ALP mRNA Research Articles

MgO-enhanced β-TCP promotes osteogenesis in both in vitro and in vivo rat models

Allogeneic bone grafts are used to treat bone defects in orthopedic surgery, but the osteogenic potential of artificial bones remains a challenge. In this study, we developed a β-tricalcium phosphate (β-TCP) formulation containing MgO, ZnO, SrO, and SiO2 and compared its bone-forming ability with that of β-TCP without biological elements. We prepared β-TCP discs with 60% porosity containing 1.0 wt% of these biological elements. β-TCP scaffolds were loaded with bone marrow-derived mesenchymal stem cells (BMSC) from 7-week-old male rats and cultured for 2 weeks. ALP activity and mRNA expression of osteogenic markers were evaluated. In addition, scaffolds were implanted subcutaneously in rats and analyzed after 7 weeks. In vitro, the MgO group showed lower Ca concentrations and higher osteogenic marker expression compared to controls. In vivo, the MgO group showed higher ALP activity compared to controls, and RT-qPCR analysis showed significant expression of BMP2 and VEGF. Histopathology, fluorescent immunostaining, and micro-CT also showed relatively better bone formation in the MgO group. β-TCP with MgO may enhance bone morphology in vitro and in vivo and improve the prognosis of patients with substantial and refractory bone defects.

Long non-coding TRPM2-AS regulates fracture healing by targeting miR-545-3p/Bmp2

ObjectiveDelayed fracture healing increases the suffering of patients. An in-depth investigation of the pathogenesis of delayed fracture healing may offer new direction for the prevention and treatment.MethodsThe study included 63 normal healing tibial fractures and 58 delayed healing tibial fractures patients. Long non-coding RNA (lncRNA)TRPM2-AS, microRNA-545-3p (miR-545-3p), bone morphogenetic protein 2 (Bmp2) mRNA and osteogenic differentiation markers, including runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), and alkaline phosphatase (Alp) mRNA expression were determined by Real-time quantitative reverse transcription-polymerase chain reaction in serum and MC3T3-E1 cells. The prediction potential of TRPM2-AS in delayed healing fracture patients was verified by receiver operating characteristic curves. The binding relationship of TRPM2-AS/miR-545-3p/Bmp2 was evaluated by dual luciferase reporter gene assay. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry.ResultsTRPM2-AS was remarkably down-regulated in patients with delayed fracture healing and could better predict the fracture healing status. TRPM2-AS downregulation inhibited osteogenic markers mRNA expression, restrained proliferation, and promoted apoptosis of MC3T3-E1 cells (p < 0.05). In delayed fracture healing, miR-545-3p was dramatically up-regulated and was negatively regulated by TRPM2-AS. Reducing miR-545-3p eliminate the negative effect of TRPM2-AS down-regulation on osteoblast proliferation and differentiation (p < 0.05). miR-545-3p targets Bmp2, which plays a positive role in osteoblast differentiation (p < 0.05).ConclusionThis study found that TRPM2-AS has the potential to be a diagnostic marker for delayed fracture healing and revealed that the TRPM2-AS/miR-545-3p/Bmp2 axis affects fracture healing by regulating osteoblast.

PPARγ inhibition promotes osteogenic differentiation of bone marrow mesenchymal stem cells and fracture healing.

This study aimed to explore the effects of peroxisome proliferator-activated receptor γ (PPARγ) inhibition on fracture healing of nonunion and the underlying mechanisms. Bone marrow mesenchymal stem cells (BMSCs) were treated with PPARγ antagonist GW9662 (5 μM, 10 μM). Alkaline phosphatase (ALP) staining and Alizarin Red S was used to assess early stage of osteogenesis and osteogenic differentiation. GW9662 (1 mg/kg/day) were administered intraperitoneally into the rats with bone fracture. Bone healing processes in the rat femur fracture model were recorded and assessed by radiographic methods on Weeks 8, 14, and 20 postoperation. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative Week 20. GW9662 treatment increased ALP activity and Alp mRNA expression in rat BMSCs. Moreover, GW9662 administration increased matrix mineralization and mRNA and protein levels of Bmp2 and Runx2 in the BMSCs. In addition, GW9662 treatment improved radiographic score in the fracture rats and increased osteogenesis-related proteins, including type I collagen, osteopontin, and osteoglycin, in the bone tissues of the fracture sites. In conclusion, PPARγ inhibition promotes osteogenic differentiation of rat BMSCs, as well as improves the fracture healing of rats through Bmp2/Runx2 signaling pathway in the rat model of bone fracture.

