Engineering Of Bone Using Calcium Phosphate Cement Scafford And Bone Marrow Mesenchymal Stem Cells And Platelet-rich Plasma For Repairing Critical-size Bone Defect

Tissue engineering field is studying the new specific therapies to meet an urgent need for tissue repair and regeneration. Bone defects caused by trauma, bone diseases, congenital malformations, infection, tumor resection is a major challenge in orthopedic and trauma surgery. In the United States700,000people suffer from fractures, musculoskeletal disorders each year at twentieth century, spend up to$215billion. With the population aging, these numbers are still increasing rapidly. More people in China suffers fracture and musculoskeletal disorders. Autologous bone graft is considered the gold standard for the treatment of bone defects, however, autologous bone transplantion has donor site infections, prolonged surgery time, restricted supply and other shortcomings. Thus, in the past few decades,the bone substitute materials for bone tissue regeneration has caused more and more attention of the medical profession.Nearly two decades study, due to its excellent biocompatibility, bioactivity and bone conduction CPC has been considered one of the most promising bone graft substitutes. In previous studies in our group, an improved CPC was confirmed to have excellent mechanical properties, a faster rate of degradation and better cell viability, as a new material and its physical properties and evaluated in vitro cell compatibility. Bone tissue engineering have three major factors:scaffolds, seeding cells, cytokines., the research of stem cell, one of the most promising seeding cells,shows that when combined with the material scaffold for bone tissue regeneration demonstrates encouraging results. Mesenchymal stem cells are considered to be a presence in the bone marrow fibroblast-like pluripotent stem/progenitor cells, with its strong growth plasticity and flexible, it can not only differentiate into bone cells, fat cells, muscle cells and other interstitial tissue, under certain in vitro conditions,but also it can be induced to differentiate into mesodermal, ectodermal and endodermal tissues and cells. Due to its characteristics:strong differentiation potential and easily to separate,amplificate and culture, genetic stability, weak immune rejection after implantation, BMMSCs increasingly widespread in tissue engineering, cell therapy, gene therapy and other fields. Unlike embryonic stem cells in the presence of ethical issues, BMMSCs are more conducive to future clinical applications.Platelet-rich plasma is a new autologous growth factors, can be prepared from autologous blood. PRP can release high concentrations of active growth factor system for regeneration of bone defect. PRP is rich in a large number of growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-1(TGF-β1), insulin-like growth factor (IGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and so on. Since1998, Marx successfully repaired mandibular continuity bone defects with autogenous bone and PRP in88patients, as a source of autologous growth factors,PRP has been got more and more attention and application.Stem cell seeding density has important significance for cell proliferation, differentiation and extracellular matrix synthesis, revious studies have shown that high cell density can increase synthesis of extracellular matrix, such as collagen. Higher seeding density also received a higher ALP and mineralized material. However, effects of different BMMSCs seeding density on CPC scaffold have not been reported, and the feasibility of constructing tissue engineering bone and bone repair effects of CPC combined with BMMSCs and PRP are still unknown, herefore, the purpose of the study was to investigate the effects of cell proliferation and osteogenic differentiation of different BMMSCs seeding density on CPC scaffold,and to attempt to find the best seeding density. On this basis, we design CPC combined PRP and BMMSCs to build tissue engineering bone, and repair the femoral condyle standard bone defects of mini-pigs in vivo studies, to determine the central role of CPC in bone tissue engineering, and to evaluate the bone repair effect of complex CPC/BMMSCs/PRP in bone tissue engineering.Based on the two objectives above,this study consists of two parts:The first part is the effect of cell seeding density on proliferation and osteodifferentiation of bone marrow mesenchymal stem cells on calcium phosphate cement scaffold,the second part is the effect of CPC bombined with BMMSCs and PRP for repairing standard femur bone defect of mini-pigs. In addition, progress in the biological characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs) were reviewed (Part Three), for reference.Part I:Effect of Cell Seeding Density on Proliferation and Osteodifferentiation of Bone marrow mesenchymal stem cells on Calcium Phosphate Cement ScaffoldObjective:To investigate the effect of cell seeding density on proliferation and osteodifferentiation of bone marrow mesenchymal stem cells on calcium phosphate cement scaffold,and to verify the biocompatibility between BMMSCs and CPC.Methods:CPC was fabricated based on equimolar amounts of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA).TTCP was synthesized by a solid-state reaction between calcium phosphate and calcium carbonate at1500℃for10h. The CPC powders were fabricated by mixing TTCP and DCPA in a molar ratio of1:1. The CPC scaffold dimension was12mm dimaterx2mm.BMMSCs were isolated from minipig and then cultured for8days on CPC disks with three different seeding densities (lmol/ml,5mol/ml,25mol/ml). At day1,4, and8, Scanning electron microscopy was used to detect cell adhesion and extension,EdU assay was used to detect cell