CN114569795B - Double-layer osteochondral scaffold material and preparation method and application thereof - Google Patents

Abstract

The invention discloses a double-layer osteochondral scaffold material and a preparation method and application thereof, wherein the scaffold material comprises a cartilage phase and a subchondral bone phase; photo-crosslinkable P constituting the cartilage phase _m L _n DMA; and P constituting the subchondral bone phase _m L _n DMA/MH nanocomposites; wherein, P is polypropylene glycol; m is the number of monomers in the polypropylene glycol; l is polylactic acid; n is the number of monomers in the polylactic acid; DMA is dimethacrylate and MH is methacrylated hydroxyapatite. The hydrophobic injectable underwater cross-linked double-layer osteochondral scaffold is quickly implemented and cross-linked and injected in a liquid environment to form a firm two-phase interface, the generation and osteogenesis of cartilage are synergistically enhanced by adjusting a physical and chemical microenvironment, and the cell function of mesenchymal stem cells can be synergistically influenced by mechanical stimulation and growth factor stimulation so as to regulate and control the differentiation of the cartilage and the osteogenesis of the mesenchymal stem cells in vitro and in vivo.

Description

Double-layer osteochondral scaffold material and preparation method and application thereof

Technical Field

The invention relates to the technical field of medical instruments, in particular to a double-layer osteochondral scaffold material and a preparation method and application thereof.

Background

The osteochondral complex is composed of cartilage and subchondral bone and has diverse biological properties in terms of cellular phenotype, extracellular matrix composition and mechanical properties. At present, when the tissue engineering scaffold is applied to treat osteochondral defects, the repair of bone and cartilage tissues is promoted by implanting a double-layer prefabricated scaffold simulating a natural osteochondral complex. This approach has certain limitations, and implanting a double-layered pre-fabricated stent is an invasive procedure with potential local tissue damage, post-operative complications, long recovery times, and risk of patient discomfort. As an alternative, the injectable adhesive has the following advantages compared to the double-layered prefabricated stent: 1. minimally invasive property: the repair can be accomplished by injection; 2. the comprehensiveness: good repair of deep regions; 3. high plasticity: can meet the requirements of repairing osteochondral defects with different shapes.

Hydrogels such as collagen, gelatin, and alginate have been used as injectable adhesives because of their excellent injectability and biocompatibility, and the ability to support biomolecules such as growth factors. However, the high hydrophilicity of the hydrogel adhesive may lead to an injection spill or failure to fill the designated defect area, which can affect the integrity of the repair area coverage after injection. At the same time, the liquid pressure environment during the operation can cause the displacement of the hydrogel adhesive and the rapid release of the biological molecules, and the defects limit the application of the hydrogel adhesive in the liquid environment. Accordingly, liquid hydrophobic materials have received attention as injectable adhesives, including polymethylmethacrylate, polypropylene fumarate, polysebacic acid glyceride and polyanhydride, and the like. When these materials are fabricated in situ as a bilayer structure, they generate a large amount of heat during crosslinking, thereby compromising their biocompatibility and ability to encapsulate bioactive molecules, while requiring a long crosslinking time. Moreover, these stents either lack strong interfacial adhesion and are prone to delamination, or require additional glue for adhesion, limiting their application in surgery. From the perspective of facilitating surgical operation, the injectable hydrophobic material can be rapidly crosslinked underwater to form a bionic anisotropic double-layer structure with strong interface combination, and the injectable hydrophobic material allows growth factors to be released for a long time, so that the injectable hydrophobic material is favorable for repairing osteochondral loss.

In conclusion, the existing osteochondral repair materials have various defects, and a novel double-layer osteochondral scaffold material needs to be developed.

Disclosure of Invention

Therefore, the invention provides a double-layer osteochondral scaffold material and a preparation method and application thereof.

