阅读说明：本技术 一种cha明胶支架 (CHA gelatin support ) 是由 杨佩 李沂阳 �田润 王坤正 孔宁 刘冠志 关焕帅 李哲 魏启鲁 焦鸣 严峻腾 于 2021-05-28 设计创作，主要内容包括：本发明公开了一种CHA明胶支架,通过以下方法制备所得：将5 g来自牛骨的B型明胶溶入50 ml含有二水合柠檬酸钠的蒸馏水中,作为分散剂；明胶完全溶胀后,加入0.37 g氢氧化钙,并使用磁力搅拌器在37℃下以200 rpm的转速混合30分钟；将0.24 g磷酸稀释在50 ml蒸馏水中,并将所得的磷酸溶液滴定到明胶水凝胶溶液中,15分钟；合成成熟度持续3小时,并定期控制pH,最终将pH调节至7.4；称取Gel‐CHA05溶液9.5g,倒入8×8 cm～(2)的溶液培养皿内,在4℃的冰箱中干燥,然后置于真空烘箱内,在142℃加热72小时,以诱导支架的交联形成,低温环氧乙烷中进行气体灭菌。(The invention discloses a CHA gelatin scaffold which is prepared by the following method: dissolving 5g of type B gelatin from bovine bone in 50 ml of distilled water containing sodium citrate dihydrate as a dispersant; after the gelatin had fully swelled, 0.37 g of calcium hydroxide was added and mixed using a magnetic stirrer at 37 ℃ for 30 minutes at 200 rpm; 0.24 g of phosphoric acid was diluted in 50 ml of distilled water, and the resulting phosphoric acid solution was titrated into a gelatin hydrogel solution for 15 minutes; the synthesis maturity lasts for 3 hours, the pH is controlled periodically, and finally the pH is adjusted to 7.4; 9.5g of Gel-CHA05 solution was weighed and poured into 8X 8 cm 2 In a hydroponic culture dish ofDried in a refrigerator at 4 ℃ and then placed in a vacuum oven heated at 142 ℃ for 72 hours to induce cross-linking of the scaffold, gas sterilized in low temperature ethylene oxide.)

1. A CHA gelatin scaffold characterized by: the preparation method comprises the following steps:

s1, dissolving 5g of B-type gelatin from ox bone into 50 ml of distilled water containing sodium citrate dihydrate to be used as a dispersing agent;

s2, after gelatin is completely swelled, 0.37 g of calcium hydroxide is added, and the mixture is mixed for 30 minutes at 37 ℃ and 200 rpm by using a magnetic stirrer;

s3, diluting 0.24 g of phosphoric acid in 50 ml of distilled water, and titrating the resulting phosphoric acid solution into a gelatin hydrogel solution for 15 minutes; the synthesis maturity lasts for 3 hours, the pH is controlled periodically, and finally the pH is adjusted to 7.4;

s4, weighing 9.5g of Gel-CHA05 solution, and pouring into 8 x 8 cm²The culture dish was dried in a refrigerator at 4 ℃ for 8-12 hours, then placed in a vacuum oven and heated at 142 ℃ for 72 hours to induce the formation of the scaffold, and gas-sterilized in low-temperature ethylene oxide.

Technical Field

The invention relates to the field of biology, and in particular relates to a CHA gelatin scaffold.

Background

In previous researches, different types of scaffolds (such as various types of metal, gel and composite material scaffolds) are often adopted for bone defects and a method for carrying seed cells (the most widely applied bone marrow mesenchymal stem cells) is adopted, but the problems that the regeneration and the poor differentiation of the seed cells in the scaffolds cause the unsatisfactory treatment effect of the seed cells exist, and the like. In order to enable seed cells in the scaffold to better differentiate and grow so as to enable the seed cells to play a better role, the mesenchymal stem cells in the scaffold are planned to obtain certain advantages in the early repair process through the covering of a layer of barrier membrane, so that the mesenchymal stem cells can be better differentiated into osteoblasts to promote osteogenesis, and meanwhile, the scaffold can also play a certain supporting role in a collagen barrier.

Disclosure of Invention

To solve the above problems, the present invention provides a CHA gelatin scaffold.

