Potential Role Of Endothelial Progenitor Cells In The Reconstruction Of Bone Defects And Possible Mechanisms

Repairing bone defects is a major clinical orthopaedic challenge, with being constrainedby the regenerative capacity of human tissue. Tissue-engineered bone has emerged as aneffective strtegy to overcome the challenge, but the vascularization of bone graft is anobstacle which restricts its performance.One possible way to address this issue is the use of endothelial progenitor cells (EPCs),which have the ability to form endothelial colonies in vitro and contribute to angiogenesis invivo. Angiogenesis is an important fundamental process during skeletal repair andregeneration because the bone is a highly vascularised tissue. Numerous studies have shownthat EPCs enhanced angiogenesis and bone formation to bridge bone defects for bone repair.However, most current studies have highlighted the importance of improving bone formation,while few studies have further elucidated the regulatory mechanism of EPC-dependentangiogenesis promoting osteogenesis.The reconstruction of bone defect is a complex and continuous biological process,which involved a variety of cells, factors and extracellular matrix. In this process, a dynamicbalance between bone-resorbing osteoclasts and bone-forming osteoblasts contributes to bonereconstruction. At early stage of the process, old or necrotic bones must be removed byosteoclasts to provide space for osteoblasts and precursors before new bone formation. At theend of bone healing, bone undergoes continuous remodeling through the osteoclast-basedbone resorption followed by osteoblast-based bone formation. Since vascular status isindispensable for bone repair, the exploration of the cross-talk between EPCs and osteoclastprecursors as well as osteoblast precursors will provided a deeper understanding of the role ofEPCs in bone regeneration and reconstruction.To explore the influence of EPCs on the reconstruction of bone defects and the possiblemechanisms underlying this process, this study first isolated, cultured and identified micebone marrow-derived EPCs, and established an indirect co-culture system of mice EPCs andRAW264.7monocyte cells to examine the effect of EPCs on the biological behavior of osteoclast precursors in vitro. Secondly, we examined the impact of EPCs on the biologicalbehavior of mesenchymal stem cells (MSCs), after we isolated and cultured EPCs and MSCsfrom the rabbit bone marrow and identified the phenotype of EPCs and MSCs. Finally, weco-seeded EPCs and MSCs onto demineralized bone matrix (DBM) as a prevascularizedtissue-engineered bone (TEB) and implanted prevascularized TEB into the radial defects inrabbits for the repair of segmental bone defects, and examine whether the addition of EPCscontributes to restoring the architectural and functional properties of newly formed bone forreconstruction of bone defects. Our data will pave the way to identify new therapeutic targetsfor developing novel regimens for the treatment of bone defects.MethodsPart I Exploration of the effect and its mechanism of EPCs on biological behaviorof osteoclast precursors in vitro.1) Mouse bone-marrow derived EPCs were isolated from the tibias and femurs ofC57BL/6mice (8wk to12wk old) and characterized by immunohistochemical staining, flowcytometry and immunofluorescence double-staining.2) An indirect co-culture system of mice EPCs and RAW264.7monocyte cells wasestablished by inserting a transwell chamber with3μm pores into12-well plates.3) The survival of RAW264.7cells was analyzed using a cell counting kit-8on days1through7and measured by microplate reader scanning at450nm.4) The effect of EPCs on the migration of RAW264.7cells was measured usingtranswell inserts with a pore size of8μm.5) The osteoclastic differentiation of RAW264.7cells co-cultured with EPCs wasevaluated by quantifying the number of TRAP positive osteoclast-like cells.6) The release of vascular endothelial growth factor-A (VEGF-A), stromal cell-derivedfactor-1α (SDF-1α), and transforming growth factor beta1(TGF-β1) was