CN115227868B - Bone defect repair material and magnesium pretreatment decellularized tissue engineering bone scaffold - Google Patents
Bone defect repair material and magnesium pretreatment decellularized tissue engineering bone scaffold Download PDFInfo
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- CN115227868B CN115227868B CN202210858161.9A CN202210858161A CN115227868B CN 115227868 B CN115227868 B CN 115227868B CN 202210858161 A CN202210858161 A CN 202210858161A CN 115227868 B CN115227868 B CN 115227868B
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Abstract
The invention provides a bone defect repair material and a magnesium pretreatment decellularized tissue engineering bone scaffold, wherein the bone defect repair material comprises a carrier and an osteogenesis promoter loaded on the carrier, the carrier adopts femur, and the osteogenesis promoter is mainly formed by magnesium ions, growth factors and BMSCs generated after the magnesium ions and magnesium rods are implanted and stimulated together. The magnesium pretreatment decellularized tissue engineering bone scaffold prepared by using the bone defect repair material can solve the problems of complex manufacturing procedure, high manufacturing cost, inaccurate treatment effect, overlong period, poor bone defect repair effect of the existing bone tissue engineering scaffold, and has the advantages of simple manufacturing procedure, low manufacturing cost, good bone formation promotion effect and the like.
Description
Technical Field
The invention relates to the technical field of bone tissue scaffolds, in particular to a bone defect repair material and a magnesium pretreatment decellularized tissue engineering bone scaffold prepared by using the bone defect repair material.
Background
Bone defects are not rare in clinic, and the reasons for the bone defects are various, including exercise or injury, debridement after bone infection, bone disconnection or bone loss of blood supply after radiotherapy or bone tumor excision, and the like, which seriously affect the life and work of patients and greatly affect the physiology and psychology of the patients.
The current clinical auxiliary treatment methods for repairing bone defects mainly comprise three methods: autologous bone grafting, allogeneic bone grafting and bone tissue engineering. Autologous bone is considered as a gold standard for treating bone defects due to good biocompatibility, strong osteogenesis ability, high osteoinductive and osteoconductive activities, however, autologous bone grafting is accompanied by a number of related complications such as hematoma of donor area, wound dehiscence, pain of donor bone area, cutaneous nerve injury, incision infection, etc. Allograft bone grafting is also a method of treating bone defects, but related complications such as immune response, infection, delayed bone healing, bone resorption, etc. also occur.
With the development of tissue engineering technology, the decellularized bone tissue engineering scaffold material is gradually applied to clinically repairing bone defects, can make up for the defects of autogenous bone and allogenic bone transplantation to a certain extent, and has wide application prospect in orthopedics clinic. However, there are still many drawbacks to existing bone tissue engineering scaffold materials that need to be overcome: 1. the manufacturing procedure is complex and the manufacturing cost is high. 2. The effects of promoting bone formation, bone conduction, bone induction, etc. are not exact. 3. The treatment time and the treatment period are longer. Therefore, it is important to design a new material that can assist in the repair of bone defects.
Research on the promotion of bone injury repair by using mesenchymal stem cells (mesenchymal stem cells, MSCs) as seed cells is receiving increasing attention. MSCs are adult stem cells which are derived from mesoderm and have multidirectional differentiation potential and self-renewal capacity, can differentiate into osteoblasts, chondroblasts and the like, and can chemotactic to damaged tissues to play roles in promoting tissue repair, secreting cytokines, playing an immunoregulatory function, promoting angiogenesis and the like when the tissues are damaged. The recruitment, proliferation and differentiation of MSCs into osteoblasts at the site of bone injury play an important role in the bone repair process, and the local recruitment of sufficient numbers of MSCs at the injury is a precondition and cytological basis for bone injury repair. However, the mere ability of seed cells is limited and insufficient to meet clinical needs, so that the treatment of seed cells and the combination of biological scaffold materials are important means for solving the current problems.
At present, in the orthopaedics field, a large number of animal experiments and a small number of precursor clinical experiments prove that the magnesium metal implant can promote the generation of surrounding new bone. Meanwhile, the research shows that magnesium ions with a certain concentration can promote proliferation and adhesion of cells in early stage. Recently, a journal of Biomaterials has shown that the expression of Calcitonin Gene Related Peptide (CGRP) and periostin can be significantly increased in bone tissue after pretreatment by magnesium rod implantation.
