CN111494719A - Novel bone tissue engineering scaffold and preparation method thereof - Google Patents

Novel bone tissue engineering scaffold and preparation method thereof Download PDF

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CN111494719A
CN111494719A CN201911408666.XA CN201911408666A CN111494719A CN 111494719 A CN111494719 A CN 111494719A CN 201911408666 A CN201911408666 A CN 201911408666A CN 111494719 A CN111494719 A CN 111494719A
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bone
solution
bone tissue
scaffold
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史强
赵瑞波
朱勇
陈灿
许琰
闫少杭
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Xiangya Hospital of Central South University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/02Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

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Abstract

The invention relates to the technical field of biomedical tissue engineering, in particular to a novel bone tissue engineering scaffold and a preparation method thereof. Comprising bone material and exosome-loaded fibrin gel complex, the bone material having pores, the gel complex being distributed in the pores. The present invention develops a bone material highly similar to natural bone matrix and having osteogenic and angiopoietic activities. The porous acellular tissue engineering scaffold is closer to normal bones, has better biomechanical properties, is suitable for repairing bone defects in a load bearing area, and retains the inherent components of the scaffold to the maximum extent. Not only can effectively remove cell components with most antigens in tissues, reduce the immunological rejection reaction of the graft, but also can maintain the approximate morphological structure of the tissues and retain most tissue matrix components and bioactive factors.

Description

Novel bone tissue engineering scaffold and preparation method thereof
Technical Field
The invention relates to the technical field of biomedical tissue engineering, in particular to a novel bone tissue engineering scaffold and a preparation method thereof.
Background
The repair and functional reconstruction of bone defects, particularly critical bone defects, caused by diseases, trauma, infection, osteonecrosis, congenital malformations, bone tumors and the like are one of the clinical problems of orthopedics. Traditional biological treatment methods for bone defect treatment include autologous bone grafting and allogeneic bone grafting.
The number of large-section bone defect patients caused by various diseases such as trauma, tumor, congenital heredity and the like in China is as high as 300 or more than ten thousand every year. At present, the clinical treatment methods for bone defects mainly comprise bone transportation, masqueret technology, autologous bone transplantation with blood vessels, autologous bone transplantation, allogeneic bone transplantation and the like, but have the defects of limited supply areas, supply area complications, long treatment period, poor operation treatment effect, poor osteogenesis capacity, rejection reaction and the like.
Over decades of development, research related to tissue engineering bone regeneration has indeed made enormous progress. However, as research continues to progress, both theoretical and technical problems are encountered: 1) the traditional tissue engineering technology needs to rely on the collection, separation and amplification of seed cells, and the extraction process is time-consuming and difficult to store; 2) the introduction of exogenous seed cells has the risk of tumorigenesis, and the seed cells are loaded on the stent in vitro, so the operation process of constructing the tissue engineering graft is complicated, and the storage cost is high; 3) the prior scaffold material has poor biological induction activity and undesirable bone tissue regeneration effect.
Disclosure of Invention
The invention aims to provide a novel bone tissue engineering scaffold.
Another object of the present invention is to provide a porous femoral bone tissue engineering scaffold.
It is another object of the present invention to provide a method for decellularizing a femur.
The invention also aims to provide a preparation method of the novel bone tissue engineering scaffold.
The invention also aims to provide a bone tissue engineering scaffold with small immune rejection and a preparation method thereof.
The invention also aims to provide a bone tissue engineering scaffold with good repairing effect and a preparation method thereof.
The above object of the present invention is achieved by the following technical means:
in one aspect, the present invention provides a bone tissue engineering scaffold comprising a bone material having pores, and an exosome-loaded fibrin gel complex distributed in the pores.
In some embodiments, the pores have a pore diameter of 50-200 μm and a pore depth of about 0.5-2 mm.
In some embodiments, the pores have a pore diameter of 80-100 μm and a pore depth of 0.5-1 mm.
In some embodiments, the holes have a pitch of 0.3-0.5 mm.
In some embodiments, the holes have a pitch of 0.4-0.5 mm.
In some embodiments, the bone material has a porosity of 30-50%.
In some embodiments, the porosity is 40-50%.
In some embodiments, the bone material is a natural femur.
In some embodiments, the bone material is a mammalian femur.
In some embodiments, the mammal is selected from a human, a mouse, or a rabbit.
