Periosteum material for promoting local endogenous bone regeneration and repair and preparation method thereof

Abstract

The embodiments of the present invention disclose a periosteal material for promoting local endogenous bone regeneration and repair, and a preparation method thereof. The method comprises: dissolving GelMA in PBS to obtain a GelMA solution; adding a photoinitiator, HAAMMA, and E7 peptide to the GelMA solution, and conducting a free radical polymerization reaction under ultraviolet light to obtain a polymer product; washing the polymer product, freeze-drying, and sterilizing to obtain the periosteal material; wherein the HAAMMA is MA-modified HAAM. The present invention provides a bone tissue engineering membrane material prepared using GelMA, HAAM, and E7 peptide as raw materials, which can promote both stem cell recruitment and osteoblast differentiation into bone in the bone defect area, i.e., a biomimetic periosteal material with osteogenesis-promoting function, to address the problems of poor graft survival, healing, and slow healing after bone transplantation in large bone defects.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedical engineering technology, and in particular to a periosteum material for promoting local endogenous bone regeneration and repair and a preparation method thereof. Background Art

[0002] In bone injuries, the healing of original bone tissue and the formation and maturation of new bone are a lengthy process. Currently, numerous bone defect repair techniques exist, including autologous bone transplantation, allogeneic bone transplantation, xenogeneic bone transplantation, bone lengthening, and periosteal induction. However, the main drawbacks of these methods include limited donor bone resources, increased pain for patients, uncertain efficacy of biomaterials, prolonged healing cycles, and high treatment costs. Natural periosteum contains a variety of mesenchymal stem cells and osteoblasts. Postoperative removal of part of the periosteum can severely impair the normal recovery of bone tissue. While constructing tissue-engineered periosteum is currently an important treatment for bone defects, well-known bone defect treatments currently focus on promoting the osteogenic differentiation of exogenous or endogenous MSCs / progenitor cells, often overlooking the impact of the locally available amount and degree of differentiation of MSCs on bone repair efficiency.

[0003] Research on the "three elements" of cells, bioactive factors, and scaffolds has made unprecedented progress in bone tissue engineering. Currently, a large number of different types of bioactive factors have been combined with scaffold materials for bone tissue engineering scaffolds. However, bone tissue engineering materials prepared by different processes have different characteristics and types. Depending on the preparation method, the added factors, and the scaffold material, bone tissue engineering materials generally have different effects. To address the survival and osteogenesis of grafts in large bone defects, periosteal tissue, which has osteogenic activity and can provide an adequate blood supply, has gradually been widely studied. Summary of the Invention

[0004] The present invention is based on the inventor's discovery and understanding of the following facts and problems:

[0005] Methacrylamide hydrogel (GelMA) is an inexpensive hydrogel with photocrosslinking properties that has been widely used in tissue engineering. It has multiple domains that bind to cell surface receptors and ECM proteins, low immunogenicity, good biocompatibility, and strong tissue adhesion. The ECM formed by GelMA provides several biochemical cues that can be found in natural tissues and has biochemical properties that can be adjusted by changing the polymer concentration, acidification level, and UV light intensity during the crosslinking process. The GelMA structure formed by UV crosslinking can be stable at physiological temperature. In recent years, GelMA has been used in tissue engineering periosteum. However, GelMA lacks biological activity and needs further improvement.

[0006] Fresh human amniotic membrane is a colorless, transparent membrane obtained from the innermost layer of the placenta. It is free of blood vessels, nerves, and lymphatic vessels. It primarily consists of an epithelial layer, a thicker basal layer, and a collagenous matrix layer devoid of blood vessels and lymphatic tissue. It exhibits low immunogenicity and inflammatory responses and has been used in a variety of clinical fields. Human acellular amniotic membrane (HAAM) is a natural extracellular matrix material obtained by removing the epithelial cells from fresh human amniotic membrane. This removal of the amniotic epithelial cells also removes immunogenic cellular components, reducing its immunogenicity while preserving the main components of the basal layer and extracellular matrix. HAAM is now widely used in corneal transplantation, skin defect repair, cartilage damage repair, and peripheral nerve regeneration, with excellent results. HAAM, a natural extracellular matrix rich in collagen, fibronectin, laminin, elastin, proteoglycans, hyaluronic acid, basic fibroblast growth factor, and transforming growth factor, can be used not only as a matrix for autologous and allogeneic cell transplantation to promote organ and tissue repair, but also as a biological carrier for cell culture, achieving significant progress in tissue engineering. In recent years, based on the excellent properties of HAAM, its applications in soft tissue repair, bone reconstruction, skin regeneration, and adhesion prevention have been increasingly studied.

