Expression Levels Of Osteocalcin Research Articles

Effects of vitamin K2 administration on guided bone regeneration in diabetic rats.

The present study aimed to investigate the histomorphometric and immunohistochemical impacts of vitamin K2 on guided bone regeneration (GBR) in calvarial critical-size defects (CSDs) in diabetic rats. A total of 30 rats were used in this study, comprising 12 non-diabetic (control) rats and 18 with streptozotocin-nicotinamide-induced experimental Diabetes mellitus (DM). In all rats, two calvarial CSDs were created: one defect was left empty (E), the other was treated with bovine-derived bone graft and collagen-based resorbable membrane (GM). Study groups were as follows: control rats administered saline (n = 6, C-E and C-GM groups) or vitamin K2 (n = 6, CK-E and CK-GM groups) and diabetic rats administered saline (n = 6, DM-E and DM-GM groups) or vitamin K2 (n = 6, DMK-E and DMK-GM groups). After 4 weeks of saline or vitamin K2 administration, the rats were euthanized. Bone defect healing and new bone formation were assessed histomorphometrically, and osteocalcin and osteopontin levels were examined immunohistochemically. Percentage of new bone formation was greater in CK-GM vs. CK-E and in DMK-GM vs. DMK-E [d = 3.86 (95% CI = 16.38-28.61), d = 1.86, (95% CI = 10.74-38.58), respectively, p < .05]. Bone defect healing scores were higher in CK-GM vs. CK-E and in DMK-GM vs. DMK-E [d = 2.69 (95% CI = -2.12 to -0.87), d = 3.28 (95% CI = 0.98-1.91), respectively, p < .05]. Osteocalcin expression levels were elevated in CK-GM vs. CK-E, in DMK-GM vs. DMK-E [d = 1.19 (95% CI = 0.08-1.41), d = 1.10 (95% CI = 0.02-1.22), respectively p < .05]. Vitamin K2 enhanced osteocalcin expression levels in DMK-E vs. DM-E [d = 2.78, (95% CI = 0.56-1.53), p < .05] and in DMK-GM vs. DM-GM [d = 2.43, (95% CI = 0.65-2.10), p < .05]. Osteopontin expression was enhanced in defects treated with GM vs. E defects [C-GM vs. C-E, d = 1.56 (95% CI = 0.38-2.01); CK-GM vs. CK-E, d = 1.91 (95% CI = 0.49-1.72); DM-GM vs. DM-E, d = 2.34 (95% CI = -1.12 to -0.50); DMK-GM vs. DMK-E, d = 2.00 (95% CI = 0.58-1.91), p < .05]. The research findings suggest that administering vitamin K2 in GBR for rats with DM favorably impacts bone healing in CSDs, presenting an adjunctive strategy for bone regeneration.

Reinforcing β-tricalcium phosphate scaffolds for potential applications in bone tissue engineering: impact of functionalized multi-walled carbon nanotubes

Beta-tricalcium phosphate (β-TCP) scaffolds manufactured through the foam replication method are widely employed in bone tissue regeneration. The mechanical strength of these scaffolds is a significant challenge, partly due to the rheological properties of the original suspension. Various strategies have been explored to enhance the mechanical properties. In this research, β-TCP scaffolds containing varying concentrations (0.25–1.00 wt%) of multi-walled carbon nanotubes (MWCNT) were developed. The findings indicate that the addition of MWCNTs led to a concentration-dependent improvement in the viscosity of β-TCP suspensions. All the prepared slurries exhibited viscoelastic behavior, with the storage modulus surpassing the loss modulus. The three time interval tests revealed that MWCNT-incorporated β-TCP suspensions exhibited faster structural recovery compared to pure β-TCP slurries. Introducing MWCNT modified compressive strength, and the optimal improvement was obtained using 0.75 wt% MWCNT. The in vitro degradation of β-TCP was also reduced by incorporating MWCNT. While the inclusion of carbon nanotubes had a marginal negative impact on the viability and attachment of MC3T3-E1 cells, the number of viable cells remained above 70% of the control group. Additionally, the results demonstrated that the scaffold increased the expression level of osteocalcin, osteoponthin, and alkaline phosphatase genes of adiposed-derived stem cells; however, higher levels of gene expersion were obtained by using MWCNT. The suitability of MWCNT-modified β-TCP suspensions for the foam replication method can be assessed by evaluating their rheological behavior, aiding in determining the critical additive concentration necessary for a successful coating process.

Based on NF-κB and Notch1/Hes1 Signaling Pathways, the Mechanism of Artesunate on Inflammation in Osteoporosis in Ovariectomized Rats was Investigated.