In order to achieve the purpose, the invention adopts the technical scheme that:

a CHA gelatin scaffold prepared by the following method:

s1, 5g of type B Gelatin from bovine bone (Nitta Gelatin Inc.) was dissolved in 50 ml of a solution containing sodium citrate dihydrate (C)₆H₅Na₃O₇·2H₂O, merck, darmstadt, germany) as a dispersant;

s2, after gelatin is fully swelled, 0.37 g calcium hydroxide (Ca (OH) is added₂) (Tokyo and Guangshi, Japan) and mixed at 37 ℃ for 30 minutes at a rotation speed of 200 rpm using a magnetic stirrer;

s3, mixing 0.24 g phosphoric acid (H)₃PO₄Merck) in 50 ml of distilled water and titrating the resulting phosphoric acid solution into a gelatin hydrogel solution for about 15 minutes; the synthesis maturity lasts for 3 hours, the pH is controlled periodically, and finally the pH is adjusted to 7.4;

s4, weighing 9.5g of Gel-CHA05 solution, and pouring into 8 x 8 cm²The culture dish was dried in a refrigerator (MPR-213F Sanyo, Osaka, Japan) at 4 ℃ for 8 to 12 hours, and then placed in a vacuum oven (VO-400, Memmert GmbH + Co., Schwarbach, Germany) and heated at 142 ℃ for 72 hours to induce scaffoldAnd (3) performing gas sterilization in low-temperature ethylene oxide.

The invention has the following beneficial effects:

by combining the collagen membrane with the hydroxyapatite-gelatin scaffold, the mechanical strength of the collagen membrane is improved, secondary operation is avoided, and the mesenchymal stem cells in the scaffold can obtain certain advantages in the early repair process depending on the selective permeability function of the collagen membrane, so that the mesenchymal stem cells can be better differentiated into osteoblasts to promote osteogenesis. At present, relevant researches prove that the collagen membrane has the capacity of promoting bone formation of the bone marrow mesenchymal stem cells, and the research innovatively adopts a mode that a hydroxyapatite-gelatin bracket carries the bone marrow mesenchymal stem cells and is combined with the collagen membrane, so that the mechanical property of the collagen membrane is improved, and the bone formation is promoted.

Drawings

FIG. 1 is an FTIR spectrum of Gel-CHA00 and Gel-CHA05 scaffolds;

in the figure: a: Gel-CHA 00; b: Gel-CHA 05.

FIG. 2 is a schematic view of a scaffold material in combination with a collagen membrane for treating a bone defect.

Fig. 3 is a photograph of mesenchymal stem cells under a light microscope.

FIG. 4 is a flow cytometry identification of surface antigens.

FIG. 5 shows the morphology of mesenchymal stem cells under electron microscope.

FIG. 6 is a schematic of HE and Masson staining of tissue at the repair of bone defects for each group.

In the figure: a, B: blank control group; c, D: a collagen membrane group; e, F: collagen membrane plus scaffold group.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Detection of membranes

First, XRD machine, FTIR spectrometer

The samples were examined using a RINT 2500V XRD machine, Fourier Transform Infrared (FTIR) spectrometer, and FTIR spectra for Gel-CHA00 and Gel-CHA05 scaffolds are shown in FIG. 1. For Gel-CHA05, the absorption peak appearing at a wavelength of 3424 cm-is related to the stretching vibration of-OH groups; the band of amide II at 1638-1655 cm-is caused by stretching vibration of the C = O bond. The characteristic absorption peak of PO 4-group V3 is at 1034cm-, and the absorption peaks caused by bending vibration of phosphate P-O bond in HA are at 565 cm-and 605cm-, indicating the existence of HA on the surface of the composite material. The appearance of an absorption peak at 1334 cm-indicates the formation of a Ca-COO chemical bond between the carboxyl group of Gel and the calcium ion of HA in the HA-Gel complex and therefore FTIR spectroscopy confirmed that CHA was completely formed during the synthesis of Gel-CHA05 scaffold involving chemical wet precipitation inside the gelatin system.

