An Experimental Study Of RFN/CDH Bio-inspired Surface Promoting Osteoblastic Adhesion And Differentiation In Vitro And Fusion Response In Vivo

The current approaches in tissue engineering have presented tremendous inspiration for developing applicable biomaterial for bone repair, and diversified substitutions have been proved to be magnificently potential in a series of studies. Even though, they didn't possess enough significance in application because of the difficulty in meeting clinical requirements. Apparently, this is due to the infertile property of materials (metal, polymer, bioceramic, etc.), and the incompetence in osteoconduction and osteoinduction. As far as we know, only the full insight into the molecular mechanism and cell interaction on the material-tissue interface, could we settle this scientific issue.Two prerequisites are necessary for delivering the osteointegration signal to the interface:(1) local cell populations must interact with the biomolecules for a period of time to initiate cellular events and (2) concentrations of bio-molecules must exceed threshold levels for cellular activity. Proceeding from this requirement, this study aimed at fabricating a scaffold with a novel recombinant fibronectin/cadherin chimera (rFN/CDH) and constructing superior microenvironment for osteoblastic adhesion, proliferation and differentiation with excellent bio-physico-chemical properties. Therefore, it can be expected to improve the repair ability of tissue engineered scaffold in vitro and in vivo by up-regulating the adhesion effect of bone seeding cells and the intensity of growth factors on the biomaterial surface, and to find an efficient solution to this traditional problem.Methods1. Engineering, cloning, expression and purification of rFN/CDH In this study, FN and CDH11, as two pre-molecules of the recombinant protein, were analysed bioinformatically to acquired the structural sequences and functional groups. The fragments of FN and CDH11 were integrated using a homology modeling strategy. The gene fragments of FNⅢ7-10 and CDH11 EC1-2 were spliced by SOE-PCR and then inserted into the expression plasmid of pET-22b for a reconstructed vector. After transforming the vector into Rsoetta-gami(DE3) strain and optimizing the experimental parameters, rFN/CDH was induced to express using IPTG and purified via nickel ion metal affinity chromatography. Further, immuno-blotting and mass spectrometry were employed to verify the identity of the protein. While centrifugal cell adhesive assay and osteoblastic induction were used to determine the bioactivity in vitro.2. Fabrication and characterization of rFN/CDH bio-inspired BCP biomimetic surface.The biomimetic surface was achieved by immobilizing rFN/CDH onto biphasic calcium phosphate ceramic (BCP) covalently, using a DTBP crosslinking method. Scanning electro-microscopy (SEM), X-ray photoelectron spectroscopy (XPS), contact angle and protein adsorption assay were introduced to determine the microstructure, chemical distribution and composition, hydrophilicity and ligand density of the surface after modification. Hereby the biocompatibility of the biomaterial was preliminarily evaluated.3. The effects of rFN/CDH-BCP biomimetic surface on cell proliferation, adhesion and differentiation:an functional and mechanism study in vitrohuman mesenchymal stem cells (hMSCs) were employed as the bone seed cells to observe the responses of adhesion, proliferation and morphology using cell centrifugal adhesive assay, MTT method and SEM, respectively. Meanwhile, the effect of rFN/CDH-BCP on differentiation were evaluated comprehensively as follows. Alkaline phosphatase (ALP) activity and osteocalcin (OCN) gene/protein expression, as the early and late markers for osteoblastic differentiation, were determined by colorimetric quantitation and real-time RT PCR/western blotting. Additionally, calcium nodules formed in extracellular matrix were visualized by alizarin red staining. Furthermore, a specific antagonist of phosphorylation of tyrosine 397 in focal adhesion kinase (FAK pY397) was adopted to block the reaction site of hMSCs, so as to explore the relationship between FAK pY397 and adhesion/differentiation of hMSCs which were mediated by rFN/CDH-BCP.4. The healing response of a tissue engineered bone of bMSCs combined rFN/CDH-BCP in a rabbit model:a functional study in vivoA tissue engineered bone was prepared with biomaterial of bMSCs loaded rFN/CDH-BCP, then implanted into the intertransverse process space of lumbar 5 to 6, to establish a rabbit lumbar fusion model for observing the healing response. Radiological methods (X plain and 3-dimensional CT remodeling) were employed to clinically evaluate the overall healing status, and 3 point bending test was introduced to determine the mechanical strength. Hematoxylin eosin (HE), Masson and Toluidine blue stainings were adopted to detect material-tissue interfacial integration.Results1. Engineering, cloning, expression and purification of rFN/CDHMolecular simulation indicated that the framework of CDH11 ECl-2-(Ser)3-FNⅢ7-10 was suitable for gene splicing. Bioinformatics suggested that a refined E. coli. system would be superior for rFN/CDH expression. Experimentally, a combined gene of 1.9 kbp was acquired by splicing the FNIII7-10 (1.1 kbp) with CDH11 EC 1-2 (0.8 kbp), which was verified as valid by double digestion and DNA sequencing. rFN/CDH was expressed high-efficiently and solubly at 30℃, with an OD value of 0.5-0.6, an final IPTG concentration of 0.4 mM, and an induction time of 4-6 hours. The purity reached 96% after chromatography. Data from MS showed that the protein corresponded to precursors of human fibronectin and cadherin. Additionally, cell centrifugal adhesive assay demonstrated that rFN/CDH coating TCPS adhered more MC3T3-E1 osteoblasts, as compared to single FN and CDH (1.4-fold and 2.9-fold, p<0.05). While results