CN108355167A - A kind of chitosan coating BCBB bone renovating bracket materials and preparation method thereof being sustained SDF-1 - Google Patents

Abstract

The present invention relates to a kind of chitosans of sustained release SDF 1 to be coated with BCBB bone renovating bracket materials and preparation method thereof, belongs to bone transplantation substitute material and bone tissue engineer technical field.The present invention is to overcome 1 bioavailabilities of SDF low, prevent the too fast release failure of 1 factors of SDF, the problem of its chemotaxis to MSCs cells can not be played, the material provided can not only preferably solve the disadvantage that SDF 1 is diluted and is enzymatically decomposed quickly locally, it also ensures and continues 1 factors of slow release SDF whithin a period of time, ensure that chemotactic MSCs participates in defect repair to SDF 1 in certain time in bone defect, the expression of local organization BMP 2 is adjusted simultaneously to increase, promote the differentiation and proliferation of MSCs, promote new bone formation and angiogenesis, and the BCBB holders for carrying SDF 1 can be degraded and absorbed within a certain period of time, and it is substituted by freshman bone tissue, to preferably repairing bone defect.

Description

A slow-release SDF-1 chitosan-coated BCBB bone repair scaffold material and its preparation method

technical field

The invention belongs to the technical field of bone graft substitute materials and bone tissue engineering, and in particular relates to a slow-release SDF-1 chitosan-coated BCBB bone repair scaffold material and a preparation method thereof.

Background technique

Clinically, bone defects often caused by trauma or surgical resection of bone tumors are very common, and bone defects often lack sufficient blood supply locally, repair the three-dimensional structure of tissue crawling growth, and lack osteoblasts involved in bone repair, so the defect Self-healing ability is very limited. The gold standard for bone defect repair is still autologous cancellous bone grafting, but autologous bone sources are limited and often cannot meet the needs of large-scale bone defect repair. Although allogeneic bone graft materials are commonly used in clinical practice, they have problems such as high cost, transmission of potential diseases, and poor long-term repair effect. Synthetic biorepair materials have the advantages of good bio-histocompatibility, strong degradability, and abundant sources, but the material has a single component, and its pore structure and composition cannot imitate the natural bone structure system, so the bone repair ability is limited and the cost is high. Bone defect repair biomaterials on the market cannot completely repair large-scale bone defects. Xenogeneic bone repair materials have attracted extensive attention due to their advantages of wide source, low cost, natural bone structure and biological characteristics. At present, there have been many research reports on new bone repair materials derived from animal bones or corals. Some heterogeneous bone repair materials, such as the BioOss ^TM bone of the Swiss company Geistler, have been used clinically for many years, and they have certain bone repair effects. However, it cannot achieve the effect of autogenous bone repair, let alone repair large-scale bone defects. A good bone repair material can not only provide three-dimensional space support for the repair tissue, but also the repair material should have the characteristics of osteoinduction and self-osteogenesis. Even if these conditions are met, it still cannot solve the problem of local repair cell shortage, microscopic Due to environmental changes and long repair time, the problem of incomplete repair or non-repair of bone defects will occur when using biomaterials alone for bone defect repair. Since the 1980s, tissue engineering technology has continued to develop, bringing new hope for human beings to repair or replace damaged bone tissue. Defective material.

The integration of repair materials and normal tissues is the key to repairing bone defects by tissue engineering. This process is closely related to three factors: ① seed cells, ② scaffold materials, and ③ growth factors. Although autologous embryonic stem cells are currently the most valuable totipotent stem cells and the best choice for tissue repair seed cells, they are difficult to promote and use in clinical work due to limited sources and complicated ethical issues. In recent years, mesenchymal pluripotent stem cells isolated from bone, cartilage, synovium, fat, heart, brain, liver, kidney and other tissues not only have a wide range of sources, but also can be expanded and cultured in vitro, and can remain intact after multiple passages. Retaining its differentiation potential has become a new focus of tissue engineering seed cells, so the existence of seed cells is a key factor affecting the repair results. Scaffold materials for bone tissue engineering are mainly divided into inorganic synthetic materials, such as: HA, β-TCP, BCP, calcium sulfate hemihydrate, titanium alloy, etc.; organic synthetic materials, such as: collagen, PLA, PGA, PLGA, etc.; biologically derived materials , such as: collagen, bone HA, coral HA, alginic acid, chitosan, etc., and mixed materials of various components. Biologically derived scaffolds not only have good biocompatibility and good biodegradability, but also have a wide range of sources. The natural bioactive groups play a good role in the adhesion, proliferation and differentiation of seed cells. Compared with artificially synthesized scaffold materials Than has unparalleled natural advantages. The process of repairing damaged tissue is a comprehensive and complex process in which multiple cytokines are simultaneously produced and functioning. The current research has not yet clarified the specific molecular biological mechanism in bone defect repair, but some cytokines such as BMP-2, SDF-1, VEGF, bFGF, etc. have been proved to be involved in the process of bone repair. With the deepening of the research on the mechanism of bone defect repair, in the future, the expression of each factor can be adjusted according to the needs of different stages of bone defect repair, so as to achieve the complete repair of large-scale bone defects.

Biphasic ceramic biological bone (Biphasic ceramic biological bone, BCBB) belongs to the category of biologically derived scaffold materials. Its production method is mainly to transform the HA part of the natural bone into β-TCP makes the material not only have a certain mechanical strength, but also easy to degrade. The material retains the porous structure of natural cancellous bone and has good osteoconduction. At the same time, the scaffold after high-temperature calcination has a certain mechanical strength, which can provide a three-dimensional supporting space structure for seed cell proliferation and repair and tissue growth. At the same time, the material It is a mixture of two phases of HA/β-TCP, and it also has certain bone conduction properties. The inorganic HA/β-TCP component has bone tissue affinity and can form a bone-material interface at the bone tissue interface, which is beneficial to cell attachment, proliferation and differentiation. Our previous BCBB scaffold repaired the rabbit radial defect model and found that the scaffold can assist in the repair of radial defects. Further research on the repair of segmental bone defects with cell-carrying BCBB found that tissue-engineered BCBB repaired bone defects better than single BCBB scaffolds. The effect is better.

The inventors of the present application found after research that a single BCBB scaffold is less effective than a BCBB scaffold compounded with cytokines or stem cells in animal bone defect repair research. In recent years, it has been reported that SDF-1, as a cytokine for mesenchyalstem cells (MSCs), has attracted much attention. It has the effect of chemoattracting cells containing CXCR4 receptors, especially MSCs, and up-regulating the CXCR4 receptor on the surface of MSCs. body expression. Studies have found that in animal models of bone defects, the expression of SDF-1 at the damaged site will increase, and the scaffold with the ability to locally release SDF-1 is better than the control group in repairing tissue damage (the control group is without release of SDF-1. ability scaffolds), the scaffolds carrying MSCs overexpressing the CXCR4 receptor were also superior to the control group in terms of tissue damage repair. Based on the above research results, the idea of in situ repair of large-scale bone defects has become possible, that is, to complete the complete repair of bone tissue in large-scale bone defects by utilizing the homing, in situ proliferation and differentiation of MSCs in vivo. At present, research at home and abroad is mainly using synthetic materials such as PLGA, PLA, synthetic HA, synthetic β-TCP, collagen porous scaffolds, etc. to combine with SDF-1, and there is no use of BCBB materials to carry SDF-1 for bone The study of defect repair. Therefore, on the basis of the previous research, the inventors of the present application combined BCBB with SDF-1 as a scaffold material to make it a bone tissue repair material carrying cytokines that induce MSCs to homing, and use their own MSCs in the body to reach bone tissue. The defect site can realize the in situ repair of the bone defect. So far, studies have shown that SDF-1 chemoattracts MSCs to participate in bone defect repair in bone defects, and at the same time regulates the increase of BMP-2 expression in local tissues, promotes angiogenesis, and promotes the differentiation and proliferation of MSCs. However, it has been reported that if the scaffold is directly loaded with SDF-1, the cytokines will be released quickly and completely, and will be decomposed by proteases in the tissue. 1. The cost will be very high, and the inventor found in the previous research that SDF-1 has a dual effect on the chemotactic effect of MSCs, and neither the concentration that is too high nor the low concentration can achieve effective chemotactic homing to MSCs effect. Therefore, improving the efficient SDF-1 sustained release system can make cytokines act on MSCs more effectively, which can enhance the ability of tissue engineered bone to repair bone defects.