Secondly, the existence of cytotoxicity

Cell seeding

Placing the scaffold in a six-well plate and immersing in a volume of 4ml of complete growth medium for 1 hour before cell inoculation to achieve optimal swelling;

② cells were inoculated into 2 experimental groups after 1 hour: cell culture was performed in stentless medium and in Gel-CHA 05-containing medium, each well plate containing 1X 10 cells⁵(ii) individual cells;

③ the medium was collected every second day for further analysis and replaced with fresh medium.

Physiological properties of the membrane:

1: cell compatibility (MTT/CCK-8)

Cutting the nanofiber membrane into a circle with the diameter of 15 mm and the thickness of 0.043 mm, and placing the circle into a 24-hole plate to sterilize for 6 hours under UV radiation before cell inoculation;

② MSC (2 is multiplied by 10)⁴Individual cells/well) were seeded onto membranes for cell proliferation and adhesion assays;

③ 3, 7 and 14 days after the culture, the cell proliferation was measured by the 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenylammonium tetrabromide bromide (MTT) method (n = 3) and passed through a microplate reader at 490 nm.

2. Detecting apoptosis:

1 is multiplied by 10⁵MSCs/hole are inoculated on the nanofiber membrane of the 6-hole plate;

② then cultured for 3 days, and then analyzed with Annexin V-EGFP/PI apoptosis kit (KeygEN BioTECH, Nanjing, China) (n = 3). Results were evaluated under a flow cytometer (BD FACSCalibur, usa);

3. morphology of the cultured MSCs was observed by SEM.

Real-time PCR

Osteogenic gene expression of Runt-related transcription factor 2 (RUNX 2, collagen α 1 (COL 1a 1) and Osteopontin (OPN) was determined by quantitative polymerase chain reaction (qPCR.) briefly, cells were harvested, total RNA was isolated, cDNA synthesis was performed, relative gene expression was calculated from the quantitative cycle (Cq) values obtained by RT-PCR based on the Δ Δ Cq method, three independent PCR reactions were performed for each sample.

1. RNA extraction

The method comprises the following steps: the TRlzol method. The main components are as follows: phenol (which acts to lyse cells and denature proteins).

Preparation before RNA extraction: preparation of reagent consumables (note RNase free), preparation of samples.

The method comprises the following steps: cracking the sample with 1ml of TRlzol at room temperature for 10min, oscillating with 200 mu L of chloroform for 30s, and standing at room temperature for 2-3 min; low temperature centrifugation at 4 ℃ (12000 g × 20 min); precipitating 400 μ L of upper water phase +600 μ L of isopropanol at room temperature for 10 min; low temperature centrifugation at 4 ℃ (12000 g × 20 min); washing with 75% ethanol; the precipitate is dried in the air and dissolved in DEPC-treated water.

2. RNA quality detection

The method comprises the following steps: spectrophotometric measurement, gel electrophoresis method

3. Reverse transcription

Different reverse transcription primers are selected according to different conditions in the process:

1. oligo dT: suitable for RNA with a PolyA tail. (prokaryotic RNA, eukaryotic rRNA and tRNA do not have PolyA tails.) Oligo dT is bound to PolyA tails, so that the quality requirement on RNA samples is high, and even a small amount of degradation can greatly reduce the synthesis amount of full-length cDNA.

2. Random primer (random primer) is suitable for RNA with long or hairpin structure. It is suitable for reverse transcription reaction of all RNAs such as rRNA, mRNA, tRNA and the like.

3. Specific primer (Specific primer) a primer complementary to the template sequence, as appropriate for the case where the sequence of interest is known.

4. PCR procedure (three-step method)

The first step is as follows: denaturation at 94 ℃ for 30 s;

second step (cycle 30-35 times): denaturation at 94 ℃ for 30s, annealing at 55-60 ℃ for 30s, and extension at 72 ℃ for 24s (time calculated from synthesis rate and length);

the third step: the extension is terminated at 72 ℃ for 5-10 min.

5. Primer and method for producing the same

Designing a primer:

finding CDs sequences of target genes (when a plurality of transcripts exist, primer design can be carried out on conserved regions of each transcript); primers were designed and evaluated using software (Oligo software); blast primer specificity.

