Composite membrane comprising a decellularized amniotic membrane and a method for preparing the same

Abstract

The present invention relates to the field of biomedical technology, and relates to a composite membrane comprising a decellularized amniotic membrane, a use of the composite membrane, and a method for preparing the composite membrane.

Claims

1. A composite membrane, comprising at least one decellularized amniotic membrane and at least one fibrous layer comprising polymer fibers, wherein the at least one fibrous layer is attached to the at least one decellularized amniotic membrane through a chemical bond: wherein the chemical bond comprises amide bonds or ester bonds.

2. The composite membrane according to claim 1, wherein the polymer fibers comprise a polymer having biocompatibility or biodegradability.

3. The composite membrane according to claim 1, wherein the polymer fibers in the at least one fibrous layer are randomly oriented, or oriented substantially in parallel to one another wherein an angle formed by any two fibers is from 0° to 10°.

4. The composite membrane according to claim 1, wherein the composite membrane has at least one of the following features: (a) the composite membrane has a strain to failure of 5% to 200%; (b) the composite membrane has an elastic modulus of 0.2 MPa to 1000 MPa; and (c) the composite membrane has a toughness of 0.5 MJ/m.sup.3 to 50 MJ/m.sup.3.

5. A method for preparing a composite membrane according to claim 1, comprising the steps of: (a) obtaining or having obtained the at least one decellularized amniotic membrane; (b) obtaining or having obtained the at least one fibrous layer, wherein the at least one fibrous layer comprises a polymer fiber sheet, wherein at least one surface of the polymer fiber sheet has a reactive group; and (c) fitting the surface of the polymer fiber sheet that has the reactive group to the decellularized amniotic membrane.

6. The method according to claim 5, wherein the decellularized amniotic membrane in step (a) is selected from (i) a de-epithelialized amniotic membrane and (ii) a fully decellularized amniotic membrane.

7. The method according to claim 5, wherein the polymer fiber sheet in step (b) is a nonwoven fiber sheet.

8. The method according to claim 5, wherein the polymer fiber sheet in step (b) is made of a polymer having biocompatibility or biodegradability.

9. The method according to claim 5, wherein step (c) comprises fitting the surface of the polymer fiber sheet that has the reactive group to the stromal side of the decellularized amniotic membrane.

10. The method according to claim 5, wherein step (c) is carried out at a temperature of 4 to 25° C.

11. An article, comprising the composite membrane according to claim 1.

12. A method for repairing a damaged soft tissue of a subject, comprising administering the composite membrane according to claim 1 or an article comprising the composite membrane according to claim 1 to the damaged soft tissue of the subject.

13. A method for treatment of an ophthalmic disease in a subject, comprising administering the composite membrane according to claim 1 or an article comprising the composite membrane according to claim 1 to an affected part in an ocular region of the subject.

14. The composite membrane according to claim 1, wherein the at least one fibrous layer is attached to a stromal side of the at least one decellularized amniotic membrane.

15. The composite membrane according to claim 1, wherein the at least one fibrous layer is a nanofiber layer or a microfiber layer.

16. The composite membrane according to claim 1, wherein the polymer fibers comprise a polymer selected from the group consisting of an aliphatic polyester, a polyester ether, a polyphosphazene, a polycarbonate, a polyamino acid, a collagen, a fibrin, a chitosan, an alginate, a hyaluronic acid, a fibronectin, a gelatin, a dextran, an elastin, a polylactic acid (PLA), a polyglycolide (PGA), a poly(D,L-lactide-co-glycolide) (PLGA), a polycaprolactone (PCL), and any combination thereof.

17. The composite membrane according to claim 1, wherein the at least one decellularized amniotic membrane is selected from (i) a de-epithelialized amniotic membrane, and (ii) a fully decellularized amniotic membrane.

18. The composite membrane according to claim 1, wherein the at least one fibrous layer is a hydrogel.

19. The composite membrane according to claim 1, wherein the at least one fibrous layer is attached directly to the decellularized amniotic membrane.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 schematically showed a method for preparation of a composite membrane, comprising: treating a polymer fiber sheet (for example PCL nanofiber membrane) to carry a carboxyl group on the fiber; activating the carboxyl group; compressing the polymer fiber sheet bearing the activated carboxyl group together with a decellularized amniotic membrane to obtain the composite membrane.

(2) FIG. 2 schematically showed a process of superimposing aligned fiber sheets in which the orientations of the fibers are different among the sheets.

(3) FIG. 3 showed the morphologies of PCL nanofibers before and after carboxylation in Example 1, in which the scale bars were 1 μm. Before and after carboxylation, the fiber surface did not change significantly.

(4) FIG. 4 showed the content of TBO in fiber membrane samples in Example 1.

(5) FIG. 5 showed the results of observation of the amniotic membrane with optical microscopes and a scanning electron microscope before and after decellularization (scale bar: 20 μm). FIGS. 5a and 5d showed the surface morphologies of the amniotic membrane observed before and after decellularization under a normal optical microscope, respectively. FIGS. 5b and 5e were the results of observation of the amniotic membrane by a fluorescence microscope before and after decellularization, in which the dots in FIG. 5b are DAPI-stained nuclei, and no nucleus is observed in FIG. 5e. FIGS. 5c and 5f showed the surface morphologies of the amniotic membrane observed under the scanning electron microscope before and after decellularization.

