An anal fistula plug stent material and a preparation method thereof

Abstract

The present application belongs to the technical field of animal-derived implantable medical devices, and particularly relates to an anal fistula plug stent material and a preparation method thereof. The anal fistula plug stent material is prepared by using a decellularized pig skin matrix as a raw material, removing cell components and DNA components, completely retaining extracellular matrix components and structures, and having a microporous structure; and by dissolving and compounding, freeze-drying and molding, packaging and sterilizing with platelet-rich autologous concentrated growth factors obtained by differential centrifugal separation and a crosslinking agent, genipin. The anal fistula plug stent material prepared by the present application uses a nearly inbred animal decellularized dermal matrix as a main raw material source, has pure, stable and unified genetic characteristics, a natural ECM three-dimensional structure, low immunogenicity and strong anti-infection ability; and is also rich in bioactive factors for promoting anal fistula tissue repair and growth. The anal fistula plug stent material of the present application can promote the healing of fistula, and ultimately achieve the purpose of minimally invasive treatment of anal fistula.

Description

Technical Field

[0001] This invention belongs to the field of animal-derived implantable medical device technology, specifically relating to an anal fistula stent material and its preparation method. Background Technology

[0002] Anal fistula is a common anorectal disease, mainly caused by infection of the rectum and perianal area. Its main characteristic is the formation of a chronic infection channel between the anus or rectum and the perineal skin. Clinically, anal fistula manifests as recurrent perianal infections, rupture, and purulent discharge. Patients with long-term non-healing fistulas may develop perianal cancer. The main goals of treating anal fistula are to remove the lesion, ensure unobstructed drainage, minimize damage to the anal sphincter, and protect anal function. Traditional treatments for anal fistula include fistula excision, fistula incision, seton placement, fistula drainage, and transanorectal mucosal flap repair. These surgical methods all have problems with anal dysfunction and recurrence. The incidence of postoperative anal incontinence is between 10% and 65%, and the recurrence rate is between 10% and 20%, resulting in a poor quality of life for patients. Currently, fistula healing and preservation of anal function are considered key indicators of treatment success. Minimally invasive biological therapy has become a major development direction in the treatment of anal fistula. Among them, fistula plug tampon therapy has advantages such as minimal surgical trauma and less damage to the anal sphincter, and has become a new approach to treating anal fistula in recent years. Fistula plugs are biomaterials used to treat anal fistulas, which stimulate the repair and reconstruction of fistula tissue and can be absorbed or degraded in vivo. In particular, fistula plugs using acellular allogeneic dermal matrix as material can induce angiogenesis and fibroblast growth, thereby filling the fistula. This method is characterized by being minimally invasive, repeatable, and causing no anal deformities.

[0003] In existing technologies, biomaterials made from porcine small intestinal mucosa are used to treat anal fistulas through a packing method. This surgery is minimally invasive, provides patients with a high quality of life, has a low recurrence rate, and a success rate of up to 80% cure rate. However, its effectiveness still needs further research. In addition, the production process of this material is complex and expensive.

[0004] Fistula healing involves three stages: the inflammatory response phase, the granulation tissue proliferation phase, and the epithelialization phase. Cytokines play a crucial role in each stage. Platelet-rich plasma (PRP), extracted from autologous blood through centrifugation, contains high concentrations of epidermal growth factor (EGF) and other bioactive growth factors. It effectively activates the EGF-ERK signaling pathway, thus playing a vital role in fistula healing and accelerating its healing process. Extracellular matrix (ECM) materials based on animal tissue, grounded in tissue engineering principles, represent a major development direction for medical implant materials. ECM is a complex, organic, three-dimensional structure composed of various macromolecules such as collagen, non-collagenous glycoproteins, glycosaminoglycans, proteoglycans, and elastin, arranged in specific proportions and structures. It provides a suitable environment for the survival and activity of various cells, regulating their growth, shape, metabolism, migration, proliferation, and differentiation, thereby modulating tissue and organ function. Summary of the Invention

[0005] Based on the aforementioned shortcomings and deficiencies in the prior art, one of the objectives of this invention is to at least solve one or more of the aforementioned problems in the prior art. In other words, one of the objectives of this invention is to provide an anal fistula stent material and its preparation method that meet one or more of the aforementioned requirements. The material is prepared by using decellularized porcine skin matrix from inbred animals as raw material, and combining it with platelet-rich autologous concentrated growth factor obtained by differential centrifugation and cross-linking agent genipin through dissolution, freeze-drying, packaging, and sterilization. The material completely retains the components and three-dimensional structure of the extracellular matrix and is rich in bioactive factors that promote the repair and growth of anal fistula tissue.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for preparing anal fistula stent material includes the following steps: (1) Dissolve the decellularized pigskin matrix in an acidic solution and perform enzymatic hydrolysis. After enzymatic hydrolysis, add hyaluronic acid and dissolve to obtain a decellularized pigskin matrix solution. (2) Add the cross-linking agent genipin to the decellularized porcine skin matrix solution to cross-link and obtain a gel; (3) The gel is added to concentrated growth factor for soaking, and then freeze-dried. After freeze-drying, it is formed into anal fistula plug stent material.