The method comprises the following steps:

the length of the primer is 15-30bp, and the most common primer is 23 bp; the Tm value of the primer is between 62 and 73 ℃, and the Tm values of the upstream primer and the downstream primer are preferably similar to ensure that the upstream primer and the downstream primer can work efficiently; the Tm value of the 3 '-end is lower than that of the 5' -end; the GC content of the primer is about 40-60%; avoiding amplification of the secondary domain of the template; secondary structure of the primer is avoided; avoiding mismatches with the target DNA.

6. PCR enzyme: ordinary Taq enzyme, Taq enzyme capable of amplifying a growing fragment or high fidelity enzyme can be selected as required.

7. And (3) PCR system: the formulations were prepared according to the chosen PCR enzyme instructions.

8. And (3) detecting a PCR product: agarose gel electrophoresis is generally used, and polyacrylamide gel electrophoresis may be used if necessary.

Western Blod

Detecting protein: osteogenic gene expression of RUNX2, collagen alpha 1 (COL 1a1,) and osteopontin (OPN,). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.

The method comprises the following operation steps:

firstly, extracting protein in a sample and measuring the concentration of the protein (comprising three types of samples and two protein concentration measuring methods); protein sample collection (protein preparation): (1) pouring out the culture solution in the cell culture bottle, pouring out the culture solution to the greatest extent, gently sucking away the residual culture solution by using a liquid-transferring gun, and ensuring that the operation time is short; (2) 3ml-4ml of 4 ℃ pre-cooled PBS (phosphate buffered saline) (0.01M pH 7.2-7.3) was added to each flask of cells. The "cross" was placed flat and gently shaken for 1min for washing, and the washing solution was discarded and repeated three times. After the PBS was discarded, the flasks were placed on ice.

(3) Mu.l of PMSF (phenylmethylsulfonyl fluoride) (100 mM) was added to 1ml of the lysate, shaken well and placed on ice, and the lysate was mixed until no crystals were present in the PMSF. (4) Adding 400 μ l of lysis solution containing PMSF into each bottle of cells, and lysing for 30min on ice, wherein the culture bottle is repeatedly shaken for fully lysing the cells; (5) after lysis, scraping the cells on one side of the flask with a clean scraper (rapid action), and then transferring the cell debris and lysate to a 1.5ml centrifuge tube with a pipette (preferably all on ice); (6) centrifuging the tube at 12000rpm at 4 deg.C for 5min (precooling the centrifuge in advance); (7) and subpackaging and transferring the centrifuged supernatant into a 0.5ml centrifuge tube, and storing the centrifuge tube in a refrigerator at the temperature of-20 ℃.

The above steps can be briefly summarized as follows: collecting cells, lysing cells, centrifuging to obtain supernatant, and storing

Note that: different companies may have different steps of methods (3) (4) for using the lysate according to the instructions for actually purchasing the lysate. 1M means 1mol/L

Preparing gel, loading and electrophoresis;

(1) and cleaning the glass plate, assembling the electrophoresis tank, namely aligning the glass plate and then clamping the glass plate by using a clamp. Then vertically placing the glass tube on a frame for glue pouring; (2) the separation gel (the larger the molecular weight, the smaller the gel concentration) was prepared, and referring to formula 1 below, approximately, different concentrations of gel were selected according to the molecular weight of the protein, and ddH2O (double distilled water) was added first, and then each component was stirred uniformly. AP (ammonium persulfate), TEMED (tetramethylethylenediamine) were added last. Here take 15ml of 12% separation gel as an example, see formulation 2; (3) and (5) after the separation glue is prepared, pouring the glue. And (5 ml of glue is sucked by a 10ml pipette and injected along the glass until the glue surface rises to the height of the middle line of the green band. A liquid-transferring gun or an injection needle is used for moving vertically to the glass plate horizontally and adding ddH2O (or isopropanol) to enable the glue to be flat and solidify fast; (4) preparing 4% of concentrated glue according to a formula 3; after the glue in the plate solidified, the upper liquid was poured off, the apparatus was tilted properly and the excess water was blotted with filter paper. The gel was poured, the comb inserted horizontally (taking care not to have air bubbles), and the gel was allowed to set for about half an hour. The inner groove is disassembled from the glue maker and is arranged in the outer groove, the upper cavity liquid and the lower cavity liquid (electrophoresis buffer liquid) are added, and then the comb is pulled out slightly upwards (preferably, the comb is pulled out after the electrophoresis liquid is added, and the comb is not required to be pulled out in a dry mode); (5) the protein supernatant with the determined protein content is treated for 5 minutes in boiling water bath or heated to 98-100 ℃ to denature the protein by taking the sample with the lowest protein concentration as a reference, adjusting the protein concentration of all samples to be consistent (adjusting the concentration by using lysis solution in a laboratory, adjusting the concentration by using buffer in some cases, and adjusting the concentration by using other reagents in some cases. After cooling, the sample can be prepared.

