Vagina-like stent capable of being transplanted in situ and preparation method thereof

Abstract

The invention provides a 3D vaginal stent capable of being transplanted in situ and a preparation method of the 3D vaginal stent, and relates to the technical field of regenerative medicine and tissue engineering. The preparation method of the 3D vaginal stent capable of being transplanted in situ comprises the following steps: mixing pig vagina acellular matrix native gel with a gelatin methacrylamide solution, performing pasteurization, adding silk fibroin, and uniformly mixing to obtain a 3D printing matrix mixture; filling the 3D printing matrix mixture into a 3D printer; and designing a model structure and parameters by applying CAD software according to a vagina structure to be printed, setting a printing system, and carrying out blue light irradiation on a printed object while printing, so as to obtain the 3D vagina stent capable of being transplanted in situ. The vagina-like stent capable of being transplanted in situ is good in biological safety and mechanical performance, especially capable of reconstructing vagina tissue, and has functional characteristics, so that in-situ transplantation of large and small animal vagina deletion models and repair of deleted vagina tissue can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of regenerative medicine and tissue engineering technology, and in particular to an in situ transplantable vaginal stent and a preparation method thereof. Background Art

[0002] A variety of congenital and acquired factors can lead to vaginal absence, such as congenital absence of vagina (MRKH syndrome), hermaphroditism, trauma, tumors, etc., which bring great physical and mental pain to patients. Traditional vaginal reconstruction techniques use non-vaginal tissues to reconstruct the vagina, which has certain functional limitations. They are also very different from normal vaginas in morphology and histology. These traditional methods may lead to many complications, including contracture, necrosis, prolapse, intestinal obstruction and malignant transformation, which require reoperation.

[0003] Traditional tissue engineering technology uses seed cells and biomaterials to construct tissue-engineered vaginas, which has achieved certain results in vaginal reconstruction, but there are disadvantages such as low cell survival rate, rough construction, and lack of personalization. With the development of technology, new biological tissue materials such as natural polymer materials, synthetic polymer materials, and natural extracellular matrix (ECM) have emerged. Among them, decellularized matrix (dECM) has become the preferred matrix for bioengineered tissues because it retains the overall structure and inherent growth factors of the natural extracellular matrix and has the characteristics of low immunogenicity. The vaginal dECM scaffold constructed using dECM materials can provide a microenvironment similar to the physiological residence of the vagina, which is conducive to the reconstruction of tissue regeneration in the physiological environment. However, there are still some limitations in the morphology and functionality of using dECM scaffolds as cell carriers to construct organic matter. First, dECM scaffolds often lack customized micro-geometry, and it is difficult to arbitrarily adjust the scaffold size according to experimental needs while retaining the original vascular system, resulting in cell distribution being mainly limited to the surface of the material, with only a small number of cells penetrating into the internal area; second, when dECM scaffolds are implanted, the survival of infiltrating or seeded cell populations mainly depends on the diffusion of oxygen and nutrients until a network supporting blood vessels is formed, making the recellularization of these organ scaffolds challenging, especially under the perfusion conditions of multiple cell types, because different cell types often require different cellular microenvironments, which makes them have higher requirements for the microenvironment, and the microenvironment provided by a single dECM scaffold is relatively simple and cannot meet these multiple needs.

[0004] Chinese patent CN112755248A discloses a 3D printed composite bio-ink based on ovarian or vaginal decellularized matrix, which is close to natural tissue matrix and can promote cell growth and tissue repair. However, the artificial vagina printed by it can only achieve subcutaneous transplantation (because it can only promote the regeneration of a small amount of vaginal epithelium and achieve vascularization effect to a certain extent) but cannot achieve in situ transplantation (because it cannot achieve muscle tissue reconstruction, that is, muscle tissue cannot regenerate and vaginal functional characteristics cannot be achieved). Summary of the invention