In some embodiments, the femur is decellularized and decalcified.
In some embodiments, the gel comprises one or more of fibrin glue, sodium alginate hydrogel, and chitosan gel.
In some embodiments, the methods of decellularization and decalcification include chemical, physical, or biological reagent methods.
In some embodiments, the exosomes overexpress DMBT 1.
In some embodiments, the exosomes are derived from hsuscs.
In some embodiments, the hUSCs are inserted into DMBT1 gene through genetic engineering, and DMBT1 is highly expressed.
In some embodiments, the hsscs are adenovirus-transfected cells that highly express DMBT 1.
In another aspect, the present invention provides a method for preparing a scaffold for bone tissue engineering, comprising the steps of:
(1) punching the bone tissue to obtain porous bone tissue;
(2) decalcifying the porous femoral tissue to obtain decalcified porous femoral tissue;
(3) decellularizing porous femoral tissue;
(4) and (3) injecting the exosome-loaded fibrin solution, and coagulating into gel.
In some embodiments, the step of obtaining porous femoral bone tissue in step (1) comprises:
(6) separating and cleaning femoral bone tissue in vitro;
(7) and punching the femoral bone tissue by using a punching device.
In some embodiments, the pores have a pore diameter of 50-200 μm and a pore depth of about 0.5-2 mm.
In some embodiments, the pores have a pore diameter of 80-100 μm and a pore depth of 0.5-1 mm.
In some embodiments, the holes have a pitch of 0.3-0.5 mm.
In some embodiments, the holes have a pitch of 0.4-0.5 mm.
In some embodiments, the bone material has a porosity of 30-50%.
In some embodiments, the porosity is 40-50%.
In some embodiments, the decalcification method comprises a chemical reagent method, a physical method or a biological reagent method.
In some embodiments, the step of decalcifying comprises: decalcifying the porous femoral bone tissue obtained in the step (1) in 5-10% EDTA buffer for 5-20 days.
In some embodiments, the step of decellularizing comprises:
(8) placing the decalcified porous femoral bone tissue in the step (2) in the solution A for 15-30 h;
(9) putting the solution B into the solution B for 8-20 h;
(10) putting the solution C for 15-30 h;
(11) putting the solution D for 8-20 h;
(12) freeze drying and gas sterilizing to obtain acellular porous femoral tissue;
the solution A is a PBS solution containing 0.1-2% (w/v) SDS and 0.1-1% (v/v) TritonX-100;
the solution B is a double-resistant PBS solution containing 0.1-2% (w/v);
the solution C is a PBS solution containing 500-800U/m L DNase Type I and 1-2mg/m L RNase;
the solution D is a double-resistant PBS solution containing 0.1-2% (w/v).
In some embodiments, the A solution is a PBS solution containing 0.1% to 0.5% (w/v) SDS and 0.1% to 0.5% (v/v) TritonX-100;
the solution B is a double-resistant PBS solution containing 0.1-1% (w/v);
the solution C is a PBS solution containing 500-600U/m L DNase Type I and 1-1.5mg/m L RNase;
the solution D is a double-resistant PBS solution containing 0.1-1% (w/v).
In some embodiments, the method for preparing the exosome-loaded fibrin solution in the step (4) is to dissolve exosomes in a fibrinogen solution, add prothrombin, and mix the mixture uniformly.
In some embodiments, the volume ratio of the fibrinogen solution to the prothrombin is 1-5: 1.
in some embodiments, the volume ratio of the fibrinogen solution to the prothrombin is 1-3: 1.
in some embodiments, the concentration of prothrombin is 500-.
In some embodiments, the concentration of prothrombin is 800-.
In some embodiments, the concentration of exosomes in the fibrin solution is 100 μ g/m L-300 μ g/m L.
In some embodiments, the concentration of exosomes in the fibrin solution is 150-250 μ g/m L.
In some embodiments, the exosomes overexpress DMBT 1.
In some embodiments, the exosomes are derived from hsuscs.
In some embodiments, the hUSCs are inserted into DMBT1 gene through genetic engineering, and DMBT1 is highly expressed.
In some embodiments, the bone material is a mammalian femur.
In some embodiments, the mammal is selected from a human, a mouse, or a rabbit.
In a further aspect, the invention provides the application of the bone tissue engineering scaffold or the preparation method in preparing bone repair materials.
