| BackgroundOsteonecrosis of the femoral head (ONFH) is a disease which was caused by the changes of the femoral head structure, collapsed of the femoral head, joint dysfunction after dynamic components death and subsequent repair by the femoral blood supply interruption or bone cell degeneration. It is not a single process, but the results of genetic factors and one or more risk factors role. It was a pathologic process caused by multi—pathogenic factors that damaged the blood supply of the femoral head, which led to the death ofinyeloid element,adipocyte and osteocytes.Osteonecrosis of the femoral head was a pathologic process caused by multi—pathogenic factors that damaged the blood supply of the femoral head, which led to the death ofinyeloid element,adipocyte and osteocytes. It is one of the common clinic diseases and mainly divided into two groups:traumatic osteonecrosis of femoral head(TONFH) and non—traumatic osteonecrosis of femoral head(NONFH). Some data indicates that ONFH has replaced the hip tuberculosis as the first place in hip diseases. Osteonecrosis of the femoral head have troubled domestic and foreign scholars for a long time.it is also called"the deadless cancer". But now, due to a lack of effective early screening and detection methods, it is rarely detected and intervented in early time. So most patients have a feel of hip’pain and limitation of hip’motion for a long time, then, think about the possibility of osteonecrosis of the femoral head. At this time, these patients had to be operated with THA because of the collapse and defomation of femoral head and osteoarthritis. It is the clinical and laboratory experts’ task to make clear its pathogenesis, search valuable biomarkers for early screening and treatment.Idiopathic femoral head necrosis (IFHN) account40%of osteonecrosis of the femoral head, including hormones and alcoholic osteonecrosis,the alcoholic osteonecrosis is more. Long-term and excessive quantities of alcoholy can make a lot of femoral intramedullary fat cells proliferated and fat cells hypertrophia,so that a relatively confined intramedullary pressure is significantly increased, tiny blood vessels become thin, blood flow is blocked. IFHN is a common disabling disease.It has a high incidence and show a gradual upward trend in recent years. If the lack of timely and effective early treatment, more than80%of patients will eventually lead to necrosis of bone and joint disease, or disability.At present, the pathogenesis of IFHN is not yet clear.There are not any similar reports at home and abroad. Osteonecrosis of the femoral head is still a major problem in the medical profession.There is an important clinical significance in the aspects of the search for effective disease prevention, early diagnosis and treatment by understanding the pathogenesis of IFHN. In recent years, scholars make more and more depth research about the pathogenesis of osteonecrosis.They proposed a variety of theories including the doctrine of lipid metabolism, the doctrine of mechanical damage to the blood circulation, intravascular coagulation theory, theory of increased pressure within the bone, cytotoxicity and cell damage theory, bone marrow mesenchymal stem cells into bone and adipogenic differentiation doctrine and so on. The doctrine of lipid metabolism was more mature than others. The pathogenesis of IFHN is extremely complex. Proliferation and hypertrophy of intramedullary fat cells, fat embolism were taken more attention in the development of osteonecrosis. The current study suggests that" the generation of fat cells," is an important mechanism for the development of osteopenia of the disease,"lipid theory" has become a hot area of IFHN disease.Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and thus rate of apoptosis of osteoporosis is responsible for the regulation of bone formation. In adults, the osteoblast, which is responsible for bone formation, is derived from multipotential mesenchymal stem cells (MSC) in bone marrow (BMSCs). Several studies have provided substantial evidence that osteoblasts and adipocytes share common progenitor:multipotential BMSCs, and the relationship between adipogenesis and osteogenesis is reciprocal with increasing adipogenesis accompanied by decreasing osteogenesis and vice versa.Habitual consumption of excessive quantities of alcohol is commonly recognized as a major factor for osteopaenia and increased incidence of bone fracture in alcoholics. The current consensus of both clinical and experimental studies is that alcohol-induced bone loss is mainly be due to the suppression of new bone formation with only relatively small changes (increase or decrease) occur in bone resorption. In adults, the osteoblast, which is responsible for bone formation, is derived from multipotential mesenchymal stem cells in bone marrow (BMSC). The capacity and dynamics of these BMSCs to differentiate into osteoblasts plays a critical role in the cellular processes involved in the maintenance of the adult human skeleton.BMSCs can exist in the bone marrow and other tissues, it has the typical characteristics of stem cells, under different conditions which can differentiate into a specific type of mesenchymal tissue, such as the regeneration of bone, cartilage, muscle and fat. Its main differentiation environment are spongy trabecular bone and bone marrow. Subjected to external stimulation, cell signaling systems of osteogenic microenvironment began to work, BMSCs