[0005] The purpose of the present invention is to provide a vaginal stent that can be transplanted in situ, which solves the technical problems that the existing vaginal stents cannot reconstruct vaginal tissue and cannot achieve vaginal functional characteristics.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A method for preparing a 3D vaginal stent that can be transplanted in situ comprises the following steps: (1) Mixing the porcine vaginal decellularized matrix native gelatin with gelatin methacrylamide solution, adding silk fibroin after pasteurization, and mixing evenly to obtain a 3D printing matrix mixture; (2) Load the 3D printing matrix mixture into the 3D printer; use CAD software to design the model structure and parameters according to the vaginal structure to be printed, including the number of layers, inner diameter, and multi-layer contour parameters, convert them into STL format and import them into the printing system; set the printing system to a layer thickness of 0.28 mm and a pinhole diameter of 0.34 mm, and check "Print Contour", "Multi-layer Contour", and "Random Starting Point" in the filling settings. While printing, irradiate the printed object with blue light to obtain a 3D vaginal stent that can be transplanted in situ.

[0007] Preferably, the mass fraction of the porcine vaginal acellular matrix native gel is 3.0 to 3.5%.

[0008] Preferably, the mass fraction of the gelatin methacrylamide solution is 8%. Preferably, the concentration of the silk fibroin in the 3D printing matrix mixture is 50 mg / mL.

[0009] Preferably, the porcine vaginal acellular matrix native gelatin and the gelatin methacrylamide solution are mixed in a volume ratio of 1:1.

[0010] Preferably, the preparation method of the gelatin methacrylamide solution is as follows: The photoinitiator is dissolved in sterile PBS to prepare a photoinitiator solution, and the filtrate is obtained by filtering; the methacrylamide-based gelatin freeze-dried powder is added to the filtrate, and the mixture is mixed to obtain a gelatin methacrylamide solution.

[0011] Preferably, the preparation method of the porcine vaginal acellular matrix native gel is as follows: (1) Grinding: Grind the freeze-dried porcine vaginal decellularized matrix into powder in a grinder with an appropriate amount of liquid nitrogen and store at -20°C for later use; The preparation of porcine vaginal acellular matrix was carried out according to the method disclosed in Chinese patent CN105079881A; (2) Digestion: Take the porcine vaginal decellularized matrix powder and place it in a pepsin solution to prepare the decellularized matrix native gel. Set the shaker at 37°C, 80 rpm, for 24 h. (3) Neutralization: Alkaline solution is added dropwise to the solution obtained in step (2) until the pH value is 7.35-7.45, thereby obtaining a fluidized decellularized matrix solution.

[0012] The present invention also provides a 3D vaginal stent that can be transplanted in situ, which is prepared using the preparation method described above.

[0013] Furthermore, the present invention also provides a method for preparing a 3D vaginal scaffold encapsulating bone marrow mesenchymal stem cells (BMSCs) that can be transplanted in situ: The porcine vaginal decellularized matrix native gelatin was mixed with gelatin methacrylamide solution, and after pasteurization, silk fibroin was added to obtain a 3D printing matrix mixture; Encapsulating the BMSCs single suspension with the 3D printing matrix mixture to obtain a 3D printing matrix mixture encapsulating BMSCs; The 3D printing matrix mixture encapsulating BMSCs is loaded into the 3D printer; the model structure and parameters, including the number of layers, inner diameter, and multi-layer contour parameters, are designed using CAD software according to the vaginal structure to be printed, and converted and saved in STL format and imported into the printing system; the printing system sets the layer thickness to 0.28 mm and the pinhole diameter to 0.34 mm, and selects "print contour", "multi-layer contour", and "random starting point" in the filling settings. While printing, the printed object is irradiated with blue light to obtain a 3D vaginal scaffold encapsulating BMSCs that can be transplanted in situ; After printing, the scaffold was placed in BMSCs culture medium at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator for 15 minutes and waited for orthotopic transplantation.

[0014] Preferably, each milliliter of the 3D printed matrix mixture encapsulates 50 microliters of the BMSCs single suspension.

