Osteochondral integrated sponge scaffold as well as preparation method and application thereof

Abstract

The invention discloses an osteochondral integrated sponge scaffold as well as a preparation method and application thereof, and belongs to the field of osteochondral repair materials. The preparation method comprises the following steps: (1) mixing a collagen dispersion liquid and a polyvinyl alcohol solution, then adding a phosphate buffer solution and beta-tricalcium phosphate, and then obtaining lower-layer hydrogel through a pouring method; (2) mixing the collagen dispersion liquid with a polyvinyl alcohol solution, then adding a phosphate buffer solution, and then obtaining upper-layer hydrogel through a pouring method; and (3) pouring the upper-layer hydrogel on the lower-layer hydrogel, constructing a sponge scaffold through a multi-time freeze-thawing method and a freeze-drying method, and finally soaking the sponge scaffold in thrombin to obtain the osteochondral integrated sponge scaffold. The scaffold can adsorb platelet-rich plasma, has certain adhesion capacity, can realize in-situ fixation and biological integration at a defect part, can well recover physical and biological dysfunctions of bone joints and osteochondral bones, and provides a potential application idea for clinical treatment of osteochondral defects.

Description

Technical Field

[0001] This invention relates to the field of osteocartilage repair materials, and in particular to an integrated osteocartilage sponge scaffold, its preparation method and application. This material can adsorb platelet-rich plasma and is particularly suitable for treating cartilage defects and osteocartilage defects. Background Technology

[0002] Osteochondrial defects are a common pathological problem in joint injuries, usually caused by trauma, degenerative diseases (such as osteoarthritis), or metabolic abnormalities, severely affecting the motor function and quality of life of hundreds of millions of patients worldwide. Cartilage defects manifest as localized damage or loss of the cartilage layer on the articular surface, while osteochondral defects further involve the subchondral bone tissue, forming complex damage across multiple layers. Characteristics of such defects include loss of articular surface mechanical properties, decreased biological lubrication, abnormal stress distribution, and secondary inflammatory responses. Because cartilage tissue lacks blood vessels and nerve supply, its self-repair capacity is extremely limited, and the defective area is prone to degenerative changes, accelerating the development of osteoarthritis.

[0003] Currently, tissue engineering-based scaffold materials have become a hot topic in clinical research for the repair of osteochondral defects. Commercially available scaffolds (such as natural polymers, synthetic polymers, or composite scaffolds) primarily promote cell adhesion, proliferation, and extracellular matrix deposition by mimicking the gradient structure of the cartilage-bone interface. However, existing scaffold designs often focus on restoring physical functions (such as mechanical support and structural biomimicry), neglecting in-situ fixation and the regulation of biological functions in cartilage-bone composite repair.

[0004] Although collagen scaffolds have good biocompatibility, the collagen currently used to prepare scaffolds is usually dispersed in acidic solutions, which leads to certain defects. Acidic environments (such as low pH) cause the triple helix structure of collagen to unwind or denature, disrupting its native conformation and thus weakening its biological activity in promoting cell adhesion and tissue regeneration. Furthermore, acidic solutions can irritate cells or tissues, especially in in vivo applications, which require additional steps (such as pH neutralization), increasing operational complexity and potential inflammatory risks.

[0005] Platelet-rich plasma (PRP) is a platelet concentrate obtained by centrifuging autologous whole blood. It is rich in high concentrations of growth factors and bioactive proteins, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF). These factors work synergistically to regulate key biological processes in cartilage repair, such as inhibiting inflammatory responses (e.g., reducing the expression of pro-inflammatory factors like IL-6 and TNF-α), promoting angiogenesis, stimulating chondrocyte migration and proliferation, and inducing mesenchymal stem cell differentiation into chondrocytes. In cartilage defect repair, PRP not only activates the synthesis of extracellular matrix (such as type II collagen and proteoglycans) in chondrocytes through growth factor release but also regulates metabolic balance in the joint microenvironment, thus delaying cartilage degeneration. For osteochondral defects involving subchondral bone, VEGF and PDGF in platelet-rich plasma can promote vascularization and bone remodeling in the defect area, while TGF-β and IGF-1 synergistically enhance the structural integration of the cartilage-bone interface. Furthermore, fibrinogen in platelet-rich plasma, after activation, forms a three-dimensional fibrin network that can temporarily fill the defect space, providing physical support for cell adhesion and tissue regeneration, while simultaneously prolonging its biological activity through the sustained release of growth factors.