Artesunate (ART) has the potential to modulate the nuclear factor kappa B (NF-κB) and Notch1/Hes1 signaling pathways, which play crucial roles in the pathogenesis of osteoporosis. This study aims to explore whether ART participates in the progression of osteoporosis by regulating these signaling pathways. In the in vitro experiments, we treated bone marrow mesenchymal stem cells (BMSCs) with different concentrations of ART (0, 3, 6, 12 µM) and evaluated osteogenic differentiation using alkaline phosphatase staining (ALP) and alizarin red S staining (ARS) staining. The expression levels of osteocalcin (OCN), RUNT-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), and receptor activator of the nuclear factor kappa ligand (RANKL) were detected by real-time quantitative PCR (RT-qPCR). The effects of ART on NF-κB p65 and Notch1 protein expression were analyzed by Western blot (WB) and immunofluorescence (IF). In the in vivo experiments, a postmenopausal osteoporosis rat model was established via ovariectomy. Bone tissue pathological injury was evaluated using hematoxylin eosin (HE) staining. Serum ALP levels were measured using a kit, bone density was determined by dual-energy X-ray absorptiometry, and serum levels of bone gla protein (BGP), OPG, RANKL, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and IL-1β were measured by enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of NF-κB p65 and Notch1 in tissues was assessed by immunohistochemistry. In vitro experiments revealed that compared to the control group, ART dose-dependently promoted BMSCs proliferation and enhanced their osteogenic differentiation capability. The expression of OCN, RUNX2, and OPG significantly increased in the ART-treated group, while RANKL expression decreased significantly (p < 0.05). ART significantly inhibited the expression of NF-κB p65 and Notch1/Hes1 signaling pathway proteins (p < 0.05). Compared to ART treatment alone, combined treatment with ART and phorbol myristate acetate (PMA) or valproic acid (VPA) resulted in increased expression of NF-κB p65 and Notch1 proteins and decreased osteogenic differentiation capability (p < 0.05). In vivo experiments showed that in rats treated with ART, bone damage was significantly reduced, bone density and mineral content were restored considerably, and the expression of inflammatory factors (TNF-α, IL-6, IL-1β) decreased significantly (p < 0.05). Additionally, ART treatment significantly reduced the expression of NF-κB p65 and Notch1 proteins, increased OPG expression, and decreased BGP and RANKL levels (p < 0.05). In summary, ART facilitates the osteogenic differentiation of BMSCs by inhibiting the NF-κB and Notch1/Hes1 signaling pathways, thereby exerting significant protective effects against osteoporosis.

GCTRP3 inhibits oophorectomy‑induced osteoporosis by activating the AMPK/SIRT1/Nrf2 signaling pathway in mice.

C1q/tumor necrosis factor‑related protein 3 (CTRP3) expression is markedly reduced in the serum of patients with osteoporosis. The present study aimed to investigate whether CTRP3 reduces bone loss in oophorectomy (OVX)‑induced mice via the AMP‑activated protein kinase (AMPK)/sirtuin 1 (SIRT1)/nuclear factor E2‑related factor 2 (Nrf2) signaling pathway. Female C57BL/6J mice and MC3T3‑E1 cells were used to construct in vivo and in vitro models of osteoporosis, respectively. The left femurs of mice were examined using micro‑computed tomography scans and bone‑related quantitative morphological evaluation was performed. Pathological changes and the number of osteoclasts in the left femurs of mice were detected using hematoxylin and eosin, and tartrate‑resistant acid phosphatase (TRAP) staining. Runt‑related transcription factor‑2 (RUNX2) expression in the left femurs was detected using immunofluorescence analysis, and the serum levels of bone resorption markers (C‑telopeptide of type I collagen and TRAP) and bone formation markers [osteocalcin (OCN) and procollagen type 1 N‑terminal propeptide] were detected. In addition, osteoblast differentiation and calcium deposits were examined in MC3T3‑E1 cells using alkaline phosphatase (ALP) and Alizarin red staining, respectively. Moreover, RUNX2, ALP and OCN expression levels were detected using reverse transcription‑quantitative PCR, and the expression levels of proteins associated with the AMPK/SIRT1/Nrf2 signaling pathway were detected using western blot analysis. The results revealed that globular CTRP3 (gCTRP3) alleviated bone loss and promoted bone formation in OVX‑induced mice. gCTRP3 also facilitated the osteogenic differentiation of MC3T3‑E1 cells through the AMPK/SIRT1/Nrf2 signaling pathway. The addition of an AMPK inhibitor (Compound C), SIRT1 inhibitor (EX527) or Nrf2 inhibitor (ML385) reduced the osteogenic differentiation of MC3T3‑E1 cells via inhibition of gCTRP3. In conclusion, gCTRP3 inhibits OVX‑induced osteoporosis by activating the AMPK/SIRT1/Nrf2 signaling pathway.