[0015] The beneficial effects of the present invention are: The present invention provides an in situ transplantable vagina-like stent and a preparation method thereof. The vagina-like stent has stable molding, certain rigidity and strength, strong manufacturability, and good biocompatibility. In particular, it can be used for in situ transplantation of vaginal loss models of large and small animals and repair of missing vaginal tissue. It can not only promote the regeneration of vaginal epithelium and muscle and the reconstruction of new blood vessels, but also directly participate in the regeneration of vaginal epithelium, blood vessels and muscles through differentiation, and finally achieve the reconstruction of a complete vagina-like stent. The obtained vagina-like stent has good shape fidelity, and the regenerated vaginal tissue thereon is similar to normal vaginal tissue in both morphological characteristics and functionality, so it can be transplanted in situ, which is of great significance for the construction of tissue engineering vagina. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a scanning electron micrograph of the native gel.

[0017] Figure 2 This is the rheological test diagram of the original rubber.

[0018] Figure 3 This is a scanning electron microscope image of the 3D printing matrix mixture.

[0019] Figure 4 This is the rheological test diagram of the 3D printing matrix mixture.

[0020] Figure 5 This is the infrared detection image of the 3D printing matrix mixture.

[0021] Figure 6 The 3D vaginal stent printing process.

[0022] Figure 7 This is a photo of the 3D SD rat vaginal scaffold.

[0023] Figure 8 This is a photo of a 3D New Zealand White Rabbit vaginal stent.

[0024] Fig. 9 This is the result of detecting cell proliferation activity using the MTT method using the 3D printing matrix mixture extract.

[0025] Fig.10 Figure 2 shows the cell viability / toxicity assay of the 3D printed matrix mixture.

[0026] Fig.11 The experimental result graph is generated for the small tube, where group A is a simple 3D printing matrix mixture extract group, and group B is a 3D printing matrix mixture extract group encapsulating BMSCs.

[0027] Fig.12The results of VEGF immunofluorescence experiment on HUVECs, where group A is a group with pure 3D printing matrix mixture extract, and group B is a group with 3D printing matrix mixture extract wrapped with BMSCs.

[0028] Fig.13 This is the result of local immune response after in situ transplantation of biological 3D printed SD rat vaginal scaffold (HE staining, 10×).

[0029] Fig.14 This is the result of local immune response after in situ transplantation of biological 3D printed SD rat vaginal scaffold (CD45 immunofluorescence staining, 10× / 20×).

[0030] Fig.15 To reconstruct the gross view of the vagina of SD rats.

[0031] Fig.16 This is a diagram to reconstruct the vaginal neovascularization evaluation of SD rats and the differentiation of rat BMSCs into CD31 and HSP47. Figure A shows the results of CD31 and HSP47 immunohistochemical staining; Figure B shows the in vivo tracing results of BMSCs; and Figure C shows the differentiation of BMSCs into CD31 and HSP47.

[0032] Fig.17 This is a diagram of the reconstruction of vaginal epithelialization and muscle regeneration (α-SAM) in SD rats. Figure A shows the results of CK14 and PCK immunostaining, and Figure B shows the results of Masson and VG staining and α-SAM immunohistochemistry.

[0033] Fig.18 This is the result of gross, morphological and functional examination of the rabbit vagina in the 3D scaffold group and the 3D scaffold cell group after 12 weeks of reconstruction.

[0034] Fig.19 The following is a diagram of epithelialization and neovascularization regeneration in SD rats reconstructed as a comparative example. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with specific examples, and reagents and operations not mentioned in the examples are performed according to conventional operations in the art. The porcine vaginal acellular matrix material of the present invention can be prepared by referring to the method disclosed in Chinese patent CN105079881A.

[0036] Example 1 Preparation of 3D vaginal stent that can be transplanted in situ A method for preparing a 3D vaginal stent that can be transplanted in situ comprises the following steps: S1: Preparation of porcine vaginal acellular matrix solution (native gel) (1) Grinding: Grind the freeze-dried porcine vaginal decellularized matrix into powder in a grinder with an appropriate amount of liquid nitrogen and store at -20°C for later use.