(6) FIG. 6 showed the DNA contents of the amniotic membrane and the decellularized amniotic membrane, respectively. The results showed that the decellularized amniotic membrane contains almost no DNA.

(7) FIG. 7 illustratively showed the composite process and the used apparatus.

(8) FIG. 8 showed the morphologies of PLA fibers and PLGA fibers before and after carboxylation in Example 4, in which the scale bar was 10 μm. As shown in the figure, there was no significant change in the surfaces of PLA fibers and PLGA fibers before and after carboxylation.

(9) FIG. 9 showed the contents of TBO in the untreated PLA fiber membrane and PLGA fiber membrane, and in the carboxylated PLA fiber membrane and PLGA fiber membrane.

(10) FIG. 10 showed the morphologies of the PCL-dAM composite membranes as prepared in Example 3, Comparative Example 1 and Comparative Example 2 after lyophilization. The results showed that exerting pressure on and treating the surface of the fiber membrane are advantageous for the stable attachment between the nanofiber membrane and the decellularized amniotic membrane.

(11) FIG. 11 showed the morphologies of the decellularized amniotic membrane and the PCL-dAM composite membranes with different thicknesses in dry and rehydrated states. As shown in the figure, the decellularized amniotic membranes were adhered together after rehydration and were difficult to unfold, while the composite membranes were easily clamped with tweezers after rehydration and were in stretched state.

(12) FIG. 12 showed the morphologies of the decellularized amniotic membrane, the PLA-dAM composite membrane, and the PLGA-dAM composite membrane in dry and rehydrated states, in which the thicknesses of said composite membranes were 80 μm. As shown in the figure, the decellularized amniotic membranes were adhered together after rehydration and were difficult to unfold, while the composite membranes were easily clamped with tweezers after rehydration and were in stretched state.

(13) FIG. 13 showed the water contents of the decellularized amniotic membrane, the PCL-dAM composite membranes (thickness: 40 μm, 80 μm, 120 μm), the surface-carboxylated PCL nanofiber membrane (thickness: 80 μm) and the untreated PCL nanofiber membrane (thickness: 80 μm).

(14) FIG. 14 showed the stress-strain curves of decellularized amniotic membrane, PLGA-dAM composite membrane, PLA-dAM composite membrane and PCL-dAM composite membrane.

(15) FIG. 15 showed the mechanical test results of decellularized amniotic membrane, PLGA-dAM composite membrane, PLA-dAM composite membrane and PCL-dAM composite membrane: (a) elastic modulus, (b) strain to failure, (c) ultimate tensile strength, and (d) toughness.

(16) FIG. 16 showed the results of suture extension test for decellularized amniotic membrane (dAM), PCL-dAM composite membrane (CM) with the thickness of 80 μm and untreated PCL nanofiber membrane (NF) with the thickness of 80 μm.

(17) FIG. 17 showed the surface structures of PCL-dAM composite membranes prepared under conditions with and without compression, as observed by scanning electron microscope, and the scale bars were 10 μm. As shown in the figure, in the composite membrane prepared under conditions without compression, the PCL nanofiber membrane (NF) could be bound to the decellularized amniotic membrane by chemical action, but did not exhibit the shape of the PCL, nanofiber on the surface of the decellularized amniotic membrane (FIGS. 17A, 17D), indicating that the combination between the decellularized amniotic membrane and PCL nanofiber membrane was weak. In the composite membrane prepared under conditions with compression, the morphology of the PCL nanofiber was clearly visible on the surface of the decellularized amniotic membrane (FIG. 17B, C, E, F), indicating that the decellularized amniotic membrane and PCL nanofiber membrane were tightly combined and formed an integrated composite structure.

(18) FIG. 18 showed the cross-section of the natural amniotic membrane and PCL-dAM composite membrane. FIGS. 18A-18C showed optical micrographs of the cross-sections after H&E staining, in which the scale bars were 20 μm. FIG. 18A showed that the natural amniotic membrane comprises a layer of epithelial cell and a sponge-like stromal structure. FIGS. 18B and 18C showed that the PCL nanofiber membrane (NF) merged into the stromal side of the decellularized amniotic membrane as observed on the cross-section of the composite membrane, and the amniotic membrane and the PCL nanofiber membrane were overlapped. FIGS. 18D to 18F were scanning electron micrographs of the cross-section, in which the scale bars were 10 μm, 2 μm, 1 μm in FIGS. 18D, 18E and 18F, respectively. The ultra-microstructure of the composite membrane showed that the PCL nanofiber membrane very closely bound to the decellularized amniotic membrane (FIGS. 18E, 18F). The location indicated by arrows showed that the decellularized amniotic membrane and PCL nanofiber membrane were tightly connected. The results showed that the PCL nanofiber membrane and the decellularized amniotic membrane successfully formed a composite membrane through physical and chemical interactions.