[0007] As a preferred embodiment, in step (1), the acidic solution is an aqueous solution of acetic acid with a concentration of 0.5–1 M; Enzymatic hydrolysis was performed using pepsin, with a pepsin mass fraction of 25–50 wt% relative to the decellularized porcine skin matrix.

[0008] As a preferred embodiment, in step (1), the mass ratio of hyaluronic acid to decellularized porcine skin matrix is ​​1:(1-2).

[0009] As a preferred embodiment, in step (2), the mass fraction of the crosslinking agent genipin relative to the decellularized porcine skin matrix is ​​0.01 to 0.1 wt%.

[0010] The present invention also provides a method for preparing anal fistula plug stent material, comprising the following steps: (1) The decellularized pigskin matrix was placed in an acidic solution for enzymatic hydrolysis. After enzymatic hydrolysis, hyaluronic acid was added and dissolved to obtain a decellularized pigskin matrix solution. (2) Add concentrated growth factor to decellularized pig skin matrix solution and stir well to obtain a mixed solution; (3) Add the crosslinking agent genipin to the mixed solution for crosslinking, and freeze-dry after crosslinking. After freeze-drying, it is formed into anal fistula stent material.

[0011] As a preferred embodiment, in step (1), the acidic solution is an aqueous solution of acetic acid with a concentration of 0.5–1 M; Enzymatic hydrolysis was performed using pepsin, with a pepsin mass fraction of 25–50 wt% relative to the decellularized porcine skin matrix. The mass ratio of hyaluronic acid to decellularized porcine skin matrix is ​​1:(1-2).

[0012] As a preferred embodiment, in step (2), the mass fraction of the concentrated growth factor relative to the decellularized porcine skin matrix is ​​10-30 wt%.

[0013] As a preferred embodiment, in step (3), the mass fraction of the crosslinking agent genipin relative to the decellularized porcine skin matrix is ​​0.01 to 0.1 wt%.

[0014] The present invention also provides an anal fistula stent material prepared by the preparation method described in any of the preceding embodiments.

[0015] As a preferred embodiment, the fistula plug support material is a hollow conical structure obtained by multi-layer molding of a membrane roll or a multi-layer strip structure formed by pressing.

[0016] Compared with the prior art, the beneficial effects of this invention are: (1) The anal fistula stent material prepared by the present invention retains the complete components and three-dimensional structure of the extracellular matrix. It is treated with acetic acid aqueous solution and pepsin enzymatic hydrolysis, which can enhance the mechanical strength of the material without destroying the biological activity of ECM and avoid premature collapse after implantation. (2) Controllable degradation rate: Genipin, as a natural cross-linking agent, has a mild and controllable degree of cross-linking, which makes the degradation rate of the scaffold material in vivo more matched with the ingrowth rate of new tissue, which is conducive to the complete healing of the fistula. (3) Significant therapeutic effect: The dual bioactive factor loading retains the inherent growth factors (such as bFGF and VEGF) of the decellularized matrix, while also compounding autologous concentrated growth factors and additionally enriching various autologous growth factors, forming a synergistic effect of "ECM structure + exogenous growth factors", which significantly promotes angiogenesis, fibroblast proliferation and fistula epithelialization; in addition, the added hyaluronic acid has anti-inflammatory, cell migration and matrix remodeling functions, and when combined with ECM and growth factors, it further accelerates the repair of fistula tissue; compared with the growth factors contained in the decellularized porcine skin matrix alone, its healing effect is significantly increased. (4) The significant synergistic effect of the triple modification (enzymatic hydrolysis-hyaluronic acid-genipin): Existing technologies simply combine decellularized matrix and PRP, while the present invention exposes more active sites through moderate enzymatic hydrolysis by pepsin, hyaluronic acid provides a hydrophilic microenvironment, and genipin achieves stable cross-linking. The synergistic effect of the three makes the scaffold material have comprehensive properties such as high porosity (>85%), suitable mechanical strength, continuous release of growth factors, and promotion of new granulation tissue growth. (5) Economic efficiency and potential for comprehensive resource utilization: By utilizing inexpensive and abundant pigskin by-products, material costs are significantly reduced. The use of low-temperature enzymatic hydrolysis, natural cross-linking agent genipin, and general-purpose freeze-drying equipment results in low energy consumption and easy large-scale production. The product of this invention can replace expensive imported anal fistula plugs and reduce postoperative complications such as incontinence and recurrence through minimally invasive treatment, shortening hospital stay and significantly reducing overall medical expenses. In addition, the low-value pigskin is transformed into Class III implantable devices, realizing the added value of agricultural by-products and conforming to the concept of green and sustainable medical device development. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of the conical anal fistula plug support of the present invention; Figure 2 This is a schematic diagram of the structure of the strip-shaped anal fistula plug support of the present invention; Figure 3 This is an electron microscope image of the surface ultrastructure of the anal fistula plug stent of Embodiment 8 of the present invention; Figure 4 This is a cross-sectional optical microscope image of the fistula plug stent of Embodiment 8 of the present invention; Figure 5 This is a cross-sectional optical microscope image of the fistula plug stent of Embodiment 8 of the present invention. Detailed Implementation