Compared with the prior art, the beneficial technical effects of one embodiment of the invention are as follows:
1. bone materials that are highly similar to natural bone matrix, having both osteogenic and angiopoietic activity, were developed.
2. The porous decellularized tissue engineering scaffold is closer to a normal bone, has better biomechanical property, is suitable for repairing bone defects in a loading area, and is subjected to hole punching and then a specific decellularization method, so that the decellularization of the bone scaffold is thorough, and the inherent components of the scaffold are reserved to the maximum extent. Not only can effectively remove cell components with most antigens in tissues, reduce the immunological rejection reaction of the graft, but also can maintain the approximate morphological structure of the tissues and retain most tissue matrix components and bioactive factors.
Drawings
Fig. 1 is a flow chart for preparing a novel bone scaffold.
FIG. 2 is a diagram showing the quantitative evaluation of the decellularization effect of the porous femoral tissue scaffold using a DNA content assay kit.
Fig. 3 is an evaluation of the decellularization effect of the porous femoral tissue engineering scaffold: h & E staining assessed the decellularization effect of the scaffolds; evaluating DNA retention after the bracket cell removal treatment by using DAPI; ABS: decellularized bone scaffold, NBT: normal bone tissue.
FIG. 4 is a Scanning Electron Microscope (SEM) evaluation of the surface topology of the porous femoral bone tissue engineering scaffold before and after decellularization.
FIG. 5 is a morphological diagram of human urinary stem cells.
FIG. 6 shows the flow-type identification results of surface markers of urinary stem cells.
FIG. 7 shows the capacity of differentiation of urinary stem cells into bone, fat and cartilage: a is the result of osteogenic staining; b is the result of fat-forming dyeing; c is the result of chondrogenic staining.
FIG. 8 is a transmission electron micrograph of urinary stem cell exosomes.
FIG. 9 is a urine-derived stem cell exosome particle size analysis.
FIG. 10 shows Western-blot results showing that the urinary stem cell exosomes highly express DMBT1 and VEGF-A proteins.
FIG. 11 is an image of a damaged femur after implantation of a composite hUSCsDMBT1-Exo porous decellularized bone scaffold:
FIG. 11A shows a day 2 DR scan (Digital Radiography) of a composite hUSCsDMBT1-Exo porous decellularized bone scaffold, and FIG. 11B shows a week DR scan after implantation of a composite hUSCsDMBT1-Exo porous decellularized bone scaffold.
FIG. 12 is an SEM image and Calcein-AM/PI/live/dead cell staining image of a multi-well decellularized bone scaffold sheet compounded with hUSCs after co-culturing with hUSCs for 3 days.
FIG. 13 shows the results of Micro CT detection at week 2 after stent implantation at the right femur defect site of a rat;
wherein FIG. 13A is a decellularized bone scaffold group; FIG. 13B is a set of porous decellularized bone scaffolds; FIG. 13C is the porous decellularized bone scaffold group of composite hUSCsDMBT 1-Exo.
FIG. 14 shows the results of Micro CT examination at week 4 after implantation of a stent at the right femur defect site of a rat;
wherein FIG. 14A is a decellularized bone scaffold group; FIG. 14B is a set of porous decellularized bone scaffolds; FIG. 14C is the porous decellularized bone scaffold group of composite hUSCsDMBT 1-Exo.
FIG. 15 shows the qRT-PCR detection of BMSCs osteogenesis specific gene (Runx-2) expression on the scaffold surface.
FIG. 16 shows the expression of three groups of osteogenic specific proteins detected by immunofluorescence:
FIG. 16A shows the results of the test in test group 1; FIG. 16B shows the results of the test in test group 2; fig. 16C shows the experimental results of the control group.
FIG. 17 is HE staining to detect regenerated new bone:
FIG. 17A is the HE staining result of experimental group 1; FIG. 17B is the HE staining result of experimental group 2; FIG. 17C shows the results of HE staining in the control group; wherein NB refers to new bone.
FIG. 18 shows the results of the biomedical test, tensile load (Failure L oad) and stiffness (stiff) test.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
As used herein, the term "high expression" is intended to mean that the expression level of DMBT1 in exosomes or stem cells is higher than the expression level of DMBT1 in exosomes in general or in mesenchymal stem cells.