[0007] In the early stage of bone defect repair, the targeted recruitment of BMSCs to the defect area is a prerequisite for the subsequent differentiation of BMSCs into cartilage and bone. In in situ tissue engineering for bone defect repair, how to efficiently and stably enable MSCs with highly activated properties to migrate and recruit to the defect site is a major challenge in bone tissue engineering. E7 peptide is a new type of short peptide with an amino acid sequence of "EPLQLKM" (glutamic acid-proline-leucine-glutamine-leucine-lysine-methionine). It has an active factor similar to the function of SDF-1, and can improve the hydrophilicity of the material and enhance the affinity of the material for cells. At the same time, it can specifically enhance the adhesion and proliferation ability of MSCs, especially with a high affinity for BMSCs. E7 peptide is considered to be a good "MSCs homing device" that has the ability to selectively capture MSCs in a flow model. At the same time, E7 peptide can accelerate the formation of blood vessels in bone defects.

[0008] To this end, the present invention provides a bone tissue engineering membrane material prepared with GelMA, HAAM and E7 peptide as raw materials, which can promote both stem cell recruitment and osteoblast differentiation into bone in the bone defect area, so as to solve the problems of difficult graft survival, healing and slow healing after bone transplantation in large bone defects.

[0009] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0010] According to a first aspect of an embodiment of the present invention, a method for preparing a periosteal material for promoting local endogenous bone regeneration and repair is provided, the method comprising: dissolving GelMA in PBS to obtain a GelMA solution; adding a photoinitiator, HAAMMA and E7 peptide to the GelMA solution, and conducting a free radical polymerization reaction under ultraviolet light to obtain a polymerization product; washing the polymerization product, freeze-drying and sterilizing to obtain the periosteal material; wherein the HAAMMA is MA-modified HAAM.

[0011] The successful implementation and application of tissue engineering requires the following three conditions: ① Suitable seed cells; ② Scaffold materials that support cell adhesion and growth; and ③ Bioactive factors that induce and regulate tissue proliferation and differentiation. This invention leverages the E7 peptide's ability to specifically recruit and proliferate BMSCs to increase their number in bone defect areas. It also leverages the effects of various growth factors in HAAM to promote osteogenesis and angiogenesis in the recruited MSCs, thereby accelerating new bone formation in the defect area.

[0012] The present invention discovered that HAAM, after being modified with MA (methacrylic anhydride), can stably bond with GelMA via MA bonds during free radical polymerization, reducing HAAM loss. The periosteal material provided by this invention can effectively promote stem cell recruitment and osteoblast differentiation into bone in bone defect areas, demonstrating excellent local bone regeneration and repair capabilities. This approach addresses the challenges of graft survival, healing, and slow healing after bone transplantation in large bone defects.

[0013] Furthermore, the mass ratio of GelMA, HAAMMA, and E7 peptide is 100,000:40,000-60,000:2.7-4. Studies have found that when GelMA, HAAMMA, and E7 peptide in this mass ratio are used as raw materials, HAAMMA can in situ expand BMSCs recruited by E7 peptide and significantly increase the number of osteoblasts in a short period of time, exerting an excellent function in promoting local bone regeneration and repair.

[0014] Furthermore, the mass ratio of GelMA, HAAMMA and E7 peptide is 100000:50000:3.

[0015] Furthermore, the photoinitiator is a free radical polymerization photoinitiator, preferably an acylphosphophosphate photoinitiator, and the mass of the photoinitiator is 0.1-0.3% of the volume of the GelMA solution.