Chitosan (CS) is a natural polysaccharide polymer, which is abundant in the natural environment and has a wide range of sources. It has good biocompatibility, strong degradability, no cytotoxicity, certain antibacterial properties, and has positive Charge, easy to attract negatively charged proteins, and has no adsorption effect on SDF-1 with positive charges. Currently, protein or drug sustained release carriers prepared with CS as sustained release carriers have been widely used in various pharmaceuticals and biological materials .

Contents of the invention

The purpose of the present invention is to address the deficiencies in the prior art, to provide a chitosan-coated BCBB bone repair scaffold material and a preparation method thereof for slow-release SDF-1, to overcome the low bioavailability of SDF-1, prevent SDF-1 The factor is too fast to be released and fails to exert its chemotactic effect on MSCs cells. This material can not only better solve the shortcomings of SDF-1 being diluted and decomposed by enzymes in the local area, but also ensure the continuous and slow release of SDF-1 factors for a period of time, ensuring that SDF-1 can remain in the bone defect for a certain period of time. Chemotactic MSCs participate in defect repair, and at the same time regulate the expression of bone morphogenic protein 2 (bonemorphogenetic protein 2, BMP-2) in local tissues, promote the differentiation and proliferation of MSCs, promote new bone formation, and promote angiogenesis, and carry SDF- 1 BCBB scaffolds will be degraded and absorbed within a certain period of time, and replaced by new bone tissue, so as to better repair bone defects.

To achieve the above object, the technical scheme adopted in the present invention is as follows:

A kind of preparation method of chitosan coating BCBB bone repair scaffold material of slow-release SDF-1, comprises the steps:

Step (1), dissolving chitosan in a volume concentration of 1% acetic acid solution to the chitosan concentration is 20mg/ml, and then adding SDF-1 to the SDF-1 concentration is 400ng/ml to obtain SDF-1 /CS sustained release solution;

In step (2), the BCBB scaffold is placed in the SDF-1/CS slow-release solution, ultrasonicated for 30 minutes, taken out and dried to obtain the chitosan-coated BCBB bone repair scaffold material (SDF-1/CS/ BCBB bracket).

Further, preferably, it is characterized in that the viscosity of the chitosan is 50-800 mpas, and the degree of deacetylation is 90-95%.

Further, preferably, when dissolving the chitosan in the acetic acid solution with a volume concentration of 1%, the chitosan is dissolved by stirring; after adding the SDF-1, stir until it is evenly mixed.

Further, preferably, the stirring time is 2 hours.

Further, preferably, the frequency of the ultrasound is 40kHz.

Further, preferably, the drying method is natural air drying.

Further, preferably, the preparation method of BCBB scaffold comprises the following steps:

Use the commercially available cancellous bone of fresh pork bone, boil it in distilled water for at least 6 hours, dry it overnight, then raise the temperature in the muffle furnace to 800°C at a rate of 10°C/min, keep the temperature for calcination for 1h, and then put it in a concentration of 0.04 Sonicate in mol/ml sodium pyrophosphate solution for 30 minutes, take it out, dry it overnight, then raise the temperature in the muffle furnace to 1150°C at a rate of 10°C/min, then keep the temperature for calcination for 1h, after natural cooling, sterilize under high temperature and high pressure at 120°C , to prepare BCBB scaffolds.

Significance of the first calcination: the organic matter, antigenicity and immunity in porcine cancellous bone are removed by high temperature, and the original trabecular bone and trabecular interstitial porous structure are retained. Significance of the second calcination: The sodium pyrophosphate covering the surface and the inorganic salt in the bone undergo a chemical reaction at high temperature to generate β-TCP and increase the β-TCP content of the BCBB scaffold. Purpose of slow temperature rise rate: The first slow temperature rise helps to remove organic matter and avoid denaturation of inorganic salts under rapid temperature changes. The second slow temperature rise helps sodium pyrophosphate and inorganic salts react completely, increasing the generation of β-TCP.

Further, preferably, before boiling, the cancellous bone of the pork bone should be prepared into a block with a size ranging from 0.5cm×0.5cm×0.5cm to 1.0cm×1.0cm×1.0cm or 1.5cm×0.5cm ×0.5cm to 2.0cm×1.0cm×1.0cm strips.

Further, preferably, the frequency of the ultrasound is 40kHz.

The invention also provides a preparation method of the slow-release SDF-1 chitosan-coated BCBB bone repair scaffold material prepared by the above preparation method.

The idea of the present invention is: in combination with the characteristics of chitosan, it is considered that if CS is used to prepare a slow-release system carrying SDF-1 cytokines in order to continuously and slowly release stromal cell-derived factor-1 (SDF-1) in bone defects, It is expected to repair bone defects well. Therefore, the inventors of the present application carried BCBB and slowly released SDF-1 to repair large bone defects and found that the scaffold has excellent osteogenic performance and biocompatibility, which is equivalent to autologous bone, and is an ideal bone graft. Defect repair materials.

Compared with the prior art, the present invention has the beneficial effects of:

(1) The present invention has the advantages of convenient preparation method of BCBB, rich sources of raw materials, availability of large quantities, and low cost; repair materials of different shapes and sizes can be made according to the shape of the defect, and the application is convenient; porcine cancellous bone is retained The inter-communicating natural network pore structure, the pore diameter is up to (514±270) μm, and the porosity is up to (60.19±6.47)%, which can provide cells with a large inner surface area for attachment space and cell proliferation space, which is conducive to the adhesion of seed cells , proliferation and differentiation, and the material has good cytocompatibility, biocompatibility and degradability, it is a good bone tissue engineering scaffold material.

(2) CS is selected as the material surface modifier because it has the advantages of low toxicity, good compatibility, and high degradability. At the same time, chitin, as the raw material of CS, has huge reserves and abundant sources on the earth. The isoelectric point of CS is 10.9, which is positively charged under in vivo conditions. After mixing with positively charged SDF-1, the same charge properties can make SDF-1 release from swollen chitosan without It is adsorbed by CS and scaffold materials, and the chemical structure of CS itself makes it have certain antibacterial properties. Its gel property provides the ability to adhere to biological materials to form a modified film. In addition, CS also has a drug sustained release function. The effective sustained release time of the SDF-1/CS sustained release system can reach more than 9 days.

(3) The product of the present invention has extremely low immunogenicity. It has been confirmed by animal experiments that the immune rejection reaction after SDF-1/CS/BBBB scaffold transplantation is low, there is no record of immune rejection reaction, and it has strong bone induction In the animal model of large bone defect, obvious bone regeneration can be seen at 12w, and the bone defect can be completely repaired at 24w. At the same time, the slow-release stent can also carry other cytokines or special antibacterial drugs to meet the needs of other special situations.

Description of drawings

Fig. 1 SDF-1/CS/BCBB scaffold material, CS/BCBB scaffold material and BCBB general view: the appearance of SDF-1/CS/BCBB scaffold material is brownish-red sponge-like, with porous structure on the surface, surface pores and deep pores The phases are connected, and the pore size is different; CS/BCBB scaffold material is light yellow, while BCBB is bright white;

Figure 2. Scanning electron microscope morphology characteristics of CS/BBBCB scaffold: SEM observation shows that the scaffold is covered with natural grid pore structures of different sizes, the pores communicate with each other without being blocked by CS, and the porous sponge-like structure CS can be seen on the surface of the pore inner wall. Evenly coated;

Fig. 3 Fluorescence imaging of FITC-labeled CS coated with BCBB scaffolds: it can be seen that the scaffold has a green porous structure, and through the pores on the surface of the scaffold, it can be seen that the CS coated on the inner wall of the deep pores emits bright green fluorescence, indicating that the ultrasonic coating method is an effective and convenient CS coating method;

Figure 4 In the cytocompatibility study, the cell proliferation was detected by CCK-8 method when co-cultured with BMSCs; the results showed that the S/BCBB scaffold had no significant effect on the proliferation of BMSCs, with low cytotoxicity and good cytocompatibility;

Fig. 5 Inverted phase-contrast microscope results of CS/BCBB scaffolds co-cultured with BMSCs for 8 days; the results show that CS/BCBB has no obvious toxicity to cells and has good cytocompatibility;

Fig. 6 Electron microscope scanning results of CS/BCBB scaffolds co-cultured with cells for 8 days; the results show that the cells adhere and grow well on the surface of the material, and have good cytocompatibility;

Fig. 7 Absorption wavelength diagram and standard curve diagram of cytochrome C; wherein, the left diagram is the absorption wavelength diagram, and the right diagram is the standard curve diagram;

Fig. 8 sustained-release concentration curve; wherein, the left figure is the sustained-release concentration curve of cytochrome C/CS/BCBB support; the right figure is the sustained-release concentration curve of SDF-1/CS/BCBB support; abscissa represents time ( d); The vertical axis represents the cumulative release amount (μg/mL);

