Identification And Purification Of Highly Isolated And Induced Rat Osteoclasts

ObjectiveIn normal bone,there were only 2-3 osteoclast cells in lmm~3 bone tissue. Although only a few in number, but steability of the number was very important, when there were so many osteoclasts, more bone resorption than bone formation, thus could cause to osteoporosis. In a contrary when there were so few osteoclasts, more bone formation than resorption, thus could cause to osteopetrosis. Osteoclasts were very important to bone formation and bone mass regulation. At present, study of osteoclasts was a key point of osteoporosis and osteopetrosis. Many person studyed the molecular and biology feature of osteoclasts. Many person islocated and cultured osteoclasts, also cultured with osteoblasts to simulate the mechanism of bone in vivo and studied the disease of bone and choosed the best medicine to cure osteopotosis. Now someone used osteoclasts to evaluate quality of tissue engineering of bone. According to different bone resorption of the same number osteoclast cells to judge which one was the best tissue engineering bone. Some reported that osteoclasts could promote the function of osteoblasts by some proteins secreted by osteoclasts in a co - culture system of osteoclasts and osteoblasts. Osteoclasts and osteoblasts could affect to each other. Now bone tissue engineering study only focused on the osteoblasts and scaffold, but neglected the promoting function of osteoclasts to osteoblasts and the remodeling of osteoclasts in scaffold. We believed that osteoclasts would have a profoundly affect to bone tissue engineering. Now osteoclasts were usually directly obtained from limbs of big animals ( rabbit and chicken) , but for little animal ( rats) was impossible to get a large amout of osteoclasts by classical methods for not so many osteoclasts in vivo. We induced marrow mononuclear cell (MMNC)to form osteoclasts. There were other adherent cells among osteoclasts. We applied the improved new technique to obtain highly enriched and purified osteoclasts in enough amount for the study of osteoclasts and the mechanism of bone resorption in vitro and further application to bone tissue engineering.MethodsRats femoral slice were stained by HE and toluidine blue, and then we ob-serverd the location shape and structure of cells in bone marrow. The classical osteoclastic isolation method from limbs of rats combined with the method of induced the same rats mononuclear cells of bone marrow was used to form osteo-clast-like cells at the presence of 1, 25 (OH)2D3. This combined method could get more osteoclasts than any single one. There were some other adherent cells among osteoclasts. For osteoclasts having more adherent ability than the other adherent cells, mature osteoclasts and osteoclast — like cells were purified by 0. 05 % trypsin /0. 02EDTA ( ethylene dinitrilo tetraacetic acid) digestion. The digested cells were induced by 1,25(OH)2D3 to form osteoclast - like cells. The osteoclasts and osteoclast - like cells were observed and counted by phase - constrast photomicroscope at different time. The osteoclasts and osteoclast - like cells also were stained by HE and Gimsa to observe the structure of osteoclasts. The osteoclasts and osteoclast - like cells were stained by tartrate -resistant acid phosphates ( TRAP) and tartrate — resistant acid adenosine triphosphatase (TrATPase) to observe the special enzyme secreted by osteoclasts. The osteoclasts were observed the three dimensional morphological feature under scanning electron microscope. We applied toluidine blue to staining and scanning electron microscope for observing the resorption lacuna on a femoral cortex slice from calf. According to the area and the deep of resorption lacuna on bone slice, we could study the degree and the ability of bone resorption a-bout the cells obtained. We use gelatin zymography to detect the expression level of gelatinase in osteoclasts culture medium, thus to find out the mechanism of bone resorption formed by gelatinase that were secreted by osteoclasts.ResultsAfter observing the femoral slices that were stained by HE and toluidine blue staining, there are some osteoclasts in the lacuna near the metaphysis and many single nucleus cells in medullary cavity of bones. 80% purified osteoclasts were obtained after 0. 05 % trypsin/0. 02EDTA digesting the other adherent cells. A large number of rats osteoclast - like cells were produced from the digested single nucleus cells at the presents of 1,25( OH)2D3 at the ninth day. The number of the osteoclast. - like cells were 11 times to that of osteoclasts after counted by phase - constrast photomicroscope . The osteoclasts and osteoclast -like cells were multiply nuclear ( more than 3 ) after observed by phase - constrast photomicroscope and stained by HE and Giemsa staining. The osteoclasts and osteoclast - like cells were all positive for TRAP and TrATPase staining and also capable of bone resorption in vitro. The bone slices cultured with the osteoclasts and osteoclast - like cells were observed the resorption lacuna after stained by toluidine blue and examined by scanning electron microscope. The area and deep of resorption lacuna were scanned by software of computer, after statistical treatment of the resorption lacuna formed by osteoclasts and osteoclast - like cells,two groups had no different. There was a significant different between 1,25 (OH)2D3 group and no 1,25(OH)2D3 group in Integrated Density Value of MMP -2 by statistical treatment( p <0.001). 1,25(OH)2D3 group highly express MMP-2 than no 1,25(OH)2D3 group.ConclusionsThere were some osteoclasts and many single nucleus cells in bone marrow. The primary culture method combined with the method of induced the single nucleus cells at present of 1,25(OH)2D3 could get a large amount of osteoclasts. All the single nucleus cells were obtained after digested the primary culture cells by 0.05 % trypsin /0. 02EDTA. The morphology between osteoclasts and osteoclast - like cells had no different after stained by HE and Giemsa, also they hadno different on special enzymes ( TRAP) and effection in resorptional lacuna. 0. 05 % trypsin /0. 02EDTA was a good method to purify osteoclasts. It had a high purifing ratio, and also convenient and economic pragmatic method. In a word, this method mentioned above could produce highly enriched and purified osteoclasts in enough amounts. The cells obtained express TRAP and TrATPase and also could form resorption lacunae on the surface of bone slices. This method could produce enough amount osteoclasts for molecular biological research. Resorption lacunae might not form by MMP -9, but MMP -2. This might be cause by some mechnism of osteoclasts. The osteoclasts might promote the other cells to secrete active MMP - 2 to form resorption lacunae.