[0016] Furthermore, the concentration of the GelMA solution is 8-12%. Studies have found that GelMA solutions with this concentration are more conducive to obtaining materials with pores of varying sizes, improving the adhesion ability to cells of different sizes, and thus improving the bone regeneration and repair function of the periosteal material. The concentration of the GelMA solution is preferably 10%.

[0017] Furthermore, the conditions of the ultraviolet light irradiation are: 395-480nm, 30-90 seconds.

[0018] Furthermore, the method further comprises: washing the mixture obtained after the free radical polymerization reaction with water, and then freeze-drying and sterilizing.

[0019] Furthermore, the freeze-drying conditions are: vacuum 15-20 Pa, temperature -78°C to -80°C, and time 10-14 hours. Studies have found that the freeze-drying time affects the pore size of the freeze-dried membrane. Under these conditions, the prepared material has pores of varying sizes, which is more conducive to the adhesion of cells of different sizes, thereby enhancing the bone regeneration and repair function of the periosteal material. The freeze-drying time is preferably 12 hours.

[0020] Furthermore, the sterilization is performed by irradiation with 60Co-γ rays, with an irradiation dose of 25-35 kGy, preferably 25 kGy.

[0021] Furthermore, the preparation method of the HAAMMA comprises:

[0022] HAAM was washed, freeze-dried, trimmed into thin slices, and then ground into HAAM powder with a particle size of less than 100 μm;

[0023] The HAAM powder is soaked in PBS containing 2-6% MA, incubated at 0-10°C for 18-36 hours, filtered with filter paper, repeatedly washed with PBS, and the solid phase obtained after centrifugation is dialyzed in an 8-14 kD dialysis bag and freeze-dried to obtain HAAMMA.

[0024] According to a second aspect of an embodiment of the present invention, there is provided a periosteum material for promoting local endogenous bone regeneration and repair, which is made by the method described in any one of the above items.

[0025] The embodiments of the present invention have the following advantages:

[0026] The present invention combines HAAM and E7 peptides for bone regeneration and repair, and the prepared material has excellent bone-promoting function.

[0027] The preparation process provided by the present invention only requires simple ultraviolet light molding and does not involve other complicated and tedious technologies. It has the advantages of simple process and strong operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0029] Figure 1 This is the general appearance of the G&E&H membrane material after freeze-drying and rehydration;

[0030] Figure 2 These are the SEM images of the freeze-dried membrane materials of the four groups: G, G&E, G&H, and G&E&H;

[0031] Figure 3 These are the infrared measurement results of the freeze-dried membrane materials of four groups: G, G&E, G&H, and G&E&H;

[0032] Figure 4 The relative growth rate of G&E&H membrane materials prepared with different weight ratios of raw materials after co-culture with cells for 3 days;

[0033] Figure 5 The results of relative proliferation rate determination after 3 days of co-culture of membrane materials with cells in groups G, G&E, G&H and G&E&H;

[0034] Figure 6 The results of 3D cell live and dead observations of the four groups of membrane materials G, G&E, G&H and G&E&H co-cultured with cells;

[0035] Figure 7 SEM photos of 3D cells co-cultured with cells in four groups of membrane materials: G, G&E, G&H, and G&E&H;

[0036] Figure 8 The results of alkaline phosphatase activity determination of cells co-cultured with membrane materials of groups G, G&E, G&H and G&E&H for 7 days;

[0037] Figure 9 The results of quantitative determination of alkaline phosphatase activity of cells co-cultured with membrane materials of groups G, G&E, G&H and G&E&H for 7 and 14 days;

[0038] Figure 10 This is the Micro-CT modeling diagram of the 8-week animal bone defect repair model;

[0039] Figure 11 Micro-CT parameter analysis: analysis results of bone volume fraction (BV / TV);

[0040] Figure 12 For Micro-CT parameter analysis: analysis results of bone mineral content (BMC);

