Decellularized membrane-carrying biological scaffold, preparation method and application

Abstract

The present invention provides a biological scaffold loaded with acellular membrane sheets, a preparation method and an application thereof. The biological scaffold loaded with acellular membrane sheets comprises: a biological scaffold, a decellular membrane sheet for promoting alveolar bone regeneration and a decellular membrane sheet for promoting periodontal ligament regeneration respectively carried on both sides of the biological scaffold. The biological scaffold loaded with acellular membrane sheets is used to prepare a periodontal regeneration module. The biological scaffold loaded with acellular membrane sheets comprises: a biological scaffold, a decellular membrane sheet for promoting alveolar bone regeneration and a decellular membrane sheet for promoting periodontal ligament regeneration respectively carried on both sides of the biological scaffold. The decellular membrane sheet for promoting alveolar bone regeneration and the decellular membrane sheet for promoting periodontal ligament regeneration are used to provide different regeneration environments on both sides of the biological scaffold, thereby providing two independent regeneration environments for periodontal ligament regeneration and alveolar bone regeneration, respectively, thereby achieving periodontal regeneration.

Description

Technical Field

[0001] The present invention relates to the field of basic biomedical research, and in particular to a biological scaffold carrying an acellular membrane sheet, a preparation method and an application thereof. Background Art

[0002] The incremental technology of periodontal defects caused by periodontitis, alveolar process fractures, bone fenestrations, bone cracks, congenital alveolar bone dysplasia and jaw deformity has always been a major clinical problem. At present, the main clinical methods used are periodontal guided bone regeneration and periodontal bone grafting to treat bone defects. However, both treatment methods have certain shortcomings. Among them, guided bone regeneration and other technologies mainly rely on the self-repair of the tissues around the lesions, which is a long process and has uncertain efficacy. In addition, guided bone regeneration mainly targets bone tissue loss, and there is no targeted solution for periodontal attachment loss and periodontal membrane structure loss.

[0003] At present, there are schemes to promote bone regeneration by decellularized extracellular matrix, which often dissolves the decellularized extracellular matrix in a polymer solution and combines it with electrospinning technology to give the extracellular matrix a certain shape and strength. However, this application method often destroys the three-dimensional structure of the extracellular matrix and causes the concentration of the extracellular matrix to decrease, thereby weakening the application effect of the extracellular matrix.

[0004] Therefore, a new solution for periodontal defects is urgently needed. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned background technology, the present invention provides a biological scaffold carrying acellular membrane, a preparation method and an application. The embodiment of the present invention adopts a biological scaffold with good biocompatibility, and directly constructs a high-quality orderly arranged cell membrane by using its cell affinity, and obtains a high-quality, structurally complete, and tightly integrated "sandwich structure" biological scaffold carrying a double-sided acellular membrane through a gentle decellularization method to repair periodontal defects.

[0006] In a first aspect, the present invention provides a decellularized membrane-carrying biological scaffold, which comprises: a biological scaffold, a decellularized membrane sheet for promoting alveolar bone regeneration and a decellularized membrane sheet for promoting periodontal ligament regeneration respectively carried on both sides of the biological scaffold.

[0007] Optionally, the biological scaffold is a three-layer structure, including: a periodontal ligament layer, an intermediate layer, and an alveolar bone layer, the periodontal ligament layer and the alveolar bone layer are ordered fibers, and the intermediate layer is disordered grid-like fibers.

[0008] Optionally, the bioscaffold is a gelatin-polycaprolactone bioscaffold.

[0009] Optionally, the alveolar bone regeneration-promoting decellularized membrane comprises any one of the following: a bone marrow mesenchymal stem cell decellularized membrane, an adipose stem cell decellularized membrane, an osteoblast decellularized membrane;

[0010] The acellular membrane sheet for promoting periodontal ligament regeneration includes any one of the following: a cell-free membrane sheet of periodontal ligament stem cells, a cell-free membrane sheet of dental follicle stem cells, and a cell-free membrane sheet of cementoblasts.

[0011] In a second aspect, the present invention provides a method for preparing a bioscaffold loaded with acellular membrane, the method comprising:

[0012] constructing biological scaffolds;

[0013] A bone marrow mesenchymal stem cell membrane sheet and a periodontal ligament stem cell membrane sheet are respectively mounted on both sides of the biological scaffold;

[0014] The bone marrow mesenchymal stem cell membrane sheet and the periodontal ligament stem cell membrane sheet respectively carried on the two sides of the biological scaffold are subjected to decellularization treatment to obtain a biological scaffold carrying a decellularized membrane sheet.

[0015] Optionally, the constructing of the biological scaffold comprises:

[0016] Prepare gelatin-polycaprolactone electrospinning solution;

[0017] The periodontal ligament layer, the middle layer and the alveolar bone layer are constructed respectively to obtain the electrospun membrane;

[0018] The electrospun membrane is cross-linked to obtain the biological scaffold.

[0019] Optionally, the bone marrow mesenchymal stem cell membrane and the periodontal ligament stem cell membrane respectively mounted on both sides of the biological scaffold include:

[0020] The alveolar bone layer of the biological scaffold is facing upwards to complete the loading of the cell membrane sheet promoting alveolar bone regeneration;

[0021] The bioscaffold is reversed so that the periodontal ligament layer of the bioscaffold faces upward, thereby completing the loading of the cell sheet for promoting periodontal ligament regeneration.

[0022] Optionally, the method further comprises:

[0023] The acellular membrane bioscaffold was immersed in a basic culture medium containing 0.5% double antibody and stored in vitro at 4° C. for a short term.