(19) FIG. 19 showed the survival rates of corneal fibroblasts on the decellularized amniotic membrane (dAM), PCL-dAM composite membrane (CM) and the PCL nanofiber membrane (NF). The results showed that none of the decellularized amniotic membrane, PCL-dAM composite membranes or the PCL nanofiber membrane had cytotoxicity.

(20) FIG. 20 showed the morphologies of corneal fibroblasts after 7 days of culture under conditions with and without serum on the decellularized amniotic membrane (dAM), the PCL-dAM composite membrane (CM), the PCL nanofiber membrane (NF) and the TCPS tissue culture polystyrene (TCPS), in which the scale bar was 50 μm. As shown in the figure, the cells showed a conventional spindle shape on the decellularized amniotic membrane, the PCL-dAM composite membrane and the cell culture plate in the serum-free medium, whereas the cells were hardly stretched on the PCL nanofiber membrane. In the serum-containing medium, the cells grew and covered the decellularized amniotic membrane, the PCL-dAM composite membrane and the cell culture plate, and formed cell layers. On the PC, fiber membrane, the cells did not form a cell layer. This result showed that similar to the decellularized amniotic membrane, the composite membrane of the present invention provided a suitable attachment surface for cell growth, which facilitates the adhesion, stretching and growth of cells.

(21) FIG. 21 showed the proliferation of cells on the decellularized amniotic membrane (dAM), the PCL composite membrane (CM), the PCL nanofiber membrane (NF) and the TCPS cell culture plate (TCPS) under serum-free culture conditions. All fluorescence detection values were compared to the values detected on the first day. The results showed that the composite membrane of the present invention well retained the nutrients in amniotic membrane and provided the nutrients for cell growth.

(22) FIG. 22 showed the expression of markers of M1 type macrophage and M2 type macrophage after culturing for 48 hours the mouse bone marrow-derived macrophages that were implanted on the decellularized amniotic membrane (dAM), the PCL-dAM composite membrane (CM), the PC, nanofiber membrane (NF), and the TCPS tissue culture polystyrene (TCPS) with inflammatory stimulating factors with different concentrations. The results represented mean±SD (n=3). *P<0.05, **P<0.01. The results showed that the composite membrane of the present invention had anti-inflammatory properties similar to that of amniotic membrane, which can promote the transformation of macrophages from pro-inflammatory type to anti-inflammatory type.

(23) FIG. 23 showed the surface morphology of EDC cross-linked aligned collagen fibers, FIG. 23A showed an image magnified 25 times, and FIG. 23B showed an image magnified 100 times. As shown in these figures, the collagen fibers were regular and arranged in parallel.

(24) FIG. 24 showed the transparencies of the collagen fiber membrane, the decellularized amniotic membrane and the collagen-dAM composite membrane, in which the membranes had a diameter of 8 mm. As shown in the figure, the letters “JHU” under the three membranes were clearly visible in both dry and rehydration conditions. The transparency of the collagen fiber membrane was even slightly higher than that of the amniotic membrane.

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

(25) While the embodiments of the present invention will be described in detail with reference to the following examples, it will be understood by those skilled in the art that the following examples are intended to be illustrative of the present invention and should not be construed as limiting the scope of the invention. When specific conditions were not given the examples, they were carried out in accordance with conventional conditions or the conditions recommended by the manufacturers. When the manufacturers of the used reagents or instruments were not indicated, they were all commercially available conventional products.

EXAMPLE 1

Preparation and Treatment of PCL Nanofiber Membrane

(26) 1. Preparation of PCL nanofiber membrane: A solution of 12 wt % polycaprolactone (PCL, MW 70 kDa, Sigma-Aldrich) was prepared. A certain amount PCL particles were weighed and dissolved in a solution of dichloromethane: methanol=4:1 at a final concentration of 12%. The PCL solution was added to a syringe, and a 27 G syringe needle was mounted. The entire syringe was placed on a motor-driven syringe pump, and was propelled with a voltage of 12 kV at an injection speed of 2.5 mL/h. A grounded tin foil paper was used to receive the ejected PCL nanofibers to form a nanofiber membrane with a membrane thickness of 80 μm, and the average diameter of the PCL nanofibers was 517 nm (517±178 nm).

(27) According to the above process, the PCL nanofiber membranes with thicknesses of 40 μm, 80 μm and 120 μm were prepared.

(28) 2. Surface treatment of PCL nanofiber membrane: the process was shown in FIG. 1.

(29) 1) The PCL nanofiber membrane was treated with a plasma cleaner (Harrick Plasma) at a moderate frequency for 10 minutes to allow the surface of the fiber to carry active oxygen groups.

(30) 2) Carboxylation: 10% acrylic acid solution was prepared with deionized water, and sodium periodate (NaIO.sub.4) was added, and the final concentration of sodium periodate was 0.5 mM. The plasma treated PCL nanofiber membrane was placed in a clean container, and the container was placed on ice. The solution of 10% acrylic acid-0.5 mM NaIO.sub.4 was added to cover the fiber membrane. UV irradiation was performed for 2 minutes (30 to 50 mW/cm.sup.2).

(31) 3) The UV treated fiber membrane was washed with deionized water for 3 to 5 times, to completely remove the residual reagents. Drying was carried out at room temperature in dark place.

