| BackgroundAlthough autologous bone is still the 'golden standard' of bone graft, the harvesting procedures are releated with severe morbidities, therefore, effective bone graft substitutes are in urgent need in clinic. Osteogenicity, osteoinductivity and osteoconductivity are key factors that influence bone healing. Because of the lack of osteogenic cells and cytokines, the osteogenic efficiency of synthetic materials (e.g., hydroxyapatite, ?-tricalcium phosphate and polymerization of lactic acid) and biomaterials (e.g., demineralized bone matrix (DBM)) are quite limited. The preparing procedures of traditional tissue engineered bone (TEB) are too complex to face the clinical need, even though it is highly effective in bone repair. Bone marrow may promote bone healing (whether mixed with osteoconductive scaffolds or directly injected) since it contains osteogenic cells and cytokines. Using the selective cell retention(SCR) strategy, osteogenic component of bone marrow might be highly concentrated in porous scaffolds, as a result, high osteogenic TEB could be constructed intraoperatively.Due to the loss of adhesive extracellular matrix, the concentrating efficiency of the commonly used scaffold—DBM is releatively limited. Proper surface modification on DBM to improve the concentrating efficiency and osteogenic activity of bone marrow enriched graft is necessary. Previously, we modified DBM scaffolds with poly-lysine and RADA-161 hydrogels. Based on their ability to decrease pore sizes and increase the surface charge, the retention efficacy and bone repair were remarkably improved in the context of SCR.However , due to the absence of adhesive ligands in poly-lysine and RADA-161, the modified scaffolds are non-specific in cell enrichment and relatively insufficient of retention efficiency,more importantly, the lack of biological stimulus may limit the performance of concentrated cells in bone reconstruction. Within the development of research on osteoinductive microenviment, bionic modification of bone substitutive scaffolds which mimic extracellular matrix or neighboring cell become a hot spot in bone tissue engineering. To avoid the shortcomings of natural protein (e.g., high cost, immunogenicity, enzymatic hydrolysis) and low specificity of short peptide, The chosen modifiers are ususlly high molecular polypeptide or mimetic peptide. In our previous studies, a recombinant fibronectin/cadherin chimera(rFN/CDH) has been constructed by integrating the functional unit of fibronectin (FN, 7-10 domain of fibronectin) and cadherin 11 (CDH, extracellular 1-2 domain of cadherin 11). The rFN/CDH exhibited nice performance in promoting cell adhesion?proliferation and osteogenic differentiation. This research aimed to improve the retention efficiency of osteogenic component in bone marrow durring SCR procedure and their performance in bone repairing by modifying DBM with rFN/CDH via a layer by layer self-assembly (LBL)technique.PurposeConstructing a multi-layered rFN/CDH composites on DBM scaffolds to improve the rentention efficiency of osteogenic cells and factors in bone marrow, investigate the underlying mechanisms of cell retention and the osteogenic effect of post-enriched graft.Method1. Fabrication and Characterization of DBM-LBL-rFN/CDH composites: poly ethylene imine (PEI) was used to develop a positive-charged precursor layer, then the multi-layered composites were fabricated by dipping the scaffolds into the rFN/CDH(poly anions) and chitosan (Chi, poly cations) solutions. The chemical components change on scaffolds surface were detected by water contact angle to determine the proper layer number of modification. The elemental compositions on the surfaces of the constructs were analysed by X-ray photoelectron spectroscopy (XPS). The morphology and pore zize were evaluated by scanning electron microscope (SEM). The porosities were determined with a modified liquid displacement method. The distribution and stablity of rFN/CDH on scaffold were observed by immunofluorescence after long term rinse with oscillation. Human bone marrow stem cells (hBMSCs) were seeded on to DBM-LBL-rFN/CDH to evaluate the influence of LBL-rFN/CDH composites on cell adhesion, proliferation and osteogenic differentiation:the rate of cells adehered to scaffolds were calculated resperatively; the effects of LBL-rFN/CDH on cell proliferation were obseved by Cytoskeleton staining and Cell Counting Kit-8 (CCK-8) tests; the protein expressions of osteoponin (OPN) and osteocalcin(OCN) were measured by western blot.2. Bone marrow retention effciency of DBM-LBL-rFN/CDH and the effect of post-enriched grafts on BMSCs migration, proliferation and osteogenic differentiation:DBM-LBL-rFN/CDH scaffolds were used to concentrate bone marrow by SCR strategy, the enriched cells were detected by flow cytometry, enriched cytokines were detected by cytokines microarray and ELISA assays. Conditioned media were harvested by diluting the protein supernatants of post-enriched grafts with different culture medium to evalulated the influences of post-enriched grafts on cell migration, proliferation and osteogenic differentiation. Cell migration of BMSCs towards conditioned media for migration were detected