Disclosure of Invention
In view of the above, the invention provides a bone defect repair material and a magnesium pretreatment decellularized tissue engineering bone scaffold, which are used for solving the problems of complex manufacturing procedure, high manufacturing cost, inaccurate treatment effect, overlong period, poor bone defect repair effect of the existing bone tissue engineering scaffold, and have the advantages of simple manufacturing procedure, low manufacturing cost, good bone formation promotion effect and the like.
The technical scheme of the invention is as follows:
the invention provides a bone defect repair material, which comprises a carrier and an osteogenesis promoter loaded on the carrier, wherein the carrier adopts femur, and the osteogenesis promoter comprises magnesium ions, and growth factors and BMSCs generated after the magnesium ions and magnesium rods are implanted and stimulated together.
Further, the vector employs a rat femur.
Further, the bone defect repair material is prepared by the following steps:
1. a retrograde intramedullary nail implantation method is adopted, a magnesium rod is implanted into a femoral intramedullary cavity through femoral intercondylar, after 2 weeks, a rat is sacrificed, the femur is taken out, and cylindrical bone tissue materials are drilled at the metaphyseal of the femur;
2. performing decellularization treatment on the rat femur after magnesium pre-stimulation to obtain a decellularized bone scaffold;
3. amplified BMSCs were inoculated into decellularized bone scaffolds.
Further, the second step comprises the following steps:
s1: washing the taken bone tissue by using phosphate buffer solution, wrapping the bone tissue by using gauze, performing freeze thawing cycle for a plurality of times, taking out a sample, putting the sample into the phosphate buffer solution added with the double antibodies, and performing oscillation rinsing at the temperature of 4 ℃;
s2: preparing 0.1-0.3% TritonX-100 solution, adding double antibody, soaking the bracket in the solution, and rinsing on a shaker at 4deg.C;
s3: placing the bracket into phosphate buffer solution with double antibodies, adding 2% sodium dodecyl sulfate, and oscillating on a shaking table in an environment of 4 ℃;
s4: after taking out the sample, putting the sample into phosphate buffer solution added with double antibodies, and rinsing in an oscillating way at the temperature of 4 ℃;
s5: adding the double antibody into 1mg/mL nuclease solution, placing the solution into incubation for 3-4 days, and performing normal temperature;
s6: adding the mixture into phosphate buffer solution of the diabody at 4 ℃ for shaking rinsing.
Further, step S6 further includes step S7: taking out the sample, freeze-drying, sterilizing with ethylene oxide, and sealing for storage.
Further, the freeze thawing cycle is to put liquid nitrogen for 10-15min, take out and put into a constant temperature water bath kettle at 37-40 ℃ for 10-15min.
Further, in step S2, the mixture is rinsed on a shaker at 4℃for 12-24 hours.
Further, in step S3, the mixture was shaken on a shaker at 4℃for 24 hours.
Further, in step S4, the rinsing is performed with shaking at 4℃for 2 to 3 hours for 3 times.
The invention also provides a magnesium pretreatment decellularized tissue engineering bone scaffold, which is prepared by applying the bone defect repair material.
The invention has the beneficial effects that:
after the magnesium rod is implanted into the body of the experimental animal, the experimental animal is taken as a bioreactor, and BMSCs in the femur of the experimental animal are stimulated to secrete CGRP, periostin, growth factors, collagen-I and the like through magnesium ions released by the magnesium rod, so that the capabilities of proliferation, adhesion, osteogenic differentiation and the like of the BMSCs in the femur can be enhanced.
The immunogenicity of the scaffold is eliminated by decellularization techniques, while retaining the naturally complex structure and fine microarchitecture, and the growth factors and various ECM components, such as fibronectin, heparin sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid, remain partially active after decellularization, while retaining their osteogenic differentiation promoting function.
The magnesium ion pre-stimulated decellularized bone scaffold can enhance biological behaviors such as adhesion, osteogenesis and the like of BMSCs. The BMSCs-loaded magnesium pre-stimulated decellularized bone scaffold can promote the repair of bone defects and shorten the treatment time, and can solve the defects of hematoma in a donor area, wound dehiscence, pain in the donor area, cutaneous nerve injury, incision infection, immune response, infection, delayed bone healing, bone resorption and the like in allogeneic bone transplantation, and the defects of complex manufacturing procedure, high manufacturing cost, inaccurate effect, long treatment period and the like of the conventional bone tissue engineering scaffold material.
