Research On Mesenchymal Stem Cells Compound Injectable Fibrinousgelatin TGF-β1 Transplantation Treatment Of Rabbit Intervertebral Disc Degeneration

Part I In vitro Culture and Identification of Marrow Mesenchymal Stem CellsObjective: We aim to develop a method for in vitro culture, proliferation and identification of the mesenchymal stem cells (MSCs) for the purpose of further application of the MSCs. Methods: The rabbit thighbone was punctured and 4 ml marrow was extracted from it. The marrow MSCs were processed by culture, proliferation and passage. The primary and passage cells were then observed under an inversion microscope for their growth characteristics. In addition, a flow cytometry was applied to assay the cell surface markers, i.e., CD44 and CD45. Finally, after induction and differentiation, the cells were processed by Collagen II immunohistochemistry, Alcian Blue stain, ALP stain and mineralization node stain. Results: We developed a method for in vitro culture and proliferation of the rabbit marrow MSCs and successfully induced osteoblast and cartilage. The marrow MSCs expressed CD44, however, did not express CD45. In addition, the results of Collagen II immunohistochemistry and staining using Alcian Blue, ALP and mineralization node stain were positive. Conclusions: The method of marrow culture can be used to effectively separate, purify and proliferate the marrow MSCs. The cultured cells have the essential phenotypes and characteristics of the marrow MSCs. PartⅡConstruction of rabbit model of intervertebral disc degeneration induced by puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discsObjective: to establish a slowly progressive reproducible rabbit model of intervertebral disc degeneration induced by puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs(IVDs). Methods: The L3/4,L4/5andL5/6 lumbar intervertebral discs of 18 New Zealand white rabbits were stabbed by 21-gauge hypodemic needle into a depth of 5mm in the antero annulus fibrosus, pulled out the needle valve, and aspirated out some nucleus pulposus tissue. Magnetic resonance imaging scans (MRI) Computed radiography (CR) immunohistochemical and histologic analyses of the stabbed discs and intact L2/3 disc were performed preoperatively and at 4, 8, 12weeks respectively after surgery. Results: In the magnetic resonance imaging the stabbed discs exhibited a progressive decrease of sigal intensity in T2-weighted images which start at 4 week after stabing and last for 12 weeks. Computed radiography analyses indicates progressive decrease of the disc height index Immunohistochemical and histologic analyses revealed progressive decrease of chondrocyte-like cells and typeⅡcollagen.Conclusion and increase of cells apoptosis(P<0.01). Conculation: The stabbing and asprirating approach results in a slowly progressive intertebral disc degeneration in rabbit model.This model is available for studying the status intervertebral disc degeneration and treatment. PartⅢA Research on Existence and Migration Capability of Rabbit Mesenchymal Stem Cells in Intervertebral Disc DegenerationObjective: to explore the existence and migration capability of rabbit mesenchymal stem cells in intervertebral disc degeneration and lay the experimental foundation for further researches. Method: with nine white New Zealand rabbits, by means of puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs, induce intervertebral disc degeneration model. After two weeks, transplant each degenerated intervertebral disc to mesenchymal stem cells fibrinousgelatin TGF-β1 complex 0.04ml with symbol of BrdU. After operation, in 2nd, 4th and 8th weeks, kill the rabbits and take nine intervertebral disc nucleus pulposus tissues of L3/4,L4/5,L5/6, carry out BrdU immunohistochemical stain and randomly take nine L2/3 nucleus pulposus as the control group. Result: From BrdU immunohistochemical stain, masculine staining cells can be seen in the 2nd, 4th and 8th weeks and cell counts are respectively 157.11±13.26, 68.56±13.16, 79.78±22.11, statistics of 4th and 8th weeks group have indifferent meanings P>0.05,and each of rest group have different meanings F=74.59, P<0.01. Along with the time, masculine staining cell count in fibrous protein implanted region decreases, until keeping a certain quantity, part of cells immigrate to nucleus pulposus tissue and there is no masculine staining cell found in normal control group. Conclusion: Rabbit mesenchymal stem cells can survive and immigrate in intervertebral disc degeneration. This lays the experimental foundation for tissue engineering researches of intervertebral disc degeneration. PartⅣA Research on Mesenchymal Stem Cells Compound Injectable Fibrinousgelatin TGF-β1 Transplantation Treatment of Rabbit Intervertebral Disc DegenerationObjective: To explore the feasibility of rabbit mesenchymal stem cell transplantation's inhibiting intervertebral disc degeneration. Method: 54 white New Zealand rabbits are randomly divided into three groups: (1) degeneration model group; (2) pure fibrinousgelatin TGF-β1 transplanted group; (3) mesenchymal stem cells TGF-β1 transplanted group. Every group has 18 rabbits. In the degeneration model group, induce and establish the degeneration model merely by means of puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs; in the pure fibrinousgelatin TGF-β1 transplanted group and the mesenchymal stem cells TGF-β1 transplanted group, 2 weeks after inducing degeneration model, transplant fibrinousgelatin TGF-β1 complex and mesenchymal stem cells fibrinousgelatin TGF-β1 complex respectively. Carry out CR, MRI and histologic examinations respectively in the 4th, 8th and 12th weeks (2nd, 6th and 10th weeks after transplant operation). Result: the disc height index (DHI) obviously decreases in the degeneration model group and the pure fibrinousgelatin TGF-β1 transplanted group, which is positively correlated with time; the normal control group and the stem cells transplanted group decrease slowly. MRI results show an increasingly aggravated degeneration in the degeneration model group and the stents transplanted group along with time, while the degeneration in the stem cells transplanted group is not obvious. An immunohistochemistry study and a histological examination indicate a decreasing quantity of the nucleus pulposus cells and the content of typeⅡcollagen in the degeneration model group, compared with that of the disc as well as an obviously increasing rate of cells apoptosis. The pure fibrinousgelatin TGF-β1 transplanted group is similar to the degeneration model group with an obviously increasing quantity of stem cells apoptosis in the stem cells transplanted group and typeⅡcollagen content compared with the degeneration group and the pure stents transplanted group while the cells apoptosis rate decreases. Conclusion: Stem cells can prohibit intervertebral disc degeneration. This lays the experimental foundation for further researches.