CN107854732A

CN107854732A - Improve space and hole promotes the compound rest and preparation method of cell adhesion rate

Info

Publication number: CN107854732A
Application number: CN201711060223.7A
Authority: CN
Inventors: 曲志伟; 肖霄; 李保强; 闫景龙; 毕佳琦
Original assignee: HARBIN FIRST HOSPITAL
Current assignee: Qu Zhiwei
Priority date: 2017-11-01
Filing date: 2017-11-01
Publication date: 2018-03-30

Abstract

The invention belongs to compound rest technical field, disclose it is a kind of improve space and hole promotes the compound rest and preparation method of cell adhesion rate, compound rest access opening and spherical pore are respectively 150 μm to 650 μm and 3 μm to 15 μm；Preparation method includes obtaining the CS/HA supports of hole and spherical pore by hybridization in situ technique and freeze drying process；CS and RGD peptide are connected by hydrogen bond, method is independently filled by electrostatic and modifies rgd peptide on CS/HA support apertures surface, prepares the how empty support of three-dimensional of the efficient adherent cells of the CS/HA containing RGD peptide.The support of the present invention has preferable cell compatibility；Compound by being carried out with RGD, effect of the three-dimensional porous CS/HA supports to the attraction to cell, induction, regulation and control growth is remarkably reinforced；The present invention combines physical absorption and immobilization chemistries, material is had more preferable adsorptivity and stability.

Description

Composite scaffold with improved gaps and pores for promoting cell adhesion rate and preparation method thereof

Technical Field

The invention belongs to the technical field of composite scaffolds, and particularly relates to a composite scaffold with improved gaps and pores for promoting cell adhesion rate and a preparation method thereof.

Background

Bone defects are a common clinical problem in orthopedics, and as many as 220 thousands of patients worldwide need bone graft treatment due to trauma, infection, tumor, and the like every year, while in the united states, approximately 60% of patients who need bone grafting adopt autologous bone grafting, 34% adopt allogeneic bone grafting, and only 7% adopt other bone grafting materials every year. Bone nonunion and bone defect caused by various bone diseases such as trauma and bone tumor are usually treated by autologous or allogeneic bone transplantation. These methods, while effective, are often still subject to insufficient material sources, risk of infection by donor diseases, difficulty in plasticity of the graft material, etc. The artificial implant is prepared by artificial synthesis, can not be limited by supply sources, avoids immunological rejection because of inert substances, but cannot completely replace biological tissues and organs in function and sometimes generates foreign body reaction. Therefore, no perfect method for completely solving the repair problem of damaged tissues and organs exists clinically so far. Thus promoting the rapid development of tissue engineering therapies to a certain extent. Tissue engineered organs will be more readily available than donor organs, tissues and organs reconstructed by tissue engineering can be mass produced as artificial substitutes, and such tissues and organs have the same functions as natural tissues and organs, and can also prevent the generation of immune rejection, which is considered as the most promising approach for thoroughly solving the problems of tissue and organ repair. Since 1995, bone tissue engineering was advanced by Crane et al, and as an important field of tissue engineering research, bone tissue engineering research has achieved exciting research results in many aspects, and has been clinically primarily applied, and is considered to be one of the most promising and feasible fields in tissue engineering. The bone tissue engineering seed cell which is most hopefully clinically widely used at present is a bone marrow stromal stem cell. It has the advantages of convenient source, simple material selection, small damage to patients and the like. BMSCs have the ability to self-renew and are capable of multipotent differentiation, which is a commonality of stem cells. Numerous studies have confirmed that BMSCs can effectively form tissue engineering bones as seed cells and successfully repair bone defects of various parts in vivo of large animals such as nude mice, mice and goats, and studies have shown that adult BMSCs can also exhibit the morphology and function of osteoblasts under appropriate induction conditions, and form tissue engineering bones in nude mice, and that BMSCs have correspondingly good therapeutic effects in the prior art. With the rapid development of bone tissue engineering in recent years, the selection and preparation of scaffold materials become hot points for research, and the ideal scaffold materials for bone tissue engineering should satisfy the following points: 1. the biocompatibility is high: besides meeting the general requirements of biological materials such as no toxicity, no aberration and the like, the degradation product also has the advantages of no toxic action on cells, no inflammatory reaction, contribution to adhesion and proliferation of seed cells and promotion on growth and differentiation of the cells; 2. the biodegradability is suitable for: the scaffold material has a degradation function, and the growth rate of bone tissue cells is adapted to the degradation rate; 3. has a suitable three-dimensional porous structure: the scaffold material has a three-dimensional structure, corresponding porosity and high specific surface area, can provide an optimal microenvironment for adhesion, proliferation, growth and function exertion of osteoblasts, and can also provide a space and a scaffold for formation of new bone tissues; 4. plasticity and suitable mechanical strength: the scaffold material has good plasticity and proper mechanical strength, and can support the new tissue until the new tissue has proper mechanical properties; 5. good scaffold-cell interface: the material should provide a good scaffold-cell interface, facilitating cell adhesion. The bioceramic is a crystalline material, and is formed by ionic bonding of metal ions and non-metal ions. According to the chemical activity of the biological ceramics in physiological environment, the biological ceramics can be divided into four types: inert bioceramics, surface active bioceramics, absorbable bioceramics and composite bioceramics. Calcium sulfate ceramics, calcium carbonate ceramics, calcium phosphate ceramics and isomers thereof belong to degradable biological ceramics. Calcium sulfate has no osteoinductive effect, and is less applicable because of its high brittleness. Cheroff extracts calcium carbonate ceramic from coral and applies to the restoration of bone defect models of thighbone and shinbone of dogs. One of the bone substitute materials widely used at present is calcium phosphate ceramics represented by hydroxyapatite, and the bone conduction performance of the calcium phosphate ceramics is very good. The application of hydroxyapatite in load bearing parts is limited by the defects of high brittleness, difficult absorption and the like. Bio-derived bone is one such class of biomaterial. After the bone tissue of human or animal is treated by a series of methods, its cell component and antigenicity are removed, and the original tissue net frame structure is completely or partially preserved. Products of biologically derived bone materials have been internationally applied to clinical applications such as Oswestry bones, bio-Os, kiel bones and the like, and have achieved good clinical effects. Li Yanlin and the like, three materials of partially decalcified bone, completely deproteinized bone and partially deproteinized bone are prepared by using pig ribs, and derived bone properties of the three materials are measured. The observation by an electron microscope shows that the reticular pore structure system with the original bone tissue is the commonality of the three materials, but the three materials have the characteristics that: the antigenicity of the completely deproteinized bone (namely calcined bone) is the minimum of three, the completely deproteinized bone has good osteogenic effect, can be clinically applied and can obtain certain curative effect, but the mechanical strength is poor and needs to be improved; the partially deproteinized bone has weak antigenicity and enough mechanical strength, but the osteogenesis effect is not ideal; the decalcified bone antigenicity is the strongest of the three, affects osteogenesis and basically cannot be applied independently. Chitin widely existing in shells of animals such as shrimps, crabs and insects is chitosan after deacetylation, and in natural organic compounds, the amount of the chitin is second to that of cellulose in nature. The chitosan is a novel natural medical biomaterial, has no toxicity and good biocompatibility, and the product of the chitosan after decomposition in vivo can be directly metabolized and is harmless to human bodies. The chitosan can guide or promote the formation of bone and has certain osteoconductivity. Chitosan is non-antigenic and can induce cell proliferation and can promote integration of the implant with host tissues. Klokkevold et al have studied the influence of chitosan on osteoblast differentiation and bone formation in vitro, and the experimental results show that chitosan can promote the differentiation of early osteoblasts and accelerate the bone formation. The percentage of the number of deacetylated links to all the number of links is the degree of deacetylation of chitosan. The deacetylation degree of chitosan affects the solubility of chitosan and directly determines the content of amino groups (NH 2) on its molecular chain, because chitosan can obtain the best solubility when its deacetylation degree is 50%. The pH of chitosan is between 6.5 and 7.3 depending on the degree of deacetylation, and the chitosan solution behaves as a weak polycationic electrolyte after protonation of the amino groups in the chitosan. The amino and hydroxyl groups on the chitosan are reactive groups, so that the chitosan can have new functions through chemical modification, such as acetification on the molecular structure of the chitosan to increase the solubility of the chitosan. Chitosan also has the property of being bacteriostatic, which inhibits the growth and reduces the activity of bacteria. However, it should be noted that the antibacterial effect is influenced by the species and molecular weight of chitosan, and by some conditions such as concentration. The biocompatibility of chitosan, whatever the biomaterial, must first be assessed. Vandervord et al, assessed by ectopic osteogenesis, first cut a porous scaffold material prepared by freeze-dried CS solution method into 1.5cm square blocks, sterilized, washed in PBS, and implanted into the back and abdomen of mice. Inflammatory responses, histological evaluation and cellular immunological responses were observed at certain time points, respectively. Finally, the stent material is found not to have inflammatory reaction in vivo; in HE staining, neutrophils were found to accumulate around the scaffold material, but gradually disappeared as the implantation time extended, and collagen was found to be produced in the scaffold pores, indicating a very low cellular immune response of the scaffold material at the initial implantation, or a surgically induced stress response. These results indicate that the CS scaffold has high biocompatibility, and can be applied as a scaffold material and an implant material in terms of biocompatibility. CS scaffolds with a certain structure are prepared by an electrospinning method, chondrocytes intercepted from joint parts of New Zealand white rabbits are planted on the CS scaffolds for co-culture, and finally, the CS scaffolds can be easily and tightly combined with the chondrocytes. There are many controversies about how chitosan is degraded in vivo, but more scholars believe that chitosan is degraded mainly by enzymes in the in vivo environment if CS is degraded more slowly in neutral aqueous media. The chitosan can be easily degraded by enzymes such as lysozyme and the like in vivo, and the final product glucosamine degraded in vivo can be completely absorbed by human bodies without toxicity. The chitosan can affect the cells of the body by adhering, activating, promoting, inhibiting and the like. Cell adhesion of chitosan has been reported in the literature to be more abundant, with osteoblast and fibroblast adhesion being the predominant ones. The chitosan and the derivatives thereof can inhibit the growth of microorganisms, stop bleeding, relieve pain, promote or inhibit the proliferation, migration, growth, activation and chemotaxis of fibroblasts, induce ordered collagen deposition and fiber arrangement, are beneficial to the activity of remodeling and construction of the structure of a new tissue and the like, and determine that the chitosan and the derivatives thereof have important application in tissue engineering. The chitosan can affect the cells of the body by adhesion, activation, promotion, inhibition and the like. Cell adhesion of chitosan has been reported in the literature to be more abundant, with osteoblast and fibroblast adhesion being the predominant ones. The development of tissue engineering puts new demands on biomaterials. It is the goal of the pursuit to develop biomaterials with the ability to produce the desired host response, starting from protein adsorption, immune responses, cytokine and growth factor release, target cell responses that occur on the surface of the material, inducing the desired healing pathway, and allowing tissue remodeling. The tissue engineering scaffold with high cell affinity is a main development direction of biological materials in the future.