[0041] Figure 13 These are the HE staining results of the four groups of membrane materials, G, G&E, G&H, and G&E&H, 8 weeks after implantation into the rat skull bone defect model. DETAILED DESCRIPTION

[0042] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0043] The HAAMMA powder used in the present invention is obtained by the following method:

[0044] (1) Preparation of HAAM

[0045] Immediately after separation from the fetus, the placenta was removed and placed in a sterile container. Blood clots were rinsed off the placenta with saline under sterile conditions. The amnion was then obtained by blunt dissection through the potential gap between the amnion and the chorion. Residual tissue and blood clots were removed using sterile PBS buffer. The amnion was then soaked in antibiotic saline (containing 1000 units / mL of gentamicin and 2.5 μg / mL of amphotericin B) for 20 minutes, digested with 0.25% trypsin for 24 hours, and rinsed 4-5 times with PBS buffer. The obtained HAAM was spread flat on a sterile filter paper sheet, epithelial side up, and cut into appropriate sizes. The cut HAAM, along with the filter paper, was soaked in sterile PBS buffer for 15 minutes. The treated HAAM was then cryopreserved using Hank's balanced salt / glycerol solution: HAAM was placed in a neutral silica glass bottle containing 4 mL of sterilized Hank's balanced salt / glycerol solution (the volume ratio of Hank's balanced salt solution to glycerol was 3:1), the bottle mouth was sealed, and the bottle was stored at -80°C. It was thawed naturally at room temperature before use. This method can preserve HAAM for up to 6 months.

[0046] (2) Preparation of HAAM powder

[0047] The preserved HAAM was taken out and then washed. After the washed HAAM was freeze-dried in a freeze dryer, it was repeatedly trimmed with tissue scissors and cut into 2mm×2mm slices. Subsequently, the trimmed freeze-dried HAAM was ground into a fine powder with a particle size of less than 100μm using a grinder at a constant temperature of 4°C.

[0048] (3) Preparation of HAAMMA powder

[0049] The resulting HAAM powder was immersed in PBS containing 4% MA (methacrylic anhydride) (v / v) and incubated at 4°C in a horizontal thermostat for 24 hours. The powder was then collected with filter paper, repeatedly washed with PBS, and centrifuged to collect the solid phase. The powder was then dialyzed in an 8-14 kD dialysis bag for 48 hours to remove residual MA. The dialyzed HAAMMA was collected by centrifugation and freeze-dried to obtain a dried HAAMMA powder. The dried HAAMMA powder was then irradiated with 60Co-γ rays at a dose of 25 kGy for sterilization and ready for use.

[0050] Example 1

[0051] 1. Preparation of Diaphragm Materials

[0052] Preparation of G membrane: GelMA (purchased from Shangpu Biotechnology Co., Ltd., model SP-BI-G01-4) was dissolved in PBS at a concentration of 10% (w / v, g / ml), and then 0.1% (w / v, g / ml) of acylphosphophotoinitiator (purchased from Shangpu Biotechnology Co., Ltd., model SP-BI-C02-2) was added to the dissolved GelMA solution to obtain a reaction solution. Then 0.2 ml of the above reaction solution was added to each well of a 48-well cell culture plate, and irradiated with 395-480 nm ultraviolet light for 60 seconds to obtain a pure G (GelMA) membrane.

[0053] Preparation of G&E membrane: 0.2 ml of the above-prepared reaction solution was added to each well of a 48-well cell culture plate. At the same time, a solution containing 0.0006 mg of E7 peptide was added to each well (E7 peptide was purchased from Beijing Shuguang Biotechnology Co., Ltd. Before use, E7 peptide was prepared into a solution with distilled water buffer at a ratio of 2 ug:2 ml according to the product instructions). After mixing evenly, the solution was irradiated with 395-480 nm ultraviolet light for 60 seconds to obtain a G&E (GelMA & E7 peptide membrane) membrane.

[0054] Preparation of G&H membrane: Add 0.2 ml of the above-prepared reaction solution to each well of a 48-well cell culture plate. At the same time, add 10 mg of freeze-dried HAAMMA powder to each well and mix well. Then irradiate with 395-480 nm ultraviolet light for 60 seconds to obtain a G&H (GelMA & HAAMMA membrane) membrane.