[0006] Currently, both scaffold and platelet-rich plasma (PRP)-only treatments for osteochondral defects have limitations in clinical practice. Scaffolds alone only provide physical support, lacking essential adhesive properties and in-situ fixation and integration capabilities, and also lack bioactive factors (such as growth factors) that promote cell proliferation and differentiation, thus affecting repair quality. On the other hand, PRP concentrations decay rapidly when used alone, making it difficult to sustainably stimulate repair, and it cannot provide a stable three-dimensional microenvironment at the defect site, resulting in low efficiency in cell migration and matrix deposition. Summary of the Invention

[0007] The purpose of this invention is to provide an integrated osteochondral sponge scaffold, its preparation method, and its application. This method uses a green and simple approach to prepare a double-layered sponge scaffold, which is then loaded with platelet-rich plasma through adsorption. The sponge scaffold of this invention can fill osteochondral defects, achieving in-situ fixation and biointegration through its adhesive properties, promoting cell adhesion and migration. Furthermore, the sustained release of growth factors from the platelet-rich plasma promotes osteochondral regeneration, effectively restoring the physical and biological functional impairments of osteochondral joints, and providing a potential application for the clinical treatment of osteochondral defects.

[0008] The objective of this invention is achieved through the following technical solutions.

[0009] A method for preparing an integrated osteochondral sponge scaffold includes the following steps: (1) Mix collagen dispersion and polyvinyl alcohol (PVA) solution, then add phosphate buffer (PBS) and β-tricalcium phosphate (β-TCP) to obtain sponge scaffold hydrogel, and then obtain the lower layer hydrogel by casting method; (2) Mix collagen dispersion and polyvinyl alcohol solution, then add phosphate buffer to obtain sponge scaffold hydrogel, and then obtain upper hydrogel by casting method; (3) The upper hydrogel of step (2) is poured on the lower hydrogel of step (1), and the sponge scaffold is constructed by multiple freeze-thaw cycles. Finally, it is soaked in thrombin and freeze-dried to obtain the final osteochondral integrated sponge scaffold.

[0010] Preferably, in steps (1) and (2), The collagen is type I collagen extracted from bovine Achilles tendon; The molecular weight of the polyvinyl alcohol is 146,000-186,000 Daltons.

[0011] Preferably, in steps (1) and (2), The collagen dispersion was prepared by adding freeze-dried collagen to phosphate buffer and homogenizing it to obtain a collagen dispersion. The polyvinyl alcohol solution is prepared by adding polyvinyl alcohol to a phosphate buffer solution and stirring until homogeneous.

[0012] Preferably, the molecular weight of the β-tricalcium phosphate in step (1) is 310.18 Daltons.

[0013] Preferably, in the lower hydrogel of step (1), the mass contents of collagen, polyvinyl alcohol and β-tricalcium phosphate are 3-5%, 0.67%-3.33% and 1-10%, respectively.

[0014] Preferably, in the upper hydrogel of step (2), the mass content of collagen and polyvinyl alcohol is 3-5% and 0.67%-3.33%, respectively.

[0015] Preferably, the multiple freeze-thaw process in step (3) involves freezing the hydrogel at -10℃ to 30℃ for 6-8 hours, thawing it at 4℃ to 6℃ for 4-6 hours, repeating this process 3-5 times, and then freeze-drying it to obtain an integrated osteochondral sponge scaffold.

[0016] Preferably, the concentration of thrombin in step (3) is 50-500 U / ml, most preferably 500 U / ml; the soaking time is 3-10 minutes, most preferably 5 minutes.

[0017] An integrated osteochondral sponge scaffold prepared by any of the above preparation methods.

[0018] The above-described application of an integrated osteochondral sponge scaffold in the preparation of osteochondral defect repair materials.

[0019] Preferably, the above-described application includes the following steps: Platelet-rich plasma (PRP) is injected into an integrated osteochondral sponge scaffold to obtain a material for repairing osteochondral defects. PRP Preparation: Rabbit PRP was prepared using a three-step centrifugation method, with the following steps: 1 ml of 2.5% sodium citrate for injection was drawn using a 10 ml syringe; 9 ml of whole blood was collected from the marginal ear vein of a New Zealand rabbit (blood volume: sodium citrate for injection ratio 9:1); the blood in the syringe was injected into a centrifuge tube, and centrifuged for the first time at 300 g for 20 min. After centrifugation, all supernatant and the intermediate layer (containing platelets, white blood cells, and a small number of red blood cells) were aspirated using a pipette and transferred to another centrifuge tube; a second centrifugation was performed at 1500 g for 10 min, and all supernatant was collected and transferred to another centrifuge tube; a third centrifugation was performed at 3000 g for 10 min, at which point all platelets had reached the bottom of the centrifuge tube. The platelets were then resuspended in 1 ml of serum to obtain PRP, which was then tested using a complete blood count.

