Tissue regeneration and repair stent and preparation method thereof

Abstract

The invention provides a tissue regeneration and repair stent and a preparation method thereof, and relates to the technical field of medical treatment. The preparation method of the tissue regeneration and repair scaffold comprises the following steps: filling a mold with a gelatinous acellular matrix, contacting the bottom of the mold with a cold source to form a directional temperature gradient field from bottom to top, and forming columnar ice crystals from the gelatinous acellular matrix along the direction of the directional temperature gradient field under the directional temperature gradient field, freeze-drying to obtain a porous matrix with directional pore channels, and cross-linking and curing the porous matrix with a cross-linking agent to prepare the tissue regeneration and repair scaffold. According to the preparation method, through a directional freezing strategy, a porous structure with directional pore channels is constructed, a tubular pore channel perpendicular to an articular surface is formed, a columnar structure of natural cartilage can be simulated, and vertical migration of bone marrow mesenchymal stem cells is facilitated.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to a tissue regeneration and repair scaffold and its preparation method. Background Technology

[0002] In the field of surgery, tissue defects are very common. Some diseases, such as cartilage defects, uterine scar rupture, and surgical loss of the meninges, exceed the body's regenerative capacity, making it difficult for damaged tissues to repair themselves to the greatest extent possible, leading to dysfunction of related organs. Using regenerative scaffold technology combined with human-derived active substances to repair and regenerate tissue is a method for repairing tissue defects that has gradually gained recognition and use in the last decade. Its main characteristic is based on a tissue regeneration scaffold prepared with specific technology, carrying active components collected from the patient's own normal tissue, such as autologous platelet-rich plasma (PRP), fat-derived active substances, and cells. The active substances are loaded onto the scaffold material according to the final required amount, and then the scaffold carrier is surgically implanted into the defect site. Under the coordinated regulation of the scaffold guidance and the active substance induction, the damaged site recruits surrounding cells to aggregate, proliferate, and differentiate at the defect site, regenerating into natural tissue similar to normal tissue, thus completing the repair of the defective tissue. Therefore, the formulation design and preparation method of the scaffold material in this strategy are crucial to determining the tissue repair effect.

[0003] An ideal tissue regeneration and repair scaffold should possess the following characteristics: (1) good bioactivity, non-toxic and teratogenic; (2) suitable biodegradability, with a degradation rate adapted to tissue growth; (3) tissue regeneration induction ability, serving as a carrier for active substances and providing a good microenvironment for cell growth and proliferation; (4) processability, facilitating the processing into the required shape; (5) structural support, with sufficient mechanical strength to maintain its shape stability for a certain period of time after implantation; and (6) porous structure, with a porosity of over 80%, which facilitates cell adhesion and ingrowth, as well as the infiltration of nutrients and the excretion of metabolic products.

[0004] Biological tissue matrix is ​​a major component of connective tissue in organisms and has good biocompatibility. Due to strict ethical regulations, the source of human tissue obtained through human donation is very limited and cannot meet the large clinical demand. Therefore, the preparation of biomaterials using xenogeneic acellular dermis, after thoroughly removing xenogeneic cells and eliminating immunogenicity, is an important direction for the development of medical devices. In 1995, Livesey et al. first reported the successful preparation of acellular dermal matrix, proving that the matrix transplantation of allogeneic dermis can have the potential for living dermal reconstruction after appropriate treatment (Transplantation, 1995, 60(1), 1-9). Since then, tissue repair scaffold materials based on acellular dermal matrix have achieved good results in the clinical application of burn wounds, oral and maxillofacial structures, skin, cartilage, etc. However, due to the limitations of acellular dermal matrix materials, its scaffolds still face problems such as poor bioactivity and unsatisfactory porosity, which seriously limits its application in the field of tissue repair. Existing patent CN102178981A discloses a method for preparing a cartilage repair scaffold using a newborn calf dermal scaffold through SDS decellularization, trypsin modification, and formaldehyde cross-linking. However, the SDS decellularization and formaldehyde cross-linking strategy may lead to difficulties in effectively removing antigens and DNA, and residual formaldehyde may inhibit cell growth. Furthermore, the issue of porosity regulation remains. Therefore, exploring a method for preparing an animal-derived decellularized dermal matrix scaffold to enhance its bioactivity and regenerative function is an important problem that needs to be solved in this field.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for preparing a tissue regeneration and repair scaffold to solve the aforementioned technical problems.

[0007] The second objective of this invention is to provide a tissue regeneration and repair scaffold that has suitable biocompatibility and degradability, a unique microstructure, can carry active substances, has a certain degree of shape plasticity, and can meet the suture strength requirements.

[0008] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing a tissue regeneration and repair scaffold, comprising the following steps: A gel-like decellularized matrix is ​​filled into a mold, and then the bottom of the mold is brought into contact with a cold source to form a directional temperature gradient field from bottom to top. Under the directional temperature gradient field, the gel-like decellularized matrix forms columnar ice crystals along the direction of the directional temperature gradient field. After freeze-drying, a porous matrix with directional channels is obtained. Then, after cross-linking and curing with a cross-linking agent, a tissue regeneration and repair scaffold is prepared.

[0009] As a further technical solution, the cold source includes liquid nitrogen; Preferably, the temperature of the cold source is -80℃ to -50℃.

[0010] As a further technical solution, the crosslinking agent includes one or more of formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0011] As a further technical solution, the porous matrix is ​​further subjected to a biological enzyme solution treatment step before crosslinking and curing; Preferably, the concentration of the bio-enzyme solution is 0.01 wt% to 0.2 wt%; the treatment time of the bio-enzyme solution is 1 to 10 min; and the bio-enzyme is selected from one or more of pepsin or trypsin.

[0012] As a further technical solution, the method for preparing the gel-like decellularized matrix includes: preparing a gel-like decellularized matrix by sequentially decellularizing animal tissue and removing antigens and exogenous DNA; The animal tissues include one or more of the pericardium or dermis.