Chitosan is formed by deacetylation of chitin in crustaceans (such as insects, shrimps, crabs and the like), is widely present in nature, and is inferior to cellulose in number in natural organic compounds. Researches find that the chitosan is a nontoxic and non-antigenic substance, has good biocompatibility, can induce the cell proliferation of organisms and promote the integration of an implant and tissues and organs, and is used in the fields of medicine, health care and the likeThe product after in vivo decomposition can be directly metabolized and is harmless to human body ^[1] . Therefore, the material can be used as a novel natural medical biomaterial for clinical application. In the aspect of bone tissue research, the chitosan can promote the differentiation of prophase osteocytes and accelerate bone formation, and has certain osteoconductivity ^[2] . In addition, chitosan can inhibit the growth of bacteria and reduce the activity of the bacteria, but different conditions, such as different types, different molecular weights and different concentrations of chitosan, have influence on the antibacterial effect.

Chitosan can affect body cells through adhesion, activation, promotion, inhibition, etc. In terms of adhesion, numerous studies have found that it has a more advanced adhesion effect on osteoblasts and fibroblasts. The chitosan and the derivatives thereof can inhibit the growth of microorganisms, stop bleeding, relieve pain, promote or inhibit the proliferation, migration, growth, activation and chemotaxis of fibroblasts, induce ordered collagen deposition and fiber arrangement, and are beneficial to the activities of structural remodeling and construction of new tissues, so the chitosan and the derivatives thereof are widely applied to tissue engineering.

The degree of deacetylation of chitosan is expressed as the percentage of the number of deacetylated links to the number of all links, which determines the solubility and the acidity or alkalinity of chitosan: when the deacetylation degree is 50%, the chitosan has the best solubility; the pH of chitosan is generally between 6.5 and 7.3. The amino and hydroxyl on the chitosan are reactive groups, and can be chemically modified to have new functions. The chitosan molecular structure is acetified to increase the solubility; when the amino group is protonated, the chitosan dissociates into polycations, and the solubility changes correspondingly.

The degradation of chitosan in organisms is still controversial at present, most scholars think that the chitosan is mainly degraded by enzymes in the organisms, although CS is slowly degraded in a neutral aqueous medium, the chitosan can be easily degraded by enzymes such as lysozyme in the organisms, and a final metabolite glucosamine can be completely absorbed by the human bodies without toxic and side effects.

Chitosan is used clinically, and its biocompatibility needs to be evaluated first. Vandervord ^[6] The evaluation is carried out by an ectopic osteogenesis method, the porous scaffold material prepared by a freeze-dried CS solution method is prepared into cube small blocks, the cube small blocks are implanted into the back and the abdomen of the mouse after being sterilized, and the scaffold material is found not to generate inflammatory reaction in vivo; collagen is generated in the pores of the scaffold around the scaffold material. Then, a CS bracket with a certain structure is prepared by an electrospinning method, chondrocytes intercepted from the joint part of a New Zealand white rabbit are planted on the CS bracket for co-culture, and finally, the bracket can be easily and tightly combined with the chondrocytes. These all indicate that the CS scaffold has high biocompatibility.

In the aspect of bone tissue engineering research in clinical at present, the bone marrow stromal stem cells are concerned about due to the advantages of convenient sources, simple material selection, small damage to patients and the like, but still in the research and exploration stage. A large number of researches prove that BMSCs can effectively form tissue engineering bones in bodies of different species of animals such as nude mice, mice and goats by taking BMSCs as seed cells, and the bone defects of all parts can be successfully repaired. Furthermore, studies have shown that adult BMSCs are capable of expressing osteoblast morphology and function under appropriate induction conditions. Stem cells have self-renewal and multipotential differentiation ability and can be used as seed cells, but how to induce differentiation and further exert biological effects is a key issue. Therefore, the problems to be solved are: how to obtain BMSCs in vitro, according to which method to induce and differentiate BMSCs, and establish a standardized mode for clinical work.