[0024] Optionally, the method further comprises:

[0025] Washing the acellular membrane bioscaffold with deionized water;

[0026] Freeze-dried and washed decellularized membrane bioscaffold;

[0027] Sterilization using ethylene oxide;

[0028] For long term storage.

[0029] In the third aspect, the present invention provides an application of a decellular membrane-carrying biological scaffold for preparing a periodontal regeneration module, wherein the decellular membrane-carrying biological scaffold comprises: a biological scaffold, a decellular membrane sheet for promoting alveolar bone regeneration and a decellular membrane sheet for promoting periodontal ligament regeneration respectively carried on both sides of the biological scaffold, the decellular membrane sheet for promoting alveolar bone regeneration and the decellular membrane sheet for promoting periodontal ligament regeneration are used to provide different regeneration environments on both sides of the biological scaffold, respectively, to support the formation of alveolar bone tissue and periodontal ligament tissue, respectively.

[0030] In the embodiment of the present invention, a biological scaffold is prepared in the form of a tissue engineering scaffold to promote the regeneration of damaged periodontal tissue. The use of electrospinning can ensure the adjustment of the pore size and diameter of the regeneration membrane and other physical and chemical properties. Thus, different microstructures can be used to guide the formation of multiple tissues in the periodontal tissue, and finally regenerate functional periodontal tissues at the periodontal defect.

[0031] The acellular membrane bioscaffold provided in the embodiment of the present invention is a bioscaffold modified by cell-specific ECM, which successfully loads electrospinning scaffolds with ordered topological structures ECM-P and ECM-B to construct a sandwich structure, thereby providing two independent regeneration environments for periodontal ligament / cementum regeneration and alveolar bone regeneration. Not only does this sandwich-shaped bioscaffold act as a barrier membrane, but the extracellular matrix components carried by it also act as a regeneration initiator.

[0032] In an embodiment of the present invention, a sandwich structure decellularized sheet from periodontal-specific stem cells (PDLSCs and BMSC) is combined with a layered electrospun gelatin basement membrane to provide a microenvironment that can guide cell growth and promote comprehensive periodontal regeneration, with ideal biocompatibility, mechanical properties, and inductivity of cementum differentiation, periodontal differentiation, and osteogenic differentiation. In addition, this sandwich structure acts as a partition to promote cementum regeneration and osteogenic regeneration in different regions, making room for two regeneration centers. In addition, the bioscaffold provided in an embodiment of the present invention has an ideal biodegradation rate, and is gradually replaced by newly formed periodontal fibers in new cementum and alveolar bone, completing periodontal tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a flow chart of a complete periodontal defect repair process in an embodiment of the present invention;

[0034] Figure 2 Schematic diagram of the structure of the three-layer gelatin / polycaprolactone electrospinning membrane prepared in Example 1 of the present invention;

[0035] Figure 3 It is an immunofluorescence image of BMSCs and PDLSCs loaded on the decellularized membrane bioscaffold prepared in Example 2 of the present invention;

[0036] Figure 4 This is a scanning electron microscope observation result diagram of BMSCs and PDLSCs loaded on the decellularized membrane bioscaffold prepared in Example 2 of the present invention;

[0037] Figure 5 The Masson's trichrome staining result of the acellular membrane-carrying bioscaffold prepared in Example 2 of the present invention used in an in vivo experiment of periodontal defects;

[0038] Figure 6 The HE staining result of the acellular membrane bioscaffold prepared in Example 2 of the present invention used in an in vivo experiment of periodontal defects;

[0039] Figure 7 The results of immunofluorescence staining of periodontal-related proteins in the control group and the experimental group after the acellular membrane-loaded bioscaffold prepared in Example 2 of the present invention was used in an in vivo experiment on periodontal defects. DETAILED DESCRIPTION

[0040] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0041] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. The reagents and other instruments used without specifying the manufacturer are all conventional reagent products that can be purchased on the market.

[0042] Periodontal defects caused by periodontitis, trauma, and tumors have always been a concern, and periodontal defects usually lead to periodontal damage, attachment loss, bone defects, and finally tooth loss. Due to the complex tissue structure of periodontal tissue, the regeneration of lost periodontal tissue is challenging. The periodontium is a complex tissue with a sandwich-like structure: alveolar bone-periodontal ligament-cementum, in which the two ends of the periodontal ligament fibers are tightly bound in the alveolar bone and cementum.

[0043] Bone marrow mesenchymal stem cells (BMSCs) from the mandible are mainly involved in the regeneration of alveolar bone, while dental follicle stem cells (PDLSCs) are mainly involved in the regeneration of periodontal ligament and cementum.

[0044] Therefore, the embodiment of the present invention proposes: providing different regeneration cues on both sides of the biomaterial for periodontal regeneration to support the formation of different tissues.

[0045] The extracellular matrix (ECM) is a dynamic and complex microenvironment with excellent biophysical, biomechanical, and biochemical properties that plays a key role in homeostasis and tissue regeneration.