(32) 4) The fiber membrane was observed under a scanning electron microscope before and after carboxylation. As shown in FIG. 3, there was no significant change on the fiber surface before and after carboxylation. The carboxyl group content of the fiber membrane was calculated according to Toluidine Blue O (TBO) reaction. FIG. 4 showed the TBO concentrations in five samples (with thickness of 70 or 80 μm, respectively). As shown in the figure, the average TBO concentration of the fiber membrane was 0.19±0.027 nmol/mm.sup.2. The ratio of carboxyl groups to TBO complexes was 1:1. Thus, the carboxyl group concentration of the fiber membrane was about 0.19±0.027 nmol/mm.sup.2. The results showed that PCL fiber surface was successfully carboxylated.

(33) 5) Activation of the carboxyl group on the surface of die PCL fiber membrane: The masses of NHS and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) required for the reaction (molar ratio COOH: NHS/EDC=1:4) were calculated from the carboxyl group content, NHS (Sigma) and EDC (Sigma) in corresponding amounts were weighed and dissolved in 50% ethanol, then added to the carboxylated fiber surface and treated at room temperature in dark place for 5 hours.

(34) 6) The fiber membrane vas washed twice with 70% ethanol.

EXAMPLE 2

Decellularization Treatment of Amniotic Membrane

(35) Fresh amniotic membrane was attached to a nitrocellulose membrane, with epithelial side up. A solution of 2.5% Dispase (Millipore) was prepared and dissolved in DMEM/F12 serum-free medium (Life Technology). The amniotic surface was covered with the enzyme solution, treated at 4° C. for 4 hours, and washed with PBS for 3 times. The amniotic membrane was placed in PBS solution and placed under a stereoscope. Epithelial cells were scraped off by using an Iris spatula from left to right, top to bottom. Under the stereoscope, it could be seen that white cell debris fell into PBS solution. The amniotic membrane was washed with PBS solution for 3 times.

(36) The thickness of the decellularized amniotic membrane was between 20 μm and 40 μm. The amniotic membrane was observed by optical microscope and scanning electron microscope before and after the decellularization, and the results were shown in FIG. 5 (scale bar: 20 μm). FIGS. 5a and 5d showed the surface morphologies of the amniotic membrane observed before and after the decellularization under the optical microscope, respectively. FIGS. 5b and 5c showed the results of observation of the amniotic membrane by a fluorescence microscope before and after the decellularization. The dots in FIG. 5b were DAPI-stained nuclei, and no nucleus was observed in FIG. 5e. FIGS. 5c and 5f showed the surface morphologies of the amniotic membrane observed under the scanning electron microscope before and after decellularization.

(37) As shown in the figures, amniotic epithelial cells were distributed over the entire surface prior to the treatment, while almost no cell remained on the amniotic membrane surface after the treatment, indicating that the amniotic epithelial cells were completely removed.

(38) The DNA contents in the amniotic membrane before and after decellularization were detected using the DNA extraction kit (DNeasy® Blood & Tissue Kit, QIAGEN). As shown in FIG. 6, the decellularized amniotic membrane contained almost no DNA.

EXAMPLE 3

Preparation of Decellularized Amniotic Membrane-PCL Nanofiber Composite Membrane (“PCL-dAM Composite Membrane” For Abbreviation)

(39) In an ultra-clean bench, the PCL nanofiber membrane was washed three times with PBS. The decellularized amniotic membrane with epithelial side down and stromal side up was spread out on the surface of Teflon (Polytetrafluoroethene, PTFE) film. The PCL nanofiber membrane was placed on the stromal side of the amniotic membrane. After that, another Teflon film was placed on the nanofiber membrane. The stacked decellularized amniotic membrane, the PCL nanofiber membrane and the Teflon films were placed between two stainless steel plates. The steel plates were placed in a vise, to which a compression of 5 to 7 MP was applied, and stayed at 4° C. for 12 hours. A condensation reaction was carried out between the carboxyl group on the surface of the PCL nanofiber membrane and the amino group on the surface of the decellularized amniotic membrane, and the reaction was more sufficiently performed by compression of the vise. After removal of the steel plates and the Teflon films, the composite membrane was taken out, lyophilized in a freeze dryer (Labconco Freezone 12L Cascade Freeze Dry System), and stored at room temperature.

(40) FIG. 7 illustratively showed the composite process and the apparatus used therein. In the figure, the decellularized amniotic membrane was laid over the PCL nanofiber membrane with activated carboxyl groups and placed between the two Teflon films; the outer side of each of the two Teflon films had a stainless steel plate, and the stainless steel plates were compressed by the vise, so as to obtain the composite membrane.

(41) The composite membranes with different thicknesses were obtained by using the PCL nanofiber membranes with different thicknesses.

(42) Comparative Example 1: By referring to the process of Example 3, the decellularized amniotic membrane was combined with the PCL nanofiber membrane under the condition without compression.

(43) Comparative Example 2: By referring to the process of Example 1, a PCL nanofiber membrane having a thickness of 80 μm was prepared without treating the membrane surface. By referring to the process of Example 3, this PCL nanofiber membrane was combined with the decellularized amniotic membrane under the condition with compression.

