| ObjectiveThe femoral-head osteonecrosis is a disabling disease, whose incidence is high. It will cause Femoral-head collapse and finally have to adopt the total hip arthroplasty. The disease mostly happens in young, which lead to prematurely total hip replacement facing much more difficult questions. Therefore, the objective of femoral-head osteonecrosis besides improving clinical symptoms, more important is to prevent femoral-head collapse, to delay disease development, to try our best to reserve sufferer's femoral-head and finally to delay and even avoid total hip replacement. The ideal treatment methods of retaining femoral-head are not only to restore the femoral mechanics performance in order to prevent femoral-head collapse but also to improve the blood supply of the femoral head, to add osteogenesis factors and finally to promote the necrotic bone to be repaired. However, the ideal methods that can achieve the above requirements are not existent. The object of the research is to explore a new way of treatment femoral-head osteonecrosis by transplanting bone marrow mesenchymal stem cells and umbrella strut bone grafting into the goats femoral-head osteonecrosis models and finally observing the restoration results, such as radiological, histology, the biomechanics results.MethodsThe research is divided into three parts.The first part:establish the goat femoral-head osteonecrosis models by the liquid nitrogen that had been improved. Methods:the femoral-head osteonecrosis models on one side are established in twelve one-year old goats using liquid nitrogen. Finally, the radiological and histology examination are made at the second day,second week,fourth week,eighth week after respectively killing the animals (three goats once) and the models have been established.The second part:to isolate and cultivate,preserve and identify the goats bone marrow mesenchymal stem cells. Methods:extracting ilium bone marrow by Ilium puncture at one-year old young Chinese goats, then isolate bone marrow mesenchymal stem cells using density gradient centrifugation method and cultivate cell by the attachment culture method. Finally, Observe morphology and growth of cells, determinate cell survival rate and ratio of cells nestin, calculate cells double time by drawing the growth curve using Continuous notation for the second, fourth, sixth generations cells. The second generation cells in semi-logarithmic growth are used to observe the changes of cell morphology about the former-later of Freeze-stored recovery in bone marrow mesenchymal stem cells. At the same time, the blue staining method is used to test survival rate. Finally, Immunocyte histochemical stain technique is used to identify the cell surface antigen marks.The third part:the research about bone marrow mesenchymal stem cells and umbrella strut bone use in treatment of femoral-head osteonecrosis. Methods:the femoral-head osteonecrosis models on one side are established in forty-four one-year old goats using liquid nitrogen. Except two goats that are randomly selected are killed, the rest are randomly classified into three groups after models have been made 8 weeks. Group A are conducted umbrella strut bone grafting and bone marrow mesenchymal stem cells transplantation. Group B are conducted simple unit side muscle bone flap grafting, and group C are conducted umbrella strut bone grafting and Cancellous bone grafting transplantation. The health side of three groups is treated as self-control. The radiological and histology examination and biomechanics examination are made at 3,6 months after the bone grafting.ResultThe first part:some bone cells in femoral-head begin to die after 2 days of operation. Myelofibrosis and bone marrow stromal cells increase around trabecula after 2 weeks of operation. Repairment may be seen at the fourth .At the same time, mild cystic changes and local bone sclerosis in the pedal area of femoral-head may be seen in the X ray. At the eighth week, the appearance becomes irregular, mild collapse, cartilage tissue become dim and articular cartilage mild collapse.The second part:primary cultured cells of bone marrow mesenchymal stem cells are just like a "baby" sample distribution, round. At the same time, there are many blood cells beyond the stem cells. When the cells are training 24 hours, part of the cells begin to adherent and the shape of cells is oval. When the cells are training 48 hours, much more cells adherent, the shape of cells are Spindle, polygonal. The cell colony is formed after 4-6 days. With the cell colony become more and more, the cells may nestin when the cell take up bottle room about 80%. The cell begin to adherent after nestin about 4 hours. The adherent rate of the 1-6 generation is above the 90% in 12 hours. The survival rate of 1-6 generation is above 95%.The shape of the nestin cells is long spindle, the membrane and the nucleus are clear. The shape of cells is radial, swirling when the cell density is high. CD29, CD44 are expressed in positive and CD34, CD45 are negative in the third generation bone marrow mesenchymal stem cells. At the same time, the third generation of bone marrow mesenchymal stem cells presents in S, which the incubation happen in 1-2 days and the logarithm growth happen after 2-3 days, plateau happen after 6-7 days. The doubling time in second, fourth, sixth generation cell is separately 25.61h,27.89h, 28.66h.. The average doubling time is (27.38±2.03) h。