[0020] Preferably, the platelet concentration in PRP is 1500-3000×10^9 / L.

[0021] The osteochondral integrated sponge scaffold of this invention utilizes its high water absorption capacity to adsorb platelet-rich plasma, repairing articular cartilage and subchondral bone defects from both physical and biological perspectives. In terms of physical function, the sponge scaffold, through its three-dimensional porous network structure and high water absorption properties, can efficiently adsorb active components from platelet-rich plasma, forming a stable fibrin-scaffold complex. Through the formed fibrin-collagen network and the adhesive properties of collagen, the scaffold achieves in-situ fixation at the defect site. Simultaneously, its porous structure can adsorb bone marrow mesenchymal stem cells from the subchondral bone, promoting cell adhesion, proliferation, and extracellular matrix deposition, restoring the mechanical stability of the osteochondral interface. In terms of biological function, the scaffold-adsorbed PRP can achieve multi-target regulation through the sustained release of various growth factors (such as TGF-β, PDGF, VEGF, etc.): TGF-β and IGF-1 can activate chondrocytes to synthesize type II collagen and proteoglycans, and inhibit the release of inflammatory factors (such as IL-1β and TNF-α); VEGF and FGF promote angiogenesis and osteogenic differentiation in the subchondral bone region, accelerating the integration and regeneration of the bone-cartilage interface. Furthermore, collagen has good biocompatibility, and its natural bioactive surface can enhance stem cell adhesion, while the fibrin network in platelet-rich plasma further forms a biomimetic microenvironment, synergistically regulating the tissue remodeling process. This can compensate for the shortcomings of current clinical treatments using scaffolds and platelet-rich plasma alone to treat osteochondral defects. This scaffold can fill defects and achieve in-situ fixation and biointegration, while also promoting osteochondral regeneration, treating osteoarthritis from both physical and biological functional perspectives.

[0022] This invention uses PBS to disperse collagen. The pH of PBS (approximately 7.4) is consistent with the body fluid environment. As a dispersion solution for collagen, it not only maintains the structural integrity of collagen but also avoids toxicity to cells or tissues, exhibiting good biocompatibility. Furthermore, the buffering capacity of PBS can stabilize the pH without subsequent adjustment, making it easy to operate.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The osteochondral integrated sponge scaffold of the present invention has adhesive properties. It achieves in-situ fixation and biointegration of the scaffold by reacting thrombin with fibrin to form a fibrin network and collagen components.

[0024] (2) The osteochondral integrated sponge scaffold of the present invention can achieve the instant formation of the "material-active factor" complex by in-situ adsorption of platelet-rich plasma, which simplifies the complexity of step-by-step operation in traditional PRP combined scaffold therapy and avoids the risk of immune rejection of allogeneic materials.

[0025] (3) The natural RGD sequence of the collagen in this invention promotes stem cell adhesion, while the porous nature of the sponge scaffold is conducive to cell adhesion. The growth factor gradient in platelet-rich plasma (such as VEGF promoting bone vascularization and TGF-β inducing cartilage differentiation) can drive the synchronous regeneration of cartilage and bone layers, realizing the functional integration of the osteochondral interface and avoiding the interface stratification problem of single platelet-rich plasma treatment or single scaffold repair.

[0026] (4) The high water absorption and fibrin network formation ability of the osteochondral integrated sponge scaffold of the present invention can deliver growth factors (such as TGF-β and VEGF) in platelet-rich plasma to the defect area in a time-controlled release manner, breaking through the limitation of rapid degradation of factors after local injection of traditional platelet-rich plasma, and significantly prolonging the biological activity period.

[0027] (5) Compared to simple scaffolds (which only provide mechanical support) or platelet-rich plasma alone (which lacks structural stability), this invention addresses the two major challenges of "insufficient structural integrity" and "lack of regeneration signal" in osteochondral defect repair by functionally coupling materials with bioactive factors, significantly improving the biomechanical properties and long-term durability of regenerated tissues. Compared to complex scaffold systems that require exogenous growth factor loading (such as microsphere scaffolds loaded with TGF-β), the autologous nature of the platelet-rich plasma in this invention significantly reduces clinical translation costs and regulatory risks. Attached Figure Description

[0028] Figure 1 The images show the microstructure of the cartilage layer sponge scaffolds obtained in Examples 1-5.

[0029] Figure 2 The swelling rate diagrams for the cartilage layer sponge scaffolds obtained in Examples 1-5 are shown at minutes 1, 3, and 10.

