CN1846793B - A kind of tissue engineering bone and its construction and application - Google Patents

Abstract

The invention belongs to the technical field of constructing artificial organs by tissue engineering method in biomedical engineering, and in particular relates to a tissue engineering bone and its construction and application. The tissue-engineered bone of the present invention includes a carrier scaffold and seed cells. The seed cells are attached to the carrier scaffold to form a complex with three-dimensional bone tissue structure and biological activity. The carrier scaffold is modified with a large pore size and The PLGA with high porosity and deacidification treatment is loaded with the cytokine bone morphogenetic protein, namely BMP; the seed cells are bone marrow stromal stem cells. The tissue engineered bone described in the invention can be used to construct a functional tissue engineered bone graft for repairing large bone defects.

Description

A kind of tissue engineering bone and its construction and application

technical field

The invention belongs to the technical field of constructing artificial organs by tissue engineering method in biomedical engineering, and in particular relates to a tissue engineering bone and its construction and application.

Background technique

In recent years, with the development of tissue engineering technology, reports on the research and development of biological substitutes that can repair, maintain or improve the function of damaged tissues have gradually increased, among which bone tissue engineering research is a hot spot, and a relatively complete system has gradually formed. Theories and technical routes on seed cells, cell carrier scaffolds and tissue construction. But it is undeniable that these achievements are still far from the final clinical application of bone tissue engineering. Therefore, it is necessary to develop a new type of tissue-engineered bone product that can be rapidly applied in clinic.

The basic method of tissue engineering artificial tissue is to disperse the tissue obtained in vivo into a single cell suspension by mechanical method or enzymatic digestion, and then incubate and culture under in vitro conditions simulating the in vivo environment, so that the cells can survive, grow and expand. Then, the cells with a certain concentration cultured in vitro are planted on a three-dimensional scaffold material with a certain spatial structure, and further cultivated. Through the mutual adhesion, growth, reproduction, and secretion of extracellular matrix between cells, a certain structure and function are formed. tissues and organs. Cells cultured in vitro should have most of the functions of cells in vivo. For example, osteoblasts cultured in vitro have high alkaline phosphatase activity, secrete osteocalcin, synthesize and secrete type I and II collagen, etc. Studies have shown that in the presence of calcification conditions (calcium ions, sodium β-glycerophosphate, vitamin C, dexamethasone, etc.), osteoblasts can form trabecular bone in culture flasks. Therefore, it is entirely feasible to inoculate osteoblasts on a certain scaffold material by using tissue engineering methods, and add some cytokines that promote bone growth and bone nerve vascularization to culture in vitro to obtain a piece of bone tissue.

The biggest problem in current bone tissue engineering research is the choice of scaffold materials. Scaffold materials required for bone tissue engineering should have the following characteristics: good biocompatibility and biodegradability, and the degradation products in the body are harmless to the human body; ② have certain osteoinductivity and osteoconductivity, which is conducive to cell attachment ③has a certain strength, maintains its own shape in the body and can resist external forces; ④easy to shape, can be processed into various shapes and sizes according to needs; ⑤has high permeability to load the largest number of cells; ⑥supports osteogenesis The surface chemical properties and microstructure of cell growth and functional differentiation; ⑦ It can be complexed with other bioactive molecules such as bone morphogenetic protein (BMP) and released to regulate the growth of seed cells; ⑧ Ease of disinfection. At present, the sources of scaffold materials for bone tissue engineering can be divided into two categories: inorganic materials and organic materials. Inorganic materials mainly refer to bioceramic materials. This type of material is mainly composed of calcium and phosphorus elements, which are similar to the main inorganic components in the human body, so they have good biocompatibility, biodegradability and osteoconductivity. However, the biggest defect of this type of material is that it is extremely difficult to degrade, which affects the formation and reconstruction of new bone. Organic materials can be divided into natural and artificial organic materials. Natural organic materials include collagen, chitosan, fibrin gel, etc. The commonality of these materials is good biocompatibility, which is beneficial to cell attachment, proliferation, and differentiation, but the mechanical strength is not enough for bone tissue engineering scaffolds, and the degradation time is difficult. control.

Bone morphogenetic protein (Bone morphogenetic protein, BMP) is an earlier bone growth factor studied, and its role in inducing osteogenesis has been confirmed by many experiments. It is the only local growth factor that can induce the formation of bone tissue alone. Under certain conditions, it can induce the transformation of undifferentiated mesenchymal cells into bone cells and promote the growth and proliferation of bone cells. At present, BMP is considered to be the most important one in promoting osteogenesis in bone tissue engineering. However, because BMP has very little content in bone tissue (about 1 mg/kg wet bone), and diffuses quickly in the body, it is easily decomposed by proteases, so it cannot continuously stimulate and induce osteogenesis in the local area. Difficult to get full play.