Note that: the loading buffer generally comprises 2X or 5X SDS-PAGE protein loading buffer. If the minimum concentration is too low, we can choose a 5 XSDS-PAGE protein loading buffer, and load more protein samples in the same volume of loading well. In addition, some laboratories after water bath or heating can carry out centrifugal operation, and the operation is selected according to actual conditions.

Optimum separation range of SDS-PAGE separating gel with different concentrations in formula 1

15ml of each component sampling amount of 212 percent separating gel in formula

Sampling amount of each component of 34% concentrated glue in formula

2.2 loading:

(1) selecting the size of the amount according to the specification by a Marker (4ul-5ul) (the function of the Marker is to display the positions of proteins with different molecular weights), and then adding samples into subsequent wells in sequence, wherein the amount of the samples is generally 10-15ul and cannot exceed 20 ul.; (2) and (5) covering the cover of the electrophoresis apparatus, connecting a power supply, and paying attention to the fact that the anode and the cathode are not connected reversely. The electrophoresis was stopped when the sample reached 80V, after which the lower gel was replaced with 120V bromophenol blue indicator to the bottom of the separation gel (about 1CM from the bottom).

Thirdly, film transferring; the pre-membrane is usually cut into gel, the gel at the position of the protein of our purpose is cut off, and the others are discarded. Nowadays, the entire block of glue is gradually spread to transfer the film (certainly, not many laboratories really doing so).

3.1 cutting the glue:

first, the length and width of the glue to be cut are calculated, and an NC film having the same length and width is cut. And then preparing filter paper, wherein the length and the width of the filter paper are slightly larger than those of the NC membrane, soaking the filter paper in the membrane transferring solution, marking the upper left corner of the NC membrane by using a gel pen, and clamping the edge of the NC membrane by using tweezers as much as possible. Combining with the marker position, cutting off the gel with proper size according to the molecular weight of the target protein, and soaking in the membrane transferring solution.

3.2 making "Sandwich"

The clamp for transferring the membrane, two sponge pads, a glass rod, filter paper and the soaked membrane are placed in a tray with electric transfer liquid. The clip of the transfer film is opened, and the transfer film is placed correctly, and the black side is kept horizontal (according to the actual condition of the laboratory device, the transfer film is correctly used), a special foam pad is padded, and a glass rod is used for rolling back and forth to remove air bubbles. The assembly was carried out in the following order:

3.3 starting to transfer the film

The prepared sandwich is clamped by a clamp and is put into a film transfer instrument. Red (white) to red and black to black, because heat is generated during the electric conversion, the temperature is reduced by adding crushed ice and water around the film converter. Generally, a constant current (such as 100mA or 350mA, and the corresponding current is selected according to the specification of a laboratory film transfer instrument) is used. After the transfer was completed, the NC film was taken out.

3.4 ponceau dyeing

The transferred film was stained with ponceau red stain for 5min (shaking on a decolorizing shaker). The membrane proteins were then visualized by rinsing the unstained dye solution with double distilled water. Note that: ponceau staining was mainly observed to be unsuccessful in protein turnover, reversible and could be washed out with appropriate solutions (e.g., PBS solutions).

Sealing;

the membrane was soaked with TBS (TRIS-HCL buffer) from below up, transferred to a container containing blocking solution, and blocked by shaking on a decolourization shaker at room temperature for 1 h. Then, the NC membrane was taken out from the blocking solution, and the membrane was washed twice with TBS (or the residual solution was blotted with absorbent filter paper without washing with TBS).

Note that: the blocking solution was purchased from a reagent company, but it may be blocked by preparing a blocking solution from skim milk powder (5% skim milk powder was added to TBST solution (TBS plus Tween-20)).

