Hydrogel-microsphere composite scaffold material for damage repair

Abstract

The invention discloses a composite scaffold material for damage repair, and belongs to the field of biological medicines.The composite scaffold material comprises self-assembled polypeptide hydrogel capable of forming a net-shaped scaffold and porous microspheres, and the self-assembled polypeptide hydrogel in the composite scaffold material can form a stable porous structure; the hydrogel-microsphere composite scaffold material has good dispersion and support effects, avoids the problem of difficult injection, and can reconstruct a tissue microenvironment, regulate cell behaviors and provide an excellent repair and regeneration microenvironment so as to induce repair and regeneration.

Description

Technical field:

[0001] The invention relates to the field of biomedical materials, in particular to a composition of self-assembling polypeptide hydrogel and porous microspheres. Background technology:

[0002] Humans live in a challenging and unexpected environment, so injury is an important aspect of human struggle against disease, and injury repair is a basic requirement for all living organisms. Improving the microenvironment at the site of injury is an important means of effectively treating injuries. After primary trauma, a variety of secondary injury complications will occur around the lesion, such as extracellular matrix (ECM) damage, severe oxidative stress and inflammation, hypoxia-ischemia, etc. In this case, it is difficult to achieve effective recovery by treating a single parameter. Therefore, comprehensive regulation of the local microenvironment is the key to injury treatment. Transplantation of biomaterials and stem cells (such as mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells) to treat injuries has been widely reported. However, the survival rate of cells at the lesion site after transplantation is impaired, which greatly reduces the efficiency of cell transplantation. And recent studies have shown that the role of mesenchymal stem cells is attributed to exosomes, which have the function of regulating the microenvironment of cells, can reduce inflammatory responses, and promote cell proliferation and migration. Injectable hydrogels are ideal materials for injury repair due to their suitable mechanical properties, good biocompatibility, and excellent encapsulation. Polylactic acid microspheres, due to their excellent biocompatibility, can provide more surface area, promoting cell adhesion and proliferation.

[0003] Therefore, it is crucial to construct a hydrogel-microsphere composite scaffold material for injury repair, which can regulate cell behavior and provide an excellent repair and regeneration microenvironment, thereby inducing repair and regeneration. Summary of the invention:

[0004] The purpose of the present invention is to provide a self-assembling peptide-microsphere system carrying different damage repair materials, which can be applied to biomedical fields such as damage repair and wound healing, in view of the fact that existing hydrogel-microsphere composite scaffold materials are difficult to disperse evenly during injection, have poor biocompatibility and cannot provide a microenvironment similar to that in the body.

[0005] The first aspect of the present invention provides a hydrogel-microsphere composite scaffold material, comprising a self-assembling polypeptide hydrogel and porous microspheres; the polypeptide hydrogel can form nano-scale fibers and present a three-dimensional network structure without chemical modification; the three-dimensional network structure can support the microspheres so that they are evenly dispersed and fixed in situ at the damaged site; and the porous microspheres can carry different damage repair materials.

[0006] The self-assembling polypeptide hydrogel in the present invention spontaneously forms a three-dimensional network structure through non-covalent interactions. The core mechanism of polypeptide self-assembly relies on hydrogen bonds, so that the peptide chain transforms from an amorphous structure to a protein secondary structure, forming a β-fold and α-helical structure, and further forming a structurally stable hydrogel three-dimensional network. Therefore, the porous microspheres carrying the damage repair material can be fixed in the three-dimensional network, achieve three-dimensional distribution and achieve in-situ fixation, reconstruct the tissue microenvironment, regulate cell behavior, and provide an excellent repair and regeneration microenvironment, thereby inducing repair and regeneration.

[0007] In a specific embodiment, in the hydrogel-microsphere composite scaffold material, the polypeptide hydrogel is selected from the following polypeptides:

[0008] SEQ ID 1, IIIIIGSIIGPGGEGPGGV

[0009] SEQ ID 2, FLIVIGSIIGPGGDGPGGV

[0010] SEQ ID 3, FLIVIGSIIGPGGEGPGGV

[0011] In a specific embodiment, in the hydrogel-microsphere composite scaffold material, the microspheres are hydrolyzed and modified; preferably, the microspheres are selected from at least one of L-polylactic acid microspheres, polycaprolactone microspheres, polylactic acid-glycolic acid microspheres, hydroxyapatite microspheres, polymethyl methacrylate microspheres and polyvinyl alcohol microspheres.