[0013] As a further technical solution, the decellularization step includes: subjecting the animal tissue matrix to freeze-thaw treatment, and then cyclically subjecting it to hypertonic solution treatment and hypotonic solution treatment; Preferably, the freeze-thaw treatment is performed 3 to 6 times; Preferably, the freeze-thaw process is followed by ultrasonic treatment; Preferably, the hypertonic solution is a NaCl solution with a concentration of 1~5 mol / L, and the hypertonic solution treatment time is 10~60 minutes; Preferably, the hypotonic solution is water, and the hypotonic solution treatment time is 10-120 minutes; Preferably, the hypertonic solution treatment and hypotonic solution treatment are repeated 1 to 5 times.

[0014] As a further technical solution, the step of removing antigens and exogenous DNA includes: soaking the decellularized matrix in a biological enzyme solution; Preferably, the bioenzyme includes one or more of pepsin or trypsin; Preferably, the mass concentration of the bio-enzyme solution is 0.1% to 2.0%; Preferably, the soaking treatment time is 1-4 hours.

[0015] As a further technical solution, the dermal tissue is prepared from skin tissue after pretreatment; The pretreatment includes hair removal; The hair removal treatment involves applying a thiosulfate solution to the skin tissue. Preferably, the concentration of the thiosulfate solution is 2g / 100ml to 5g / 100ml, and the pH is 8 to 10; Preferably, the temperature for treatment with the thiosulfate solution is 20~30℃; Preferably, the treatment time of the thiosulfate solution is 2 to 6 hours.

[0016] As a further technical solution, the step of loading platelet-rich plasma or bioactive factors is also included. The bioactive factors include one or more of TGF-β, IGF-1, or BMP-2; Preferably, the platelet-rich plasma or bioactive factor is loaded via physical adsorption or chemical bonding.

[0017] Secondly, the present invention provides a tissue regeneration and repair scaffold, which is prepared by the above-described preparation method.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The method for preparing a tissue regeneration and repair scaffold provided by the present invention constructs a porous structure with directional channels through a directional freezing strategy, forming tubular pore channels perpendicular to the joint surface. This can simulate the columnar structure of natural cartilage and is more conducive to the vertical migration of bone marrow mesenchymal stem cells.

[0019] The tissue regeneration and repair scaffold prepared by this invention has suitable biocompatibility and degradability, unique microstructure, can carry active substances, has a certain degree of shape plasticity, can meet the suture strength, and can be used for the repair of tissue defects such as cartilage. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a scanning electron microscope (SEM) image of the decellularized matrix from Example 1; Figure 2 This is a scanning electron microscope (SEM) image of decellularized matrix in Comparative Example 3. Detailed Implementation

[0022] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] In a first aspect, the present invention provides a method for preparing a tissue regeneration and repair scaffold, comprising the following steps: 1. Surface treatment 1.1 Raw material pretreatment and gentle peeling: 1) Select fresh animal skin (such as calf pericardium, pig skin or cow skin, 1-2mm thick, from healthy animals aged 6-12 months); 2) Mechanical removal of subcutaneous fat and connective tissue (using a peeling machine, such as model Dermatome GD-100, speed of 200-500 rpm, pressure of 0.5-1 MPa, processing time of 5-15 min). 3) Rinse with running water (if using tap water or deionized water, flow rate 1-2L / min, time 10-20min), preserving the dermis and subdermal fat layer.

[0024] 4) Physical peeling machine removes most of the epidermal layer (speed 300-400rpm, blade thickness 0.2-0.5mm, processing time 10-30min). 5) Soak in sodium thiosulfate depilatory solution for chemical hair removal and removal of residual epidermis.

[0025] In this invention, the concentration of the sodium thiosulfate hair removal solution is 2 g / 100ml to 5 g / 100ml (for example, but not limited to 2 g / 100ml, 3 g / 100ml or 5 g / 100ml), the pH is 8-10 (the pH is adjusted to 8, 9 or 10 using NaOH), the solvent is deionized water, the treatment temperature is 20-30℃ (for example, but not limited to 20℃, 25℃ or 30℃), the treatment time is 2-6 hours (for example, but not limited to 2 hours, 4 hours or 6 hours), and preferably the hair removal treatment is carried out under oscillation conditions at a speed of 100-200 rpm.

[0026] 6) Wash with PBS (pH 7.4, concentration 0.01M) 3-5 times, 10-20 min each time.

[0027] Compared to traditional strong alkali (NaOH) or sodium sulfide hair removal, the method of this invention can more gently protect the collagen structure of the dermal matrix, avoid collagen denaturation, achieve a collagen retention rate of >90% (verified by the hydroxyproline assay), and reduce environmental pollution.

[0028] 1.2 Decellularization and Antigen / DNA Removal: A combined decellularization strategy using physical ultrasonic freeze-thaw and osmotic pressure cyclic shock was employed.

[0029] 1.2.1 The tissue matrix after epidermal removal is subjected to freeze-thaw treatment: 1) Repeated freeze-thaw cycles (freezing temperature -80℃ to 50℃, frozen storage for 12-24 hours; thawing temperature 20℃, thawing for 30-60 minutes, 3-6 cycles, preferably 4-5 cycles).

[0030] 2) Then perform ultrasonic oscillation (power 200W~500W, preferably 300W~400W, frequency 20-40kHz, time 10-40min, intermittent mode: 10s working / 5s pausing, solution volume 5-10 times the tissue mass, using PBS as medium).

[0031] 1.2.2 Decellularize the frozen-thawed tissue matrix using high and low osmotic pressures: 1) The matrix is ​​immersed in NaCl solution, and the cells are severely dehydrated and shrunken by high osmotic pressure, which causes the cell membrane to be physically separated from the extracellular matrix (ECM).

[0032] In this invention, the NaCl solution concentration is 1 mol / L to 5 mol / L (preferably 3 mol / L to 4 mol / L), and the solution is shaken for 10 to 60 minutes (preferably 30 to 40 minutes).