The preferred embodiments of the present invention and their advantageous effects will be described in further detail with reference to specific embodiments.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Example 1
The invention provides a bone defect repair material, which comprises a carrier and an osteogenesis promoter loaded on the carrier. Preferably, the carrier adopts rat femur, and has the advantages of low cost and easy implantation operation. The following description will be given by taking a rat femur as an example. It will be appreciated that the vector is not limited to the femur of a rat, but may be used with xenogeneic bones from rabbits, dogs, pigs, etc. The osteogenesis promoter is mainly formed by magnesium ions, protein components such as growth factors generated after the magnesium ions and the magnesium rod are implanted and stimulated together, and BMSCs.
The bone defect repair material provided by the invention is prepared by the following steps:
1. SD rats with proper age are selected, a retrograde intramedullary nail implantation method is adopted, a magnesium rod is implanted into a femoral bone marrow cavity (the length of the magnesium rod is about 2.5 cm) through femoral intercondylar, and after 2 weeks, the rats are sacrificed, and the femur is taken out. According to clinical and experimental requirements, a dental drill with a corresponding diameter is selected, and cylindrical bone tissue material is drilled at the metaphyseal of femur.
2. And (3) performing decellularization treatment on the rat femur after magnesium pre-stimulation to obtain the decellularized bone scaffold. The method comprises the following specific steps:
(1) The removed bone tissue was washed 3 times for 10 minutes with Phosphate Buffered Saline (PBS). Then wrapping with gauze, and performing five freeze thawing cycles (one cycle after placing into liquid nitrogen for 10min and placing into a constant temperature water bath at 37 ℃ for 10min after taking out). After taking out the sample, the sample is put into PBS solution added with double antibodies, and the sample is rinsed for 3 times under the condition of 4 ℃ in an oscillating way, and each time is 2 hours.
(2) Preparing 0.1% TritonX-100 solution, adding double antibody, soaking the stent, and rinsing on a shaker at 4deg.C for 12 hr.
(3) The scaffolds were placed in PBS with double antibody, 2% Sodium Dodecyl Sulfate (SDS) was added and shaken on a shaker at 4℃for 24 hours.
(4) After taking out the sample, the sample is put into PBS solution added with double antibodies, and the sample is rinsed for 3 times under the condition of 4 ℃ in an oscillating way, and each time is 2 hours.
(5) Adding the diabody into 1mg/mL nuclease solution, placing and incubating for 3 days at normal temperature.
(6) The solution was rinsed by shaking in PBS at 4deg.C for 2 hours each time and repeated 3 times.
(7) Taking out the sample, freeze-drying, sterilizing with ethylene oxide, and sealing for storage.
3. Amplified mouse BMSCs were inoculated into decellularized bone scaffolds. A mouse bone defect model is constructed, a BMSCs-loaded decellularized bone scaffold is implanted into a bone defect position, and imaging and histological analysis are carried out 2 weeks and 4 weeks after operation.
Example two
The invention provides a bone defect repair material, which comprises a carrier and an osteogenesis promoter loaded on the carrier. Preferably, the carrier adopts rat femur, and has the advantages of low cost and easy implantation operation. The following description will be given by taking a rat femur as an example. It will be appreciated that the vector is not limited to the femur of a rat, but may be used with xenogeneic bones from rabbits, dogs, pigs, etc. The osteogenesis promoter is mainly formed by magnesium ions, protein components such as growth factors generated after the magnesium ions and the magnesium rod are implanted and stimulated together, and BMSCs.
The bone defect repair material provided by the invention is prepared by the following steps:
1. SD rats with proper age are selected, a retrograde intramedullary nail implantation method is adopted, a magnesium rod is implanted into a femoral bone marrow cavity (the length of the magnesium rod is about 2.5 cm) through femoral intercondylar, and after 2 weeks, the rats are sacrificed, and the femur is taken out. According to clinical and experimental requirements, a dental drill with a corresponding diameter is selected, and cylindrical bone tissue material is drilled at the metaphyseal of femur.
2. And (3) performing decellularization treatment on the rat femur after magnesium pre-stimulation to obtain the decellularized bone scaffold. The method comprises the following specific steps:
(1) The removed bone tissue was washed 3 times for 15 minutes with Phosphate Buffered Saline (PBS). Then wrapping with gauze, and performing five freeze thawing cycles (placing into liquid nitrogen for 12min, taking out, and placing into a 37 ℃ constant temperature water bath for 13min, which is one cycle). After the sample was taken out, it was put into a PBS solution to which a double antibody was added, and rinsed with shaking at 4℃for 3 times, each for 2.5 hours.