Icariin promotes bone marrow mesenchymal stem cells osteogenic differentiation via the mTOR/autophagy pathway to improve ketogenic diet-associated osteoporosis

BackgroundIcariin, a traditional Chinese medicine, has demonstrated anti-osteoporotic properties in ovariectomized mice. However, its effectiveness in preventing bone loss induced by ketogenic diet (KD), which mimics osteoporosis in human, remains unexplored. This study aims to investigate icariin’s impact on KD-induced bone loss in mice.MethodsThirty mice were divided into: sham, KD, and KD + icariin groups. Post a 12-week intervention, evaluation including bone microstructures, serum concentrations of tartrate-resistant acid phosphatase (TRAP) and bone-specific alkaline phosphatase (ALP), and femoral tissue expression levels of osteocalcin (OCN) and TRAP. The expression levels of mammalian target of rapamycin (mTOR), ALP, peroxisome proliferator-activated receptor gamma (PPAR-γ), phosphorylated mTOR (p-mTOR), and the autophagy adaptor protein (p62) were also analyzed. Alizarin granule deposition and cellular ALP levels were measured following the induction of bone marrow mesenchymal stem cells (BMSCs) into osteogenesis.ResultsThe study found that KD significantly impaired BMSCs' osteogenic differentiation, leading to bone loss. Icariin notably increased bone mass, stimulated osteogenesis, and reduced cancellous bone loss. In the KD + icariin group, measures such as bone tissue density (TMD), bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were significantly higher than in the KD group. Additionally, bone trabecular separation (Tb.Sp) was markedly lower in the KD + icariin group. Moreover, icariin increased OCN and ALP levels while suppressing PPAR-γ, TRAP, p62, and p-mTOR. In cellular studies, icariin encouraged osteogenic development in BMSCs under KD conditions.ConclusionsIcariin effectively counteracts bone thinning and improves bone microstructure. Its mechanism likely involves stimulating BMSCs osteogenic differentiation and inhibiting bone resorption, potentially through mTOR downregulation. These findings suggest icariin's potential as an alternative treatment for KD-induced bone loss.

Effect of strontium ranelate and cerium oxide addition in the diet on bone quality and expression level of osteocalcin and alkaline phosphatase genes in broiler chicken.

In the modern broiler industry, leg and gait disorders are considerable problems. Fast-growing broilers are especially susceptible to bone abnormalities, causing major problems for broiler producers. Strontium ranelate (SrR) has been used successfully for the treatment of osteoporosis in humans. In addition, cerium oxide (CeO) is an anti-stress agent applied in the biological system. This study was conducted to investigate the effect of SrR, CeO, and their combinations on tibia quality in broilers. A total of 384 one-day-old Ross chicks were divided into six treatments, with four replicates per treatment (16 birds per replicate). The control group was fed a standard diet, and other groups were fed SrR at levels 450, 900mg/kg feed, CeO at levels 300 and 600mg/kg feed and a combination of 450 SrR + 300 CeO mg/kg feed. Bone mineral density (BMD), bone mineral content (BMC), bone strength (BS), tibia area, tibia weight, bone Length, bone diameter, minerals in tibia bone of male broilers, alkaline phosphatase gene (ALP) and osteocalcin gene (OC) in male broilers were analysed. The results showed that the addition of SrR and CeO had no significant influence (p > 0.01) on BMD, BMC, BS, bone weight, bone length and bone diameter. While there was a significant interaction between sex and treatments, especially in the combination group, BS in females significantly (p < 0.01) increased compared to the control group. Generally, females were found to be more responsive to treatments than males. Significant increases in gene expression were noticed in OC with the addition of low levels of SrR and CeO and mixed group compared to the control. The gene expression of ALP was increased significantly only in a combination group compared to the control group. It is concluded that SrR and CeO can be used as beneficial additives in the feed to improve the tibia quality of broilers.

Assessment of the Influence of Various Concentrations of Sodium Hypochlorite on Stem Cell Derived From Human Exfoliated Deciduous Teeth (SHED) Proliferation and Differentiation.

