The Neuropeptide Spexin Promotes the Osteoblast Differentiation of MC3T3-E1 Cells via the MEK/ERK Pathway and Bone Regeneration in a Mouse Calvarial Defect Model

BACKGROUND@#The neural regulation of bone regeneration has emerged recently. Spexin (SPX) is a novel neuropeptide and regulates multiple biological functions. However, the effects of SPX on osteogenic differentiation need to be further investigated. Therefore, the aim of this study is to investigate the effects of SPX on osteogenic differentiation, possible underlying mechanisms, and bone regeneration. @*METHODS@#In this study, MC3T3-E1 cells were treated with various concentrations of SPX. Cell proliferation, osteogenic differentiation marker expressions, alkaline phosphatase (ALP) activity, and mineralization were evaluated using the CCK-8 assay, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), ALP staining, and alizarin red S staining, respectively. To determine the underlying molecular mechanism of SPX, the phosphorylation levels of signaling molecules were examined via western blot analysis. Moreover, in vivo bone regeneration by SPX (0.5 and 1 lg/ll) was evaluated in a calvarial defect model. New bone formation was analyzed using micro-computed tomography (micro-CT) and histology. @*RESULTS@#The results indicated that cell proliferation was not affected by SPX. However, SPX significantly increased ALP activity, mineralization, and the expression of genes for osteogenic differentiation markers, including runt-related transcription factor 2 (Runx2), Alp, collagen alpha-1(I) chain (Col1a1), osteocalcin (Oc), and bone sialoprotein (Bsp). In contrast, SPX downregulated the expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). Moreover, SPX upregulated phosphorylated mitogen-activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2). in vivo studies, micro-CT and histologic analysis revealed that SPX markedly increased a new bone formation. @*CONCLUSION@#Overall, these results demonstrated that SPX stimulated osteogenic differentiation in vitro and increased in vivo bone regeneration via the MEK/ERK pathway.

Orthognathic surgery for patients with fibrous dysplasia involved with dentition / 대한악안면성형재건외과학회지

BACKGROUND: Fibrous dysplasia (FD) is characterized by the replacement of normal bone by abnormal fibro-osseous connective tissue and typically treated with surgical contouring of the dysplastic bone. When dysplastic lesions involve occlusion, not only is surgical debulking needed, orthognathic surgery for correction of dentofacial deformity is mandatory. However, the long-term stability of osteotomized, dysplastic bone segments is a major concern because of insufficient screw-to-bone engagement during surgery and the risk of FD lesion re-growth. CASE PRESENTATION: This case report reviewed two patients with non-syndromic FD that presented with maxillary occlusal canting and facial asymmetry. Le Fort I osteotomy with recontouring of the dysplastic zygomaticomaxillary region had been performed. The stability of osseous segments were favorable. However, dysplastic, newly formed bone covered the previous plate fixation site and mild bony expansion was observed, which did not influence the facial profile. Including the current cases, 15 cases of orthognathic surgery for FD with dentition have been reported in the literature. CONCLUSION: The results showed that osteotomy did not appear to significantly reduce the long-term stability of the initial fixation insufficiency of the screw to the dysplastic bone. However, based on our results and those of the others, long-term follow-up and monitoring are needed, even in cases where the osteotomized segment shows stable results.

Extracellular Calcium-Binding Peptide-Modified Ceramics Stimulate Regeneration of Calvarial Bone Defects

Secreted protein, acidic, cysteine-rich (SPARC)-related modular calcium binding 1 (SMOC1) has been implicated in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). In this study, we found that a peptide (16 amino acids in length), which is located in the extracellular calcium (EC) binding domain of SMOC1, stimulated osteogenic differentiation of human BMSCs in vitro and calvarial bone regeneration in vivo. Treatment of BMSCs with SMOC1-EC peptide significantly stimulated their mineralization in a dose-dependent manner without changing their rate of proliferation. The expression of osteogenic differentiation marker genes, including type 1 collagen and osteocalcin, also increased in a dose-dependent manner. To examine the effect of the SMOC1-EC peptide on bone formation in vivo, the peptide was covalently immobilized onto hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) particles. X-ray photoelectron spectroscopy analysis showed that the peptide was successfully immobilized onto the surface of HA/β-TCP. Implantation of the SMOC1-EC peptide-immobilized HA/β-TCP particles into mouse calvarial defects and subsequent analyses using microcomputed tomography and histology showed significant bone regeneration compared with that of calvarial defects implanted with unmodified HA/β-TCP particles. Collectively, our data suggest that a peptide derived from the EC domain of SMOC1 induces osteogenic differentiation of human BMSCs in vitro and efficiently enhances bone regeneration in vivo.