Chitosan (CS) has excellent biocompatibility, low toxicity and biodegradability, and is a newly developed biomedical material. In its polysaccharide side chain, it contains numerous amino and hydroxyl groups, which can be a convenient chain for surface modification. Therefore, it has potential applications in various tissue engineering fields. Furthermore, hydroxyapatite (HA) HAs been widely used in orthopaedics as a common biomaterial with similar bone mineral chemical composition to facilitate bone growth and osseointegration. Recently, it is considered that a single material cannot satisfy the requirements of the scaffold for bone tissue engineering, and as a result, the concept of a composite material has been introduced in order to enhance the excellent properties of a single material. In the literature, there are numerous studies on the synthesis of CS/HA complexes by wet chemical methods, such as S-sol-gel, co-precipitation, in situ synthesis and electrochemical deposition, and this HAs demonstrated that CS/HA complexes can accelerate osseointegration.

It is considered to be a crucial scaffold for promoting cell adhesion, e.g. cell differentiation and proliferation can only occur on cells adhering to a biocompatible surface. Therefore, there is a need to further improve scaffold-cell interactions. In this regard, modification of the surface of scaffold materials using signaling molecules, such as arginine-glycine-aspartic acid (RGD) peptide and cell growth factors, has been claimed to enhance the ability of cells to adhere. However, some researchers have also reported that RGD peptides do not always enhance cell attachment and inhibit overall bone formation to some extent. In order to deeply understand the influence of RGD polypeptide surface modification on a CS/HA complex and the application potential thereof in bone tissue engineering, in the invention, an RGD modified CS/HA (RGD-CS/HA) complex is prepared, and the interaction characteristics of material-cells and the osseointegration capability of an RGD-CS/HA scaffold are analyzed.

Any biological material applied to clinic must be surface modified, so that the biological material has good biocompatibility and cell affinity. The biocompatibility of the material includes two principles: (1) the principle of "biological safety" is to eliminate the toxic and side effects (such as cytotoxicity and carcinogenicity) of biological materials on individual organs. (2) The "biofunctionality" principle, i.e., the ability to "provoke the host to respond properly" in a particular application. With the development of the tissue engineering concept, a biocompatible material which has a certain number of viable cells and is suitable for cell growth is required, and the safety and functionality of the material are comprehensively evaluated according to the requirements.

Current surface modification methods include: surface modification, chemical modification, plasma method, hybrid modification, etc., wherein the surface modification is most commonly used to increase the biocompatibility of the material by fixing certain proteins and active molecules on the surface of the material or by changing the local structural characteristics of the material ^[27-30] There are various methods such as physical coating, physical entrapment, chemical knots, etc. ImmobilizerThe simplest method of bioactive factors is physical adsorption, which includes electrostatic adsorption and intermolecular adsorption, but bioactive molecules cannot act on the surface of the material for a long time during physical adsorption and are easy to separate. The chemical bonding method can make up for the above disadvantages due to its high stability. The chemical fixing method requires that the material surface has reactive groups such as hydroxyl, shuttle, amino and the like.

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells, have a strong dividing ability, and can differentiate and develop into many cell lines having specific functions. When bone tissues in vivo are stimulated by wound or local osteogenesis 'microenvironment' changes to promote signal transduction changes, bone marrow mesenchymal stem cells proliferate and finally differentiate into osteoblasts to participate in tissue regeneration and wound repair. Because the mesenchymal stem cells have the potential of differentiating into tissues such as bones, cartilages, fat, muscles, tendons and the like, the mesenchymal stem cells can be better applied to the research of tissue engineering, and have the following advantages: (1) The mesenchymal stem cells can be obtained by bone marrow puncture, so the material is convenient to obtain and the organism damage is small. (2) Mesenchymal stem cells can be obtained from autologous bone marrow, are non-immunogenic, and induce immune rejection of the resulting tissue after transplantation. (3) Mesenchymal stem cells can differentiate into multiple tissue types and can be used to treat a variety of traumatic diseases. In addition, researches find that the MSCs also have the characteristics of strong in vitro culture and proliferation capacity, capability of stably expressing osteoblast phenotype, feasibility of autologous transplantation for continuous osteogenesis, no tumorigenicity and the like, and are ideal bone tissue engineering seed cells. However, the content of MSCs in bone marrow is very small, accounting for about 1/10 ten thousand of nucleated cells in bone marrow, and a large amount of MSCs is required for clinical bone defect repair, so that how to perform primary cell subculture under ex vivo conditions causes a significant problem of proliferation of bone marrow MSCs. The current method is that Percoll separating fluid is separated after density gradient centrifugation, but the operation is relatively complex and takes a long time, and a large amount of cells are easily lost due to careless operation.

Good biocompatibility is required for the scaffold for tissue engineering or the bone graft material, which affects physiological characteristics such as proliferation and differentiation of seed cells on the scaffold material, and the characteristics are achieved by adhesion of the seed cells and the scaffold material. In vivo, adhesion factors mediate this process. Arginine-glycine-aspartic acid (RGD) is a minimal recognition short peptide sequence of many extracellular matrix proteins (e.g. collagen), and can specifically bind to integrin receptors on the surface of cell membranes to form adhesive spots, which can also mediate the transmission of extracellular growth signals to the inside of cells, influence the growth and differentiation of the cells, and promote cell adhesion. Ho et al measured the adhesion rate for 1-4 weeks using murine osteosarcoma cells and found that the adhesion rate of CS-RGD scaffolds was significantly higher than that of the CS scaffolds alone.

In summary, the problems of the prior art are as follows: at present, most of researches on bone tissue engineering scaffolds need to be carried out through long-time in-vitro cell and scaffold culture, so that the treatment time is prolonged, the treatment cost is increased, and the pain of patients is increased.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a composite scaffold with improved gaps and pores for promoting cell adhesion rate and a preparation method thereof. The application promotes the adhesion rate of cells on the stent and the growth of the cells by improving the rules of the gaps and the pores of the composite stent, and can enable blood vessels in tissues to grow into the stent rapidly along the pores in the stent.

The composite scaffold with the improved gaps and the improved pores for promoting the cell adhesion rate is characterized in that the channel pores and the spherical pores of the composite scaffold with the improved gaps and the improved pores for promoting the cell adhesion rate are respectively 150-650 mu m and 3-15 mu m; and (3) chitosan: the mass ratio of the hydroxyapatite is 7:3.

another object of the present invention is to provide a method for preparing a composite scaffold with improved void and pore cell adhesion promoting rate, comprising the following steps:

obtaining CS/HA scaffolds with pores and spherical pores by in situ hybridization and freeze-drying;

the CS and the RGD peptide are connected through hydrogen bonds, the RGD polypeptide is modified on the surface of a CS/HA bracket pore channel through an electrostatic self-assembly method, and the three-dimensional porous bracket containing the RGD peptide and having CS/HA high-efficiency adhesion cells is prepared.

Further, the influence of the composite scaffold with the improved gap and pore cell adhesion promoting rate on the short-term adhesion rate of the MSCs cells and the adhesion microscopic morphology of the cells and the composite scaffold with the improved gap and pore cell adhesion promoting rate on the in-vitro composite cell culture of the MSCs is evaluated by improving the gap and pore cell adhesion promoting rate of the composite scaffold.

Further, after the RGD polypeptide is modified on the surface of the CS/HA bracket pore channel,

cell growth on RGD/CS/HA material was quantitatively detected by PicoGreen cell propagation.

