CN112370572A

CN112370572A - Bone repair material for treating large bone defect and preparation method and application thereof

Info

Publication number: CN112370572A
Application number: CN202011319177.XA
Authority: CN
Inventors: 邢军超; 贺思豪; 侯天勇; 窦策; 周江玲; 谭玖林; 许建中
Original assignee: First Affiliated Hospital of PLA Military Medical University
Current assignee: Nanfang Hospital; First Affiliated Hospital of PLA Military Medical University
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2021-02-19

Abstract

The invention discloses a preparation method of a bone repair material for treating large bone defect, which comprises the following steps: 1) preparing a biotinylated decalcified bone matrix scaffold; 2) preparing a biotinylated human IL-4 protein solution; 3) soaking a biotinylated decalcified bone matrix scaffold in an avidin solution, and then soaking the biotinylated decalcified bone matrix scaffold in a biotinylated IL-4 solution to obtain the decalcified bone matrix scaffold coupled with IL-4 protein; 4) soaking the decalcified bone matrix scaffold coupled with the IL-4 protein in a sodium alginate solution containing recombinant human IFN-gamma protein, transferring the decalcified bone matrix scaffold coupled with the IL-4 protein into a calcium oxide solution for soaking after a certain time, repeating the soaking for a plurality of times until light white floccules are attached to the surface of the scaffold, and then washing with deionized water to obtain the bone repair material. The bone repair material provided by the invention can sequentially release inflammatory factors and promote angiogenesis.

Description

Bone repair material for treating large bone defect and preparation method and application thereof

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a bone repair material for treating large bone defect and a preparation method and application thereof.

Background

The major bone defect is a difficult problem in clinical treatment of bone tissue injury, and one of the important reasons is lack of effective and reliable bone repair materials with tissue reconstruction function. The research and development of bone repair materials focuses on the osteogenesis capacity, and the basic condition of vascularization of the osteogenesis activity is often ignored. Studies have shown that the osteogenic activity of bone grafts is always closely related to the process of vascularization, the extent of which even directly determines the success or failure of a bone graft. Early good vascularization not only provides the necessary conditions for oxygen and nutrients delivery, metabolic waste removal, and migratory colonization of host osteogenic-related cells such as MSCs^[5,6]. Macrophage (Mp) polarization, which is regulated by the synergistic effect of local inflammatory factors, plays an important role in vascular and bone remodeling in ischemic areas of bone defects. Macrophages are classified into classical activated macrophage (classical activated macrophage), i.e., M1 type macrophage, and alternative activated macrophage (alternative activated macrophage), i.e., M2 type macrophage, and in the early stage of tissue repair, M1 type macrophage exerts a pro-inflammatory function, and in the later stage, M2 type macrophage exerts effects of anti-inflammation, promotion of damaged tissue repair, and vascular growth. Currently, no bone repair material based on macrophage polarization regulation mechanism exists, and the condition of failure of bone grafting often occurs due to unsatisfactory vascularization process.

Disclosure of Invention

The invention aims to provide a bone repair material which can sequentially release inflammatory factors and promote vascularization and is used for treating large bone defects.

The invention provides a preparation method of a bone repair material for treating large bone defect, which comprises the following steps:

1) soaking the decalcified bone matrix scaffold in Biotin-sulfo-LC-LC-NHS for a certain time, washing and sterilizing, and then cleaning to obtain a biotinylated decalcified bone matrix scaffold;

2) adding Sulfo-NHS-Biotin powder into the recombinant human IL-4 protein solution, carrying out protein biotinylation at room temperature, dialyzing, and carrying out sterile filtration to obtain a biotinylated IL-4 solution;

3) soaking a biotinylated decalcified bone matrix scaffold in an avidin solution, taking out and washing, soaking the biotinylated decalcified bone matrix scaffold in a biotinylated IL-4 solution, and washing after a certain time to obtain the decalcified bone matrix scaffold coupled with IL-4 protein;

4) soaking the decalcified bone matrix scaffold coupled with the IL-4 protein in a sodium alginate solution containing recombinant human IFN-gamma protein, transferring the decalcified bone matrix scaffold coupled with the IL-4 protein into a calcium oxide solution for soaking after a certain time, repeating the soaking for a plurality of times until light white floccules are attached to the surface of the scaffold, and then washing with deionized water to obtain the bone repair material.