from osteoblastic induction indicated that rFN/CDH further promoted ALP activity and OCN gene expression (p<0.05).2. Fabrication and characterization of rFN/CDH bio-inspired BCP biomimetic surface.Data from XPS displayed that two spectra of 164.0 eV and 401.0 eV appeared on the biomimetic surface, representing the existence of sulfur and nitrogen elements and suggesting the validity in surface fabrication. SEM observations revealed an irregular distribution of macro/micro-pores among the surface and no obvious influence after modification. XPS results also showed that additional N spectrum and C-N peak for C appeared on modified surface. Besides, the chemical composition exhibited increase for C and N contents while decrease for P and Ca contents. For contact angle measurement, the 0 angle was significantly decreased after modification (45°→31°, p<0.05), indicating increased hydrophilicity. As for protein adsorption assay, adsorption of rFN/CDH onto BCP was concentration-dependent within a certain range, and the saturation concentration of rFN/CDH was 5μg/ml while ligand density was 1700 fmol/cm2.3. The effects of rFN/CDH-BCP biomimetic surface on cell proliferation, adhesion and differentiation:an functional and mechanic study in vitroThe proliferation rate were significantly higher within the first 5 days, as compared to negative and positive controls (p<0.01, p<0.05). SEM observations found that the hMSCs of rFN/CDH-BCP evenly dispersed on the surface as a rounded or polygonal morphology, exhibiting better status for cell ingrowth. Cell centrifugal adhesive assay indicated the adherent hMSCs on rFN/CDH-BCP was 4.9-fold and 1.52-fold higher at the ligand density of 1500 fmol/cm2 when compared to positive controls (p<0.05). The results unveils biomimetic surface possesses excellent biocompatibility, which is convenient for cell adhesion, proliferation and growth.As for differentiation, the ALP activity on rFN/CDH-BCP surface was the highest among all samples at the 10th day after induction (p<0.05). Correspondingly, the expression level of OCN gene and protein on rFN/CDH-BCP surface also exhibited the highest manifestation at the 14th day (p<0.05, p<0.05). At the 21st day, alizarin red staining showed that the oval-shaped and orange-red nodules, either the number or the area, distributed wider on rFN/CDH-BCP surface. These results suggest that the biomimetic surface could substantially promoted hMSCs'differentiation towards osteo-lineage.After blocking the signal transduction with specific FAK pY397 antibody, the above indices were suppressed to some extent, but not completely inhibited. This implies that the the mechanism by which the rFN/CDH-BCP promotes adhesion and ossification is not fully dependent on the phosphorylation of tyrosine 397 of FAK, but other unknown phosphorylated sites or signal molecules might be involved in this process.4. The healing response of a tissue engineered bone of bMSCs combined rFN/CDH-BCP in a rabbit model:a functional study in vivoA rabbit lumbar fusion model was established by implanting bMSCs combined rFN/CDH-BCP into intertransverse process space of L5-L6. Radiologically, the results from X plain and 3-dimensional reconstruction after 3 months revealed a fuzzy gap between material and bone bed, and higher cover rate of intertransverse process space with new bone deposition on rFN/CDH-BCP surface (p<0.05). Mechanically, a stress-strain curve was set up to illustrate the changes of mechanic load, flexural strength, and elastic modulus. The results demonstrated that the bending stiffness at the fusion site of rFN/CDH-BCP group was significantly higher than that in BCP group (0.044±0.0059 GPa vs 0.026±0.0058 GPa, p<0.01), while slightly improved as compared with bMSCs combined BCP group (0.044±0.0059 GPa vs 0.041±0.0093 GPa, p>0.05). However, no comparability can be found when compared to autogenous iliac implanting group (0.044±0.0059 GPa vs 0.071±0.0119, p<0.01). Histologically, rFN/CDH-BCP revealed incorporating bone islands consistent with autogenous iliac group, which exhibited as interlacing bone trabecula bridging biomaterial suface and cortical bone of transverse process continously. The bone mass was even more as compared with autograft, while the maturation was significantly higher than the groups of BCP and bMSCs loaded BCP groups (p<0.01).The comprehensive data reveal that when loaded with MSCs, rFN/CDH-BCP demonstrates superior characteristics of osteoconduction and osteoinduction, and substantially enhances healing capacity in vivo.Conclusions:1. The rFN/CDH chimera prepared in this study possesses excellent capacity in promoting osteoblastic adhesion and ossification, which achieves the functional integration of FN and CDH11.2. The rFN/CDH bio-functionalized BCP ceramic demonstrates superior characteristics of bio-physico-chemstry such as roughness, micro-topography, hydrophilicity, chemical composition and controllable surface density, indicating an excellent biocompatibility.3. rFN/CDH-BCP exhibits superior characteristics in promoting hMSCs'adhesion, proliferation and viability, up-regulating ALP and OCN expression, and advancing calcium nodule formation and matrix mineralization.4. Specific ITG-FAK pY397 pathway plays an important role in mediating cell adhesion and differentiation on rFN/CDH-BCP surface, and may be a regulatory and controllable target in the process.5. rFN/CDH-BCP significantly promotes bone repair efficiency in a rabbit lumbar fusion model, as evidence by the enhanced healing manifestation in radiological, mechanical and histological integration, and may be an applicable candidate for autograft.6. rFN/CDH-BCP is a novel and promising bone substitution derived from tissue engineering strategy, and can be greatly expected for repair/fusion in non-load bearing regions. However, its mechanical property should be improved as an ideal substiution for autograft.