A Study Of The P15Peptide And Its Mechanism On The Enhancement Of Bone Formation

Back groundCell–matrix interactions are crucial for the regulation of cytoskeletal structure, growthand differentiation. Cell survival depends on cellular interaction with extracellular matrix(ECM), with other cells and with soluble growth factors in the serum. If these interactionsare prevented, normal cells may undergo apoptosis, a physiological form of programmedcell death. Apoptosis provides a vital mechanism for maintaining cell number and tissuestructure with complex mechanisms of regulation. Certain cell–ECM interactions aremediated by integrins, a major family of cell surface receptors. Most integrins mediate celladhesion to matrix proteins, including collagen, fibronectin, and vitronectin. Integrins cangenerate intracellular signals that regulate cell adhesion, morphology, differentiation,proliferation and apoptosis.Integrins can interact with several ECM molecules including; fibronectin, osteopontin,bone sialoprotien, collagen I, II and X. As each of these proteins contain specific aminoacid sequences, multiple types of integrin alpha and beta subunits exist and formnoncovalent heterodimers to conform to each different binding site thereby modulatingcellular response. P15is the common name of a synthetic15amino acid peptide(GTPGPQGIAGQRGVV) whose sequence is taken from the cell-binding domain ofhuman collagen type I protein. In this study, we explore covalently attaching the P15peptide to a titanium surface to evaluate whether it will enhance osteoblastic adhesion,proliferation and differentiation. Additionally, we determine if integrin activation isinvolved in this process to evaluate the feasibility of P15peptide as an effectively bioactivemolecule in the use of bone tissue regeneration.Bone formation is complex but the three-dimensional positioning of cells andmatrices is straightforward. Normal bone develops using only2mechanisms:Intramembranous bone formation and endochondral bone formation. Intramembranousbone formation is mediated by the inner periosteal osteogenic layer with bone synthesizedinitially without the mediation of a cartilage phase. Endochondral bone formation describesthe synthesis of bone on a mineralized cartilage scaffold after epiphyseal and physealcartilage have shaped and elongated the developing organ. These mechanisms are alsoused in fracture and osteotomy repair with the specific mechanism dependent on the mechanical environment provided during repair. With intramembranous bone repair,mesenchymal cells differentiate along a preosteoblast to osteoblast line while endochondralbone repair is characterized by the initial synthesis of cartilage followed by theendochondral sequence of bone formation. This study investigate the effect ofexogenously added P-15peptide on chondrocyte morphology, alkaline phosphatase levels,and alteration of chondrocytic gene and protein expression to analyze whether theendochondral bone formation is involed in the mechanism of P15peptide in the promotionof bone formation.The autologous bone graft is considered to be the “gold standard” bone graftingmaterial to enhance bone regeneration and repair bone defects, its harvesting is associatedwith donor-site morbidity and restricted availability. Furthermore, the alternative use ofallografts has also certain drawbacks, including high cost, issues of processing, sterilizationand storage, and most importantly potential immunogenic response by the host to theforeign tissue and disease transmission. Overall, the requirements for bone graftingmaterial have increased significantly within the recent years, mainly due to the increasingnumber of procedures requiring bone augmentation, such as spinal fusion procedures,revision arthroplasties and limb salvage procedures secondary to trauma, tumour orskeletal abnormalities. Ongoing research to overcome the aforementioned limitations ofautologous and allogeneic bone grafts led to development of new “orthobiologic” materialseither as adjuncts or as alternatives to the “traditional” grafting materials for themanagement of any clinical condition-requiring enhancement of bone regeneration.Anorganic bovine-derived hydroxyapatite （ABM） is a natural microporous HAcompound that is approved for the filling of periodontal defects. HA has excellentbiocompatibility and its porous structure serves as a scaffold and allows bone ingrowth.However, HA is rather inert and has