In one embodiment according to the present invention, the decalcified bone matrix scaffold in step 1) is prepared by a method comprising the steps of:

taking a cancellous bone block at the near-far end of a tibia or a femur of a pig, putting the cancellous bone block into a closed glass container of 0.6M hydrochloric acid, soaking for 24 hours at normal temperature, replacing the hydrochloric acid every 8 hours until the cortical bone is in a semitransparent flexible elastic state, taking out the cancellous bone block when the cancellous bone block is automatically restored after being compressed in a spongy manner, and repeatedly washing and soaking the cancellous bone block by sterile distilled water until the PH value is 7 to obtain the decalcification bone matrix support.

In one embodiment according to the present invention, the concentration of Biotin-sulfo-LC-LC-NHS in step 1) is 10 mM; preferably, the disinfection is performed by soaking in a 70% ethanol solution for 10 minutes; the rinsing and washing was carried out by PBS rinsing.

In one embodiment according to the invention, the final concentration of Sulfo-NHS-Biotin in step 2) is 10 mM; preferably, the dialysis is performed in PBS.

In one embodiment according to the invention, the concentration of the avidin solution is 100-250. mu.g/ml.

In one embodiment according to the invention, the IFN- γ concentration in step 4) is 1-2mg/ml and the sodium alginate concentration is 60-90 mg/ml.

In one embodiment according to the invention, the calcium chloride solution has a concentration of 10.1 mg/ml.

In one embodiment according to the present invention, in step 4), after the decalcified bone matrix scaffold coupled with the IL-4 protein is soaked in the sodium alginate solution containing the recombinant human IFN-gamma protein for 30min, the decalcified bone matrix scaffold coupled with the IL-4 protein is transferred to the calcium oxide solution and soaked for 10min, and the process is repeated for 3 times.

The invention also provides the tissue engineering bone scaffold prepared by the preparation method.

The invention further provides application of the bone repair material in preparing a material for treating large bone defects.

The invention has the beneficial effects that: the bone repair material for treating large bone defect provided by the invention is based on the important functions of Mp in immune defense, angiogenesis and osteogenesis and different physiological functions thereof under different polarization states, and improves the vascularization level in the transplantation environment by regulating the polarization state of the Mp of the host. The bone repair material provided by the invention can sequentially and controllably activate M1 and M2 Mp, can fully adjust the anti-infection and tissue injury repair capability of a host, and can form a cascade amplification regulation effect on angiogenesis and bone repair by sequentially regulating the secretion function of the Mp.

Drawings

FIG. 1 is a graph of the general view of a material and the result of a scanning electron microscope (n-5);

figure 2 is a line graph of IFN- γ and IL-4 sustained release kinetics (n-5);

FIG. 3 is a graph comparing the change in mRNA expression (n-5) in Mp cells; wherein, a is P <0.05, compared with Ctrl group; p <0.05, compared to IL-4 group; p <0.05, compared to IFN group;

fig. 4 is a graph comparing changes in Mp cellular protein expression (n-5); wherein, a is P <0.05, compared with Ctrl group; p <0.05, compared to IL-4 group; (ii) a P <0.05, compared to IFN group.

FIG. 5 is a Transwell cell migration experiment (n-5); a result graph; wherein P <0.05, compared to Ctrl group;

fig. 6 is a graph showing the results of the lumen formation experiment (n ═ 5).

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly define the scope of the invention.

Materials and reagents

Human bone marrow derived Endothelial Progenitor Cells (EPC) were purchased from ATCC (PCS-800-012) of the United states;

human peripheral blood-derived mononuclear macrophages were purchased from ATCC (CRL-9855) of USA;

endothelial Cell Medium (ECM), 0.25% trypsin, Fetal Bovine Serum (FBS), macrophage colony stimulating factor (M-CSF), IMDM, DMEM/F12 medium were purchased from HyClone, USA;

sodium alginate, Dil-ac-LDL, FITC-UEA-1, Biotin-sulfo-LC-LC-NHS, ELISA kit and Takara reverse transcription kit are purchased from Sigma company in the United states;

recombinant human IL-4, IFN-gamma proteins were purchased from PeproTec, USA; inhibitors were purchased from seleck, usa.