[0055] Preparation of G&E&H membrane: Add 0.2 ml of the above-prepared reaction solution to each well of a 48-well cell culture plate. Simultaneously, add a solution containing 0.0006 mg of E7 peptide and 10 mg of freeze-dried HAAMMA powder to each well, mix thoroughly, and irradiate with 395-480 nm UV light for 60 seconds to obtain a G&E&H (GelMA & E7 peptide & HAAMMA membrane) membrane. Wash and freeze-dry all membranes for later use.

[0056] 2. Sterilization of Preparation Materials

[0057] The obtained G, G&E, G&H and G&E&H membrane materials were sterilized by 60Coγ rays with an irradiation dose of 25 kGy.

[0058] Test Example 1

[0059] Performance characterization of diaphragm materials

[0060] Determination of physical and chemical characteristics of diaphragm materials

[0061] 1. Porosity determination

[0062] Take the G, G&E, G&H, and G&E&H membrane materials prepared in Example 1 and calculate their volumes (V = a × b × c, where a represents length, b represents width, and c represents height). Place the membranes in a graduated container and soak for several hours until they are saturated with water (the container is pre-filled with a certain volume of glycerin). This provides the calculated volume (V1) before and after rehydration. Porosity = (1 - V1 / V) × 100%. Ten measurements were taken and the average value was taken.

[0063] The results showed that the porosity of the G, G&E, G&H, and G&E&H membrane materials varied somewhat, with the multi-element materials having slightly lower porosity than the single-element materials. The porosity was 74.6% ± 2.4%, 72.8% ± 3.1%, 68.1% ± 3.6%, and 67.9% ± 4.2%, respectively. The porosity of the G, G&E, G&H, and G&E&H membrane materials prepared according to the procedure was stable and suitable for further experiments.

[0064] 2. Pore diameter measurement

[0065] After the surfaces of the prepared G, G&E, G&H and G&E&H freeze-dried membrane materials were sprayed with gold, the pore sizes were measured using a scanning electron microscope.

[0066] Figure 1 The gross appearance of the G&E&H membrane material after freeze-drying and rehydration was observed. The results showed that the freeze-dried G&E&H membrane had a uniform thin sheet appearance, and there was no significant difference between the membrane state after rehydration and the freeze-dried state. Figure 2SEM and electron microscopy results for the freeze-dried membranes of G, G&E, G&H, and G&E&H show distinct and uniform pores in the G and G&E membranes, while pores are present but uneven in the G&H and G&E&H membranes. In quantitative experiments, the pore sizes of the freeze-dried membranes of G, G&E, G&H, and G&E&H were nearly identical, with pore sizes of 396.56±35.05μm, 391.75±33.77μm, 367.42±62.57μm, and 373.55±59.73μm, respectively. The pore sizes of the multi-element materials were slightly smaller than those of the single-element materials, with the order of pore size being: G > G&E > G&H > G&E&H. The addition of HAAMMA resulted in smaller and more uniform pores in the hydrogels, which facilitated the adhesion of BMSCs of varying sizes to the membrane scaffolds.

[0067] 3. Material composition determination

[0068] The prepared G, G&E, G&H and G&E&H freeze-dried membrane materials were subjected to Fourier transform infrared analysis.

[0069] The results are as follows Figure 3 As shown, compared with G, G&E, G&H and G&E&H freeze-dried membrane materials have the following infrared characteristics at 2500cm -1 There are obvious peak changes in the four curves, which shows that HAAM and GelMA are evenly distributed and firmly connected to the corresponding positions of the material by forming stable covalent bonds through MA grafting.

[0070] 4. Material tensile strength test

[0071] Prepared G, G&E, G&H, and G&E&H freeze-dried membranes were soaked in PBS for several hours to achieve saturation. The compressive and tensile strength of the membranes was measured on a universal electronic testing machine. Ten samples of each type were tested, with a uniform load of 0.1 N / s and the pressure at which the sample failed was accurately read.