[0030] Figure 3 The diagram shows the water absorption rate of the cartilage layer sponge scaffolds obtained in Examples 1-5.

[0031] Figure 4 The images show the microscopic morphology of the subchondral bone layer sponge scaffolds obtained in Examples 8-10.

[0032] Figure 5 The swelling rate diagrams for the subchondral bone layer sponge scaffolds obtained in Examples 8-10 are shown at minutes 1, 3, and 10.

[0033] Figure 6 The diagram shows the water absorption rate of the subchondral bone layer sponge scaffolds obtained in Examples 8-10.

[0034] Figure 7 This is a schematic diagram of the integral osteochondral sponge scaffold obtained in Example 13.

[0035] Figure 8 The fracture elongation, tensile strength, and Young's modulus diagrams are shown for the osteochondral integrated sponge scaffold of Example 13, the subchondral bone layer sponge scaffold of Example 8, and the cartilage layer sponge scaffold of Example 2.

[0036] Figure 9 This is a microscopic morphology image of platelets obtained in Example 14 on an integrated osteochondral sponge scaffold.

[0037] Figure 10 This is a graph showing the release of various growth factors at different time points in platelet-rich plasma obtained in Example 15 with and without a sponge scaffold.

[0038] Figure 11 This study examines the adhesion properties of the integrated osteochondral sponge scaffold obtained in Example 13 and its in-situ fixation capability in osteochondral defects.

[0039] Figure 12 This refers to the adhesion ability of the osteochondral integrated sponge scaffold.

[0040] Figure 13 Immunofluorescence image of the osteochondral integrated sponge scaffold's effect on cell adhesion in animals.

[0041] Figure 14 The image shows the adhesion function of platelet-rich plasma to cells in the osteochondral integrated sponge scaffold and the identification of the adherent cells.

[0042] Figure 15 The image shows the cell migration of bone marrow mesenchymal stem cells obtained in Example 19 in the control group, the osteochondral integrated sponge scaffold group, the platelet-rich plasma group, and the osteochondral integrated sponge scaffold group loaded with platelet-rich plasma.

[0043] Figure 16 These are gross images of the control group, the osteochondral integrated sponge scaffold group, the platelet-rich plasma group, and the platelet-loaded plasma-loaded osteochondral integrated sponge scaffold group after rabbit osteochondral defect modeling obtained in Example 20 at 6 and 12 weeks.

[0044] Figure 17 Micro-CT images of the control group, the osteochondral integrated sponge scaffold group, the platelet-rich plasma group, and the platelet-loaded plasma-loaded osteochondral integrated sponge scaffold group after rabbit osteochondral defect modeling in Example 20 at 6 weeks and 12 weeks. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the embodiments, but the present invention is not limited to these embodiments.

[0046] Example 1 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS, a sponge scaffold hydrogel with a final collagen concentration of 4% and a final PVA concentration of 0.67% was obtained. After being cast in a mold, the hydrogel was frozen at -20℃ for 8 hours, then thawed at 4℃ for 4 hours. After three cycles, a cartilage layer sponge scaffold with a porous structure was obtained by freeze-drying.

[0047] Example 2 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS, a sponge scaffold hydrogel with a final collagen concentration of 4% and a final PVA concentration of 1.33% was obtained. After being cast in a mold, the hydrogel was frozen at -20℃ for 8 hours, then thawed at 4℃ for 4 hours. After three cycles, a cartilage layer sponge scaffold with a porous structure was obtained by freeze-drying.

[0048] Example 3 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS, a sponge scaffold hydrogel with a final collagen concentration of 4% and a final PVA concentration of 2% was obtained. After being cast in a mold, it was frozen at -20℃ for 8 hours, then thawed at 4℃ for 4 hours. After three cycles, a cartilage layer sponge scaffold with a porous structure was obtained by freeze-drying.

[0049] Example 4 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS, a sponge scaffold hydrogel with a final collagen concentration of 4% and a final PVA concentration of 2.67% was obtained. After being cast in a mold, the hydrogel was frozen at -20℃ for 8 hours, then thawed at 4℃ for 4 hours. After three cycles, a cartilage layer sponge scaffold with a porous structure was obtained by freeze-drying.

[0050] Example 5 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions, a sponge scaffold hydrogel with a final collagen concentration of 4% and a final PVA concentration of 3.33% was obtained. After being cast in a mold, the hydrogel was frozen at -20℃ for 8 hours, then thawed at 4℃ for 4 hours. After three cycles, a cartilage layer sponge scaffold with a porous structure was obtained by freeze-drying.