Herein, hUSCs refer to human urinary stem cells.
In the examples of the present application, the procedure for preparing the novel bone scaffold is shown in fig. 1.
In the examples, the fibrinogen solution and thrombin solution were purchased from Sigma.
Example 1 preparation and evaluation of decellularized porous femur
1. Porous femoral tissue harvesting
After euthanasia of adult (12-week-old) SD rats, the right femoral mid-section tissue was collected, trimmed to a length of 10 mm, washed with 1% double-resistant PBS to remove bone marrow and blood, and the bone tissue was perforated using a perforating device (device reference patent application CN201520084830.7) and decalcified with 10% EDTA buffer for 15 days.
Wherein the pores of the femoral bone tissue obtained after perforation have an average pore diameter of 100 μm, a pore depth of about 1mm, a pitch of the pores is 0.5mm on average, and the porosity of the bone material is 50%.
2. Cellular femoral tissue decellularization
The porous femoral bone tissue is placed in PBS solution containing 0.1% (w/v) SDS + 0.1% (v/v) Triton X-100, slowly shaken for 24h at 4 ℃, rinsed for 12h by 1% (w/v) double-antibody PBS solution, treated for 24h by ribozyme solution (PBS solution containing 500U/m L DNase TypeI and 1mg/m L RNase) to remove DNA and RNA, rinsed for 12h by 1% (w/v) double-antibody (penicillin-streptomycin) PBS solution, freeze-dried and sterilized by gas, and the porous decellularized bone scaffold is prepared.
3. Evaluation of cell depletion Effect
The decellularization effect was quantitatively evaluated using a DNA content assay kit (Qiagen, Germany), and the results are shown in FIG. 2.
The H & E staining and DAPI staining qualitatively assessed the effect of decellularization, and the results are shown in FIG. 3.
From the experimental results, it was shown that after the femur was decellularized, the DNA was not substantially retained and the cells in the femur were completely decellularized. But the structure of the femoral bone tissue is preserved relatively intact after decellularization.
4. Topology estimation
Scanning Electron Microscopy (SEM) was performed on a 1cm × 1cm femoral structure slice to evaluate the surface topology of the scaffold before and after decellularization, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the structure of the femoral bone tissue remains relatively intact after decellularization, and the cells remain almost free.
EXAMPLE 2 preparation of novel scaffolds
1. Isolation culture of human urinary stem cells (hUS C s)
Collecting 200m L of 3 healthy adult sterile fresh middle section urine, adding 5ml streptomycin, mixing uniformly, subpackaging into 4 tubes of 50m L centrifuge tubes, centrifuging at 400 rpm for 10min, discarding the supernatant, adding 20m L PBS, blowing, mixing uniformly, continuing to centrifuge at 400 rpm for 10min, discarding the supernatant, resuspending the hUSCs culture medium, inoculating in 0.1% gelatin coated 6-well plate, standing and culturing at 37 ℃ and 5% CO2 incubator hUSCs culture medium components of DMEM/F12, 2% fetal calf serum, 10ng/ml Epidermal Growth Factor (EGF), 2ng/ml Platelet Derived Growth Factor (PDGF), 1ng/m L Transforming Growth Factor (TGF), 2ng/ml recombinant human fibroblast growth factor (hEGF), 0.5 mmol/L hydrocortisone, 24 mg/ml insulin, 20mg/m L transformin, 549/m epinephrine, primary thyroid stimulating factor L, 125ng/m L, thyroid stimulating factor, 3.5 mmol/m, 7% primary thyroid stimulating factor, culturing with 10% trypsin, observing the growth rate after 7-80% trypsin is added, and culturing with 7.7% of the USCs.
2. Identification of hUSCs
Cell proliferation assay by selecting the aforementioned hUSCs according to 3 × 103The culture medium of the wells to be detected is sucked out, CCK-8(10 mu L/well) is added, the culture is cultured for 4 hours at 37 degrees, an optical density value (OD value) is measured on a microplate reader at a wavelength of 450nm, the surface marker detection is that after the hUSCs are digested by 0.25 percent trypsin, the α -MEM complete culture medium stops digestion, adherent cells are blown by a pasteur pipette to form a cell suspension, the cell suspension is centrifuged at 1000rpm for 5 minutes, a supernatant is discarded, the count is carried out after the PBS is washed once, the incubation is carried out for 30 minutes according to the count of flow type antibodies, and then the flow type cytology detection is carried out.