[0072] The results show that the tensile strength of the G, G&E, G&H, and G&E&H diaphragm materials varies. The addition of HAAMMA powder slightly enhances the tensile strength of the materials, with the maximum tensile strengths being 0.33±0.06N, 0.45±0.03N, 0.66±0.08N, and 0.67±0.07N, respectively. The compressive strength of the materials is in the order: G&E&H > G&H > G&E > G.

[0073] 5. Determination of E7 peptide release rate from membrane materials

[0074] A batch of biomimetic periosteum containing fluorescent E7 peptide was prepared according to the above procedure using fluorescent E7 peptide (Beijing Zhongke Yaguang Biotechnology Co., Ltd., C0868404) instead of E7 peptide. The prepared G, G&E, G&H, and G&E&H fluorescent membrane materials were placed in SBF simulated body fluid for incubation. The fluorescent E7 peptide concentration in the incubated SBF solution was measured using the BCA protein quantification method on days 1, 3, 5, 7, and 9 to determine the extent of E7 peptide release.

[0075] The results showed that the G&E&H fluorescent membrane material did release E7 peptide slowly in simulated body fluids, but compared with the burst release of previous materials, this composite material could ensure that the E7 peptide remained on the membrane for at least 9 days, or even longer.

[0076] Test Example 2

[0077] In vitro cell experiments on membrane materials

[0078] Effects of the dosage of various raw materials of membrane materials on the proliferation of BMSCs

[0079] BMSCs were used to evaluate the cell compatibility of the material. GelMA:HAAMMA:E7 was used with a weight ratio of 100000:50000:0, 100000:50000:1, 100000:50000:2, 100000:50000:3, 100000:50000:4, and 100000:50000:5. Different membrane materials were prepared according to the preparation method of G&E&H membrane in Example 1, and freeze-dried and irradiated for sterilization. The prepared G&E&H freeze-dried membrane material was placed in the culture medium to reach a saturated state of rehydration. BMSCs were routinely cultured with α-MEM medium containing 10% fetal bovine serum. When the cells were 80% fused, 0.25% trypsin was added for digestion. After centrifugation, the cells were resuspended in α-MEM medium containing 10% fetal bovine serum, and the cell density was adjusted to 2×10 4 2 ml of the culture medium was seeded into 12-well cell culture plates containing rehydrated G&E&H membrane materials at different ratios. After 3 days of co-culture with the G&E&H membrane materials, the cells were discarded and washed once with PBS. α-MEM medium supplemented with 10% CCK-8 was added. Incubation was continued at 37°C for 2 hours. The incubation medium was then transferred to a 96-well plate, with 100 μl per well inoculated. Absorbance was measured at 450 nm.

[0080] The results are as follows Figure 4As shown in the figure, the relative growth rates of G&E&H membrane materials with different weight ratios were greater than 1 in 3 days compared with the empty group without E7 peptide (100000:50000:0), among which the proliferation peak appeared between 100000:50000:2.7-100000:50000:4. According to the changes in the curve in the picture, it can be seen that the optimal weight ratio of raw materials for the membrane to promote BMSCs proliferation is 100000:50000:3.

[0081] Test Example 3

[0082] In vitro cell experiments on membrane materials

[0083] 1. Cytocompatibility testing

[0084] The cytocompatibility of the materials was evaluated using BMSCs. The prepared G, G&E, G&H, and G&E&H freeze-dried membranes were placed in culture medium until saturated with water. BMSCs were routinely cultured in α-MEM medium containing 10% fetal bovine serum. When the cells reached 80% confluency, 0.25% trypsin was added for digestion. After centrifugation, the cells were resuspended in α-MEM medium containing 10% fetal bovine serum and the cell density was adjusted to 2 × 10 4 2 ml of the culture medium was seeded into 12-well cell culture plates containing rehydrated G, G&E, G&H, and G&E&H membrane materials, respectively. After 1, 3, and 5 days of co-culture with the G, G&E, G&H, and G&E&H membrane materials, the cells were discarded, washed once with PBS, and supplemented with α-MEM medium containing 10% CCK-8. The cells were incubated at 37°C for 2 hours. The incubation medium was then transferred to a 96-well plate, 100 μl per well, and absorbance was measured at 450 nm.