[0051] Example 6 The swelling properties of the five cartilage layer sponge scaffolds obtained in Examples 1-5 were tested. A known mass (W1) of cartilage layer sponge scaffold was immersed in PBS for 1, 3, and 10 minutes respectively. The surface moisture of the swollen scaffold was removed with absorbent paper and weighed as W2. The swelling rate of the cartilage layer sponge scaffold was calculated as follows: Swelling rate = (W2-W1) / W1*100%.

[0052] Depend on Figure 2 It can be seen that the cartilage layer sponge scaffold has rapid swelling characteristics and can reach swelling equilibrium in 1 minute. Among them, the cartilage layer sponge scaffold obtained in Example 2 has the best swelling performance.

[0053] Example 7 The water absorption properties of the five cartilage layer sponge scaffolds obtained in Examples 1-5 were tested. A known mass (W1) of cartilage layer sponge scaffold was immersed in PBS until constant weight. The swollen scaffold was then weighed after removing surface moisture with absorbent paper. The swelling rate of the cartilage layer sponge scaffold was calculated as follows: water absorption rate = (W2-W1) / W2*100%.

[0054] Depend on Figure 3 It can be seen that the cartilage layer sponge scaffold obtained in Example 2 has the best water absorption performance, with a water absorption rate of up to 93%.

[0055] Example 8 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS and β-TCP, a sponge scaffold hydrogel with a final collagen concentration of 4%, a final PVA concentration of 1.33%, and a final β-TCP concentration of 1% was obtained. After being cast in a mold, the hydrogel was frozen at -20℃ for 8 hours, then thawed at 4℃ for 4 hours. After three cycles, a subchondral bone layer sponge scaffold with a porous structure was obtained by freeze-drying.

[0056] Example 9 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS and β-TCP, a sponge scaffold hydrogel with a final collagen concentration of 4%, a final PVA concentration of 1.33%, and a final β-TCP concentration of 5% was obtained. After being cast in a mold, the hydrogel was frozen at -20℃ for 8 hours, then thawed at 4℃ for 4 hours. After three cycles, a subchondral bone layer sponge scaffold with a porous structure was obtained by freeze-drying.

[0057] Example 10 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS and β-TCP, a sponge scaffold hydrogel with a final collagen concentration of 4%, a final PVA concentration of 1.33%, and a final β-TCP concentration of 10% was obtained. After being cast in a mold, the hydrogel was frozen at -20℃ for 8 hours, then thawed at 4℃ for 4 hours. After three cycles, a subchondral bone layer sponge scaffold with a porous structure was obtained by freeze-drying.

[0058] Example 11 The swelling properties of the three groups of subchondral bone layer sponge scaffolds obtained in Examples 8-10 were tested. A known mass (W1) of subchondral bone layer sponge scaffolds was immersed in PBS for 1, 3, and 10 minutes respectively. The surface moisture of the swollen scaffolds was removed with absorbent paper and weighed as W2. The swelling rate of the subchondral bone layer sponge scaffolds was calculated as follows: Swelling rate = (W2-W1) / W1*100%.

[0059] Depend on Figure 5 It can be seen that the subchondral bone layer sponge scaffold has rapid swelling characteristics and can reach swelling equilibrium in 1 minute. Among them, the subchondral bone layer sponge scaffold obtained in Example 8 has the best swelling performance.

[0060] Example 12 The water absorption properties of the three groups of subchondral sponge scaffolds obtained in Examples 8-10 were tested. A known mass (W1) of subchondral sponge scaffold was immersed in PBS until constant weight. The swollen scaffold was then weighed after removing surface moisture with absorbent paper. The swelling rate of the subchondral sponge scaffold was calculated as follows: water absorption rate = (W2-W1) / W2*100%.

[0061] Depend on Figure 6 It can be seen that the subchondral bone layer sponge scaffold obtained in Example 8 has the best water absorption performance.

[0062] Example 13 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS and β-TCP, a sponge scaffold hydrogel with a final collagen concentration of 4%, a final PVA concentration of 1.33%, and a final β-TCP concentration of 1% was obtained. The hydrogel was then cast into a mold to form the lower layer. 6g of freeze-dried collagen was weighed and mixed with 100ml of PBS in a homogenizer to obtain a 6% collagen solution. 4g of PVA was weighed and mixed with 100ml of PBS at 90℃ for two hours to obtain a 4% PVA solution. By mixing the two solutions and adding PBS, a top hydrogel with a final collagen concentration of 4% and a final PVA concentration of 1.33% was obtained. The top hydrogel was poured on top of the bottom hydrogel and frozen at -20℃ for 8 hours. After thawing at 4℃ for 4 hours, the cycle was repeated 3 times. The sponge scaffold was obtained by freeze-drying. Finally, the sponge scaffold was soaked in thrombin (concentration of 500 U / ml) for 5 minutes and then freeze-dried to obtain the final osteochondral integrated sponge scaffold.

