Experiment Study Of Porous Tantalum And Mesenchymal Stem Cells Co-culture For The Treatment Of Bone Necrosis

Research Source:The bone necrosis is often caused by trauma, alcohol, hormones and some other causes, the position of femoral head have the highest incidence to become necrosis, and it can cause local pain and activity dysfunction after femoral head necrosis occurring, if the disease cann’t get treatment timely and effectively, it will make the femoral head collapse and the joint space narrow, lead to osteoarthritis, loss of function and disability caused by paralysis at last. The treatment result of femoral head necrosis makes a big difference with the severity, discovered sooner or later, and the course of the disease stage, the earlier the lesions detected and the more lighter of the condition, the better the treatment, therefore, osteonecrosis should be done by early diagnosis and early treatment, in recent years, more and more research about the early treatment of the disease are emphasized. For the treatment, the correct diagnosis and stage of the osteonecrosis from clinic is the key to treatment decisions. There are many staging of osteonecrosis, the earliest stage is Ficat, it had divided the osteonecrosis into four stages based on X-ray findings which has clinical symptoms and has been confirmed by biopsy. There are also Ficat four divided stage, Marcus six of points system, but these methods have their limitations.1997World Bone Cycle Research Institute (Association Research Circulation Osseous, ARCO) established the International Standard osteonecrosis, referred to ARCO staging. The stage offers a comprehensive and accurate reflection of osteonecrosis area, location and prognosis,and has an important reference value in guiding clinical treatment. ARCO stages I, II are pre-collapse stage, with the shape of the femoral head intact, then timely intervention may terminate the course, but when there was a slight collapse at phase III, preserveing heads therapeutic effects is worse than before, and at the stage IV, the has formed osteoarthritis, joint replacement therapy can only be choosen. The young patients may have to face several renovations joint replacement prosthesis implantation, a large loss of bone mass, make renovations more difficult, therefore, early treatment is the key to avoiding the aggravation of osteonecrosisand disability.In recent years, the application of bone marrow stem cells (BMSCs) becomes hot to treat early osteonecrosis, easily obtained, proliferation and osteogenic ability higher are its advantages in different physical and chemical environment and cytokine induction, BMSCs may be directed multi-directionally to osteogenic cell, osteogenic cartilage cell, fat cell and fiber cell and so on, such as isolation, amplification, and is easy to be infected by exogenous gene, then express stablly, so it is ideal target cells to gene therapy and tissue engineering applications, it is generally regarded as the best bone tissue engineering seed cells,lots of the early treatment of osteonecrosis with core decompression surgical procedures applied BMSCs, and achieved good clinical results, it could survival in the medium, but for the reason that the lack of attachment,causing heavy loss.With the rapid development of tissue engineering, looking for the ideal stent become enthusiastic about the research. As their own drawbacks about then appeared demineralized bone matrix, polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers, bio ceramic materials, etc. it is too early to be applied clinically, with increasing levels of industrial technology, research and development of artificial biomaterials metal has also been developing rapidly, manufacture porous tantalum implant becomes success for the body to add a new choice. Tantalum metal’s color is grey, light, texture is very hard. The melting point of Tantalum metal is3000℃, and chemical properties of Tantalum metal are very stable, tantalum metal could also be hardly corroded even put in the acid alkaline. Tantalum are inert in organisms, so it is insoluble in water and acid and alkaline environment, and it does not produce any chemical reaction under room temperature, or stimulate tissue with erosion, these make tantalum has good biocompatibility. Tantalum metal became to be the clinical application of surgical choice of implanted materials because of characters of high melting point, high hardness, wear resistance and good physical properties, chemical properties of corrosion resistance and good biocompatibility. In the medical field, the history of the application of tantalum metal is more than half a century, it is mainly used in cardiac pacemaker, skull defect repair materials, vascular