Further, the preparation of the CS/HA scaffold by the in situ hybridization method comprises the following steps:

adding a certain weight of Ca (NO) ₃ ) ₂ ·4H ₂ O and KH ₂ PO ₄ Adding the mixture into an acetic acid solution with the volume ratio of 2% for mixing; then stirring for 3 hours by using a magnetic stirrer until the deposited calcium salt is completely dissolved to prepare HA precursor solution; adjusting the pH value of the liquid to 4.0;

adding CS powder into the solution, stirring for 4 hr at room temperature with a magnetic stirrer to obtain a CS solution with a mass ratio of 4%;

then injecting 2ml of CS solution into a mould to form a layer of solidified CS film, then injecting 10ml of the settled chitosan and hydroxyapatite precursor solution into the inner surface of the mould to cast and mould, standing for 12h at room temperature, and then soaking the mould in 5wt% of sodium hydroxide solution to form hydrogel; washing the hydrogel rod with deionized water until the pH value is 7, and then placing the hydrogel rod in a 60 ℃ oven to be air-dried for 8-10h; stopping drying when the diameter of the gel rod gradually becomes 10mm, and freeze-drying the gel rod in a freeze dryer to obtain a rod-shaped scaffold material; sterilizing at high temperature and high pressure for later use.

Further, CS and RGD peptide are connected through hydrogen bonds, RGD polypeptide is modified on the surface of a CS/HA bracket pore channel through an electrostatic self-assembly method, and the three-dimensional porous bracket of CS/HA high-efficiency adhesion cells containing the RGD peptide is prepared, and specifically comprises the following steps:

different doses of RGD were dissolved in PBS to make up different concentration reagents: from 10mg/L to 100mg/L, in 10mg/L increments, with spectrophotometric measurements;

and (3) drawing a standard curve Ci of the RGD concentration by using the absorbance, wherein the correlation between the Ci and the absorbance is shown as the formula (1): absorbance =0.002 × Ci +0.0036 (R2 =0.992, which is a slope and is obtained by measurement) (1);

after being disinfected, the CS/HA scaffold is respectively placed in RGD solutions with different concentrations of 50mg/L and 100mg/L for soaking for 24 hours, the CS/HA scaffold containing RGD peptide is prepared, washed for 3 times by PBS to remove free molecules, and finally dried in the air; recovering the flushing liquid into the original RGD solution, and diluting to the required concentration; and measuring the absorbance again, and calculating the attachment amount of RGD in the stent, wherein the formula (2):

the spectrophotometric values of RGD in the recovered solution were 0.0694 and 0.0325, C0=100mg/L, and Ci100 and Ci50 were 32.9 and 14.4mg/L, respectively.

Further, a method for determining the short-term adhesion rate of MSCs cells by a composite scaffold with improved void and pore cell adhesion promoting rate comprises:

each well contains a CS/HA or CS/HA-RGD disk, and 1X 10 of the magnetic particles are added into the well ⁶ ml volume of cells; diluting the wells by adding 1ml volume of 10% PBS; the composite scaffold with the improved gaps and pores for adhering cells to promote the cell adhesion rate is placed in a culture medium for 4 hours, and is washed for 3 times by PBS (phosphate buffer solution) to remove the cells which are not adhered; the formula for calculating the cell adhesion rate is shown in formula (3):

the invention has the advantages and positive effects that:

the invention promotes the adhesion rate of cells on the scaffold by improving the rules of the gaps and the pores of the composite scaffold to be expected to be improved by two hundred percent, and stem cells growing in the composite scaffold by utilizing special pores are more easily differentiated into similar bone tissues, the differentiation rate is expected to be higher than that of normal culture by 50 percent, and blood vessels in the tissues can rapidly grow along the pores in the scaffold.

The invention produces a composite bracket which can adhere cells with higher quantity and quality in a short time and is constructed by a cell-carrier, thereby achieving the purpose of quickly treating bone defects and meeting the clinical requirement.

The scaffold has no obvious toxicity to cells and has better cell compatibility. Through compounding with RGD, the three-dimensional porous CS/HA scaffold HAs obviously enhanced effects on attraction, induction and growth regulation of cells.

In the invention, the chitosan molecule of the scaffold contains hydroxyl and amino groups, thereby meeting the requirement of chemical bonding. Therefore, the invention combines physical adsorption and chemical fixation methods to make the material have better adsorbability and stability. The RGD polypeptide scaffold loading rate is found to be high, and through tests, the release amount of RGD in 24h and 48h is found to be low, which indicates that the RGD polypeptide has a stable scaffold adsorption function, and the simple physical adsorption is not enough to maintain high adsorption amount and stable adsorption force, so that the complex chemical adsorption exists in addition to the physical adsorption (electrostatic adsorption and intermolecular action force) in the process of loading the scaffold and the RGD polypeptide, and the process is dominant.

The present invention adopts in vitro composite cell culture method to avoid the interference of various in vivo body fluid components. The invention adopts GENMED PicoGreen cell propagation quantitative detection, and can accurately detect trace cells and slow down mitotic cell propagation by an immunofluorescence method. Therefore, the technology is suitable for primary cells of various animal or human bodies, cells in extracellular matrixes and cells growing very slowly and in a trace amount. In addition, different from the method for measuring cell growth through cell metabolism, the PicoGreen fluorescent dye can be specifically combined with double-stranded DNA, the technology can detect the change of 0.5 nanogram double-stranded DNA or 100 cell magnitudes, the fluorescence signal intensity or the obvious increase of a relative fluorescence unit can be caused by the micro increase of the cell number, the repeatability standard difference is low, the interference of chemical components of a sample is avoided, and the method has the advantages of simplicity, convenience, high sensitivity, small cytotoxicity damage and the like.

The present invention adopts whole bone marrow method to separate and culture, uses 10% of fetal bovine serum MEM as culture solution, and separates MSC by replacing culture solution. The cells isolated by this method have the following characteristics: (1) the morphology of MSC is characterized by small cell volume, large nucleus, fine and dispersed chromatin, obvious nucleolus and the like. (2) The cells proliferate vigorously. The cell attachment time was found to be 2-3 days shorter than that of density gradient centrifugation during the culture, probably because the whole bone marrow method preserved abundant adhesion molecules and various growth factors in the bone marrow environment. In addition, compared with primary cells, the proliferation speed of the MSCs after passage is higher, the wall adhesion time is shortened, the growth latency period is shorter, and the bottom surface of the culture bottle can be fully paved in about 7-9 days per generation. Therefore, we believe that the improved osteoblast bone marrow culture method not only improves the isolation rate and proliferation capacity of MSCs, but also shortens the isolation time, reduces the chance of contamination, and improves the working efficiency.

In the present invention, the third-generation MSCs were cultured together with the two scaffolds for 4 hours and the cell adhesion rate was measured, and the CS/HA-RGD 4-hour adhesion rate was approximately one-fold higher than that of the CS/HA group (80.7% vs 54.7%). Furthermore, the 4 hour adhesion on CS/HA scaffolds was 80.7%, significantly higher than 10% of the first week adhesion reported by Ho et al (according to which the reported scaffold volume was 0.05 cm) ³ Cell adhesion density was calculated to be 5 × 103cells/cm 3), indicating that the specific pore structure of the scaffold enhances cell adhesion efficiency, and the loading of RGD polypeptide increases the adhesion of MSCs.