And (3) detecting the trilinear differentiation capacity: after the hUSCs are subjected to osteogenic induction for 21 days, alizarin red is stained and observed; after the hUSCs are centrifugally deposited at the bottom of the tube, chondrogenic induced differentiation is carried out for 21 days, and the hUSCs are stained and observed with the Aglaia blue; after 21 days of adipogenic induction of the hUSCs, the hUSCs were stained with oil red O and observed. The results are shown in FIGS. 7A-7C.
3. Construction of hUSCs line with high expression of DMBT1
Constructing an adenovirus vector (Ad-DMBT 1-GFP) with DMBT1 overexpression by Shanghai Jikai gene company, searching for the optimal multiplicity of infection (MOI) by referring to an operation manual, and transfecting hUSCs (hUSCs transfected with empty vector adenovirus are used as a control) with a target gene; after culturing for 3-5 days, detecting the expression of DMBT1 by qRT-PCR and Western-Blot; thereby establishing a hUSCs line (DMBT1-hUSCs) with high expression of DMBT 1.
4. Extraction and identification of hUSCs exosome (hUSCsDMBT1-Exo) rich in DMBT1
Collecting DMBT1-hUSCs culture supernatant, extracting purified exosome by ultracentrifugation, and observing the form under an electron microscope, as shown in figure 8. Particle size analysis is carried out by detecting exosome marker proteins CD63, CD9, CD81, TSG101 and Nanosight through Western-blot, and the content of DMBT1 in hUSCsDMBT1-Exo is detected through Western-blot.
The results of the particle size analysis of the urine-derived stem cell exosomes are shown in fig. 9.
The Western-blot result is shown in FIG. 10, and shows that the urinary stem cell exosomes highly express DMBT1 and VEGF-A proteins.
5. Preparation of exosome-loaded fibrin gel complex
The method comprises the following specific steps of dissolving the separated and purified hUSCsDMBT1-Exo solution into a fibrinogen solution (200U/m L) at a ratio of 200 mu g/m L to obtain a mixed solution, mixing the obtained mixed solution with thrombin (the concentration is 1000U/m L) according to a volume ratio of 1: 1, injecting the solution of 5m L into a porous decellularized femur scaffold by using a Y-shaped injector, and preparing the fibrin gel compound porous decellularized femur scaffold containing a loaded exosome after the fibrin gel compound porous decellularized femur scaffold is solidified into gel, thus obtaining the novel vascularization effect porous decellularized bone scaffold.
The carrier can well compound exosome in protein gel to be in a solidified state and is placed in the hole of the bone scaffold.
Example 3 evaluation of in vivo Effect of decellularized bone scaffolds
1. Establishment of SD rat femoral shaft bone defect model and surgical treatment
Taking adult male SD rats, performing abdominal anesthesia with 0.3% sodium pentobarbital, removing hair of right lower limb, sterilizing with iodophor, and spreading sterile towel and hole towel. Taking a minimally invasive longitudinal incision on the outer side of the femur, exposing the femoral shaft, sawing off bone tissues with the length of 0.8cm by using a small animal, implanting and trimming the length of the novel bracket, and placing the novel bracket into the femoral shaft defect. Then, selecting a lateral incision of the patella of the right knee joint to cut the skin, separating layer by layer, exposing the femoral condyle, reversely drilling a 2.0mm Kirschner wire into a femoral medullary cavity through an intercondylar fossa of the femur with an electric drill, enabling the proximal end to penetrate out of the greater trochanter skin of the femur, embedding the distal end below the surface of the articular cartilage, cutting and bending the Kirschner wire at the proximal end to 90 degrees to prevent the proximal end from falling off, and repeatedly washing the wound with iodophors and biological saline and suturing the skin.
The animals were kept in an SPF-grade environment and were free-living. Buprenorphine (0.12mg/kg) analgesia administered immediately after surgery; carprofen (4mg/kg) was administered orally once a day after surgery for 7 consecutive days; cephalosporin (10 mg/kg) was orally administered once a day for 5 days as a prophylactic antibiotic. The life conditions of the mice were closely observed during the experimental period.
The experimental groups are shown in table 1.
