Co-culture Of Peripheral Blood CD34-positive Cells And Mesenchymal Stem Cells Enhances Bone Formation In A Rabbit Calvarial Critical-size Defect Model

The calvarial skull defect caused by trauma, intracranial tumors and cerebral vascularaccident surgery is a common problem in craniofacial surgery and plastic surgery.Patientswith large area skull defect are susceptible to a range of neurological deficits, such asheadaches, dizziness, memory decline, irritable, depressed, clinically referred to skulldefect syndrome, severe impacting patients’ quality of life. Moreover, loss of the skullprotecting, the brain is vulnerable to external forces. Skull defects also cause seriousaesthetic problems for patients. Therefore, how to repair skull defect becomes an important issue for clinical workers.Traditional methods of treatment of skull defects, including bone autograft andallograft bone transplantation, held obvious shortcomings. Development of bone tissueengineering provided a new way for repairing skull defect. Bone tissue engineeringincluded the seed cells, scaffold and cell factor. The selection of excellent seed cells fortissue engineering was the premise and basis. Bone marrow-derived mesenchymal stemcells (BM-MSC) was the most widely used as seed cells and its osteogenic ability wereconfirmed by a series of in vitro and in vivo studies. However, BM-MSC also held anumber of disadvantages, in particular failure to promote new blood vessels, in case of thedetriment of repairing large bone defects.Peripheral blood CD34+(PB-CD34+) cell contained hematopoietic stem cells (HSC)and endothelial progenitor cells (EPC). In recent years, the PB-CD34+cells’ role inpromoting neovascularization had been confirmed by multiple experiments. PB-CD34+cells could also provide the osteoblast differentiation and promote the formation of newbone. Therefore, we assumed, will the PB-CD34+cells co-cultured with the BM-MSC asa co-culture system? What is the biological characteristics of co-cultured cells, particularlythe in vitro and in vivo osteogenetic ability? Can co-cultured cells serve as a new seedcells for clinical use? In order to answer these questions, this study established PB-CD34+cells together with BM-MSC co-culture systems, and through a series of in vitro and invivo experimental verification the capability of co-cultured cells as a new type of seedcells for bone tissue engineering.Objectives1. To separate PB-CD34+cells and BM-MSC, establishment of a PB-CD34+cells andBM-MSC co-culture system.2. To explore all the biological characteristics of co-cultured cells, in particular in vitroand in vivo osteogenic ability, and do comparison between co-cultured cells andBM-MSC. 3. To explore the prospects of clinical application of co-cultured cells, co-cultured cellsheet composite with hydroxyapatite (HA) scaffold was used to repair a rabbitcalvarial critical-size defect model.Methods1. Application flow cytometry points selected rabbit PB-CD34+cell; application densitygradient centrifugal combined posted wall training law separation BM-MSC, and usesflow cytometry technology detection proceeds cell of surface molecular expression,clone set falls formed, and MTT law detection cell proliferation, and for osteogenesisand adipogenesis differentiation induced, to identification proceeds cell for BM-MSC;finally to directly contact way on PB-CD34+cell and the BM-MSC for co-culture,establish co-culture system, and detect the constitute of co-culture cells.2. Analysis of biological characteristics of cultured cells: detect cell cycle and apoptosis,cells osteogenic differentiation in vitro, induce cell sheet, transplant cell sheetcomposited with HA in nude mice, to detect osteogenic differentiation in vivo, moretest items are designed to compare, in contrast to simple BM-MSC.3. A rabbit calvarial critical-size defect model was established. Respectively the use ofco-cultured cells and BM-MSC cell sheet composite to repair the skull defect. Afterthe execution of animals, respectively by radiographic, histological and molecularbiology test were made to compare the two methods.Results1. PB-CD34+cells were separated using flow cytometry, and the separation rates was0.8%. The separated the BM-MSC, using flow cytometry to identify their highexpression of mesenchymal stem cell markers, without expression of hematopoieticstem cell markers. Through clone experiments and MTT assay for detection offorming cell proliferation, we confirmed the separated cells with self-renewalcapacity; by osteoblasts, adipogenic differentiation experiment we confirmed theisolation cells had multipotent differentiation of capacity. Establishment of co-culture system, the system includes a large number of BM-MSC, including adherentPB-CD34+cells.2. All biological characteristics of cultured cells compared with BM-MSC: flowcytometry cell cycle and apoptosis of found, a total of proliferation of co-culturedcells had a higher capacity and a relatively small rate of early apoptotic; and the invitro and in vivo osteogenic ability and osteogenic potential of co-cultured cells werestronger than BM-MSC.3. Transplantation co-culture cell and BM-MSC cell sheet composited with HA repair arabbit calvarial critical-size defect model: Live general CT and the micro-CT scandisplayed, co-cultured cell group experimental animal of repair effect was better;histological detection and the quantitative analysis displayed, co-cultured cell groupof new bone volume and bone repair ratio were better than BM-MSC group; real-timequantitative PCR and the Western blot detection found, co-cultured cells stimulatedand upregulated the expression of osteogenic and angiogenesis-related factors betterthan BM-MSC group.Conclusion1. Co-cultured PB-CD34+cells with BM-MSC by direct contact, we can establish aspecial cell culture system.2. Co-cultured cells demonstrated higher osteogenic differentiation capacity thanBM-MSC in vitro and in vivo.3. Co-cultured cells improve bone formation in a rabbit calvarial defect model betterthan BM-MSC, holding an promising clinical application in future.