Oyster Shell Engineering Scaffold And The Role Of Rab5 In The Osteogenesis

BackgroundBone defects and nonunion are the problems most frequently encountered in modern reconstructive orthopedic surgery. Autografting and allografting are the "gold standard" for treating these diseases. However, these bone grafting techniques cannot be applied widely due to their intrinsic drawbacks. For example, autografting and allografting are severely restricted by the scarce supply of donors. Moreover, great concerns exist regarding the pain and complications such as infection and hematoma caused by donor harvesting and immune response. In addition, the grafting techniques require critical defect sizes and host-bed viability. Therefore, several factitial bone substitutes with excellent biocompatibility have been developed as implantable grafts.Osteoporosis places a huge burden to the present medical system. At the molecular level, osteoporosis is accompanied by impaired osteogenic differentiation and enhanced osteoclast activity. Since the identification of osteoporosis as a major health issue in aging populations and the subsequent development of the first treatment modalities for its management, considerable progress has been made in our understanding of the mechanisms controlling bone turnover and disease pathophysiology, thus enabling the pinpointing of new targets for intervention. Rab5 is a member of the GTP (guanosine-5’-triphosphate) binding protein superfamily involved in the early period of endocytosis and in cell motility, regulating endosomal trafficking and maturation.In human cells, three distinct isoforms exist (Rab5A, Rab5B, and Rab5C) which present overlapping localization and redundant obligation in endocytosis. Rab5 is mainly localized to early endosomes and controls their fusion, docking, and movement on microtubules. During its active GTP-bound form, downstream effectors (APPL1 and APPL2) are recruited that, in return, are required to distinct aspects of early endosome function from signal transduction to selection and transport of cargoes. Rab5, in Caenorhabditis elegans, is also responsible for maintaining the morphology of the endo-plasmic reticulum and control the kinetics of nuclear envelope disassembly. In our study, we explored if the Rab5 protein is involved in age-associated bone remodeling and try to ascertain the mechanistic role of Rab5 in in the regulation of osteogenesis and bone growth.This study is divided into four parts:1). The Preparation and Characteristics of Oyster Shell Engineering Scaffold; 2). The Bio-properties of Oyster Shell Engineering Scaffold; 3). The role of Rab5 in the process of Osteognesis; 4). The Effects of Rab5 knockdown/overexpression on Osteogenesis.Part Ⅰ:The Preparation and Characteristics of Oyster Shell Engineering ScaffoldObjective:Engineering scaffolds combinging natural bio-mineral and artificially synthesized material hold promising potential for bone tissue regeneration. To develop one type of engineering scaffold composited with oyster shell and synthesized calcium sulfate.Method:A salt solution method was used to prepare the calcium sulfate/oyster shell (CS/OS) engineering composites. The obtained CS/OS composites were examined using X-ray diffraction (XRD, Bruker D8 Advance, Germany) with Cu radiation at a scanning rate of 15°/min over the 2θ range from 10° to 90°. The morphologies of the composites were investigated using a field emission scanning electron microscope (FE-SEM, S-4800, Japan). The setting properties of the pastes were measured using a Vicat needle according to ISO9597-1989E. The compressive strengths of the specimens were tested using an Instron (Instron 5800, America) at a loading rate of 0.5 mm/min. The in vitro bioactivity of the CS/OS composites was evaluated using the simulated body fluid (SBF) method. In vitro degradation test was carried out. Then Ca2+ concentrations and pH values of the soaking solutions were examined using inductively coupled plasma-atomic emission spectrometry (ICP-AES, Optima 2100, U.S.A.) and an electrolyte-type pH meter (FE20K, Mettler Toledo, Switzerland).Result:In this study, novel bioactive calcium sulfate/oyster shell (CS/OS) composites were prepared. Comparing