a weak osteoinductive activity. In order to enhancebone ingrowth and to induce new bone formation, we added the cell-binding peptide (P-15)to HA. P-15is a synthetic clone of15-amino acid sequence of type I collagen, which ismainly involved in the adhesion/junction of cells, primarily fibroblasts and osteoblasts.P-15has been shown to have the ability to support the intracellular adhesion; moreover itsupports the adhesion of cells on HA-surface. In this study, we analyze and evaluate theefficacy and safety of the ABM/P15as the bone graft substitute in the mouse subdermalmodel and sheep vertebral defect model. Objective1. To examine the effect of exogenously added P-15peptide on chondrocyte morphology,alkaline phosphatase levels, and alteration of chondrocytic gene and protein expression.2. To investigate the role of P-15peptide on activation of specific integrins.3. An in vivo murine subdermal endochondral ossification model was utilized to evaluateeffect of P-15peptide on MSC attraction, chondrocytic differentiation, and subsequentossification and mineralization.4. To investigated osteoblast and mesenchymal cell (MSC) adhesion, proliferation andmaturation on covalently attached P15-titanium (Ti-P15) surfaces.5. The evaluation of the efficacy of the P15/ABM in the bone repair and the cellularcytotoxicity of P-15peptide.Methods1. The chondrogenic micromass culture and staining were used to observe the effects ofP15peptide on the cellular growth and differentiation.2. The double immunofluorescence was used to test the coexpression of P15peptide andα5integrin. The western blot analysis and immunohistochemistry were performed to testthe expression of chondrogenic genes in mouse stem cells cultured with P15peptide.3. The biological matrigel containing P15peptide was injected subcutaneously to themouse. Then, the microCT, HE and alcian blue and immunohistochemical staining wereused to analyze the effects of P15peptide on bone formation.4. P15peptide was covalently bonded to titanium alloy surfaces. LDH and MTT assaywere used to evaluate the cellular cytotoxity of titanium alloy surface covalently bondedwith P15peptide. The real-time qPCR, western blot and fluorescent immunohistochemistrywere performed to measure osteoblast gene expression and differentiation of the culturedcells.5. The role of the P-15peptide in bone formation was investigated using in vivo sheepvertebral defect and murine subdermal endochondral ossification models. In addition,western blot and fluorescent immunohistochemistry were performed to evaluate the effects of P-15peptide on the human articular chondrocytes and fibroblasts. LDH and MTT assaywere also used to investigate the cellular cytotoxicity of P-15peptide.Results1. The proliferation, growth and differentiation of the cells in P15group are significantlyenhanced compared with control group.2. The coexpression of the P15peptide and α5integrin was observed in P15group. Theexpression of chondrogenic genes in P15group was significantly higher than the controlgroup.3. The mouse subdermal endochondral ossification model showed that the volume of thenewly formed bone in P15group was significantly higher than the control group. The P15group has more newly formed cartilage and higher expression of the chondrogenic genescompared with control group.4. The LDH and MTT results showed no statistical difference between the cells growed onthe P15-titanium surface and those on the titanium surface (P＞0.05), and the P15peptidecan enhance the expression of the osteogenic genes in cells grown on the P15-titaniumsurface.5. Large amount of newly formed bone was observed in the defect area of the sheep spinein P15/ABM group without the presence of inflammatory response. The LDH and MTTtests showed no statistical difference between the fibroblasts in P15group and those incontrol group (P＞0.05). No statistical difference of the expression of apoptosis genes wasfound between the P15and control group (P＞0.05).Conclusion1. P15peptide can enhance the proliferation, growth and chondrogenic differentiation ofMSCs.2. P15peptide can promote the bone growth by the endochondral bone formation.3. The P15-titanium has no cellular cytotoxicity to the MSCs and can elevate theexpression of osteogenic genes of MSCs. 4. P15/ABM can facilitate the bone repair in the spinal defect area without theinflammatory response. P15peptide has no cellular cytotoxicity to the fibroblasts andcannot induce the apoptosis.