proliferation.Live/Dead fluorescent staining assay was used to detect cell viability, and RT-PCR was used to detect ALP,OC,Coll-1,Runx-2mRNA expression.Results:Scanning electron microscopy showed that the adhesion and expansion of BMMSCs on the CPC scaffold is good; The EdU results show that the BMMSCs proliferation rate reduced o with prolonged growth time,at1d the differences between1,5,25mol/mL was statistically significant (F=56.262, P=0.000), and further LSD method showed5mol/mL and25mol/mL group had no statistical significance (P=0.090), similar results were found at8d; LIVE/DEAD staining showed that with the time going, the number of live cells and live cells per unit area increased rapidly, the percentage of live cells increased more than93%at8d, the number of viable cells per unit area increased to558cells/mm2. the percentage of live cells between5mol/mL and25molmL group using LSD method showed no statistically significant difference(P=0.215) at the time of4d,8d were found similar results (P=0.230). An8d the number of live cells per unit area between5mol/mL group and25mol/mL group LSD method showed no significant difference (P=0.199); Alizarin red staining showed that a layer of new minerals matrix on CPC surface was Alizarin Red dyed red, and red gradually became deeper with the initial seeding density increases; ALP gene peaked at day4,at this time, ALP gene expression with seeding density of5M/ml and25M/ml was2.6fold and2.8fold the ALP at1M/ml respectively, but the difference between5M/ml and25M/ml was not statistically significant(P=0.746). OC, Coll I, and Runx2were significantly higher at day8, but the difference between5M/ml and25M/ml was not statistically significant (F=1.131, p=0.383; F=3.940, p=0.081; LSD P=0.211)Conclusion:The higher cell seeding density might not be advantageous, and an intermediate, optimal seeding density yielded the best proliferation, differentiation and ECM formation.The best Cell Seeding Density was between5mol/ml and25mol/ml.Part II:Engineering of bone using porous calcium phosphate cement and bone marrow mesenchymal stem cells and platelet-rich plasma for repairing femur critical-size bone defect in mini-pigsObjective:To determine the central role of CPC in bone tissue engineering, and to evaluate the bone repair effect of complex CPC/BMMSCs/PRP in bone tissue engineering.Methods:The CPC powder was made according to the first part, after mixed with deionized water a8mm diameter,10mm height cylindrical macropores CPC scafford was produced.1×105cells of the third generation of osteogenic BMMSCs were resuspended in100μl osteogenic culture medium,then slowly dropped to the top of the CPC scafford, incubated for24hours before implantation in vivo. The fresh PRP was made by cephalic vena cava before surgery with density gradient centrifugation methord, after activation.10Tibet mini-pigs was used, five mini-pigs were observed at each8weeks and12weeks observation time point. Four legs of each mini-pigs were randomly selected as a blank control group, CPC group, CPC/BMMSCs group, CPC/BMMSCs/PRP group, the corresponding biomaterial were implanted into bone defects. At8weeks and12weeks, animals were killed after taking digital X-ray, microscopy CT scanning were observed to analyze material degradation and bone regeneration. The histological analysis was used to calculate the percentage of new bone stained area in total area.Compared the differences among the four groups.Results:Aftetr cultured14days in osteogenic medium, dark brown calcium deposition was found with alkaline phosphatase staining, red calcium nodules accumulation was found with Alizarin red staining; X-ray results showed a marked biological response between the material and the host bone tissue at8weeks after implantation. At12weeks, most of the material was degraded,absorbed and new bone bridge formated; Whether at8weeks or12weeks, from CPC group, CPC/BMMSCs group to CPC/BMMSCs/PRP group,the CPC degradation extent increased seriously. The volume fraction of residual material by quantitative analysis among three experimental groups there was no statistically significant difference (F=0.458, P=0.670) at8weeks,12weeks were found similar results (F=3.136, P=0.117). New bone volume increased with time going, at8weeks the CPC group and control group had significant difference (LSD method, P=0.156), while the CPC/BMMSCs group, CPC/BMMSCs/PRP group comparted to the control group were significantly differentce(P=0.022and P=0.010), Showed that the addition of stem cells (CPC/BMMSCs group) or growth factor (CPC/BMMSCs/PRP group) to CPC had better repair bone defects ability than CPC alone (Blank group); Histological quantitive analysis found new bone volume fraction increased from11.51%to16.88%among three experimental groups at8weeks, CPC/BMMSCs group and CPC/BMMSCs/PRP group compared to the CPC group were statistically significant respectively (P=0.048, P=0.008). At week12,CPC group new bone volume fraction of15.46%, BMMSCs/PRP/CPC group of up to26.00%, and the two groups compared with the CPC group differences were statistically significant(P=0.014, P=0.001). Differences from8weeks to12weeks only CPC group was statistically significant (P=0.041), two other groups were not statistically significant (P=0.169, P=0.252). These results showed BMMSCs and PRP helped the healing of bone defect, after complexed with CPC a better result achieved than a simple CPC repair.Conclusion:CPC is an effective bone substitute for treatment of critical bone defects, the in vivo experiments demonstrated its excellent osteoconductive and supportion of bone regeneration. Combine use BMMSCs and PRP, the degradation rate of the CPC improved and new bone formation promoted. This method may be widely used in the future of bone tissue reconstruction.