Novel Biofunctional Calcium Phosphate Cement Composite For Bone Tissue Engineering Research

BackgroudThe skeleton system of Human was to bear the body movement, support the weight and protect organs. Bone defects are a common clinical problem cause by congenital, trauma, infection and tumors etc. According to data estimates, the consumption for bone disease in United States every year is more than hundreds of billions dollars. In China, the number of patient who requiring bone graft surgery keep increasing in past decade. Currently, autogenous bone graft, allograft and substitute bone material is a common bone tissue repair methods, but these treatments have many shortcomings. Autogenous bone graft: for pain, blood loss, infection, structural damage function of a limited number of bone graft; allograft bone:immune rejection, disease transmission, substitute bone material is limit to use for bone defect filling and supporting, butlack of osteoinductive capability. Therefore, how better to repair bone defects has been attracted extensive attention in the past years.Recently, with the application of biology and engineering technology, new methods to develop tissue engineering bone for bone defect repair was available. The hot issues in the field of orthopedics research are to demonstrate the mechanism of bone regeneration and investigate a new bone substitute material for bone tissue engineering. To develop a biological bone substitute materials with similar composite and structure with the natural bone is considered to be one effective way to solve the puzzle. Calcium phosphate cement (CPC) as a non-self-curing hydroxyapatite ceramic-like material, has good biocompatibility and bone conduction, biocompatibility, easy for shaping, moderate temperature during the curing process,is considered an ideal bone substitute biomaterials. However, it is still no clear of the mechanism to make a biofunctional bone substitute material. The osteogenesis and angiogenesis of biomaterial remain a challenge in the field of bone tissue engineering research.Nowaday, bone tissue engineering has formed a rich theories and methods of combination of seed cells, bioactive factors and biomaterial scaffolds. Mesenchymal stem cells (Mesenchymal stem cells, MSCs) can proliferate and maintain a non-differentiated phase in vitro and possess the capacity to differentiate into:bone cartilage, fat, tendon, muscle, leather and bone marrow stromal and other mesodermal tissues.It has been reported that MSCs could be successfully extracted from various body organizations. MSCs are easily obtained, proliferation, fast, stable differentiation potential, easy to vaccination. As a result, MSCsare considered an optimal seed cells for bone regeneration. Among the multi-derived MSCs, the Bone Marrow Mesenchymal Stem Cells (BMSCs) are the most extensive application in bone tissue engineering.Previous studies have demonstrateed that BMSCs can enhance osteogenic capacity of the biomaterial and promote new bone formation. Furthermore, various bioactive factors have been widely used for enhance the ostogenesis and angiogenesis of biomaterials. Platelet-rich plasma (PRP) as a convenient derived platelet concentrate can release a variety of growth factorsafter activation whichcould promote osteocytes and osteoblasts to proliferation, differentiation and tissue formation. Since Marx the first time to use PRP composite with bone graft to repair mandibular defects, PRP has been gradually applied to the oral tissue repair, orthopedics, and neurosurgery.However, the mechanism of seed cells to promote bone formation when combine use with biomaterial scaffold still need to be clarified. In addition, it still controversial of PRP’saffect on osteogenesis and angiogenesis.Therefore, it is necessary to combined use BMSCs and PRPwith novel CPC scaffold for repair critical bone defect and investigate the outcome of CPC, BMSCs and PRP for bone regeneration. By evaluating the new bone formation and degradation rate of CPC in vivo, it could help to evaluate the valueof clinical application of the novel CPC. Furthermore, to clarify the affect of BMSCs and PRP could provide a new treatment strategy for bone repair in clinic.Objective1) To evaluate the ability of calcium phosphate cement (CPC) scaffolds to enable the adhesion, proliferation, and