Figure 9 Transwell chamber method to detect scaffold chemotaxis BMSCs experiment diagram;

Figure 10 Transwell chamber method detection scaffold chemotaxis BMSCs transmembrane cell comparison diagram; where A is the BCBB scaffold group, B is the CS/BCBB scaffold group, C is the SDF-1/CS/BCBB scaffold group;

Fig. 11 The histogram of chemotaxis BMSCs cell count of three groups of scaffold groups; the abscissa is the grouping situation; the ordinate indicates the number of cells;

Figure 12 BCBB stent, CS/BCBB stent, SDF-1/CS/BCBB stent implanted into the hind leg muscle pocket of rats; the left picture is the experimental animal, and the right picture is the schematic diagram of the stent implantation;

Figure 13 The general view of the stent and surrounding muscle tissue at 4 and 8 weeks after implantation of BCBB stent, CS/BCBB stent, and SDF-1/CS/BCBB stent;

Figure 14 The general appearance of BCBB stent, CS/BCBB stent, and SDF-1/CS/BCBB stent after sampling at 4 and 8 weeks;

Figure 15 Microscopic observation results of HE and Masson staining on decalcified sections of BCBB scaffolds, CS/BCBB scaffolds, and SDF-1/CS/BCBB scaffolds implanted into muscle bags for 4 weeks;

Figure 16 Microscopic observation results of HE and Masson staining of decalcified sections when BCBB scaffolds, CS/BCBB scaffolds, and SDF-1/CS/BCBB scaffolds were implanted into muscle bags for 8 weeks;

Fig. 17 Microscopic observation of the tissue blood vessels growing into BCBB stents, CS/BCBB stents, and SDF-1/BCBB stents after implantation for 8 weeks;

Figure 18 Microscopic observation of type Ⅰ collagen immunohistochemical staining results of BCBB stent implantation after 4 weeks;

Fig. 19 Microscopic observation of type Ⅰ collagen immunohistochemical staining results of decalcified section after SDF-1/CS/BCBB stent implantation 4w;

Fig. 20 Microscopic observation of type Ⅰ collagen immunohistochemical staining results of decalcified section 8 weeks after BCBB stent implantation;

Fig. 21 Microscopic observation of type Ⅰ collagen immunohistochemical staining results of SDF-1/CS/BCBB stent implantation 8 weeks after decalcification section;

Figure 22 Microscopic observation of HE staining results of hard tissue sections after BCBB stent implantation 8w; S indicates the stent; black arrows indicate the collagen tissue;

Figure 23 Microscopic observation of the results of HE staining of hard tissue sections after SDF-1/CS/BCBB stent implantation 8 weeks; S indicates the scaffold; black arrows indicate collagen tissue; white arrows indicate immature cartilage tissue; white arrows on black background indicate blood vessels;

Fig. 24 Photoshop CS6 software calculates the histogram of tissue ingrowth in each group; the abscissa indicates the grouping situation; the ordinate indicates the tissue ingrowth rate (%);

Figure 25 HE staining results of hard tissue sections after 4 weeks of ectopic osteogenesis;

Figure 26 HE staining results of hard tissue sections after 8 weeks of ectopic osteogenesis;

Figure 27 HE staining results of hard tissue sections after ectopic osteogenesis for 12 weeks;

Figure 28 VG staining results of hard tissue sections after 4 weeks of ectopic osteogenesis;

Figure 29 VG staining results of hard tissue sections after 8 weeks of ectopic osteogenesis;

Figure 30 VG staining results of hard tissue sections after 12 weeks of ectopic osteogenesis;

Figure 31 Histogram of ectopic osteogenesis measurement analysis;

Figure 32 is a diagram of preparing a rabbit radius 1.5cm defect model;

Figure 33 Gross specimen macroscopic view of each group at 2 weeks;

Figure 34 Gross specimen macroscopic view at 4 weeks in each group;

Fig. 35 Gross specimen macroscopic view at 8 weeks in each group;

Figure 36 Gross specimen macroscopic view at 12 weeks in each group;

Figure 37 Gross specimen macroscopic view at 24 weeks in each group;

Figure 38 Microscopic observation of the immunohistochemical results of the blank group at 4 weeks, no positive cells were seen;

Figure 39 Microscopic observation of the immunohistochemical results of the autologous bone group at 4 weeks, a large number of positive cells can be seen (indicated by white arrows);

Figure 40 Microscopic observation of the immunohistochemical results of the BCBB group at 4 weeks, occasionally positive cells were distributed along the material-tissue interface (indicated by white arrows);

Figure 41 Microscopic observation of the immunohistochemical results of the SDF-1/CS/BCBB scaffold group at 4 weeks, it can be seen that many positive cells are distributed along the material-tissue interface (indicated by white arrows);

Figure 42 X-ray results of the blank group at 2 weeks, 4 weeks, 8 weeks, 12 weeks, and 24 weeks. The translucent areas of bone defects can be seen in each group. As time goes by, the fracture end gradually absorbs and hardens;

Figure 43 X-ray results of autogenous bone group at 2 weeks, 4 weeks, 8 weeks, 12 weeks, and 24 weeks. The bone is basically absorbed, the shape is similar to the radius, and the medullary cavity is basically recanalized;

Figure 44 X-ray results of the BCBB group at 2 weeks, 4 weeks, 8 weeks, 12 weeks, and 24 weeks. The material fit well with the host bone, and callus appeared after 4 weeks. The material was partially absorbed at 8 weeks, and new growth appeared at some positions at 12 weeks Bone, at 24 weeks, the material is integrated with the host bone, and there is a boundary between the two;

Figure 45 X-ray results of the SDF-1/CS/BCBB group at 2 weeks, 4 weeks, 8 weeks, 12 weeks, and 24 weeks. The material fits well with the host bone, is closely connected with the host bone at 4 weeks, and is partially absorbed at 8 weeks , most of the material was replaced by new bone tissue at 12 weeks, and the defect was completely repaired at 24 weeks, which was close to the normal radius.

Fig. 46 Histograms of X-ray scores in each group;

Figure 47 HE staining results of hard tissue sections in each group at 2 weeks;

Figure 48 HE staining results of hard tissue sections in each group at 4 weeks;

Figure 49 HE staining results of hard tissue sections in each group at 8 weeks;

Figure 50 HE staining results of hard tissue sections in each group at 12 weeks;

Figure 51 HE staining results of hard tissue sections in each group at 24 weeks;

Figure 52 VG staining results of hard tissue sections in each group at 4 weeks;

Fig. 53 High-power microscope observation results of VG staining of hard tissue sections in groups C and D at 4 weeks;

Figure 54 VG staining results of hard tissue sections in each group at 8 weeks;

Fig. 55 High-power microscope observation results of VG staining of hard tissue sections in groups C and D at 8 weeks;

Figure 56 VG staining results of hard tissue sections in each group at 12 weeks;

Figure 57 VG staining results of hard tissue sections in each group at 24 weeks;

Figure 58 calculates bone mass with the histogram function of Photoshop CS6 software;

Figure 59 Histogram of bone mass calculation results for each component.

Detailed ways

The present invention will be further described in detail below in conjunction with the examples.

Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The materials or equipment used are not indicated by the manufacturer, and they are all conventional products that can be obtained through purchase.

In the present invention, unless otherwise stated, percentages represent mass percentages.

Experimental example 1:

1. Preparation of BCBB scaffolds

(1) Use commercially available fresh pork bones, take the cancellous bone, and prepare them into 0.5cm×0.5cm×0.5cm to 1.0cm×1.0cm×1.0cm blocks or 1.5cm×0.5cm×0.5cm to 2.0cm ×1.0cm×1.0cm strip;

(2) Boil in distilled water for 6 hours and dry overnight;

(3) Calcination in a muffle furnace at 800°C for 1 hour, with a heating rate of 10°C/min;

(4) placed in 0.04mol/ml sodium pyrophosphate solution, ultrasonically vibrated at 40KHz for 30min, and dried overnight;

(5) Calcining in a muffle furnace at 1150°C for 1 hour again, with a heating rate of 10°C/min;

(6) High temperature and high pressure sterilization at 120°C to obtain BCBB stents (Fig. 1).

2. Detection of CS coated with BCBB, changes in coating quality, cytocompatibility, and sustained release ability:

(1) Add 200mg CS (viscosity 50-800mpas, deacetylation degree 90-95%) into 10ml 1% v/v acetic acid solution, stir for 2 hours to fully dissolve to obtain 2% CS solution, and then place BCBB stent in it , 40KHz ultrasonic vibration in the glass test tube for 30min to make the CS solution enter the pores of the BCBB scaffold, take it out and air dry naturally, it can be seen that the SDF-1/CS/BCBB scaffold is brownish red, and the CS/BCBB scaffold is pale yellow (Figure 1).