Although great progress has been made in the development of tissue engineered bone, and some products have entered the stage of clinical trials, these products are still not perfect, and have defects in many aspects such as mature methods, development cycle and product cost, especially for large bone defects in clinical practice. Repair still has no obvious effect.

Contents of the invention

The object of the present invention is to provide a tissue engineered bone. The tissue-engineered bone has no rejection reaction after being transplanted into the human body, can be completely degraded and absorbed within a certain period of time, has an obvious osteogenic effect, can repair large bone defects, and has low cost.

A tissue engineered bone according to the present invention comprises a carrier scaffold and seed cells, the seed cells are attached to the carrier scaffold to form a complex with three-dimensional bone tissue structure and biological activity, and the carrier scaffold is modified with a large The PLGA with high porosity and deacidification treatment is loaded with the cytokine bone morphogenetic protein, namely BMP; the seed cells are bone marrow stromal stem cells (bone marrow stem cells, BMSCs).

The seed cells are bone marrow stromal stem cells of the third generation obtained from the same bone marrow, separated, expanded and cultured in vitro, with a cell density of 1×10 ⁶ -1×10 ⁷ cells/ml.

The pore diameter of the PLGA carrier bracket is 150-200um, and the pore diameter ratio is 85-95%.

The invention also provides the method for constructing the tissue engineered bone.

The construction method of tissue engineered bone of the present invention comprises the following steps:

A. Using modified PLGA with large pore size and high porosity and deacidification treatment as raw material, adding cytokine bone morphogenetic protein, namely BMP, during the construction of tissue engineering bone carrier scaffold;

B. Digest and centrifuge bone marrow stromal stem cells taken from the same bone marrow that were isolated, expanded and cultured in vitro to the third passage, and inoculated on the PLGA carrier scaffold at a concentration of 1×10 ⁶ -1×10 ⁷ /ml;

C. Put the PLGA carrier scaffold compounded with cells into the incubator for 3-6 hours, add DMEM conditioned medium, put it back into the incubator and continue to culture for 3-5 days, and obtain the tissue engineered bone.

In the above construction method, the seed cells, namely bone marrow stromal stem cells, are constructed through the following steps:

(1) Acquire cancellous bone marrow, and gradually wash away red blood cells in the process of changing the medium with the whole bone marrow culture method to obtain bone marrow stromal stem cells;

(2) Digest with 0.25% trypsin and pass to the third generation bone marrow stromal stem cells;

(3) When the 3rd generation cells are transferred, the DMEM conditioned medium is used to change the medium to induce osteoblast orientation, and it is ready to use after 3-5 days of culture.

The DMEM conditioned medium contains 15% serum, 50 μg/ml ascorbic acid, 10 ⁻⁸ mol/L dexamethasone, and 10 mmol/L β-sodium glycerophosphate.

In the above construction method, the PLGA support is constructed through the following steps:

a. Using modified PLGA with large pore size and high porosity as raw material, add powdered cytokine bone morphogenetic protein (BMP), and trim into shape;

b. Ultrasonic vibration cleaning;

c. Surface deacidification treatment;

d. Soak in ethylene oxide fumigation and sterilization at 37°C;

e. Add DMEM complete medium containing 15% serum and soak for 2-3 days;

f. Add 10% polylysine solution and soak for 1-2 days;

g. Dry under UV light at 15-25°C and store for future use.

The raw material of the carrier bracket used in the present invention is polylactic-glycolic acid (polylactic-glycolic acid, PLGA), which is a derivative of artificial organic material PLA, has good biocompatibility, non-toxic degradation products, easy processing, certain strength, etc. The advantages make the tissue engineered bone of the present invention widely used.

The cytokine used in the present invention is bone morphogenetic protein (BMP), which can not only accelerate bone regeneration, but also accelerate the degradation of scaffold materials.

The invention optimizes the three-dimensional structure of the PLGA scaffold material, and can maximize the biological efficacy of the BMP. Using modified PLGA with large pore size and high porosity and deacidification treatment as raw material, the preferred PLGA carrier bracket has a pore size of 150-200um and a pore size of 85-95%, and can be arbitrarily processed according to experimental requirements. Various specifications.

The present invention also provides the use of said tissue engineered bone.

The tissue engineered bone described in the invention can be used to construct a functional tissue engineered bone graft for repairing large bone defects. This use has been established through animal bone defect repair tests.