osteogenic differentiation of bone mesenchymal stem cells(MSCs) derived from minipig bone marrow;2) To investigate the outcome of autologous bone marrow mesenchymal stem cells (BMSCs) and platelet-rich plasma (PRP) in combination with calcium phosphate cement (CPC) scaffold to reconstruct femoral condyle critical bone defects of minipig.Methods1) BMSCsof minipig proliferation and differentiation on CPC ScaffoldMSCs were harvested from minipig bone marrow. High-temperature calcinationdicalcium phosphate with a Ca/P ratio of approximately1:9was mixed with calcium carbonate in the molar ratio of1:1for calcium phosphate cement (CPC) powder. Each CPC paste was placed into a circular mold of12mm diameter and2mm thickness to make disks scaffold for cell culture. The scaffolds were placed individually in24-well plates. Then BMSCs in passage3was seeded dropwise on the top of the CPC scaffold at0.3x105cells/scaffold and cultivated with osteogenic media contain100nM dexamethasone,10mM P-glycerophosphate,0.05mM ascorbic acid, and10nM1α,25-dihydroxyvitamin. As for control group, the BMSCs were cultured with normal medium. After1,4,7and14days, cell viability was assessed using the live/dead viability/cytotoxicity kit. The expression levels of the collagen type I (Col I), Runx-2, osteocalcin (OC) and ALP genes were quantified by the quantitative real-time polymerase chain reaction technique (QT-PCR). The alkaline phosphatase (ALP) activity and mineralization of BMSCs in both groups were also measured2) Combined use of cCPC, BMSC and PRP for bone regeneration in critical-size defect of the mini-pig femoral condyle12Tibetan minipigs were included in the study, among the12animals,6were included into the subgroup for6weeks observation. And another6were for12weeks observation. In each group,2critical bone defects were created at each femoral condyle of the animals, the12bone defects of total were randomly filled with:1) CPC only (Group CPC),2) CPC with BMSCs (Group CPC-SC),3) CPC with BMSCs and PRP (Group CPC-SC&P). Radiographic evaluation was performed immediately and every2weeks post-surgery. At the time points of6weeks and12weeks,6animals were separately scarified and a total of24block section samples were obtained for Micro-CT, histological and histomorphometricAnalyses.ResultsLive/dead staining showed that the viable cell density was increasing over time in both group. At day14, cell density of osteogenic group and control group was1115cells/mm2and1116cells/mm2respectively. The percentage of live cells increased to94.8%and95.2%in control and osteogenic and group. There was no significantly difference of cell density andpercentage of live cells of two groups at each time point. The ALP activity of osteogenic group significantly increased on day, peak at day14with a value of777±48(μM pNPP/min)/(mgProtein), increased about7times from109±24(μMpNPP/min)/(MgProtein) of day1. The ALP activity ofday7, day14and day21in osteogenic group were significantly higher than that in control group (p<0.01). ALP and OC in gene expression peaks at4days and8days in osteogenic group, achieved a4-fold increase than that at day1. Mineral synthesis by BMSCs of soteogenic group at7、14and21days were2.32±0.41mM,3.92±0.17mM and5.59±0.2Mm, significantly higher than that of the control group at respectively time point.MICRO-CT results showed,12weeks post-opertion, the RMVF of CPC-SC&P group was25.45±1.74%, and38.33±3.49%of CPC group, there was significantly difference between two groups (P<0.05).12weeks post-operation, NBV in CPC-SC&P group was78.03±3.19mm3, compared with the CPC group of53.36±5.36mm3. There was significantly difference, however, the NBV of CPC&BMSCs group was69.65±1.99mm3, there was no significantly difference with the other two groups. Histometrically, PRP/BMSCs group showed a significant increase of new bone at each time point compared with the CPC-alone group (P <0.05). However, there is no significant difference of the new bone between PRP&BMSCs-group and BMSCs-group.ConclusionCPC scaffold was enable for adhesion, proliferation, and differentiation of bone marrow mesenchymal stem cells.The novel CPC was a promising biomaterial for bone regeneration. The strategy of CPC in combination with BMSCs and PRP might be an effective method to repair bone defect in orthopaedic surgery.