Collagen Peptide Upregulates Osteoblastogenesis from Bone Marrow Mesenchymal Stem Cells through MAPK- Runx2.
ABSTRACT: Collagen is the most abundant extracellular fibrous protein that has been widely used for biomedical applications due to its excellent biochemical and biocompatibility features. It is believed that the smaller molecular weight collagen, i.e., collagen peptide (CP), has more potent activity than native collagen. However, the preparation of CP from fish bone collagen is a complex and time-consuming process. Additionally, the osteogenic effect of CP depends on its molecular weight and amino acid composition. Considering the above concept, the present work was undertaken to extract the CP directly from Mahi mahi fish (Coryphaena hippurus) bones and test its osteogenic potential using bone marrow mesenchymal stem (BMMS) cells. The hydrolyzed collagen contained triple alpha chains (110 kDa) and a peptide (~1 kDa) and the peptide was successfully separated from hydrolyzed collagen using molecular weight cut-off membrane. CP treatment was up-regulated BMMS cells proliferation and differentiation. Interestingly, CP accrued the mineral deposition in differentiated BMMS cells. Protein and mRNA expression revealed that the osteogenic biomarkers such as collagen, alkaline phosphatase, and osteocalcin levels were significantly increased by CP treatment in differentiated BMMS cells and also further elucidated the hypothesis that CP was upregulated osteogenesis through activating Runx2 via p38MAPK signaling pathway. The above results concluded that the CP from Mahi mahi bones with excellent osteogenic properties could be the suitable biomaterial for bone therapeutic application.
Project description:Deproteinization is an indispensable process for the elimination of antigenicity in xenograft bones. However, the hydrogen peroxide (H2O2) deproteinized xenograft, which is commonly used to repair bone defect, exhibits limited osteoinduction activity. The present study was designed to develop a new method for deproteinization and compare the osteogenic capacities of new pepsin deproteinized xenograft bones with those of conventional H2O2 deproteinized ones.Bones were deproteinized in H2O2 or pepsin for 8 hours. The morphologies were compared by HE staining. The content of protein and collagen I were measured by the Kjeldahl method and HPLC-MS, respectively. The physical properties were evaluated by SEM and mechanical tests. For in vivo study, X-ray, micro-CT and HE staining were employed to monitor the healing processes of radius defects in rabbit models transplanted with different graft materials.Compared with H2O2 deproteinized bones, no distinct morphological and physical changes were observed. However, pepsin deproteinized bones showed a lower protein content, and a higher collagen content were preserved. In vivo studies showed that pepsin deproteinized bones exhibited better osteogenic performance than H2O2 deproteinized bones, moreover, the quantity and quality of the newly formed bones were improved as indicated by micro-CT analysis. From the results of histological examination, the newly formed bones in the pepsin group were mature bones.Pepsin deproteinized xenograft bones show advantages over conventional H2O2 deproteinized bones with respect to osteogenic capacity; this new method may hold potential clinical value in the development of new biomaterials for bone grafting.
Project description:Collagen is the most abundant fibrous protein in animal's body and is widely used for biomedical and pharmaceutical applications. The principal sources of this protein are bovine, porcine and fish skin and bones. In Colombia, bovine bones are waste from meat industry, this material have potential as an alternative source of collagen isolation. The aim of this study was to evaluate the composition and some properties of type I collagen (COL I) extracted of bovine bones of Zebu-Bos Primigenius Indicus and its use as textile finishing to modify two types of fabrics: first a taffeta weave and the second a single jersey knit, both 100% cotton. The extracted bone collagen showed the main characteristic bands of this material in the FTIR spectra, corresponding to amide A, I, II and III. Gel electrophoresis (SDS-PAGE) presented the main bands of α1 and α2 chains characteristic of COL I with a molecular weight of approximately 120 kDa and the amino acid profile of hydrolyzed protein evaluated by amino acid analysis showed 9.4% of hydroxyproline, 10.3% proline and 16.9% of glycine content. Two traditional methods of applying finished textiles were evaluated to modify both fabrics with COL I, exhibiting better attachment through PAD method compared with exhaustion method. These results suggest that bone is an alternative source for type I collagen extraction, which can be applied as a functional textile finishing for traditional fabrics for implementation in healthtech field.