EXAMPLE 4

Preparation of Decellularized Amniotic Membrane-PLA Fiber

(44) Composite Membrane (“PLA-Dam Composite Membrane” for Abbreviation) and Decellularized Amniotic Membrane-PLGA Fiber Composite Membrane (“PLGA-Dam Composite Membrane” for Abbreviation)

(45) 1. Preparation of polylactic acid (PEA) fiber membrane: A solution of 9 wt % PEA (PLA, Mw=78 kDa, Mn=48 kDa, Nature Works LLC) was prepared, A certain amount of PLA was weighed and dissolved in a solution of trichloromethane-dimethylformamide at a final concentration of 9%. The PLA solution was added to a syringe and a 27 G syringe needle was mounted. The entire syringe was placed on an electric syringe pump and propelled by using a voltage of 16 kV at an injection speed of 0.5 mL/h. A grounded tin foil paper was used to receive the ejected PLA fibers to form a fiber membrane with a thickness of 80 μm, and the diameter of the fibers was 935 nm (935±218 nm).

(46) 2. Preparation of Poly(D,L-lactide-co-glycolide) (PLGA) fiber membrane: 15 wt % polylactic acid-glycolide random copolymer (number ratio of repeating units was: lactic acid: glycolide=50: 50, MW 30 to 60 kDa, Sigma-Aldrich) solution was prepared. A certain amount of PLGA was weighed and dissolved in 1,1,1,3,3,3-hexafluoroisopropanol at a final concentration of 15%. The PLGA solution was added to a syringe and a 27 G needle was mounted. The entire syringe was placed on an electric syringe pump and propelled with a voltage of 6.8 kV at an injection speed of 0.5 mL/h. A grounded tin foil paper was used to receive the ejected PLGA fibers to form a fiber membrane with a thickness of 80 μm, and the diameter of the fibers was 1260 nm (1260±132 nm).

(47) 3. The surface treatment process of the PLGA and PLA fiber membranes was similar to that of the PCL fiber membrane. The fiber membranes before and after carboxylation were observed by scanning electron microscope. As shown in FIG. 8, the surface of the PLA and PLGA fibers did not change significantly before and after carboxylation.

(48) The TBO concentrations of the fiber membranes after carboxylation were detected by TBO assay. The results were shown in FIG. 9, indicating that after carboxylation, the average TBO concentration on the PLA fiber membrane with a thickness of 80 μm was 0.22±0.031 nmol/mm.sup.2 and the average TBO concentration on the PLGA fiber membrane with a thickness of 80 μm was 0.21±0.018 nmol/mm.sup.2.

EXAMPLE 5

Performance Test of Composite Membranes

(49) (1) Stability

(50) FIG. 10 showed the morphologies of PCL-dAM composite membranes as prepared in Example 3, Comparative Example 1 and Comparative Example 2 after lyophilization. As shown in the figure, in the composite membrane prepared in Example 3, the decellularized amniotic membrane and the PCL nanofiber membrane were very closely combined together. In the composite membrane prepared under conditions without compression (Comparative Example 1). It could be seen that the decellularized amniotic membrane was detached from the nanofiber membrane at the edge. In the composite membrane prepared with the untreated PCL nanofiber membrane (Comparative Example 2), the decellularized amniotic membrane could be easily peeled off from the untreated PCL nanofiber membrane. The results show that exerting compression on and treating the surface of the fiber membrane were advantageous for the stable attachment between the nanofiber membrane and the decellularized amniotic membrane.

(51) (2) Operability

(52) The operability of the composite membrane was evaluated by observing the state of the composite membrane when it was and clamped with tweezers in a dry and rehydrated states.

(53) FIG. 11 showed the morphologies of the decellularized amniotic membrane and PCL-dAM composite membranes with different thicknesses in dry and rehydrated states. FIG. 12 showed the morphologies of PLA-dAM composite membrane, and PLEA-dAM composite membrane in dry and rehydrated states. As shown in the figures, the decellularized amniotic membranes were adhered together after rehydration and were difficult to unfold, while the composite membranes were easily clamped with tweezers after rehydration and were in stretched state.

(54) (3) Hydrophilicity

(55) The water contents of the decellularized amniotic membrane, the PCL-dAM composite membranes (thickness: 40 μm, 80 μm, 12.0 μm), the surface-carboxylated and activated PCL nanofiber membrane (thickness: 80 μm) and the untreated PCL nanofiber membrane (thickness: 80 μm) were determined by a process as follows: The freeze-dried membranes were cut, each sample had a size of 1 cm×1 cm, weighed (Wd), and then placed in double distilled water at 37° C. for 10 minutes. The membranes were attached to glass slides, placed vertically for 1 min to drip the surface water, removed from the glass slides and weighed again (Ws).

(56) Water contents were calculated according to the following formula: Water content (%)=[(Ws−Wd)/Ws]×100. FIG. 13 showed the results. It can be seen from the figure that the composite membrane had a water content similar to those of the decellularized amniotic membrane and the surface-carboxylated PCL nanofiber membrane (Treated NF), and their water contents were about 80%, indicating that the composite membrane exhibited good hydrophilicity during rehydration.

