Extracellular matrix component is degraded by enzymes of thematrix metalloproteinases(MMPs). MMPs are produced by both hemopoietic and structural cells. Increased activity of MMP-3 in periodontium is strongly associated with inflammatory periodontal disease. The purpose of the present study was to estimate the effect of BMP-7 on regeneration of periodontium. The optical density was measured by microwell plate reader at 450 nm.The difference of the optical density and the relative activity ofMMP-3 according to the concentration were statistically analyzed by one way ANOVA. The results were as follows: 1. Tetracycline-HCl showed the tendency to inhibit the activity of MMP-3 at the concentration lower than $25{\mu}g/ml$. 2. Doxycycline-HCl inhibited significantly the activity of MMP-3 at the concentration lower than $100{\mu}g/ml$. 3 . Minocycline-HCl inhibited the activity of MMP-3 at the concentration in the range of 10 to 200${\mu}g/ml$. Within the limit of the present study, the above results suggested that bone morphogenetic protein-7 may play a important role in development of periodontium.
In this study, we investigated primary biocompatibility and osteogenic gene expression of porous granular BCP bone substitutes with or without strontium (Sr) doping. In vitro biocompatibility was investigated on fibroblasts like L929 cells and osteoblasts like MG-63 cells using a cell viability assay (MTT) and one cell morphological observation by SEM, respectively. MTT results showed a cell viability percent of L929 fibroblasts, which was higher in Sr-BCP granules (98-101%) than in the non-doped granules (92-96%, p < 0.05). Osteoblasts like MG-63 cells were also found to proliferate better on Sr-doped BCP granules (01-111%) than on the non-doped ones (92-99%, p < 0.05) using an MTT assay. As compared with pure BCP granules, SEM images of MG-63 cells grown on sample surfaces confirmed that cellular spreading, adhesion and proliferation were facilitated by Sr doping on BCP. Active filopodial growth of MG-63 cells was also observed on Sr-doped BCP granules. The cells on Sr-doped BCP granules were well attached and spread out. Gene expression of osteonectin, osteopontin and osteoprotegrin were also evaluated using reverse transcriptase polymerase chain reaction (RT-PCR), which showed that the mRNA phenotypes of these genes were well maintained and expressed in Sr-doped BCP granules. These results suggest that Sr doping in a porous BCP granule can potentially enhance the biocompatibility and bone ingrowth capability of BCP biomaterials.
Purpose: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. Methods: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn-($1{\mu}M$ Zn) or Zn+($15{\mu}M$ Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. Results: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn-. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn-. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn-, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn-, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn-. Conclusion: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.
Adel-Khattab, Doaa;Afifi, Nermeen S.;el Sadat, Shaimaa M. Abu;Aboul-Fotouh, Mona N.;Tarek, Karim;Horowitz, Robert A.
Journal of Periodontal and Implant Science
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제50권6호
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pp.418-434
/
2020
Purpose: The purpose of the present study was to evaluate the effect of silica-calcium phosphate composite (SCPC) granules on bone regeneration in extraction sockets. Methods: Ten patients were selected for a split-model study. In each patient, bone healing in SCPC-grafted and control ungrafted sockets was analyzed through clinical, radiographic, histomorphometric, and immunohistochemical assessments 6 months postoperatively. Results: A radiographic assessment using cone-beam computed tomography showed minimal ridge dimension changes in SCPC-grafted sockets, with 0.39 mm and 1.79 mm decreases in height and width, respectively. Core bone biopsy samples were obtained 6 months post-extraction during implant placement and analyzed. The average percent areas occupied by mature bone, woven bone, and remnant particles in the SCPC-grafted sockets were 41.3%±12%, 20.1%±9.5%, and 5.3%±4.4%, respectively. The percent areas of mature bone and woven bone formed in the control ungrafted sockets at the same time point were 31%±14% and 24.1%±9.4%, respectively. Histochemical and immunohistochemical analyses showed dense mineralized bundles of type I collagen with high osteopontin expression intensity in the grafted sockets. The newly formed bone was well vascularized, with numerous active osteoblasts, Haversian systems, and osteocytes indicating maturation. In contrast, the new bone in the control ungrafted sockets was immature, rich in type III collagen, and had a low osteocyte density. Conclusions: The resorption of SCPC granules in 6 months was coordinated with better new bone formation than was observed in untreated sockets. SCPC is a resorbable bone graft material that enhances bone formation and maturation through its stimulatory effect on bone cell function.
To investigate the effect of Bombycis Corpus on male osteoporosis, we performed Dual Energy X-Ray Absorptiometry(DEXA) and histochemical methods. The animals were used ICR-based male mice of 8 weeks and 50 weeks, respectively. ICR male mice at 8 weeks were used in the control group, and ICR male mice at 50 weeks were used in aging group and Bombycis Corpus group(BC group). In the aging group, 0.5 ml of distilled water was administered once a day for 6 months. In BC group, Bombycis Corpus(0.78g/kg) was dissolved in distilled water for 6 months once a day. As a result, Bombycis Corpus decreased bone loss, increased bone density by reducing the loss of bone matrix in the femur due to aging, and increased osteoblast - induced osteopontin(OPN) and osteocalcin(OPC) positivite reaction. In addition, administration of Bombycis Corpus decreased Reaction of activation of nuclear factor kappa B ligand(RANKL) positive reaction, increased osteoprotegerin(OPG) positive reaction, and decreased matrix metalloproteinase-3(MMP-3) and 8-hydroxy-2'-deoxyguanosine(8-OHdG) positivite reaction. Taken together, Bombycis Corpus increases the activity of osteoblasts, inhibits osteoclast function, promotes osteoblast function, inhibits bone tissue degradation, and inhibits bone loss due to oxidative stress. It was observed that Bombycis Corpus reduced bone loss and increased bone density caused by aging to improve male osteoporosis. Therefore, Bombycis Corpus may be used as a preventive and therapeutic agent for male osteoporosis.