[0033] 2) The matrix is ​​transferred to deionized water (hypotonic environment). Due to the osmotic pressure difference, water rushes into the shrunken cells, causing the cell membrane to "burst".

[0034] In this invention, the soaking time in deionized water is 10 to 120 minutes (preferably 40 to 80 minutes).

[0035] 3) After undergoing high and low osmotic pressure circulation treatment 3 to 5 times, it is then cleaned with deionized water circulation 3 to 5 times.

[0036] The decellularization method of the present invention, compared with the traditional decellularization strategy that relies on chemical reagents and surfactants, can significantly reduce chemical reagent residues and reduce toxicity, while protecting collagen fibers from long-term enzyme / strong alkali damage.

[0037] 1.2.3 The tissue matrix after physical ultrasonic freeze-thaw-osmotic pressure cyclic impact treatment was subjected to biochemical enzymatic hydrolysis to ultimately form a gel-like decellularized matrix: 1) Cut the decellularized matrix into 3cm×3cm×1cm pieces (using sterile scissors); 2) Deeply remove antigens and exogenous DNA using a bio-enzyme complex solution (condition parameters such as temperature 37℃, time 1-4h, and shaking 100-150rpm), loosen collagen fiber bundles, and remove immunogenic telopeptides; finally forming a gel-like decellularized matrix.

[0038] In this invention, the bioenzyme composite solvent contains one or both of pepsin and trypsin, with a concentration of 0.1wt%~2.0wt% (preferably 0.5wt%~1.0wt%), a pH of 1~4 (pepsin) or 7~10 (trypsin), and an enzyme activity >100U / mg. When the pH is 1~4, the concentration of acid in the bioenzyme composite solvent is 0.01mol / L~0.5mol / L (preferably 0.05mol / L~0.1mol / L), and the acid is selected from HCl, acetic acid, etc. When the pH is 7~10, an alkaline solution is used to adjust the pH.

[0039] 3) Wash the gel-like decellularized matrix with PBS to remove biological enzymes and acids (3-5 times, 15-30 min each time, volume ratio 1:20).

[0040] The entire process ensures a cell removal rate of >99% (no cell nuclei observed by DAPI staining) and DNA residue of <50 ng / mg.

[0041] 1.3 Directional Microstructure Reshaping and Forming Crosslinking Directional channel reconstruction: In a specific mold, a gel-like decellularized matrix is ​​used to construct a porous structure with directional channels through a directional freezing strategy. This forms tubular pore channels perpendicular to the joint surface, mimicking the columnar structure of natural cartilage, which is more conducive to the vertical migration of bone marrow mesenchymal stem cells.

[0042] In this invention, liquid nitrogen or a -80°C cold source is placed at the bottom of a mold filled with a gel-like decellularized matrix. The sidewalls of the mold are wrapped with insulating material to ensure that the freezing front advances from bottom to top, forming a vertically oriented temperature gradient field. Before placing the decellularized matrix into the mold, the mold is pre-cooled to ensure the stability of the vertically oriented temperature gradient. The gel-like decellularized matrix crystallizes into columnar ice crystals along the vertical temperature gradient direction. Loose collagen fibers are squeezed into the gaps between the ice crystals under the directional crystallization, forming an ordered collagen fiber structure.

[0043] The decellularized matrix after directional freezing is freeze-dried to form a porous matrix with directional channels: 1) The preset cold trap temperature is -80℃ to -50℃; 2) Pre-freezing temperature -50℃, pre-freezing time 2~4h, vacuum degree <10Pa; 3) Sublimation temperature -20℃, time 24~48h; 4) The total drying time is 48~72 hours.

[0044] 1.4 Directional cryogenic matrix vertical surface etching The freeze-dried, oriented porous decellularized matrix was placed in a bio-enzyme solution and gradually immersed in the solution. A short-term treatment with a low concentration of bio-enzyme was used to create nanoscale "marks" on the surface of collagen fibers in the decellularized matrix, increasing the specific surface area, which is more conducive to cell adhesion and improves the cell adhesion rate.

[0045] In this invention, the concentration of the bio-enzyme is 0.01wt%~0.2wt% (preferably 0.05wt%~0.1wt%), the treatment time of the bio-enzyme is 1~10min (preferably 4~8min), and the treatment temperature of the bio-enzyme is 4~40℃ (preferably 25~37℃).

[0046] The bioenzyme is selected from one or both of pepsin or pancreatic enzyme, with pepsin being preferred.

[0047] 1.5 Matrix Crosslinking Treatment 1) The etched matrix is ​​immersed in a crosslinking agent solution to carry out a crosslinking reaction, so that the scaffold has an adjustable porosity (70%~95%), suitable mechanical strength and degradation resistance, which is conducive to the ingrowth of chondrocytes and the transport of nutrients.

[0048] In this invention, the crosslinking agent may be formaldehyde (mass concentration 0.1~2%, preferably 0.5~1%; pH 4~8, preferably 5~7, adjusted using NaOH; crosslinking temperature 20~40℃, preferably 25~37℃). The crosslinking agent can be glutaraldehyde (mass concentration 0.5~3%, preferably 1~2%; pH 4~8, preferably 5~7; crosslinking temperature 20~40℃, preferably 25~37℃). The crosslinking agent can be EDC / NHS (EDC:NHS ratio 5:1~2:1, preferably 4:1~3:1; mixed concentration 0.3~3%, preferably 1~2%; solvent 0.1M MES buffer, pH 4~8, preferably 5~7; crosslinking temperature 20~40℃, preferably 25~37℃; addition order: first dissolve EDC in buffer and stir for 5 min, then add NHS, freshly prepared, and completed within 30 min before use).