(2) Preparing 0.2% TritonX-100 solution, adding double antibody, soaking the stent, and rinsing on a shaker at 4deg.C for 18 hr.
(3) The scaffolds were placed in PBS with double antibody, 2% Sodium Dodecyl Sulfate (SDS) was added and shaken on a shaker at 4℃for 24 hours.
(4) After the sample was taken out, it was put into a PBS solution to which a double antibody was added, and rinsed with shaking at 4℃for 3 times, each for 2.5 hours.
(5) Adding the diabody into 1mg/mL nuclease solution, placing and incubating for 4 days at normal temperature.
(6) The solution was rinsed by shaking in PBS at 4deg.C, and repeated 3 times for 2.5 hours each.
(7) Taking out the sample, freeze-drying, sterilizing with ethylene oxide, and sealing for storage.
3. Amplified mouse BMSCs were inoculated into decellularized bone scaffolds. A mouse bone defect model is constructed, a BMSCs-loaded decellularized bone scaffold is implanted into a bone defect position, and imaging and histological analysis are carried out 2 weeks and 4 weeks after operation.
Example III
The invention provides a bone defect repair material, which comprises a carrier and an osteogenesis promoter loaded on the carrier. Preferably, the carrier adopts rat femur, and has the advantages of low cost and easy implantation operation. The following description will be given by taking a rat femur as an example. It will be appreciated that the vector is not limited to the femur of a rat, but may be used with xenogeneic bones from rabbits, dogs, pigs, etc. The osteogenesis promoter is mainly formed by magnesium ions, protein components such as growth factors generated after the magnesium ions and the magnesium rod are implanted and stimulated together, and BMSCs.
The bone defect repair material provided by the invention is prepared by the following steps:
1. SD rats with proper age are selected, a retrograde intramedullary nail implantation method is adopted, a magnesium rod is implanted into a femoral bone marrow cavity (the length of the magnesium rod is about 2.5 cm) through femoral intercondylar, and after 2 weeks, the rats are sacrificed, and the femur is taken out. According to clinical and experimental requirements, a dental drill with a corresponding diameter is selected, and cylindrical bone tissue material is drilled at the metaphyseal of femur.
2. And (3) performing decellularization treatment on the rat femur after magnesium pre-stimulation to obtain the decellularized bone scaffold. The method comprises the following specific steps:
(1) The removed bone tissue was washed 3 times for 20 minutes with Phosphate Buffered Saline (PBS). Then wrapping with gauze, and performing five freeze thawing cycles (placing into liquid nitrogen for 15min, taking out, and placing into a constant-temperature water bath at 37 ℃ for 15min, which is one cycle). After the sample was taken out, it was put into a PBS solution to which a double antibody was added, and rinsed 3 times with shaking at 4℃for 3 hours each.
(2) Preparing 0.3% TritonX-100 solution, adding double antibody, soaking the stent, and rinsing on a shaker at 4deg.C for 24 hr.
(3) The scaffolds were placed in PBS with double antibody, 2% Sodium Dodecyl Sulfate (SDS) was added and shaken on a shaker at 4℃for 24 hours.
(4) After the sample was taken out, it was put into a PBS solution to which a double antibody was added, and rinsed 3 times with shaking at 4℃for 3 hours each.
(5) Adding the diabody into 1mg/mL nuclease solution, placing and incubating for 4 days at normal temperature.
(6) The solution was rinsed by shaking in PBS at 4deg.C, and repeated 3 times for 3 hours each.
(7) Taking out the sample, freeze-drying, sterilizing with ethylene oxide, and sealing for storage.
3. Amplified mouse BMSCs were inoculated into decellularized bone scaffolds. A mouse bone defect model is constructed, a BMSCs-loaded decellularized bone scaffold is implanted into a bone defect position, and imaging and histological analysis are carried out 2 weeks and 4 weeks after operation.
The experimental results of the three embodiments can show that the technical scheme of the invention has the following beneficial effects:
after the magnesium rod is implanted into the body of the experimental animal, the experimental animal is taken as a bioreactor, and BMSCs in the femur of the experimental animal are stimulated to secrete CGRP, periostin, growth factors, collagen-I and the like through magnesium ions released by the magnesium rod, so that the capabilities of proliferation, adhesion, osteogenic differentiation and the like of the BMSCs in the femur can be enhanced.