Objectives Previous research has established that sodium hypochlorite (NaOCl) has a detrimental effect on dental stem cell viability and maturation. However, a review of the literature revealed no study evaluating the response of NaOCl to the survival of stem cells derived from human exfoliated deciduous teeth (SHEDs). Hence, the aim of the present trial was to assess the influence of various dilutions of NaOCl on SHEDproliferation and differentiation. Materials and Methods The 5.25% NaOCl solutions at concentrations of 0.5, 0.1, 0.025, 0.0125, and 0.005 mg/ml were used to assess the response to SHEDproliferation and differentiation through methyl tetrazolium (MTT) assay, alkaline phosphatase (ALP) assay, and reverse transcription polymerase chain reaction gene expression analysis at various time point intervals. Results MTT assay showed that the viability of SHEDs decreased with an increase in the concentration of NaOCl and an increase in incubation time. The ALP activity decreased with an increase in the concentration of NaOCl up to 14 days of incubation. However, the ALP activity of all the test specimens further decreased after 14 days of incubation. The gene expression levels of osteocalcin, dentin sialophosphoprotein, and STRO-1 were statistically significant when compared to the control after one, three, and seven days of incubation. Conclusion Different doses of NaOCl other than 0.5 mg/ml revealed encouraging outcomes in terms of proliferation, long-term ALP functioning, and odontogenic differentiation potential when cultivated in SHEDs.

Expression profile analysis of long noncoding RNA and messenger RNA during mouse cementoblast mineralization.

Emerging evidence has uncovered that long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) exert biofunctions on cellular mineralization and bone formation. In this study, we aimed to identify lncRNA-mRNA expression profiles and expression patterns, and explore their underlying biofunctions during cementoblast mineralization. Cementoblasts were cultured in mineralized medium for 0, 7, and 14 days. We used quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) to detect expression levels of osteocalcin (OCN), bone sialoprotein (BSP), and Osterix (Osx). Alkaline phosphatase (ALP) staining and alizarin red staining (ARS) were conducted to detect ALP activity and number of mineralized nodule. Total RNA was extracted from cells and used for high-throughput sequencing. EBSeq package was applied to analyze differentially expressed genes. Mfuzz R package was used to identify gene expression patterns. The weighted gene co-expression network analysis (WGCNA) was performed to explore co-expressed mRNAs of differentially expressed lncRNAs (DElncRNAs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were adopted by Clusterprofile R package. Cementoblasts were successfully induced by osteogenic medium. Compared with those on day 0, 384 DElncRNAs and 4255 differentially expressed mRNAs (DEmRNAs), respectively, were found on day 7. Meanwhile, 645 DElncRNAs and 4717 DEmRNAs were detected on day 14. Both DElncRNAs and DEmRNAs were classified into six clusters with different expression patterns. DEmRNAs and co-expressed mRNA of DElncRNAs were predominantly related to cell process, binding, phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathway, hypoxia-inducible factor-1 (HIF-1) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and hippo signaling pathway. The results demonstrated that both noncoding and coding RNAs were involved in the process of mineralization in cementoblasts, which may provide a new database for further study.

Lactate inhibits osteogenic differentiation of human periodontal ligament stem cells via autophagy through the MCT1-mTOR signaling pathway.

Periodontal ligament stem cells (PDLSCs) play a crucial role in periodontal bone regeneration. Lactate used to be considered a waste product of glucose metabolism. In recent years, a few pieces of evidence revealed its roles in regulating the osteogenic differentiation of stem cells, but the standpoints were controversial. This study aims to investigate the effects and the mechanisms of lactate on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The hPDLSCs were treated with different concentrations of lactic acid and lactate to differentiate the effects of the acidic PH and ion lactate. Proliferation and cytotoxicity were measured by Cell Counting Kit-8 (CCK8) assay and Live/Dead assay. The osteogenic differentiation of hPDLSCs was analyzed by alizarin red staining, alkaline phosphatase (ALP) staining, and then osteogenic proteins and genes were measured by western blot and reverse transcription-quantitative PCR (qRT-PCR). To investigate the potential signaling pathways, MCT1 inhibitor, G-protein inhibitors, and rapamycin were used, and then autophagy-related proteins and osteogenic proteins were measured by western blot. The inhibition of lactic acid on the osteogenic differentiation of hPDLSCs was more significant than lactate at the same concentration. Lactate inhibited the expression of ALP which can be rescued by Gα inhibitor. Alizarin red staining, the protein expression levels of osteocalcin (OCN), osteoprotegerin (OPN), osterix (OSX), and beclin1, LC3-II/LC3-I were decreased by lactate and partly rescued by MCT1 inhibitor. Rapamycin restored the protein expression levels of beclin1, LC3-II/LC3-I and OCN, OPN, OSX under the high lactate conditions. Lactate inhibits the expression of ALP via Gα subunit signaling, and inhibits mineralized nodules formation and the expression of osteogenic-related proteins via reducing autophagy through the MCT1-mTOR signaling pathway.