In order to achieve the above purpose, the invention provides the following technical scheme:

the embodiment of the invention provides a double-layer osteochondral scaffold material, which comprises a cartilage phase and a subchondral bone phase;

make up thePhoto-crosslinkable P of the cartilage phase _m L _n DMA; and

p constituting the subchondral bone phase _m L _n DMA/MH nanocomposites;

wherein, P is polypropylene glycol; m is the number of monomers in the polypropylene glycol; l is polylactic acid; n is the number of polylactic acid in the monomer; DMA is dimethacrylate and MH is methacrylated hydroxyapatite.

In one embodiment of the invention, the photocrosslinkable P _m L _n DMA is through P _m L _n DMA, hydroxyethyl methacrylate and a photoinitiator.

In one embodiment of the invention, the photocrosslinkable P _m L _n In the DMA, the mass percent of each component is as follows: p _m L _n 90wt% of DMA, 9wt% of hydroxyethyl methacrylate and 819 wt% of photoinitiator.

In one embodiment of the invention, the scaffold material further comprises TGF-. Beta.1 and/or BMP-2.

In one embodiment of the present invention, said P _m L _n The DMA/MH nano composite material is prepared by mixing P _m L _n DMA is mixed with hydroxyethyl methacrylate functionalized nano-hydroxyapatite with different concentrations to prepare the nano-hydroxyapatite.

In one embodiment of the present invention, said P _m L _n DMA is P ₇ L ₄ DMA、P ₇ L ₂ DMA、P ₁₇ L ₂ DMA、P ₁₇ L ₄ DMA、P ₃₄ L ₂ DMA、P ₃₄ L ₄ DMA、P ₆₈ L ₂ DMA or P ₆₈ L ₄ DMA。

In another aspect, the present invention also provides a method for preparing the double-layered osteochondral scaffold material, the method comprising:

subjecting the photocrosslinkable P _m L _n DMA and P _m L _n And carrying out photopolymerization on the DMA/MH nano composite material to obtain the double-layer osteochondral scaffold material.

In one embodiment of the invention, the photopolymerization reaction is carried out in a liquid environment to carry out curing and crosslinking to form the double-layer osteochondral scaffold material.

In one embodiment of the present invention, the method further comprises adding growth factors at different concentrations to the photocrosslinkable P _m L _n DMA

And/or adding growth factors to the different concentrations of P _m L _n DMA/MH nanocomposites;

the growth factor is TGF-beta 1 and/or BMP-2.

The application of the double-layer osteochondral scaffold material in preparing products for treating or repairing cartilage injuries or beautifying the skin also belongs to the protection scope of the invention.

In the process of reconstructing and repairing osteochondral bone, the mechanical property of the adjustable material is not only beneficial to the operability in the operation, but also beneficial to the adjustment of the repairing performance of self cells. Research has proved that the mechanical property of the biomaterial can regulate and control the osteogenic and chondrogenic differentiation of the mesenchymal stem cells. When the mechanical property of the extracellular mechanism is matched with that of natural tissues, the differentiation of mesenchymal stem cells can be guided, for example, the elastic modulus of the natural cartilage at 1-10MPa can induce cartilage differentiation, and the bone tissue at 100-1000MPa can induce osteogenic differentiation. In addition to biophysical stimuli, growth factors may also serve as important biochemical stimuli to regulate the osteochondral microenvironment, stimulating cartilage and bone repair.

For example, transforming growth factor-beta 1 (TGF-beta 1) plays an important role in inhibiting early inflammation (inflammation may lead to bone/cartilage degeneration), attenuating osteoclastogenesis, and enhancing cartilage formation. In addition, bone morphogenetic protein-2 (BMP-2) is the only FDA approved osteogenic growth factor, and it becomes an important regulatory factor for osteogenesis by up-regulating the Smad pathway. Therefore, the adhesive which is matched with natural tissues and has the physical and chemical stimulation capability can guide the mesenchymal stem cells to differentiate into bones and cartilages and is beneficial to the repair of osteochondral injury. Therefore, the invention develops an injectable hydrophobic double-layer osteochondral scaffold material, which can be rapidly implemented in water, is crosslinked by ultraviolet light to form an anisotropic double-layer structure, has good interface bonding force, forms a double-layer structure similar to natural osteochondral, can release target growth factors for a long time, and regulates and controls the repair of osteochondral injury by physicochemical and chemical dual stimulation.