Example 1 Decalcified Bone Matrix (DBM) Material preparation

Relying on a southwest hospital bone bank, a DBM bracket is prepared from proximal and distal cancellous bone blocks of tibia and femur of Yunnan Xiaoxiang pigs, and the size of the DBM bracket is about 3x3x3mm³. Firstly, decalcifying DBM, adding 0.6M salt into a sealed glass containerAnd (3) putting the DBM scaffold into the acid for soaking. Soaking at normal temperature for 24 hours, changing hydrochloric acid once every 8 hours, when cortical bone is in a semitransparent elastic state capable of bending, automatically recovering the cancellous bone after the cancellous bone is compressed in a spongy manner, taking out the bone, repeatedly washing and soaking by sterile distilled water until the pH value is 7, sucking out soaking liquid, placing the decalcified DBM support material in a centrifuge tube, centrifuging for 5min at 300g, carrying out irradiation treatment on the decalcified DBM, drying by a sterilization vacuum freeze dryer, subpackaging, soaking the DBM in 10mM Biotin-sulfo-LC-NHS for 1 hour, washing by PBS for three times, soaking in PBS for 24 hours at 4 ℃ for later use.

Example 2 protein biotinylation and scaffold conjugation

Preparation of DBM: the scaffold was prepared according to the method described above: the scaffold material is prepared from proximal and distal cancellous bone blocks of tibia and femur of Yunnan miniascape by relying on a bone bank of a first subsidiary hospital of army medical university. 0.6M hydrochloric acid is added into a closed glass container, and the bracket is put into the closed glass container to be soaked for decalcification. Soaking at normal temperature for 24h, replacing hydrochloric acid once every 8h, when cortical bone is in a semitransparent elastic state capable of bending, spongy bone blocks are automatically restored after being compressed in a spongy manner, taking out the bone blocks, repeatedly washing and soaking the bone blocks by sterile distilled water until the pH value is 7, sucking out soaking liquid, placing DBM support materials in a centrifuge tube, centrifuging the bone blocks for 5min at 300g, performing irradiation treatment on the DBM for sterilization, and subpackaging the DBM for later use after being dried by a vacuum freeze dryer.

Biotinylation of DBM: taking a DBM bracket, soaking the DBM bracket in PBS, and trimming the DBM bracket to be about 3x3x3mm in size³The membrane was soaked in 10mM Biotin-sulfo-LC-LC-NHS for 1 hour, washed 3 times with PBS to remove non-attached Biotin, soaked in 70% ethanol solution for 10 minutes, washed with PBS, soaked in PBS, and soaked at 4 ℃ for 24 hours.

Biotinylation of the protein: the concentration of the recombinant human IL-4 protein solution is 1mg/ml, 10mMSulfo-NHS-Biotin powder is added into the solution, the solution is placed at room temperature for 1h to complete protein biotinylation, PBS dialysis is carried out to remove unattached Biotin, and the solution is stored at 4 ℃ for standby after sterile filtration.

Scaffold-protein coupling: the biotinylated DBM scaffolds were soaked in 172ug/ml avidin solution for 1h, washed 3 times with PBS, and then the IL-4 and combo groups were soaked in biotinylated IL-4 solution for 1h, the IFN and Ctrl groups were soaked in PBS solution for 1h, and washed three times with PBS. Since avidin provides 4 high affinity biotin binding sites for its specific binding, the non-covalent linkage between IL-4 and the scaffold is very strong.

Constructing a composite material: preparing a sodium alginate solution containing recombinant human IFN-gamma protein (the concentration of IFN-gamma is 1mg/ml, the concentration of sodium alginate is 80mg/ml), soaking the IL-4 coupled scaffold material for 30min, soaking a Ctrl group and an IL-4 group in the sodium alginate solution without IFN-gamma for 30min, soaking in a 10.1mg/ml calcium chloride solution for 10min, repeating the steps for three times, observing the microstructure of the material under an electron microscope, wherein light white floccule is attached to the surface of the material, washing with deionized water, freezing and drying at-80 ℃ for later use. The grouping is as follows:

TABLE 1 grouping of materials

Example 3 Slow Release Power detection

Placing the 4 th group of composite materials in 10ml of DMEM/F12 culture medium, placing the DMEM/F12 culture medium in a 37 ℃ incubator, replacing liquid every day and collecting the culture medium, centrifuging the culture medium at 10000r/min for 10 minutes, taking supernate, measuring the concentrations of IL-4 and IFN-gamma through an ELISA kit, and calculating the cumulative release amount.