[0018] The following provides a detailed description of the anal fistula stent material and its preparation method according to the present invention.

[0019] The fistula stent material of the present invention uses decellularized porcine skin matrix from inbred pigs, autologous concentrated growth factors, cross-linking agent genipin, and hyaluronic acid as raw materials; The residual amount of matrix DNA in decellularized porcine skin was ≤100 pg / g; The above-mentioned autologous concentrated growth factor was prepared by differential centrifugation of porcine autologous peripheral blood, as follows: The differential centrifugation preparation method is as follows: about 10 mL of peripheral blood is drawn and placed in a sterile test tube without anticoagulant. Centrifugation is started immediately, accelerated for 30 seconds, and then centrifuged sequentially at 2600 rpm for 2 min, 2200 rpm for 4 min, 2600 rpm for 4 min, and 3200 rpm for 3 min. Finally, the centrifugation is decelerated to stop for 36 seconds. After standing still, the liquid in the test tube is divided into three layers: the top layer is platelet-poor plasma, the middle layer is autologous concentrated growth factor, and the bottom layer is red blood cell.

[0020] The method for preparing the anal fistula stent material of the present invention includes the following steps: (1) Add decellularized porcine skin matrix and pepsin to an acid solution for enzymatic hydrolysis to obtain decellularized porcine skin matrix solution; The ratio of decellularized porcine skin matrix to acid solution is 1-2g:100mL, and the specific ratio can be determined according to actual application requirements. The mass fraction of the above-mentioned pepsin relative to the decellularized porcine skin matrix is ​​25-50 wt%; the specific mass fraction can be determined according to the actual application requirements. The above-mentioned acid solution is an aqueous solution of acetic acid with a concentration of 0.5–1 M. The specific concentration can be determined according to the actual application requirements. (2) The enzymatically hydrolyzed decellularized pigskin matrix solution was mixed and dissolved with hyaluronic acid to obtain an enhanced solution; The mass ratio of hyaluronic acid to decellularized porcine skin matrix is ​​1:(1-2), and the specific mass ratio can be determined according to the actual application requirements. (3) Add the cross-linking agent genipin to the reinforcing solution to cross-link and obtain a gel; The mass fraction of the cross-linking agent genipin relative to the decellularized porcine skin matrix is ​​0.01–0.1 wt%, and the specific mass fraction can be determined according to the actual application requirements. (4) The gel was soaked in autologous concentrated growth factor and then freeze-dried to form anal fistula plug stent material. The autologous concentrated growth factor can be diluted in water or PBS buffer for soaking. The aforementioned fistula plug support material is a hollow conical structure obtained by multi-layer molding of membrane rolls or a multi-layer strip structure obtained by compression molding; (5) The anal fistula stent material is packaged under sterile conditions. The inner layer is made of Tyvek paper and the outer layer is made of polyethylene plastic. After packaging, it is sterilized with ethylene oxide.