by trans-well assays. The effect of the protein supernatants on BMSCs proliferation were detected by CCK-8 tests. After cultured in conditioned osteogenic media, the mRNA expressions of OPN,OCN and alkaline phosphatase (ALP) were detected by real-time reverse transcriptionpolymerase chain reaction (RT-PCR),the ALP activity and the protein expressions of OPN and OCN were measured with an ALP assay kit and western blot.3. Bone repairing potential of post-enriched DBM-LBL-rFN/CDH scaffolds and the recruitment of host cells : The mouse femoral critical size defect models were established to evaluate the osteogenic efficacy of post-enriched DBM-LBL-rFN/CDH composites, bone healing were evaluate by micro-CT,biomechanical test, HE and Masson stains at 4 weeks postoperatively. GFP labelled mouse BMSCs (GFP+ mBMSCs) were injected via tail vein immediately after implantation. The recruited cells measure with immunofluorescence of GFP and small animal imaging system, and local concentration of stromal cell-derived factor-1? (SDF-1?) were detected by ELISA assays at 3 days postoperatively.Results1. A regular variation of contact angle around 34°(rFN/CDH) and 45°(Chi) were detected after 8 layer's modification, suggesting at least 8 layers are needed to construsts a stable interface, a 12 layers complexs-PEI-rFN/CDH-(Chi-rFN/CDH)5 was chosen as the final modifier. XPS demonstrated that the major atomic composition of the DBM surfaces was carbon and the levels of nitrogen and oxygen significantly increased after PEI modification.The percentages of carbon, nitrogen and oxygen were 51.63±0.64%, 15.42±0.2% and 32.31 ±0.35% when the outermost layer was Chi,and they became 45.91±0.61%,18.47±0.29%and 34.40±0.19% when the outermost layer was rFN/CDH. The surfaces of DBM scaffolds were smooth, due to the formation of irregular bulges and new pores, the pore size of scaffolds decreased from 463±110 ?m (DBM) to 246±87?m. There was no difference in porosity of DBM-LBL-rFN/CDH (0.630±0.148) and DBM (0.660±0.153).The immunofluorescence of rFN/CDH demonstrated that larger amounts of rFN/CDH were distributed on DBM-LBL-rFN/CDH than single-layered DBM-rFN/CDH both before and after powerful rinse, suggesting the modification is effective and stable. After hBMSCs seeding, the rate of cells adehered to scaffolds was remarkablly higher in DBM-LBL-rFN/CDH group (71.2±5.9%) when compared to DBM (54.0±3.7%),indicating DBM-LBL-rFN/CDH are high adhesive scaffolds. More cells were observed on DBM-LBL-rFN/CDH than DBM at both 3 days and 7days after seeding by cytoskeleton staining and CCK-8 tests, implying LBL-rFN/CDH composites promote cell proliferation.BMSCs expressed more OCN and OPN protein when culture on DBM-LBL-rFN/CDH compared to DBM, indicating LBL-rFN/CDH provided beneficial microenvironments for osteogenic differentiation.2. The retention rate and enriching multiple of karyocytes was significantly higher in DBM-LBL-rFN/CDH group (29.35±1.69% and 2.80±0.16) when compared to DBM group(18.55± 1.47% and 1.69±0.13),and decreased to 23.70±1.87% and 2.29±0.18 after RGD specific integrins blocking. The percentage of fibronectin specific integrins(?3+ and ?V+)were 60.51±4.11% and 32.72±3.89% in DBM-LBL-rFN/CDH concentrated karyocytes,significantly higher than DBM group (48.13±2.89% and 21.89±2.26%) and integrins blocking group (49.97±5.04% and 25.38±2.42%). More over,same changes were observed in osteogenic cells(e.g., monocytes, hematopoietic stem cells and BMSCs). Theses results demonstrated that DBM-LBL-rFN/CDH can selectively concentrate osteogenic cells in bone marrow via specific fibronectin-integrins recogonition. Cytokines microarray and ELISA assays demonstrated that the concentration of many osteogenic cytokines (e.g., CXCL3,IFN-?, bFGF, BMP-2, BMP-6, MDC, SDF-1?, PDGF-BB) are higher in post-enriched DBM-LBL-rFN/CDH then those of DBM and DBM/SAP. Protein supernatants of post-enriched DBM-LBL-rFN/CDH showed better performance in promoting BMSCs migration, proliferation and osteogenic differentiation.3. Immunofluorescence of GFP and small animal imaging demonstrated that moreGFP+ mBMSCs injected via tail vein were recruited to the sites of DBM-LBL-rFN/CDH , and the local concentration of SDF-1? in DBM-LBL-rFN/CDH was remarkablly higher then DBM and DBM/SAP, indicating post-enriched DBM-LBL-rFN/CDH composites may recruit host cells to ehance bone repairing. As revealed by imaging examination, biomechanical testing and histological observation, the post-enriched DBM-LB L-rFN/CDH manifested distinctive advantages in treating critical-sized bone defects.ConclusionDBM scaffolds could be stably modified with a multi-layered rFN/CDH composites via LBL technique. The DBM-LBL-rFN/CDH composites exhibited satisfactory capacities for enriching BMSCs, monocytes, HSCs, and bioactive factors within the bone marrow. The post-enriched DBM-LBL-rFN/CDH composites could provide a favourable microenvironment to recruit MSCs and further support their proliferation and osteogenic differentiation. As a result,bone repair was significantly promoted. Overall, the DBM-LBL- rFN/CDH composites might present a novel bone substitution for clinic use. |
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