[0046] Based on this, after a series of explorations, the present invention proposes a new solution for periodontal regeneration, such as Figure 1 As shown, a schematic flow diagram of the periodontal regeneration strategy provided by an embodiment of the present invention is shown, including three parts: (A) construction of a biological scaffold (GP-TLS scaffold), (B) construction of an ordered periodontal ligament stem cell (PDLSC) / bone marrow mesenchymal stem cell (BMSC) membrane sheet, and construction and application of a double-loaded ordered PDLSC / BMSC membrane sheet extracellular matrix scaffold. Among them, the biological scaffold includes: a periodontal ligament layer with an ordered fiber surface, an intermediate layer with a porous structure, and an alveolar bone layer with ordered fibers, and the material of the biological scaffold is gelatin / polycaprolactone. After completing the construction of the biological scaffold, BMSCs are inoculated in the alveolar bone layer of the biological scaffold, and then the biological scaffold is turned over, and PDLSC is inoculated in the periodontal ligament layer, thereby completing the construction of the cell membrane sheet and obtaining an ordered PDLSC / BMSC membrane. Further, the biological scaffold loaded with an ordered PDLSC / BMSC membrane is decellularized using a decellularization technique to obtain a double-loaded ordered PDLSC / BMSC membrane sheet extracellular matrix scaffold, the periodontal ligament side of the scaffold is loaded with PDLSC extracellular matrix, and the alveolar bone side is loaded with BMSC extracellular matrix. The double-loaded ordered PDLSC / BMSC membrane extracellular matrix scaffold was transplanted into the periodontal defect to complete the repair of the periodontal defect.

[0047] Specifically, an embodiment of the present invention provides a decellularized membrane-carrying biological scaffold, which includes: a biological scaffold, a decellularized membrane sheet for promoting alveolar bone regeneration and a decellularized membrane sheet for promoting periodontal ligament regeneration respectively carried on both sides of the biological scaffold.

[0048] The biological scaffold is a three-layer structure, including: a periodontal membrane layer, an intermediate layer, and an alveolar bone layer. The periodontal membrane layer and the alveolar bone layer are ordered fibers, and the intermediate layer is disordered grid-like fibers.

[0049] Specifically, the biological scaffold is a gelatin-polycaprolactone biological scaffold.

[0050] The alveolar bone regeneration-promoting decellularized membrane sheet includes any one of the following: a bone marrow mesenchymal stem cell decellularized membrane sheet, adipose stem cell decellularized membrane sheet, and osteoblast decellularized membrane sheet.

[0051] In the embodiments of the present invention, bone marrow mesenchymal stem cells, adipose stem cells or osteoblasts with alveolar bone regeneration ability can be used as the source of the decellularized membrane for promoting alveolar bone regeneration.

[0052] The acellular membrane sheet for promoting periodontal ligament regeneration includes any one of the following: a cell-free membrane sheet of periodontal ligament stem cells, a cell-free membrane sheet of dental follicle stem cells, and a cell-free membrane sheet of cementoblasts.

[0053] In the embodiments of the present invention, periodontal ligament stem cells, dental follicle stem cells, and cementoblasts that have the ability to regenerate periodontal ligament can all be used as sources of acellular membrane sheets that promote periodontal ligament regeneration.

[0054] Simple application of pure ECM often fails to reconstruct defective structures. This may be caused by the complex composition and structure of periodontal tissue, which is an organic combination of soft and hard tissues, while ECM is usually too fragile to provide the required stiffness and mechanical transduction of load forces. Decellularized membranes are too fragile to form reliable 3D structures with the desired shape and size, and cannot be maintained in certain periodontal induced structures.

[0055] In the embodiment of the present invention, a biological scaffold is prepared in the form of a tissue engineering scaffold to promote the regeneration of damaged periodontal tissue. The use of electrospinning can ensure the adjustment of the pore size and diameter of the regeneration membrane and other physical and chemical properties. Thus, different microstructures can be used to guide the formation of multiple tissues in the periodontal tissue, and finally regenerate functional periodontal tissues at the periodontal defect.

[0056] The acellular membrane-carrying bioscaffold provided in the embodiment of the present invention is a bioscaffold modified by cell-specific ECM, which successfully loads an electrospinning scaffold with an ordered topological structure of PDLSC extracellular matrix (ECM-P) and BMSC extracellular matrix (ECM-B) to construct a sandwich structure, thereby providing two independent regeneration environments for periodontal ligament regeneration and alveolar bone regeneration, respectively. This sandwich-shaped bioscaffold acts as a barrier membrane, and the extracellular matrix it carries also acts as a regeneration initiator.

[0057] In an embodiment of the present invention, a sandwich structure decellularized sheet from periodontal-specific stem cells (PDLSCs and BMSC) is combined with a layered electrospun gelatin basement membrane to provide a microenvironment that can guide cell growth and promote comprehensive periodontal regeneration, with ideal biocompatibility, mechanical properties, and inductivity of cementum differentiation, periodontal differentiation, and osteogenic differentiation. In addition, this sandwich structure acts as a partition to promote cementum regeneration and osteogenic regeneration in different regions, making room for two regeneration centers. In addition, the bioscaffold provided in an embodiment of the present invention has an ideal biodegradation rate, and is gradually replaced by newly formed periodontal fibers in new cementum and alveolar bone, completing periodontal tissue regeneration.

[0058] The present invention also provides a method for preparing a bioscaffold carrying acellular membrane, the method comprising the following steps:

[0059] S1, construction of biological scaffolds.

[0060] Specifically, S1 includes the following sub-steps:

[0061] S11, prepare gelatin-polycaprolactone electrospinning solution: 3.6 g gelatin / 0.4 g polycaprolactone (90:10), a total of 4 g, are dissolved in 40 mL trifluoroethanol and incubated in a water bath at 37° C. overnight.