[0021] Another method for preparing the anal fistula plug support material of the present invention includes the following steps: (1) Add decellularized porcine skin matrix and pepsin to an acid solution for enzymatic hydrolysis to obtain decellularized porcine skin matrix solution; The ratio of decellularized porcine skin matrix to acid solution is 1-2g:100mL, and the specific ratio can be determined according to actual application requirements. The mass fraction of the above-mentioned pepsin relative to the decellularized porcine skin matrix is ​​25-50 wt%; the specific mass fraction can be determined according to the actual application requirements. The above-mentioned acid solution is an aqueous solution of acetic acid with a concentration of 0.5–1 M. The specific concentration can be determined according to the actual application requirements. (2) The enzymatically hydrolyzed decellularized pigskin matrix solution was mixed and dissolved with hyaluronic acid to obtain an enhanced solution; The mass ratio of hyaluronic acid to decellularized porcine skin matrix is ​​1:(1-2), and the specific mass ratio can be determined according to the actual application requirements. (3) Add the concentrated growth factor to the decellularized pig skin matrix solution and stir until homogeneous to obtain a mixed solution; The concentrated growth factor has a mass fraction of 10–30 wt% relative to the decellularized porcine skin matrix, and the specific mass fraction can be determined according to the actual application requirements. (4) Add the crosslinking agent genipin to the mixed solution for crosslinking, and freeze-dry after crosslinking. After freeze-drying, it is formed into anal fistula plug stent material; The mass fraction of the cross-linking agent genipin relative to the decellularized porcine skin matrix is ​​0.01–0.1 wt%, and the specific mass fraction can be determined according to the actual application requirements. The aforementioned fistula plug support material is a hollow conical structure obtained by multi-layer molding of membrane rolls or a multi-layer strip structure obtained by compression molding; (5) The anal fistula stent material is packaged under sterile conditions. The inner layer is made of Tyvek paper and the outer layer is made of polyethylene plastic. After packaging, it is sterilized with ethylene oxide.

[0022] The following specific embodiments further explain the material and preparation method of the anal fistula plug stent of the present invention.

[0023] Example 1: The method for preparing the decellularized porcine skin matrix in this embodiment includes the following steps: (1) Identification of animal source and pretreatment and initial washing of pig skin tissue; The inbred Wuzhishan miniature pig was selected as the animal source. The skin tissue of freshly slaughtered inbred Wuzhishan miniature pigs was cleaned and rinsed three times with water for injection. (2) Virus inactivation; The virus was inactivated using a peracetic acid-ethanol solution method. This step was carried out in a constant-temperature ultrasonic cleaner with an oscillating tank. The volume percentage of peracetic acid was 0.1%, the inactivation time was 1 hour, and the temperature was 25°C. Then, the material was washed three times in phosphate buffer for 15 minutes each time. The pH value of the phosphate buffer after washing was measured. When the pH reached 6.5-7.5, the material was washed with flowing water for injection. The process was terminated when the conductivity reached below 1.5 μm / s. The oscillation frequency of the cleaning tank is 200 rpm, and the ultrasonic frequency is 45 kHz. (3) Decellularization; This step is performed in a constant-temperature ultrasonic cleaner with an oscillating cleaning tank. First, the material is placed in the cleaning tank, and then sodium hydroxide solution is injected into the cleaning tank. The cleaner is turned on and the cleaning time is 20 minutes. The concentration of sodium hydroxide solution is 10 mmol / L. Then, the cleaner is turned off, the sodium hydroxide solution is poured out, and phosphate buffer solution is injected for cleaning. The cleaner is turned on and the cleaning time is 15 minutes. The phosphate buffer solution is used to repeat the cleaning 3 times. The pH value of the phosphate buffer solution after cleaning is detected. When the pH reaches 6.5-7.5, the material is cleaned with flowing water for injection. The cleaning is terminated when the conductivity reaches below 1.5 μm / s. The cleaning tank has an oscillation frequency of 200 rpm and an ultrasonic frequency of 45 kHz. (4) DNA removal treatment; This step is carried out in a constant temperature ultrasonic cleaner with an oscillating cleaning tank. Sodium chloride solution is injected into the cleaning tank, the cleaner is turned on, the cleaning time is 20 minutes, the concentration of sodium chloride solution is 0.015 mol / L, the pH value does not exceed 7.8, and then the material is cleaned with flowing water for injection. The cleaning is terminated when the conductivity reaches below 1.5 μm / s. The cleaning tank has an oscillation frequency of 200 rpm and an ultrasonic frequency of 45 kHz. (5) Drying to obtain decellularized pigskin matrix.