(2) BCBBs coated with CS were randomly selected, dehydrated and freeze-dried with gradient alcohol concentration, sprayed with gold and placed in a scanning electron microscope. Observe the morphological changes of BCBB after coating, and porous CS can be seen on the surface of BCBB scaffold (Fig. 2).

Wherein, the specific steps of alcohol concentration gradient dehydration freeze-drying and sticky sample gold spraying are: ethanol gradient dehydration (respectively 30%, 50%, 70%, 85%, 90% ethanol once each, 100% ethanol twice, 15min /Second-rate). The samples were respectively frozen at 20°C, 40°C and 80°C for 12 hours, dried in a freeze dryer for 12 hours, and set aside. Take out the dried bracket with tweezers, adhere the bracket to the aluminum tray with conductive glue, and coat a layer of metal film with a thickness of 1500nm on the surface of the sample with an ion sputtering coating device.

(3) Place the BCBB scaffold in the pre-prepared 100 μg/ml FITC green fluorescently labeled CS solution (this part of CS is added with FITC fluorescently labeled, written as FITC-CS/BCBB, only this part uses FITC-CS/ BCBB), ultrasonic 30min, make the chitosan solution completely enter the BCBB pores. Air dry overnight in a clean bench. Under an inverted fluorescence microscope, CS coated on the pore surface of BCBB and emitting bright green fluorescence can be seen (Figure 3), indicating that the method of ultrasonic vibration can effectively and uniformly coat CS on the porous inner wall of BCBB scaffold.

(4) Randomly select 5 BCBB scaffolds, record the weight of each scaffold after weighing, and record it as W ₀ , then coat it with CS, dry it in a blast oven at 50°C overnight, weigh it again and record it as W ₁ , ΔW= (W _{1 -} W ₀ )/W ₀ ×100%, and statistical analysis was carried out, the measured average mass increase of BCBB after coating was 53.2±9.63% (Table 1).

Table 1 Comparison of scaffold quality before and after coating with chitosan (n=5, )

Among them, T: T test (Student's t test) is mainly used for normally distributed data with a small sample size (for example, n=5 in this case) and an unknown population standard deviation σ.

P: P value (P value) is the probability of the sample observation or more extreme results obtained when the null hypothesis is true. If the P value is small, it means that the probability of the occurrence of the null hypothesis is very small.

S: Standard Deviation, which is the square root of the arithmetic mean of the square of the mean deviation, reflecting the degree of dispersion of a data set.

(5) Cytocompatibility study: the experiment was divided into 3 groups, the experimental group A was the BCBB scaffold; the experimental group B was the CS/BCBB scaffold group; Put into four 96-well culture plates (in this experiment, each group has 20 samples, and each group of 5 samples is put into 1 plate, that is, each plate has 5 samples of groups A, B, and C, totaling 4 plate), each group had 5 replicate wells, cultured in complete LG-DMEM medium for 24 hours, no bacterial contamination was found. After aspirating the medium, BMSCs were inoculated into three groups according to the above corresponding groups at a density of 5× ¹⁰⁴ /mL 100 μL, cultured in a 37°C, 5% _CO2 incubator, cultured in complete LG-DMEM medium, Change the medium every 2 days. Take out a culture plate on the 2nd, 4th, 6th, and 8th day after culture respectively, suck off the medium, add 100 μl of new medium to each well, add 10 μl of CCK-8 liquid to each well under the condition of avoiding light, and put it into the incubator After incubation for 2 h, the OD value was detected, and the growth curve was drawn and statistically analyzed. It was observed by an inverted phase-contrast microscope that the cells co-cultured with the scaffold grew well on the 8th day, and the cells were well adhered to the CS/BCBB scaffold on the 8th day by the electron microscope (Fig. 5, Fig. 6). The CCK-8 method of cell proliferation detection showed that there was no significant difference in OD value among the groups after the co-culture of cells and scaffolds for 2 days (P>0.05). Statistical analysis of the OD values of the three groups after the 8th day of culture showed that there was no significant statistical difference between the BBCB stent group and the CS/BBBC stent group (P>0.05), but there was a statistical difference between the CS/BBBC stent group and the blank control group (P <0.05) (Figure 4).

(6) The sustained-release function of the coating layer was detected by cytochrome C release assay.

1) Dissolve 25mg of cytochrome C in 10ml of double distilled water to obtain the mother liquor, take 0.1ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml of the mother liquor and dilute to a total volume of 2ml to obtain the concentration of cytochrome C 62.5μg/ml, 250μg/ml, 357μg/ml, 500μg/ml, 625μg/ml, 750μg/ml solution, carry out ultraviolet spectroscopic absorbance detection and draw a standard curve (Figure 7), the maximum ultraviolet absorption wavelength of cytochrome C is 410nm , the absorbance is measured at this wavelength.

2) Take 1.6ml of 2.5mg/ml cytochrome C solution and add it to 10ml 2% CS solution to obtain a cytochrome C/CS solution with a concentration of about 400μg/ml. Put a piece of BCBB scaffold into it, take it out after 40KHz ultrasonic vibration for 30min, and air-dry overnight to obtain cytochrome C/CS/BCBB scaffold. The air-dried scaffolds were pre-wetted with PBS for 2 hours, and put into a six-well culture plate according to the number of one per well. After adding 4.5ml of PBS solution to each well, they were placed in a cell incubator at 37°C and humidity 97% to incubate in a simulated in vivo environment. The soaking liquid was sucked out on the 1st day, 2nd day, 3rd day, 6th day, and 9th day, and then 4.5ml of PBS was added to continue soaking. Add the soaking liquid to 3 cuvettes, add 1ml to each cuvette, measure the absorbance of ultraviolet spectrometer, and calculate the average value of the obtained concentration as the cumulative release curve. From the release curve, it can be seen that the 1-3 days after soaking is cytochrome C. The rapid release period, and then the release rate gradually slowed down, and the release of cytochrome C could still be detected on the 9th day (Figure 8).

(7) According to the detection instructions of the SDF-1 kit, standard curves were drawn for the concentrations of 0 ng/ml, 1.0 ng/ml, 2.0 ng/ml, 4.0 ng/ml, 8.0 ng/ml, and 16 ng/ml. SDF-1 with a concentration of 1000ng/ml was selected as a slow-release factor and coated on BCBB, and the production method was the same as that of cytochrome C coating. The specific production method is as follows:

Put 3 pieces of BCBB scaffolds whose size is 0.5cm×0.5cm×0.5cm into SDF-1/CS solution (the preparation method of SDF-1/CS solution is to take 1.6ml of 2.5mg/ml SDF-1 solution, add to 10ml of 2% CS solution to obtain SDF-1/CS solution), 40KHz ultrasonic vibration for 30min, take out, and air-dry for 4h naturally to obtain SDF-1/CS/BCBB scaffold. The air-dried scaffolds were pre-wetted with PBS for 2 hours, put 1 piece per well into a six-well culture plate, added 4.5ml of PBS solution to each well, and placed in a cell culture incubator at 37°C and humidity 97% for static incubation. The soaking liquid was sucked out on the 1st day, 2nd day, 3rd day, 5th day, and 7th day, and then 4.5ml PBS was added to continue soaking the stent. After diluting the collected soaking solution 5 times with PBS, detect the OD value at the wavelength of 450nm of the microplate reader according to the operation manual of the 96-well microplate SDF-1ELISA kit, and calculate the average concentration value multiplied by the dilution factor as the cumulative concentration Release curve, it can be seen from the release curve that the first 1-3 days after soaking is the rapid release period of SDF-1, and the release rate slows down after that, and the release of SDF-1 can be detected at 7 days. It shows that the CS sustained-release system can slowly release SDF-1 into the solution, and the SDF-1/CS system has a sustained-release function (Figure 8).

The present invention uses cytochrome C to replace SDF-1 when detecting. This is because cytochrome C is a compound with the same charge property and similar molecular size as SDF-1, and its price is relatively low. Many studies use it as SDF-1. The substitute of 1 was used for related detection of SDF-1 function, so cytochrome C was used as the substitute protein of SDF-1 to detect the slow-release function of CS/SDF-1SDF-1/CS. Cytochrome C has a maximum absorption wavelength of 410nm, and when cytochrome C is mixed with CS, the maximum absorbance wavelength is still 410nm. Transwell chemotaxis chamber is often used in experiments related to cell migration and invasion, so this chamber can be used to detect the chemotaxis performance of BCBB scaffolds carrying SDF-1 in CS to BMSCs.