[0062] S12, constructing a periodontal membrane layer, an intermediate layer, and an alveolar bone layer respectively to obtain an electrospun membrane:

[0063] "Periodontal membrane layer" electrospinning: using 28-30KV voltage and 1600-1800rpm rotation speed, obtain the "periodontal membrane layer" orderly electrospinning layer; simulate the orderly surface structure of the periodontal membrane;

[0064] "Middle layer" electrospinning: using 28-30KV voltage and 500-600rpm rotation speed to obtain a disordered electrospinning layer; providing anisotropy to maintain mechanical strength and avoid weak spinning structure; promoting tissue ingrowth and material degradation;

[0065] Electrospinning of “alveolar bone layer”: Using 28-30KV voltage and 1600-1800rpm rotation speed, an orderly electrospinning layer of “alveolar bone layer” is obtained.

[0066] S13, cross-linking the electrospun membrane to obtain the bioscaffold.

[0067] Specifically, S13 includes the following sub-steps:

[0068] S131, preparing a cross-linking liquid: thoroughly dissolving 0.47 g of EDC and 0.28 g of NHS in 5 mL of deionized water, and transferring to 95 mL of anhydrous ethanol to obtain a cross-linking liquid;

[0069] S132, cross-linking: at room temperature, place the Bio-House in the cross-linking solution and shake it in a low-speed horizontal shaker overnight;

[0070] S133, rehydration: put the cross-linked bioscaffold into 500 mL of deionized water, change it every 4 hours, and shake it on a low-speed horizontal shaker for 24 hours. After that, transfer the bioscaffold to a -20°C refrigerator for 2 hours, and then transfer the bioscaffold to a -80°C refrigerator overnight;

[0071] S134, freeze drying: freeze drying for 12 h until the bioscaffold is completely dry;

[0072] S135, sterilizing the freeze-dried bioscaffold using ethylene oxide and storing it.

[0073] S2, respectively mounting a bone marrow mesenchymal stem cell membrane sheet and a periodontal ligament stem cell membrane sheet on both sides of the biological scaffold.

[0074] The step S2 specifically includes the following sub-steps:

[0075] Step S21, the alveolar bone layer of the biological scaffold is facing upward to complete the loading of the cell membrane sheet promoting alveolar bone regeneration.

[0076] Specifically, step S21 includes:

[0077] The bone side of the biological scaffold is facing upwards, and gravity is used to complete the loading of the alveolar bone regeneration cell membrane. For example, bone marrow mesenchymal stem cells can be used as the alveolar bone regeneration cells. 4*10 5 BMSCs were inoculated for 2 days.

[0078] Step S22, inverting the bioscaffold so that the periodontal ligament layer of the bioscaffold faces upward, completing the loading of the cell sheet promoting periodontal ligament regeneration.

[0079] Specifically, step S22 includes:

[0080] Reverse the periodontal ligament so that the periodontal ligament side faces upward to achieve periodontal ligament stem cell membrane loading. Taking the periodontal ligament regeneration cells as periodontal ligament stem cells as an example, 1*10 5 The co-culture of BMSC and PDLSC was completed using a common culture medium to obtain a double-sided membrane sheet scaffold.

[0081] S3, performing decellularization treatment on the bone marrow mesenchymal stem cell membrane sheet and the periodontal ligament stem cell membrane sheet respectively carried on both sides of the biological scaffold to obtain a biological scaffold carrying a decellularized membrane sheet.

[0082] Specifically, the S3 includes: 1% tritonx-100 (1 mL) + 4 mmol / L ammonia water (1 mL) treatment for 15 minutes, then sterile saline 2 mL shaker washing 3 times, each time for 10 minutes; cycle 3 times; a-MEM medium balancing 3 times, each time for 1 hour; 0.02 mg / mL DNA enzyme incubation at 37 ° C overnight; a-mem rinse 3 times, each time for 15 minutes.

[0083] In the embodiment of the present invention, after the decellularized membrane sheet bioscaffold is prepared, the decellularized membrane sheet bioscaffold can also be stored in vitro, specifically including: placing the decellularized membrane sheet bioscaffold in a basic culture medium containing 0.5% double antibody to inhibit bacterial growth and maintain the scaffold morphology, and storing it at 4°C for in vitro storage. In the embodiment of the present invention, under this storage condition, the decellularized membrane sheet bioscaffold can be stored in vitro for two weeks.

[0084] In the embodiment of the present invention, the acellular membrane bioscaffold can also be stored for a long time, specifically including:

[0085] Washing the acellular membrane bioscaffold with deionized water;

[0086] Freeze-dried and washed decellularized membrane bioscaffold;

[0087] Sterilization using ethylene oxide;

[0088] For long term storage.

[0089] An embodiment of the present invention also proposes an application of a decellular membrane-carrying biological scaffold for preparing a periodontal regeneration module. The decellular membrane-carrying biological scaffold includes: a biological scaffold, a decellular membrane sheet for promoting alveolar bone regeneration and a decellular membrane sheet for promoting periodontal ligament regeneration respectively carried on both sides of the biological scaffold. The decellular membrane sheet for promoting alveolar bone regeneration and the decellular membrane sheet for promoting periodontal ligament regeneration are used to provide different regeneration environments on both sides of the biological scaffold, respectively, to support the formation of alveolar bone tissue and periodontal ligament tissue, respectively.

[0090] In order to enable those skilled in the art to better understand the present invention, the following specific examples are used to illustrate the acellular membrane bioscaffold, preparation method and application provided by the present invention.