[0024] Example 2: The method for preparing the anal fistula stent in this embodiment includes the following steps: (1) Take 1.5g of decellularized porcine skin matrix and 0.5g of pepsin from Example 1, add them to 100mL of 0.5M acetic acid aqueous solution at 4℃ for enzymatic hydrolysis, and stir at 300r / min for 48h to obtain decellularized porcine skin matrix solution; (2) After freezing the decellularized porcine skin matrix solution, freeze-dry it for 24 hours. Then freeze-dry it according to the pre-designed freeze-drying process: pre-freeze to -25℃ and keep warm for 2 hours, raise the temperature to -15℃ and keep warm for 12 hours, raise the temperature to 15℃ and keep warm for 2 hours, raise the temperature to 25℃ and keep warm for 4 hours. The freeze-drying is completed to obtain the decellularized porcine skin matrix membrane. (3) Hollow conical structure anal fistula stent material obtained by pressing decellularized porcine skin matrix membrane into 8-layer strips or 4-layer rolls, such as Figure 1 or Figure 2 As shown; (4) The anal fistula stent material is packaged under sterile conditions. The inner layer is made of Tyvek paper and the outer layer is made of polyethylene plastic. After packaging, it is sterilized with ethylene oxide.

[0025] Example 3: The difference between the preparation method of the anal fistula stent in this embodiment and that in embodiment 2 is that hyaluronic acid is added before the freezing treatment in step (2); Specifically, 1.5g of hyaluronic acid was added to the decellularized porcine skin matrix solution and dissolved at room temperature. The solution was stirred at 300r / min for 2h to obtain a mixed solution. After freezing treatment, the solution was freeze-dried for 24h. The freeze-drying process was carried out according to the pre-designed freeze-drying procedure: pre-freeze to -25℃ and hold for 2h, raise the temperature to -15℃ and hold for 12h, raise the temperature to 15℃ and hold for 2h, raise the temperature to 25℃ and hold for 4h. The freeze-drying was completed to obtain the decellularized porcine skin matrix membrane. The other steps are the same as in Example 2.

[0026] Example 4: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 3 in that: 0.01 wt% of the natural crosslinking agent genipin (0.15 mg) was added to the mixed solution of Example 3 to carry out the crosslinking reaction. The crosslinking was carried out at 37°C in the dark for 5 h to obtain a gel. After freezing treatment, the gel was freeze-dried for 24 h according to the pre-designed freeze-drying process: pre-freeze to -25°C and hold for 2 h, raise the temperature to -15°C and hold for 12 h, raise the temperature to 15°C and hold for 2 h, raise the temperature to 25°C and hold for 4 h. The freeze-drying was completed to obtain a decellularized porcine skin matrix membrane. The other steps are the same as in Example 3.

[0027] Example 5: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 3 in that: 0.03 wt% of the natural crosslinking agent genipin (0.45 mg) was added to the mixed solution of Example 3 to carry out the crosslinking reaction. The crosslinking was carried out at 37°C in the dark for 5 h to obtain a gel. After freezing treatment, the gel was freeze-dried for 24 h according to the pre-designed freeze-drying process: pre-freeze to -25°C and hold for 2 h, raise the temperature to -15°C and hold for 12 h, raise the temperature to 15°C and hold for 2 h, raise the temperature to 25°C and hold for 4 h. The freeze-drying was completed to obtain a decellularized porcine skin matrix membrane. The other steps are the same as in Example 3.

[0028] Example 6: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 3 in that: 0.05 wt% of the natural crosslinking agent genipin (0.75 mg) was added to the mixed solution of Example 3 to carry out the crosslinking reaction. The crosslinking was carried out at 37°C in the dark for 5 h to obtain a gel. After freezing treatment, the gel was freeze-dried for 24 h according to the pre-designed freeze-drying process: pre-freeze to -25°C and hold for 2 h, raise the temperature to -15°C and hold for 12 h, raise the temperature to 15°C and hold for 2 h, raise the temperature to 25°C and hold for 4 h. The freeze-drying was completed to obtain a decellularized porcine skin matrix membrane. The other steps are the same as in Example 3.

[0029] Example 7: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 3 in that: 0.1 wt% of the natural crosslinking agent genipin (1.5 mg) was added to the mixed solution of Example 3 to carry out the crosslinking reaction. The crosslinking was carried out at 37°C in the dark for 5 h to obtain a gel. After freezing treatment, the gel was freeze-dried for 24 h according to the pre-designed freeze-drying process: pre-freeze to -25°C and hold for 2 h, raise the temperature to -15°C and hold for 12 h, raise the temperature to 15°C and hold for 2 h, raise the temperature to 25°C and hold for 4 h. The freeze-drying was completed to obtain a decellularized porcine skin matrix membrane. The other steps are the same as in Example 3.