[0037] (2) Digestion: Take 0.1 g of the above powder and place it in 3 mL of 0.01 mol / L hydrochloric acid solution containing 30 mg (10 mg / mL), 45 mg (15 mg / mL), 60 mg (20 mg / mL) and 75 mg (25 mg / mL) of pepsin, respectively, to prepare a 3.33% decellularized matrix solution, i.e., native gelatin. Set the shaker to 37°C, 80 rpm, for 24 h.

[0038] (3) Neutralization: Add about 1.0 mL of 0.01 mM NaOH solution to the above solution to change its acidic state (pH 3.2-3.5) to neutral state (pH 7.35-7.45), and the gel solid state to a fluid state, which is irreversible.

[0039] The bottle inversion method was used to test the native gels made from four different protease concentrations. The first three concentrations met the characteristics of the hydrogel, but only the hydrogel made from 20 mg / mL pepsin did not have any lumpy undigested matter, avoiding the situation where undigested matter blocked the pinholes during 3D printing. Therefore, it was selected as the hydrogel for subsequent 3D printing.

[0040] The original rubber prepared above was identified: (1) Scanning electron microscopy observation of the ultrastructure of native rubber. Native rubber presents a porous network structure ( Figure 1 ).

[0041] (2) Rheological test of raw rubber The prepared raw rubber was placed on the sample stage of Anton Paar's MCR302 advanced rotational rheometer for rheological characterization. The changes of G' (storage modulus) and G" (loss modulus) with amplitude, frequency and time were measured. The amplitude scan results showed that G' was much higher than G", and the material showed solid characteristics ( Figure 2 ).

[0042] S2: Preparation of GelMA Solution Take 15 mg of LAP and add it to 3 ml of sterile PBS (W / V 5%), place it in a 50℃ shaker, melt it for 20 minutes, and filter it through a 0.22μm filter; add 240 mg of GelMA lyophilized powder to the above filtrate, mix it well, and place it in a 50℃ shaker for 30 minutes. The GelMA lyophilized powder has a degree of substitution of 90, a mass fraction of 8% (50℃, 30 minutes melting), and a mass fraction of the photoinitiator (LAP) of 5%.

[0043] S3: Preparation of 3D printing matrix mixture The porcine vaginal decellularized matrix native gel prepared in step 1 was mixed with the GelMA solution prepared in step 2 at a volume ratio of 1:1, pasteurized, and silk fibroin (50 mg / mL) was added and fully dissolved to prepare a matrix mixture for 3D printing. The mixture was placed in a 4°C refrigerator for 3D printing.

[0044] Furthermore, the 3D printing matrix mixture prepared above was freeze-dried in a -80°C freeze dryer for 24 h and observed by scanning electron microscopy. Figure 3 As shown, it can be seen that the 3D printing matrix mixture is more uniform and dense than the original glue.

[0045] The 3D printing matrix mixture was further subjected to rheological testing, such as Figure 4 As shown, it can be seen that the mechanical strength of the 3D printing matrix mixture is also better than that of the original glue.

[0046] The 3D printing matrix mixture was further subjected to infrared detection, as shown in Table 1 and Figure 5 As shown, the 3D printing matrix mixture without UV irradiation is attributed to 1635 cm -1 The -C=C- at the bottom comes from the acrylate double bond in LAP GelMA; after UV irradiation, it disappears, indicating that the double bond undergoes polymerization reaction, and it is speculated that the polymerization forms a three-dimensional network structure; the -OH peak intensity in the 3D printed matrix mixture before and after UV irradiation is greatly reduced and the position moves, which is due to the lower absorption intensity of the sample and the generation of hydrogen bonds after photopolymerization curing; 1527 cm-1 in the 3D printed matrix mixture without UV irradiation and 3187 cm-1 in the 3D printed matrix mixture with UV irradiation are both attributed to -NH in LAPGelMA. For the fingerprint region 400-1300cm -1 Range, 1256, 1077 and 764 cm -1 They belong to -CH-, -CN and -COC- and -CH- vibrations respectively.