[0091] Example 1: Preparation of three-layer gelatin / polycaprolactone electrospun membrane (TLS)

[0092] A transparent solution of gelatin / polycaprolactone (3.6 g / 0.4 g) dissolved in trifluoroethanol (TFE, 40 ml) doped with acetic acid (HAc) (HAc / TFE 0.1%) was delivered to a blunt metal needle (18G) used as a spinneret by a syringe pump at a feed rate of 1.0 ml / h. Different rotation rates were applied to form a layered pattern. Specifically, the electrospinning was collected at a speed of 1600 rpm for 3 hours to obtain an electrospinning membrane sheet of a certain thickness, forming a periodontal membrane layer with ordered fibers, 600 rpm for 4 hours to form a porous middle layer with randomly distributed fibers, and collected at a speed of 1600 rpm for 3 hours to form an alveolar bone layer with ordered fibers to obtain an ideal three-layer electrospinning membrane.

[0093] In an embodiment of the present invention, a 28.0 kV potential of a high voltage power supply (TXR1020N30-30, Teslaman, Dalian, China) is applied between the spinneret and an aluminum foil (150 mm * 300 mm) mounted on the surface of an adjustable laboratory jack, and a gap distance of 15 cm is maintained from the tip of the spinneret. The prepared gelatin / polycaprolactone fiber membrane was dried in a vacuum oven at room temperature for 3 days to remove the residual solvent before subsequent use. The prepared three-layer electrospun membrane was cross-linked, specifically comprising: dissolving 1.91 grams of EDC and 1.15 grams of NHS in 100 mL of anhydrous ethanol, and then immersing the three-layer electrospun membrane in the solution for 24 hours in a 37 ° C water bath for shaking. The three-layer electrospun membrane can be customized into different shapes later as a biological scaffold.

[0094] Morphological studies Scanning electron microscopy (SEM) observations of the gold-sprayed samples were performed using a SEM instrument (S-4800, Hitachi, Japan) at an accelerating voltage of 5 kV. Approximately 150 fibers and 500 pores were measured by image acquisition and analysis software (NanoMeasurer1.2), and the distribution of fiber diameter and pore size was determined. The results were presented as mean diameter ± standard deviation (M ± SD).

[0095] In the embodiment of the present invention, a three-layer gelatin / polycaprolactone electrospinning membrane (TLS) is constructed by adjusting the electrospinning speed. Figure 2 As shown, Figure 2 The schematic diagram of the structure of the three-layer gelatin / polycaprolactone electrospun membrane prepared in the embodiment of the present invention is shown. The left side shows the cross-sectional view and surface SEM micrographs of the electrospun membrane. From top to bottom, they are: periodontal membrane layer, porous inner layer, alveolar bone layer; white arrows indicate the fiber direction. The average fiber diameter and average pore size of each membrane are also given on the right.

[0096] Example 2: Preparation of acellular membrane-loaded bioscaffold

[0097] Human periodontal ligament samples from 10 individuals (15-18 years old) were scraped from the middle 1 / 3 of the root surface of healthy premolars extracted for orthodontic treatment. PDLSCs were isolated as follows: First, the tissue samples were cut into 1 mm 3The pieces were sliced ​​and washed in phosphate buffered saline (PBS); subsequently, the excised fragments were digested with a solution of 2 mg / ml collagenase I (Sigma) at 37°C for 10 minutes; after a brief digestion, the tissue fragments were cultured in α-MEM containing 10% fetal bovine serum (FBS) and 100 U / ml penicillin / streptomycin at 37°C and 5% CO2 incubator culture environment. Once cells were observed to grow out of the tissue pieces, they were digested and collected with 0.25% trypsin; the passaged cells were cultured in basal medium (α-MEM containing 10% FBS). PDLSCs between passages 3-5 were used for the following experiments.

[0098] Human bone marrow mesenchymal stem cells were purchased from Celgene Corporation and cultured in α-MEM containing 10% fetal bovine serum (FBS) and 100 U / ml penicillin / streptomycin. PDLSCs between passages 4 and 6 were used in the following experiments.

[0099] The three-layer gelatin / polycaprolactone electrospun membrane (TLS) prepared in Example 1 was cut to obtain a biological scaffold, and 5*10 4 After 24 hours, the bioscaffold was flipped over and 5*10 BMSCs were inoculated on the surface of the periodontal ligament. 4 PDLSCs were cultured on both sides of the membrane. The cells were cultured in the same medium with the same medium components. After 48 hours, ascorbic acid at a concentration of 100ug / mL was added to the culture medium to induce the cells to form specific cell sheets on each side of the electrospun membrane to obtain a cell-loaded membrane bioscaffold. Scanning electron microscopy and immunofluorescence staining were used to characterize the cell-loaded membrane bioscaffold.

[0100] Immunofluorescence staining was used to detect cell morphology and compare the distribution of cells on ordered and random fiber membranes. Fixed cells were permeabilized with 0.5% Triton X-100 for 15 minutes (no permeabilization of cell surface antigens). The cells were then blocked in 5% bovine serum albumin in PBS containing 0.1% Triton X-100 for 1 hour at room temperature. The cells were stained with phalloidin (CST, #8953) at room temperature for 30 minutes and then washed three times with PBS before DAPI staining (Solarbio, China).

[0101] Immunofluorescence images of BMSCs and PDLSCs Figure 3 As shown in Figure 3, the morphology of the three-layer scaffold is suitable for the preparation of cell sheets derived from BMSCs and PDLSCs on the bone side and PDL side of the three-layer GPA scaffold, respectively. Cell spreading along the fibers was also observed by SEM, as shown in Figure 3. Figure 4As shown, the results confirmed that the three-layer gelatin / polycaprolactone electrospun membrane (TLS) is an ideal scaffold for growing orderly cell sheets on each side.