[0012] Preferably, the particle size of the microspheres is 150-300 μm;

[0013] Different microspheres have their optimal particle size. Preferably, the particle size of polylactic acid microspheres is 150-300 μm, the particle size of polycaprolactone microspheres is 150-250 μm, the particle size of polylactic acid-glycolic acid microspheres is 150-250 μm, the particle size of hydroxyapatite microspheres is 150-250 μm, and the particle size of polymethyl methacrylate microspheres is 150-250 μm. The second aspect of the present invention provides the aforementioned hydrogel-microsphere composite scaffold material, wherein the scaffold material is a solution, a lyophilized powder, or a suspension.

[0014] In a specific embodiment, the solution in the hydrogel-microsphere composite scaffold material is water, or physiological saline, or phosphate buffer.

[0015] When the solution preparation or the freeze-dried powder preparation is used, the concentration of the microspheres is controlled at 10wt%-40wt%; and the concentration of the polypeptide is controlled at 1.5%-3%.

[0016] The third aspect of the present invention provides the use of a hydrogel-microsphere composite scaffold material, wherein the hydrogel-microsphere composite scaffold material is used for regenerative medicine and tissue regeneration; drug delivery; wound healing; implantable materials; gene therapy; and stem cell therapy.

[0017] The present invention provides a hydrogel-microsphere composite scaffold material. The self-assembling peptide hydrogel in the composite material forms a three-dimensional network structure in an aqueous solution, which plays a role in dispersing support and local fixation of porous microspheres carrying damage repair materials. At the same time, after the composite material is injected into damaged tissue, it can reconstruct the tissue microenvironment, regulate cell behavior, and provide an excellent repair and regeneration microenvironment, thereby inducing repair and regeneration.

[0018] When the hydrogel-microsphere composite scaffold material is used, injection water, physiological saline or phosphate buffer solution is first added to the freeze-dried polypeptide powder and mixed evenly. The dissolved polypeptide self-assembles to form a three-dimensional network structure under the action of non-covalent bonds. The network structure formed by the self-assembled polypeptide supports the microspheres carrying the damage repair material in a gel state, so that the microspheres are evenly distributed in three dimensions in the polypeptide self-assembled network material, which can regulate cell behavior and provide an excellent repair and regeneration microenvironment, thereby inducing repair and regeneration.

[0019] The self-assembling polypeptide hydrogel of the present invention can form a stable porous structure through intermolecular interactions, so it has excellent mechanical properties and can stably support porous microspheres carrying different repair materials; and the hydrogel has excellent shear thinning and self-healing properties. Under 100% strain, the storage modulus G' quickly drops below the loss modulus G", turning it into a solution, indicating that the original network scaffold is severely damaged due to the large number of non-covalent bonds broken therein. After the high strain ends, G' quickly returns to its original level. The rapid and repeated automatic reconstruction of the previously shear-damaged network scaffold shows the recoverability and fatigue resistance of the hydrogel. Based on this phenomenon, it can be concluded that the hydrogel has excellent injectability.

[0020] In recent years, composite scaffold materials of self-assembling peptide hydrogels and porous microspheres have shown significant advantages in the field of injury repair, especially in tissue engineering and regenerative medicine. They can effectively promote the repair and regeneration of damaged areas, providing new ideas for the treatment of injuries such as bones, soft tissues and nerves.

[0021] Self-assembling peptide hydrogels have good biocompatibility, they can be completely degraded without producing any toxic degradation products. They also have the ability to transmit signal molecules and functional proteins. Due to their stable network structure, self-assembling peptide hydrogels provide the possibility of slow and sustained release of damage repair materials. In addition, self-assembling peptide hydrogels can highly simulate the extracellular matrix, forming a microenvironment suitable for cell growth and the action of functional proteins, which has a significant beneficial effect on bone and cartilage regeneration, wound healing, and drug delivery.

[0022] The composite scaffold material of self-assembling peptide hydrogel and porous microspheres can promote cell growth and proliferation. The structure of self-assembling peptide hydrogel can be designed by controlling the amino acid sequence and the arrangement of peptide chains to specifically bind to cell receptors, thereby promoting cell adhesion, proliferation and differentiation. For example, some self-assembling peptides can mimic the components in the extracellular matrix (ECM) to enhance the adhesion and growth ability of cells on the scaffold. The porous microspheres provide a structured support framework. The porous microspheres can provide more surface area and microenvironment, allowing cells to grow and expand on their surface and form a three-dimensional structure in the scaffold. More importantly, the porosity and size of the microspheres can be regulated to optimize the microenvironment required for cell growth.