[0047] Table 1 S4: 3D printed vaginal stent Use CAD software to draw a 3D vaginal structure, convert it into STL format, and use the second-generation bioprinting system of Genefi for printing (extrusion type). Take 1mL of the above 3D printing matrix mixture and put it into a sterile barrel (specification: 5mL), with a needle diameter of 0.34mm, embed it into a low-temperature nozzle, and fix it on the bioprinter. The nozzle temperature is maintained at 20°C and the printing table temperature is controlled at 4°C. The printing process is: model import → parameter setting (number of layers, layer thickness, pinhole diameter, air pressure, wire speed, etc.) → equipment calibration (platform height measurement, nozzle height measurement) → start printing ( Figure 6). The 3D printing matrix mixture can form uniform lines, and UV irradiation can be applied while printing to print out the designed bracket.

[0048] 1. Design and printing of 3D vaginal scaffolds for 8-10 week old SD rats First, import the circular tube model drawn by CAD software, and set the parameters as follows: needle diameter 0.34mm, layer thickness 0.28mm, number of layers 35 (bracket height is about 1cm), bracket size X 6mm Y 6mm Z 19mm (Z is larger than the actual printed bracket height), check "Print outline", "Multi-layer outline", "Random starting point" in the filling settings, the inner diameter of the circular tube to be printed is 2mm, according to the ductility characteristics of the 3D printing matrix mixture, the inner diameter is theoretically designed to be 3-3.7mm, and the multi-layer outline parameters are set to 1.15-1.5mm ( Figure 7 ).

[0049] 2. Design and printing of 3D vaginal stents for 8-month-old New Zealand white rabbits The model is a double-ring circular model. Inner ring: X8 Y8 Z19 (inner ring diameter is about 7mm), needle diameter, layer thickness, and number of layers are the same as above, check "Print outline" and "Random starting point"; outer ring: X13.6 Y13.6 Z19 (outer ring diameter is about 16mm), needle diameter, layer thickness, and number of layers are the same as above, check "Print outline", "Multi-layer outline", and "Random starting point", and the multi-layer outline parameters are set to 1mm. The length of the rabbit vagina is 4-5cm, and 4-5 of the above double-ring circular models are stacked together to form the required stent ( Figure 8 ).

[0050] Example 2 Vaginal scaffold encapsulating rat BMSCs / New Zealand white rabbit BMSCs In another embodiment, a 3D printing matrix mixture is prepared according to the above steps S1-S3, and then 1 mL of the 3D printing matrix mixture is encapsulated with 50 μL of a single cell suspension (2×10 6 1×10 SD rat BMSCs or 1×10 7 New Zealand white rabbit BMSCs) to obtain a 3D printed matrix mixture encapsulating rat BMSCs; The vaginal-like scaffolds encapsulating rat BMSCs / New Zealand white rabbit BMSCs were printed according to the above step S4.

[0051] Example 3 In vitro biocompatibility testing Extract MTT method to detect cell proliferation activity: Referring to the extract cytotoxicity test standard, 1 mL of sterile 3D printing matrix mixture was added to a 6-well cell culture plate, UV irradiation turned it into a gel, 3-5 mL of bone marrow mesenchymal stem cell (BMSCs) culture medium was added to the well, and placed in a 37°C 5% incubator. After 72 h, the liquid was taken out as the extract, filtered and sterilized with a 0.22μm filter and stored at 4°C for use. The cells were divided into 3 groups based on different extract concentrations: Group 1 was cultured with BMSCs culture medium alone, and Groups 2-3 were cultured with 25% and 50% extracts by volume. Different groups of material extracts were added to each well of the 96-well plate where BMSCs had grown, with 5 samples in each group. MTT solution was added at D1, D3, D5, and D7, and the supernatant was discarded after incubation for 4 h. 150 μL DMSO was added to each well and shaken for 10 min to fully dissolve the crystals.