began to proliferate and differentiate, from primary cells to osteoblasts, involved in updating tissue and repairing wounds,particularly in repairing the bone tissue injury, BMSCS plays a particularly important role. Even in vitro, mechanism of osteoblasts or adipogenic induced BMSCs differentiation is also involved in several respects. Osteogenesis in the periosteum can not be produced in the femoral head because of the special structure of human femoral head around that femoral head and neck lacks of periosteal structure, coupled with the ability of cartilage into bone was very weak in this place which made osteogenesis of BMSCS has the dominant position in osteogenic mechanisms of the femoral bone. Research shows that BMSCS can be a lot of adipogenic differentiation in the induction of alcohol, however, osteogenic differentiation was inhibited which revealed a new pathogenesis of alcoholic ONFH. Relevant experiments studies had shown that mature osteoblasts can promote the proliferation of bone marrow stromal cells through direct contact with the role of cells. Large number of clinical and experimental studies had confirmed that osteoblasts have secretory action,they could secrete b-FGF, IGF, PDGF, BMP and so on. They were considered as BMSCs bone differentiation-inducing factor.The surface receptor of BMSCs after combined with these growth factors, intracellular signaling receptor mediated effect was changed which to stimulate osteoblast-specific transcription factor Osf2/Cbfql or Cbfql/Runx2, then the proliferation and osteogenic differentiation of BMSCs was accelerated.BMP and TGF plays on BMSCs were induced bone and proliferation.The BMSCs have been proven to differentiate into multiple lineages and generate progenitors committed to one or more cell lines. Among these multiple lineages, those of adipogenesis and osteogenesis are the most closely related:Several studies have provided substantial evidence that osteoblasts and adipocytes share a common progenitor:multipotential BMSCs. An inverse relationship has been demonstrated between osteogenesis and adipogenesis suggesting the possible reciprocal relationship between the two differentiation pathways. There has been evidence for the differentiation switching between these two cell lineages, suggesting a large degree of plasticity between osteoblasts and adipocytes. Because the relationship between adipogenesis and osteogenesis is reciprocal and the adipocytic and osteogenic cells share a common lineage, it is possible that inhibition of adipogenesis may provide an approach to prevent or treat osteoporosis or other bone diseases. Thus, the signal transduction pathways implicated in these processes are evaluated as potential targets for therapeutic intervention of osteopenic disorders.After consumption, alcohol is readily distributed throughout the body in the blood stream and crosses biological membranes, affecting virtually all biological processes inside the cell. Excessive alcohol consumption substantially promotes adipogenesis and inhibits osteogenesis. Moreover, both chronic and binge consumptions of ethanol are known to cause suppression of bone formation and enhancement of bone resorption. Thus, alcohol is considered a major risk factor for osteoporosis, a disease particularly prevalent in women worldwide. Alcohol and hormones are powerful factors of the pathogenesis of IFHN, but what channels how do they through to make the disease? Or a variety of ways to jointly work? or a new disease process? There is no clear answer. To find therapeutic targets according to elucidate the pathogenesis IFHN remains an important task for the present study.Microenvironment is a functional environment which was composed including two aspects:microamount physical and chemical factors of the surrounding cells which can excite or inhibit the action of cells by Close-up action and regulation,and special structures and receptors which are formed in the process of evolution and differentiation of the cell. Many studies have shown that the role of hormones and partial adjustment factors on osteoblasts, BMSC, hematopoietic stem cells and the role of vivo bone reconstruction is extremely complex which can be overlapping, collaborative, antagonistic or short-lived. Many kinds of systemic and local signal was received,conducted and integrated by the cell in the net, and further stimulate the release of other members of the network, so that a signal amplification cascade. In this process. not only the cycles of the local cell signal within the bone microenvironment link, but is also involved in the regulation of its physiological activities. Thus, the coupling within the bone marrow microenvironment and stability between bone resorption and bone formation, perhaps the result of this regulatory network via signal contact between cells in the bone marrow variety of dynamic regulation.Several studies have provided evidence that alcohol shifts lineage commitment and differentiation of BMSCs from osteogenesis to adipogenesis by upregulation of gene expression for peroxisome proliferator-activated receptor gamma (PPARy); however, the molecular mechanism underlying the reciprocal relationship is not yet well understood.Through this project,we make a research about whether the existence of regulation of the the differentiation direction of BMSC by the influence of alcohol, the ability of osteoblasts and fat cell function, as well as the system of fat cells+BMSC, fat