Related Research Of Constructing A New Bone Filling Material For Kyphoplasty

Background:With the population growing aging,Senile osteoporosis is receiving increasing attention, is common and frequently-occurring diseas, the clinical manifestation is pain, shorter length, kyphosis, fractures,decreased respiratory function, etc. It is estimated that osteoporosis in our country there are over 90 million patients and it will reach 110 million by 2015.Reduced bone mass, bone micro-structure degradation and bone brittleness increasing is three main features, often leads to multiple complications, vertebral compression fracture(VCF)is one of its serious complications, that is OVCF-osteoporotic vertebral compression fractures, of which 1 / 3 can cause long-term chronic thoracic back pain,and the incidence is 20% in the 70 years old people, 16% in postmenopausal women. Due to lack of effective treatment, it resulting in a sharp decline in the quality of life of patients, lead to various complications; advanced lesions further vertebral compression injury, pain and kyphosis significantly increase, resulting in thoracic volume cardiopulmonary dysfunction caused by narrowing or even death. Traditional surgery with lager traumatic or patients have more special cases that unable to surgery. Today,under the development of percutaneous vertebroplasty( PVP), percutaneous balloon kyphoplasty ( PKP) has become recognized worldwide and an effective standard treatment, but in a growing number of applications it can be found that: (a) The curing heat and toxicity can cause cardiac arrest and spinal cord,nerve root injury when perfusing the bone cement (PMMA) in low-pressure;(b) Easy leakage of bone cement caused blood clots and pulmonary embolism; (c) bone cement can not be degraded, poor biocompatibility that resulting in a permanent residual foreign for body; (d)It is difficult to deal with the disease of vertebral fractures at later stage and the long-term effect was not obvious. To some extent, limit the popularization and application of the advanced treatment means.Therefore,the development of a new bone filling material that having a good biocompatibility, high security performance, can replace the ordinary cement (PMMA) , must become a major trend of osteoporotic vertebral compression fracture .According to the principle of tissue engineering using bone marrow mesenchymal stem cells (BMSCs) and bone morphogenetic protein -2 (BMP-2) loaded by a new type of biodegradable injectable calcium phosphate cement (CPC) to build a certain bio-mechanical strength, good biological activity of biodegradable injectable tissue-engineered bone as percutaneous kyphoplasty ideal filling material for bone tissue engineering ,is one of the general direction of research in the field at present. The purpose of this study for isolation and culture of BMSCs in vitro and the biological characteristics of BMSCs, CPC biocompatibility and co-culture with BMSC,etc, the experimental results will provide new scientific basis for developing ideal filling material of kyphoplasty.Objective:1, looking for a simple and reliable method to isolate , culture and identify human bone marrow mesenchymal stem cells in vitro, and to study its biological characteristics and inducing differentiation conditions ,and to provide a large number of seed cells for bone tissue engineering.2, Evaluation the cytocompatibility of new injectable and degradable calcium phosphate cement, to provide a theoretical basis for experiments in vitro and in vivo.3, To explore the feasibility of co-culture with new injectable calcium phosphate bone cement and bone marrow-derived mesenchymal stem cells, for provide a scientific basis of building tissue engineered bone .Methods:1,Take a small amount of healthy bone marrow using Pecoll liquid separation density gradient centrifugation to obtain mononuclear cells, containing 10%FBS with L-DMEM in adherent culture to separat and purifica, amplifica a large number of hBMSCs that observating the cell morphology and growth characteristics,identificating by flow cytometry and respectively osteoinducting by osteoblast-induced fluidand BMP-2;2, Using extracts prepared CPC extraction, adding to the human bone marrow mesenchymal stem cells's culture system, MTT determinating the relative proliferation rate of cells and cell toxicity classification; flow cytometry detecting the cell cycle of hBMSCs that cultured by CPC extraction; Heparin fresh healthy human blood be diluted into a 2% suspension of human blood by saline, joined the CPC saline extraction(Liquid extract group, 50% Solution Group),normal saline (negative control group)and Double distilled water (positive control group)respectively in the centrifuge tube that measured by spectrophotometer absorbance value, calculated the rate of red blood cell lysis after bath 60 min by 37℃Water;5ml CPC extraction will be slowly injected into the ear vein of New Zealand White Rabbit,recorded temperature values.3, The new type calcium phosphate bone cement were added into BMSC concentration of 1×107 / L 6-hole plate at different time,the daily observation of the growth of adherent cells at the edge of the material. Observing by scanning electron microscope after cultured a week; Taking the third generation of BMSC, checking the density to 1×107 / L and added to 6 hole culture plates, injected calcium phosphate cement paste (as Mission), then terminating the culture and observing by scanning electron microscope after cultured 1 day, 3 days ,5days and 1 week.Results:1, By density gradient centrifugation adherent culture method can be more easily amplified to obtain a large number of high-purity BMSC, their cell morphology, differentiation and phenotypic characteristics are in line with the characteristics of stem cells and BMSCs can differentiate into osteoblasts under specific culture conditions (OS, or BMP-2 ).2, New type of calcium phosphate cement for the cell toxicity 0～Ⅰlevel, it is basically non-toxic; of hemolysis on human blood rate <5%, that is, no in vitro hemolysis; rabbit body temperature fluctuations <1℃, no significant effect caused by heat. 3, With the inverted light microscope, we can see cells growth well in the edge of material, no cell deformation, necrosis;By the scanning electron microscope, we can see the cells that were irregularly polygonal and nearly circular grow well, a large number of cells growing close to the material, and with other cells linked to integration into tablets in the bone cement surface.Conclusion:1, BMSCs can easily drawn,less trauma to body, easy to isolate and amplify, using density gradient centrifugation + adherent culture can access a large number of high-purity BMSCs; cell have proliferation ability,the technology that orientation-induced differentiation are so mature that could easily induce to the osteoblast. Because of its good performance of osteoblaststo ,it suitable for the ideal seed cells of tissue engineering bone.2, The new injectable calcium phosphate cement has a good biocompatibility and in line with the cytotoxicity request of implant materials in vivo, does not cause any significant adverse impact to body, can transit to clinical application.3, Bone marrow mesenchymal stem cells can co-cultured with new injectable and degradable calcium phosphate bone cement in the conventional culture system, and the next step experiment that building tissue engineered bone could be carry out in animal.