clamp, the femoral prosthesis handle, bone defect filler material manufacturing and so on. Studies have shown that phagocytic cells are the main material of removal of tantalum in the body. Experiments showed that phagocytic cells are kept active and no cell degeneration, only accompanied by glucose oxidation increased significantly in the body in contact with tantalum dust after1hour, and under the same conditions with silica dust can cause a serious phagocytes cytoplasm degeneration and death, which suggests that tantalum metal has no cytotoxicity and toxic to the human body. In1997, the American FDA approved of porous tantalum metal applied to clinical, the material looks like cancellous bone structure under the microscope, its pore size is between400-600microns, the whole inter connected porosity is up to75%-85%, significantly higher than the other biological fixation materials, such as cobalt, chromium, titanium which with porosity of30%～50%. Porous tantalum of three-dimensional porous structure has more advantageous to the adhesion, differentiation and growth of osteoblast, promotes the growth of bone, so as to strengthen the connection between the implant and bone, to achieve the effect of biological fixation, as well as to the moisture and nutrients in implanted transmission. Porous tantalum metal has good elastic modulus, it can reduce the stress shelter of implants, porous design also make its surface has a higher coefficient of friction than other materials, early implantation is also more conducive to regional stability, the porous structure is advantageous to the bone ingrowth. Although in a separate application of porous tantalum metal despite better mechanical support, due to the reduce or death of local bone cell activity caused by the occurrence of osteonecrosis, it still lacks of enough seed cells, thus we can conceive of BMSCs adhesion to porous tantalum metal micro hole, after being BMSCs proliferation to a certain number, make them joint into the part of osteonecrosis, let porous tantalum metal have the effect of support, provide bone ingrowth space, and have enough seed cells, repair necrotic areas around the porous tantalum metal and micro hole to form a healthy bone growth environment, repair osteonecrosis, to prevent further collapse of femoral head.Purpose and meaning:Investigate the porous tantalum metal used on the effects of BMSCs proliferation, discusses the application of porous tantalum metal joint BMSCs curative effect for the treatment of osteonecrosis. Osteonecrosis is currently one of the common bone disease, the disease has brought the huge influence to people’s life and necrosis are generally first occurs within the femoral head, femoral head necrosis process will gradually collapse, severe pain and dysfunction make patients with walking difficulties, loss of labor, serious life cannot walk who can only just stay in bed. Osteonecrosis ARCO stage guide the treatment of the clinic disease, commonly in clinical phase Ⅰ and phase Ⅱ is the early stage of the collapse, that is the early cases, active therapy may terminate illness development, the general treatment method is pulp core decompression, pulp core decompression with stem cell therapy, with vascular pedicle bone flap transfer operation, etc., but there are all have some different faults. Commonly pulp core decompression surgery lack the support of the structure of the subchondral bone, that have the risk of subchondral bone fracture, stem cell therapy of stem cells that is the liquid, which inject the parts of the osteonecrosis is not fixed, and the support with vascular pedicle bone flap transfer operation is not well. The period of Ⅲstage with femoral head is collapse, even after surgery osteogenesis performance, also difficult to stop further development to osteoarthritis pathological changes. Protect the head and joint replacement treatment have different standard, but a unified point of view is confirmed head treatment effect is worse than the treatment before the head collapse. In the stage of IV, collapse of the femoral head and osteoarthritis, have joint replacement has become a consensus, joint pain, joint function after replacement are can be significantly improved, that have great significance to improve the quality of people’s life, but the wear and tear of prosthetic joint, the occurrence of osteoporosis, etc all can cause joint prosthesis has the limitation of use fixed number of year, this is a joint replacement surgery in a short period of time can’t solve the problem. Therefore, how to active treatment in front of the collapse of the femoral head necrosis, decrease complications and increase the success rate of surgery is the meaning of the experiment.Methods:BMSCs of healthy