[0063] Tensile tests were conducted on the osteochondral integrated sponge scaffold of Example 13, the subchondral bone layer sponge scaffold of Example 8, and the cartilage layer sponge scaffold of Example 2 to evaluate the interfacial structural stability of the double-layer scaffold.

[0064] Depend on Figure 8 It can be seen that the tensile test of the interface of the osteochondral integrated sponge scaffold of Example 13 showed that its elongation at break, tensile strength and Young's modulus were all between those of the subchondral bone layer sponge scaffold of Example 8 and the cartilage layer sponge scaffold of Example 2. The results showed that the interface structure of the osteochondral integrated sponge scaffold was stable. It is considered that a continuous porous network can be formed by freeze-thaw method to promote the structural fusion of the cartilage layer and the bone layer.

[0065] Example 14 Preparation of rabbit platelet-rich plasma (PRP): Draw 1 ml of 2.5% sodium citrate for injection using a 10 ml syringe; collect 9 ml of whole blood from the marginal ear vein of a New Zealand rabbit (blood volume: sodium citrate for injection ratio 9:1); inject the blood from the syringe into a centrifuge tube; centrifuge for the first time at 300 g for 20 min. After centrifugation, use a pipette to aspirate all the supernatant and intermediate layer (containing platelets, white blood cells, and a small number of red blood cells) and transfer them to another centrifuge tube; perform a second centrifugation at 1500 g for 10 min, collect all the supernatant, and transfer it to another centrifuge tube; perform a third centrifugation at 3000 g for 10 min. At this point, all platelets are at the bottom of the centrifuge tube. Resuspend the platelets in 1 ml of serum to obtain PRP.

[0066] Table 1

[0067] By testing the number of red blood cells, white blood cells, and platelets using a routine blood test, Table 1 shows that PRP with low white blood cell count, low red blood cell count, and high platelet count can be stably prepared using a three-step centrifugation method. At the same time, the platelet-rich plasma extracted has a platelet content that is more than 5 times that of whole blood, proving the successful extraction of PRP.

[0068] The prepared PRP was injected into the osteochondral integrated sponge scaffold obtained in Example 13. After incubation for 1 hour, it was washed three times with PBS for 5 minutes each time, fixed with paraformaldehyde for 3 hours, and then dehydrated in gradients of 50%, 75%, 90%, and 100% ethanol for 15 minutes each. After freeze-drying, it was sprayed with gold and subjected to electron microscopy to obtain osteochondral defect repair material.

[0069] Figure 9 This image shows the microscopic morphology of platelets on an integrated osteochondral sponge scaffold, a material used for repairing osteochondral defects. Platelets are observed to aggregate on the scaffold.

[0070] Example 15 The platelet-rich plasma obtained in Example 14 was added to the osteochondral integrated sponge scaffold obtained in Example 13, and the growth factor sustained-release effect with and without the scaffold was compared using an ELISA kit.

[0071] Figure 10 The figure shows the effect of the presence or absence of a sponge scaffold on the sustained release behavior of growth factors. As can be seen from the figure, platelet-rich plasma loaded on a sponge scaffold has a better sustained release function of growth factors.

[0072] Example 16 Platelet-rich plasma was injected into the osteochondral integrated sponge scaffold obtained in Example 14, and then bonded to a rabbit-derived bone block. The bonded scaffold and bone block were then soaked in water for 48 hours. Figure 11 It can be seen that the stent loaded with platelet-rich plasma has good adhesion ability, and it still has adhesion ability after being soaked in water for 48 hours.

[0073] The osteochondral integrated sponge scaffold obtained in Example 14 was implanted into the osteochondral defect site, and then... Figure 11 It can be seen that even when the entire bone block is inverted, the implanted scaffold can still be well fixed at the defect site, mainly because the scaffold's adhesive ability achieves in-situ fixation.

[0074] Platelet-rich plasma was injected into the thrombin-containing and thrombin-free stents obtained in Example 14, rinsed with water, and then... Figure 11It is known that a large number of platelets adhere to the surface of the stent containing thrombin, forming a dense fibrin network. The surface of the stent is thickly covered by the platelet-fibrin complex, while the microstructure of the stent material itself can still be seen in the stent without thrombin.

[0075] Example 17 Pig skin was degreased, and a gelatin coating was prepared by coating glass slides with a 20% gelatin solution.