Example 4 Effect of composite materials on macrophage polarization

Taking Mp of M-CSF for 5 days, and stimulating at 1 × 10⁶The concentration of the solution/ml is inoculated to each group of materials by adopting a double-sided standing inoculation method, a culture medium is added, and the culture medium is replaced after 3 days of culture. The culture medium was collected at 1, 4 and 7 days of culture, and the protein content thereof was measured by ELISA. Taking materials of each group at the same time point, grinding by liquid nitrogen, extracting mRNA, carrying out reverse transcription according to the instruction of a Takara reverse transcription kit, and then carrying out Real-time PCR. The primer sequences and the lengths of the amplification products are shown in Table 2. The amplification conditions were 95 ℃ for 30s, [95 ℃ for 5s (denaturation), [ 57 ℃ for 20s (annealing), ] and 72 ℃ for 15s (extension)]X 40 cycles, 95 ℃ 30s, 57 ℃, 30s, 95 ℃ 30 s.

TABLE 2 RT-PCR primer sequences

Example 5 method and results for examining osteogenic Effect

1. Transwell experiment

Taking EPC and re-suspending the EPC by ECM to prepare single cell suspension, and adjusting the cell concentration to be 1 multiplied by 10⁴And/ml. Transwell chambers were assembled in a 24-well plate, and 700. mu.l of each of the culture media after 3 days of culture was added to each of the chambers in the lower part according to experimental groups. 200 μ l of single cell suspension containing single cell suspension, axitinib (VEGFR2 inhibitor) or SB225002(IL-8 inhibitor) pretreated EPC were added to the transwell upper chamber according to experimental groups. The transwell chamber was placed in an incubator at 37 ℃ under 5% CO2 and was terminated after 6 hours of migration. The upper layer of carbonate membrane was wiped with a sterile cotton swab to remove non-migrated cells. The carbonate membrane was washed 3 times with PBS. Fixed with 4% paraformaldehyde for 15min, and washed 3 times with PBS. 0.5% Triton-X100 membrane rupture for 10min, washing with PBS for 3 times, staining with DAPI working solution (1 μ g/ml) in dark for 5-8min, and washing with PBS for 3 times. 10 high power fields (200X) were randomly selected for each carbonate membrane under a fluorescence microscope and counted, and the number of cell migration in each group was counted and compared.

Effect of composite materials on endothelial progenitor cell migration

The results of Transwell cell migration show (FIG. 5) that IFN-gamma and Combo group conditioned media significantly enhanced EPC migration; although the Combo group has high VEGF expression level, no significant change of EPC migration is observed after VEGF receptor (Axitinib) is blocked; while blocking IL-8(SB225002) significantly inhibited the recruitment of EPC by Combo group conditioned media.

2. Tube cavity formation experiment

The Matrigel gel was slowly thawed overnight at 4 ℃, added to a 96-well plate (50 ul/well), allowed to stand at 37 ℃ for 30 minutes, and the media was prepared at a concentration of 2X 10 for 7 days of incubation in each group⁵50ul of EPC single cell suspension was added to each well, incubated at 37 ℃ in an incubator for 8h and then subjected to microscopic examination.

The results of the tube cavity formation experiments (fig. 6) show that the IL-4 and Combo group conditioned medium can significantly promote tube cavity formation; the VEGF receptor (Axitinib) blocking effect can obviously inhibit the angiogenesis promoting effect of the Combo group conditioned medium, and the IL-8(SB225002) blocking effect on the angiogenesis promoting effect of the Combo group conditioned medium is little.

3. Statistical analysis

Data are expressed as mean ± standard error, SPSS 11.0 statistical software is used to perform one-way analysis of variance and Least Significant Difference (LSD), P <0.05 is the difference with statistical significance.

Example 6 Material Properties

The appearance of DBM is shown in FIG. 1A, and a pale gel is formed on the surface of the stent pores after repeated soaking of IFN-gamma alginate mixed solution and calcium chloride solution (FIG. 1B). Scanning electron microscope results show that the Ctrl group scaffold has uniform porosity and smooth micropore surface (figure 1C), IL-4 and IFN-gamma group visible protein matrix are uniformly shrunk and distributed on the scaffold surface (figures 1D and E), and IFN-gamma group and Combo group form gel-like folds due to the reaction of IFN-gamma alginate mixed solution and calcium chloride solution (figure 1F).