[0049] 2) After crosslinking, remove residual crosslinking agent by ethanol gradient dehydration (concentration 70%-95%, soaking for 1-2 hours at each gradient) and repeated washing with deionized water (3-5 times, 20-30 minutes each time, volume ratio 1:20); 3) Finally, a second freeze-drying process is performed to obtain the molded support.

[0050] 1.6 Functionalized Composite During or after scaffold fabrication, bioactive components are loaded via physical adsorption or chemical bonding: 1.6.1 The formed stent is immersed in a solution containing bioactive components (platelet-rich plasma, bioactive factors) to achieve loading.

[0051] The platelet-rich plasma (PRP) solution has a concentration of 10%-50%, is separated from autologous blood, and has a platelet count >10. 6 / μL, pH 7.4); bioactive factors include, but are not limited to, TGF-β (concentration 1-100ng / mL), IGF-1 (concentration 10-200ng / mL), and BMP-2 (concentration 5-50ng / mL); the pH of the bioactive factor solution is 7.4.

[0052] Vacuum loading adsorption: Moisture is removed by vacuuming to achieve loading. Condition parameters: Vacuum degree 0.05~0.1MPa, temperature 4~25℃, time 1~4h.

[0053] Chemical bonding method: The bioactive component is activated by a cross-linking agent and then forms a chemical bond with the scaffold. Activation can be performed by EDC / NHS (EDC:NHS ratio 5:1~2:1, preferably 4:1~3:1; mixed concentration 0.3~3%, preferably 1~2%).

[0054] By incorporating PRP or specific bioactive factors, the scaffold is endowed with the ability to actively induce cartilage regeneration, significantly shortening the repair cycle.

[0055] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0056] The test results of the following examples and comparative materials are shown in Table 1. The test methods are as follows: 1. Porosity a) The average pore size and porosity were tested using SEM image analysis combined with the Archimedes drainage method; b) For the etched structure in the sidewall of the directional channel, the sidewall sample is observed after vertical cutting of the matrix.

[0057] 2. Structural strength (compression test) a) A universal testing machine is used, and the Instron 6800 / 5940 series or MTS Bionix can be selected.

[0058] b) The testing standard adopted is ASTM D695.

[0059] c) Based on tests conducted at a loading rate of 2 mm / min.

[0060] 3. In vitro cell culture a) Rabbit primary chondrocytes are commonly used as model cells in cell culture. These cells are derived from the knee cartilage of young rabbits (usually 2-4 weeks old New Zealand white rabbits), obtained through enzymatic digestion, and possess good cartilage phenotypic expression capabilities, making them suitable for evaluating the cell attachment, proliferation, and cartilage differentiation induction performance of scaffolds. Reference standard: ISO 10993:2009 "Bioevaluation of medical devices".

[0061] b) Cell isolation and primary culture: Tissue blocks were excised from the knee joint cartilage of young rabbits, washed with PBS, and digested sequentially with 0.25% trypsin (37°C, 30 min) and 0.2% type II collagenase (37°C, 4–6 h). Cells were collected by centrifugation (1000 rpm, 5 min), resuspended in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and cultured at a density of 5 × 10⁶ cells / mL. 4 / cm² inoculated into culture flasks, incubated at 37℃ with 5% CO2, and passaged to P1-P2 generations for use (to avoid phenotypic loss).

[0062] c) Stent pretreatment: The stent is cut to a suitable size (e.g., 6 mm in diameter, 1-2 mm in thickness) and sterilized (Co). 60 Irradiation or soaking in 70% ethanol followed by PBS washing, then soaking in culture medium for 24 h to promote cell attachment.

[0063] d) Cell culture in scaffolds: Pretreated scaffolds were placed in 24-well plates at a density of 1×10⁶ cells / well. 5 Inoculate the scaffold surface with a cell suspension at a density of / cm², and let stand for 2-4 hours to promote initial attachment. Then, add culture medium until the scaffold is submerged. Culture conditions: 37°C, 5% CO2, saturated humidity; change the medium every 2-3 days, for a total culture time of 7 days.

[0064] e) Testing and evaluation: The activity of the material was detected using CCK-8 assay. qPCR detection and analysis of SOX9 / COL2A1 gene expression; Cell fluorescence staining was used to observe cell adhesion to the matrix sidewalls.

[0065] 4. Animal experiments with New Zealand white rabbits a) Select healthy New Zealand white rabbits (weight 2.5~3.5 kg, age 3~6 months, male or female), fast for 12 hours before surgery, and acclimatize to the environment for 1 week.

[0066] b) Intravenous injection of sodium pentobarbital (30 mg / kg) or isoflurane inhalation anesthesia, followed by shaving and disinfection of the knee joint area.

[0067] c) Make a medial paramedian incision to expose the femoral condyle of the knee joint and drill a hole to create a full-thickness cartilage defect (4 mm in diameter, 2-3 mm in depth, to the subchondral bone but not through).

[0068] c) Sterilized stents (size-matched to the defect) were press-fitted and implanted into the defect area, while the control group was blank or sham surgery; the joint capsule and skin were sutured.

[0069] d) Observation and sampling: Gait was observed 4-12 weeks postoperatively (12 weeks is common); animals were euthanized, knee joint specimens were collected, and gross observation, HE / Masson staining, immunohistochemistry and scoring (repair rate, vascularization, cartilage integration) were performed. The experimental results were compared with the blank control group to analyze the vascularization.

[0070] Example 1 1. Raw material pretreatment and gentle peeling: 1.1 Select fresh calf pericardium, 2mm thick, aged 10 months.

[0071] 1.2 Subcutaneous fat and connective tissue were mechanically removed using a peeling machine at a speed of 300 rpm, a pressure of 0.6 MPa, and a processing time of 10 min.

[0072] 1.3 Rinse with running water (flow rate 1L / min, 15min) to preserve the dermis and subdermal fat layer.

[0073] 1.4 Peeling machine removes most of the epidermis (speed 300 rpm, blade thickness 0.3 mm, processing time 15 min).