BACKGROUND: Bioprinting has recently appeared as a powerful tool for building complex tissue and organ structures. However, the application of bioprinting to regenerative medicine has limitations, due to the restricted choices of bio-ink for cytocompatible cell encapsulation and the integrity of the fabricated structures. METHODS: In this study, we developed hybrid bio-inks based on acrylated hyaluronic acid (HA) for immobilizing bio-active peptides and tyramine-conjugated hyaluronic acids for fast gelation. RESULTS: Conventional acrylated HA-based hydrogels have a gelation time of more than 30 min, whereas hybrid bio-ink has been rapidly gelated within 200 s. Fibroblast cells cultured in this hybrid bio-ink up to 7 days showed >90% viability. As a guidance cue for stem cell differentiation, we immobilized four different bio-active peptides: BMP-7-derived peptides (BMP-7D) and osteopontin for osteogenesis, and substance-P (SP) and Ac-SDKP (SDKP) for angiogenesis. Mesenchymal stem cells cultured in these hybrid bio-inks showed the highest angiogenic and osteogenic activity cultured in bio-ink immobilized with a SP or BMP-7D peptide. This bio-ink was loaded in a three-dimensional (3D) bioprinting device showing reproducible printing features. CONCLUSION: We have developed bio-inks that combine biochemical and mechanical cues. Biochemical cues were able to regulate differentiation of cells, and mechanical cues enabled printing structuring. This multi-functional bio-ink can be used for complex tissue engineering and regenerative medicine.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
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제47권6호
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pp.454-464
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2021
Objectives: This study aimed to investigate the in vitro osteoinductivity of the combination of bone morphogenetic protein-2 (BMP-2) and nanohydroxyapatite (nHAp) and the in vivo effects of implants coated with nHAp/BMP-2. Materials and Methods: To evaluate the in vitro efficacy of nHAp/BMP-2 on bone formation, bone marrow-derived mesenchymal stem cells (BM-MSCs) were seeded onto titanium disks coated with collagen (Col), Col/nHAp, or Col/nHAp/BMP-2. Protein levels were determined by a biochemical assay and reverse transcriptase-polymerase chain reaction. Stem cell differentiation was analyzed by flow cytometry. For in vivo studies with mice, Col, Col/nHAp, and Col/nHAp/BMP-2 were injected in subcutaneous pockets. Titanium implants or implants coated with Col/nHAp/BMP-2 were placed bilaterally on rabbit tibias and evaluated for 4 weeks. Results: In the in vitro study, BM-MSCs on Col/nHAp/BMP-2 showed reduced levels of CD73, CD90, and CD105 and increased levels of glycosaminoglycan, osteopontin, and alkaline phosphatase activity. After 4 weeks, the Col/nHAp/BMP-2 implant showed greater bone formation than the control (P=0.07), while no differences were observed in bone implant contact and removal torque. Conclusion: These results suggest that a combination of BMP-2 and an nHAp carrier would activate osseointegration on dental implant surfaces.
Radhika Adhikari;Saugat Shiwakoti;Eunmin Kim;Ik Jun Choi;Sin-Hee Park;Ju-Young Ko;Kiyuk Chang;Min-Ho Oak
Biomolecules & Therapeutics
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제31권5호
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pp.515-525
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2023
The most common heart valve disorder is calcific aortic valve stenosis (CAVS), which is characterized by a narrowing of the aortic valve. Treatment with the drug molecule, in addition to surgical and transcatheter valve replacement, is the primary focus of researchers in this field. The purpose of this study is to determine whether niclosamide can reduce calcification in aortic valve interstitial cells (VICs). To induce calcification, cells were treated with a pro-calcifying medium (PCM). Different concentrations of niclosamide were added to the PCM-treated cells, and the level of calcification, mRNA, and protein expression of calcification markers was measured. Niclosamide inhibited aortic valve calcification as observed from reduced alizarin red s staining in niclosamide treated VICs and also decreased the mRNA and protein expressions of calcification-specific markers: runt-related transcription factor 2 and osteopontin. Niclosamide also reduced the formation of reactive oxygen species, NADPH oxidase activity and the expression of Nox2 and p22phox. Furthermore, in calcified VICs, niclosamide inhibited the expression of β-catenin and phosphorylated glycogen synthase kinase (GSK-3β), as well as the phosphorylation of AKT and ERK. Taken together, our findings suggest that niclosamide may alleviate PCM-induced calcification, at least in part, by targeting oxidative stress mediated GSK-3β/β-catenin signaling pathway via inhibiting activation of AKT and ERK, and may be a potential treatment for CAVS.
Background: Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. Objectives: Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. Methods: VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. Results: Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca++-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runtrelated transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca2+-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. Conclusions: This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.
Purpose: The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs). Methods: The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7. Results: Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control. Conclusions: These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.
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