Inhibition of Poly (ADP-Ribose) Glycohydrolase Accelerates Osteoblast Differentiation in Preosteoblastic MC3T3-E1 Cells.

Poly ADP-ribosylation (PARylation) is a post-translational modification catalyzed by poly (ADP-ribose) polymerase (PARP) family proteins such as PARP1. Although PARylation regulates important biological phenomena such as DNA repair, chromatin regulation, and cell death, little is known about the relationship between osteoblast differentiation and the PARylation cycle involving PARP1 and the poly (ADP-ribose)-degrading enzyme poly (ADP-ribose) glycohydrolase (PARG). Here, we examined the effects of PARP inhibitor olaparib, an approved anti-cancer agent, and PARG inhibitor PDD00017273 on osteoblast differentiation. Olaparib decreased alkaline phosphatase (ALP) activity and suppressed mineralized nodule formation evaluated by Alizarin Red S staining in preosteoblastic MC3T3-E1 cells, while PDD00017273 promoted ALP activity and mineralization. Furthermore, PDD00017273 up-regulated the mRNA expression levels of osteocalcin and bone sialoprotein, as osteoblast differentiation markers, and osterix as transcription inducers for osteoblast differentiation, whereas olaparib down-regulated the expression of these genes. These findings suggest that PARG inhibition by PDD00017273 accelerates osteoblast differentiation in MC3T3-E1 cells. Thus, PARG inhibitor administration could provide therapeutic benefits for metabolic bone diseases such as osteoporosis.

MicroRNA-582-3p regulates osteoporosis through regulating homeobox A10 and osteoblast differentiation

Objective Recent studies have demonstrated that micro RNAs (miRNAs) are involved in bone formation and bone cell differentiation, but the role of miR-582-3p in osteoporosis is unclear. We want to study the mechanism of miR-582-3p on osteogenic differentiation. Method The expression of miR-582-3p and homeobox (Hox) A10 were analyzed by quantitative RT-PCR. The expression levels of HOXA10 protein were determined by Western blot. The target of HOXA10 was identified by bioinformatics and luciferase reporter gene assay. Results The results showed that miR-582-3p was up-regulated in OP tissues and down-regulated in osteogenic differentiated C2C12 cells compared with that in the control group. Overexpression of miR-582-3p resulted in reduced expression levels of osteocalcin (OC), alkaline phosphatase (ALP), and collagen, type I, α1 (COL1A1). miR-582-3p had a potential binding site with HOXA10. Moreover, miR-582-3p inhibited the expression of HOXA10, and overexpression of HOXA10 reduced the effect of miR-582-3p on osteoblast markers. HOXA10 was the target gene of miR-582-3p, which could inhibit the expression of HOXA10. Furthermore, HOXA10 reduced the role of miR-582-3p in osteoblast markers. miR-582-3p inhibited the development of osteoporosis by regulating HOXA10 and osteoblast differentiation. Conclusions miR-582-3p may be a therapeutic target of osteoporosis treatment.

Positive Osteocalcin Expressions in Periodontium Regenerated by Nano Bioceramics after Orthodontic Tooth Movement

The research is to analyse the immunohistochemical reaction of orthodontic force on the periodontium reformed by nanobiphasic calcium phosphate ceramics (nBCP). Two third incisors were selected randomly and operated as experimental groups in 2 Beagle dogs. In the labial aspects of the third incisors, alveolar bone defects were surgically made and implanted with NBCP. The contralateral teeth in the same jaw did not receive any treatment as control. After 24 weeks, all the third incisors were moved labially. The dogs were euthanized 4 weeks later. The expression levels of osteocalcin were detected by immunohistochemical staining. Positive osteocalcin expressions in regenerated periodontium were observed and compared with the normal periodontium in the control groups. There were no significant differences within and between them. It means the periodontium regenerated by nBCP can bear orthodontic forces with a normal function. Based on these findings, we concluded that nBCP may offer a new bone graft choice for periodontic disease patients who have demands for orthodontic treatment.

Long non-coding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 regulates the proliferation and osteogenic differentiation of human periodontal ligament stem cells by targeting miR-24-3p.