[0074] 1.5 Soak in 3% (g / 100ml) sodium thiosulfate depilatory solution (pH 9) for 3 hours to perform chemical hair removal and remove residual epidermis.

[0075] 1.6 Wash with PBS 4 times, 15 min each time.

[0076] 2. Decellularization treatment: 2.1 Repeatedly freeze and thaw 4 times (freezing temperature -80℃, 18h; thawing temperature 20℃, 45min), then ultrasonically vibrate (power 350W, frequency 30kHz, time 25min).

[0077] 2.2 The matrix was immersed in NaCl solution (3 mol / L) and shaken for 30 min, then transferred to deionized water for 60 min. This process was repeated 4 times.

[0078] 2.3 The solution is cleaned 4 times using deionized water circulation.

[0079] 2.4 Cut the decellularized matrix into small pieces (using sterile scissors), and enzymatically hydrolyze it with an acid-enzyme complex solution (0.75% pepsin, 0.1 mol / L acetic acid, pH 3) at 37°C for 3 h to remove antigens and exogenous DNA. Then wash with PBS 4 times for 20 min each time.

[0080] 2.5 Ultimately, a gel-like decellularized matrix is ​​formed.

[0081] 3. Directional microstructure reshaping and molding crosslinking 3.1 In a specific mold, liquid nitrogen is placed at the bottom of the mold, and a porous structure with directional channels is constructed through a directional freezing strategy, in which collagen fibers are oriented and orderly arranged.

[0082] 3.2 The directional porous matrix was freeze-dried, pre-frozen at -50℃ (3h), and sublimated at -20℃ (36h) to form a porous sponge-like matrix.

[0083] 3.3 The freeze-dried directional pore structure decellularized matrix was soaked in pepsin solution with a concentration of 0.05 wt% for 5 min at a temperature of 25 ℃. This created nanoscale "marks" on the surface of collagen fibers in the decellularized matrix, increasing the specific surface area, which is more conducive to cell adhesion and improving the cell adhesion rate.

[0084] 3.4 After etching, the substrate was immersed in 1% formaldehyde (pH 6, 30℃) for 4 hours, then dehydrated by gradient immersion in 80% ethanol for 1 hour, and then repeatedly washed with deionized water 4 times to remove residual crosslinking agent.

[0085] 3.5 Finally, a second freeze-drying process is performed to obtain the molded support.

[0086] 4. Functional composite: 4.1 The molded scaffold was immersed in a 30% PRP solution at 15°C and vacuum-loaded (vacuum degree 0.07MPa) for 2 hours.

[0087] 4.2 Then, 50 ng / mL of TGF-β solution was added, and after activation by EDC / NHS at pH 7.4, it chemically bonded to the scaffold and was soaked for 60 min.

[0088] 5. Material testing: 5.1 Upon testing, the pore structure of the support material is as follows: Figure 1As shown, the porosity is 88%; the structural compressive modulus is 1.3 MPa, and its sidewall structure is rich in etched micropores, which promotes cell adhesion.

[0089] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 2.8.

[0090] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 was upregulated by 2.8 times and COL2A1 was upregulated by 2.6 times.

[0091] 5.4 Cell ingrowth and vascularization were observed in the defective area of ​​the rabbit knee joint, with a repair rate of 85%.

[0092] Example 2 1. Raw material pretreatment and gentle peeling: 1.1 Select fresh pig skin, 1.8 mm thick, from pigs aged 8 months.

[0093] 1.2 Subcutaneous fat and connective tissue were removed mechanically using a peeling machine at a speed of 350 rpm, a pressure of 0.7 MPa, and a processing time of 12 min.

[0094] 1.3 The cleaning process is the same as in Example 1.

[0095] 1.4 Peeling machine removes most of the epidermis (speed 350 rpm, blade thickness 0.4 mm, processing time 20 min).

[0096] 1.5 Soak in 4% (w / v) sodium thiosulfate depilatory solution (pH 9.5) for 4 hours to perform chemical depilation and remove residual epidermis.

[0097] 1.6 Wash with PBS 5 times, 18 min each time.

[0098] 2. Decellularization treatment: 2.1 Repeatedly freeze and thaw 5 times (freezing temperature -80℃, 20h; thawing temperature 20℃, 50min), then ultrasonically vibrate (power 400W, frequency 35kHz, time 30min).

[0099] 2.2 The matrix was immersed in NaCl solution (3 mol / L) and shaken for 30 min, then transferred to deionized water for 60 min. This process was repeated 4 times.

[0100] 2.3 The solution is cleaned 4 times using deionized water circulation.

[0101] 2.4 Cut the decellularized matrix into small pieces (using sterile scissors), and enzymatically hydrolyze it with an acid-enzyme complex solution (0.75% pepsin, 0.1 mol / L acetic acid, pH 3) at 37°C for 3 hours to remove antigens and exogenous DNA. Then wash it with PBS 5 times for 25 minutes each time, and then freeze-dry it to form an antigen-free and DNA-free decellularized matrix material.

[0102] 2.5 Ultimately, a gel-like decellularized matrix is ​​formed.

[0103] 3. Stent molding and crosslinking: 3.1 In a specific mold, liquid nitrogen is placed at the bottom of the mold, and a porous structure with directional channels is constructed through a directional freezing strategy, in which collagen fibers are oriented and orderly arranged.

[0104] 3.2 The directional porous matrix was freeze-dried, pre-frozen at -50℃ (3h), and sublimated at -20℃ (36h) to form a porous sponge-like matrix.

[0105] 3.3 Same surface etching as in Example 1.

[0106] 3.4 The freeze-dried matrix was soaked in 1.5% glutaraldehyde (pH 6.5, 32℃) for 5 hours, then dehydrated by gradient soaking in 85% ethanol for 1.5 hours, and then repeatedly washed with deionized water 5 times to remove residual crosslinking agent.