to CS scaffold, the CS/OS composites with a controllable degradation rate displayed prolonged setting time with 11-28 mins. CS/OS showed the best mechanical property among all the groups. CS/OS gained the bioactivity in vitro with simulated body fluid (SBF) method by exhibiting new apatite layers on the surface of grains. Furthmore, CS/OS engineering scaffold presented more controllable rate of degradation which could be beneficial to clinic use.Conclusion:This study represents the first time that natural biomineralized oyster shell could be combined with artificially synthesized calcium sulfate material to create a novel bioactive composite.Part Ⅱ:The Bio-properties of Oyster Shell Engineering ScaffoldObject:In order to evaluate the effect of engineering scaffold to cell proliferation and osteogenic differentiation, we co-cultureed the extract of scaffold with MG63 cells then recorded. We used femroal condyle defect model to evaluate the bone repaire ability of oyster shell engineering scaffold.Methods:MG63 cell line was used in vitro biocompatibility assay. After treaded with exact from scaffold, MG63 cells were detected with cell proliferation by Cell Counting Kit-8 (CCK-8). Alkaline phosphatase staining (ALP staining) and calcium node staining (Von Kossa staining) were carried out to evaluate the osteogenic differentiation. Western Blot analysis was proceeded to test the type 1 collagen (COLI) and bone morphogenetic protein-2 (BMP-2) protein level. Femoral condyle defect animal model was carried tout to evaluate the bone repair ability of CS/OS engineering scaffold.Results:Comparing to CS scaffold, the CS/OS engineering scaffold group with a controllable degradation rate presented elevated mineral nodule formation, higher alkaline phosphate (ALP) activity and enhanced proliferation rate while treated with osteocytes MG63. In CS/OS group, elevated mRNA levels of key osteogenic genes including runt-related transcription factor 2 (Runx2), bone morphogenetic protein-2 (BMP-2), osterix (Osx), and osteocalcin (OCN) were observed. Furthermore, the up-regulation of type I collagen (COL-I) and BMP-2 was observed in CS/OS group relative to the CS group. Engineering scaffolds were implanted into rabbits critical-sized femur cavity defects in order to investigate the osteogenic capacity of the CS/OS in vivo. Then based on the HE staing, CS/OS group showed showed nearly complete osseous repair of defect, with a typical organized mature bone morphology and noticeable marrow spaces.Conclusions:With proper suitable times and mechanical strength, the CS/OS scaffolds strongly enhanced osteogenic tissue regeneration capacity relative to the regeneration ability of CS scaffolds, as presented by the results of histological staining. These results suggest that the OS-modified CS engineering scaffolds with improved mechanical properties and bioactivity would facilitate the development of a new strategy for clinic bone defect regeneration.Part Ⅲ The role of Rab5 in the process of OsteognesisObject In order to study the role of Rab5 in the peocess of osteoporosis, we collected clinic samples from osteoporosis patient. Emplying the MC3T3 osteogenic differentiation model, the dynamically changed osteogenesis-related genes were tested and the amount of Rab5 was measured both in mRNA and protein level.Methods Immunohistochemistry for Rab5 and Sp7 protein was performed on sections of distal femur tissues obtained from patients (27y-65y-85y) undergoing total knee replacement. Then we use osteoblast precursor cells (MC3T3) for osteogenic differentiation model, employing Western Blot for detection of osteogenic differentiation markers Runt-related transcription factor 2 (Runx2), Osterix (Osx), Osteopontin (Opn), Type I collagen (Coll), and Osteocalcin (Ocn). At the same time, we observed the expression of APPL1/2 (two Rab5 effectors) and Rab5 level during osteogenic differentiation. Alkaline phosphatase (ALP), Alizarin red (AR), and Von Kossa (VonK) staining were used to detect the osteogenic differentiation in MC3T3. Use Confocal microscopy to observe markers change during osteogenic differentiation. Construct aging osteoblasts(MC3T3) in vitro to study cellular osteoporosis model.Results