This study aims to explore the effect and molecular mechanism of long non-coding RNA (lncRNA) potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) on proliferation and osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs). The hPDLSCs of normal periodontal tissues were isolated and cultured. The mineralized solution induced the osteoblast differentiation of hPDLSCs. The down-regulation of lncRNA KCNQ1OT1, the overexpression of anti-miR-24-3p on the proliferation and the levels of osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) of hPDLSCs were investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA KCNQ1OT1, miR-24-3p, OCN, OPN, and ALP. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell viability and activity. Cell proliferation was evaluated by MTT. Western blot was used to detect protein expression. The targeted relationship between lncRNA KCNQ1OT1 and miR-24-3p was detected by double-luciferase experiment. The expression level of lncRNA KCNQ1OT1 increased, and that of miR-24-3p decreased during the osteogenesis of hPDLSCs (P<0.05). The down-regulation of lncRNA KCNQ1OT1 inhibited cell proliferation and reduced the mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). LncRNA KCNQ1OT1 targeted and regulated miR-24-3p. The overexpression of miR-24-3p inhibited cell proliferation and reduced the mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). Inhibition of miR-24-3p reversed the effect of the down-regulation of lncRNA KCNQ1OT1 on cell proliferation and mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). Down-regulation of lncRNA KCNQ1OT1 inhibited the proliferation and osteogenic differentiation of hPDLSCs by targeting the up-regulated expression of miR-24-3p.

Comparison Capacity of Collagen Hydrogel and Collagen/Strontium Bioglass Nanocomposite Scaffolds With and Without mesenchymal Stem Cells in Regeneration of Critical Sized Bone Defect in a Rabbit Animal Model.

Bone self-healing is limited and requires additional or external intervention to promote and accelerate bone regeneration. Therefore, the aim of this study was to investigate the potential capacity of hydrogel collagen (Co) nanocomposite alone, and in combination with 2% strontium (Co/BGSr2%) in presence of mesenchymal stem cells (MSCs) in full-thickness bone defect regeneration in the rabbit animal model. A total of 72 New Zealand white rabbits were randomly divided in 6 groups of 12 rabbits with full-thickness bone defect. In five groups, the bone defect was treated with MSC, Co, Co/BGSr2%, Co + MSCs, and Co/BGSr2% + MSCs. No treatment was done in the control group. The treatments were assessed radiographically, histopathologically, and immunohistochemically on days 14, 28, 42, and 56 post-treatment. The highest radiographical and histological scores were belonged to the Co/BGSr2% + MSC followed by Co + MSCs, Co/BGSr2%, Co, MSC, and the control groups. The highest and lowest mean expression level of osteocalcin was detected in the Co/BGSr2% + MSC and control groups by 28th dayof post-implantation, respectively. In contrast, the highest and lowest mean expression level of osteocalcin on day 56 post-implantation was belonged to the control and Co/BGSr2% + MSC, respectively. The Co/BGSr2% nanocomposite scaffold seeded with MSC can accelerate bone regeneration resulted from osteoblastic production of osteocalcin protein. Therefore, collagen hydrogel combined with 2% strontium in nanocomposite form is a suitable candidate scaffold for bone tissue engineering.

LncRNA LINC00963 promotes osteogenic differentiation of hBMSCs and alleviates osteoporosis progression by targeting miRNA-760/ETS1 axis

Although long non-coding RNA LINC00963 has been reported to play a crucial regulatory role in osteoporosis (OP), its specific mechanism has not been well studied. Cell viability of human bone marrow mesenchymal stem cells (hBMSCs) transfected with short hairpin RNA targeting LINC00963 (sh-LINC00963) and negative control (sh-NC) was analysed by cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity in hBMSCs transfected with sh-LINC00963 and sh-NC after induction by osteogenic medium (OM) on day 7 was detected. The protein expression levels of osteocalcin (OCN) and osteopontin (OPN) in hBMSCs transfected with sh-LINC00963 and sh-NC during OM induction on day 3 were detected by western blot. The relationship among LINC00963, miR-760, and E26 transformation specific-1 (ETS1) was determined by bioinformatics analysis, luciferase reporter assay, and RNA-binding protein immunoprecipitation (RIP) assay. A rat model with OP was established to confirm the role of LINC00963 in vivo. The expression level of LINC00963 was much lower in hBMSCs isolated from the discarded femoral head tissues of OP patients compared with that in health patients. Meanwhile, the expression level of LINC00963 was significantly increased and the expression level of miR-760 was decreased in hBMSCs during osteogenic induction. LINC00963 could bind to the 3′-untranslated region (3′-UTR) of miR-760 and negatively regulate the expression of miR-760, then promote the osteogenic differentiation in hBMSCs. ETS1 was identified as a target of miR-760. Moreover, overexpression of LINC00963 obviously reduced bone mineral density (BMD) of the left femur in OP rats and alleviated OP progression in vivo. Our results demonstrated that LINC00963 positively regulated the expression of ETS1 by directly targeting miR-760, and then promoted osteogenic differentiation of hBMSCs in vitro, and also attenuated OP progression in vivo, suggesting that LINC00963 might be a potential therapeutic target for OP.