(57) (4) Tensile and Toughness Performance

(58) The stress-strain curves of the decellularized membrane (dAM), as well as PCL-dAM composite membrane, PLA-dAM composite membrane and PLGA-dAM composite membrane which all had a thickness of 40 μm, were measured by the process as follows the membranes after lyophilization were cut to obtain dumbbell-shaped membrane samples, which both ends had a size of 1 cm×1 cm, and the center part had a size of 1 mm×2 mm. The both ends of the I-shaped membrane sample were fixed to metal clamps of a mechanical tester (BOSE Enduratec ELF 3200), the sample was immersed in a phosphate buffer for 2 minutes and then stretched at a rate of 0.5 mm/min. The measurement yielded stress-strain curves, elastic modulus, strain to failure, ultimate tensile strength, and toughness.

(59) The stress-strain curves of the decellularized amniotic membrane and the composite membranes were shown in FIG. 14. The stress-strain curve of PLGA-dAM composite membrane increased and decreased sharply, and exhibited the highest curve peak; the curve of PLA-dAM composite membrane increased and decreased slowly; the curve of PCL-dAM composite membrane increased and decreased more slowly; and the curve of the decellularized amniotic membrane increased slowly, but its curve peak was significantly lower than those of the three composite membranes. These indicate that the decellularized amniotic membrane had good elasticity, but its ultimate tensile strength was the lowest.

(60) FIG. 15 showed the results of other mechanical tests: (a) elastic modulus, (b) strain to failure, (c) ultimate tensile strength, and (d) toughness. As shown in the figures, the decellularized amniotic membrane after rehydration had the lowest elastic modulus compared to the three composite membranes, indicating that its elasticity was best, but its ultimate tensile strength and toughness were the lowest, and significantly lower than those of the three composite membranes, indicating that the tensile properties and toughness of the composite membranes were better than those of the amniotic membrane.

(61) (5) Resistance to Suture Extension

(62) The suture extension test was performed on the decellularized amniotic membrane, PCL-dAM composite membrane with the thickness of 80 μm and untreated PCL nanofiber membrane with the thickness of 80 μm. The procedure was as follows: the membranes after lyophilization were cut to get samples in size of 1 cm×2 cm. One end of the membrane sample was attached to a metal clamp of a mechanical tester (BOSE Enduratec ELF 3200) and the other end was pierced into a 7-0 nylon suture at a distance of 2 mm from the center. A section of the suture was fixed to the metal clamp on the other side. After the membranes were immersed with a phosphate buffer for 2 min, the membranes were stretched at a rate of 0.2 mm/s.

(63) FIG. 16 showed the test results. As shown in the figure, the individual decellularized amniotic membrane was torn apart h the suture when the suture displacement was less than 2 mm. For PCL-dAM composite membrane (CM) and the PCL nanofiber membrane (NF), the pinholes become longer and longer as the suture displacement increased, but these membranes were not torn apart. The results show that the composite membranes were well resistant to suture extension.

EXAMPLE 6

Microtopographic Characterization of PCL-dAM Composite Membrane

(64) FIG. 17 showed the surface structures of PCL-dAM composite membranes prepared under conditions with and without composition, as observed by a scanning electron microscope. The scale bars were 10 μm. As shown in the figure, in composite membrane prepared without compression, the PCL nanofiber membrane (NE) could be combined with the decellularized amniotic membrane by chemical action, but the morphology of the PCL nanofiber did not present on the surface of the decellularized amniotic membrane (FIGS. 17A, 17D), indicating that the combination between the decellularized amniotic membrane and the PCL nanofiber membrane was relatively weak. In the composite membrane prepared with compression, the morphology of the nanofiber was clearly visible on the surface of the decellularized amniotic membrane (FIGS. 17B, C, E, F), indicating that the decellularized amniotic membrane and the PCL nanofiber membrane were tightly connected and formed an integrated composite structure.

(65) FIG. 18 showed the cross-section of the natural amniotic membrane and PCL-dAM composite membrane. FIGS. 18A-18C showed optical micrographs of the cross-section after H&E staining, with a scale bar of 20 μm. FIG. 18A showed that the natural amniotic membrane composed of a layer of epithelial cells and a sponge-like stromal structure. FIGS. 18B and 18C showed that, as observed on the cross-section of the composite membrane, the PCL nanofiber membrane (NF) had been merged into the stroma of the decellularized amniotic membrane, and the decellularized amniotic membrane and the PCL nanofiber membrane were overlapped. FIGS. 18D to 18F showed scanning electron micrographs of the cross-section, in which FIGS. 18D, 18E and 18F had a scale bar of 10 μm, 2 μm and 1 μm, respectively. The ultra-microstructure of the composite membrane showed that the PCL nanofiber membrane connected to the decellularized amniotic membrane very closely (FIGS. 18E, 18F). The results show that the PCL nanofiber membrane and the decellularized amniotic membrane successfully form a composite membrane through physical and chemical interactions.