Inhibitory effect of (-)-epigallocatechin gallate on titanium particle-induced TNF-alpha release and in vivo osteolysis

Tumor necrosis factor-alpha (TNF-alpha) and inflammatory cytokines released from activated macrophages in response to particulate debris greatly impact periprosthetic bone loss and consequent implant failure. In the present study, we found that a major polyphenolic component of green tea, (-)-epigallocatechin gallate (EGCG), inhibited Ti particle-induced TNF-alpha release in macrophages in vitro and calvarial osteolysis in vivo. The Ti stimulation of macrophages released TNF-alpha in a dose- and time-dependent manner, and EGCG substantially suppressed Ti particle-induced TNF-alpha release. Analysis of signaling pathway showed that EGCG inhibited the Ti-induced c-Jun N-terminus kinase (JNK) activation and inhibitory kappaB (IkappaB) degradation, and consequently the Ti-induced transcriptional activation of AP-1 and NF-kappaB. In a mouse calvarial osteolysis model, EGCG inhibited Ti particle-induced osteolysis in vivo by suppressing TNF-alpha expression and osteoclast formation. Therefore, EGCG may be a potential candidate compound for osteolysis prevention and treatment as well as aseptic loosening after total replacement arthroplasty.

Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells

Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 1, 3, 15, 25 uM) along with a normal osteogenic medium (OSM) as control for 1, 5, 10 days. As measured by MTT assay for mitochondrial metabolic activity, cell proliferation was stimulated even at low zinc treatment (1-3 micrometer) compared to OSM, and it was stimulated in a zinc concentration-dependent manner during 5 and 10 days, with the most pronounced effect at 15 and 25 uM Zn. Cellular (synthesized) alkaline phosphatase (ALP) activity was increased in a zinc concentration-dependent manner, so did medium (secreted) ALP activity. Cellular collagen concentration was increased by zinc as time went by, therefore with the maximum zinc stimulatory effect in 10 days, and medium collagen concentration showed the same pattern even on 1 and 5 day. This zinc stimulatory effect of collagen synthesis was observed in cell matrix collagen staining. The study results imply that zinc can increase osteogenic effect by stimulating cell proliferation, ALP activity and collagen synthesis in osteoblastic cells.

A comparative study of various type of autogeneous bone graft on the rabbit-skull defect healing

INTRODUCTION: Piezosurgery device is one of the most commonly used instrument on the intraoral surgery such as maxillary sinus lift and autogeneous bone graft. Piezosurgery instrument also contains the tips that are manufactured especially for the convenient bone graft, which now many surgeons apply them for collecting bone graft materials in the curettage method for the restoration of skull defects. However, objective data has not been shown concerning the effects about bone graft with using Piezosurgery. Therefore we investigated the effects of Piezosurgery on the rabbit-skull defect healing. MATERIALS & METHODS: To investigate the regeneration of the bony defect with various bone graft, 10 adult New Zealand white rabbits (average weight : 2.8+/-0.3kg, about 12weeks) were used. The four circular bony defects measuring 6mm in diameter were made with Piezosurgery device on each rabbit cranial bone. The harvested bone tissues during defect formation were also used for autogeneous bone graft. They were grafted into the defects in a various type; block type (Group 1), particulated type by the bone mill (Group 2), chopped type by curette shaped Piezosurgery tip (Group 3), the defect without any graft was served as control (control group). The animals were sacrificed after 6 weeks and bone regeneration capacity was evaluated histomorphometrically. RESULT & CONCLUSION: Autogeneous bone graft harvested using a Piezosurgery instrument showed satisfactory bone regeneration. There was no conspicuous difference bone prepared amomg by bone mill or Piezosurgery and block bone graft. Therefore, the bone harvested from the intraoral site near the operation field using the piezosurgery device can be a feasible and reliable graft for intraoral bony defects.

Expression patterns of beta ig-h3 in chondrocyte differentiation during endochondral ossification

beta ig-h3 is a TGF-beta-induced extracellular matrix protein which is expressed in many tissues including bones and cartilages. In previous reports, we showed that beta ig-h3 mediates cell adhesion and migration and, especially in bones, negatively regulates the mineralization in the end stage of endochondral ossification. Here, to elucidate the expression pattern and role of beta ig-h3 in chondrocyte differentiation, ATDC5 chondrocytes and embryonic and postnatal mice were used for in vitro differentiation studies and in vivo studies, respectively. beta ig-h3 was strongly induced by the treatment of TGF-beta1 and the expression level of beta ig-h3 mRNA and protein were highly expressed in the early stages of differentiation but decreased in the late stages in ATDC5. Furthermore, the patterns of TGF-beta1, -beta2, and -beta3 mRNA expression were concurrent with beta ig-h3 in ATDC5. beta ig-h3 was deeply stained in perichondrium (PC), periosteum (PO), and prehypertrophic chondrocytes (PH) through the entire period of endochondral ossification in mice. beta ig-h3 was mainly expressed in PC and PH at embryonic days and obviously in PH in postnatal days. These results suggest that beta ig-h3 may play a critical role as a regulator of chondrogenic differentiation in endochondral ossification.