3. The chemotaxis effect of SDF-1/CS/BBBCB scaffold with sustained release function on BMSCs in vitro:

(1) Add 200mg CS (viscosity 50-800mpas, deacetylation degree 90-95%) into 10ml 1% v/v acetic acid solution, stir for 2 hours to fully dissolve and mix evenly to obtain CS liquid; after that, place the BCBB stent in It has been put into the CS solution of the glass test tube, and the mouth of the tube is closed to ensure the sterility of the product. In the glass test tube, 40KHz ultrasonic vibration is used for 30 minutes to make the CS solution enter the material pores, and the CS/BCBB scaffold is obtained by taking it out and air-drying naturally.

(2) Select SDF-1 with a concentration of 400ng/ml. Take 4 μl of 1 μg/μl SDF-1, dissolve it in 10ml CS solution, stir at room temperature for 2 hours and mix evenly to obtain the SDF-1/CS slow-release solution, put the BCBB scaffold into the SDF-1/CS slow-release solution and put it in glass The test tube, with the tube mouth closed, was coated with 40kHz ultrasonic vibration for 30 minutes to obtain the SDF-1/CS/BCBB bracket, placed in a sterile orifice plate and air-dried for 4 hours under an ice bath on an ultra-clean table for subsequent experiments (the whole process was carried out under aseptic operation) )

(3)(3) The experiment was divided into 3 groups, group A was BCBB scaffold group, group B was CS/BCBB scaffold group, group C was SDF-1/CS/BCBB scaffold group, and each group had 3 parallel samples. Wet the three groups of scaffolds with PBS in advance for 30 minutes, and then put the scaffolds into the bottom center of different wells of the 24-well plate, and placed the chemotaxis chamber above the scaffolds, digested the BMSCs into a single cell suspension, and washed them with 2% FBS. Adjust the density of LG-DMEM to 2×10 ⁵ cells/mL, take 200 μl of BMSCs single-cell suspension and add it to the upper layer of the chemotaxis chamber, and add 500 μl of 2% FBS LG-DMEM to the lower layer. Place them in a 37°C, 97% humidity cell culture incubator to incubate (Figure 9).

(4) After being cultured in the incubator for 36 hours, take out the support, remove the medium (the method of removing the medium is to wash the medium three times with PBS), fix with 4% paraformaldehyde for 15 minutes, and wash twice with PBS , add 1ml of 0.1% crystal violet solution for staining for 10min, wash with PBS for 3 times, use a medical cotton swab dipped in water to wipe off the non-penetrating cells in the upper layer of the chamber, observe the penetrating cells under an inverted phase-contrast microscope, and randomly select 5 fields of view at 200 times to count the cells. Statistical analysis. By comparing the number of BMSCs chemotaxis of BCBB scaffold, CS/BCBB scaffold and SDF-1/CS/BCBB scaffold, it was found that SDF-1/CS/BCBB scaffold had obvious chemotactic effect on BMSCs (Figure 10), statistical analysis results It showed that the number of chemotactic cells on the SDF-1/CS/BCBB scaffold was significantly different from that of other groups (P<0.05) (Figure 11). It shows that CS can be used as a slow-release carrier of SDF-1 to coat the surface of the material, and the SDF-1 mixed in CS can be slowly released into the liquid environment, and the released SDF-1 still has chemotactic BMSCs activity.

Wherein, 4% paraformaldehyde is gained by dissolving 4g paraformaldehyde into 100ml PBS;

1% crystal violet solution is obtained by dissolving 2 g of crystal violet in 20 mL of 95% ethanol and 80 mL of 1% ammonium oxalate aqueous solution.

4. Biodegradability experiment of SDF-1/CS/BCBB scaffold with sustained release function:

Eighteen SD rats were randomly divided into 3 groups, 6 rats in each group. Group A was the BCBB scaffold group, Group B was the CS/BCBB group, and Group C was the SDF-1/CS/BCBB group. 3% sodium pentobarbital was anesthetized by 0.3ml/100g intraperitoneal anesthesia. After the anesthesia was successful, the thigh muscles of the rats were selected, hair removed, and disinfected with povidone iodine. After incision of the skin, the muscles were bluntly separated to make muscle bags, and the stents of groups A, B, and C were implanted into the bilateral femoral muscle bags (Figure 12). The local wounds were disinfected with povidone iodine again, and the muscle bags were sutured sequentially with silk thread. Suture the incision. Intraperitoneal injection of 400,000 units of penicillin for 3 days to prevent wound infection. The specimens were collected at 4w and 8w respectively, and were subjected to HE staining, Masson staining, type I collagen staining, and histological examination of hard tissue sections.

Wherein 3% pentobarbital sodium is pentobarbital sodium 3g is dissolved in 100ml physiological saline gained pentobarbital sodium solution.

(1) General observation of materials:

The stents were implanted into the hind leg muscle bag of the rats, and the samples were taken at 4w and 8w respectively. The wounds of the rats were completely healed, and the stents in each group had good compatibility with the surrounding muscle tissue. There was no obvious scar wrapped around the stents. Good, no scar sclerosis and tissue necrosis. After 4 weeks of implantation, it can be seen that there is no obvious scar hardening and tissue necrosis between the scaffolds and the muscle bed in each group. The pores on the surface of BCBB scaffolds and CS/BCBB scaffolds are still clearly visible, with some tissue filling in between. The color of CS/BCBB scaffolds is yellowish, which is shell Glycans were coated with color, while the surface of the SDF-1/CS/BCBB scaffold was covered with white fibrous tissue, and no pores were found on the surface of the scaffold (Figure 13). After 8 weeks of implantation, it can be seen that there is no obvious scar sclerosis and tissue necrosis between the stent and the muscle bed, the blood supply around the stent is richer than that of 4 weeks, and the stents in each group are more closely connected with the surrounding muscle tissue. SDF-1/CS/BCBB The surrounding tissues on the surface of the stents were pink, with blood supply on the surface, and no pores were found on the surface of the stents in each group (Fig. 13). There were tissues growing into the surface pores of the stent implanted at 4w and 8w, and with the prolongation of implantation time, the amount of new tissue on the surface of the stents in the BCBB group and the CS/BCBB group gradually increased, and the gaps in the SDF-1/CS/BCBB group The amount of tissue ingrowth was more than that of other groups at 4w and 8w (Fig. 14).

(2) Histological observation:

Group A: 4 weeks after the BCBB scaffold was implanted, a small amount of muscle tissue and new fibrous tissue grew into the pores of the material, and Masson stained the collagen fiber tissue in red, and the muscle tissue in blue (Figure 15). After 8 weeks of implantation, it was observed that the amount of tissue growing into the pores increased compared with before, the collagen fiber tissue was pink in HE staining, blue in Masson staining, and the collagen fibers were arranged in disorder (Figure 16). The results of HE staining at 8w after implantation showed the tissue wrapped around the stent and the tissue growing into the pores, and no new small blood vessels were seen in the BCBB stent group (Figure 17). Immunohistochemical results of type Ⅰ collagen after implantation for 4w showed positive reactions in the extracellular matrix and individual cells in the new tissue in the pores (Figure 18), and the immunohistochemical results of implantation for 8w showed more positive reactions (Fig. 20), mainly concentrated in the interface between the new tissue and the material in the pores. The hard tissue section of the BCBB scaffold 8 weeks after implantation showed that a small amount of disordered collagen tissue was formed on the edge of the new tissue and the material growing into the gap. HE staining was pink, the collagen was arranged in disorder, and there were no orderly arranged osteoblasts on the surface. No osteoid or calcified tissue was formed (Fig. 22).

Group B: The results of HE staining after 4 weeks of CS/BCBB stent implantation showed that CS still remained inside the stent, and the amount of new tissue in the gap was less than that of groups A and C. The results of Masson staining showed that the part stained red was muscle tissue. The part stained in blue is the collagen tissue (Figure 15). After 8 weeks of implantation, the coated CS component in the material basically disappeared, but the amount of new tissue in the pores was less than that of groups A and C. The results of Masson and HE staining suggested that the growth into the tissue was collagen (Figure 16). The results of HE staining at 8 weeks after implantation showed that there was no obvious small blood vessel formation in the tissue wrapped by the scaffold and the new tissue in the space (Figure 17).