TABLE 1
Figure BDA0002349377240000081
Wherein, the blank porous decellularized bone scaffold group 1 is the scaffold prepared in example 1;
the porous decellularized bone scaffold compounded with the hUSCsDMBT1-Exo (exosomes derived from hUSCs and rich in DMBT 1) is the porous decellularized bone scaffold compounded with the hUSCsDMBT1-Exo prepared in example 2;
the control group was injected subcutaneously with 200. mu.g/m L-labeled exosome in PBS.
Micro CT pictures of the porous decellularized bone scaffold group of composite hscsdmbt 1-Exo are shown in fig. 11, where fig. 11A is a DR scan at day 2 of the porous decellularized bone scaffold implanted with composite hscsdmbt 1-Exo, and fig. 11B is a DR scan one week after the porous decellularized bone scaffold implanted with composite hscsdmbt 1-Exo. As can be seen in FIG. 11, bone mass increased at the femoral defect site after implantation of the hUSCsDMBT 1-Exo-complexed porous decellularized bone scaffold.
In addition, 50 μm sheets of porous decellularized bone scaffolds complexed with hUSCs DMBT1-Exo were co-cultured with hUSCs for 3 days, and the SEM image obtained is shown in FIG. 12.
As can be seen in fig. 12, the hsscs entered the interior of the scaffold through the pores of the scaffold and grew well inside the scaffold. And fig. 12 also shows that the spacing between pores of the scaffold was 0.5mm on average, the porosity was 50%, the pore diameter of the pores was 100 μm on average, and the pore depth was about 1 mm.
2. Ex vivo evaluation
Animals were euthanized at 2 weeks and 4 weeks post-surgery, and fresh bone from the right femoral defect site was collected for Micro CT scan detection. The results are shown in FIG. 13 and FIG. 14.
As can be seen from FIGS. 13 and 14, the bone mass of the porous decellularized bone scaffold group was increased to some extent, and the bone mass of the porous decellularized bone scaffold group in which hUSCsDMBT1-Exo was compounded was increased most. Has excellent treatment effect.
Through various tests, the pore diameter and the porosity of the porous femur are set to be 0.3-0.5mm and 30-50%, so that the mechanical property can be maintained to the maximum extent, the cell adhesion proliferation is promoted, the bone repair capacity is good, and the characteristics of the scaffold can be influenced when the pore diameter and the porosity are beyond or less than the range.
Comparative example 1
A comparison experiment is carried out on the novel bone tissue engineering scaffold (the porous decellularized bone scaffold compounded with the hUSCsDMBT1-Exo) and the bone tissue engineering scaffold (the porous bone scaffold compounded with hUSCs up to the expression of DMBT 1).
Experimental group 1: compounding a hUSCs porous bone scaffold up to the expression of DMBT 1;
experimental group 2: a porous decellularized bone scaffold complexed with hUSCsDMBT1-Exo (prepared in example 2);
control group: decellularized bone scaffold group (prepared in example 1).
Experimental group 1 differs from experimental group 2 in that experimental group 1 is hsscs highly expressing DMBT 1; experimental group 2 is an exosome (derived from hUSCs) highly expressing DMBT 1.
1. qRT-PCR detection
According to the detection of the conventional qRT-PCR experimental method, after bone marrow mesenchymal stem cells (BMSCs) and three groups of scaffolds are cultured for 14 days, the expression change of osteogenic genes is shown in figure 15, and the result shows that the expression of BMSCs osteogenic specific genes (Runx-2) on the surface of the scaffold of the experimental group 2 is obviously increased, and the expression of BMSCs osteogenic specific genes (Runx-2) on the surfaces of the scaffolds of the experimental group 1 and the control group is not obvious.
2. Immunofluorescence detection
After bone marrow mesenchymal stem cells (BMSCs) and scaffolds of each phase are cultured for 14 days according to a conventional immunofluorescence method, and then subjected to immunofluorescence detection and semi-quantitative analysis, the expression condition of the osteogenic specific protein is shown in figure 16, the expression of the BMSCs osteogenic specific protein on the surface of the scaffold of the experimental group 2 is obviously increased, and the expression of the BMSCs osteogenic specific protein on the surfaces of the scaffolds of the experimental group 1 and the control group is not obvious.