[0030] Example 8: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 3 in that: 0.3 g of autologous concentrated growth factor was added to the mixed solution of Example 3, and stirred at 300 r / min for 1 h at room temperature until the solution was uniformly mixed; then 0.03 wt% of the natural cross-linking agent genipin (0.45 mg) was added, and cross-linking was carried out at 37 °C in the dark for 5 h. After freezing treatment, the solution was freeze-dried for 24 h according to the pre-designed freeze-drying process: pre-freeze to -25 °C and hold for 2 h, raise the temperature to -15 °C and hold for 12 h, raise the temperature to 15 °C and hold for 2 h, raise the temperature to 25 °C and hold for 4 h. The freeze-drying was completed to obtain the decellularized porcine skin matrix membrane. The other steps are the same as in Example 3; In this embodiment, the decellularized porcine skin matrix membrane is pressed into an 8-layer strip-shaped or 4-layer hollow conical structure anal fistula plug scaffold material, such as... Figure 4 and Figure 5 As shown.

[0031] Example 9: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 3 in that: 0.3g of autologous concentrated growth factor was added to the mixed solution of Example 3, and stirred at 300r / min for 1h at room temperature until the solution was uniformly mixed; then 0.05wt% of the natural cross-linking agent genipin (0.75mg) was added, and cross-linking was carried out at 37℃ in the dark for 5h. After freezing treatment, the solution was freeze-dried for 24h according to the pre-designed freeze-drying process: pre-freeze to -25℃ and hold for 2h, raise the temperature to -15℃ and hold for 12h, raise the temperature to 15℃ and hold for 2h, raise the temperature to 25℃ and hold for 4h. The freeze-drying was completed to obtain the decellularized porcine skin matrix membrane. The other steps are the same as in Example 3.

[0032] Example 10: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 3 in that: 0.3 g of autologous concentrated growth factor was added to the mixed solution of Example 3, and stirred at 300 r / min for 1 h at room temperature until the solution was uniformly mixed; then 0.1 wt% of the natural cross-linking agent genipin (1.5 mg) was added, and cross-linking was carried out at 37 °C in the dark for 5 h. After freezing treatment, the solution was freeze-dried for 24 h according to the pre-designed freeze-drying process: pre-freeze to -25 °C and hold for 2 h, raise the temperature to -15 °C and hold for 12 h, raise the temperature to 15 °C and hold for 2 h, raise the temperature to 25 °C and hold for 4 h. After freeze-drying, decellularized porcine skin matrix membrane was obtained. The other steps are the same as in Example 3.

[0033] Example 11: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 5 in that: Add 0.3g of autologous concentrated growth factor to 100mL of water, mix well, and then surface-load the gel of Example 5 for 1h. After freezing treatment, freeze-dry for 24h. Freeze-dry according to the pre-designed freeze-drying process: pre-freeze to -25℃ and hold for 2h, raise the temperature to -15℃ and hold for 12h, raise the temperature to 15℃ and hold for 2h, raise the temperature to 25℃ and hold for 4h. After freeze-drying, decellularized porcine skin matrix membrane is obtained. The remaining steps are the same as in Example 5.

[0034] Example 12: The method for preparing the fistula stent in this embodiment differs from that in Embodiment 6 in that: Add 0.3g of autologous concentrated growth factor to 100mL of water, mix well, and then surface-load the gel of Example 6 for 1h. After freezing treatment, freeze-dry for 24h. Freeze-dry according to the pre-designed freeze-drying process: pre-freeze to -25℃ and hold for 2h, raise the temperature to -15℃ and hold for 12h, raise the temperature to 15℃ and hold for 2h, raise the temperature to 25℃ and hold for 4h. After freeze-drying, decellularized porcine skin matrix membrane is obtained. The remaining steps are the same as in Example 6.

[0035] Example 13: The difference between the preparation method of the anal fistula stent in this embodiment and that in embodiment 7 is as follows: Add 0.3g of autologous concentrated growth factor to 100mL of water, mix well, and then surface-load the gel of Example 6 for 1h. After freezing treatment, freeze-dry for 24h. Freeze-dry according to the pre-designed freeze-drying process: pre-freeze to -25℃ and hold for 2h, raise the temperature to -15℃ and hold for 12h, raise the temperature to 15℃ and hold for 2h, raise the temperature to 25℃ and hold for 4h. After freeze-drying, decellularized porcine skin matrix membrane is obtained. The remaining steps are the same as in Example 7.

[0036] The following tests were conducted on the physicochemical properties, histological characteristics, growth factors, and biological properties of the anal fistula plug stent material prepared in the above embodiments: 1. Porosity determination; Porosity determination: The porosity of the material was determined by mercury intrusion porosimetry and compared with that of the BiodesignSurgisis product. Results: The porosity of the sample provided in Example 7 was 87.8 ± 2.51, while the porosity of the BiodesignSurgisis product was 78.3 ± 6.38. The porosity of the other examples was lower than that of Example 7.