Group C: After implantation of SDF-1/CS/BCBB scaffolds for 4 weeks, the new tissue in the pores was significantly more than that of the other two groups, and the results of HE and Masson found no residual CS coating, and the nature of the new tissue in the pores was collagen fiber components ( Figure 15). The amount of ingrowth into the tissue after 8 weeks of implantation was significantly higher than that at 4 weeks, which was composed of collagen fibers (Figure 16). The results of HE staining at 8 weeks after implantation showed that there were many small blood vessels formed in the tissue wrapped with the stent and the new tissue in the pores (Figure 17). The results of type Ⅰ collagen immunohistochemistry showed that the amount of type Ⅰ collagen in the SDF-1/CS/BCBB scaffold group was large and in a sheet shape, and it grew into the new tissue in the pores (Fig. 19, Fig. 21), which has the characteristics of intramembranous osteogenesis. The result of HE staining of the hard tissue section of the SDF-1/CS/BCBB scaffold implanted at the same time for 8 weeks showed that there were pink collagen fibers at the junction edge between the new tissue and the material in the pores, and cells arranged in rows on the surface of the collagen tissue could be seen , has the characteristics of intramembranous osteogenesis, and the amount of small blood vessels in the new tissue in the pores is more than that of the simple BCBB scaffold group. At the same time, immature bone tissue colored blue can be seen inside the new tissue in the pores, which has the characteristics of endochondral bone formation, but No calcified tissue was found in the new tissue in the pores (Fig. 23).

(3) Calculation results of tissue ingrowth rate in the material

The obtained digital photos were analyzed with the histogram function of Photoshop CS6 software to measure the area of tissue ingrowth, that is, the rate of tissue ingrowth=pixels of tissue ingrowth÷pixels of the whole picture×100%. The results were statistically analyzed by SPSS 17 software, and the tissue ingrowth rate of different groups of scaffolds was calculated by calculation. Statistical analysis (Table 2), as shown in Figure 24, the tissue ingrowth rate after implantation for 8 weeks was higher than that of 4 weeks; The tissue ingrowth rate among the three groups was statistically different at the same time point (P<0.05); the tissue ingrowth rate of the CS/BCBB stent group was the lowest, and it was statistically different from other groups (P<0.05); SDF- The rate of tissue ingrowth in the 1/CS/BCBB scaffold group was higher than that of the other two groups, which was statistically different from the other two groups (P<0.05).

Table 2 Comparison of tissue ingrowth rates of three groups of materials at different time points (n=6, )

4w 8w Group A 14.97±0.67 34.57±0.54 Group B 12.32±0.88 29.38±0.75 Group C 18.21±0.86 51.06±2.09 F value 40.037 220.182 P value 0.000 0.000

The F value indicates the significance of the entire fitting equation, and the larger the F, the more significant the equation and the better the fitting degree. The P value indicates the degree to which the null hypothesis is not rejected, and P<0.5 indicates that the hypothesis is more likely to be correct, otherwise it may be wrong

5. Ectopic osteogenesis experiment of SDF-1/CS/BCBB scaffold with sustained release function:

Twelve SD rats were randomly divided into 2 groups, 6 rats in each group. Group A was implanted with BCBB stents, and group B was implanted with SDF-1/CS/BCBB stents. 3% sodium pentobarbital was anesthetized by 0.3ml/100g intraperitoneal anesthesia. After the anesthesia was successful, the meat of the femur of the rat was selected, hair removed, and disinfected with povidone iodine. After incision of the skin, the muscles were bluntly separated to make muscle bags. The scaffolds of groups A and B were respectively implanted into the bilateral femoral muscle bags. The local wounds were consumed again with povidone iodine, and the muscle bags were sutured sequentially with silk thread to close the incision. Intraperitoneal injection of 400,000 units of penicillin for 3 days to prevent wound infection. Samples were collected at 4w, 8w, and 12w, respectively, and histological examination of hard tissue sections was performed.

(1) Hard tissue section observation:

Group A: BCBB scaffolds were implanted for 4 weeks, and both HE staining and VG staining showed that the pores were filled with fibrous tissue and cells (Fig. 25, Fig. 28). After 8 weeks of implantation, it was clearly observed that the amount of tissue growing into the pores increased and the blood vessel components were less (Fig. 26). In the VG staining, the collagen fibers were homogeneously stained blue, and small blood vessels were seen in the pores (shown by black arrows). The fiber arrangement is disordered (Fig. 29). 12 weeks after implantation, VG staining results showed that there were blue-stained collagen substances on the surface of the scaffold (indicated by white arrows) (Figure 30), and red staining under HE staining, distributed along the surface of the material (Figure 27), which is the early manifestation of endochondral ossification .

Group B: The HE staining and VG staining results of SDF-1/CS/BCBB stent implantation 4w showed that the amount of new tissue in the scaffold pores was more than that of group A, and blue-stained collagen was distributed along the surface of the material under VG staining (Fig. 28), in HE staining, neovascularization in the tissue (shown by the white arrow) was more than that in group A at the same period (Fig. 25). After 8 weeks of implantation, the amount of new tissue in the pores was more than that of group A. HE staining showed cell aggregation on the surface of the material (black arrows), and many small blood vessels in the pores (white arrows) (Fig. 26). The obviously red-stained osteoblast tissue (shown by the white arrow) indicated that ectopic osteogenesis occurred at 8w (Fig. 29). The results of HE staining at 12 weeks after implantation showed that there were red osteogenic tissues on the material surface (shown by white arrows) (Fig. 27), and the results of VG staining showed that the red-stained osteogenic tissues increased (Fig. 30).

(2) Osteogenesis calculation results

The obtained digital photos were analyzed with the histogram function of Photoshop CS6 software to measure the area of tissue growth, that is, the amount of new bone tissue = new bone tissue area pixels ÷ pixels of the whole picture × 100%. The results were statistically analyzed by SPSS 17 software (Table 3), and the tissue ingrowth rates of different groups of scaffolds were calculated by calculation. As can be seen in Figure 31, the bone formation in group B after implantation for 4w, 8w, and 12w was higher than that of Group A, and there was a significant statistical difference (P<0.05).

Table 3 Comparison of tissue ingrowth rates at different time points between the two groups (n=6, )(%)

4w 8w 12w Group A 0.09±0.06 0.46±0.05 5.96±0.80 Group B 0.71±0.33 25.59±4.43 42.97±6.25 F value 20.555 193.117 206.968 P value <0.05 <0.05 <0.05

The F value indicates the significance of the entire fitting equation, and the larger the F, the more significant the equation and the better the fitting degree. The P value indicates the degree to which the null hypothesis is not rejected, and P<0.5 indicates that the hypothesis is more likely to be correct, otherwise it may be wrong.

6. Comparison experiment between SDF-1/CS/BCBB scaffold and blank group, autologous bone group, and BCBB group in repairing large segmental bone defects in rabbits:

40 healthy adult Japanese big-eared white rabbits were randomly divided into A (blank group), B (autologous bone group), C (BCBB group), D (SDF-1/CS/BCBB group), with 10 rabbits in each group . The radial center of the bilateral forelimbs was used as the surgical site; 10% chloral hydrate was anesthetized by intraperitoneal injection of 3ml/kg; after anesthesia, the rabbit was fixed on the animal operating table, the forelimbs were fully depilated, and the surgical site was exposed, and 0.5% iodophor was used to Disinfect repeatedly 3 times; spread sterile drapes, use a scalpel to cut a 2.0cm skin incision at the middle of the radial radius of the forelimb, separate the skin, subcutaneous tissue, fat, fascia, muscle, and blood vessels layer by layer, carefully stop bleeding, and expose the radius ; Determine the position of the middle part of the radius, peel off the periosteum on the bone surface with a periosteal stripper, measure the length of the proposed osteotomy site with a stainless steel ruler, and ensure that a 1.5cm-long radial defect is made (Figure 32); take out the autogenous bone with a bone length of 1.5cm; place it on the defect site Implant materials: no special treatment in group A, autologous contralateral radius implanted in group B, BCBB stent implanted in group C, SDF-1/CS/BCBB stent implanted in group D, and the implants were fixed in place; 1ml of pre-prepared eGFP-BMSCs at a concentration of 10 ⁷ /mL was infused along the ear vein, and 400,000 U of penicillin was injected intramuscularly every day for 3 consecutive days to prevent infection of the surgical port, and the dressing was changed every other day. The samples were collected at 2w, 4w, 8w, 12w, and 24w for general observation, X-ray examination, hard tissue section observation and GFP protein immunohistochemical staining observation at 4w.

Wherein, 10% chloral hydrate is the chloral hydrate solution obtained by dissolving 10 g of chloral hydrate in 100 ml of physiological saline.