[0023] The composite scaffold material of self-assembled polypeptide hydrogel and porous microspheres can provide suitable mechanical properties and flexibility. Self-assembled polypeptide hydrogels usually have high softness, which can be similar to human tissues and adapt to the flexibility requirements during tissue damage repair. The soft nature of the hydrogel enables it to better integrate with tissues when used in soft tissue repair, wound healing and other fields, and does not cause discomfort during local mechanical deformation. Although porous microspheres have strong mechanical strength, they often need to have sufficient support during the repair process to withstand the stress and load in the body. Porous microspheres provide this support, and the pore structure of the microspheres can be adjusted to achieve ideal physical properties and ensure mechanical stability during the repair process.

[0024] The composite scaffold material of self-assembling peptide hydrogel and porous microspheres has a controlled release function. The self-assembling peptide hydrogel and porous microspheres have a highly controlled release function. In the composite scaffold, drugs, growth factors or antibacterial agents can be loaded to provide sustained drug release. The molecular structure in the self-assembling peptide hydrogel can be designed to have the function of loading drugs, thereby helping the sustained release of drugs, slowing down the rate of drug concentration decline, and improving the therapeutic effect.

[0025] Glossary:

[0026] Hydrogel refers to a class of substances composed of natural or synthetic polymer materials that can absorb water and swell and maintain their three-dimensional network structure in water. Its main feature is that it has a high water content, which can usually reach more than 90% of its weight, so its appearance is soft and elastic like water. The physical and chemical properties of hydrogel make it widely used in many fields, such as biomedicine, environmental protection, food industry and cosmetics.

[0027] Hydrogels have the following properties:

[0028] 1. High Water Content: The greatest feature of hydrogels is their high water content, which makes them very effective in simulating the environment of human tissue.

[0029] 2. Softness and elasticity: Hydrogels have similar softness and elasticity to biological tissues and are often used to simulate biological tissues in medical and biological experiments.

[0030] 3. Biocompatibility: Many hydrogel materials have good biocompatibility and can be used in the biomedical field for drug delivery, wound healing, and tissue engineering.

[0031] In the present invention, when a peptide having both a hydrophilic surface and a hydrophobic surface structure self-assembles under the conditions described in the present invention, especially under physiological conditions, a hydrogel is obtained by encapsulating water.

[0032] Self-assembling peptides refer to short peptides that spontaneously assemble into aggregates or materials with specific three-dimensional structures through non-covalent interactions between peptide chains (such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, etc.). Self-assembling peptides are usually composed of amino acid sequences. Through careful design and optimization of the arrangement order of their amino acids, they can spontaneously form nanoscale structural units under specific conditions. These peptide molecules do not rely on external templates or catalysts, but through their own physical and chemical properties, they spontaneously organize and assemble under appropriate environmental conditions to form ordered aggregates. Self-assembling peptides usually have good biocompatibility, easy synthesis, and strong structural adjustability, which makes them widely used in drug delivery, tissue engineering, biosensors, nanomaterials and other fields.

[0033] Self-assembling peptides have the following properties:

[0034] 1. Spontaneity: Self-assembling peptides can spontaneously assemble into stable structures through the interaction forces of the peptide molecules themselves (such as hydrophobicity, hydrogen bonds, van der Waals forces, etc.) without external stimulation.

[0035] 2. Nanoscale structure: Self-assembling peptides can often self-assemble into nanoscale structures, such as nanofibers, nanoparticles or micelles, which have excellent biological and physical properties.

[0036] 3. Biocompatibility and biodegradability: Since self-assembling peptides are usually composed of natural amino acids, they have good biocompatibility, and many peptides can be degraded by enzymes in the body, so they have broad application prospects in the fields of medicine and biology.

[0037] 4. Structural adjustability: By changing the amino acid sequence of the peptide chain, or by regulating environmental factors such as pH, temperature, and ionic strength, the self-assembly behavior and final structure of the peptide can be precisely controlled, giving the self-assembling peptide multiple functions.