[0076] Experimental grouping: This experiment was divided into two groups: the unsupported group and the supported group.

[0077] Scaffold-free group: Platelet-rich plasma and thrombin were simultaneously injected into the surfaces of pigskin and glass slides using a syringe to form a gel, which was then pressed and adhered to the pigskin and glass slides respectively.

[0078] The osteochondral integrated sponge scaffold obtained in Example 14 was placed on pigskin and a glass slide. Platelet-rich plasma was injected into the osteochondral integrated sponge scaffold, and then pigskin and a glass slide were covered and pressed together.

[0079] A shear test was conducted using a universal testing machine. Figure 12 It can be seen that the adhesion performance of the stent group is higher than that of the group without stent.

[0080] Example 18 Establishment of a rat osteochondral defect model: After anesthetizing the rats, the knee joint was disinfected, draped with surgical towels, and exposed after shaving. A 1 cm long incision was carefully made on the medial side of the knee joint with a scalpel. After cutting the joint capsule and exposing the patella, the patella was everted and fixed to expose the patellar trochlea. Then, a circular osteochondral defect with a diameter of 3 mm and a depth of 2 mm was constructed in the trochlear groove using an electric drill.

[0081] Experimental Groups: This experiment was divided into two groups: the osteochondral integrated sponge scaffold group and the osteochondral integrated sponge scaffold group loaded with platelet-rich plasma.

[0082] Treatment methods: Osteochondrial integrated sponge scaffold group: the defect is filled with an osteochondral integrated sponge scaffold with a diameter of 3mm and a height of 2mm; Platelet-rich plasma-loaded osteochondral integrated sponge scaffold group: the defect is first filled with an osteochondral integrated sponge scaffold with a diameter of 3mm and a height of 2mm, and then platelet-rich plasma is injected onto the sponge scaffold.

[0083] Five days later, the sponge scaffold was removed and subjected to 4',6-diamidin-2-phenylindole (DAPI) nuclear blue staining and F-actin (F-actin) cytoskeleton red staining, and observed under a confocal microscope.

[0084] The sponge scaffolds were removed at 7, 14, and 21 days for 6-diamidinyl-2-phenylindole (DAPI) nuclear blue staining, F-actin (F-actin) cytoskeleton red staining, and CD44 green staining, and observed under a fluorescence microscope.

[0085] Figure 13 Two-dimensional results showed that a large number of cells adhered to the osteochondral integrated sponge scaffold. This is likely due to the porous structure, which allows the scaffold to adsorb cells from the defect site. The large number of cells adhering to the scaffold also demonstrates its biocompatibility. Figure 13 The three-dimensional results showed that the cells were evenly distributed on the osteochondral integrated sponge scaffold, which could promote the uniformity of tissue regeneration.

[0086] Figure 14 The results showed that, compared with the osteochondral integrated sponge scaffold group, the osteochondral integrated sponge scaffold loaded with platelet-rich plasma had more cell adhesion, and platelet-rich plasma could further promote cell adhesion and growth. CD44 staining results showed that the adhered cells were bone marrow mesenchymal stem cells.

[0087] Example 19 Cell scratching inserts were placed on blank wells, wells containing integrated osteochondral sponge scaffolds, wells containing platelet-rich plasma, and wells containing integrated osteochondral sponge scaffolds loaded with platelet-rich plasma, respectively. Bone marrow mesenchymal stem cells were injected at a rate of 1×10⁻⁶. 6 Cells / mL. Take 70 μL of cell suspension and add it to the two wells of the plug. After all the cells have adhered to the wall, use sterile forceps to hold one corner of the plug and peel it off. Perform calcein AM staining at 0, 12 and 24 hours and observe under an inverted fluorescence microscope.

[0088] Figure 15 Regarding cell migration, the results showed that bone marrow mesenchymal stem cells migrated faster in the platelet-rich plasma group and the osteochondral integrated sponge scaffold group loaded with platelet-rich plasma, demonstrating that platelet-rich plasma promotes cell migration.

[0089] Example 20 Establishment of a rabbit osteochondral defect model: After anesthetizing New Zealand rabbits, the knee joint was disinfected, draped with surgical towels, and exposed after shaving. A 1 cm long incision was carefully made on the medial side of the knee joint with a scalpel. After cutting the joint capsule and exposing the patella, the patella was everted and fixed to expose the patellar trochlea. Then, a circular osteochondral defect with a diameter of 4 mm and a depth of 3 mm was constructed in the trochlear groove using an electric drill.

[0090] Experimental Groups: This experiment was divided into four groups: control group, osteochondral integrated sponge scaffold group, platelet-rich plasma group, and osteochondral integrated sponge scaffold group loaded with platelet-rich plasma.