cells+osteoblast cells. We observed the expression of PPARy and cbfal which are the key gene of adipogenic Osteogenic differentiation differentiation under different conditions, detect osteogenesis-related indicators,such as ALP, OCN, I collagen and fat cells LPL. Explore the pathways of the risk factors, and its influence to intramedullary BMSC cells, osteoblasts, adipocytes. Whether it will produce local effects after the fat cells are effected by causative factors, further clarify the contribution and mechanisms of the bone microenvironment of the bone marrow in the pathogenesis of IFHN. Objective1To elucidate lipid abnormalities which was caused by the risk factors effected the BMSC adipogenic and osteogenic differentiation of the Intramedullary cell pool in the pathogenesis of IFHN.2To discuss functional changes of osteoblasts and fat cells afer effectd3To investigate the adjustment of fat cells’ changes to osteoblasts and BMSC4To analyse the effection of alcohol on co-culture system which was Consisted of fat cells+BMSC and fat cells+osteoblastsMethods1We isolated and cultured marrow BMSC, fat cells and osteoblasts from the New Zealand rabbits;made an identification about BMSC differentiation potential,the surface shape of marrow osteoblasts and fat cellcells.2To observe the effection to the differentiation of BMSC, function of fat cells and osteoblasts after applicating of the role of alcohol in BMSC, fat cells and osteoblasts.3After alcohol acted on fat cells, we Preparated the conditioned medium,then acted on intramedullary osteoblasts and BMSC,observed the contribution after the fat cells was effected.4We made a co-culture system which was Consisted of fat cells+BMSC and fat cells+osteoblasts, observed the circumstances change of cell differentiation and osteogenic index in the co-culture system and the change after acted by alcohol.5We measured changes of genes related to endoplasmic reticulum (ER) stress, adipogenic markers and osteogenic markers using quantitative real-time RT-PCR and Western blot analysis. We performed Alizarin red staining for osteogenesis. We also conducted assays for osteogenic biomarkers alkaline phosphatase, collagen-I and osteocalcin.We also conducted caspase3activity assay to assess BMSC apoptosis. ResultsAfter12days of culture, ALP staining was performed on two groups of cells, the experimental group showed that the cytoplasmic ALP staining was positive, the control group, experimental group cells staining, alizarin red staining after calcified nodules number and larger, control group with a small amount of calcium nodules, but relatively small, experimental group standard calcium nodule count significantly higher than the control group differences were statistically significant (P<0.05). We trained the BMSCs of rabbit in logarithmic growth phase for induction, when the confluence reached70-80%, added osteogenic induction medium. With time, the number of osteoblasts of the experimental group was significantly more than the control group. The number of Calcium nodules in experimental groups of3,6,9d, respectively (200.83±24.73)、(1112.00±77.56)、(1199.00±44.91)个/cm2; control group, respectively.(99.75±10.15)、(790.00±94.13)、(1059.00±26.39)个/cm2(P<0.001).With time, the number of fat cells of the experimental group was significantly more than the control group. Two groups of small lipid droplets are gradually increasing and enlarging, but the experimental group was significant. The number of experimental groups in the the intramedullary fat cells of4,6,8,10d, respectively (200.83±24.78)、(1102.00±76.21)、(1160.00±28.83)、(1199.00±44.51)/cm2; control group, respectively.(99.75±10.67)、(790.00±94.63)、(1000.00±41.30)、(1059.00±26.54)/cm2, the number of fat cells increased with time of the influence of alcohol (P<0.001). We showed here that chronic exposure of BMSCs to alcohol induced adipogenesis and disrupted osteogenesis as indicated by upregulation of adipogenic markers (PPARy2and aP2), downregulation of osteogenic markers (Osf2/Cbfal), and accumulation of lipid droplets. Long-term excessive drinking could lead to femoral intramedullary fat cells rapidly proliferate, meanwhile,the fat cells were hypertrophy than normal, so that the relatively closed intramedullary pressure increased significantly, tiny blood vessels become thinner due to pressure, eventually leading to disruption or interruption of blood flow, thereby forming osteonecrosis of the femoral head.ConclusionsBy density gradient centrifugation and differential velocity adherent method the obtained BMSCs in conventional culture or inducing culture can be showed osteogenic potential of bone induction ability cultivation.lt shows that BMSCs can provide a reliable source of seed cells for bone tissue engineering research. Alcohol can restrain BMSC into osteoblasts and promote them into adipocytes; Alcohol can cause the osteoblasts to death,promote the intramedullary fat cells to increase and enlarge.Innovation1.This study proposes that alcohol can promote intramedullary fat cells proliferation and hypertrophy and suppress osteogenic differentiation potential of BMSCs2.This study presents that the influence of alcohol on the metabolites of fat cells have certain extent to the proliferation of mesenchymal stem cells and bone marrow cells.3. This study established the fat cells+bone marrow mesenchymal stem cells, adipocytes+osteoblasts co-culture system. |
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