted collagen matrix please refer to FIG. 2, which is a schematic cross-sectional view of a biological scaffold according to an embodiment of the present invention. As shown above, the non-reconstituted collagen matrix 10a is composed of a plurality of bundled collagen fibrils which includes a plurality of pores 10b. The biological material 10c is absorbed into the non-reconstituted collagen matrix 10a by the principle of siphon and is coated on the outer surface of the bundled collagen fibrils. That is, the inner walls of the pores 10b are all coated with biological material 10c, which is more conductive for cells to enter the pores and adhere to the biological material for subsequent applications.

[0106] In a preferred embodiment of the present invention, although not shown in FIG. 2, after the biological material is coated on the outer surfaces of the bundled collagen fibrils, cells can be introduced into the biological scaffold.. A cell culture medium or a solution containing cells is injected into the pores 10b to allow the cells to enter the pores of the non-reconstituted collagen matrix. That is, in a preferred embodiment of the present invention, the biological scaffold provided by the present invention further includes cells, as shown in FIG. 13A. Referring to FIG. 13B, which is a partially enlarged view of the dotted lined circle in FIG. 13A, in which the cells 30 are attached to the biological material 10c in the pore 10b. Preferably, the cells may be stem cells, satellite cells, precursor cells, progenitor cells, or tissue cells, and the tissue cells may also include skin tissue, cartilage tissue, or adipose tissue, and so on.

[0107] Table 2 below shows the comparison of the number of adhered cells of a non-reconstituted collagen matrix without biological material and non-reconstituted collagen matrix with biological material in different fields of vision. As can be seen from Table 2, the non-reconstituted collagen matrix itself is a hydrophobic material, which is not conductive for adherence of cells into the pores. However, when the present invention further coats the surface of the non-reconstituted collagen matrix with a cell attachment material, it was shown to be beneficial for subsequent cells to enter these pores and adhere to the biological scaffold.

TABLE-US-00002 TABLE 2 Cell number Non-reconstituted Non-reconstituted collagen matrix without collagen matrix coated Field biological material with biological material 1 1 24 2 4 24 3 1 10 4 0  5 5 3  0 6 1  3 Average 1.67 11.00 Standard 1.51 10.58 Deviation

[0108] Next, water absorption of a non-reconstituted collagen matrix (A1) without biological material, a non-reconstituted collagen matrix (A2) coated with hyaluronan (once), and a non-reconstituted collagen matrix (A3) coated with hyaluronan (five times) were further compared. See FIGS. 14A to 14C. The three figures show the water absorption conditions of A1, A2, and A3, respectively. Therefore, it was demonstrated that the non-reconstituted collagen matrix coated with more layers of biological materials has better water absorption capacity.

[0109] The effect of the biological scaffold used in wound dressings provided by the present invention was confirmed. See FIGS. 10B and 11. FIG. 10B compares the wound healing time of different biological scaffolds, and FIG. 11 compares the wound healing status of different biological scaffolds.

[0110] First, FIG. 10B shows different treatments of the wound, which were divided into groups A, B, C, D, and E to observe the relationship between the wound area and healing time. Among them, the initial wound area was 100%, and it was measured every two days until the wounds were completely healed. A represented an untreated wound, B represented a wound treated with a porous collagen matrix (PCM), C represented a wound treated with CNBr-activated hyaluronan (aH) combined porous collagen matrix (PCM-aH), D represented a wound treated with PCM-aH with an additional 5 mg/mL of hyaluronan, and E represented a wound treated with Integra® artificial skin. As shown in the figure, the wounds of group B, group C and group D all healed in about 30 days, but the wounds treated with artificial skin (group E) took 44 days to heal. Although group A healed slightly faster, it showed wound contraction and other conditions.

[0111] As shown in FIG. 10B, after 12 days, the fibroblast proliferation status of group C (wound treated with PCM-aH) did indeed exceed that of group E (wound treated with artificial skin). It was thus shown that the above-mentioned main body 10 combined with biological materials can effectively promote cell proliferation without causing wound contraction and can heal wounds more quickly than the artificial skin of the conventional technology.

[0112] Next, in another embodiment, as shown in FIGS. 15A-15D, the biological scaffold was coated with different antibacterial materials, such as with anti-microbial peptide Pexiganan in FIGS. 15A and 15B and with neomycin in FIGS. 15C and 15D. The results showed that the antibacterial effect was concentration-dependent, and the biological scaffolds coated with antibacterial material had a good antibacterial effect.

[0113] In summary, a purpose of the present invention is to provide a biological scaffold that can be used in wound dressings. In one embodiment, one side of the main body is coated with a layer of biological material, and another side is coated with a tissue adhesive layer which allows cells to move within the main body based on the effect of concentration gradients. Furthermore, because the main body contains hydrophilic biological materials, it can further facilitate the movement and attachment of cells. Therefore, in addition to effectively blocking foreign bacterial invasion to prevent wound infection, the biological scaffold provided by the present invention can also be used to accelerate cell growth and promote the speed of wound healing.

[0114] Furthermore, although it is known in conventional techniques to add minced collagen to biological material (such as hyaluronan) and then cross-linking them to coagulate into a structure, as collagen and biological materials become intertwined, the outside of the biological scaffold is not entirely hydrophilic. In addition, if a reconstituted collagen scaffold is first made ready (as shown in FIG. 3) and then subjected to the method provided by the present invention, the support force of the reconstituted collagen matrix would be insufficient (see FIG. 9 and the related description above) to withstand the external pressure exerted by the vacuuming step, which is required in the subsequent coating of the scaffold with a biological material. As described above, the principle of siphon is applied so that the biological material can be completely absorbed or introduced into the non-reconstituted collagen matrix and uniformly coats the surface, so that the surface of the biological scaffold is favorable for cell infiltration and attachment. This biological scaffold can be widely applied in surgery, repair of human organs and tissues, regenerative medicine, skin transplantation or cosmetic medicine, and can also be used for surgical repair mesh fabric, dental repair mesh fabric, human skin substitute, artificial meningeal substitute, orthopedic repair substitute or wound dressing substitute.

[0115] The above detailed description is the specific description of certain feasible embodiments of the present invention, but these embodiments are not intended to limit the patent scope of the present invention. Any equivalent implementation or change that does not depart from the technical spirit of the present invention should be included within the patent scope of the present invention. All publications cited herein are herein incorporated by reference in their entirety.

DESCRIPTION OF SYMBOLS

[0116] 1 biological scaffold

[0117] 10 main body

[0118] 10a non-reconstituted collagen matrix

[0119] 10b pores

[0120] 10c biological material

[0121] 20 biological material layer

[0122] 30 tissue adhesive layer

[0123] 2 tissue

[0124] S100-S105 Method for preparing non-reconstituted collagen matrix

[0125] S100-S104b Method for preparing non-reconstituted collagen matrix

[0126] S106-S109 Preparation of biological scaffold