[0102] Construction of acellular membrane bioscaffold: The culture was treated with PBS containing 0.5% TritonX-100 (Sigma-Aldrich) and 2mM ammonium hydroxide at 37°C for 15 minutes to lyse the cell membrane, and the cell components were harvested and removed by cyclic washing. Specifically, the culture was washed with PBS 5 times, each time for 5-10 minutes, and finally the acellular membrane bioscaffold was obtained.

[0103] Example 3: In vivo testing

[0104] In order to characterize the osteogenic / fibrogenic differentiation ability of the decellularized membrane bioscaffold, alveolar bone defects were modeled in vivo, and the size of each hole was 3 mm (L) × 2 mm (W) × 1 mm (D). Rats were randomly divided into 6 groups (n = 4 in each group). The groups were divided into: (1) blank: the control group had alveolar bone defect but did not receive any treatment; (2) biological scaffold: the experimental group had alveolar bone defect and was treated with biological scaffold; (3) ECM-P-scaffold: the experimental group had alveolar bone defect and was treated with PDLSC-ECM scaffold; (4) ECM-B-scaffold: the experimental group had alveolar bone defect and was treated with ECM-B-scaffold; (5) double ECM-scaffold: the experimental group had alveolar bone defect and was treated with double ECM scaffold, using ECM-P on the PDL side of the defect and ECM-B on the BMSC side; (6) r-Bi-ECM-scaffold: the experimental group had alveolar bone defect and was treated with reverse-laid double ECM scaffold; (5) and (6) were performed on either side of the mandible of the same individual.

[0105] After 6 weeks, the rats were killed, and the mandibles were harvested and fixed with 10% formaldehyde. Micro-CT tomography was used. 3D images were reconstructed by NRecon software. The region of interest (ROI) was selected with a range of 3 mm (length) × 2 mm (width) × 1 mm (depth) corresponding to the defect.

[0106] Afterwards, the mandibles were decalcified in 10% disodium ethylenediaminetetraacetic acid (EDTA, Sigma, USA), dehydrated and embedded in paraffin. The paraffin samples were prepared into 5 μm thick sections and subjected to hematoxylin and eosin (H&E), Masson's trichrome (Basso Diagnostics, China) and immunofluorescence (IF) staining. The primary antibodies used included OPN (1:400, Abcam, UK), OCN (1:300, Zhengneng, China), Periostin (1:200, Abcam, UK) and RUNX2 (1:400, Abcam, UK). Images were acquired by confocal microscopy (Olympus, Japan), and the results are shown in the figure. Figure 5-7 As shown, Figure 5 The results of Masson's trichrome staining. Figure 6 HE staining results, Figure 7 This is the result of immunofluorescence staining of periodontal-related proteins.

[0107] In the embodiment of the present invention, the function of the decellularized membrane bioscaffold (TLS loaded with ECM-B / P) in supporting cell endo-growth and in situ reconstruction of new tissues was studied. In view of the fact that ECM-B plays a favorable role in bone regeneration, and ECM-P has advantages in both cementum regeneration and periodontal regeneration, the ECM-P in Bi-ECM-TLS in contact with the root surface is used for periodontal ligament regeneration, and ECM-B in contact with the periosteum is used for alveolar bone regeneration. In another group, the decellularized membrane bioscaffold was placed in the opposite way to study whether ECM from different sources plays different roles in the regeneration of soft and hard tissues. In addition, TLS loaded with only one decellularized membrane sheet was used to study the effects of ECM-P or ECM-B on PDL and periodontal regeneration. A bare TLS scaffold was set up, compared with a control group in which the periodontal defect was not treated.

[0108] H&E staining of the periodontal tissue containing the implants was performed six weeks after implantation in rat periodontal defects. In all cases, the defects were covered by fibrous / bone tissue. Most of the residual defect area in the control group was invaded by loosely organized fibrous connective tissue, with visible hyperemia and edema. However, the control group showed minimal new bone formation, and the residual defect area was filled with connective tissue without a clear PDL boundary. The bare scaffold TLS seemed to provide support for tissue regeneration.

[0109] Although there was no significant difference between the two single ECM-loaded groups (ECM-B and ECM-P), the dual-loaded ECM scaffolds had a better periodontal regeneration effect, showing more bone regeneration. In addition, according to the results of micro-CT evaluation, even though less bone regeneration was detected in the reversed bi-ECM (r-Bi-ECM), even though it showed better periodontal regeneration, such as Figure 6 As shown in the figure, various cell-loaded extracellular matrix scaffolds have different periodontal regeneration performances after being used in periodontal defects. Figure 7Hematoxylin and eosin (H&E) staining results are shown. Although the new bone was the thickest among the six groups, it can be seen that there is a fibrous structure in the middle of the new bone, and new bone structure can be observed in the new periodontal ligament. The affinity of fibroblasts for ECM-P seems to make it easier for PDLSC and fibroblasts to penetrate into the matrix, resulting in the thickest fibrous tissue of all groups, and more fibrous tissue grows in the defect where alveolar bone should exist, resulting in an arc-shaped gap on the cortical bone surface. Although the new PDL tends to be healthier, with a thinner PDL width and good fiber quality, ECM-P is in contact with the root surface. This may be due to the induction of ECM-P and the upregulation of PLAP-1 on PDLSC. When the periodontium is damaged under pathological conditions, the upregulation of PLAP-1 may hinder bone regeneration analysis The current data suggest that PLAP-1 may have a negative impact on this process, indicating that the presence of ECM-P may have a negative impact on bone regeneration.