Fifthly, primary anti-incubation;

5.1 incubation

Preparing antibodies, diluting the antibodies with TBST according to requirements (such as the ratio of an antibody stock solution to TBST is 1: 500), incubating the diluted primary antibodies with NC membranes, adding about 2ml of diluted antibodies into each membrane, and standing overnight at 4 ℃ (for saving cost, the primary antibodies can be recycled until the antibodies cannot be incubated out).

5.2 washing:

the membranes were removed from the primary antibody, placed in fresh TBST solution (2 ml per membrane), shaken rapidly on a shaker for 5min, and washed five times in total (or 3 times in total, 10 minutes each).

Sixthly, incubating with a second antibody;

secondary antibody and TBST ratios of 1:1000 and 1:5000 are common, diluted as required. The membrane was incubated with the secondary antibody for 1 hour (some laboratories were also incubated for 2 hours, as the case may be).

Note that the secondary antibody is often coupled to alkaline phosphatase, and the source of the secondary antibody should match the primary antibody, i.e., specifically bind.

Washing: the membranes were removed from the secondary antibody, placed in fresh TBST solution (2 ml per membrane), shaken rapidly on a shaker for 5min, and washed five times in total (or 3 times in total, 10 minutes each).

The color development exposure comprises two modes of traditional darkroom color development and instrument exposure.

7.1 darkroom color development:

(1) mixing the luminous liquid A and the luminous liquid B on the preservative film in a ratio of 1: 1; after 1min, the membrane protein is faced downwards and fully contacted with the mixed solution (80-100 ul of each membrane); after 1min, transferring the film to another preservative film, removing residual liquid, packaging, and placing into an X-ray film holder; (2) in a dark room, respectively putting 1 × developing solution and fixing solution into a plastic tray; opening a red light, taking out an X-ray film under the red light, and cutting the film to a proper size (the size is 1cm larger than the length and the width of the film) by using a paper cutter; opening the X-ray film holder, placing the X-ray film on the film, once the X-ray film is placed on the film, the X-ray film holder cannot be moved, closing the X-ray film holder, and starting timing; properly adjusting the exposure time according to the intensity of the signal, generally 1min or 5min, and optionally pressing for multiple times at different times to achieve the best effect; after exposure, opening the X-ray film clamp, taking out the X-ray film, quickly immersing the X-ray film in a developing solution for development, and stopping development immediately after an obvious strip appears. The developing time is generally 1-2min (20-25 ℃), and when the temperature is too low (lower than 16 ℃), the developing time needs to be properly prolonged; after the development is finished, immediately immersing the X-ray film into the fixing solution, wherein the fixing time is generally 5-10min, and the film is transparent; after washing off the residual fixer with tap water, the plate was dried at room temperature. Scanning the exposed film with a scanner and performing gray value analysis with an image processing system

7.2 color development and exposure of instrument:

the formula is as follows:

folin-phenol reagent formula

Reagent A: comprises (a) and (b) solutions, wherein (a) 10 g Na₂CO₃2 g NaOH and 0.25 g sodium potassium tartrate (KNaC)₄H₄O₆-4H₂O). Dissolving in 500 ml of distilled water; (b) 0.5 g copper sulfate (CuSO)₄-5H₂O) is dissolved in 100 ml of distilled water, and 50 parts of (a) and 1 part of (b) are mixed before each use, so that the reagent A is obtained.

And a reagent B: 100 g of sodium tungstate (Na) was added to a 2 liter ground reflux flask₂WO₄-2H₂O), 25 g of sodium molybdate (Na)₂MOO₄-2H₂O) and 700 ml of distilled water, then adding 50 ml of 85 percent phosphoric acid and 100 ml of concentrated hydrochloric acid, fully mixing, connecting with a return pipe, refluxing for 10 hours by soft fire, and adding 150 g of lithium sulfate (Li) when the reflux is finished₂SO₄) 50 ml of distilled water and several drops of liquid bromine, boiling was continued for 15 minutes at the opening to drive off excess bromine. After cooling, the solution was yellow (if still green, the dropwise addition of liquid bromine was repeated). Diluted to 1 liter, filtered and the filtrate was stored in a brown reagent bottle. When in use, standard NaOH is used for titration, phenolphthalein is used as an indicator, and then appropriate dilution is carried out, about 1 time of water is added, so that the final acid concentration is about 1N (the H + concentration is 1 mol/L). (in the experiment, the required standard protein solution, crystalline bovine serum albumin or G-globulin is accurately weighed and dissolved in distilled water at a concentration of about 250 mg/ml. if the bovine serum albumin is dissolved in water and is turbid, 0.9% NaCl solution can be used instead)

Alizarin red staining

The purpose is as follows: the mineralization potential of the engineered membrane was assessed quantitatively and qualitatively using alizarin red staining (ARS, Sigma-Aldrich).