Using osteoporosis patient samples, reduced bone density was found with the increasing age of clinic patient keen samples. Importantly, Rab5 immunoreactivity was found in trabecular bone and cortex, and appeared to be inversely proportional to patient age in a limited sample. After osteogenic differentiation, MC3T3 expressed mature osteoblast characteristic and enhanced expression of osteogenic markers, which began to rise in 3rd day, and maintain a high level on 21th day. Crucially, in vitro osteogenesis, APPL1, the effector of Rab5, also increased dramatically. In vitro osteogenesis assay, senescence of MC3T3 pre-osteoblasts by serial passaging was associated with increased P-galactosidase activity and decreased expression of both Rab5 and its effectors APPL1 and APPL2, as compared to non-senescence cells.Conclusions We speculate that decreasing Rab5 protein levels with age may potentially contribute to age-related changes in bone mass. Based on our observation, we envisage that Rab function enhancement can be utilized as a potential therapeutic strategy in degenerative bone diseases.Part Ⅳ:The Effects of Rab5 knockdown/overexpression on OsteogenesisObject siRNA was used to knockdown the expression of Rab5, then started the osteogenic differentiation to evaluate the effect of Rab5 in it. Rab5 overexpression plasmid was constructed and osteogenic differentiation was carried out after that. After injection of siRNA to the neonatal mouse skull, the effect of Rab5 on the osteogenosis was evaluated.Methods Design siRNA for MC3T3 cells for interference Rab5 to knockdown Rab5 protein during differentiation. Then we made PXJ-40-GFP-Rab5 plasmid overexpression of Rab5. All the osteogenic markers and Rab5 were detected after the KD and overexpression of Rab5, along with the osteogenic staining-ALP, AR and VonK. Take newborn fetal mice injected siRNA to knock down calvarial Rab5, starting from the postnatal2 (P2), use fluorescent dye Calcein and μCT technical reconstruction of the mice skull.Results Overexpression of Rab5 in the aged MC3T3 cells resulted in their enhanced osteogenic differentiation, alkaline phosphatase, alizarin red staining and calcified nodules support these results, in part via the Akt pathway, indicating a pro-osteogenic effect of Rab, and its possible mechanistic role in age-dependent osteoporosis possibly through AKT pathway. The main form of Rab5 in MC3T3 cells which can be interfered is Rab5a/c. SiRNA mediated inhibition of Rab resulted in decrease of the osteogenic transcription factors, Runx2 and osterix, as well as multiple extracellular matrix genes, like collagens, osteopontin and osteocalcin. Injection of specific siRNA into neonatal mice skulls at P2 effectively suppressed Rab5 expression, with a significant delay in bone growth when assessed at P13.Conclusions:In summary, our results revealed an important role of the intracellular endosomal trafficking proteins Rab5a/c in promoting and maintaining bone development. We found that the endosomal trafficking protein Rab5 plays an important role in osteogenesis and osteoblast differentiation in a partly APPL1/2- and Akt-dependent manner. In vivo suppression of Rab5a/c was associated with impaired skull morphogenesis. Rab5a/c may represent a target for the therapeutic modulation of bone growth.The APPL family of proteins are involved in linking, trafficking and signaling downstream of tyrosine kinase receptors. APPL1, also designated adaptor protein containing pH domain, PTB domain and leucine zipper motif 1; APPL; or DCC interacting protein, and APPL2, also designated adaptor protein containing pH domain, PTB domain and leucine zipper motif 2 or DCC interacting protein, are involved in the coupling of epidermal growth factor (EGF) signaling and chromatin remodeling in the nucleus. They associate with GTPase Rab 5 and are released from the plasma membrane and translocated to the nucleus. In the nucleus, APPL1 and APPL2 associate with NuRD/MeCP1 and are essential for cell growth and proliferation. APPL1 is also involved in Akt regulation, binding the kinase domains of Aktl and Akt2; APPL1 is highly expressed in heart, ovary, skeletal muscle and pancreas. APPL1 shares 54% homology with APPL2.