[0110] Furthermore, in the present invention, immunofluorescence staining was performed to verify periodontal regeneration by labeling specific proteins in the new periodontal ligament and new alveolar bone. In the double ECM group, higher expression of ligament-specific markers (Periostin) and osteogenic markers (RUNX2, OCN, OPN) was observed (e.g. Figure 7 ). Periostin is an extracellular matrix protein that supports the adhesion and migration of fibroblasts and osteoblasts and is thought to promote periodontal regeneration by recruiting PDL fibroblasts and osteoblasts for new PDL and bone formation, respectively. High expression of Periostin was observed on the bi-ECM loaded (Bi-ECM) scaffolds, which was consistent with the HE and Masson results, showing more oriented fibers and new alveolar bone connected to the ligament structure, suggesting stronger ligament attachment. In addition, osteogenic differentiation-related factors OCN, OPN, and RUNX2 were highly expressed in the Bi-ECM scaffold group, which indicated the mineralization of the bone extracellular matrix. In summary, these findings suggest that the selected ECM promotes periodontal regeneration through a synergistic combination of enhancing cell viability, migration, proliferation, matrix synthesis, and differentiation to form new bone and PDL attachment through specific cell-ECM interactions.

[0111] It has been confirmed in the relevant art that ECM can affect cell chemotaxis and direct cell differentiation, and induce constructive host tissue remodeling. The above effects can be attributed to the 3D structure, surface topology and composition of ECM. Integrin can receive signals from ECM or ECM-derived mechanical stimulation and transduce them to downstream signaling pathways that regulate cell viability. When grown on the surface of a fiber scaffold with ideal physicochemical properties, BMSC cell sheets are formed in an osteogenic microenvironment, which can ensure that the ECM-B obtained after decellularization maintains osteogenic ability. However, PDLSC cell sheets are cultured in a fibrotic microenvironment topology with an ordered fiber topology, and ECM-P formation has fibrogenic differentiation ability, which may be more likely to promote cell adhesion and ligamentogenesis. ECM extracted from cells with fibrogenic potential is more likely to promote fiber regeneration. Therefore, the ordered ECM-P topology can be used as a regenerative template for PDLSC periodontal tissue.

[0112] In the embodiment of the present invention, a TLS with an ideal topological structure is manufactured, which helps to change the ECM structure and has good biocompatibility, and helps to integrate ECM-B and ECM-P to form a 3D structure for periodontal regeneration. The ECM extracted from the MSC sheet maintains an ordered structure according to the topological structure of the electrospun biocompatible TLS. The interaction between the ordered bioscaffold topological structures promotes the MSC sheet to produce more ECM with certain topological structures to maximize the regeneration induction effect of ECM derived from PDLSC and BMSC. In vitro studies have shown that ECM-P and ECM-B derived from decellularized cell sheets are biocompatible after treatment with mild decellularization agents, and improve the proliferation, adhesion, migration and differentiation of PDLSCs and BMSCs. In addition, the embodiment of the present invention verifies the importance of periodontal-specific MSC-derived ECM for biocompatible scaffolds to stimulate periodontal regeneration. In addition, the embodiment of the present invention further verifies the different responses of MSCs to ECM from different sources. It is evident that ECM-B exerts a significant promoting effect on adhesion, as evidenced by upregulation of integrin β1 and general activation of the integrin-linked kinase (ILK) pathway to promote bone regeneration. This was further demonstrated by mRNA expression and protein expression of osteogenic genes. However, ECM-P was also shown to have cell-specific effects on PDLSCs, with effects on cell proliferation, migration, ligamentogenesis, and cementum-based potential of PDLSCs. The rat buccal defect model demonstrated that the Bi-ECMs scaffold maximized the benefits of ECM in promoting periodontal regeneration through the formation of new ligamentous attachments via two carefully constructed periodontogenic centers. Notably, a ligamentous structure called neoperiodontium was naturally formed with the degradation of the Gel / PCL scaffold and the invasion of new periodontal fibers and alveolar bone.

[0113] For the method embodiments, for the sake of simplicity, they are all described as a series of action combinations, but those skilled in the art should know that the present invention is not limited by the order of the actions described, because according to the present invention, some steps can be performed in other orders or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and components involved are not necessarily required by the present invention.

[0114] The above is a detailed introduction to a decellularized membrane bioscaffold, preparation method and application provided by the present invention. Specific examples are used in this article to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea; at the same time, for general technicians in this field, according to the idea of ​​the present invention, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A bioscaffold loaded with acellular membrane, characterized in that: The acellular membrane-carrying biological scaffold comprises: a biological scaffold, an alveolar bone regeneration-promoting acellular membrane and a periodontal ligament regeneration-promoting acellular membrane respectively carried on two sides of the biological scaffold; The biological scaffold is a three-layer structure, including: a periodontal ligament layer, an intermediate layer, and an alveolar bone layer, wherein the periodontal ligament layer and the alveolar bone layer are ordered fibers, and the intermediate layer is disordered grid-like fibers; The preparation method of the acellular membrane-carrying biological scaffold comprises: constructing biological scaffolds; A bone marrow mesenchymal stem cell membrane sheet and a periodontal ligament stem cell membrane sheet are respectively mounted on both sides of the biological scaffold; Decellularization is performed on the bone marrow mesenchymal stem cell membrane sheet and the periodontal ligament stem cell membrane sheet respectively carried on both sides of the biological scaffold to obtain a biological scaffold carrying the decellularized membrane sheet; A bone marrow mesenchymal stem cell membrane and a periodontal ligament stem cell membrane are respectively mounted on both sides of the biological scaffold, including: Inoculate 4*10 5 Bone marrow mesenchymal stem cells were inoculated into the periodontal ligament layer at a rate of 1*10 5 The periodontal ligament stem cells were co-cultured with bone marrow mesenchymal stem cells and periodontal ligament stem cells using a common culture medium to obtain a double-sided membrane sheet scaffold.

