Effects Of EPCs On Biological Characteristics Of Rat Bone Marrow Mesenchymal Stem Cells

Chronic metabolic bone disease, such as osteoporosis and osteogenesis imperfecta, arethe most common causes of bone tissue destruction, which will severely damage the teeth,periodontal tissue, and the jaw bone. Some studies demonstrate that the systemic bonemetabolism disorder can impair microenvironment of the stem cell and impact the stemcell‘s characteristics and functions, which may reduce repairing capability of stem cell.Bone marrow mesenchymal stem cells (BMSCs), a population of multipotent stem cellsthat can self-renew and differentiate into osteogeniclineage, have been applied to boneregeneration for the repair of bone defect. However, when repair the bone defects withmetabolism disorder, the cellular activity and function of BMSCs will be decreasedbecause of unhealthy microenvironment. Recently, Endothelial Progenitor Cells (EPCs)have shown played an important role in promoting angiogenesis and improvingmicroenvironment. Thus in our study, EPCs were co-cultured with BMSCs to attempt to further promote the stem cell‘s function and activity, which would help us to find a bettermethod of bone regenerating with BMSCs and develop promising stragegies to treat bonedefects with osteoporosis.Objective1. To determine whether EPCs can affect phenotype and characteristics of rBMSCs in theindirect co-culture system.2. To investigate the effect of EPCs on regulating pluripotency of rBMSCs in the indirectco-culture system.3. To study the role of EPCs on proliferation, apoptosis, and differentiation of rBMSCs inthe indirect co-culture system.4. To explore the function of EPCs in regulating the bone repair ability of rBMSCs afterindirect co-culture.Methods1. Using the density gradient centrifugation combined with whole bone marrow adherentmethod to isolate and culture rBMSCs. cell morphology were observed by usinginverted microscope. Phenotypes of rBMSCs were analyzed by flow cytometry.rBMSCs proliferation activity were detected by colony formation and MTT. Themulti-differentiation potential of rBMSCs were determined by osteogenic andadiopogenic differentitation.2. Mononuclear cells were separated and purified from the rat bone marrow by densitygradient centrifugation. And then cultured the cells with endothelial cell growthmedium-2to obtained EPCs in vitro. The sequential morphology changes of primarypassage cells were observed by using inverted microscope. The mRNA expression ofEPCs surface molecules were determined by RT-PCR. The cultured cells were furtheridentified by immunofluorescent assay and tube formation experiment in vitro.3. Using transwell inserts to establish EPCs and BMSCs indirect co-culture system, andRT-PCR, flow cytometry, DiI-acLDL immunofluorescent staining were uesd toinvestigate the effect of EPCs on phenotype and characteristics of rBMSCs.4. After indirect co-cultured with EPCs, RT-PCR and real-time PCR were used to analyse regulator‘s gene expression of rBMSCs pluripotency. EDU immunofluorescent assay,incidence of CFU-F, and flow cytometry were applied to detect BMSCs proliferationand apoptosis. In order to study the effect of EPCs on the differentitation of BMSCs,the cells were induced to osteogenic and adipogenic differentiation with inducingmedium under indirect co-culture condition. ALP and Alizarin red S staining and ALPassay were used to determine ALP enzymatic activity and calcium phosphate formationability. Oil red O staining and optical density (OD) assay were used to determineadipogenic ability. Real-time PCR and Western blot were used to analyses osteogenicand adipogenic marker genes expression.5. Established the rat alveolar bone defect model and collected the BMSCs that obtainedfrom indirect co-culture system, and then immediately implanted the combination ofcell and fibrin glue into the bone defects. Immunofluorescent tracer technique wereemployed to trace the implant. Micro-CT, histological HE stain were used to observeand value the osteogenesis.Results1. Cultured rBMSCs showed elongated spindle or rhomboid-shaped and displayed arather homogeneous confluent population growth. MTT assay and clone colony showedthat rBMSCs have the ability of self-renewal. The cells were immunopositive forsurface markers of mesenchymal, but were negative for the hematopoietic markers.Osteogenic and adipogenic induction results showed that the cells could differentiateinto adipocytes and osteoblasts when cultured in induction media.2. After separating by density gradient centrifugation and cultivating short time underspecial inductive culture media, enough quantity of EPCs can be obtained in vitro. Thesequential morphology observation showed that the early adherent EPCs displayedelongated spindle shape, and then gradually changed into cobblestone appearance. Thecells expressed surface markers of haematopoietic stem cell, and they could alsoexpressed endotheliocyte markers. EPCs were cultured on Matrigel in EGM-2, andresulted in the formation of tube-like structures. immunofluorescent assay indicatedthat the cultured cells have characteristics of endothelial cell. 3. After indirect co-culture with EPCs in vitro, the morphology and surface markers of ratBMSCs did not change compared with control group. It suggested that EPCs can notaffect the phenotype profile of BMSCs under indirect cells contact.4. Under indirect transwell co-culture condition, EPCs could maintaine the pluripotencygene expression of rBMSCs and significantly increased BMSCs proliferation, while ithad a slight but non-significant increase effect on the BMSCs apoptosis. Moreover,indirectly contacted with EPCs could up-regulate osteogenic and adipogenicdifferentiation of BMSCs in vitro.5. Gross anatomy observation, Micro-CT, and histological HE staining results togethershowd that indirect co-cultured BMSCs markedly promote the bone repair ability invivo.Conclusions1. Indirectly co-cultured with EPCs could not change the characteristic of rBMSCs.2. In indirect transwell co-culture condition, EPCs could enhance the pluripotency,proliferation and differentiation of BMSCs in vitro. Further more, after indirectlycontacted with EPCs, BMSCs displayed great enhancement of the new bone formationin vivo.