The method comprises the following steps:

1 × 10 per membrane⁴MSC were seeded at a density, with fresh medium replacing the medium every 3 days;

② on day 14 and 21, cells were washed with PBS (2X);

③ fixing the mixture in 4 percent formaldehyde for 30 minutes at room temperature;

fourthly, washing for 20 minutes by PBS (2 x);

fifthly, staining the cells with 40 mM ARS, and incubating the plate for 30 minutes at room temperature;

sixthly, washing with deionized water (4 x), drying and scanning with an Epson Perfection V50 photo scanner and converting into binary images;

after that, the ARS is decolorized using 10% acetic acid and ammonium hydroxide;

(iii) absorbance of an aliquot (using a microplate reader (Spectra iD 3)) the supernatant (. mu.L) was measured at 405 nm.

Micro computer tomography (micro-CT)

The region of interest (ROI) of each specimen surrounding the defect was analyzed to calculate Tissue Volume (TV), Bone Volume (BV) and bone volume score (BVF, BV/TV).

Histological analysis

Bone samples were decalcified with 10% formic acid solution. The decalcified samples were dehydrated in ascending order, then infiltrated and embedded in paraffin, and then cut into 4 μm sections. Sections were stained with hematoxylin and eosin (H & E) or Masson trichrome for microscopic examination. The stained sections were observed with an optical microscope equipped with a digital camera. H & E staining revealed a cellular response indicating bone formation; trichrome staining by Masson was used to identify mineralized bone (blue) and osteoid (red) and the results are shown in fig. 5.

Cell culture

Rabbit primary BMSCs were purchased. Then, the cells were cultured at 37 ℃ in α -MEM supplemented with 10% fetal bovine serum. The media was changed every three days. After the cells reached 90% confluence, they were trypsinized for passaging. The third BMSC passage was incubated with a fluorescently labeled antibody (Beyotime, shanghai, china) for 30 minutes, then washed with PBS. The expression levels of different cell surface markers including CD29, CD90, CD11b and CD45 were examined by flow cytometry. Cells from the third generation were used in subsequent experiments. (self-isolation: taking 7-month old male rabbits from New Zealand white rabbits of young age, 7 months old, removing peripheral muscles, cutting both ends, repeatedly flushing the marrow cavity with alpha-MEM containing 10% fetal bovine serum by a sterile syringe, culturing the flushing fluid under the same cell culture conditions), the results are shown in FIG. 3.

Animal model

Male 7-month old New Zealand white rabbits (2.5-3.5 kg) were kept pathogen-free. In order to evaluate the bone repair capacity of the collagen and the scaffold, 3% sodium pentobarbital is adopted for ear margin intravenous injection anesthesia, the dose is 3mg/kg, iodophor is used for sterilizing 3 common surgical drapes after defecation, 2-3cm long longitudinal incisions are made along the outer side of the distal end of the femur, the distal end part of the femur is exposed, 3-4mm of the distal joint surface of the femur of each big rabbit is the center of the condyle of the femur, cylindrical bone defect cavities with the diameter of 6mm and the depth of 8mm are drilled by an electric drill, and the bone defect groups are treated respectively by flushing with physiological saline; bone defect + collagen + scaffold; bone defect + scaffold. 2 of each group were sacrificed at weeks 4 and 8 after drilling. Peripheral blood was collected from each group of rats and Osteopontin (OPN), osteocalcin (BGP) (spare) was detected by ELISA. All tests were performed according to the manufacturer's instructions. Collecting thighbone, decalcifying after micro-CT detection, and performing histological detection. (for example, the femoral head necrosis can be done by changing the position). The results are shown in FIG. 2.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.