EXAMPLE 7

Evaluation of the Safety of the Composite Membrane and its Effect on Cell Morphology and Proliferation

(66) (1) Safety Test

(67) The decellularized amniotic membrane, the PCL-dAM composite membrane prepared in Example 3 and the PCL nanofiber membrane, were cut into a size of 1 cm×1 cm, and each of them was immersed into 200 μL of E12/DMEM low-glucose medium (containing 10% FBS and 1% penicillins/streptomycin) for 48 hours to obtain a leachate of membrane material. Corneal fibroblasts were implanted into a 96-well plate at an amount of 10.sup.3 cells per well and cultured for 24 hours. The cell culture medium was replaced with the membrane leachate, or fresh medium (positive control), or 5% DMSO (negative control). After 72 hours of incubation, the medium was discarded and 10% alamar Blue® (Invitrogen) reaction solution was added, and incubated in a 5% CO.sub.2, 37° C. incubator for 2 hours. Absorbance was detected at 570 nm. FIG. 19 showed the survival rate of each group of cells. The results showed that none of the decellularized amniotic membrane (dAM), PCL-dAM composite membrane (CM) or the PCL nanofiber membrane (NF) has cytotoxicity.

(68) (2) Cell Morphology Detection

(69) Corneal fibroblasts were implanted on the decellularized amniotic membrane, PCL-dAM composite membrane prepared in Example 3, the PCL nanofiber membrane, and the Tissue Culture Polystyrene (TCPS) cell culture plate, and cultured with serum-free medium or 10% serum medium. After 7 days of incubation, the cells were fixed with 4% paraformaldehyde for 10 min. After being washed with phosphate buffer, treatment with 0.1% Triton X-100 was carried out for 5 min. After being washed with phosphate buffer, Fluorescent Phallotoxins was added to the solution (Alexa Fluor® 546 Phalloidin, Invitrogen), and incubation was performed at room temperature for 20 min. After being washed with phosphate buffer, DAPI dye liquor (Invitrogen) was added and incubation was performed at room temperature for 10 min. After being washed with phosphate buffer, the cells were sealed for observation.

(70) FIG. 20 showed the morphologies of corneal fibroblasts after 7 days of culture under conditions with and without serum on the decellularized amniotic membrane (dAM), the PCL-dAM composite membrane (CM), the PCL nanofiber membrane (NF) and the TCPS cell culture plate (TCPS), in which the scale bar was 50 μm. As shown in the FIG. 20, the cells showed a conventional spindle shape on the decellularized amniotic membrane, the PCL-dAM composite membrane and the cell culture plate in the serum-tree medium, whereas the cells were hardly stretched on the PCL nanofiber membrane. In the serum-containing medium, the cells grew and covered the decellularized amniotic membrane, the PCL-dAM composite membrane and the cell culture plate, and formed cell layers. On the PCL fiber membrane, the cells did not form a cell layer. This result shows that similar to the decellularized amniotic membrane, the composite membrane of the present invention provides a suitable attachment surface for cell growth, which facilitates the adhesion, stretching and growth of the cells.

(71) (3) Cell Proliferation Test

(72) The proliferation of cells on the decellularized amniotic membrane, the PCL-dAM composite membrane prepared in Example 3, the PCL nanofiber membrane, and the TCPS cell culture plate under serum-free culture conditions was studied. The process was as follows:

(73) CellCrown™ insert was purchased from Sigma. The membrane was placed between the cylindrical holder and the round sleeve chuck of the CellCrown™ insert to form a cylindric membrane support. Cells were grown on different membrane supports at a density of 5×10.sup.3 cells/cm.sup.2 under serum-free culture conditions. The activity of the cells was examined at the 1.sup.st, 3.sup.rd, 5.sup.th, and 7.sup.th day, respectively. The medium was discarded and 10% alamar Blue® (lnvitrogen) reaction solution was added, Incubation was performed in a 5% CO.sub.2, 37° C. incubator for 2 hours. Absorbance was detected at 570 nm.

(74) FIG. 21 showed the proliferation of cells on different membranes under serum-free culture conditions. All fluorescence detection values were compared to the values detected on the first day. As shown in the figure, the cell viability gradually decreased on the PCL nanofiber membrane (NF) and the TCPS cell culture plate (TCPS) from the 1.sup.st day to the 7.sup.th day under serurn-free culture conditions. However, the cell viability increased from the 1.sup.st day to the 3.sup.rd day when the cells were cultured on the surface of the decellularized amniotic membrane (dAM), began to decline 3 days later. On the PCL-dAM composite membrane (CM), the situation was similar to that of the amniotic membrane, i.e., the cell viability increased from the 1.sup.st day 1 to the 5.sup.th day, decreased from the 5.sup.th day, and there was no net growth from the 7.sup.th day. This suggests that the composite membrane of the present invention retains well the nutrients in the amniotic membrane and provides nutrients for cell growth.