Example 7 sustained Release kinetics of composite materials

As shown in FIG. 2, the composite material was stable in binding to IFN-. gamma.and IL-4. The composite material achieves the effect of early release by an alginate slow-release system and IFN-gamma outer layer load, the IFN-gamma is stably and rapidly released in the first 4 days, and the release is nearly complete in 4 days; the composite material realizes the inner layer coupling of the IL-4 through a biotin-avidin system to achieve the slow release effect, the IL-4 is not obviously released in the first 3 days, is quickly and stably released in the 4 th to 7 th days, and is released a little every day till the complete release in the 13 th day.

Example 8 Effect of composite materials on Mp polarization State

The PCR results show (FIG. 3) that the early release of IFN- γ polarizes Combo and IFN group macrophages to M1 type, and on day 1 of incubation IFN- γ induces Mp to release M1 marker TNF- α, showing a partial trend of M1 type polarization; on day 4, IFN- γ was released such that Mp expressed the M1 markers CCR7, TNF- α and IL-1 β; on day 7, there was no significant difference in the expression level of the M1 type marker compared to the control group. IL-8 was highly expressed by cells in all three groups coupled with inflammatory factors on days 1 and 4, and IL-8 expression was not significantly different between groups on day 7. The stable release of IL-4 on days 4-7 polarizes IL-4 group macrophages to M2 type, and IL-4 group cells highly express M2 type marker CD206 on day 1 of incubation, which confirms that IL-4 can induce M2 type cell polarization; on day 4, the IL-4 group cells had significantly increased CD206 expression, while the composite group was not significantly different from the control; on day 7, the IL-4 group cells showed no significant difference in CD206 and Ctrl, while the composite group CD206 expression was significantly elevated; on days 4 and 7, the composite material group has a significant increase in the expression level of the M2 type marker CCL18 and PDGF-BB due to the release of IL-4. For VEGF expression, the Combo group cells expressed VEGF at day 1, significantly higher than that of the IL-4 group, comparable to the IFN group levels, with VEGF expression peaking at day 4, still high at day 7, and significantly higher than that of the control group.

ELISA results showed (FIG. 4), that on days 1, 4 of incubation, IFN-. gamma.release resulted in the high expression of Mp the M1-type markers TNF-. alpha., IL-1. beta., and the chemokine IL-8; the release of IL4 leads to the high expression of Mp in M2 type marker group CCL18, PDGF-BB; at day 4 and day 7, the composite significantly promoted secretion of the M2-type polarization marker of Mp due to release of IL-4. For the expression change trend of VEGF, the ELISA result is more consistent with the PCR result.

The above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements may be made by those skilled in the art within the spirit and principles of the invention. The scope of the invention is to be determined by the appended claims.

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Claims

1. A method of preparing a bone repair material for treating a large bone defect, comprising:

2. The method for preparing a bone repair material according to claim 1, wherein the decalcified bone matrix scaffold in the step 1) is prepared by a method comprising the steps of:

3. The method for preparing a bone repair material according to claim 1, wherein the concentration of Biotin-sulfo-LC-LC-NHS in step 1) is 10 mM; preferably, the disinfection is performed by soaking in a 70% ethanol solution for 10 minutes; the rinsing and washing was carried out by PBS rinsing.

4. The method for preparing a bone repair material according to claim 1, wherein the final concentration of Sulfo-NHS-Biotin in step 2) is 10 mM; preferably, the dialysis is performed in PBS.

5. The method for preparing bone repair material according to claim 2, wherein the concentration of the avidin solution is 100 μ g/ml.

6. The method for preparing a bone repair material according to claim 1, wherein the concentration of IFN- γ in step 4) is 1-2mg/ml and the concentration of sodium alginate is 60-90 mg/ml.

7. The method of preparing a bone repair material according to claim 1, wherein the calcium chloride solution has a concentration of 10.1 mg/ml.

8. The method for preparing a bone repair material according to claim 1, wherein in the step 4), the decalcified bone matrix scaffold coupled with the IL-4 protein is soaked in a sodium alginate solution containing recombinant human IFN- γ protein for 30min, and then the decalcified bone matrix scaffold coupled with the IL-4 protein is transferred to a calcium oxide solution and soaked for 10min, and the process is repeated 3 times.

9. Bone repair material for the treatment of large bone defects prepared by the preparation method according to any one of claims 1 to 8.

10. Use of a bone repair material according to claim 8 in the preparation of a material for the treatment of a large bone defect.