[0038] "Amino acid" includes those naturally occurring as well as non-naturally occurring amino acids, such as D-natural amino acids, beta and gamma derivatives. According to standard terminology, amino acid residue sequences may be named using either a three-letter or a one-letter code, e.g., alanine (Ala, A); arginine (Arg, R); asparagine (Asp, N); aspartic acid (D), cysteine ​​(C); glutamine (Q); glutamic acid (E); glycine (Gly, G); histidine (His, H), isoleucine (Ile, I); leucine (Leu, L), lysine (L), methionine (Met, M); phenylalanine (Ala, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W), tyrosine (Tyr, Y); valine (Val, V); selenocysteine ​​(Sec, U); hydroxyproline (Hyp, O).

[0039] In the present invention, a "peptide" is an amino acid chain. In particular, a peptide is 2 to 40 amino acids in length.

[0040] In the present invention, "self-assembly" refers to the aggregation of polypeptides into an ordered structure under normal environmental conditions.

[0041] In addition, the preparation method of the obtained polylactic acid porous microspheres is simple, the conditions are mild, and the operation is easy. The raw material source is low-toxic or non-toxic, which is conducive to large-scale production and application.

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] 1. The self-assembling polypeptide of the present invention can form a hydrogel with excellent mechanical properties, excellent shear thinning and self-repairing properties without complicated cross-linking process and expensive 3D printing equipment. It is easy to be injected in situ and forms a microenvironment similar to that in vivo locally.

[0044] 2. The polylactic acid porous microspheres of the present invention have cheap raw materials and a simple preparation process. More importantly, the polylactic acid porous microspheres have excellent biocompatibility and can provide a larger surface area for cells to attach and grow, promote cell proliferation, and accelerate damage repair.

[0045] 3. The hydrogel-microsphere composite scaffold material of the present invention has appropriate biodegradability and allows degradation and resorption to match tissue regeneration after in situ injection.

[0046] 4. The hydrogel-microsphere composite scaffold material of the present invention has appropriate porosity to allow cell migration and proliferation, as well as the transportation of nutrients and metabolic waste. Description of the drawings:

[0047] Figure 1 This is a picture of the self-assembling polypeptide hydrogel prepared by the present invention;

[0048] Figure 2 The microstructure pictures of the self-assembling polypeptide hydrogel prepared by the present invention. (A): Scanning electron microscope (SEM) picture, (B): Transmission electron microscope (TEM) picture;

[0049] Figure 3 The rheological results of the self-assembling polypeptide hydrogel prepared by the present invention. (A): mechanical strength test results, (B): self-healing performance test results, (C): stability test results;

[0050] Figure 4 The Fourier transform infrared spectrum test results of the self-assembling polypeptide hydrogel prepared by the present invention. (A): Fourier transform infrared spectrum, (B): Fourier transform infrared spectrum of amide I region, (C): Pie chart of polypeptide secondary structure;

[0051] Figure 5 This is a Congo red staining image of the self-assembling polypeptide hydrogel prepared by the present invention;

[0052] Figure 6 The polylactic acid porous microspheres prepared by the present invention. (A): optical image of polylactic acid porous microspheres, (B): scanning electron microscope image of polylactic acid porous microspheres, (C): particle size statistics of polylactic acid porous microspheres, (D): pore size statistics of polylactic acid porous microspheres;

[0053] Figure 7 This is a scanning electron microscope image of exosomes adsorbed on the surface of polylactic acid porous microspheres in the present invention;

[0054] Figure 8The diagram of the hydrogel-microsphere composite scaffold material carrying exosomes prepared by the present invention. (A): 3D distribution of exosomes in polylactic acid porous microspheres, (B): Picture of self-assembled polypeptide hydrogel supporting polylactic acid porous microspheres and exosomes, (C): Picture of the injectable hydrogel-microsphere system carrying exosomes;

[0055] Fig. 9 This is the recovery status of different groups after 7 days of spinal cord injury repair treatment in the present invention.

[0056] Fig.10 The morphological distribution of bone marrow mesenchymal stem cells in the hydrogel-microsphere composite scaffold material of the present invention. (A): overall picture, (B): partial picture.

[0057] Fig.11 The surgical process of implanting the hydrogel-microsphere composite scaffold material carrying bone marrow mesenchymal stem cells into a rat. (A) Before the scaffold is injected; (B) During the scaffold is injected; (C) After the scaffold is injected.