EXAMPLE 8

Evaluation of the Anti-Inflammatory Effects of the Composite Membrane

(75) Furthermore, the anti-inflammatory effects of the composite membrane was evaluated to examine whether the composite membrane had the effect of promoting macrophage to transit from M1 type to M2 type, as the amniotic membrane. The procedure was as follows:

(76) The macrophages derived from mouse bone marrow mesenchymal cells were first isolated and cultured, then the macrophages were grown on the decellularized amniotic membrane, the PCL-dAM composite membranes prepared in Example 3, the PCL nanofiber membrane and TCPS cell culture plate, and inflammatory stimulating factors (LPS and IFN-γ) were added. After 48 hours, real-time Q-PCR was used to detect the expression of markers of M1 and M2.

(77) FIG. 22 showed the expression of markers of M1 type macrophage and M2 type macrophage after culturing for 48 hours the mouse bone marrow-derived macrophages that were implanted on the decellularized amniotic membrane (dAM), the PCL-dAM composite membrane (CM), the PCL nanofiber membrane (NE), and TCPS cell culture plate (TCPS) with inflammatory stimulating factors with different concentrations. As shown in the figure, the markers of M1 and M2 had similar expression on different substrates when no stimulatory factor was added (LPS: 0 ng/ml; IFNγ: 0 ng/ml). After the stimulating factors LPS and IFNγ were added, the expression of M1 markers (IL1b, IL6, iNOS and CD86) increased with the increase of concentration of the stimulating factors, while the expression of M2 marker CD206 gradually decreased.

(78) The expression of M1 marker iNOS in the cells cultured on the decellularized amniotic membrane and the composite membrane in the presence of low concentration of stimulating factors decreased significantly in comparison with the cells cultured on TCPS. In the presence of high concentration of stimulating factors, the expression of M1 markers IL1b, iNOS and CD86 all decreased significantly. The expression of M2 markers Argl and CD206 all increased significantly in the cells cultured on the decellularized amniotic membrane and the composite membrane in the presence of either lower or higher concentration of stimulating factors, in comparison with the cells cultured on the cell culture plate. Hence, in the inflammatory environment, the decellularized amniotic membrane and the PCL-dAM composite membrane could significantly reduce the expression of proinflammatory factors, improve the expression of anti-inflammatory factors. This suggests that the composite membrane of the present invention has anti-inflammatory properties similar to that of amniotic membrane, and can promote the transformation of macrophages from proinflammatory type to anti-inflammatory type.

EXAMPLE 9

Obtaining an Aligned Electrospun Hydrogel Fiber Membrane and Preparation of an Decellularized Amniotic Membrane-Collagen Composite Membrane (“Collagen-dAM” for Abbreviation)

(79) Similar to the PCL fiber membrane, a hydrogel electrospun fiber membrane (such as electrospun collagen fiber membrane) could also form a composite membrane with the decellularized amniotic membrane. The gelling material was dissolved in a water-soluble solution and then electrospun, in which a cross-linking agent was added for crosslinking.

(80) Collagen was taken as an example. First, 1% (wt %) solution of collagen (Elastin Products Company) was prepared. A certain amount of collagen was weighed and dissolved in 75 mM citric acid solution (pH=3.7). The collagen solution was added to a syringe and a 27 G needle was mounted. The entire syringe was placed on a motor-driven syringe pump, and the electrospun fibers were sprayed into a disc collection device containing 50 mg/mL of EDC cross-linker. EDC could quickly cross-link the amino groups and carboxyl groups distributed in the collagen to further form microfibers. The disc collection device had a rotational speed of 50 RPM. The electric syringe pump was moved on an electric translation stage to obtain an aligned fiber membrane with a width of 2 cm. The collected hydrogel fiber membrane was dehydrated by dehydration reagent (for example ethanol) with gradient concentrations (0 to 100%).

(81) FIG. 23 showed the surface morphology of EDC cross-linked aligned collagen fibers, FIG. 23A showed an image magnified 25 times, and FIG. 23B showed an image magnified 100 times. As shown in these figures, the collagen fibers are regular and arranged in parallel.

(82) The dehydrated collagen fiber membrane was treated with 50 mM EDC/NHS at room temperature for 2 hours, and afterwards, washed thoroughly with PBS. The decellularized amniotic membrane with epithelial side up and stromal side down was spread out on a Teflon film. The collagen fiber membrane was placed on the stromal side of the decellularized amniotic membrane. Another Teflon film was placed on the collagen fiber membrane. The entire composition was placed between two steel plates. The steel plates were placed in the jaws of a vise. The claw beam of the vise was turned to exert a compression of 1 MP. After standing and reacting at VC for 12 hours, the steel plates and the Teflon films were removed, and the composite membrane was freeze dried under vacuum.

EAMPLE 10

Test for Transparency of the Collagen-dAM Composite Membrane

(83) The transparencies of the collagen fiber membrane, the decellularized amniotic membrane and the collagen-dAM composite membrane were measured. As shown in FIG. 24, the letters “JHU” under the three membranes were clearly visible in both dry and rehydrated states. The transparency of the collagen fiber membrane was even slightly stronger than that of the amniotic membrane. This suggests that the collagen fiber membrane played a role as physical support to the decellularized amniotic membrane, and does not weaken the transparency of the amniotic membrane.

(84) While the specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and variations of the details may be made in accordance with all teachings already disclosed and that such changes are within the scope of the present invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.