[0052] MTT solution colorimetric detection: Select 490 nm wavelength and measure the absorbance value of each group on an enzyme-linked immunosorbent assay. Fig. 9 It can be seen that the cell proliferation rates of the three groups were similar.

[0053] Cell live / death experiment: The vaginal scaffolds encapsulating rat BMSCs were taken and three samples were set up. LIVE / DEAD cell viability / toxicity tests were performed on D1, D4, and D7, and two-photon microscope films were taken. The cells in the vaginal scaffolds were observed to be evenly distributed, with regular spherical shapes, no swelling or shrinkage; live cells showed green fluorescence, and dead cells showed red fluorescence. The number of cells at D7 was similar to that at D1 ( Fig.10 ).

[0054] The above experiments show that the vaginal stent of the present invention has good biocompatibility.

[0055] Example 4 In vitro culture to promote angiogenesis experimental detection 1. Tubule formation experiment The extract of the simple 3D printing matrix mixture (liquid A), encapsulated rat BMSCs (4×10 per ml of 3D printing matrix mixture) were prepared respectively. 6 3D printing matrix mixture extract (liquid B) was prepared in the same way as above. Human umbilical vein endothelial cells (HUVECs) were purchased and cultured in the same way as conventional cells. P2-HUVECs (human umbilical vein endothelial cell line) were cultured and subcultured with 1 mL of liquid A / liquid B / PBS+4 mL of fetal bovine HUVECs culture medium to obtain P3-HUVECs, and each well of a 24-well plate covered with Matrigel was inoculated with 4×10 4Each well of the HUVECs was filled with 1 mL of culture medium (200 μL of A solution / B solution / PBS + 800 μL of fetal bovine HUVECs culture medium), and placed in a 37°C, CO2 cell culture incubator. Angiogenesis was observed at 4 h and 7 h, respectively.

[0056] like Fig.11 As shown, the results showed that both the 3D printed matrix mixture and BMSCs have the ability to promote angiogenesis, and the 3D printed matrix mixture encapsulating BMSCs is superior to the simple 3D printed matrix mixture in terms of the number of cavities and the number of branches.

[0057] 2. VEGF immunofluorescence experiment on HUVECs The above P3-HUVECs were seeded in 6-well plates (3×10 5 Each well was filled with 2 mL of culture medium (500 μL of A solution / B solution / PBS+1500 μL of fetal bovine HUVECs culture medium), and VEGF immunofluorescence staining was performed when HUVECs grew to 80%-90%. Fig.12 As shown, it can be seen that the 3D printed matrix mixture of the present invention can increase VEGF expression.

[0058] Example 5 Detection of local immune response in rat vaginal stent orthotopic transplantation A rat vaginal defect model was constructed and randomly divided into a normal control group (12 rats), a SIS control group (16 rats), a 3D scaffold group (16 rats), and a 3D scaffold cell group (16 rats). Three animals were randomly selected from each group at 1, 2, 4, and 8 weeks after surgery to collect vaginal tissue. The tissues were waxed, sectioned, and stained with HE and CD45 antibody immunofluorescence chemical staining, and observed under a light microscope. It was found that all rats survived within 8 weeks after implantation without complications. Hematoxylin-eosin staining (HE) ( Fig.13 ) and immunofluorescence ( Fig.14 ) The results showed that the number of inflammatory cells gradually increased one week after implantation, while the number of inflammatory cells gradually decreased from 2 to 8 weeks.

[0059] Example 6 Histomorphological and functional evaluation of vaginal reconstruction in SD rats and New Zealand white rabbits using vaginal stents A rat / rabbit vaginal loss model was constructed, and SD rats were randomly divided into a control group (10 rats), a 3D scaffold group (16 rats), a 3D scaffold cell group (3D scaffold encapsulating BMSCs) (16 rats), and a SIS group (16 rats). Three animals were randomly selected from each group to collect vaginal tissues at 2 and 4 weeks after surgery, respectively. New Zealand white rabbits were randomly divided into a control group (5 rats), a 3D scaffold group (5 rats), and a 3D scaffold cell group (5 rats) to collect samples at 12 weeks after surgery. The SIS group is a commercial scaffold constructed using a decellularized matrix of the small intestinal mucosa.