(1) General observation

Group A: The bone defect can be clearly seen in each group. At 2 weeks, the broken end of the bone was obvious, a large amount of interstitial fluid exuded from the broken end and new tissue was formed, and there was no sign of infection (Fig. 33); at 4 weeks, the exudation decreased, the medullary cavity was sealed by fibrous tissue, and fibrotic adhesions formed in the surrounding tissue (Fig. 34 ); at 8 weeks, the broken end of the bone formed a bony seal, surrounded by a large amount of fibrous tissue, which was difficult to separate from the surrounding tissue and muscle, and the exudation between the broken ends was reduced and absorbed, forming a fibrous scar (Fig. 35); Scar adhesion was formed in the surrounding fibrous tissue (Fig. 36); there was no significant difference between 24 weeks and 12 weeks, and the defect still existed (Fig. 37).

Group B: At 2 weeks, the broken end of the bone was neat, and a large amount of new fibrous tissue was seen in the gap (Fig. 33); at the 4th week, the broken end of the bone was wrapped with new fibrous callus, and periosteum-like tissue was seen on the surface of the autologous bone (Fig. 34); after 8 weeks The autologous bone and the host bone formed bony union, the callus grew significantly, and the grafted bone resorbed and remodeled (Fig. 35); at 12 weeks, the autologous bone and the host bone were completely fused, covered with periosteum-like tissue and rich in blood supply (Fig. 36) ; At 24 weeks, the shape of the defect was close to the normal radius (Fig. 37).

Group C: At 2 weeks, the boundary between the implanted material and the broken end of the bone can be observed clearly, filled with fibrous tissue, and there is no obvious sign of infection (Fig. 33); at 4 weeks, the surface of the material is complete, tightly combined with the host bone tissue, and new growth around the surface of the material Surrounded by callus (Fig. 34); at 8 weeks, the outline of the material was complete, tightly integrated with the host bone, the surface of the material and in the pores were filled with tissue, and the material was absorbed and degraded to varying degrees (Fig. 35); at 12 weeks, the boundary between the material and bone tissue was not clear , formed a firm healing, and the repair segment was well shaped (Fig. 36); at 24 weeks, the surface tissue of the material was tightly coated, rich in blood supply, and the boundary with the host bone tissue was unclear, and some materials remained (Fig. 37).

Group D: There was no significant difference from Group C at 2 weeks, the structure of the material was complete, the gap between the bone and the host was filled with fibrous tissue, new tissue grew into the internal pores of the material, and no obvious signs of infection were seen (Figure 33); at 4 weeks, the surface of the material The structure is complete, closely combined with the host bone, new callus is formed around it, and the blood supply is abundant (Fig. 34); at 8 weeks, the structure of the material is relatively complete, a large number of callus can be seen on the surface, the material is tightly combined with the host bone, and the material is partially absorbed (Fig. 35); after 12 weeks, the material and the host bone formed a solid union, the boundary was unclear, most of the material was absorbed, the new bone grew in, and the defect part was shaped (Fig. 36); at 24 weeks, the repair segment was well shaped and had no shape with the host bone Significant difference (Figure 37).

(2) Histological observation

Immunohistochemical staining of GFP protein was carried out at 4w. In group A, a large number of fibrous tissue hyperplasia and cell infiltration were seen in the tissue, and positive cells were rarely seen (Figure 38); in group B, a large number of round and oval positive cells (white Arrow), which is the largest number among the four groups (Fig. 39); BCBB residues in group C are fine granules, the pores are filled with tissue, and the cells are numerous and distributed like fiber strands, occasionally a small amount of stained positive cells (white arrow (shown) (Fig. 40); group D has more chitosan residues, fine particle-like distribution, abundant cells in the pores, mixed positive cells and negative cells, which can be seen distributed at the material-tissue interface (shown by white arrows), and The number is more than that of group C (Figure 41).

(3) X-ray results and scoring

Group A: Lucent areas of bone defects were observed at each time point. At 2 weeks, the bone defect was obvious and translucent, filled with fibrous tissue, and the edge of the defect was neat; at 4 weeks, a small amount of bone resorption and remodeling appeared at the defect end; at 8 weeks, the defect end had obvious bone absorption and remodeling, and the medullary cavity was closed; at 12 weeks, the defect The end bone cortex resorbed and hardened; X-rays showed no obvious changes at 24 weeks and 12 weeks, and the defect still existed (Figure 42)

Group B: The gap between the defect end and the implanted material was clear at 2 weeks; the boundary between the defect end and the autogenous bone was blurred at 4 weeks; bony connection was formed between the defect end and the implanted bone at 8 weeks, and the bone cortex was thickened at the bony connection, and the medullary cavity part Recanalization; at 12 weeks, the defect end was connected with the implanted bone, and the medullary cavity was connected, and the brightness of the cortical bone at the connection was increased, and the shape was rough; at 24 weeks, the cortical bone of the defect site was continuous and complete, and the medullary cavity was remodeled, which was no different from that of the normal radius (Fig. 43).

Group C: The implanted material fit well with the defect end at 2 weeks, without significant displacement, and the shape of the material was complete, and the gap with the broken end of the host bone was clearly visible. Plastic, the edge of the implanted material is slightly blurred, and the material is completely visible; at 8 weeks, the edge of the implanted material is degraded and absorbed to varying degrees, and the medullary cavity is not closed; at 12 weeks, the density of the material is mostly reduced, and it still appears as a high-brightness shadow, which is similar to the density of the radius , partially surrounding the new bone; the density of the material was still high at 24 weeks, and the density of the connection part with the defect area was similar to that of the radius, and the connection with the bone was tight (Fig. 43).

Group D: At 2 weeks, the implanted material was in good alignment with the defect end, without significant displacement, and the shape of the material was complete, with a clear gap with the broken end of the host bone; at 4 weeks, the shape of the material was complete, and the density of the contact surface with the broken end of the bone was still high. The material was partially covered by callus; the boundary between the material and the host bone was not clear at 8 weeks, the density was similar to that of the host bone, and the pore structure in the material was not clear; at 12 weeks, the material was partially absorbed, shortened and narrowed, and bony healed with the defect end, repaired The density of the area was similar to that of the radius, and the boundary was unclear. The reconstruction of the medullary cavity was completed at 24 weeks, and the bone cortex in the repaired area was continuous and smooth, which was similar to that of the radius.

The X-rays were scored according to the X-ray scoring standard of Lane et al. The evaluation content included the healing of the material and the host bone, the formation of the medullary cavity, and the degree of remodeling of the medullary cavity. Using SPSS17.0, analysis of variance was used to analyze and compare the results. At 2 weeks, because the score of each group was 0, it was not included in the statistical scope; because the score of group A was 0 at each time point, it was not included in the statistical scope. After analysis of variance, there was a difference between group B and group C at the same time point of 8-24 weeks (P<0.05), there was a difference between group B and group D at the time point of 8 weeks (P<0.05), and there was no significant difference after 12 weeks There was a significant difference (P>0.05) between groups C and D at the time point of 24 weeks (P<0.05). After 12 weeks, there was no significant difference in the X-ray results of the bone repair effect between group B and group D (Table 4, Figure 46). It shows that the SDF-1/CS/BCBB scaffold is close to autogenous bone.

The X-ray score (n=3, )

time Two weeks 4 weeks 8 weeks 12 weeks 24 weeks Group A 0±0 0±0 0±0 0±0 0±0 Group B 0±0 2.33±1.54 6.33±0.58 ^□■ 7.33±1.53 ^□ 10.33±1.53 ^□ Group C 0±0 2.00±1.00 3.67±0.58 ^□ 5.00±1.00 ^□ 5.33±1.53 ^▲ Group D 0±0 2.33±0.58 4.00±1.00 ^■ 6.33±0.58 8.67±1.12 ^▲ f - 0.125 11.400 3.364 9.722 P - 0.885 0.000 0.105 0.002

Note: ^□ Compared between group B and C, P<0.05; ^■ Compared between group B and D, P<0.05; ^▲ Compared between group C and D, P<0.05.

The F value indicates the significance of the entire fitting equation, and the larger the F, the more significant the equation and the better the fitting degree. The P value indicates the degree to which the null hypothesis is not rejected, and P<0.5 indicates that the hypothesis is more likely to be correct, otherwise it may be wrong.

(4) Hard tissue section observation

Group A: The defect end was neat at 2 weeks, with fibrous tissue proliferation and cell infiltration around the defect end, and multinucleated cells could be seen around the defect end, participating in bone resorption, without obvious signs of infection (Figure 47); bone resorption and remodeling around the defect end at 4 weeks (Fig. 48), VG staining showed that a large area of purple-stained bone formation gradually closed the medullary cavity (Fig. 52); the texture of trabecular bone at the defect end was clear at 8 weeks, the range of surrounding bone formation area was reduced, and the trabecular bone in the area of bone resorption and remodeling Disorder (Fig. 49, Fig. 54); the medullary cavity was closed at 12 weeks, and the defect was filled with fibrous tissue (Fig. 50, Fig. 56); at 24 weeks, multinucleated cells could be seen beside the bone of the medullary cavity, and the bone resorption was mutated, and the defect remained exists (Figure 51, Figure 57).