The immunogenicity of the scaffold is eliminated by decellularization techniques, while retaining the naturally complex structure and fine microarchitecture, and the growth factors and various ECM components, such as fibronectin, heparin sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid, remain partially active after decellularization, while retaining their osteogenic differentiation promoting function.
The magnesium ion pre-stimulated decellularized bone scaffold can enhance biological behaviors such as adhesion, osteogenesis and the like of BMSCs. The BMSCs-loaded magnesium pre-stimulated decellularized bone scaffold can promote the repair of bone defects and shorten the treatment time, and can solve the defects of hematoma in a donor area, wound dehiscence, pain in the donor area, cutaneous nerve injury, incision infection, immune response, infection, delayed bone healing, bone resorption and the like in allogeneic bone transplantation, and the defects of complex manufacturing procedure, high manufacturing cost, inaccurate effect, long treatment period and the like of the conventional bone tissue engineering scaffold material.
Example IV
The invention needs to provide a magnesium pretreatment decellularized tissue engineering bone scaffold which is prepared from the bone defect repair materials in the first, second and third embodiments.
The foregoing is only illustrative of the present invention and is not to be construed as limiting thereof, but rather as various modifications, equivalent arrangements, improvements, etc., within the spirit and principles of the present invention.
Claims (9)
1. The bone defect repairing material is characterized by comprising a carrier and an osteogenesis promoter loaded on the carrier, wherein the carrier adopts femur, and the osteogenesis promoter comprises magnesium ions, growth factors and BMSCs generated after the magnesium ions and magnesium rods are implanted together for stimulation;
the bone defect repair material is prepared by the following steps:
1. a retrograde intramedullary nail implantation method is adopted, a magnesium rod is implanted into a femoral intramedullary cavity through femoral intercondylar, after 2 weeks, a rat is sacrificed, the femur is taken out, and cylindrical bone tissue materials are drilled at the metaphyseal of the femur;
2. performing decellularization treatment on the rat femur after magnesium pre-stimulation to obtain a decellularized bone scaffold;
3. amplified BMSCs were inoculated into decellularized bone scaffolds.
2. The bone defect repair material of claim 1, wherein the carrier is a rat femur.
3. The bone defect repair material of claim 1, wherein step two comprises the steps of:
s1: washing the taken bone tissue by using phosphate buffer solution, wrapping the bone tissue by using gauze, performing freeze thawing cycle for a plurality of times, taking out a sample, putting the sample into the phosphate buffer solution added with the double antibodies, and performing oscillation rinsing at the temperature of 4 ℃;
s2: preparing 0.1-0.3% TritonX-100 solution, adding double antibody, soaking the bracket in the solution, and rinsing on a shaker at 4deg.C;
s3: placing the bracket into phosphate buffer solution with double antibodies, adding 2% sodium dodecyl sulfate, and oscillating on a shaking table in an environment of 4 ℃;
s4: after taking out the sample, putting the sample into phosphate buffer solution added with double antibodies, and rinsing in an oscillating way at the temperature of 4 ℃;
s5: adding the double antibody into 1mg/mL nuclease solution, placing the solution into incubation for 3-4 days, and performing normal temperature;
s6: adding the mixture into phosphate buffer solution of the diabody at 4 ℃ for shaking rinsing.
4. The bone defect repair material of claim 3, further comprising step S7 after step S6: taking out the sample, freeze-drying, sterilizing with ethylene oxide, and sealing for storage.
5. The bone defect repair material according to claim 3, wherein the freeze-thawing cycle is performed by placing in liquid nitrogen for 10-15min, taking out, and placing in a constant temperature water bath kettle at 37-40 ℃ for 10-15min.
6. The bone defect repair material of claim 3, wherein in step S2, the material is rinsed on a shaker at 4 ℃ for 12-24 hours.
7. The bone defect repair material of claim 3, wherein in step S3, the vibration is performed on a shaker at 4 ℃ for 24 hours.
8. The bone defect repair material according to claim 3, wherein in step S4, rinsing is performed with shaking at 4 ℃ for 3 times, each for 2 to 3 hours.
9. A magnesium pretreatment decellularized tissue engineering bone scaffold prepared by applying the bone defect repair material of any one of claims 1-8.
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