Benign fibrous histiocytoma of mandible: a case report

Zinc deficiency negatively affects alkaline phosphatase and the concentration of Ca, Mg and P in rats

Zn is an essential nutrient that is required in humans and animals for many physiological functions, including immune and antioxidant function, growth, and reproduction. The present study evaluated whether Zn deficiency would negatively affect bone-related enzyme, ALP, and other bone-related minerals (Ca, P and Mg) in rats. Thirty Sprague Dawley rats were assigned to one of the three different Zn dietary groups, such as Zn adequate (ZA, 35 mg/kg), pair fed (PF, 35 mg/kg), Zn deficient (ZD, 1 mg/kg) diet, and fed for 10 weeks. Food intake and body weight were measured daily and weekly, respectively. ALP was measured by spectrophotometry and mineral contents were measured by inductively coupled plasma-mass spectrophotometer (ICP-MS). Zn deficient rats showed decreased food intake and body weight compared with Zn adequate rats (p<0.05). Zn deficiency reduced ALP activity in blood (RBC, plasma) and the tissues (liver, kidney and small intestine) (p<0.05). Also, Zn deficiency reduced mineral concentrations in rat tissues (Ca for muscle and liver, and Mg for muscle and liver) (p<0.05). The study results imply the requirement of proper Zn nurture for maintaining bone growth and formation.

gp130 is important for the normal morphogenesis of Meckel's cartilage and subsequent mandibular development

gp130-mediated signaling is involved in both chondrogenesis and osteogenesis, but its direct role in the formation of embryonic Meckel's cartilage and associated mandibular development has not yet been elucidated. In this study, we examined the influence of gp130 ablation on the developing mandibular Meckel's cartilage by evaluating the morphological and histological changes as well as the gene expression patterns in developing embryonic gp130-/- mice. The ablation of the gp130 gene showed no change in region-specific collagen mRNA expression except for a slight delay in its expression but caused shortened embryonic Meckel's cartilage, delayed hypertrophic chondrocyte maturation and subsequent bony replacement with characteristic bending of the intramandibular Meckel's cartilage. The bending of Meckel's cartilage led to a narrow mandibular arch at the rostral area with poor cortical plate formation. These findings indicate that gp130-mediated signaling is important for the normal morphogenesis of Meckel's cartilage and subsequent mandibular development.

Interrelationship of Matrix Metalloproteinase and TNF-alpha in Human Gingiva with Chronic Periodontitis associated to Type 2 Diabetes Mellitus / 대한치주과학회지

No abstract available.

Methionine synthase reductase polymorphisms are associated with serum osteocalcin levels in postmenopausal women

Homocysteine (Hcy) is thought to play an important role in the development of osteoporosis and fracture. Methionine synthase reductase (MTRR) is an enzyme involved in the conversion of Hcy to methionine. We hypothesized that certain genetic polymorphisms of MTRR leading to reduced enzyme activity may cause hyperhomocysteinemia and affect bone metabolism. We therefore examined the associations of the A66G and C524T polymorphisms of the MTRR gene with bone mineral density (BMD) and serum osteocalcin levels in postmenopausal women. Although we did not detect any significant associations between MTRR polymorphisms and BMD or serum osteocalcin levels, we found that the 66G/524C haplotype, which has reduced enzyme activity, was significantly associated with serum osteocalcin levels in a gene-dose dependent manner (P=0.002). That is, the highest osteocalcin levels (34.5+/-16.8 ng/ml) were observed in subjects bearing two copies, intermediate osteocalcin levels (32.6+/-14.4 ng/ml) were observed in subjects bearing one copy, and the lowest levels of osteocalcin (28.8+/-10.9 ng/ml) were observed in subjects bearing no copies. These results suggest that the 66G/524C haplotype of the MTRR gene affect bone turn over rate.

Cellular Biocompatibility and Stimulatory Effects of Calcium Metaphosphate on Osteoblastic Differentiation of Human Bone Marrow-derived Stromal Cells / 대한정형외과연구학회지