[0107] 3.5 Secondary freezing, same as in Example 1. 4. Functionalized composite: 4.1 The molded scaffold was immersed in a 40% PRP solution at 20°C and vacuum-loaded (vacuum degree 0.08MPa) for 3 hours.

[0108] 4.2 Then, 70 ng / mL of IGF-1 solution was added, and after activation by EDC / NHS at pH 7.4, it chemically bonded to the scaffold and was soaked for 90 min.

[0109] 5. Material testing: 5.1 The porosity of the support material is 90%; the structural compressive modulus is 1.5 MPa.

[0110] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 3.0.

[0111] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 was upregulated by 3.0-fold and COL2A1 was upregulated by 3.2-fold.

[0112] 5.4 Cell ingrowth and vascularization were observed in the defective area of ​​the rabbit knee joint, with a repair rate of 90%.

[0113] Example 3 (Increasing the number of freeze-thaw cycles and optimizing physical decellularization) 1. Raw material pretreatment and gentle peeling: Same as Example 1.

[0114] 2. Decellularization treatment: 2.1 Repeated freeze-thaw cycles 6 times (freezing temperature -80℃, 22h; thawing temperature 20℃, 55min), followed by ultrasonic vibration (power 375W, frequency 32kHz, time 28min).

[0115] 2.2 The matrix was immersed in NaCl solution (3 mol / L) and shaken for 30 min, then transferred to deionized water for 60 min. This process was repeated 4 times.

[0116] 2.3 The solution is cleaned 4 times using deionized water circulation.

[0117] 2.4 Cut the decellularized matrix into small pieces (using sterile scissors), and enzymatically hydrolyze it with an acid-enzyme complex solution (0.75% pepsin, 0.08 mol / L acetic acid, pH 3) at 37°C for 3 h to remove antigens and exogenous DNA. Then wash with PBS 4 times for 20 min each time.

[0118] 2.5 Ultimately, a gel-like decellularized matrix is ​​formed.

[0119] 3. Stent molding and crosslinking: 3.1 In a specific mold, liquid nitrogen is placed at the bottom of the mold, and a porous structure with directional channels is constructed through a directional freezing strategy, in which collagen fibers are oriented and orderly arranged.

[0120] 3.2 The directional porous matrix was freeze-dried, pre-frozen at -50℃ (3h), and sublimated at -20℃ (36h) to form a porous sponge-like matrix.

[0121] 3.3 Same surface etching as in Example 1.

[0122] 3.4 The freeze-dried matrix was soaked in 0.8% formaldehyde (pH 5.5, 28℃) for 3.5h, then dehydrated by gradient soaking in 75% ethanol for 1.2h, and then repeatedly washed with deionized water 4 times to remove residual crosslinking agent.

[0123] 3.5 Secondary freeze-drying, same as in Example 1. 4. Functionalized compound: 4.1 The molded scaffold was immersed in a 35% PRP solution at 18°C ​​and vacuum-loaded (vacuum degree 0.075MPa) for 2.5h.

[0124] 4.2 Then, add 50 ng / mL of BMP-2 solution, activate it via EDC / NHS at pH 7.4, and chemically bond it with the scaffold. Soak for 75 min.

[0125] 5. Material testing: 5.1 The porosity of the support material is 92%; the structural compressive modulus is 1.2 MPa.

[0126] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 2.9.

[0127] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 was upregulated by 2.9 times and COL2A1 was upregulated by 2.8 times.

[0128] 5.4 Cell ingrowth and vascularization were observed at the defect site of the rabbit knee joint, with a repair rate of 92%.

[0129] Example 4 (Reduced enzyme concentration, gentle biological treatment) 1. Raw material pretreatment and gentle peeling: Same as Example 2.

[0130] 2. Decellularization treatment: 2.1 Repeatedly freeze and thaw 3 times (freezing temperature -80℃, 16h; thawing temperature 20℃, 40min), then ultrasonically vibrate (power 300W, frequency 28kHz, time 20min).

[0131] 2.2 The matrix was immersed in NaCl solution (3 mol / L) and shaken for 30 min, then transferred to deionized water for 60 min. This process was repeated 4 times.

[0132] 2.3 The solution is cleaned 4 times using deionized water circulation.

[0133] 2.4 Cut the decellularized matrix into small pieces (using sterile scissors), and enzymatically digest it with an acid-enzyme complex solution (0.3% pepsin, 0.1 mol / L acetic acid, pH 3) at 37°C for 1.5 h to remove antigens and exogenous DNA. Then wash with PBS 4 times for 20 min each time.

[0134] 2.5 Ultimately, a gel-like decellularized matrix is ​​formed.

[0135] 3. Stent molding and crosslinking: 3.1 In a specific mold, liquid nitrogen is placed at the bottom of the mold, and a porous structure with directional channels is constructed through a directional freezing strategy, in which collagen fibers are oriented and orderly arranged.

[0136] 3.2 The directional porous matrix was freeze-dried, pre-frozen at -50℃ (3h), and sublimated at -20℃ (36h) to form a porous sponge-like matrix.

[0137] 3.3 Same surface etching as in Example 1.

[0138] 3.4 The freeze-dried matrix was soaked in 0.5% formaldehyde (pH 5, 25℃) for 3 hours, then dehydrated by gradient soaking in 70% ethanol for 1 hour, and then repeatedly washed with deionized water 3 times to remove residual crosslinking agent.

[0139] 3.5 The freeze-drying process is the same as in Example 1.

[0140] 4. Functional composite: 4.1 The molded scaffold was immersed in a 25% PRP solution at 10℃ and vacuum-loaded (vacuum degree 0.06MPa) for 1.5h.

[0141] 4.2 Then, 40 ng / mL of TGF-β solution was added, and after activation by EDC / NHS at pH 7.4, it chemically bonded to the scaffold and was soaked for 45 min.

[0142] 5. Material testing: 5.1 The porosity of the support material was found to be 85% and the structural compressive modulus was 1.1 MPa.

[0143] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 2.5.