In order to ensure that a local environment enriched with a large number of cells is formed around the implant, the proliferation activity must be expressed as soon as possible after the cells are attached and spread on the surface of the material. According to the invention, the cell growth curve on the RGD/CS/HA material is always higher than that of a CS/HA bracket through the quantitative detection of PicoGreen cell propagation, the existence of granular bone not only improves the adhesion rate of MSCs, but also ensures that bone cells which survive per se do not exist in a small number and exist for a long time, and can be used as seed cells to participate in osteogenesis. In the later stage of in vitro culture, the proportion of living cells is increased compared with that of the pure scaffold group from the overall view of the composite material. In the invention, MSCs are directly inoculated to the RGD/CS/HA composite material and the CS/HA scaffold for in vitro culture, the cells are well attached to the surface of the material by electron microscope observation, the cells are changed from a circle to a polygon along with the prolonging of the culture time, extend out of pseudopodia, are mutually connected, secrete matrix and cover the surface of the material, the condition is good, and the scaffold HAs no obvious toxicity to the cells and HAs better cell compatibility. Through compounding with RGD, the three-dimensional porous CS/HA scaffold HAs obviously enhanced effects on attraction, induction and growth regulation of cells.

Drawings

FIG. 1 is a flow chart of a method for preparing a composite scaffold with improved void and pore cell adhesion promotion rates according to embodiments of the present invention.

Fig. 2 is a mechanical property analysis diagram of a stent provided by an embodiment of the present invention.

FIG. 3 is an electron-scanning and pore analysis diagram of a CS/HA cross section of a scaffold material provided by an embodiment of the present invention.

In the figure: a. the width of the CS/HA scaffold channel hole is 400 mu m; b. interconnecting the unified graphs; c. the width of the CS/HA stent channel hole is 400 mu m; d. the unified map is interconnected.

Fig. 4 is a standard graph of an RGD polypeptide-loaded CS/HA scaffold provided by an embodiment of the present invention.

FIG. 5 is a 24h scanning electron microscope image of composite material planted MSCs provided by the embodiment of the invention.

In the figure: a. cells were attached to the RGD modified CS/HA scaffold surface; b. fewer cells and cellular pseudopodia attach to the surface.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The chitosan and the derivatives thereof can inhibit the growth of microorganisms, stop bleeding, relieve pain, promote or inhibit the proliferation, migration, growth, activation and chemotaxis of fibroblasts, induce ordered collagen deposition and fiber arrangement, are beneficial to the activity of remodeling and construction of the structure of a new tissue and the like, and determine that the chitosan and the derivatives thereof have important application in tissue engineering. The development of tissue engineering puts new demands on biomaterials. It is the goal of our pursuit to develop biomaterials with the ability to produce the desired host response, starting from protein adsorption, immune responses, cytokine and growth factor release, target cell responses that occur on the surface of the material, inducing the desired healing pathway, and allowing tissue remodeling. The tissue engineering scaffold with high cell affinity is a main development direction of biological materials in the future.

The invention discloses a three-dimensional porous scaffold for efficiently adhering cells, which is prepared by preparing a chitosan/nano-hydroxyapatite composite porous material with double pores by using an in-situ hybridization method and modifying RGD polypeptide on the surface of the material pores by using an electrostatic self-assembly method. And the influence of the scaffold on the short-term adhesion rate of the MSCs cells and the adhesion microscopic morphology of the cells and the scaffold is evaluated by the composite culture of the scaffold and the MSCs. Therefore, the artificial bone biomaterial is designed and manufactured to meet the clinical application.

In the initial stage of cell seeding, the attachment of a large number of cells to the scaffold is essential for bone tissue repair engineering. In order to achieve a high adhesion rate of the scaffold cells within a few hours, the chitosan/hydroxyapatite bioskeleton with channels/pores obtained by the in situ hybridization technique combined with lyophilization is generated by transportation, which uniformly disperses the hydroxyapatite in the chitosan. The sizes of chitosan/hydroxyapatite (CS/HA) channel pores and spherical pores are 150-650 μm and 3-15 μm respectively. The compressive strength and the porosity are respectively 3.54 +/-0.32 MPa and 88.4 percent. The nitrogen content was increased by 7.5% compared to CS/HA scaffolds without the arginine-glycine-aspartic acid (RGD) component. In a PBS solution, more than 67 percent of RGD is dissolved in a CS/HA scaffold by self, and the effect of RGD in the CS/HA scaffold on bone marrow Mesenchymal Stem Cells (MSCs) is further analyzed by measuring the cell adhesion rate, the alkaline phosphatase (ALP) activity and mineralized calcium nodules. The cell adhesion rates of CS/HA scaffolds with different RGD concentrations (50,100mg/L) were (71.6 + -8.5)% and (80.7 + -9.7)%, respectively. After 4 hours of culture, the p is obviously increased by less than 0.05 compared with the simple CS/HA scaffold group (54.7 +/-6.4). The expression level of alkaline phosphatase (ALP) in CS/HA scaffolds containing RGD peptide was 107.7% of that in CS/HA scaffolds alone (191. + -. 6U/g protein vs 92. + -. 9U/g protein, p < 0.05). In addition, the CS/HA scaffold containing RGD peptide HAs higher content of mineralized calcium nodules (the CS/HA scaffold shows reddish brown color) than the pure CS/HA scaffold group. The CS/HA scaffold containing RGD peptide can not only promote high adhesion of cells in a short time, but also increase the adhesion capacity of cells and promote differentiation of mesenchymal stem cells into osteoblasts.

The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.

1. The composite scaffold with the improved gaps and the improved pores for promoting the cell adhesion rate is provided by the embodiment of the invention, and the channel pores and the spherical pores of the composite scaffold with the improved gaps and the improved pores for promoting the cell adhesion rate are respectively 150-650 μm and 3-15 μm. And (3) chitosan: the mass ratio of the hydroxyapatite is 7:3.

as shown in fig. 1, the embodiment of the present invention provides a method for preparing a composite scaffold with improved void and pore cell adhesion promoting rate, comprising the following steps:

s101: obtaining CS/HA scaffolds with pores and spherical pores by in situ hybridization and freeze-drying;

s102: the RGD peptide is modified on the surface of a CS/HA bracket pore channel by an electrostatic self-assembly method through connecting CS and RGD peptide by hydrogen bonds to prepare the three-dimensional porous bracket of CS/HA high-efficiency adhesion cells containing the RGD peptide.

The application of the principles of the present invention will now be described in further detail with reference to specific embodiments.

The composite scaffold with improved gaps and pores for promoting the cell adhesion rate provided by the embodiment of the invention comprises:

2. materials and methods

2.1 materials

2.2 preparation of CS/HA scaffolds by in situ hybridization

Adding a certain weight of Ca (NO) ₃ ) ₂ ·4H ₂ O and KH ₂ PO ₄ Adding the mixture into an acetic acid solution with the volume ratio of 2%, mixing, and stirring for 3 hours by using a magnetic stirrer until the deposited calcium salt is completely dissolved to prepare an HA precursor solution. At this point the pH of the liquid was adjusted to 4.0. Then, the CS powder was added to the solution, stirred at room temperature for 4 hours using a magnetic stirrer to obtain a CS solution with a mass ratio of 4%, and then transferred to a clean beaker, left to stand for 24 hours for deaeration, to form a precursor solution of a gel bar with CS uniformly aligned with HA. And then 2ml of CS solution is injected into a mould to form a layer of solidified CS film, then the standing chitosan and hydroxyapatite precursor solution (10 ml) are injected into the inner surface of the mould to cast and mould, and after standing for 12h at room temperature, the mould is immersed in 5wt% of sodium hydroxide solution to form hydrogel. The hydrogel stick is washed with deionized water until the pH value is about 7, and then the hydrogel stick is placed in an oven at 60 ℃ for air drying for 8-10h. The gel stick gradually becomes thinner and smaller, and the drying is stopped when the diameter of the gel stick is 10mm, and the gel stick is put into a freeze dryer for freeze drying to obtain the rod-shaped scaffold material. According to the specification, the disc-shaped porous bracket is made into the disc-shaped porous bracket with the diameter of 10mm and the thickness of 1.5 mm. Sterilizing at high temperature and high pressure for later use.