After 3 months:(1)X-ray diffraction imaging shows that the treated lateral femoral-head shape restore, cystic cnlow-density area disappeared, the original collapse have repaired, and no further collapse of the femoral-head shaped for both group A and B. Bone fusion is good and bone column shadow have been vague for group A, while the bone fusion heal somewhat less and bone column shadow still clear for group B. Group C have irregular contour of femoral head, flat collapse, uneven density, and fracture cracks can be seen at weight-bearing area of femoral head, obviously is the poorest therapy effective in contrast with group A and B. (2)The histological observations show that the trabecular bone have different states: mature and arrange rules for group A, childish and irregular arrangement for group B, thinning, sparse and fracture for group C.①.The percentage of trabeculae area in three groups of operation and normal is respectively(40.18±2.71),(36.21±2.46),(31.42±2.74) %; (41.90±2.52),(41.56±2.35),(41.90±2.48) %. The percentage of trabeculae area in group A is higher than group B and group C (P<0. 05) and the difference between in operation side and normal side is not obvious (P> 0.05).②.The percentage of empty lacunae counts in three groups of operation and normal is respectively (12.01±1.53),(18.35±1.51),(22.23±1.12) %; (11.53±1.32),(11.68±1.21),(11.47±1.26) %.The percentage of empty lacunae counts in group A is lower than group B and group C (P<0.05) while the difference between in operation side and normal side is not obvious (P>0.05) (3).After 3 months, the biomechanics examination show that:the maximum compressive strength and average elastic modulus of sub-chondral bone and cancellous bone of three groups are respectively (77.43±2.65 and 120.60±4.18) (64.43±1.55 and 84.85±2.31),(42.10±1.25 and 54.72±2.09) MPa; (46.63±1.32 and 81.74±3.96),(39.48±0.75 and 63.78±1.27),(30.83±0.73 and 45.90±2.34) MPa. The maximum compressive strength and average elastic modulus of sub-chondral bone and cancellous bone in group A are higher than group B and group C and the difference is statistically significant (p<0.05) while the difference is not statistically significant comparing to its healthy side (p>0.05).6 months later, the radiological examination shows that:group A and group B have been no significant difference comparing to normal femoral heads, while the femoral head of group C collapse and disappear completely. The histology examination shows that:Group A and group B are no difference comparing the healthy side (P>0.05) in the area fraction of trabecualr bone and percentage of empty lacunae counts, neither it is between group A and group B (P>0.05). The biomechanics test shows that:Group A and group B are no difference comparing the healthy side (P>0.05) in the maximum compressive strength and average elastic modulus of sub-chondral bone and cancellous bone,neither it is between group A and group B (P>0.05)ConclusionThe first part:the liquid nitrogen way that have been improved may produce the femoral-head osteonecrosis animal models accompanying Joint collapse.The way that is simple and the traumatic is smaller. It does not damage the articular surface and the Stability of hip and it is much more coincident with the process of femoral-head osteonecrosis.The second part:the purity of bone marrow mesenchymal stem cells that is obtained using the density gradient centrifugation method combined with attachment culture method is higher and the vegetative state is very well. The way of Separation and purification, training, proliferation and freeze-stored recovery is feasible. It can provide the excellent seed cells for the tissue engineering and cell engineering, etc.The way of umbrella strut bone grafting and bone marrow mesenchymal stem cells transplantation may promote the repairment of femoral-head osteonecrosis. At the same time, it can restore the biomechanical strength in femoral-head and prevent the femoral-head collapse. |
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