2. The acellular membrane bioscaffold according to claim 1, characterized in that: The biological scaffold is a gelatin-polycaprolactone biological scaffold.

3. The acellular membrane bioscaffold according to claim 1, characterized in that: The alveolar bone regeneration-promoting decellularized membrane sheet includes any one of the following: a bone marrow mesenchymal stem cell decellularized membrane sheet, adipose stem cell decellularized membrane sheet, and osteoblast decellularized membrane sheet; The acellular membrane sheet for promoting periodontal ligament regeneration includes any one of the following: a cell-free membrane sheet of periodontal ligament stem cells, a cell-free membrane sheet of dental follicle stem cells, and a cell-free membrane sheet of cementoblasts.

4. A method for preparing a bioscaffold loaded with acellular membrane, characterized in that: The method comprises: Constructing a biological scaffold, wherein the biological scaffold has a three-layer structure, including: a periodontal ligament layer, an intermediate layer, and an alveolar bone layer, wherein the periodontal ligament layer and the alveolar bone layer are ordered fibers, and the intermediate layer is disordered grid-like fibers; A bone marrow mesenchymal stem cell membrane sheet and a periodontal ligament stem cell membrane sheet are respectively mounted on both sides of the biological scaffold; Decellularization is performed on the bone marrow mesenchymal stem cell membrane sheet and the periodontal ligament stem cell membrane sheet respectively carried on both sides of the biological scaffold to obtain a biological scaffold carrying the decellularized membrane sheet; A bone marrow mesenchymal stem cell membrane and a periodontal ligament stem cell membrane are respectively mounted on both sides of the biological scaffold, including: Inoculate 4*10 5 Bone marrow mesenchymal stem cells were inoculated into the periodontal ligament layer at a rate of 1*10 5 The periodontal ligament stem cells were co-cultured with bone marrow mesenchymal stem cells and periodontal ligament stem cells using a common culture medium to obtain a double-sided membrane sheet scaffold.

5. The preparation method according to claim 4, characterized in that: The construction of the biological scaffold comprises: Prepare gelatin-polycaprolactone electrospinning solution; The periodontal ligament layer, the middle layer and the alveolar bone layer are constructed respectively to obtain the electrospun membrane; The electrospun membrane is cross-linked to obtain the biological scaffold.

6. The preparation method according to claim 5, characterized in that: The bone marrow mesenchymal stem cell membrane and the periodontal ligament stem cell membrane are respectively mounted on both sides of the biological scaffold and include: The alveolar bone layer of the biological scaffold is facing upwards to complete the loading of the cell membrane sheet promoting alveolar bone regeneration; The bioscaffold is inverted so that the periodontal ligament layer of the bioscaffold faces upward, thereby completing the loading of the cell sheet that promotes periodontal ligament regeneration.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The method further comprises: The acellular membrane bioscaffold was immersed in a basic culture medium containing 0.5% double antibody and stored in vitro at 4° C. for a short term.

8. The preparation method according to any one of claims 4 to 6, characterized in that: The method further comprises: Washing the acellular membrane bioscaffold with deionized water; Freeze-dried and washed decellularized membrane bioscaffold; Sterilization using ethylene oxide; For long term storage.

9. An application of a bioscaffold carrying acellular membrane, characterized in that: Applicable to the preparation of a periodontal regeneration module, the acellular membrane-carrying biological scaffold comprises: a biological scaffold, an alveolar bone regeneration-promoting acellular membrane and a periodontal ligament regeneration-promoting acellular membrane respectively carried on both sides of the biological scaffold, the alveolar bone regeneration-promoting acellular membrane and the periodontal ligament regeneration acellular membrane are used to provide different regeneration environments on both sides of the biological scaffold, respectively, to support the formation of alveolar bone tissue and periodontal ligament tissue respectively; the biological scaffold is a three-layer structure, including: a periodontal ligament layer, an intermediate layer, and an alveolar bone layer, the periodontal ligament layer and the alveolar bone layer are ordered fibers, and the intermediate layer is disordered grid-like fibers; The preparation method of the acellular membrane-carrying biological scaffold comprises: constructing biological scaffolds; A bone marrow mesenchymal stem cell membrane sheet and a periodontal ligament stem cell membrane sheet are respectively mounted on both sides of the biological scaffold; Decellularization is performed on the bone marrow mesenchymal stem cell membrane sheet and the periodontal ligament stem cell membrane sheet respectively carried on both sides of the biological scaffold to obtain a biological scaffold carrying the decellularized membrane sheet; A bone marrow mesenchymal stem cell membrane and a periodontal ligament stem cell membrane are respectively mounted on both sides of the biological scaffold, including: Inoculate 4*10 5 Bone marrow mesenchymal stem cells were inoculated into the periodontal ligament layer at a rate of 1*10 5 The periodontal ligament stem cells were co-cultured with bone marrow mesenchymal stem cells and periodontal ligament stem cells using a common culture medium to obtain a double-sided membrane sheet scaffold.

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