2.3 uptake of RDG and composition of CS/HA scaffolds

Different doses of RGD were dissolved in PBS to make up different concentration reagents: from 10mg/L to 100mg/L, in 10mg/L increments, spectrophotometry was measured with a spectrophotometer (Smart SpecTM 3000, bioRad, USA). And (3) drawing a standard curve (Ci) of the RGD concentration by using the absorbance, wherein the correlation between the Ci and the absorbance is shown in the formula (1): the absorbance =0.002 × Ci +0.0036 (R2 = 0.992) (1).

And after the CS/HA scaffold is disinfected, respectively placing the CS/HA scaffold in RGD solutions (3 ml) with different concentrations of 50mg/L and 100mg/L for soaking for 24 hours, completing the preparation of the CS/HA scaffold containing RGD peptide, washing the CS/HA scaffold for 3 times by using PBS to remove free molecules, and finally air-drying the CS/HA scaffold. The rinse was recovered in the original RGD solution and diluted to 3ml in total volume. And measuring the absorbance again, and calculating the attachment amount of RGD in the stent, wherein the formula (2):

the spectrophotometric values of RGD in the recovered solution were 0.0694 and 0.0325, C0=100mg/L, ci100 and Ci50 were 32.9 and 14.4mg/L, respectively, with a volume of 3ml and a scaffold weight equal to the CS/HA scaffold weight of 0.0254g.

2.4 proliferation and amplification of MSCs

Selecting male New Zealand rabbits with the weight of 300g to 400g, following the animal health care and use guidelines of national institutes of health, extracting animal bone marrow, killing the male rabbits with carbon dioxide, taking femurs and tibias, flushing with 1:1 equal volume of penicillin/streptomycin (Gibco) culture solution, cutting off epiphyses at the ends of the femurs and the tibias, extracting 10ml of flushing fluid by using a syringe, flushing and collecting bone marrow in the femurs and the tibias. The bone marrow cells were filtered using a 70 mm cell filter and then centrifuged for 8 minutes (300 g). The resulting cell pellet was resuspended in 10mL of DMEM, 10% fetal bovine serum was added, placed in a T-75 polystyrene flask to promote stem cell attachment, the culture medium was changed to remove unattached cells, and finally, bone marrow cell isolation was completed.

2.5 culture of bone marrow Stem cells in CS/HA and RGD-CS/HA scaffolds

The cell standard culture solution is composed of: DMEM was supplemented with 10% FBS and 1% penicillin/streptomycin. Bone culture medium composition: osteogenic supplements (10 mM Na-. Beta. -glycerophosphate, 50 mg/L-ascorbic acid and 10-8M dexamethasone, sigma) were added to the standard culture medium. Cell culture in 5% CO ₂ The temperature in the incubator was 37 ℃ and the culture medium was changed every 2 days. Cell passaging was performed with 0.05% trypsin-EDTA (Gibco), and the bone marrow stem cell-attached flasks were rinsed with 2ml of PBS. Incubation with 2ml Trypsin/EDTA at 37 deg.CIncubate for 2 min to release cells. Trypsin was neutralized with 4ml of 10% fbs-added DMEM solution, the cell lysate was placed in a 50ml centrifuge tube, 100ul of the reagent was taken out, and the cell number was counted using a hemocytometer. The cells were centrifuged at 300g for 8 minutes, the cell pellet was resuspended in DMEM osteogenic medium, and 10% FBS was added. Prior to cell culture, CS/HA and CS/HA-RGD scaffolds were placed in an autoclave (VP-P8037, changchun Baiao Bio-appaatus Company), sterilized at 121 ℃ for 30 minutes, and disks (disks) were rinsed with Dulbecco's PBS prior to cell experiments.

2.6 determination of initial cell adhesion Rate on scaffolds

Each well contained a CS/HA or CS/HA-RGD disk, and 1X 106ml volumes of cells were added to the wells. The wells were diluted by adding 1ml volume of 10% PBS. The cell-attached scaffolds were placed in culture medium for 4 hours and washed 3 times with PBS to remove non-attached cells. The cell attachment rate was calculated at 450nm using Multiskan Mk3 type (Finland thermal electric laboratory system). Measuring absorbance values of the initial cell solution (Ai) and the cell solution (Afree) which is not attached, and calculating the cell attachment rate according to the formula (3):

2.7 alkaline phosphatase Activity and staining

After 14 days, the culture medium was removed, CS/HA and CS/HA-RGD were transferred to a new well plate, 0.5ml of TritonX-100 (Sigma) was added to each well, bone marrow stem cells were scraped from the surface of a disk (disk) with a cell scraper, and the disk (disk) and 0.5ml of cell lysate were placed in a 1.5ml centrifuge tube. Sampling: the cell membrane was disrupted by 2 freeze-thaw cycles (-70 ℃ and room temperature, 45 min each) and the intracellular proteins were extracted. Alkaline phosphatase (ALP) activity in each disc was analyzed using an alkaline phosphatase detection kit (BioSino Biotechnology and Science inc.). ALP was determined using a histochemical staining method. After 14 days of cell growth in the scaffolds, the cells were detached by incubating in 2ml of trypsin/EDTA at 37 ℃ for 2 minutes, after which the cells were incubated in a 37 ℃ incubator containing 5% CO2 for 48 hours. Cells were stained with alkaline phosphatase staining solution (Nanjing Jianche Bioengineering Institute) and recorded by photography. Cells were evaluated by the quality and intensity of the intracellular pellet staining and were graded from 0 to 4.

2.8 morphological and statistical analysis of bone marrow stem cells in scaffolds as shown in FIG. 2.

After culturing bone marrow stem cells in CS/HA and CS/HA-RGD for 3 days, the cells were washed with normal saline, added with 1% paraformaldehyde, dehydrated with gradient ethanol, rinsed with PBS, sputter-coated with gold, and then observed with a scanning electron microscope (SEM, S-3400N). All experiments were repeated 3 times. The results are expressed as means ± sd, and P <0.01 is considered statistically different using one-way anova.

2.9 ectopic osteogenesis in rat

Fisher rats 18, 6 months old and 100-150g in weight are taken, and males are divided into two groups. The materials were divided into RGD/CS/HA group and CS/HA group. The 10% chloral hydrate solution is used for abdominal cavity anesthesia, back depilation, fixation, and iodine disinfection, then the single laying is carried out, 3 skin incisions with the length of 2cm are formed beside the back vertebra, subcutaneous tissues and fascia are separated, the 3 groups of materials are respectively implanted into the paravertebral muscle bags, the wounds are closed layer by layer, and the single breeding is carried out after 8 thousands of gentamicin is injected into the muscle.