Description of drawings

Fig. 1 is a schematic diagram of the structure of tissue engineered bone according to the present invention, wherein (a) is a partially cut-away schematic diagram, and (b) is a cross-sectional view along A-A in (a).

Fig. 2 is a phase-contrast microscope observation (100×) diagram of the tissue engineered bone according to the present invention after 3 days of culture.

Fig. 3 is a HE-stained section view of the broken end of the bone defect 4 weeks after the tissue engineered bone of the present invention was used to repair the defect of the rabbit radius.

Fig. 4 is a local X-ray diagram of 4, 8, and 12 weeks after repairing the radial bone defect of rabbits with the tissue engineered bone according to the present invention, respectively 4, 8, and 12 weeks from left to right.

Detailed ways

The present invention and implementation effect will be further described in conjunction with experiment now.

As shown in Figure 1, a kind of tissue engineered bone according to the present invention includes a carrier scaffold and seed cells, and the seed cells are attached to the carrier scaffold to form a complex with bone tissue three-dimensional structure and biological activity, and the carrier scaffold It is modified PLGA with large pore size and high porosity and deacidification treatment, on which is loaded with cytokine bone morphogenetic protein, namely BMP; the seed cells are bone marrow stromal stem cells.

Figure 1 is a microscopic schematic diagram, and its proportion does not represent the proportion of the actual product.

Example 1: In vitro culture, expansion and directional induction of bone marrow stromal stem cells

According to the whole bone marrow cell culture method, the bone marrow puncture needle was inserted into the bone marrow cavity of the ilium, a total of 3-5ml of bilateral bone marrow was aspirated, mixed into DMEM medium, anticoagulated with 500U/ml heparin solution, mixed and then centrifuged at 800r/min at low speed for 5min , remove the supernatant, resuspend the culture medium and inoculate through a 90-mesh filter. Culture in a 37°C, 5% _CO2 incubator, change the medium in half (DMEM complete medium, 15% serum) after 4 days, and change the medium once in the next 2 to 3 days, observe daily with an inverted microscope, and wait until the cells confluence into a monolayer After that, they were digested with 0.25% trypsin, counted, subcultured, and the medium was changed once every 3 days. When the cells were transferred to the third generation, they were replaced with DMEM conditioned medium (containing 15% serum, 50 μg/mi ascorbic acid, 10 ^-8 mol/L dexamethasone, 10 mmol/L β-sodium glycerophosphate) for later use.

Example 2: Loading of PLGA scaffolds and cytokines

(1) Using modified PLGA with large pore size and high porosity as raw material, adding powdered cytokine bone morphogenetic protein, namely BMP, and trimming into shape;

(2) Ultrasonic vibration cleaning: use triple distilled water to clean with ultrasonic vibration, and dry naturally

(3) Surface deacidification treatment: immerse in 1% NaOH ₂ for 2 hours, wash with PBS buffer, wash with 50% ethanol for 2 hours, wash with PBS buffer;

(4) Sterilize by ethylene oxide fumigation at 37°C;

(5) Add DMEM complete medium containing 15% serum and soak for 2-3 days;

(6) Add 10% polylysine solution and soak for 1-2 days;

(7) Dry under ultraviolet light at 15-25°C and store for future use.

In this embodiment, the PLGA scaffold is in the shape of a 4×4×2 mm block, with a pore diameter of 150-200 um and a pore diameter ratio of 85-95%.

Example 3: Construction of Tissue Engineered Bone Loaded with Cytokines

Trypsinize and centrifuge BMSCs that have passed the third passage according to the method described in Example 1 and have grown well after being induced by conditioned medium, and use a micropipette at a concentration of 1×10 ⁶ -1×10 ⁷ /ml Inoculate the cell suspension onto the material constructed in Example 2, put it in a 37°C, 5% _CO2 incubator for 4 hours, add an appropriate amount of conditioned medium, and put it back into the incubator to continue culturing for 3-5 days, that is, Obtain the tissue-engineered bone and save it for future use.

Embodiment 4: Animal bone defect repair test

New Zealand big-eared white rabbits, 2 months old, weighing 1.5-2.0 kg, male or female, were purchased from the Animal Center of Southern Medical University (formerly First Military Medical University). Under sterile conditions, standard bone defects with a length of 15 mm were constructed in the middle segment of the right radius, and the tissue engineered bone constructed in Example 3 was implanted. The incisions were closed layer by layer, and the animals moved normally after operation. The control groups were the blank PLGA group and the PLGA+BMSCs group. Animals were sacrificed at 4, 8, and 12 weeks, and the following tests were performed: (1) Gross and X-ray observation: observe animal activities, gait, wound healing, and defect repair, and compare the optical density values of new bone in the bone defect area. (2) Histological observation: Bone specimens were taken from the midsection of the double radius, fixed with 10% paraformaldehyde, decalcified, routinely embedded in paraffin, stained with HE, and observed the growth of new bone with a light microscope. (3) Biomechanical tests were performed on the new bone in the bone defect area and the normal bone at the same site, including compressive strain and three-point bending tests.

result:

Gross observation: The bone defects in the tissue engineered bone group were completely repaired and the appearance was normal.

Phase-contrast microscope observation: As shown in Figure 2, BMSCs and BMP-loaded PLGA scaffolds were cultured for 3 days. It can be seen that the cells and materials were well attached, and a large amount of cell matrix was secreted, and the intercellular space was not clear.

Histological observation: As shown in Figure 3, it can be seen that the PLGA scaffold is degraded into a mesh-like shape, and chondrocytes and a large amount of cartilage matrix are formed. HE staining showed that a large amount of new bone had formed at 4 weeks, and the bone formation at each time period was significantly better than that of the control group.

X-ray observation: as shown in Figure 4(a), X-rays at the 4th week showed that there was obvious new bone growth in the bone defect area, and the interface between the implanted tissue engineered bone and the implanted bone became blurred; as shown in Figure 4(b) It was shown that continuous bone callus appeared in the bone defect area at the 8th week, and the density was significantly increased; as shown in Figure 4(c), the bone defect was basically completely repaired after the 12th week, and the medullary cavity recanalized.

Optical density detection: it shows that the tissue engineered bone composition of the present invention has the largest bone mass.

Biomechanical test: It showed significant differences in load and bending stress among the groups.

Claims (6)

1. A tissue engineered bone, comprising a carrier support and seed cells, the seed cells are attached to the carrier support to form a complex with bone tissue three-dimensional structure and biological activity, characterized in that: the carrier support is modified with PLGA with large pore size and high porosity and deacidification treatment, the PLGA carrier bracket has a pore size of 150-200um and a porosity of 85-95%, and the PLGA carrier bracket is loaded with cytokines, which are bone The morphogenic protein is BMP; the seed cells are third-generation bone marrow stromal stem cells derived from the same bone marrow, and the cell density is 1×10 ⁶ -1×10 ⁷ cells/ml. 2. The construction method of tissue engineering bone as claimed in claim 1, is characterized in that, comprises the following steps: A. Using modified PLGA with large pore size and high porosity and deacidification treatment as raw material, adding cytokine bone morphogenetic protein, namely BMP, during the construction of tissue engineering bone carrier scaffold; B. Digest and centrifuge bone marrow stromal stem cells taken from the same bone marrow that were isolated, expanded and cultured in vitro to the third passage, and inoculated on the PLGA carrier scaffold at a concentration of 1×10 ⁶ -1×10 ⁷ /ml; C. Put the PLGA carrier scaffold compounded with cells into the incubator for 3-6 hours, add DMEM conditioned medium, put it back into the incubator and continue to culture for 3-5 days, and obtain the tissue engineered bone. 3. The construction method of tissue engineered bone according to claim 2, characterized in that, said bone marrow stromal stem cells are constructed through the following steps: (1) Acquire cancellous bone marrow, and gradually wash away red blood cells in the process of changing the medium with the whole bone marrow culture method to obtain bone marrow stromal stem cells; (2) Digest with 0.25% trypsin and pass to the third generation bone marrow stromal stem cells; (3) When the 3rd generation cells are transferred, the DMEM conditioned medium is used to change the medium to induce osteoblast orientation, and it is ready to use after 3-5 days of culture. 4. The construction method of tissue engineered bone according to claim 3, characterized in that, the DMEM conditioned medium comprises 15% serum, 50 μg/ml ascorbic acid, 10 ⁻⁸ mol/L dexamethasone and 10 mmol/L β - Sodium glycerophosphate. 5. the construction method of tissue engineering bone according to claim 2, is characterized in that, described PLGA scaffold is to construct by following steps: a. Using modified PLGA with large pore size and high porosity as raw material, add powdered cytokine bone morphogenetic protein (BMP), and trim into shape; b. Ultrasonic vibration cleaning; c. Surface deacidification treatment; d. Soak in ethylene oxide fumigation and sterilization at 37°C; e. Add DMEM complete medium containing 15% serum and soak for 2-3 days; f. Add 10% polylysine solution and soak for 1-2 days; g. Dry under UV light at 15-25°C and store for future use. 6. The use of the tissue engineered bone as claimed in claim 1 in constructing a functional tissue engineered bone graft for repairing large bone defects. the

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