[0058] Fig.12 These are images taken four weeks after the hydrogel-microsphere composite scaffold material carrying bone marrow mesenchymal stem cells was implanted into the bone defect site. (A) Micro-CT image of the regenerated proximal femoral defect in a rat; (B) an image after the sample was placed in a Micro-CT scanner for scanning and three-dimensional reconstruction. Specific implementation method:

[0059] The technical scheme of the present invention is further described below in conjunction with the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Given the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0060] Example 1. Synthesis of polypeptides (i.e., self-assembling peptides)

[0061] Three peptides were synthesized by a standard solid phase peptide synthesis method. The amino acid sequences are shown in Table 1 below. The amino acid sequences of the synthesized peptides were confirmed to be correct by high performance liquid chromatography and mass spectrometry.

[0062] Table 1. Peptide sequences

[0063]

[0064] Example 2. Preparation of self-assembling polypeptide hydrogel

[0065] Taking Seq 1 (IIIIIGSIIGPGGEGPGGV) as an example, the self-assembled polypeptide lyophilized powder was dissolved in phosphate buffer, mixed by pipetting, and fully dissolved by vortexing to prepare a mother solution with a mass concentration of 5.0 wt.%. When used, it was diluted to a mass concentration of 1.5 wt.%, and allowed to stand at room temperature for 15 min to form a stable gel.

[0066] The structure and properties of the self-assembling polypeptide hydrogel prepared above were characterized, and the characterization results are as follows:

[0067] Figure 1 This is a picture of the self-assembling polypeptide hydrogel prepared in this example. It can be seen from the figure that after standing at room temperature for 15 minutes, the self-assembling polypeptide hydrogel with a mass concentration of 1.5wt.% can self-assemble to form a stable gel.

[0068] Figure 2 (A) is a scanning electron micrograph of the self-assembled polypeptide hydrogel prepared in this example, which shows a porous scaffold similar to the natural extracellular matrix, capable of accommodating polylactic acid porous microspheres, cells, and exosomes, while allowing metabolic molecules and nutrients to be transported between cells. Figure 2 (B) Transmission electron microscopy image of the self-assembled polypeptide hydrogel prepared in this example, showing that the hydrogel is a porous, highly organized dispersed network topology.

[0069] Figure 3 (A) is an evaluation of the mechanical strength of the self-assembled polypeptide hydrogel prepared in this example. 200 μl of the self-assembled polypeptide hydrogel with a mass concentration of 1.5 wt.% was aspirated for rheological testing. The storage modulus (G′) and the loss modulus (G″) both showed a parallel state and the storage modulus (G′) was much larger than the loss modulus (G″). This phenomenon shows that the hydrogel prepared in this example has a stable structure. Figure 3 (B) Shear thinning test of the self-assembling polypeptide hydrogel prepared in this example. The injectability of the hydrogel was evaluated by performing a recovery test after cyclic shear thinning. At 100% strain, G′ dropped rapidly below G″, turning it into a solution, indicating that the original network scaffold was severely damaged due to the large number of non-covalent bonds broken therein. After the high strain ended, G′ quickly recovered to its original level. The rapid and repeated automatic reconstruction of the previously shear-damaged network scaffold showed the recoverability and fatigue resistance of the hydrogel. Based on this phenomenon, it can be concluded that the hydrogel has excellent injectability. Figure 3 (C) is a stability test of the self-assembling polypeptide hydrogel prepared in this example. The hydrogel is relatively stable between 0.1-10 Hz, indicating that the internal structure of the hydrogel remains stable during the test.

[0070] Figure 4The Fourier transform infrared spectrum test results of the self-assembling polypeptide hydrogel prepared in this example are as follows. The amide I region is further processed to obtain the protein secondary structure distribution of the self-assembling polypeptide hydrogel. Figure 5 This is a Congo red staining image of the self-assembling polypeptide hydrogel prepared in this example. Congo red clearly displays the β-folding structure. The results show that the self-assembly of the self-assembling polypeptide hydrogel is time-dependent.