rabbit were extracted by whole bone marrow adherent method, cultured with Alpha-MEM, containing10%FBS,1%PSN, identify stem cell properties by lipoblast experiment, osteogenesis experiment and colony forming experiment in particular inducer, Detect the cytotoxicity of porous tantalum metal by MTT method, compared the cell activity in more porous tantalum metal group and titanium group and control group respectively, Observe adhesion and proliferation of BMSCs on porous tantalum metal with GFP staining and electron microscope in the second day, five days and seven days after BMSCs jointed porous tantalum metal, detect porous tantalum metal influenced on the distribution of BMSCs induced osteogenic by PCR. Osteonecrosis model is set up, according the methods by professor Qinling,16healthy adult New Zealand white rabbit were selected,6months of age, muscle injection e. coli endotoxin with10g/kg dose, the injection interval time is24h, muscular injection methylprednisolone with20mg/kg dose3times, the injection interval time is24h, all experimental animals were no death. start experiments after models were successful built in2weeks. It is divided into two groups, each group of eight, they were implanted into pure porous tantalum metal and BMSCs jointed porous tantalum metal, judge the therapeutic effect of osteonecrosis with BMSCs jointed porous tantalum metal.by assessing the new bone formation around porous tantalum metal.Results:we induced BMSCs which extracted by adherent separation by a concomitant, it confirmed that the cells can change to fat cells by the application of oil red staining, it confirmed the cells can transform to the osteoblast by application of alizarin red staining and cobalt calcium alkaline phosphatase staining,cell colony forming experiment indicates that the cells are colony growth, these all show that extracted cells are BMSCs.Detect cytotoxicity by MTT method on24hours and48hours comparing two time points, the cell of activity control group and experimental group were no statistical significance (P>0.05),it showed that porous tantalum metal as a implanted material has good biocompatibility. BMSCs were distributed evenly on surface of tantalum metal in GFP dyeing when BMSCs and porous tantalum metal culturing two days. Different cell morphology under microscope shows that these cells is not on a flat surface, some of them grow into the pores. At the fifth day, more and more cells proliferate and Passage on porous tantalum metal with pseudopodia adhere to the metal. At the seventh day, the three-dimensional structure of porous tantalum metal is full of BMSCs. Under scanning electron microscope, a few cells adhere to porous tantalum metal surface with varied shapes at the second day, at the fifth day cells clearly increased and extended into micro-pore, At the seventh day, pseudopodia of cells stuck out towards all directions and their vigor were nice. After co-culture two weeks, osteocalcin (OCN), osteopontin (OPN) and mRNA transcription factors Runx-2obviously increases determined with PCR, The co-work of tantalum and osteoblast inducing conditional media can enhance the expression of the OCN expression (A), inhibit the expression of OPN (B), and not affect the expression of Runx-2(C); Gene expression of the cell around tantalum material is similar. As a result, we judged that tantalum can promote differentiation of BMSCs to osteoblasts as a porous scaffolds. At3week after surgery, new bone can be observed in two groups around tantalum metal formed, but can not be in microporous. There was no statistical significant differences of average width of new bone in the two group (P>0.05).At6week after surgery, the new bone increased obviously, the difference were significant in the two group (P<0.05); by Hard tissue section fluorescence technology, both groups have new bone growing into porous tantalum metal, and the depth of group adding BMSCs is greater than the group not adding. The difference was statistically significant (P<0.05).Conclusion:Porous tantalum metal has no cytotoxicity. It is free of negative effect on the growth and proliferation of BMSCs with no negative impact. There is no significant difference compared with the control group. BMSCs grown on the surface of porous tantalum metal has increased, proliferated and migrated into the microporous. there is more new bone around the porous tantalum metal in the treatment of osteonecrosis using BMSCs and porous tantalum metal, which show the proliferation and differentiation to osteoblasts of BMSCs in the body can speed up the formation of new bone and the repairment of osteonecrosis. The two functions make the repairment of osteonecrosis and the growth of bone into porous tantalum metal even faster.