Effects Of Injecting Mesenchymal Stem Cells To The Rats Degenerative Intervertebral Disc

Objective:To isolate culture and identify the bone marrow derived mesenchymalstem cells(MSCs).To build and verify the disc degeneration model induced bypercutaneous needle puncture in the rat tail.To explore the effects of injecting MSCs tothe rat model of degenerated intervertebral disc.Methods:Rat bone marrow was collected by aspiration from the femur and tibia.MSCs were isolated by the gradient isolation of mononuclear cells and cell attachmentto tissue culture plastic.The MSCs were proliferated in vrtro,and the growth curves ofprimary cells,third-generation cells and fifth-generation cells were drawed.The cellsurface markers CD90,CD 45 and CD 34 were checked by flow cytometry.The MSCswere also checked for multi-linage differentiation by adipogenic and osteogeniedifferentiation assays.Forty 3-month-old male Sprague Dawley rats were randomdevided into four groups:8 in normal control group;8 in degeneration group;8 inmedium injection group and experiment group with 16 rats.Degeneration was inducedin degenerative group,medium injection group and experiment group.Theintervertebral discs at regions Co7/Co8,Co8/Co9 and Co9/Co10 were puncturedpercutaneously using a 20-gauge needle with half penetration.2 weeks after induction,MSCs were injected into the inducted discs of all rats in experiment group,and mediumfor MSCs culture was injected into the inducted discs of all rats in medium injectiongroup.At 1,2,3 and 4 weeks after injection,4 rats from experiment group and 2 ratseach from normal control group,degeneration group and medium injection group wereeuthanized with an excess dose of sodium pentobarbital and their spines harvested. Before harvesting,anteroposterior plain radiographs were taken.Co7/Co8 discs wereisolated with both upper and lower vertebral bodies completely attached for histologicalevaluation by staining with safranin o-fast green and hematoxylin,and hematoxylin andeosin respectively.Co8/Co9 discs were harvested for estimating GAG andhydroxyproline content by using 1-9 dimethylmethylene blue(DMMB)binding assaykit and colorimetric assay kit with chloramine T and dimethylaminobenzaldehyde(DMBA).Co9/Co10 discs were used to extracting total RNAs for quantitative PCR ofaggrecan expression.Results:The MSCs derived from bone marrow were easy to harvest,isolate and grow.These cells expressed CD90 but were negative for CD34 and CD45.Differentiationassay results demonstrated sufficient differentiation of MSCs in adipogenic andosteogenic lineages.Needle punctures with half penetration caused significant discspace narrowing,progressive histological changes of degeneration and decrease ofGAG content and aggrecan expression after injury.Compared with degeneration group,these indexes of experiment group showed trends of improvement.Significant changein GAG content between degeneration group and experiment group was observed 4weeks after injection.The hydroxyproline content of the discs did not changeappreciably.Conclusions:The MSCs are easy to harvest,isolate and grow,with minimuminvolvement of in vitro techniques.Tail disc percutaneous needle puncture is a simpleand effective method for inducing disc degeneration.Injection of MSCs can slow theintervertebral disc degeneration process.