[0071] Example 3 Preparation of polylactic acid porous microspheres

[0072] This embodiment relates to the preparation of polylactic acid porous microspheres, which specifically includes the following steps:

[0073] 1) Dissolve 320 mg of polylactic acid in 8 mL of dichloromethane (DCM) as the oil phase;

[0074] 2) Then, 5 mL of 15 wt.% NH4HCO3 aqueous solution was added as the internal aqueous phase;

[0075] 3) stirring the primary W1 / O emulsion at a set power using an ultrasonic dispersion device in an ice-water bath for 3 minutes to form a W1 / O primary emulsion;

[0076] 4) Pour the colostrum into 300 mL of 0.7% (w / v%) polyvinyl alcohol (PVA) aqueous solution and stir at 500 rpm for 4 hours;

[0077] 5) After stirring, the microspheres are collected and washed with distilled water for multiple times to obtain polylactic acid porous microspheres;

[0078] 6) The polylactic acid porous microspheres were immersed in 75% anhydrous ethanol at 4° for 4-6 hours for sterilization, rinsed with PBS buffer 2-3 times, and stored in serum for future use.

[0079] The polylactic acid porous microspheres prepared above were characterized, and the characterization results are as follows:

[0080] Figure 6 (A) and (C) are optical images of the polylactic acid porous microspheres prepared in this example and their particle size distribution diagrams, indicating that the prepared polylactic acid porous microspheres have good morphology, uniform size, and an average diameter of about 251 μm. Figure 6 (B) and (D) are scanning electron microscope images and pore size statistics of the polylactic acid porous microspheres prepared in this example, indicating that the prepared polylactic acid porous microspheres have a higher porosity and can provide cells with a larger surface area for attachment and growth, and the average pore size is about 24 μm.

[0081] Example 4 Construction of exosome-carrying hydrogel-microsphere composite scaffold material

[0082] Experimental method: Taking Seq 2 (IIIIIGSIIGPGGEGPGGV) and polylactic acid porous microspheres as an example, 15 mg of polylactic acid porous microspheres were weighed using a precision balance. After sterilization, exosomes derived from mesenchymal stem cells were added, and then hydrogel with a concentration of 1.5 wt% was added. After blowing and mixing, the hydrogel supported the microspheres to suspend.

[0083] Experimental results: Scanning electron microscope images of exosomes were observed, and the results showed that exosomes have a typical cup-shaped or spherical structure ( Figure 7 ), polylactic acid porous microspheres and exosomes can be well supported by self-assembled polypeptide hydrogels ( Figure 8 A), the hydrogel-microsphere composite scaffold material loaded with exosomes has good injectability ( Figure 8 B).

[0084] Example 5 Application of exosome-loaded hydrogel-microsphere composite scaffold material in spinal cord injury repair

[0085] Experimental method: A spinal cord injury model was constructed using mice, and then the exosome-loaded hydrogel-microsphere composite scaffold material constructed in Example 4 was injected into the injury site. At the same time, physiological saline and polypeptide hydrogel were injected as controls, and the spinal cord injury repair was observed after 7 days.

[0086] Experimental results: Fig. 9 As shown in the figure, compared with the control group and the hydrogel group, the improvement of the hydrogel-microsphere composite scaffold material loaded with exosomes was more obvious, with no obvious inflammatory reaction, and the cavity in the injury site was significantly reduced, and the injury surface was smoother. This shows that the hydrogel-microsphere composite scaffold material loaded with exosomes has the function of promoting the proliferation and migration of neural stem cells and promoting the repair of spinal cord injuries.

[0087] Example 6 Construction of Hydrogel-Microsphere Composite Scaffold Material Carrying Bone Marrow Mesenchymal Stem Cells

[0088] Experimental method: Taking Seq 3 (FLIVIGSIIGPGGDGPGGV) and polylactic acid porous microspheres as an example, 15 mg of polylactic acid porous microspheres were weighed using a precision balance and sterilized. Bone marrow mesenchymal stem cells were added to allow the cells to adhere to the surface of the polylactic acid porous microspheres and grow. After culturing for 1 day, 1.5 wt% hydrogel was added and mixed by blowing to suspend the microspheres.

[0089] Experimental results: Fig.10 This is a morphological distribution picture of bone marrow mesenchymal stem cells attached to polylactic acid porous microspheres in this example. After 1 day of culture, bone marrow mesenchymal stem cells can attach to the surface of the microspheres and grow with good cell morphology, indicating that polylactic acid porous microspheres have excellent biocompatibility.

[0090] Example 7 Application of hydrogel-microsphere composite scaffold material carrying bone marrow mesenchymal stem cells in bone repair

[0091] Experimental method: A bone defect model was constructed using mice, and then the hydrogel-microsphere composite scaffold material loaded with bone marrow mesenchymal stem cells constructed in Example 6 was injected into the bone defect. At the same time, physiological saline and polylactic acid porous microspheres loaded with bone marrow mesenchymal stem cells were injected as controls, and the bone defect damage repair was observed after 4 weeks.