[0091] Treatment methods: Control group: the wound was sutured after defect modeling; Osteochondrial integrated sponge scaffold group: the defect was filled with an osteochondral integrated sponge scaffold with a diameter of 4 mm and a height of 3 mm; Platelet-rich plasma group: platelet-rich plasma and thrombin were injected into the defect simultaneously using a syringe to form a gel to fill the defect; Platelet-rich plasma-loaded osteochondral integrated sponge scaffold group: the defect was first filled with an osteochondral integrated sponge scaffold with a diameter of 4 mm and a height of 3 mm, and then platelet-rich plasma was injected onto the sponge scaffold.

[0092] The animals were euthanized at 6 and 12 weeks, and tissue samples were harvested from the knee joints for gross examination of the repaired defects. Results were as follows... Figure 16 As shown, the osteochondral sponge scaffold group loaded with platelet-rich plasma showed better repair results than other groups. The repaired joint surface was smooth and similar to the surrounding tissue. Micro-CT was used to observe the subchondral bone repair, and the results are as follows. Figure 17 As shown, the osteochondral sponge scaffold group loaded with platelet-rich plasma showed better subchondral bone repair than other groups. The subchondral bone regeneration in the osteochondral sponge scaffold group loaded with platelet-rich plasma was good, and the subchondral bone was almost completely repaired at 12 weeks.

Claims

1. A method for preparing an integrated osteochondral sponge scaffold, characterized in that, Includes the following steps: (1) Mix collagen dispersion and polyvinyl alcohol solution, then add phosphate buffer and β-tricalcium phosphate to obtain sponge scaffold hydrogel, and then obtain lower layer hydrogel by casting method; (2) Mix collagen dispersion and polyvinyl alcohol solution, then add phosphate buffer to obtain sponge scaffold hydrogel, and then obtain upper hydrogel by casting method; (3) The upper layer of hydrogel in step (2) is poured on the lower layer of hydrogel in step (1), and the bone sponge scaffold is constructed by multiple freeze-thaw cycles. Finally, it is soaked in thrombin and freeze-dried to obtain the final bone and cartilage integrated sponge scaffold.

2. The method for preparing an integrated osteochondral sponge scaffold according to claim 1, characterized in that, In steps (1) and (2), The collagen is type I collagen extracted from bovine Achilles tendon; The molecular weight of the polyvinyl alcohol is 146,000-186,000 Daltons.

3. The method for preparing an integrated osteochondral sponge scaffold according to claim 1, characterized in that, In steps (1) and (2), The collagen dispersion was prepared by adding freeze-dried collagen to phosphate buffer and homogenizing it to obtain a collagen dispersion. The polyvinyl alcohol solution is prepared by adding polyvinyl alcohol to a phosphate buffer solution and stirring until homogeneous.

4. The method for preparing an integrated osteochondral sponge scaffold according to claim 1, characterized in that, The molecular weight of the β-tricalcium phosphate in step (1) is 310.18 Daltons.

5. The method for preparing an integrated osteochondral sponge scaffold according to claim 1, characterized in that, In the lower hydrogel described in step (1), the mass contents of collagen, polyvinyl alcohol, and tricalcium β-phosphate are 3-5%, 0.67%-3.33%, and 1-10%, respectively.

6. The method for preparing an integrated osteochondral sponge scaffold according to claim 1, characterized in that, In the upper hydrogel described in step (2), the mass contents of collagen and polyvinyl alcohol are 3-5% and 0.67%-3.33%, respectively.

7. The method for preparing an integrated osteochondral sponge scaffold according to claim 1, characterized in that, The multiple freeze-thaw method in step (3) involves freezing the hydrogel at -10℃ to 30℃ for 6-8 hours, thawing it at 4℃ to 6℃ for 4-6 hours, repeating this cycle 3-5 times, and then freeze-drying it to obtain an integrated osteochondral sponge scaffold. The concentration of thrombin in step (3) is 50-500 U / ml, and the soaking time is 3-10 minutes.

8. An integrated osteochondral sponge scaffold prepared by the preparation method according to any one of claims 1-7.

9. The application of the osteochondral integrated sponge scaffold according to claim 8 in the preparation of osteochondral defect repair materials.

10. The application of the integrated osteochondral sponge scaffold according to claim 9 in the preparation of osteochondral defect repair materials, characterized in that, Includes the following steps: Platelet-rich plasma was injected into an integrated osteochondral sponge scaffold to obtain a material for repairing osteochondral defects. The platelet concentration in the platelet-rich plasma is 1500-3000×10^9 / L.

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