Comparison of characteristics of mesenchymal stem cells from bovine

Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and thus hold therapeutic implications in field of regenerative medicine and reproductive biotechnology. In the present study, we compared the stem cell properties of bovine ear skin tissue (ESK)- and nasal mucosa (NM)-derived MSCs. Bovine ESK-MSCs and NM-MSCs were successfully isolated by collagenase digestion and maintained proliferative capacity during the 20 consecutive passages. Both ESK-MSCs and NM-MSCs showed similar morphology and expressed common cell surface markers (CD29, CD44, CD90, and CD105). Also, we compared differentiation potentials of bovine ESK-MSCs and NM-MSCs into osteogenic, adipogenic, and chondrogenic lineages through specific staining and quantitative real-time RT-PCR. As results, bovine ESK-MSCs and NM-MSCs could differentiate into mesodermal cell lineages. However, bovine ESK-MSCs and NM-MSCs exhibited difference in expression of differentiation-related specific markers. Specifically, NM-MSCs exhibited increased expression levels of osteocalcin, peroxisome proliferator-activated receptor gamma, and aggrecan compared to ESK-MSCs. Also, ESK-MSCs exhibited increased expression levels of collagen type I, II, and lipoprotein lipase compared to NM-MSCs. We suggest that the nasal mucosa of bovine could be used as a source of bovine MSCs.

Dietary Supplementation of Calcium Propionate and Calcium Butyrate Improves Eggshell Quality of Laying Hens in the Late Phase of Production.

The aim of this study was to evaluate the effects of dietary supplementation of calcium propionate and calcium butyrate on the laying performance, eggshell quality, and expression of genes related to calcium and phosphorus metabolism in the tibia. One hundred and twenty 70-week-old Isa Brown hens were randomly assigned to three treatments, and each treatment had four replicates of 10 birds fed a basal diet (control) or a basal diet supplemented with 0.5% calcium propionate (CP) or 0.5% calcium butyrate (CB) for 8 weeks. The CB and CP treatments had no significant effect (P>0.05) on the laying rate, egg production, egg weight, and feed efficiency. The eggshell percentage was increased from week 2 (P<0.05) and eggshell thickness was elevated at week 8 (P<0.01) by both CP and CB treatments. Compared to the control treatment, the CB treatment increased serum calcium and phosphorus levels at week 4 (P<0.05), whereas the CP and CB treatments decreased serum phosphorus at weeks 6 and 8, respectively (P<0.05). Dietary supplementation had no effect on the bone index and bending strength of the tibia (P>0.05). The calcium and phosphorus content of the tibia was decreased by the CB treatment (P<0.05). In the spleen, NF-κB and IL-6 transcript levels were not influenced (P>0.05) but TNF-α transcript levels were decreased by the CP treatment (P<0.05). In the tibia, the expression levels of NF-κB, TNF-α, and IL-17 were not affected by the CP or CB treatment (P>0.05). The CP and CB treatments had no significant effect on the transcript levels of RANKL, OPG, RNUX2, OPN, α-Clotho, and VDR (P>0.05). In contrast, PHEX transcript levels were increased by the CP treatment (P<0.05). The expression levels of osteocalcin (P=0.094) and FGF23 (P=0.087) tended to decrease under the CB treatment. In conclusion, dietary supplementation of 0.5% calcium butyrate or 0.5% calcium propionate improved the eggshell quality of aged laying hens, possibly as a result of decreased deposition or enhanced mobilization of bone calcium and phosphorus.

Evaluation of Preosteoblast MC3T3-E1 Cells Cultured on a Microporous Titanium Membrane Fabricated Using a Precise Mechanical Punching Process.

The surface topography of Titanium (Ti) combined toughness and biocompatibility affects the attachment and migration of cells. Limited information of morphological characteristics, formed by precise machining in micron order, is currently available on the Ti that could promote osteoconduction. In the present study, a pure Ti membrane was pierced with precise 25 μm square holes at 75 μm intervals and appear burrs at the edge of aperture. We defined the surface without burrs as the “Head side” and that with burrs as the “Tail side”. The effects of the machining microtopography on the proliferation and differentiation of the preosteoblasts (MC3T3-E1 cells) were investigated. The cells were more likely to migrate to, and accumulate in, the aperture of holes on the head side, but grew uniformly regardless of holes on the tail side. The topography on the both surfaces increased osteopontin gene expression levels. Osteocalcin expression levels were higher on the head side than one on the blank scaffold and tail side (p < 0.05). The osteocalcin protein expression levels were higher on the tail side than on the head side after 21 days of cultivation, and were comparable to the proportion of the calcified area (p < 0.05). These results demonstrate the capacity of a novel microporous Ti membrane fabricated using a precise mechanical punching process to promote cell proliferation and activity.