3. Histological Examination (HE)
After 8 weeks of treatment with the porous bone scaffold according to the conventional HE staining method in the SD rat femoral shaft bone defect model, H & E staining results are shown in fig. 17, and it was revealed that regenerated new bone formation was observed in three groups of specimens 8 weeks after the operation. Among them, the experiment group 2 showed the most significant increase in new bone.
4. Biomechanical testing
The SD rat femoral dry bone defect model is treated by a porous bone scaffold, the fracture load and rigidity of three groups of samples after 8 weeks and 16 weeks are obtained through biomechanical detection, and the result is shown in figure 18, and the fracture load and rigidity of the experimental group 2 are obviously higher than those of the experimental group 1 and the control group.

Claims (10)

1. The bone tissue engineering scaffold is characterized by comprising bone materials and exosomes, wherein the bone materials are provided with holes, and the exosomes are distributed in the holes through loading gel.
2. The scaffold for bone tissue engineering according to claim 1, wherein said pores have a pore size of 50-200 μm and a pore depth of about 0.5-2 mm;
preferably, the aperture of the hole is 80-100 μm, and the hole depth is 0.5-1 mm;
or preferably, the distance between the holes is 0.3-0.5 mm;
or preferably, the bone material has a porosity of 30-50%.
3. The bone tissue engineering scaffold according to claim 1, wherein said bone material is a mammalian femur;
preferably, the mammal is selected from a human, a mouse or a rabbit;
or preferably, the femur is decellularized and decalcified.
4. The bone tissue engineering scaffold according to claim 1, wherein said gel comprises one or more of fibrin glue, sodium alginate hydrogel and chitosan gel.
5. The bone tissue engineering scaffold according to claim 1, wherein said exosomes overexpress DMBT 1;
preferably, the exosomes are derived from hsuscs;
preferably, the hUSCs are inserted into the DMBT1 gene through genetic engineering, and the DMBT1 is highly expressed.
6. The preparation method of the bone tissue engineering scaffold is characterized by comprising the following steps:
(1) punching the bone tissue to obtain porous bone tissue;
(2) decalcification of porous bone tissue;
(3) decellularizing the porous bone tissue;
(4) injecting the exosome-loaded fibrin solution, and coagulating into gel;
preferably, the aperture of the hole is 50-200 μm, and the depth of the hole is 0.5-2 μm;
more preferably, the aperture of the hole is 80-100 μm, and the hole depth is 0.5-1 mm;
or preferably, the distance between the holes is 0.3-0.5 mm;
or preferably, the bone material has a porosity of 30-50%.
7. The method of claim 6, wherein said decalcification method comprises a chemical reagent method, a physical method or a biological reagent method;
preferably, the decellularizing step comprises:
(8) placing the porous bone tissue in the step (2) in the solution A for 15-30 h;
(9) putting the solution B into the solution B for 8-20 h;
(10) putting the solution C for 15-30 h;
(11) putting the solution D for 8-20 h;
(12) freeze drying and sterilizing to obtain acellular porous bone tissue;
the solution A is a PBS solution containing 0.1-2% (w/v) SDS and 0.1-1% (v/v) TritonX-100;
the solution B is a double-resistant PBS solution containing 0.1-2% (w/v);
the solution C is a PBS solution containing 500-800U/m L DNase Type I and 1-2mg/m L RNase;
the solution D is a double-resistant PBS solution containing 0.1-2% (w/v).
8. The method according to claim 6, wherein the exosome-loaded fibrin solution in the step (4) is prepared by dissolving exosomes in a fibrinogen solution, adding prothrombin, and mixing well;
preferably, the volume ratio of the fibrinogen solution to the prothrombin is 1-5: 1;
or preferably, the concentration of the prothrombin is 500-1200U/m L;
preferably, the concentration of exosomes in the fibrin solution is 100 mug/m L-300 mug/m L;
preferably, the exosomes overexpress DMBT 1;
preferably, the exosomes are derived from hsuscs;
preferably, the hUSCs are inserted into the DMBT1 gene through genetic engineering, and the DMBT1 is highly expressed.
9. The method of claim 6, wherein the bone material is a mammalian femur;
preferably, the mammal is selected from human, murine or rabbit.
10. Use of the bone tissue engineering scaffold according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9 for the preparation of a bone repair material.
CN201911408666.XA 2019-12-31 2019-12-31 Novel bone tissue engineering scaffold and preparation method thereof Pending CN111494719A (en)

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