Group B: Fibrous tissue connection between the autologous bone and the host bone at 2 weeks, with a large number of cell infiltrations (Figure 47); at 4 weeks, the autologous bone and the host bone were tightly integrated, and there were a large number of blue-stained fibrous callus mixed with red-stained bone callus (Fig. 48, Fig. 52); at 8 weeks, autologous bone marrow cavity recanalized, new bone trabecular texture was arranged irregularly, and it was clearly distinguished from the host bone (Fig. 49, Fig. 54); at 12 weeks, autologous bone marrow cavity recanalized, and cells in the medullary cavity side gathered , bone resorption and remodeling in the medullary cavity were seen, and the texture of the trabecular bone in the repair area was regular (Fig. 50, Fig. 56). At 24 weeks, the structure of the cortical bone was clear, the trabecular bone was arranged regularly, and the remodeling of the medullary cavity was completed (Fig. 51, Fig. 57).

Group C: At 2 weeks, new blood vessels and fibrous tissue grew into the material, less than that of group D, and the broken ends of the bones were neat, without obvious signs of infection (Figure 47); at 4 weeks, more blood vessels and fibrous tissue grew into the material, and a small amount of bone appeared. Formation, fibrous tissue connection between the material and the host bone, blood vessels and fibrous tissue grow into the material (Fig. 48, Fig. 52, Fig. 53); red-stained osteogenic tissue increased in the material at 8 weeks, and osteoblasts formed collagen tissue on the surface of the material (Fig. 49, Fig. 54, Fig. 55); red-stained bone tissue increased in the material at 12 weeks, distributed along the surface of the material, blood vessels increased, and collagen tissue formed around blood vessels (Fig. 50, Fig. 56); most of the material was absorbed at 24 weeks , the interior is filled with new bone tissue, the bone tissue is rich in blood vessels, the material is osseointegrated with the new bone tissue, and the texture is disordered (Fig. 51, 57).

Group D: New blood vessels and fibrous tissue grew into the material at 2 weeks, more than that of group C, a large number of cells infiltrated, and no obvious signs of infection (Figure 47); at 4 weeks, more blood vessels and fibrous tissue grew into the material, and new bone was seen in the material Tissue and collagen are distributed along the surface of the material (Fig. 48, Fig. 52, Fig. 53); at 8 weeks, new bone increased and gradually grew from both ends of the defect, and red-stained osteoblast tissue appeared in the material, with large osteoblast cells and deep staining. Mesenchymal cells have shallow nuclei, multiple protrusions, and are distributed along the edge of the material (Fig. 49, Fig. 54, and Fig. 55); at 12 weeks, the new bone increased, the material was partially degraded and absorbed, the arrangement of the new bone trabeculae was disordered, and the cells were active in osteogenesis. The bone was osseointegrated (Fig. 50, Fig. 56); at 24 weeks, all the materials were absorbed, the new bone trabeculae were arranged neatly, the calcification was good, and the medullary cavity was remodeled (Fig. 51, Fig. 57).

(5) Calculation of bone formation area

The obtained digital photos were analyzed with the histogram function of Photoshop CS6 software to measure the area of tissue ingrowth (Figure 61), that is, the rate of tissue ingrowth=pixels of tissue ingrowth ÷ pixels of the whole picture×100%. The results were statistically analyzed by SPSS 17 software, and the tissue ingrowth rate of different groups of scaffolds was calculated by calculation. Statistical analysis (Table 4), there were differences in the amount of bone formation in each group at different time points (P<0.05), 4 There was no significant difference in bone mass between group C and group D before 4 weeks (P>0.05), but after 4 weeks, the bone mass of group D was more than that of group C. There was always a difference between group C and group B during the observation period (P<0.05), and there was no significant difference between group D and group B at 12 weeks (P>0.05). There was no significant difference in bone formation between group B and group D after 12 weeks, indicating that the final bone formation effect of the two groups was equivalent (Table 5, Figure 59).

Table 5 Osteogenesis area comparison at different time points in each group ( n=12)(%)

time Two weeks 4 weeks 8 weeks 12 weeks 24 weeks Group A 0±0 0±0 0±0 0±0 0±0 Group B 7.68±2.59 ^□■ 9.57±1.55 ^□■ 39.88±5.65 ^□■ 48.30±7.78 ^□ 71.92±6.39 ^□ Group C 3.15±1.94 ^□ 4.07±1.12 ^□ 4.88±0.12 ^▲ 7.37±3.42 ^□▲ 34.66±4.41 ^▲ Group D 3.36±1.78 ^■ 5.70±1.27 ^■ 26.56±5.21 ^▲ 42.88±8.59 ^▲ 67.81±3.57 ^▲ f 8.661 27.324 93.778 60.871 30.145 P <0.05 <0.05 <0.05 <0.05 <0.05

Note: Compared with group B and C, P<0.05; ^■ Compared with group B and D, P<0.05; ^▲ Compared with group C and D, P<0.05;

The F value indicates the significance of the entire fitting equation, and the larger the F, the more significant the equation and the better the fitting degree. The P value indicates the extent to which the null hypothesis is not rejected, and P<0.5 indicates that the hypothesis is more likely to be correct, otherwise it may be wrong.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Variations and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. a preparation method of chitosan-coated BCBB bone repair scaffold material of slow-release SDF-1, is characterized in that, comprises the steps: Step (1), dissolving chitosan in 1% acetic acid solution by volume until the chitosan concentration is 20 mg/ml, and then adding SDF-1 to it until the SDF-1 concentration is 400 ng/ml to obtain SDF-1 /CS sustained release solution; In step (2), the BCBB scaffold was placed in the SDF-1/CS slow-release solution, ultrasonicated for 30 minutes, taken out and dried to obtain the chitosan-coated BCBB bone repair scaffold material with slow-release SDF-1. 2. the preparation method of the chitosan coating BCBB bone repair support material of slow release SDF-1 according to claim 1, it is characterized in that, described chitosan viscosity is 50～800mpas, and deacetylation degree is 90 ~95%. 3. the preparation method of the chitosan-coated BCBB bone repair support material of slow-release SDF-1 according to claim 1, is characterized in that, when chitosan is dissolved in volume concentration and is in 1% acetic acid solution, adopts Stir to dissolve chitosan; after adding SDF-1, stir until it is evenly mixed. 4. The preparation method of the chitosan-coated BCBB bone repair scaffold material of the slow-release SDF-1 according to claim 2, wherein the stirring time is 2h. 5. the preparation method of the chitosan-coated BCBB bone repair scaffold material of sustained-release SDF-1 according to claim 1, is characterized in that, the frequency of ultrasound is 40kHz. 6. The preparation method of the chitosan-coated BCBB bone repair scaffold material of sustained-release SDF-1 according to claim 1, characterized in that, the drying method is natural air drying. 7. the preparation method of the chitosan-coated BCBB bone repair scaffold material of slow release SDF-1 according to claim 1, is characterized in that, the preparation method of BCBB support comprises the steps: Use the commercially available cancellous bone of fresh pork bone, boil it in distilled water for at least 6 hours, dry it overnight, then raise the temperature in the muffle furnace to 800°C at a rate of 10°C/min, keep the temperature for calcination for 1h, and then put it in a concentration of 0.04 Sonicate in mol/ml sodium pyrophosphate solution for 30 minutes, take it out, dry it overnight, then raise the temperature in the muffle furnace to 1150°C at a rate of 10°C/min, then keep the temperature for calcination for 1h, after natural cooling, sterilize under high temperature and high pressure at 120°C , to prepare BCBB scaffolds. 8. the preparation method of the chitosan-coated BCBB bone repair support material of slow-release SDF-1 according to claim 7, is characterized in that, before poaching, the cancellous bone of pig bone needs to be prepared into size range earlier 0.5cm×0.5cm×0.5cm to 1.0cm×1.0cm×1.0cm blocks or 1.5cm×0.5cm×0.5cm to 2.0cm×1.0cm×1.0cm strips. 9. The preparation method of the chitosan-coated BCBB bone repair scaffold material of sustained-release SDF-1 according to claim 7, wherein the frequency of ultrasound is 40kHz. 10. the chitosan of the slow-release SDF-1 described in any one of claim 1-9 is coated with the preparation method of BCBB bone repair support material The chitosan of slow-release SDF-1 that the preparation method makes is coated with BCBB bone repair support The method of preparation of the material.