Maternal high-fat diet promotes calcified atherosclerotic plaque formation in adult offspring by enhancing transformation of vascular smooth muscle cells to osteochondrocytic-like phenotype

Abstract Background and objective Maternal high-fat diet (HFD) has been shown to modulate vascular function and remodeling in adult offspring. Atherosclerotic vascular calcification is closely associated with the onset of cardiovascular event. We therefore investigated the impact of maternal HFD on calcification of atherogenic plaques. Methods and results Eight-week-old female apo-E−/− mice (C57BL/6) were fed an HFD or a normal diet (ND) one week prior to mating, and the diet was continued throughout gestation and lactation. Offspring of both groups were fed a high-cholesterol diet (HCD) from 8 weeks of age. Ex vivo osteogenic activity of aortic root and aortic arch was analyzed using in vivo imaging system (IVIS) with OsteoSense 680. Sixteen-week-old male offspring of HFD-fed dams (O-HFD) showed a 1.4-fold increase in fluorescent intensity compared with those of ND-fed dams (O-ND) (p&amp;lt;0.05). Likewise, female O-HFD showed a significantly increased osteogenic activity in aortic arch (154%, p&amp;lt;0.05). Percentages of plaque area and oil red O-positive area were comparable between O-ND and O-HFD of both genders, suggesting that augmented osteogenic activity in O-HFD is not dependent on the plaque size. To investigate the underlying mechanism of augmented calcified plaque formation in O-HFD, vascular smooth muscle cells (VSMCs) of thoracic aorta form 8-week-old male offspring were primarily cultured and VSMCs calcification was induced by treatment with calcification media supplemented with phosphate (2.6 mM). Alizarin-red-positive area upon 10 days stimulation showed a 3.4-fold increase in VSMCs from O-HFD compared with that from O-ND (p&amp;lt;0.01). Consistently, western blotting analysis revealed that expression level of osteocalcin was significantly higher in O-HFD than O-ND, suggesting that osteochondrocytic transformation of VSMCs is augmented in O-HFD. Conclusion Our findings demonstrate that maternal HFD accelerates the development of atherogenic calcification independent of plaque size. In vitro transformation to osteochondrocytic-like cells is enhanced in VSMCs from offspring of HFD-fed dams. Inhibition of VSMCs skewing toward osteochondrocytic-like cells could be a potential therapeutic target for preventing the development of atherosclerotic vascular calcification. Funding Acknowledgement Type of funding source: None

Effectiveness of mesenchymal stem cell-seeded onto the 3D polylactic acid/polycaprolactone/hydroxyapatite scaffold on the radius bone defect in rat

PurposeThe importance of regeneration in large bone defects forces the orthopedic surgeons to search for a proper methodology. The present experiment evaluated the capability of polylactic acid/polycaprolactone/hydroxyapatite (PLA/PCL/HA) scaffold loaded with and without mesenchymal stem cells (MSCs) on bone regeneration. MethodsFourier transform infrared spectrometry, X-ray diffraction, scanning electron microscopy, and rheology methodologies were used to characterize the scaffold. Forty Wistar rats were randomly divided into the four groups including the untreated defects as the control group and three other groups in which the bone defects were treated with autologous bones (autograft group), the PLA/PCL/HA scaffolds (PLA/PCL/HA group), and the MSCs-seeded scaffolds (MSCs-seeded PLA/PCL/HA group). ResultsBased on the qRT-PCR results, significantly higher expression levels of osteocalcin, osteopontin, and CD31 were seen in the cell-seeded scaffold group compared to the control group (P < 0.05). The CT scanning and radiographic images depicted significantly more newly formed bonny tissue in the MSCs-loaded scaffold and autograft groups than the untreated group (P < 0.001). The immunohistochemistry, biomechanical, histopathologic, and histomorphometric evaluations demonstrated significantly improved regeneration in the autograft and MSCs-loaded scaffold groups compared to the non-treated group (P < 0.05). There were significant differences between the scaffold and untreated groups in all in vivo evaluations (P < 0.05). ConclusionThe MSCs enhanced bone healing potential of the PLA/PCL/HA scaffold and the MSCs-seeded scaffold was comparable to the autograft as the golden treatment regimen (P > 0.05).