Project description:Defects in craniofacial bones occur congenitally, after high-energy impacts, and during the course of treatment for stroke and cancer. These injuries are difficult to heal due to the overwhelming size of the injury area and the inflammatory environment surrounding the injury. Significant inflammatory response after injury may greatly inhibit regenerative healing. We have developed mineralized collagen scaffolds that can induce osteogenic differentiation and matrix biosynthesis in the absence of osteogenic media or supplemental proteins. The amniotic membrane is derived from placentas and has been recently investigated as an extracellular matrix to prevent chronic inflammation. Herein, we hypothesized that a mineralized collagen-amnion composite scaffold could increase osteogenic activity in the presence of inflammatory cytokines. We report mechanical properties of a mineralized collagen-amnion scaffold and investigated osteogenic differentiation and mineral deposition of porcine adipose-derived stem cells within these scaffolds as a function of inflammatory challenge. Incorporation of amniotic membrane matrix promotes osteogenesis similarly to un-modified mineralized collagen scaffolds, and increases in mineralized collagen-amnion scaffolds under inflammatory challenge. Together, these findings suggest that a mineralized collagen-amnion scaffold may provide a beneficial environment to aid craniomaxillofacial bone repair, especially in the course of defects presenting significant inflammatory complications.
Project description:Deer sinew (DS) has been used traditionally for various illnesses, and the major active constituent is collagen. In this study, we assessed the effects of collagen peptide from DS on bone loss in the ovariectomized rats. Wister female rats were randomly divided into six groups as follows: sham-operated (SHAM), ovariectomized control (OVX), OVX given 1.0?mg/kg/week nylestriol (OVX + N), OVX given 0.4?g/kg/day collagen peptide (OVX + H), OVX given 0.2?g/kg/day collagen peptide (OXV + M), and OVX given 0.1?g/kg/day collagen peptide (OXV + L), respectively. After 13 weeks of treatment, the rats were euthanized, and the effects of collagen peptide on body weight, uterine weight, bone mineral density (BMD), serum biochemical indicators, bone histomorphometry, and bone mechanics were observed. The data showed that BMD and concentration of serum hydroxyproline were significantly increased and the levels of serum calcium, phosphorus, and alkaline phosphatase were decreased. Besides, histomorphometric parameters and mechanical indicators were improved. However, collagen peptide of DS has no effect on estradiol level, body weight, and uterine weight. Therefore, these results suggest that the collagen peptide supplementation may also prevent and treat bone loss.
Project description:Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4-/- bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4-/- BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.
Project description:In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1 and Msx2 conditionally in neural crest. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx1/2 alleles, the large defect is filled in with mispatterned bone consisting of ectopic islands of bone between the reduced frontal bones, just anterior to the parietal bones. The bone is derived from neural crest, not mesoderm, and, from DiI cell marking experiments, originates in a normally non-osteogenic layer of cells through which the rudiment elongates apically. Associated with the heterotopic osteogenesis is an upregulation of Bmp signaling in this cell layer. Prevention of this upregulation by implantation of noggin-soaked beads in head explants also prevented heterotopic bone formation. These results suggest that Msx genes have a dual role in calvarial development: They are required for the differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of pathological conditions in which calvarial bone development is compromised.
Project description:Bone regeneration, following fracture, relies on autologous and allogenic bone grafts. However, majority of fracture population consists of older individuals with poor quality bone associated with loss and/or modification of matrix proteins critical for bone formation and mineralization. Allografts suffer from same limitations and carry the risk of delayed healing, infection, immune rejection and eventual fracture. In this work, we apply a synergistic biomimetic strategy to develop matrices that rapidly form bone tissue - a critical aspect of fracture healing of weight bearing bones. Collagen matrices, enhanced with two selected key matrix proteins, osteocalcin (OC) and/or osteopontin (OPN), increased the rate and quantity of synthesized bone matrix by increasing mesenchymal stem/stromal cell (MSC) proliferation, accelerating osteogenic differentiation, enhancing angiogenesis and showing a sustained bone formation response from MSC obtained from a variety of human tissue sources (marrow, fat and umbilical cord). In vivo assessment of OC/OPN mineralized scaffolds in a critical sized-defect rabbit long-bone model did not reveal any foreign body reaction while bone tissue was being formed. We demonstrate a new biomimetic strategy to rapidly form mineralized bone tissue and secure a sustained bone formation response by MSC from multiple sources, thus facilitating faster patient recovery and treatment of non-union fractures in aging and diseased population. Acellular biomimetic matrices elicit bone regeneration response from MSC, obtained from multiple tissue sources, and can be used in variety of scaffolds and made widely available.