[0037] 2. Chemical performance testing, including tests for viruses, pH, endotoxins, and residual DNA. (1) Preparation of test solution: Take the uniformly thick part of the sample from Example 1 and cut it into 1cm pieces.2 The fragments were washed with water and dried, then added to a glass container, according to the total internal and external surface area of ​​the sample (cm²). 2 Add water at a ratio of 5:1 (mL) to the sample, cover, and place in a pressure steam sterilizer. Heat at 121℃±1℃ for 30 min. After heating, separate the sample from the liquid and cool to room temperature to prepare the test solution. Take the same volume of water and place it in a glass container to prepare a blank control solution using the same method. (2) Virus detection: Method: Pseudorabies virus was selected as the indicator virus, and the viral DNA copy number was detected by real-time quantitative PCR. Three batches of samples were tested. Result: Viral DNA copy number 0; (3) pH test: The test shall be conducted in accordance with the method specified in 5.4.1 of GB / T14233.1 (Test Methods for Medical Infusion, Transfusion and Injection Equipment Part 1: Chemical Analysis Methods). The result shall be that the pH difference between the test solution and the blank control solution shall not exceed 1.5. (4) Endotoxin: Press 6cm 2 The sample was mixed with 1 ml of extraction medium and incubated at 37±1℃ for 72±2 h to prepare the test solution. The extraction medium was physiological saline. The test was conducted according to the method specified in GB / T14233.2-2005 (Medical Infusion, Transfusion and Injection Equipment Test Methods Part 2: Biological Test Methods), and three batches of samples were tested. Results: Endotoxin content was less than 5 EU / g. (5) DNA Residual Detection: Based on the method for detecting residual DNA in biological agents (Chinese Pharmacopoeia 2010, Appendix IX-B, Determination of Exogenous DNA Residual Content), the residual DNA content of the sample provided in Example 1 was detected using fluorescence staining and compared with the BiodesignSurgisis product. Results: The residual DNA content of the sample provided in Example 1 was 120±15 pg / g, while the residual DNA content of the BiodesignSurgisis product was 250±45 pg / g.

[0038] 3. Histological examination; (1) Optical microscopy observation: The paraffin-coated material was stained with hematoxylin and eosin and observed under an inverted phase-contrast microscope. Results: No cells or cell debris remained; the collagen was microscopically continuous without breakage; the cross-section of the fistula plug stent was as shown in the figure. Figure 4 , 5 As shown; (2) Observation of ultrastructure. Results: The material exhibits a porous structure with no fiber breakage, uniform pore size, an average pore size of 200 μm, and a porosity greater than 85%. Figure 3 As shown.

[0039] 4. Growth factor detection; Press 6cm 2The sample was mixed with 1 ml of extraction medium and incubated at 37±1℃ for 72±2 h to prepare the test solution. The extraction medium was physiological saline. The contents of basic growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the extract were detected by ELLISA. Results: The bFGF content was 121.8±2.683 ng / L, and the VEGF content was 93.8±3.033 ng / L.

[0040] 5. Biological performance testing, including cytotoxicity, delayed-type hypersensitivity, and intradermal reaction; (1) Cytotoxicity: at 6cm 2 The sample was mixed with 1 ml of extraction medium and incubated at 37±1℃ for 24±2 h to prepare the test solution. The extraction medium was MEM culture medium containing serum. The test solution was then tested according to the test method specified in GB / T16886.5-2003 (Biological Evaluation of Medical Devices Part 10: Stimulation and Delayed Hypersensitivity Tests). Table 1. Cytotoxicity test results of Examples 2-13 ; As shown in Table 1, the results indicate that different treatment steps significantly improve biosafety, and the stronger the synergy of the treatment measures, the more prominent the optimization effect: the cell viability of decellularized porcine skin matrix hydrolyzed alone was 100%, and compared with the control group, it did not have the advantage of promoting cell growth; when using the simple process of "decellularized porcine skin matrix + hyaluronic acid", the cell viability was 102%, and the optimization range was limited; when genipin crosslinking agent was added, cell viability was significantly improved, with the most significant improvement at a concentration of 0.03 wt%. However, as the genipin content increased, cell viability decreased instead. This is because genipin has a protective and promoting effect on cells at low concentrations, reducing oxidative stress damage by activating the AMPK pathway and protecting cells from apoptosis. At high concentrations, genipin exhibits inhibitory and toxic effects on cells. Subsequently, based on Examples 5-7, autologous concentrated growth factor was added. In Example 8, the low concentration of genipin combined with the proliferation-promoting autologous concentrated growth factor showed the greatest enhancement of cell activity. For Examples 9 and 10, with higher genipin concentrations, the addition of autologous concentrated growth factor partially compensated for the inhibitory effect of genipin. In the treatment of Examples 11, 12, and 13, autologous concentrated growth factor was soaked and loaded onto the cross-linked material. Since the autologous concentrated growth factor is the first to exert its effect, it is equivalent to providing a "protective shield" for the cells, allowing them to quickly adapt to the high-concentration genipin environment, truly achieving a synergistic effect. In summary, the anal fistula stent obtained by the preparation method of Example 13 achieves the maximum synergistic effect of the three components, representing the optimal technical path for promoting cell proliferation.