[0144] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 was upregulated by 2.4 times and COL2A1 was upregulated by 2.2 times.

[0145] 5.4 Cell ingrowth and vascularization were observed at the defect site of the rabbit knee joint, with a repair rate of 89%.

[0146] Example 5 (Two biological factors combined for synergistic induction) 1. Raw material pretreatment and gentle peeling: Same as Example 1.

[0147] 2. Decellularization treatment: Same as Example 1.

[0148] 3. Stent molding and crosslinking: Same as Example 1.

[0149] 4. Functional composite: 4.1 The molded scaffold was immersed in a 35% PRP solution at 15℃ and vacuum-loaded (vacuum degree 0.07MPa) for 2 hours.

[0150] 4.2 Then, a mixed solution of 40 ng / mL TGF-β and 30 ng / mL IGF-1 was added, and after activation by EDC / NHS at pH 7.4, it chemically bonded to the scaffold and was soaked for 75 min.

[0151] 5. Material testing: 5.1 The test results showed that the porosity of the support material was 89% and the structural compressive modulus was 1.4 MPa.

[0152] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 2.9.

[0153] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 was upregulated by 3.1 times and COL2A1 was upregulated by 3.0 times.

[0154] 5.4 Cell ingrowth and vascularization were observed at the defect site of the rabbit knee joint, with a hyaline cartilage formation rate of 85%.

[0155] Example 6 (Crosslinking agent combination, low toxicity optimization) 1. Raw material pretreatment and gentle peeling: Same as Example 2.

[0156] 2. Decellularization treatment: Same as Example 2.

[0157] 3. Stent molding and crosslinking: 3.1 Same as Example 1.

[0158] 3.2 Same as Example 1.

[0159] 3.3 Same as Example 1.

[0160] 3.4 The freeze-dried matrix was soaked in a mixture of EDC / NHS (ratio 3:1, concentration 1.5%, pH 5.5, 30℃) for 4 hours, then dehydrated by gradient soaking in 80% ethanol for 1 hour, and then repeatedly washed with deionized water 4 times to remove residual crosslinking agent.

[0161] 3.5 Freeze-drying is the same as in Example 1. 4. Functional compounding: Same as Example 1.

[0162] 5. Material testing: 5.1 The test results showed that the porosity of the support material was 90% and the structural compressive modulus was 1.6 MPa.

[0163] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 3.0.

[0164] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 was upregulated by 3.0 times and COL2A1 was upregulated by 2.8 times.

[0165] 5.4 Cell ingrowth and vascularization were observed at the defect site of the rabbit knee joint, with a cartilage formation rate of 93%.

[0166] Example 7 (High concentration of PRP, accelerating vascularization).

[0167] 1. Raw material pretreatment and gentle peeling: Same as Example 1.

[0168] 2. Decellularization treatment: Same as Example 1.

[0169] 3. Stent molding and crosslinking: 3.1 In a specific mold, liquid nitrogen is placed at the bottom of the mold, and a porous structure with directional channels is constructed through a directional freezing strategy, in which collagen fibers are oriented and orderly arranged.

[0170] 3.2 The directional porous matrix was freeze-dried, pre-frozen at -50℃ (3h), and sublimated at -20℃ (36h) to form a porous sponge-like matrix.

[0171] 3.3 Same surface etching as in Example 1.

[0172] 3.4 The freeze-dried matrix was soaked in 1% formaldehyde (pH 6, 30℃) for 4 hours, then dehydrated by gradient soaking in 80% ethanol for 1 hour, and then repeatedly washed with deionized water 4 times to remove residual crosslinking agent.

[0173] 3.5 Finally, a second freeze-drying process is performed to obtain the molded support.

[0174] 4. Functional composite: 4.1 The molded scaffold was immersed in a 50% PRP solution at 20°C and vacuum-loaded (vacuum degree 0.09MPa) for 3.5h.

[0175] 4.2 Then, add 100 ng / mL of BMP-2 solution, activate it with EDC / NHS at pH 7.4, and chemically bond it with the scaffold. Soak for 90 min.

[0176] 5. Material testing: 5.1 The porosity of the support material was found to be 87% and the structural compressive modulus was 1.2 MPa.

[0177] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 3.2 c).

[0178] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 was upregulated by 3.3 times and COL2A1 was upregulated by 3.1 times.

[0179] 5.4 Cell ingrowth and vascularization were observed at the defect site of the rabbit knee joint, with a cartilage formation rate of 95%.

[0180] Comparative Example 1 (without functionalization) 1. Raw material pretreatment and gentle peeling: Same as Example 1.

[0181] 2. Decellularization treatment: Same as Example 1.

[0182] 3. Stent molding and crosslinking: Same as Example 1.

[0183] 4. Functional composite: The difference from Example 1 is that there is no PRP vacuum loading and no added bioactive factors.

[0184] 5. Material testing: 5.1 The porosity of the support material was found to be 88% and the structural compressive modulus was 1.3 MPa.

[0185] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 1.3.

[0186] 5.3 After 7 days of cell culture, the qPCR results for SOX9 and COL2A1 were both 1.

[0187] 5.4 In rabbit knee joint defects, cell ingrowth was minimal, vascularization was not obvious, and the repair rate was 60%.

[0188] Comparative Example 2 (using traditional strong alkaline hair removal, without using weak alkaline chemical treatment) 1. Raw material pretreatment and gentle peeling: The skin selection is the same as in Example 1.

[0189] The peeling method is the same as in Example 1. The cleaning method is the same as in Example 1. The hair was removed and residual epidermis was removed by treating the hair with a 5% sodium hydroxide strong alkaline solution (pH 12) for 3 hours, followed by washing with PBS 4 times for 15 minutes each time.

[0190] 2. Decellularization treatment: Same as Example 1.

[0191] 3. Stent molding and crosslinking: Same as Example 1.

[0192] 4. Functional composite: Same as Example 1.