Project description:Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.
Project description:As a culmination of efforts over the last years, our knowledge of the embryonic origins of the mammalian frontal and parietal cranial bones is unambiguous. Progenitor cells that subsequently give rise to frontal bone are of neural crest origin, while parietal bone progenitors arise from paraxial mesoderm. Given the unique qualities of neural crest cells and the clear delineation of the embryonic origins of the calvarial bones, we sought to determine whether mouse neural crest derived frontal bone differs in biology from mesoderm derived parietal bone.BrdU incorporation, immunoblotting and osteogenic differentiation assays were performed to investigate the proliferative rate and osteogenic potential of embryonic and postnatal osteoblasts derived from mouse frontal and parietal bones. Co-culture experiments and treatment with conditioned medium harvested from both types of osteoblasts were performed to investigate potential interactions between the two different tissue origin osteoblasts. Immunoblotting techniques were used to investigate the endogenous level of FGF-2 and the activation of three major FGF signaling pathways. Knockdown of FGF Receptor 1 (FgfR1) was employed to inactivate the FGF signaling.Our results demonstrated that striking differences in cell proliferation and osteogenic differentiation between the frontal and parietal bone can be detected already at embryonic stages. The greater proliferation rate, as well as osteogenic capacity of frontal bone derived osteoblasts, were paralleled by an elevated level of FGF-2 protein synthesis. Moreover, an enhanced activation of FGF-signaling pathways was observed in frontal bone derived osteoblasts. Finally, the greater osteogenic potential of frontal derived osteoblasts was dramatically impaired by knocking down FgfR1.Osteoblasts from mouse neural crest derived frontal bone displayed a greater proliferative and osteogenic potential and endogenous enhanced activation of FGF signaling compared to osteoblasts from mesoderm derived parietal bone. FGF signaling plays a key role in determining biological differences between the two types of osteoblasts.
Project description:Postmenopausal osteoporosis (PMOP) is a severe health issue faced by postmenopausal women. microRNA-128 (miR-128) is associated with aging, inflammatory signaling, and inflammatory diseases, such as PMOP. It has also been reported to modulate in vitro osteogenic/adipogenic differentiation. However, its function in osteoclast formation is unknown. Methods: First, the expression of miR-128 and nuclear factor of activated T cells 1 (Nfatc1, bone resorption master marker) was investigated in bone tissues derived from PMOP patients, while their correlation to each other was also investigated. The levels of miR-128 and Nfatc1 in bone specimens and bone marrow-derived macrophages (BMMs) from mice subjected to ovariectomy (OVX) were also assayed. Next, we employed mice BMMs modified for overexpression and inhibition of miR-128 levels to determine its effect on osteoclast differentiation. Moreover, we generated osteoclastic miR-128 conditional knockout (miR-128Oc-/- ) mice and isolated miR-128 deletion-BMMs to observe its biological function on bone phenotype and osteoclastogenesis in vivo, respectively. The miR-128Oc-/- BMMs were used to explore the downstream regulatory mechanisms using pull-down, luciferase reporter, and western-blotting assays. Finally, the impact of miR-128 deficiency on OVX-induced bone loss in mice was evaluated. Results: The miR-128 level was found to be positively correlated with the increase in Nfatc1 level in mouse/human bone specimens and mouse primary BMMs. In vitro experiments demonstrated miR-128 levels that were dependent on activity of osteoclast differentiation and miR-128 overexpression or inhibition in BMMs significantly increased or decreased osteoclastogenesis, respectively. In vivo, we revealed that osteoclastic miR-128 deletion remarkedly increased bone mass through the inhibition of osteoclastogenesis. Mechanistically, we identified sirtuin 1 (SIRT1) as the direct target of miR-128 at the post-transcriptional level during osteoclast differentiation. Increased levels of SIRT1 reduced nuclear factor ?B (NF-?B) activity by decreasing the level of acetylation of Lysine 310, as well as inhibiting tumor necrosis factor-? (Tnf-?) and interleukin 1 (IL-1) expressions. Lastly, osteoclastic deletion of miR-128 significantly suppressed OVX-triggered osteoclastogenesis and exerted a protective effect against bone loss in mice. Conclusions: Our findings reveal a critical mechanism for osteoclastogenesis that is mediated by the miR-128/SIRT1/NF-?B signaling axis, highlighting a possible avenue for the further exploration of diagnostic and therapeutic target molecules in PMOP.