[0041] In addition, high concentrations of genipin can first enhance the mechanical properties of the fistula stent, then significantly reduce the degradation rate. Since fistula healing is a relatively long process, it may eventually make the circumferential structure of the stent more compact, providing more sites for cell proliferation.

[0042] (2) Delayed-type hypersensitivity reaction: Press 6cm 2 The sample was mixed with 1 ml of extraction medium to prepare the test solution. The extraction medium consisted of physiological saline and cottonseed oil. The test was conducted according to the test method specified in CB / T16886.10-2005 (Biological Evaluation of Medical Devices Part 10: Stimulation and Delayed-Type Hypersensitivity Test). Results: No delayed-type hypersensitivity reactions were observed in any samples.

[0043] (3) Intradermal reaction: Press 6cm 2 The sample was mixed with 1 ml of extraction medium to prepare the test solution, which was prepared at 37±1℃ for 72±2 h. The extraction medium consisted of physiological saline and cottonseed oil. The test was conducted according to the test method specified in CB / T16886.10-2005 (Biological Evaluation of Medical Devices Part 10: Stimulation and Delayed-Type Hypersensitivity Tests). Results: The difference in average score between the test sample and the solvent control was less than 1.0.

[0044] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing anal fistula stent material, characterized in that, Includes the following steps: (1) Dissolve the decellularized pigskin matrix in an acidic solution and perform enzymatic hydrolysis. After enzymatic hydrolysis, add hyaluronic acid and dissolve to obtain a decellularized pigskin matrix solution. (2) Add the cross-linking agent genipin to the decellularized porcine skin matrix solution to cross-link and obtain a gel; (3) The gel is added to concentrated growth factor for soaking, and then freeze-dried. After freeze-drying, it is formed into anal fistula plug stent material.

2. The preparation method according to claim 1, characterized in that, In step (1), the acidic solution is an aqueous solution of acetic acid with a concentration of 0.5–1 M; Enzymatic hydrolysis was performed using pepsin, with a pepsin mass fraction of 25–50 wt% relative to the decellularized porcine skin matrix.

3. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of hyaluronic acid to decellularized porcine skin matrix is ​​1:(1-2).

4. The preparation method according to claim 1, characterized in that, In step (2), the mass fraction of the crosslinking agent genipin relative to the decellularized porcine skin matrix is ​​0.01 to 0.1 wt%.

5. A method for preparing anal fistula plug stent material, characterized in that, Includes the following steps: (1) The decellularized pigskin matrix was placed in an acidic solution for enzymatic hydrolysis. After enzymatic hydrolysis, hyaluronic acid was added and dissolved to obtain a decellularized pigskin matrix solution. (2) Add concentrated growth factor to decellularized pig skin matrix solution and stir well to obtain a mixed solution; (3) Add the crosslinking agent genipin to the mixed solution for crosslinking, and freeze-dry after crosslinking. After freeze-drying, it is formed into anal fistula stent material.

6. The preparation method according to claim 5, characterized in that, In step (1), the acidic solution is an aqueous solution of acetic acid with a concentration of 0.5–1 M; Enzymatic hydrolysis was performed using pepsin, with a pepsin mass fraction of 25–50 wt% relative to the decellularized porcine skin matrix. The mass ratio of hyaluronic acid to decellularized porcine skin matrix is ​​1:(1-2).

7. The preparation method according to claim 5, characterized in that, In step (2), the mass fraction of concentrated growth factor relative to decellularized porcine skin matrix is ​​10-30 wt%.

8. The preparation method according to claim 5, characterized in that, In step (3), the mass fraction of the crosslinking agent genipin relative to the decellularized porcine skin matrix is ​​0.01 to 0.1 wt%.

9. The anal fistula stent material prepared by the preparation method according to any one of claims 1-8.

10. The anal fistula plug support material according to claim 9, characterized in that, The fistula plug support material is a hollow conical structure obtained by multi-layer molding of membrane rolls or a multi-layer strip structure formed by compression molding.

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