measured in10×concentrated culture supernatants by ELISA.7) Total protein were extracted from the RAW264.7monocyte cells with or withoutEPC co-culture for7days, and the expression of CXCR4, p-VEGFR-2, p-Akt, p-ERK1/2,p-Smad2/3, p-p38MARK in osteoclasts was detected. Part II Exploration of the effect and its mechanism of EPCs on biological behaviorof osteoblast precursors in vitro.1) Rabbit bone-marrow derived EPCs and MSCs were isolated from iliac crests ofhealthy New-Zealand white rabbits and characterized by immunohistochemical staining, andimmunofluorescence double-staining.2) Bone marrow-derived EPCs and MSCs were co-cultured indirectly at ratios of2:1(2×10~5EPCs with1×10~5MSCs),1:1(1.5×10~5EPCs with1.5×10~5MSCs) and1:2(1×10~5EPCswith2×10~5MSCs), respectively.3) The cell viability of MSCs with three kinds of ratios was compared to determine theoptimal cell-seeding ratio for delivery.4) The survival and osteoblastic differentiation of MSCs co-cultured with EPCs at theratio of1:1was evaluated by CCK-8and reverse transcription PCR.5)5×10~5EPCs and5×10~5MSCs were co-seeded onto DBM as a prevascularized TEB,10×10~5MSCs were co-seeded onto DBM as a non-prevascularized TEB.6) The seeding efficiency of prevascularized TEB and non-prevascularized TEB wascompared to exmine the the effect of co-seedding approach on seeding efficiency of seedingcells on DBM scaffold.Part III Exploration of the effect and its mechanism of EPCs on the reconstructionof bone defects1) Diaphyseal defects (15mm long) were created on the bilateral radii of24healthyNew-Zealand white rabbits under aseptic conditions after the rabbits were anesthetizedintravenously with3%sodium pentobarbital.2)48radial diaphyseal defects were randomized into3groups as follows. In group A,prevascularized TEB (EPCs+MSCs+DBM) was implanted into the bone defects (n=16). Ingroup B, non-prevascularized TEB (MSCs+DBM) was implanted (n=16). In group C, DBMwithout cells was implanted for control (n=16).3) The rabbits were radiographed in the prone position at2-12weeks postoperatively toobserve the osteogenesis and recanalization of the medullary cavity. Each radiograph wasexamined by two independent observers who were blind to treatment type and given a scorebased on a radiographic scoring system (Lane and Sandhu).4) The morphological and histological characteristics of newly formed bone wereevaluated by Chinese ink microangiography, immunohistochemistry and HE staining.5) The hemodynamic and mechanical of newly formed bone were evaluated by radionuclide bone imaging, biomechanical and BMD evaluation.ResultsPart I Co-culture with EPCs promotes survival, migration, and differentiation ofosteoclast precursors1) EPCs enhanced survival, migration, and differentiation of RAW264.7cells.2) VEGF-A, SDF-1α, and TGF-β1from EPCs are involved in the regulation of RAW264.7cells.3) Presence of EPCs increased expression of VEGFR-2, CXCR4, Smad2/3, Akt, andMAPKs in RAW264.7cells.Part II Co-culture with EPCs promotes survival, adhesion, and differentiation ofosteoblast precursors1) Rabbit bone-marrow derived EPCs and MSCs were isolated and characterizedsuccessfully.2) The most proliferative activity was achieved when EPCs and MSCs were cultured atthe1:1ratio.3) EPCs could improve in vitro cell survival of MSCs and promote osteogenicdifferentiation of MSCs in vitro after72h of co-culture.4) The seeding efficiency of prevascularized TEB was significantly higher thannon-prevascularized TEB.Part III Prevascularization with EPCs improved restoration of the architecturaland functional properties of newly formed bone.1) EPC-based prevascularized TEB significantly accelerated the process ofreconstruction of bone defects, including bone repair and remodelling.2) The restoration of the intraosseous vasculature and medullary cavity was improvedmarkedly compared to the non-prevascularized groups.3) The blood supply, biomechanical strength, and bone mineral density of theprevascularized group were significantly higher than those of the non-prevascularized groupsduring bone reconstruction.