PURPOSE: The in vitro biocompatibility of Calcium Metaphosphate (CMP) with human bone marrow stromal cells (HBMSCs) and its effect on osteoblastic differentiation have been evaluated. MATERIALS AND METHODS: The effects of CMP on the HBMSCs undergoing osteoblastic differentiation were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Morphologies of the HBMSCs were examined using scanning electron microscopy and confocal laser scanning microscopy. Osteoblastic differentiation of the HBMSCs was analyzed by alkaline phosphatase (ALP) staining and RTPCR. RESULTS: The CMP powder and disk did not exert cytotoxic effect on the HBMSCs. In addition, the HBMSCs were adhered on the surface of CMP disk as successfully as on the culture plate or HA disk and displayed similar actin arrangement and cellular phenotypes. Furthermore, the HBMSCs grown on three different matrices were able to support osteoblastic differentiation of the HBMSCs as accessed by ALP staining. However, the CMP disk compared to the HA disk has a better ability to induce expression of osteoblast-related genes such as ALP, osteopontin (OPN) and osteoprotegerin (OPG). CONCLUSION: The results demonstrate that, in addition to biocompatibility of the CMP with the HBMSCs, the CMP has an ability to stimulate osteoblastic differentiation of the HBMSCs in vitro.

Spatio-temporal expression patterns of Runx2 isoforms in early skeletogenesis

Skeletogenesis occurs through either intramembranous or endochondral ossification. In addition, some parts of the skeletal components maintain their cartilaginous characteristics throughout life without mineralization. Runx2 is known to be a pivotal transcription factor for all skeletogenic processes. In this study, we examined the expression patterns of two major isoforms of Runx2 in early skeletogenesis. During intramembranous bone formation, Runx2-type I (Runx2-I) was widely expressed in osteoprogenitor cells and active osteoblasts, while Runx2-type II (Runx2-II) expression was stringently restricted to cells lining mineralized bones. Cells in permanent cartilage expressed collagen type II (Col-II) but never expressed Runx2 or Col-X. These permanent cartilages were well circumscribed by Runx2-I positive cells, in which Runx2-II was negative. In endochondral bone formation, Runx2 expression temporarily disappeared in Col-II-positive proliferating chondrocytes, but a secondary surge of Runx2-I expression occurred in the prehypertrophic zone before the mineralization of cartilage. Collectively, both Runx2 isoforms showed very similar expression patterns in active bone forming areas; however, Runx2-I has an exclusive role in the early commitment stage of intramembranous or endochondral bone forming processes or in cells surrounding permanent cartilage.

Localization of bone matrix gene mrna in regenerating bone tissue during the guided bone regeneration

The investigation of microvasculature changes in osseous regeneration by guided tissue regeneration procedure

mRNA Expression of Cytokines and Release of Metalloproteinases around Loose Cemented Total Hip Arthroplasty / 대한정형외과학회잡지

The aim of the present study was to investigate the mRNA expression of several cytokines which were not reported previously from interface tissues around loose cemented acetabulum to obtain better understanding of the biological mechanisms connected with aseptic loosening and osteolysis of THA. We investigated mRNA expression for several cytokines (interleukin-1 alpha [IL-l~a], IL-lp, IL-2, IL-2 receptor[2R], IL-4, IL-5, IL-8, IL-10, transforming growth factor-beta [TGF-p], and interferon- gamma [IFN-y]) by reverse transcription-polymerase chain reaction (RT-PCR) and release of metalloproteinase (MMP)-2 and MMP-9 from the cement-bone interface tissues around five loose polyethylene acetabular components. We did not include TNF-a and IL-6 because the biologic effect of the former is so similar to that of IL-1, and the latter fails to stimulate prostaglandin E, or collagenase production by fibroblsts or synovial cells. Expression of mRNA for IL-1p was detected in four, IL-2R and IL-8 in three, IL-10 and TGF-p in two of five interface tissues .No expression of mRNA for IL-la, IL-2, IL-4, IL-5, and IFN-p was detected. Zymographic analysis for gelatinase/type IV collagenase revealed gelatinolytic bands corresponding to metalloproteinase(MMP)-2 and MMP-9 in cemenl-bone interface tissues. Activated cells phagocytose particles in cement-bone interface tissues expressed more cytokines mRNA than previously known to be related to periprosthetic bone resorption, and secreted metalloproteinases associated with extracellular matrix degradation and fibrosis.

EVALUAT10N OF ECTOPIC BONE FORMATION EFFECT BY DECALCIFIED DEGREE OF ALLOGRAFTS

Homologous fibronectin enhances healing of excised wounds in rats

In order to evaluate the effects of a topical application of homologous fibronectin on the healing of skin wounds, we made 2 excisional wounds on the back skin of each rat, applied ointment with or without fibronectin purified from citrated homologous plasma, and evaluated the effect according to wound size and microscopic findings. Excised lesions treated with carrier alone, but the difference was significant only in the early phase of wound healing, 2 and 3 days, according to wound size and microscopic changes. A significant decrease in wound size could be found in both groups, treated with ointment containing and not containing fibronectin, between day 4 and 9 when wound contraction was a major contributor to wound closure. Therefore it can be concluded that topical application of fibronectin has a beneficial effect on wound healing during its early phase, but no significant influence on wound contraction.