The materials are respectively taken at 2.4.6 weeks, the taken materials are fixed in formaldehyde with volume fraction of 10%, EDTA is decalcified, dehydrated and transparent, and are continuously sliced after paraffin embedding, the thickness is 5 mu m, and the change condition of the materials is observed by light microscope observation (HE staining).

3. Results

3.1 mechanical and physical properties of CS/HA scaffold materials:

the stress and strain of the CS/HA scaffold material are shown in FIG. 2. The collapsed portion of the two-hole structure is indicated by the arrows. The stress thresholds at which the two-pore structure starts to collapse were 3.4 and 2.6MPa, indicating that the CS/HA scaffold HAs good toughness due to the fact that it HAs channel voids. The CS/HA stent HAs elasticity and mobility (flexibility) with a maximum bending (strain) range of 10% to 20%.

The CS/HA scaffold is obtained by freeze-drying, so that the water dispersion and the CS gel network play a role of a pore-foaming agent. The porosity was 88.4% by analysis of the CS/HA pore and dense structure. The pore structure and particle size distribution results are shown in FIG. 3. FIGS. 3a and 3c illustrate the CS/HA scaffold channel pore width of 400 μm, uniform interconnection (FIGS. 3b,3 d). The highly porous scaffold has an open structure, can promote better bone oxygenation and angiogenesis, and therefore can be used as a good substitute for bone. The major pore connections range from 3 μm to 15 μm and from 150 μm to 650 μm in diameter, and theoretically, such a structure can satisfy cell migration and tissue attachment between pores. The diameter of the channel hole is about 150-650 μm, which can provide framework for bone growth in the hole, thereby meeting the requirements of nutrition supply and waste removal required by cell growth in the bracket. Spherical pores smaller than 10 μm can satisfy the requirements of capillary growth and cell matrix interaction.

3.2 absorption and composition of RGD in CS/HA scaffolds

By means of physical absorption, CS provides the major amino uptake for RGD. XPS can analyze the content of nitrogen in the CS/HA scaffold, and after the CS/HA-RGD group is soaked in an RGD PBS solution for 24 hours, the molar percentage content of nitrogen in the CS/HA-RGD group is increased by 7.5 percent compared with that in the CS/HA group, which shows that a certain content of RGD is combined with CS in a hydrogen bond mode and attached to the CS/HA scaffold. In addition, the molar ratio of Ca/P of HA was 1.58 and the presence of some decalcified HA synthesis was determined by in situ hybridization.

As shown by the calibration curve (fig. 4), the concentration-dependent absorbance analysis of RGD with a concentration threshold was used in this experiment, and the content of RGD in PBS solution was determined by comparison with the calibration curve after CS/HA scaffold infiltration, as shown by the arrow in fig. 4. Due to the fact that CS and RGD are combined through hydrogen bonds, RGD can be attached to a CS/HA support in a physical adsorption mode, so that the physical structure of the CS/HA support is kept, and active peptide components are added to enhance the attaching and differentiating capacity of bone marrow stem cells.

3.3 initial bone marrow mesenchymal Stem cell adhesion of CS/HA scaffolds

The CS/HA scaffold improved by RGD HAs higher cell attachment rate. The 4-hour cell attachment rates of the improved CS/HA scaffolds with different RGD concentrations (50, 100mg/L) are respectively (71.6 +/-8.5)% and (80.7 +/-9.7)%, and are obviously improved (P is less than 0.05) compared with the cell attachment rate (54.7 +/-6.4)% of the CS/HA scaffolds.

It was found that both CS/HA scaffolds and RGD-modified CS/HA scaffolds promoted the attachment and spreading of bone marrow stem cells at 4 hours of stem cell attachment. The RGD modified CS/HA scaffold HAs a higher cell attachment rate, so that the modified scaffold HAs a higher cell density in the initial culture stage.

FIG. 5 illustrates the molecular imaging performance of CS/HA scaffolds and RGD-modified CS/HA scaffolds for cell attachment. FIG. 5a illustrates cell attachment to RGD-modified CS/HA scaffold surface; figure 5b illustrates less cells and cell pseudopodia attached to the surface. Pictures are 3 days after cells were seeded in the scaffolds. Cell attachment was determined by morphological manifestation of bone marrow stem cell attachment and spreading. When bone marrow stem cells adhere to the scaffold, the morphology becomes flatter. The cells climb along the channel holes, are attached to the hole walls, are dispersed in the RGD modified CS/HA scaffold in various pseudo-podded forms, and are fixedly planted in micro-pores with the pore diameter of 3-15 mu m. This shows that the pore structure of the channel with the diameter of 150-650 μm can promote the cell to climb in the bracket, and the spherical pore structure is favorable for the attachment of the cell pseudopodia. Without the RGD molecular structure, the cell adhesion is reduced, which is not favorable for the adhesion and fixation of the cell pseudopodia (FIG. 5 b).

3.4ALP Activity and staining

The rate of p-nitrophenol formation was determined to measure ALP activity following the ALP detection kit protocol. After the bone marrow stem cells are cultured in the bracket with the osteogenic filling for 2 weeks, the ALP activity expression in the RGD modified CS/HA bracket is obviously increased, the average is 191 +/-6U/gprotein, the ALP activity expression is increased by 107.7 percent compared with the CS/HA bracket (92 +/-9U/gprotein), and the P is less than 0.05. Increased ALP activity was associated with increased cell density, and also demonstrated that more bone marrow stem cells differentiated into osteoblasts than CS/HA. In addition, both the CS/HA scaffold and the RGD-modified CS/HA scaffold have good biocompatibility. Therefore, the RGD-modified CS/HA scaffold can better promote the differentiation of bone marrow stem cells to osteoblast lineages and HAs a high level of ALP as a bone marker.

Under appropriate culture conditions, a proportion of bone marrow cells can differentiate and develop into osteoblastic morphology, e.g., expressing ALP and calcification. After 2 weeks, different differentiated cells were subjected to ALP staining. Bone marrow osteoblasts at different stages were stained and the RGD modified CS/HA scaffold was graded as IV. Places with phosphatase activity are shown as dark red particles, medium to large numbers. RGD modified CS/HA was dark red indicating it had higher ALP. However, the CS/HA group stained less strongly, and the CS/HA staining was of grade I. The RGD-modified CS/HA HAs higher ALP content and osteogenic activity.

3.5 histological observation of ectopic bone formation

The scaffolding material remains after the decalcification treatment and is replaced with red-stained areas. Neogenetic tissue becomes the major visible component.

The tissue cells of the RGD group are tightly combined with the scaffold at 2 weeks, the gap between the tissue without the RGD group and the scaffold is large, and the RGD polypeptide has a certain adhesion effect on the tissue cells. Cells were seen to have grown into the scaffold at 4 weeks, and some of the scaffold began to degrade. And most of the scaffolds are degraded in 6 weeks, and the degradation degree of the RGD group scaffold is greater than that of the RGD-free group scaffold.

4. Discussion of the related Art

With the rapid development of bone tissue engineering, it has become an important method for repairing bone defects today. Recently, research interest has been directed towards the preparation of composite scaffolds and their osteointegrative capabilities. In our previous publications, CS/HA complexes, were successfully prepared by a combination of in situ synthesis and freeze-drying. Preliminary results indicate that the CS/HA composite is characterized by the formation of both slotted pores (average particle size: 400 microns) and spherical pores (average particle size: 7.8 microns) with a porosity of about 88.4%. Highly permeable porous scaffolds of open structure are considered to be the best bone substitutes because they promote bone oxygenation and angiogenesis. The slot-shaped pores may provide a framework for bone ingrowth into the pores, and such spherical pores are suitable for capillary ingrowth and extracellular matrix interaction. In the present invention, the CS/HA complex is modified in which an RGD peptide is used to further improve the cell affinity of the material. From the point of view of the interaction of RGD-CS/HA scaffold-bone marrow stromal cells (including cell adhesion rate, cell viability, cell morphology and ALP activity) and osteointegrative capacity (including ex-situ ossification in vivo and repair of bone defects in vivo), it is suggested that the application of RGD-CS/HA scaffold for bone tissue engineering would be a promising scaffold.