Due to the hydrophobic nature of the material, it was found that the two phases could be confined to the defect site without being dispersed in the body fluid. P photocrosslinkable by UV irradiation _m L _n DMA and P _m L _n The DMA/nHAMA nano composite material can be used as an adhesive, and the adhesive can be crosslinked and solidified within 200 seconds to form a double-layer osteochondral scaffold material. Due to P _m L _n DMA and P _m L _n There is "C = C" in DMA/nHAMA, and these two layers can form strong covalent bonds at the interface. By systematically adjusting P _m L _n Composition of DMA (m and n), and MH and P _m L _n The proportion of DMA will accurately control the mechanical properties of the two phases to match the native tissue.

The invention has the following advantages:

the hydrophobic injectable underwater cross-linked double-layer osteochondral scaffold material is quickly implemented and cross-linked and injected in a liquid environment to form a firm two-phase interface, cartilage generation and osteogenesis are synergistically enhanced by adjusting a physical and chemical microenvironment, and the cell function of mesenchymal stem cells can be synergistically influenced by mechanical stimulation and growth factor stimulation to regulate and control the cartilage and osteogenic differentiation of the mesenchymal stem cells in vitro and in vivo.

The two components of the double-layer osteochondral scaffold material are matched with the mechanical properties of cartilage and subchondral bone, different growth factors are loaded at the same time, the physical properties and the biochemical properties of the double-layer osteochondral scaffold material are matched with natural osteochondral tissues, the long-term release of the growth factors can be realized, the repair of osteochondral injury is accelerated, the double-layer osteochondral scaffold material can be injected, and the requirements of orthopedic arthroscopic surgery application can be met.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, the proportions, the sizes, and the like shown in the specification are only used for matching with the contents disclosed in the specification, so that those skilled in the art can understand and read the present invention, and do not limit the conditions for implementing the present invention, so that the present invention has no technical essence, and any modifications of the structures, changes of the proportion relation, or adjustments of the sizes, should still fall within the scope of the technical contents disclosed in the present invention without affecting the efficacy and the achievable purpose of the present invention.

FIG. 1 is a schematic diagram showing the results of mechanical property tests of a double-layered osteochondral scaffold material provided by an embodiment of the present invention, wherein PmLnDMA-D and PmLnDMA/MH-D are the mechanical properties of cross-linking under dry conditions; pmLnDMA-W and PmLnDMA/MH-W are the mechanical properties of crosslinking under a wet environment (W);

FIG. 2 shows a photo-crosslinkable P composition according to an embodiment of the present invention ₇ L ₂ DMA and P ₇ L ₂ DMA/50% MH binder may form different shapes of double-layered osteochondral scaffold material;

FIG. 3 is a schematic diagram illustrating the results of an interfacial force test of a double-layered osteochondral scaffold material prepared by performing photopolymerization reaction through underwater injection of the double-layered osteochondral scaffold material provided by an embodiment of the invention;

FIG. 4 is a schematic diagram illustrating the detection result of interfacial force of the double-layered osteochondral scaffold material according to the embodiment of the present invention;

FIG. 5 shows a photo-crosslinkable P composition according to an embodiment of the present invention _m L _n DMA and P _m L _n Schematic diagram of the heat release and growth factor release test results in DMA/MH crosslinking process;

FIGS. 6 and 7 are schematic diagrams illustrating the results of the good biocompatibility test of the double-layered osteochondral scaffold material provided by the embodiment of the invention;

FIG. 8 is a graph showing the results of the double-layered osteochondral scaffold material promoting cartilage differentiation provided by the present invention;

FIG. 9 is a schematic diagram showing the results of a rabbit osteochondral defect repairing mold with a double-layer osteochondral scaffold material according to an embodiment of the present invention.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the present invention, P _m L _n DMA: poly (lactic acid-propylene glycol) dimethacrylate; MH: methacrylated hydroxyapatite; p _m L _n DMA/MH: polymeric injectable adhesive, P constituting the subchondral bone phase _m L _n DMA/MH nanocomposites.

Example 1, P _m L _n Preparation of DMA

Provision of P of the embodiment _m L _n The preparation method of DMA comprises the following steps: wherein P represents propylene glycol, and m is the number of propylene glycol in the monomer; l represents polylactic acid, n is the number of polylactic acid in the monomer, and DMA is dimethacrylate. In the invention, the polylactic acid-propylene glycol dimethacrylate is P ₇ L ₄ DMA、P ₇ L ₂ DMA、P ₁₇ L ₄ DMA or P ₃₄ L ₄ DMA。

Wherein, P ₇ L ₄ The preparation process of the DMA comprises the following steps:

step one, 34.4g of polypropylene glycol (PPG, average molecular weight 425, 0.08mol) and 46g of lactide (LA, 0.32 mol) are subjected to ring-opening polymerization reaction for 6 hours at 150 ℃ by taking stannous octoate as a catalyst in a nitrogen-containing environment to obtain P ₇ L ₄ ；

Step two, weighing 10.01g P ₇ L ₄ (0.01 mol) in 100ml of dichloromethane, and then 4.22g of methacryloyl chloride (MAC) and 4.05 g of Triethylamine (TEA) are added dropwise, alternately at 0 ℃ in Dichloromethane (DCM)To remove the triethylamine/hydrochloric acid formed, the product was dissolved in 200mL of diethyl ether, filtered under vacuum, and finally diluted with 200mL of hydrochloric acid (0.1 mol/L) and 200mL of NaHCO ₃ The solution (0.1 mol/L) was washed with 200mL of deionized water, and the final oil-solvent phase was recovered from the mixture through a separation funnel and then rotary evaporated for 2 hours to completely remove the residual solvent.

P ₇ L ₂ The preparation method of the DMA comprises the following steps:

step one, 34.4g of polypropylene glycol (PPG, average molecular weight 425, 0.08mol) and 23g of lactide (LA, 0.16 mol) are subjected to ring-opening polymerization reaction for 6 hours at 150 ℃ by taking stannous octoate as a catalyst in a nitrogen-containing environment to obtain P ₇ L ₂ ；

Step two, weighing 7.13g P ₇ L ₂ (0.01 mol) in 100mL of dichloromethane, then 4.22g of methacryloyl chloride (MAC) and 4.05 g of Triethylamine (TEA) are added dropwise alternately at 0 ℃ in Dichloromethane (DCM), the two reagents are diluted, in order to remove the triethylamine HCl formed, the product is dissolved in 200mL of diethyl ether, filtered under vacuum and finally treated with 200mL of hydrochloric acid solution (0.1 mol/L), 200mL of NaHCO ₃ The solution (0.1 mol/L) was washed with 200mL of deionized water, and the final oil-solvent phase was recovered from the mixture through a separation funnel and then rotary evaporated for 2 hours to completely remove the residual solvent.

P ₁₇ L ₄ The preparation method of the DMA comprises the following steps:

step one, carrying out ring opening polymerization reaction on 40g of polypropylene glycol (PPG, average molecular weight 1000, 0.04mol) and 23g of lactide (LA, 0.16 mol) at 150 ℃ for 6 hours in a nitrogen-containing environment by using stannous octoate as a catalyst to obtain P ₁₇ L ₄ ；

Step two, weighing 15.75g P ₁₇ L ₄ (0.01 mol) in 100mL of dichloromethane, and then at 0 ℃ by alternately dropwise addition of 4.22g of methacryloyl chloride (MAC) and 4.05 g of Triethylamine (TEA), both reagents being diluted in Dichloromethane (DCM), the product being dissolved in 200mL of diethyl ether in order to remove the triethylamine HCl formed, filtered off in vacuo and finally treated with 200mL of hydrochloric acid solution (0.1 mol/L), 200mL of hydrochloric acid solution (0.1 mol/L)mL NaHCO ₃ The solution (0.1 mol/L) was washed with 200mL of deionized water, and the final oil-solvent phase was recovered from the mixture through a separation funnel and then rotary evaporated for 2 hours to completely remove the residual solvent.

P ₃₄ L ₄ The preparation method of DMA comprises the following steps:

step one, 40g of polypropylene glycol (PPG, average molecular weight 2000, 0.02mol) and 11.5g of lactic acid (LA, 0.08 mol) are subjected to ring-opening polymerization reaction for 6 hours at 150 ℃ by taking stannous octoate as a catalyst in a nitrogen-containing environment to obtain P ₃₄ L ₄ ；

Step two, weighing 31.52g P ₃₄ L ₈ (0.01 mol) in 100mL of dichloromethane, and then at 0 ℃ by alternately dropwise addition of 4.22g of methacryloyl chloride (MAC) and 4.05 g of Triethylamine (TEA), both reagents being diluted in Dichloromethane (DCM), the product being dissolved in 200mL of diethyl ether in order to remove the triethylamine HCl formed, filtered off in vacuo and finally treated with 200mL of hydrochloric acid solution (0.1 mol/L), 200mL of NaHCO ₃ The solution (0.1 mol/L) was washed with 200mL of deionized water, and the final oil-solvent phase was recovered from the mixture through a separation funnel and then rotary evaporated for 2 hours to completely remove the residual solvent.

Example 2, P _m L _n Preparation of DMA/MH nano composite material

This example provides P _m L _n The preparation method of the DMA/MH nano composite material comprises the following steps:

step one, preparation of methacrylic acid hydroxyapatite (MH)

The source of the methacrylated hydroxyapatite in the present invention is not particularly limited, and for example, it can be prepared by the following method:

20g HA was thoroughly dried at 120 ℃ for 48 hours, dissolved in 300mL of Dimethylformamide (DMF) under nitrogen protection, then 4mL HMDI and 0.4mL dibutyltin dilaurate (DBTDL) were added as a catalyst, reacted at 50 ℃ for 24 hours, then 8mL HEMA was added, reacted under the same conditions for 5 hours, and finally, 400mL methanol was added to stop the reaction, and the HAMA obtained by the reaction was centrifuged, washed three times with Dichloromethane (DCM), and dried at room temperature for 96 hours.

Step two, P _m L _n DMA/MH nanocomposite

P prepared in example 1 _m L _n Mixing DMA and methacrylic acid hydroxyapatite (MH) prepared in the step one according to a proportion to obtain P _m L _n DMA/MH nanocomposites, i.e., photopolymerizable injectable adhesives.

Example 3 preparation of double-layered osteochondral scaffold Material and mechanical Property test

This example provides a bilayer osteochondral scaffold material comprising photocrosslinkable P's constituting the cartilage phase _m L _n DMA; and P constituting subchondral bone phase _m L _n DMA/MH nanocomposites;

wherein P is photocrosslinkable _m L _n DMA is through the exchange of P _m L _n DMA (90 wt%) was mixed with hydroxyethyl methacrylate (9 wt%) and photoinitiator 819 (1 wt%) to prepare photocrosslinkable P as the cartilage phase _m L _n DMA。

P _m L _n DMA/MH nanocomposites are prepared by reacting P _m L _n DMA was prepared as the subchondral bone phase mixed with different concentrations of MH (e.g. 10, 30, 50, 70 wt.%).

Separately, 15. Mu.g of transforming growth factor-. Beta.1 (TGF-. Beta.1) and bone morphogenetic protein-2 (BMP-2) powders were mixed with 500mg of P _m L _n DMA and P _m L _n In DMA/MH, ultraviolet irradiation is carried out, and then the double-layer osteochondral scaffold material loaded with growth factors can be prepared.

The test method of the double-layer osteochondral scaffold material comprises the following steps: compression test of P crosslinkable by light _m L _n DMA and P _m L _n Injecting DMA/MH nano composite material into Teflon mold in air or underwater

In the presence of ultraviolet light, and then subjecting the mixture to ultraviolet irradiationThe line irradiation was carried out for 200 seconds. Then, the sample was compressed at a rate of 1mm/min until breakage. Three replicates were tested for each material formulation.

As shown in fig. 1, P of the present invention ₇ L ₄ DMA and P ₃₄ L ₄ DMA has the highest and lowest compressive modulus (49.3. + -. 5.2MPa vs. 5.6. + -. 0.6 MPa), respectively.

P ₇ L ₂ The compression modulus of the DMA group is 36.5 +/-3.2 MPa, the tensile modulus is 16.5 +/-2.3 MPa, and the DMA group is mechanically matched with natural cartilage. Furthermore, there was no significant difference between the samples crosslinked under underwater or dry conditions, indicating that the underwater crosslinking process did not compromise the mechanical properties of the double-layered osteochondral scaffold material. Thus, P is selected ₇ L ₂ DMA is used as photo-crosslinkable PmLnDMA, i.e. cartilage phase binder, constituting the cartilage phase.

Incorporation of MH into P ₇ L ₂ After DMA, P ₇ L ₂ The compression and tensile modulus of DMA/MH increased significantly. 50% the highest compression and tensile moduli of the MH group were 382.2. + -. 24.9MPa and 173.2. + -. 14.9MPa, respectively, comparable to natural cancellous bone, and could provide sufficient support for the upper cartilage layer and surrounding tissues, and could serve as P constituting the subchondral bone phase _m L _n DMA/MH nanocomposites, subchondral bone phase cement.

Example 4 double layer osteochondral scaffold Material Molding layered Structure

This example prepares teflon moulds of different shapes (round, moon, star, triangle) into which are successively injected the photocrosslinkable P ₇ L ₂ DMA and P ₇ L ₂ DMA/50% MH adhesive 500. Mu.L was irradiated with 365nm wavelength ultraviolet light for 200s to form different shapes of double-layered osteochondral scaffold material.

As shown in FIG. 2, P which is photo-crosslinkable ₇ L ₂ DMA and P ₇ L ₂ DMA/50% MH binder can form a good layered structure double-layered interbonding material and fill different shaped molds.

Example 5 Underwater photopolymerization of double-layer osteochondral scaffold Material

This example provides photocrosslinkable P ₇ L ₂ DMA and P ₇ L ₂ DMA/50% MH the photopolymerization reaction process under water:

injection of P in 20mL Water ₇ L ₂ DMA and P ₇ L ₂ DMA/50% MH binder, or P was injected sequentially under water in a 5mm round PDMS mold ₇ L ₂ DMA and P7L2DMA/50% MH adhesive 500. Mu.L, irradiated with 365nm wavelength ultraviolet light for 200 seconds.

As shown in figure 3, the double-layer osteochondral scaffold material can be injected underwater without dispersion and can be rapidly cross-linked at the defect to form a double-layer structure with strong interfacial force, and the requirement of underwater injection filling of an arthroscope can be met.

Example 6 interface force test of double-layer osteochondral scaffold Material

This example will P _m L _n DMA and P _m L _n DMA/MH was injected into a 20mm by 20mm Teflon mold and then photocrosslinked to form a double-layered osteochondral scaffold material. The double layer osteochondral scaffold material was then adhered to a glass slide by 3M scotch tape to assess shear strength. Both normal and shear forces are performed. The separation speed will be set to 1cm/min and the adhesion at the adhesive interface will be determined by the critical peel separation strength. This example prepares three parallel samples for each material formulation to ensure reproducibility.

As shown in fig. 4, the interfacial force detection of the double-layered osteochondral scaffold material detects the adhesion force in the vertical direction and the tangential direction, respectively, and there is no significant difference in the cross-linking interfacial force in the dry and wet environments, and meanwhile, the interfacial force formed by the double-layered osteochondral scaffold material of this embodiment is significantly higher than that of the commercial 3M glue.

Example 7 sustained Release Performance of double layer osteochondral scaffold

In this example, 15. Mu.g of transforming growth factor-. Beta.1 (TGF-. Beta.1) and bone morphogenetic protein-2 (BMP-2) powders were mixed with 500mg of P _m L _n DMA and P _m L _n In DMA/MH, a double-layer bone cartilage adhesive loaded with growth factors, namely a double-layer bone cartilage scaffold material, is prepared.

Crosslinked samples were incubated at room temperature with 10mL of phosphate buffer saltIncubate in water (PBS). At different predetermined time points, 1mL of PBS was collected from the culture medium and the same volume of fresh PBS was added. The amount of TGF-. Beta.1 released was then analyzed by TGF-. Beta.1 ELISA kit. Similarly, BMP-2 loaded P ₇ L ₂ DMA/50HAMA (7/2/50MH @ BMP-2) was prepared using the same method, and the amount of released BMP-2 was analyzed by ELISA kit.

As shown in FIG. 5, the photo-crosslinkable P of the present invention _m L _n DMA and P _m L _n The DMA/MH crosslinking process has no obvious heat release, the transforming growth factor-beta 1 is loaded in the cartilage phase, the osteogenesis protein 2 is loaded in the subchondral bone phase, the growth factor can be stably released in 60 days along with the material, and the release amount is more than 60 percent.

Example 8 double layer osteochondral scaffold Material compatibility

In this example, 15. Mu.g of transforming growth factor-. Beta.1 (TGF-. Beta.1) and bone morphogenetic protein-2 (BMP-2) powder were mixed with 500mg of P _m L _n DMA and P _m L _n In DMA/MH, preparing a growth factor-loaded osteochondral adhesive, namely a double-layer osteochondral scaffold material, and preparing different disc-shaped double-layer osteochondral scaffold material samples

By subjecting rat mesenchymal stem cells (rBMSCs) to a density of 1X 10 ⁴ Individual cell/cm ² The biocompatibility of the double-layer osteochondral scaffold material was evaluated by inoculating the double-layer osteochondral scaffold material.

As shown in FIG. 6, the double-layer osteochondral scaffold material has good biocompatibility and can support the adhesion and spreading of the transplanted rat bone marrow mesenchymal stem cells.

As shown in fig. 7, the double-layered osteochondral scaffold material has good biocompatibility, can provide good proliferation of the transplanted rat bone marrow mesenchymal stem cells, and has a cell survival rate of more than 90%.

Example 9 double layer osteochondral scaffold Material promoting cartilage differentiation

This example will yield 15. Mu.g of transforming growth factor-. Beta.1 (TGF-. Beta.1) and bone morphogenetic protein-2 (BMP-2)) Mixing the powder with 500mgP _m L _n DMA and P _m L _n In DMA/MH, the bone cartilage adhesive loaded with growth factors, namely a double-layer bone cartilage scaffold material, is prepared.

Preparing different disc-shaped double-layer osteochondral scaffold material samples

By subjecting rat mesenchymal stem cells (rBMSCs) to a density of 4X 10 ⁴ Individual cell/cm ² The double-layer osteochondral scaffold material is inoculated to evaluate the differentiation induction capability of the adhesive on stem cells, and chondrogenesis experiments and osteogenesis experiments respectively use corresponding chondrogenesis and osteogenesis induction culture medium to intervene.

As shown in fig. 8, the cartilage phase matched with the mechanical properties of the double-layered osteochondral scaffold material of the present embodiment is loaded with transforming growth factor- β 1 to promote chondrogenic differentiation of rat mesenchymal stem cells, and the fast green turning over to red O and the staining of a Li Xinlan prove that the cartilage scaffold material can promote chondrogenic differentiation of stem cells.

Mechanical property matched P of the embodiment _m L _n The bone formation generation protein 2 is loaded on the DMA/MH subchondral bone phase to promote the osteogenic differentiation of rat mesenchymal stem cells, and the staining of alkaline phosphatase and alizarin red proves that the subchondral bone adhesive can promote the osteogenic differentiation of the stem cells.

Example 10 double layer osteochondral scaffold materials promoting cartilage repair

This example provides new zealand white rabbits (male, body weight 3.0-3.5 kg) randomly divided into 5 groups: blank group, 7/2 group (fill P) ₇ L ₂ DMA Adhesives), 7/2/50MH groups (fill P) ₇ L ₂ DMA/50%), BL group (filled with double-layer osteochondral scaffold material) and BL/GFs group (filled with double-layer osteochondral scaffold material with TGF-1 and BMP-2 incorporated in different layers). A full thickness osteochondral cylindrical defect (5 mm diameter, 4 mm depth) was formed on the trochlear groove at the distal end of the rabbit femur using a dental drill.

Then, the defect was filled with a double-layered osteochondral scaffold material composed at different ratios, and then irradiated with ultraviolet rays for 200 seconds. Next, the surgical knee joint was sutured with intramuscular antibiotic sutures (4-0 thread). After 8 and 12 weeks of implantation, the rabbits were sacrificed and femoral samples were collected. And evaluating the cartilage damage repair result through ICRS international cartilage repair score, and evaluating the regeneration repair of subchondral bone through micro-CT scanning.

As shown in fig. 9, in this example, it is proved by a rabbit osteochondral defect model that a double-layered osteochondral scaffold material can promote repair and regeneration of cartilage and subchondral bone of rabbit osteochondral defects, cartilage regeneration of defect regions can be observed in rabbit femoral specimens after 8 weeks and 12 weeks, and subchondral bone regeneration is observed in CT scan results. The growth factor-loaded and mechanical property-matched two-phase adhesive can synchronously promote the repair of rabbit bone cartilage.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (8)

1. A double-layer osteochondral scaffold material, wherein the scaffold material comprises a cartilage phase and a subchondral bone phase;

photo-crosslinkable P constituting the cartilage phase _m L _n DMA; and

p constituting the subchondral bone phase _m L _n DMA/MH nanocomposites;

wherein, P is polypropylene glycol; m is the number of monomers in the polypropylene glycol; l is polylactic acid; n is the number of polylactic acid in the monomer; DMA is dimethacrylate, MH is methacrylated hydroxyapatite;

the photocrosslinkable P _m L _n DMA is through P _m L _n DMA, hydroxyethyl methacrylate and a photoinitiator are mixed to prepare the UV curing coating;

the P is _m L _n The DMA/MH nano composite material is prepared by mixing P _m L _n DMA and hydroxyethyl methacrylate functionalized nano-hydroxyl phosphorus with different concentrationsAnd mixing the limestone.

2. The double-layered osteochondral scaffold material of claim 1,

the photocrosslinkable P _m L _n In the DMA, the mass percent of each component is as follows: p _m L _n 90wt% of DMA, 9wt% of hydroxyethyl methacrylate and 819 wt% of photoinitiator.

3. The double-layered osteochondral scaffold material of claim 1,

the scaffold material also includes TGF-beta 1 and/or BMP-2.

4. The double-layered osteochondral scaffold material of claim 1,

the P is _m L _n DMA is P ₇ L ₄ DMA、P ₇ L ₂ DMA、P ₁₇ L ₂ DMA、P ₁₇ L ₄ DMA、P ₃₄ L ₂ DMA、P ₃₄ L ₄ DMA、P ₆₈ L ₂ DMA or P ₆₈ L ₄ DMA。

5. A method for preparing the double-layered osteochondral scaffold material of any one of claims 1-4, comprising:

subjecting the photocrosslinkable P _m L _n DMA and P _m L _n And carrying out photopolymerization on the DMA/MH nano composite material to obtain the double-layer osteochondral scaffold material.

6. The method of claim 5,

the photopolymerization reaction is carried out in a liquid environment to form the double-layer osteochondral scaffold material through curing and crosslinking.

7. The method of claim 5,

the method further comprises adding growth factors at different concentrationsTo said photocrosslinkable P _m L _n DMA

And/or adding growth factors to the different concentrations of P _m L _n DMA/MH nanocomposites;

the growth factor is TGF-beta 1 and/or BMP-2.

8. Use of the double-layered osteochondral scaffold material of any one of claims 1-4 in the manufacture of a product for treating or repairing cartilage damage or for cosmetic use.

Images