[0060] The expression of specific proteins in each tissue layer was evaluated by immunohistochemical staining of CD31, HSP47, PCK14, and α-SAM. Fig.15 The gross appearance of the reconstructed SD rat vagina is shown in Figure 1. The shape fidelity is very good. CD31 and HSP47 immunohistochemical staining showed that vaginal neovascularization was present in the reconstructed rat vaginal tissue. The positive signals of CD31 and HSP47 were more in the 3D scaffold cell group and the 3D scaffold group than in the SIS group ( Fig.16 A). Observation of the in vivo tracking of BMSCs labeled with CM-Dil revealed that BMSCs can differentiate into CD31 and HSP47 ( Fig.16 B), indicating that the mixture of BMSCs and acellular matrix-derived 3D-printed matrix has a synergistic effect on vascularization in vaginal reconstruction.

[0061] The reconstructed tissues were further immunostained for CK14 and PCK to evaluate epithelial regeneration. Compared with the normal group, more positive signals were detected in both the 3D scaffold cell group and the 3D scaffold group, especially at 4 weeks after transplantation ( Fig.17 A). Masson and VG staining showed that 4 weeks after transplantation, the area of ​​collagen fibers in the reconstructed vagina increased and the muscle fibers became more obvious, especially in the 3D scaffold cell group ( Fig.17 B). The results of α-SAM immunohistochemistry showed that the muscle fiber density of the 3D scaffold cell group was higher than that of the 3D scaffold group and SIS group ( Fig.17 B).

[0062] HE, Masson, and VG staining were used to evaluate the morphological characteristics of the reconstructed vaginal tissues in each group. It can be seen that the surface of the rabbit vaginal tissue 12 weeks after reconstruction is rich in blood vessels, and many wrinkles can be seen on the mucosal surface. The texture is tough and elastic, similar to normal vaginal tissue, especially the 3D scaffold cell group. Specifically, in terms of morphology, HE staining, Masson staining, and VG staining of normal vaginal tissue can be seen to have stratified squamous epithelium, and under the epithelium is a loose submucosal layer rich in blood vessels and collagen fibers. The submucosal layer is a smooth muscle layer with an inner circular and an outer longitudinal shape, and blood vessels and collagen fibers are interlaced in it. Fig.17 As shown in B, Masson and VG staining showed that 12 weeks after surgery, several layers of epithelial cells were visible in the 3D scaffold cell group and the 3D scaffold group reconstructed by the present invention, arranged in a scale-like manner, which was a stratified squamous epithelium. Abundant blood vessels were seen running between the fibers in the submucosal layer, and red (Masson staining) / yellow (VG staining) muscle fibers were also seen around the vascular wall. In addition, obvious muscle fibers were seen to be distributed longitudinally in an inner ring and an outer ring. The results showed that a large number of new blood vessels were generated in the vagina reconstructed by the 3D scaffold cell group and the 3D scaffold group, and the epithelium and muscle layer were formed at the same time, and the structure was similar to that of the normal vagina.

[0063] PAS staining and scanning electron microscopy were used to evaluate the function of the reconstructed tissue. Compared with the normal vagina, a large number of red positive cells were observed in the vaginal tissue of the 3D scaffold cell group and the 3D scaffold group after 12 weeks of reconstruction, indicating a large amount of glycogen synthesis, especially the 3D scaffold cell group was closer to the normal group. Scanning electron microscopy showed that the reconstructed vaginal epithelial mucosa surface was smooth, mucosal folds could be observed, and the epithelial cells were plump and arranged regularly, with no significant difference from normal epithelial tissue ( Fig.18 ).