The initial cell attachment rate on the scaffold is critical for subsequent cell proliferation and differentiation, which affects the morphology of the artificial tissue. In this study, the rate of bone marrow stromal stem cell adhesion on RGD-CS/HA scaffolds was as high as 80.7% after 4 hours of culture. Although the adhesion rate decreases to 71.6% the lower the concentration of the CS solution, it is also much higher than the values reported in other documents. A study by Ho et al showed that after one week of culture, the rate of cell adhesion of rat osteosarcoma cells on RGD-modified CS scaffolds was only 10% ^[49] . The differences in seed cell usage may be the cause of the differences, but it is believed that the unique surface morphology of the RGD-CS/HA scaffold plays a more important role in the channel-shaped pores providing a viable pathway for bone marrow stromal cells to enter the interior of the scaffold. This has been well established by observation of cell viability assays, which confirm the presence of bone marrow stromal cells in the slotted pores of the scaffold. Furthermore, the morphology of bone marrow stromal stem cells from SEM images after 48 hours of culture showed many cell-cell interactions, and the ALP content of bone marrow stromal stem cells on the RGD-CS/HA scaffold after 14 days of culture was much higher than previously reported values. In addition, the in vivo ectopic ossification and in vivo bone defect repair experiments also show that the RGD-CS/HA scaffold HAs satisfactory osseointegration capability and similar biomechanical properties compared with the radius of a normal human. All these results support the good biocompatibility, cell compatibility and histocompatibility of the RGD-CS/HA scaffold organized in this study in the application potential in the field of bone tissue engineering.

Scaffold design and fabrication with excellent cell affinity is considered a key area of bone tissue engineering. Generally, the scaffold should carry a three-dimensional porous structure, which provides a pathway for nutrient supply and promotes cell adhesion and proliferation. In addition, surface treatments of scaffolds with specific extracellular matrices, such as fibronectin and collagen, can also enhance scaffold-cell interactions. It has been demonstrated in previous studies that RGD-modified scaffolds can improve cell adhesion, spreading and cytoskeletal organization. The rationale can be attributed to the formation of focal adhesions between RGD peptides and certain integrins on the cell membrane. In this study, the RGD peptide was uniformly immobilized on the CS/HA complex by physical adsorption, which is considered to be superior to the chemical grafting method reported in the literature.

With in vivo implantation, the RGD-CS/HA scaffold undergoes CS degradation, HA adsorption and osteointegration. Studies demonstrated that from in vivo ectopic ossification experiments, after implantation of RGD-CS/HA scaffolds and CS/HA scaffolds, the X-ray optical density values were lowest at week 4 and greatest at week 6. Assuming that the rate of CS degradation and HA adsorption is faster early after implantation (4 weeks) than osteointegration, a decreasing optical density value is produced. After 4 weeks, the osteointegrative process controls the degradation of CS and HA adsorption, and the optical density value rises again to a high value. Experiments on in vivo bone defect repair have shown that this RGD-CS/HA scaffold HAs optimal osteointegrative capacity, which can be attributed to the slot-shaped and spherical pores, which stimulate differentiation of bone marrow stromal cells into osteoblasts and chondrocytes and new bone tissue formation. In addition, a high calcium, phosphorus level environment is formed during HA adsorption, which enhances the osteointegrative capacity of differentiated osteoblasts.

5. Conclusion

The CS/HA scaffold with channel/spherical pores can be obtained by in-situ hybridization technology and freeze-drying method, wherein the channel pores and the spherical pores are respectively 150-650 μm and 3-15 μm; the compressive strength and the porosity of the material are respectively 3.54 +/-0.32 MPa and 88.4 percent, and the structure is similar to that of cancellous bone. CS/HA scaffold containing RGD peptide can be prepared by hydrogen bond connection of CS and RGD peptide and physical absorption method, i.e. the physical structure of the scaffold is maintained, and the attachment and differentiation of bone marrow stem cells are promoted due to the existence of bioactive peptide. SEM results show that the CS/HA scaffold containing RGD can improve the attachment of bone marrow stem cells, and the channel pore structure in the scaffold can promote cell migration; meanwhile, the spherical pores are favorable for cell attachment.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. The composite scaffold with the improved gap and pore cell adhesion promoting rate is characterized in that the channel pores and the spherical pores of the composite scaffold with the improved gap and pore cell adhesion promoting rate are respectively 150-650 μm and 3-15 μm; and (3) chitosan: the mass ratio of the hydroxyapatite is 7:3.

2. a method for preparing a composite scaffold with improved void and pore cell adhesion rate according to claim 1, comprising the steps of:

3. The method according to claim 2, wherein the effect of the composite scaffold with improved gap and pore adhesion rate on the short-term adhesion rate of MSCs cells and the effect of the adhesion microscopic morphology of the cells to the composite scaffold with improved gap and pore adhesion rate is evaluated by culturing the composite scaffold with improved gap and pore adhesion rate and MSCs in vitro composite cells.

4. The method for preparing a composite scaffold with improved gap and pore cell adhesion rate according to claim 2, wherein the RGD polypeptide is modified on the surface of CS/HA scaffold pore channels, and then the cell growth on the RGD/CS/HA material is quantitatively detected by PicoGreen cell propagation.

5. The method of claim 2, wherein the step of preparing the CS/HA scaffold by in situ hybridization comprises:

6. The method for preparing the composite scaffold capable of improving the gap and pore space and promoting the cell adhesion rate according to claim 2, wherein the CS and RGD peptides are connected by hydrogen bonds, the RGD peptides are modified on the surfaces of the pores of the CS/HA scaffold by an electrostatic self-assembly method, and the three-dimensional porous scaffold containing the CS/HA high-efficiency adhesion cells of the RGD peptides is prepared, which specifically comprises the following steps:

the standard curve Ci for RGD concentration was plotted as absorbance, the correlation between Ci and absorbance:

absorbance =0.002 × Ci +0.0036;

after being disinfected, the CS/HA stent is respectively placed in RGD solutions with different concentrations of 50mg/L and 100mg/L for soaking for 24 hours, the CS/HA stent containing RGD peptide is prepared, washed by PBS for 3 times to remove free molecules, and finally air-dried; recovering the flushing liquid into the original RGD solution, and diluting to the required concentration; and measuring the absorbance again, and calculating the RGD attachment amount in the stent:

7. The method according to claim 3, wherein the method for determining the short-term adhesion rate of the composite scaffold with improved void and pore cell adhesion rate to MSCs comprises:

each well contains a CS/HA or CS/HA-RGD disk, and 1X 10 of the magnetic particles are added into the well ⁶ ml volume of cells; diluting the wells by adding 1ml volume of 10% PBS; the composite scaffold with the improved gaps and pores for adhering cells to promote the cell adhesion rate is placed in a culture medium for 4 hours, and is washed for 3 times by PBS (phosphate buffer solution) to remove the cells which are not adhered; the calculation formula of the cell adhesion rate is as follows: