| Background:Intervertebral disc degeneration is one of the important reasons causing spinal anatomical and functional disorder, is a series of pathologic basis of spinal degenerative diseases.As the population ages and people life rhythm speeding up, the incidence of spinal diseases becomes more and more high, which also are a heavy burden to individuals, families and society.Therefore, more and more researcher pay attention to the treatment of intervertebral disc degeneration, and intervertebral disc degeneration research become the focus in the multiple disciplines.Although the extensive and in-depth basic and clinical research of occurrence, development and treatment of intervertebral disc degeneration was carried out both at home and abroad, as a result of intervertebral disc degeneration is a complex process, there is no effective method to prevent its occurrence and development in clinical, and no ideal treatment of intervertebral disc degeneration itself.Traditional treatment including conservative treatment and surgical treatment, conservative treatment is given priority to with symptomatic treatment and curative effect is limited;Surgical treatment include simple removal of nucleus pulposus or chemical dissolving of nucleus pulposus tissue, bone graft fusion, artificial disc replacement and so on, these methods to a certain extent, ease the symptoms caused by intervertebral disc degenerative diseases, but at the same time caused damage of anatomic structure and stability of the spine and the loss of the segmental activity function, increase of adjacent segment degeneration, and may cause many other complications, and the long-term effect is still not sure.A number of studies suggest that intervertebral disc degeneration begins with nucleus pulposus. nucleus pulposus degeneration is result in the decrease of the number nucleus pulposus cells and the decline in vigor. The reduction of secretion of extracellular matrix lead to nucleus pulposus water content decreased, the loss of nucleus pulposus elasticity and the ability to buffer pressure, reduced intervertebral disc height, increase pressure on the fiber ring, which some severe cases can cause deformation of fiber ring, intervertebral disc herniation or tear formation, therefore, to study ways to repair the degenerative nucleus pulposus, perhaps is the key to the repair of degenerative intervertebral disc.In recent years, with the continuous development and innovation of tissue engineering techniques and methods at home and abroad, to constrct the biological function of nucleus pulposus through the use of tissue engineering technology, perhaps will bring a new way for the treatment of intervertebral disc degenerative disease.Currently the research of nucleus pulposus tissue engineering scaffolds are variety, mainly includes:1. The synthetic material support, such as PLA, PGA, gelatin, etc.;2. Single natural ingredients such as hyaluronic acid, collagen type II, chitosan, etc.;3. Artificial-natural compound composition material, such as gelatin-chitosan composite scaffolds.Above scaffolds are still many deficiencies, because natural nucleus pulposus composition is complicated, it is difficult to complete simulation its’composition and micro environment. Extracellular matrix is extremely close to the normal nucleus pulposus, has been mad many deeper research and significant progress in some arease, for example in the bladder, blood vessels, the dermis, heart valves, and cartilage and so on, some even approved for use in clinical.It also have a lot of research in cartilage, but rarely report in the field of tissue engineering nucleus pulposus by retrieval of literature at home and abroad. We suppose nucleus pulposus extracellular matrix as material source of nucleus pulposus scaffold can also successfully building tissue engineering nucleus pulposus? On this basis, rabbit bone marrow mesenchymal stem cells were seeded on nucleus pulposus extracellular matrix-derived three-dimensional porus scaffold (NP ECM-derived 3D porus scaffolds), to construct tissue engineering nucleus pulposus in vitro.There is a lot of decellularized method at home and abroad, such as enzyme elimination, freeze drying, detergent processing method, etc., our previous study carried out extensive experimental research, summed up a set of ideal methods of decellularization, but we found that nucleus pulposus extracellular matrix exists obvious flaws, namely dense structure, porosity and pore diameter are unsatisfactory. So we further adopt a series of processing system of physical crushing, dissolved, centrifugal, freeze drying forming, physical and chemical crosslinking to make a nucleus pulposus matrix 3D porous scaffolds, it in addition to the nucleus pulposus cells and immune protein source sex small molecules, and thus has good biological compatibility* and lower cytotoxicity, basic retain the natural extracellular matrix of the nucleus pulposus original ingredients and local microenvironment.It owned a three-dimensional structure, good mechanical properties, good microenvironment, good porosity and pore diameter size.It is very beneficial to adhesion and migration of seeded cells.Rabbit nucleus pulposus are conveniently to collected, and fabrication techniques of rabbit intervertebral disc degeneration model are mature.Bone marrow mesenchymal stem cells are a class of widely exists in the hematopoietic system which has the ability of multi-directional differentiation of adult stem cells, also has a widely sources, easy to proliferation in vitro and small antigenicity and strong tissue repair capacity, it also facilitate genetic modification and owned ability of multi-directional differentiation which many literatures have reported bone marrow mesenchymal stem cells differentiate into the nucleus pulposus cells, therefore it is an ideal seed cells.Purpose:Prepare nucleus pulposus extracellular matrix using method of detergent-nuclease. Further physical crushing, dissolved, centrifugal, freeze drying forming, physical and chemical crosslinking and irradiation disinfection are preparared to fabricate NP ECM-derived porus scaffolds. rBMSCs were seeded into NP ECM-derived porus scaffolds, then the cell-scaffold complex were incubated in low oxygen environment and induced medium containing TGF-beta1 in vitro;The complex were transplanted into subcutaneous of nude mice for observation and evaluation of complex conditions in vivo. To evaluate decellularization degrees of scaffold and cell proliferation, migration, extracellular matrix secretion in scaffold by histopathology, biological chemistry, molecular biology technology.To evaluate biomechanical properties of the complex through biomechanical evaluation. To observe macrostructure and microstructure of scaffold and cell-scaffold complex by naked eye and scanning electron microscope.Cell proliferation were judged by the fluorescence quantitative analysis of total DNA. r to To evaluate proteoglycan and collagen type II mRNA content by real-time fluorescent quantitative analysis.A comprehensive evaluation of NP ECM-derived porus scaffolds and feasibility of its application were performed in the nucleus pulposus tissue engineering.Methods:1. Preparation of NP ECM-derived porus scaffolds.Select 2.5-3 kg of 36 New Zealand white rabbit as the source of nucleus pulposus, the detergent-nucleic acid enzymatic on rabbit nucleus pulposus cells treated, according to early research team concluded the scheme effective removal of nucleus pulposus cells, and extracellular matrix components loss less.In cell-free scheme after processing the original innovation, further will go to the cell nucleus pulposus substrate further adopting physical crushing, dissolved, centrifugal, freeze drying, forming, physical and chemical crosslinking, irradiation sterilization, and a series of processing system have to go to the cell nucleus pulposus matrix source three-dimensional porous scaffolds. To evaluate decellularization degrees of scaffold and cell proliferation, migration, extracellular matrix secretion in scaffold by HE staining, DAPI staining, scaffold morphology and pores size observed under SEM. Water absorption and porosity were Measured through water absorption test. Rabbit bone marrow mesenchymal stem cells were cultivated at different concentration of scaffold leaching solution and complete medium.The cell proliferation condition reflects scaffold cytotoxicity.2. rBMSCs were seeded into NP ECM-derived porus scaffolds and cultivated in vitro. Cell-scaffold complex were incubated in 5% O2,5% CO2 anaerobic incubator and in the induced medium for 4 days,2 weeks,4 weeks respectively. Gross morphology change of cell-scaffold complex by naked eyes; Cell proliferation were judged by the fluorescence quantitative analysis of total DNA;The adhesion, migration, proliferation and extracellular matrix secretion of seeded cell on the scaffold were observed by SEM.The survival of the cells in the scaffold were observed by Live/Dead cell staining; After the cell-scaffold complex induced cultivated for 2 weeks,4 weeks, the specimens sections were observed by HE staining, DAPI staining, alcian blue staining and collagen type Ⅱ immunohistochemical staining, and mRNA real-time fluorescence quantitative analysis of collagen type Ⅱ, aggrecan in vitro.Above results are evaluated the morphological changes, cell proliferation, differentiation and secretion of extracellular matrix of cell-scaffold complex.3.After 2 weeks induced cultivated, the NP ECM-derived porus scaffolds (blank control group) and cell-scaffold complex were transplanted into subcutaneous and muscle membrane layer of nude mice, after transplantation of cell-scaffold complex into nude mice for 4 weeks,the nude mice were detected in vivo imaging;The transplanted 2 weeks,4 weeks of complex samples were taken out for observation. It include naked observation of gross morphology changement, HE staining, DAPI staining, alcian blue staining, collagen type Ⅱ immunohistochemical staining and mRNA real-time fluorescence quantitative analysis of collagen type Ⅱ and proteoglycan.Above results can evaluated cell proliferation, differentiation and secretion of extracellular matrix of complex.Results:The first part:The gross morphology and basic character of NP ECM-derived porus scaffolds. It is cylindrical, milky white, elastic, like a sponge, porous structure by optical microscopy. The water absorptivity of the scaffold was 272.16 ± 33.30% and the porosity up to 81.28 ± 4.10%.SEM results shows that the pore size of scaffold range from 45um to 450μm, which was very close to the ideal size of TE (100-500um). However, the pore size of it are irregular and it lead to unsuitable for calculating the precise pore size. HE staining shows no cells or cell fragments were present in the scaffold. DAPI staining also reflects almost no cell nucleus left.The second part:Property evaluation of rBMSCs-scaffolds cultivated in vitro. Cells-scaffolds complex volume become smaller, more dense with the extension of incubation time in naked eye,and have formed a solid circle pie which is similar to nucleus pulposus tissue at 2 weeks. The amount of DNA was significantly increased over the period of culture from 1 day to 3 weeks. rBMSCs could survive well in the scaffolds by Live/Dead staining. The SEM imagesrevealed that rBMSCs could spread, grow and secrete abundant ECM. Compressive elastic modulus after 4 weeks of culture reached to 0.12 MPa which was significantly higher compared to 0.07 MPa of 2 week culture and 0.04 MPa of control. Compared with 2 weeks, H&E staining at week 4 showed a large number of cells distributed inside the pores of the scaffolds. alcian blue staining revealed that NP-like cells secreted a large amount of PGs and immunohistochemical staining was positive for collagen Ⅱ.The third part:Property evaluation of rBMSCs-scaffolds cultivated in vivo. CM-DIL dye was successfully labeled on the cells membranes. For the implanted scaffolds seeded CM-DIL-labeled rBMSCs cells, strong fluorescence could still be detected at the site of implantation after 4 weeks. H&E staining at week 4 showed a large number of cells distributed inside the pores of the scaffolds, alcian blue staining revealed that NP-like cells secreted a large amount of PGs and immunohistochemical staining was positive for collagen Ⅱ compared with 2 weeks. The quantitative analysis of samples cultured in vivo by real-time RT-PCR showed that both PGs and Type Ⅱ collagen contents increased with increase of culture time, and had a similar increasing tendency.Conclusion:1. rBMSCs owned fast proliferation, widely souces, can be differentiate into nucleus pulposus cells when incubated in low oxygen environment and induced medium containing TGF-betal.It can secreate a large amount of extracellular matrix of nucleus pulposus, and can be used as an ideal sources of seed cells; 2. The NP ECM-derived 3D porous scaffold was successfully fabricated by the combination detergent-nuclease methods and freeze-drying methods.It owned porous three-dimensional structure, good biological mechanics performance, no cytotoxicity, and retained the most nucleus pulposus extracellular matrix components. It can be used as a source of nucleus pulposus tissue engineering scaffold; 3. rBMSCs compound NP ECM-derived 3D porous scaffold cultured in induction mediumin containing TGF-betal in vitro can be successfully to the nucleus pulposus-like tissue under the condition of low oxygen; 4. rBMSCs compound NP ECM-derived 3D porous scaffold cultivated in vivo of nude mice, seed cells in scaffold can proliferate, distribution uniformity, form cell clusters in some part, and syntheze a large number of proteoglycan and collagen type Ⅱ. The complex still showed strong fluorescence in vivo imaging after 4 weeks. Finally, after seeded rBMSCs into scaffold, nucleus pulposus-like tissue were successfully formed. |
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