Research On Adult MSCs Differentiation Into Osteoblasts In Vitro And MSCs Activity Of Steroid-induced Osteonecrosis Of Femoral Head

Objective1. Though with the potential of self-proliferation and multi-directional differentiation, mesenchymal stem cells (MSCs) are few in adult bone marrow. Getting enough, high purity, well growth condition of the adult MSCs in vitro is very important for each research. This subject tried to establish a simple and reliable system of adult MSCs in vitro separation, purification, cultivation, amplification and identification, and induced the differentiation of MSCs into osteoblasts. It can be used to guide clinical research by understanding some biological characteristics of adult MSCs, and this subject laid an experimental basis for the subsequent tissue engineering combined with MSCs in the repair of osteonecrosis of femoral head(ONFH).2. Although obvious achievements have been made by Chinese Medicine in treating ONFH by using the "concept of wholism", but currently, the cognition of the "concept of wholism on ONFH" is still restricted in subjective comments or boring Basic Theory of TCM, lacking of convincing and objective experimental evidences, especially the cytological changes closely related to pathogenesis of ONFH is still uncertain. Therefore, the "concept of wholism" is not extensively accepted. This subject tried to provide an objective experimental evidence for the "concept of wholism on ONFH" according to study the MSCs activity of patients with steroid-induced ONFH.3. The pathogenesis of steroid-induced ONFH is still not entirely clear. Recognizing the pathogenesis is very important for judging prognosis and choosing appropriate treatments for the sick hip. This subject tried to investigate and improve the pathogenesis of steroid-induced ONFH by studying the proliferation activity and osteoinduction of adult MSCs, and provide experimental evidences. Methods1. Extracted the bone marrow from a healthy adult's anterior superior iliac, combining density gradient centrifugation with adhesion method, then isolated, purified, cultured and amplified the MSCs. Observing morphological changes under phase-contrast inverted microscope, determining the cell ultrastructure by electron microscopy, assaying the proliferation activity of different passages by MTT method, drawing the growth curve of MSCs, detecting cell surface antigens of the 3rd passage with flow cytometry.2. The second part of this subjects was based on the first part of the experiment. Prepared osteogenic medium by mixing appropriate amount of dexamethasone, ascorbic acid C andβ-glycerophosphate. Induced MSCs to differentiate into osteoblasts. Detected ALP activity at the 4th,8th,12th and 16th day after osteoinduction, and identified osteoblasts by ALP staining and alizarin red staining.3. The third part of this subjects was based on the previous parts. Adapted the methods of combined clinical case-control study and experimental research. Patients were recruited strictly in the light of diagnostic criteria, inclusion criteria and exclusion criteria devised in the project started from May 2010. The osteonecrosis group included patients with steroid-induced ONFH, while the normal control group included patients with normal bone marrow and no osteonecrosis. Extracted the bone marrow from anterior superior iliac of the two groups, and isolated, cultured, amplifed the MSCs, and then induced to differentiate into osteoblasts. Comparing the cellular morphology, using MTT method to assay the proliferation activity of the 2 passage MSCs, drawing the cell growth curve of each group. Induced the 3 passage MSCs of each group to differentiate into osteoblasts. Detected ALP activity at the 4th,12th day after osteoinduction. All the above items were compared between the two groups.Results1. Adult MSCs presented the typical long fusiform or cambiform shapes. Primary culture presented colony-like growth in early stage, and the cells growed into rapid proliferation phase at 8-11 days. After then, the cells gradually integrated into flake, arranged in a certain direction.2. Growth curves of passage MSCs showed that growth latency at 0-2 days, increased logarithmic phase at 3-7 days, platform phase at 8-9 days. The growth curves presented "S" shape, and the MSCs had good growth characteristics before the 5 passage. Cell proliferation and growth gradually became slow along with increased passages.3. Many evections were observed on the rough cell surface. The cell nuclear is bigger and irregular, while abundant cytoplasm, prosperous rough endoplasmic reticulum and plenty of protein excretion were found inside MSCs under electron microscopes.4. Flow cytometry was applied to detect cell surface antigens of MSCs. It showed that the relatively specific antigen CD44 was 94.81% positive expression, and CD90 was 93.15%, while the hematopoietic cell surface antigens of CD34,CD45 were almost negative(2.87%,2.02%).5. The MSCs morphology became irregular after osteoinduction, and MSCs gathered into clumps at about 7 days, connected each other closely, and formed calcified nodules after 14-20 days.6. The ALP activity leveled up with osteoinduction time. It increased from the 4th day and reached the summit at the 12th day. The ALP activities of osteoinduction group were higher than the blank control group at each different time(P<0.01). ALP staining and alizarin red staining of the osteoblasts were all positive.7. The MSCs growth states between the osteonecrosis group and the normal control group were significantly different. The osteonecrosis group's amounts of primary cell clonies were more than the normal control group, and its cell density was more intensive, doubling speed was faster. While the normal control group's cell clonies were sparse and the doubling speed was slower. The aggregates of normal control group formed at about 7th day after osteoinduction, while the osteonecrosis group was later and the aggregates formed at about 10th day.8. The MSCs average proliferation activity of the osteonecrosis group were lower than the normal control group at each different time, and had significant difference from the 3rd day(P<0.05). The average growth curves of the two groups showed that, the osteonecrosis group MSCs proliferation speed was slow, the logarithmic phase was short, and the platform phase came early.9. Both two groups'ALP activities were gradually increasing day by day after osteoinduction, and this phenomenon consisted with the feature that MSCs differentiated into osteoblasts. While at every specific time, the average ALP activities of osteonecrosis group were lower than the normal control group (P<0.05).Conclusions1. We can obtain high purity mononuclear cells by using density gradient centrifugation. According to the adhesion property, MSCs can be further purified after changing medium, digestion and passaging. MSCs can also be differentiated into osteoblasts. Using this established experimental system of in vitro cultivation, identification and osteoinduction, can obtain high purity and good osteogenic differentiation of MSCs.2. Compared with the normal, the decreased MSCs proliferation activity and osteoinduction capability of osteonecrosis group play important roles in the pathogenesis of ANFH.3. The MSCs abnormal activity of steroid-induced ONFH might be systemic while not confined in the surrounding of femoral head. ANFH could be a local reflection of systemic pathological change which caused by steroid. The "concept of wholism on ONFH" can be supported by objective celluar experiment evidence.