[0193] 5. Material testing: 5.1 The porosity of the support material was found to be 80% and the structural compressive modulus was 1.0 MPa.

[0194] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 2.0.

[0195] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 decreased by 0.7 and COL2A1 decreased by 0.8.

[0196] 5.4 Cell ingrowth and vascularization were observed at the defect site of the rabbit knee joint, but the immune rejection reaction was obvious, and the repair rate was 35%.

[0197] Comparative Example 3 (without directional freezing treatment) 1. Raw material pretreatment and gentle peeling: Same as Example 1.

[0198] 2. Decellularization treatment: Same as Example 1.

[0199] 3. Stent molding and crosslinking: Using the traditional freeze-drying method, the substrate is pre-frozen at -50℃ (3h) and sublimated at -20℃ (36h) to form a porous sponge-like matrix.

[0200] The crosslinking method is the same as in Example 1.

[0201] 4. Functional composite: Same as Example 1.

[0202] 5. Material testing: 5.1 Testing revealed that the porosity of the scaffold material was 88%; the structural compressive modulus was 0.7 MPa. Scanning electron microscopy observation showed... Figure 2 As shown, the matrix has a porous structure and does not have directional structural features.

[0203] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 2.3.

[0204] 5.3 After 7 days of cell culture, the qPCR test results (compared to control group 1) showed that SOX9 was upregulated by 2.3 times and COL2A1 was upregulated by 2.1 times.

[0205] 5.4 In rabbit knee joint defects, cell ingrowth was minimal, vascularization was not obvious, and the repair rate was 70%.

[0206] Comparative Example 4 (no matrix etching performed) 1. Raw material pretreatment and gentle peeling: Same as Example 1.

[0207] 2. Decellularization treatment: Same as Example 1.

[0208] 3. Stent molding and crosslinking: The directional freezing method is the same as in Example 1.

[0209] No vertical surface etching of the substrate is performed.

[0210] The matrix crosslinking method is the same as in Example 1.

[0211] 4. Functional composite: Same as Example 1.

[0212] 5. Material testing: 5.1 The porosity of the support material was found to be 84% and the structural compressive modulus was 1.4 MPa.

[0213] 5.2 After 48 hours of cell culture, the OD value of CCK-8 was 2.5.

[0214] 5.3 After 7 days of cell culture, the SOX9 and COL2A1 results of qPCR were 3.5 and 3.3, respectively.

[0215] 5.4 In rabbit knee joint defects, cell ingrowth was minimal, vascularization was not obvious, and the repair rate was 75%.

[0216] Table 1

[0217] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a tissue regeneration and repair scaffold, characterized in that, Includes the following steps: A gel-like decellularized matrix is ​​filled into a mold, and then the bottom of the mold is brought into contact with a cold source to form a directional temperature gradient field from bottom to top. Under the directional temperature gradient field, the gel-like decellularized matrix forms columnar ice crystals along the direction of the directional temperature gradient field. After freeze-drying, a porous matrix with directional channels is obtained. Then, after cross-linking and curing with a cross-linking agent, a tissue regeneration and repair scaffold is prepared.

2. The preparation method according to claim 1, characterized in that, The cold source includes liquid nitrogen; Preferably, the temperature of the cold source is -80℃ to -50℃.

3. The preparation method according to claim 1, characterized in that, The crosslinking agent includes one or more of formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

4. The preparation method according to claim 1, characterized in that, Prior to the cross-linking and curing, the process further includes a step of treating the porous matrix with a biological enzyme solution. Preferably, the concentration of the bio-enzyme solution is 0.01wt%~0.2wt%; the treatment time of the bio-enzyme solution is 1~10min; and the bio-enzyme is selected from one or more of pepsin or pancreatin.

5. The preparation method according to claim 1, characterized in that, The method for preparing the gel-like decellularized matrix includes: preparing a gel-like decellularized matrix by sequentially decellularizing animal tissue and removing antigens and exogenous DNA; The animal tissues include one or more of the pericardium or dermis.

6. The preparation method according to claim 5, characterized in that, The decellularization step includes: freezing and thawing the animal tissue matrix, followed by repeated treatment with hypertonic and hypotonic solutions. Preferably, the freeze-thaw treatment is performed 3 to 6 times; Preferably, the freeze-thaw process is followed by ultrasonic treatment; Preferably, the hypertonic solution is a NaCl solution with a concentration of 1~5 mol / L, and the hypertonic solution treatment time is 10~60 minutes; Preferably, the hypotonic solution is water, and the hypotonic solution treatment time is 10-120 minutes; Preferably, the hypertonic solution treatment and hypotonic solution treatment are repeated 1 to 5 times.

7. The preparation method according to claim 5, characterized in that, The steps for removing antigens and exogenous DNA include: soaking the decellularized matrix in a biological enzyme solution; Preferably, the bioenzyme includes one or more of pepsin or trypsin; Preferably, the mass concentration of the bio-enzyme solution is 0.1% to 2.0%; Preferably, the soaking treatment time is 1-4 hours.

8. The preparation method according to claim 5, characterized in that, The dermal tissue is prepared from skin tissue after pretreatment; The pretreatment includes hair removal; The hair removal treatment involves applying a thiosulfate solution to the skin tissue. Preferably, the concentration of the thiosulfate solution is 2g / 100ml to 5g / 100ml, and the pH is 8 to 10; Preferably, the temperature for treatment with the thiosulfate solution is 20~30℃; Preferably, the treatment time of the thiosulfate solution is 2 to 6 hours.

9. The preparation method according to claim 1, characterized in that, It also includes the step of loading platelet-rich plasma or bioactive factors; The bioactive factors include one or more of TGF-β, IGF-1, or BMP-2; Preferably, the platelet-rich plasma or bioactive factor is loaded via physical adsorption or chemical bonding.

10. A tissue regeneration and repair scaffold, characterized in that... It is prepared by the preparation method according to any one of claims 1 to 9.

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