[0092] Experimental results: The surgical implantation process was as follows Fig.11 As shown in the figure, the hydrogel-microsphere composite scaffold material carrying bone marrow mesenchymal stem cells was directly injected into the femoral defect without the need for additional surgical fixation, which facilitated minimally invasive implantation. For comparison, microspheres carrying bone marrow mesenchymal stem cells and normal saline were also injected into the femoral defect. Fig.12 These are micro-CT images of the 4th week after the hydrogel-microsphere composite scaffold material carrying bone marrow mesenchymal stem cells was implanted into the bone defect. There was no obvious bone regeneration in the control group after 4 weeks, while new bone appeared around the defect in the groups injected with microspheres carrying bone marrow mesenchymal stem cells (PLLA-MS) and hydrogel-microsphere composite scaffold material carrying bone marrow mesenchymal stem cells (P-PLLA-MS). The cavity volume in the defect center of the P-PLLA-MS group was smaller than that of the PLLA-MS group, proving that the 3D culture system created by the P-PLLA-MS group can more effectively exert cellular effects, repair bone defects and promote bone regeneration.

[0093] With the above ideal examples of the present invention as inspiration, through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the content of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A hydrogel-microsphere composite scaffold material for damage repair, comprising a self-assembling polypeptide hydrogel and porous microspheres; characterized in that: The polypeptide hydrogel can form nano-scale fibers and present a three-dimensional network structure; the three-dimensional network structure can support the microspheres so that they are evenly dispersed, three-dimensionally distributed and fixed in situ at the damaged site; and the porous microspheres can carry different damage repair materials.

2. The hydrogel-microsphere composite scaffold material according to claim 1, characterized in that: The polypeptide hydrogel is selected from the following polypeptides: SEQ ID 1, IIIIIGSIIGPGGEGPGGV SEQ ID 2, FLIVIGSIIGPGGDGPGGV SEQ ID 3, FLIVIGSIIGPGGEGPGGV.

3. The porous microsphere according to claim 1, characterized in that The porous microspheres are modified by hydrolysis; preferably, the microspheres are selected from at least one of L-polylactic acid microspheres, polycaprolactone microspheres, polylactic acid-glycolic acid microspheres, hydroxyapatite microspheres, polymethyl methacrylate microspheres and polyvinyl alcohol microspheres.

4. Damage repair according to claim 1, characterized in that The repair materials include but are not limited to exosomes and stem cells.

5. The porous microsphere according to any one of claims 1 to 3, characterized in that: The particle size of the microspheres is 150-250 μm; Preferably, the particle size of polylactic acid microspheres is 150-250 μm, the particle size of polycaprolactone microspheres is 150-250 μm, the particle size of polylactic acid-glycolic acid microspheres is 150-250 μm, the particle size of hydroxyapatite microspheres is 150-250 μm, and the particle size of polymethyl methacrylate microspheres is 150-250 μm.

6. A hydrogel-microsphere composite scaffold material according to any one of claims 1 to 4, characterized in that: The composite material is a solution, or a freeze-dried powder, or a suspension.

7. The composite scaffold material according to claim 6, characterized in that: The solution or the suspension is water, or physiological saline, or phosphate buffer.

8. The composite scaffold material according to claim 7, characterized in that: In the composite scaffold material, the concentration of microspheres is controlled at 10wt%-40wt%; and the concentration of the polypeptide is controlled at 1.5%-3%.

9. Use of the composite scaffold material according to any one of claims 1 to 4, characterized in that: The composite scaffold material is used for regenerative medicine and tissue regeneration; drug delivery; wound healing; implantable materials; gene therapy; and stem cell therapy.

10. The use according to claim 9, characterized in that: When the composite scaffold material is used, injection water, physiological saline or phosphate buffer solution are first added to the freeze-dried polypeptide powder and mixed evenly. The dissolved polypeptide self-assembles to form a three-dimensional network structure under the action of non-covalent bonds. The network structure formed by the self-assembled polypeptide supports the porous microspheres carrying the repair material, which are injected in situ into the damaged area to regulate cell behavior, provide an excellent repair and regeneration microenvironment, and thus induce repair and regeneration.

Images