[0085] The results are as follows Figure 5 As shown in the data, the relative growth rates of G, G&E, G&H and G&E&H membrane materials were all greater than 0.75 at 1, 3 and 5 days compared with the blank plate group. Among them, at 3 days, the relative growth rates of the four groups were 1.00, 1.18, 1.39 and 1.41, respectively. The trend of promoting cell growth was G&E&H>G&H>G&E>G.

[0086] 2. Cell live-dead staining experiment

[0087] BMSCs cells were co-cultured with rehydrated G, G&E, G&H, and G&E&H membrane materials for 3 days. The original culture medium was discarded, and the cells were washed once with PBS. The co-culture scaffolds were stained with a live-dead staining reagent and then observed under a fluorescence microscope.

[0088] The results are as follows Figure 6As shown, when G, G&E, G&H and G&E&H membrane materials were co-cultured with cells for 3 days, live and dead cell photography showed that the number of cells was G&E&H>G&H>G&E>G, proving that HAAM and E7 peptide have the effect of promoting cell proliferation, and the cell morphology is normal without specific changes.

[0089] 3 SEM observation

[0090] BMSCs were co-cultured with rehydrated G, G&E, G&H, and G&E&H membrane materials for 3 days. After discarding the original culture medium, the cells were washed once with PBS, fixed with 2.5% glutaraldehyde for 4 hours, freeze-dried, and sprayed with gold. The adhesion of cells on the materials was observed by SEM.

[0091] The results are as follows Figure 7 The results showed that when the rehydrated G, G&E, G&H and G&E&H membrane materials were co-cultured with cells for 3D, SEM photography of the cells showed that the cells grew and adhered normally on the scaffold, and the cell morphology did not change and was stably attached; G&H and G&E&H with the addition of HAAM group showed significantly improved efficiency in cell proliferation and growth.

[0092] 4. Alkaline phosphatase (ALP) activity in cells

[0093] BMSCs were co-cultured with rehydrated G, G&E, G&H, and G&E&H membrane materials for 7 and 14 days, respectively. The original culture medium was discarded, the cells were rinsed once with PBS, lysed, stained with an ALP kit, and ALP was quantitatively detected.

[0094] The results are as follows Figure 8 As shown in Figure 2, rehydrated G, G&E, G&H, and G&E&H membrane materials promoted the expression of alkaline phosphatase activity at 7 days, and the intensity of the promotion effect was G&E&H>G&H>G&E>G. Figure 9 As shown in the results, the rehydrated G, G&E, G&H, and G&E&H membrane materials all promoted the quantitative expression of alkaline phosphatase activity at 7 and 14 days. Furthermore, the alkaline phosphatase activity was particularly evident in the G&E&H and G&H groups containing HAAM.

[0095] Test Example 4

[0096] In vivo animal experiments on diaphragm materials

[0097] Evaluation of rat skull defect repair

[0098] G, G&E, G&H, and G&E&H membrane materials were prepared according to the procedure and then rehydrated and saturated for rat skull bone defect repair experiments. All rats were intraperitoneally injected with 3% sodium pentobarbital. After the rats were anesthetized, the skin on the top of the skull was incised and the unilateral skull was completely exposed. A circular full-thickness defect with a diameter of 5 mm was drilled in the rat skull with a round drill bit. The surgical area was rinsed to create a bone defect model. The rehydrated membrane material was mixed with normal saline and implanted into the bone defect of the experimental rat. The membrane was fixed and sutured layer by layer to cover the material and the wound. At the same time, a blank experimental group of the bone defect model was retained as a control. Samples were collected and observed 4 and 8 weeks after surgery.

[0099] Observation indicators:

[0100] Gross observation: observe the animal's wound for swelling, redness, and secretions after surgery. After the animal is sacrificed, observe whether there is an inflammatory reaction in the bone graft area.