[0064] Comparative Example In this comparative example, an artificial vagina was printed according to the method disclosed in Chinese patent CN112755248A, and its effect was evaluated. The experiment found that the artificial vagina scaffold can only be transplanted subcutaneously, but not in situ. Specifically, the artificial vagina scaffold was transplanted subcutaneously in SD rats, and the epithelial regeneration was evaluated by HE staining, and the neovascularization was evaluated by CD31 immunohistochemical staining. The test results are as follows: Fig.19 As shown, it can be seen that it can achieve the vascularization effect and there is vaginal epithelial regeneration, but the amount of vaginal epithelium is very small, there is no muscle tissue, and it does not have the functional characteristics of the vagina, which is far from normal vaginal tissue (normal vaginal tissue includes epithelial tissue, muscle tissue and new blood vessels, etc.).

Claims

1. A method for preparing a 3D vaginal stent that can be transplanted in situ, It is characterized in that The following steps are involved: The porcine vaginal decellularized matrix native gelatin and gelatin methacrylamide solution were mixed, and after pasteurization, silk fibroin was added and mixed evenly to obtain a 3D printing matrix mixture; The 3D printing matrix mixture was loaded into the 3D printer; the model structure and parameters, including the number of layers, inner diameter, and multi-layer contour parameters, were designed using CAD software according to the vaginal structure to be printed, and converted and saved in STL format and imported into the printing system; the printing system set the layer thickness to 0.28 mm and the pinhole diameter to 0.34 mm, and checked "print contour", "multi-layer contour", and "random starting point" in the filling settings. While printing, the printed object was irradiated with blue light to obtain a 3D vaginal stent that can be transplanted in situ.

2. The preparation method according to claim 1, It is characterized in that The mass fraction of the porcine vaginal acellular matrix native gel is 3.0-3.5%.

3. The preparation method according to claim 1, It is characterized in that The mass fraction of the gelatin methacrylamide solution is 8%.

4. The preparation method according to claim 1, It is characterized in that The porcine vaginal acellular matrix native gelatin and gelatin methacrylamide solution were mixed in a volume ratio of 1:

1.

5. The preparation method according to claim 1, It is characterized in that The concentration of the silk fibroin in the mixture is 50 mg / mL.

6. The preparation method according to claim 1, It is characterized in that The preparation method of the gelatin methacrylamide solution is as follows: The photoinitiator is dissolved in sterile PBS to prepare a photoinitiator solution, and the filtrate is obtained by filtering; the methacrylamide-based gelatin freeze-dried powder is added to the filtrate, and the mixture is mixed to obtain a gelatin methacrylamide solution.

7. A 3D vaginal stent that can be transplanted in situ. It is characterized in that It is prepared by the preparation method described in any one of claims 1 to 6.

8. A method for preparing a 3D vaginal scaffold encapsulating bone marrow mesenchymal stem cells that can be transplanted in situ, It is characterized in that The porcine vaginal decellularized matrix native gelatin was mixed with gelatin methacrylamide solution, and after pasteurization, silk fibroin was added to obtain a 3D printing matrix mixture; Encapsulating a single suspension of bone marrow mesenchymal stem cells with a 3D printing matrix mixture to obtain a 3D printing matrix mixture encapsulating bone marrow mesenchymal stem cells; The 3D printing matrix mixture encapsulating bone marrow mesenchymal stem cells was loaded into the 3D printer; the model structure and parameters, including the number of layers, inner diameter, and multi-layer contour parameters, were designed using CAD software according to the vaginal structure to be printed, and converted and saved in STL format and imported into the printing system; the printing system set the layer thickness to 0.28 mm and the pinhole diameter to 0.34 mm, and checked "print contour", "multi-layer contour", and "random starting point" in the filling settings. While printing, the printed object was irradiated with blue light to obtain a 3D vaginal scaffold encapsulating bone marrow mesenchymal stem cells that can be transplanted in situ.

9. The preparation method according to claim 8, It is characterized in that Each milliliter of 3D printed matrix mixture encapsulates 50 microliters of bone marrow mesenchymal stem cell suspension.

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