[0101] Microscopic morphological observation: After sampling, the materials were fixed with 4% paraformaldehyde and scanned, modeled and quantitatively analyzed using microCT.

[0102] The results are as follows Figure 10 As shown in the 8-week animal bone defect repair model, biomimetic periosteum materials loaded with HAAM and / or E7 peptides can promote the progress of bone repair. Figure 11 、 Figure 12 Quantitative analysis of bone volume fraction (BV / TV) and bone mineral content (BMC) revealed that the G&E&H group produced the most bone mass and the highest bone mineral content over the same period of time. This result suggests that HAAM and E7 peptides synergize during osteogenesis. The bone repair-promoting efficacy of the four membrane materials was ranked as follows: G&E&H > G&H > G&E > G.

[0103] Histomorphological observation: Part of the bone tissue was decalcified and dehydrated, embedded in paraffin, sliced ​​with a tissue slicer, and stained with HE for histomorphological observation. The new bone formation in the implanted area was observed under a microscope. Figure 13 As shown, after 8 weeks of implantation into a rat calvarial bone defect model, the G, G&E, G&H, and G&E&H membrane materials prepared according to the procedure all demonstrated stable bone repair as determined by HE staining. Furthermore, in terms of repair thickness and repair area, the repair effect ranked G&E&H > G&H > G&E > G.

[0104] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing a periosteum material for promoting local endogenous bone regeneration and repair, characterized in that: The method comprises: Dissolve GelMA in PBS to obtain GelMA solution; adding a photoinitiator, HAAMMA and E7 peptide to the GelMA solution, and performing a free radical polymerization reaction under ultraviolet light to obtain a polymer product; washing, freeze-drying and sterilizing the polymerized product to obtain the periosteum material; Among them, HAAMMA is MA-modified HAAM.

2. The method for preparing a periosteum material for promoting local endogenous bone regeneration and repair according to claim 1, characterized in that: The mass ratio of the GelMA, HAAMMA and E7 peptide is 100000:40000-60000:2.7-4.

3. The method for preparing a periosteum material for promoting local endogenous bone regeneration and repair according to claim 1, characterized in that: The mass ratio of the GelMA, HAAMMA and E7 peptide is 100,000:50,000:

3. 4 . The method for preparing a periosteum material for promoting local endogenous bone regeneration and repair according to claim 1 , wherein the concentration of the GelMA solution is 8-12%.

5. The method for preparing a periosteum material for promoting local endogenous bone regeneration and repair according to claim 1, characterized in that: The photoinitiator is a free radical polymerization photoinitiator, and the mass of the photoinitiator is 0.1-0.3% of the volume of the GelMA solution.

6. The method for preparing a periosteum material for promoting local endogenous bone regeneration and repair according to claim 1, characterized in that: The conditions of the ultraviolet irradiation are: 395-480nm, 30-90 seconds.

7. The method for preparing a periosteum material for promoting local endogenous bone regeneration and repair according to claim 1, characterized in that: The freeze-drying conditions are: vacuum degree 15-20 Pa, temperature -78°C to -80°C, and time 10-14 hours.

8. The method for preparing a periosteum material for promoting local endogenous bone regeneration and repair according to claim 1, characterized in that: The sterilization is performed by irradiation with 60Co-γ rays, and the irradiation dose is 25-35 kGy.

9. The method for preparing a periosteum material for promoting local endogenous bone regeneration and repair according to claim 1, characterized in that: The preparation method of the HAAMMA comprises: HAAM was washed, freeze-dried, trimmed into thin slices, and then ground into HAAM powder with a particle size of less than 100 μm; The HAAM powder is soaked in PBS containing 2-6% MA, incubated at 0-10°C for 18-36 hours, filtered with filter paper, repeatedly washed with PBS, and the solid phase obtained after centrifugation is dialyzed in an 8-14 kD dialysis bag and freeze-dried to obtain HAAMMA.

10. A periosteum material for promoting local endogenous bone regeneration and repair, characterized in that: The invention is prepared by the method according to any one of claims 1 to 9.

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