A sericin hydrogel scaffold for sustained dexamethasone release modulates macrophage polarization to promote mandibular bone defect repair in rats

To evaluate the efficacy of a modified sericin hydrogel scaffold loaded with dexamethasone (SMH-CD/DEX) scaffold for promoting bone defect healing by stimulating anti-inflammatory macrophage polarization. The light-curable SMH-CD/DEX scaffold was prepared using dexamethasone-loaded NH2-β-cyclodextrin (NH2-β-CD) and sericin hydrogel and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), biocompatibility assessment and drug release test. THP-1 macrophages incubated with the scaffold were examined for protein expressions of iNOS and Arg-1, mRNA expressions of IL-6, Il-10, Arg-1 and iNOS, and surface markers CD86 and CD206 using Western blotting, RT-qPCR, and flow cytometry. In a co-culture system of human periodontal ligament stem cells (HPDLSCs) and THP-1 macrophages, the osteogenic ability of the stem cells incubated with the scaffold was evaluated by detecting protein expressions of COL1A1 and Runx2 and expressions of ALP, Runx2, OCN and BMP2 mRNA, ALP staining, and alizarin red staining. In a rat model of mandibular bone defect, the osteogenic effect of the scaffold was assessed by observing bone regeneration using micro-CT and histopathological staining. In THP-1 macrophages, incubation with SMH-CD/DEX scaffold significantly enhanced protein expressions of Arg-1 and mRNA expressions of IL-10 and Arg-1 and lowered iNOS protein expression and IL-6 and iNOS mRNA expressions. In the co-culture system, SMH-CD/DEX effectively increased the protein expressions of COL1A1 and Runx2 and mRNA expressions of ALP and BMP2 in HPDLSCs and promoted their osteogenic differentiation. In the rat models, implantation of SMH-CD/DEX scaffold significantly promoted bone repair and bone regeneration in the bone defect. The SMH-CD/DEX scaffold capable of sustained dexamethasone release promotes osteogenic differentiation of stem cells and bone defect repair in rats by regulating M2 polarization.

The direct impact of pegvisomant on osteoblast functions and bone development.

Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly. We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae. We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (-30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5days post-fertilization. This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.

Structural identification of an polysaccharide isolated from Epimedium brevicornum and its beneficial effect on promoting osteogenesis in osteoblasts induced by high glucose

AimDiabetes osteoporosis (DOP) is a chronic bone metabolic disease induced by diabetes, whose morbidity continues to increase. Epimedium brevicornum Maxim (EB), a popular Chinese traditional medicine, has been used to treat bone diseases in China for thousands of years. But its material basis and specific mechanism of action are not clear. MethodsEpimedium brevicornum crude polysaccharide (EPE) is the main component, in this research the characterized the structure of EBPC1 purified from EPE was detected and its effects on cell proliferation, differentiation, and cytoskeletal in osteoblasts induced by high glucose. ResultsThe molecular weight of EBPC1 was 10.5 kDa. It was mainly comprised of glucose and galactose, and the backbone of EBPC1 was→4)-α-D-Galp-(1→4)-α-D-Galp-(1→6)-β-D-Galp-(1→6)-β-D-Galp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→. The results from in vitro experiments revealed that EBPC1 significantly increased alkaline phosphatase (ALP) activity and mineralized nodule formation in primary osteoblasts, also significantly up-regulated expression of Alp mRNA and Runx2 mRNA in the presence of EBPC1 pretreatment. Moreover, EBPC1 modulated apoptosis via the regulation of Bax/Bcl2. ConclusionThese results indicate that EBPC1 treatment can promote osteogenesis during DOP, which can ameliorate apoptosis by regulating Bax/Bcl2 and accelerating osteogenesis in osteoblasts.

Attenuative effects of collagen peptide from milkfish (Chanoschanos) scales on ovariectomy-induced osteoporosis.

Osteoporosis is characterized by low bone mass, bone microarchitecture disruption, and collagen loss, leading to increased fracture risk. In the current study, collagen peptides were extracted from milkfish scales (MS) to develop potential therapeutic candidates for osteoporosis. MS was used to synthesize a crude extract of fish scales (FS), collagen liquid (COL), and hydroxyapatite powder (HA). COL samples were further categorized according to the peptide size of total COL (0.1 mg/mL), COL < 1 kDa (0.1 mg/mL), COL: 1-10 kDa (0.1 mg/mL), and COL > 10 kDa (0.1 mg/mL) to determine it. Semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and immunofluorescence labeling were used to assess the expression levels of specific mRNA and proteins in vitro. For in vivo studies, mice ovariectomy (OVX)-induced postmenopausal osteoporosis were developed, while the sham surgery (Sham) group was treated as a control. Collagen peptides (CP) from MS inhibited osteoclast differentiation in RAW264.7 cells following an insult with nuclear factor kappa-B ligand (RANKL). CP also enhanced osteoblast proliferation in MG-63 cells, possibly through downregulating NFATc1 and TRAP mRNA expression and upregulating ALP and OPG mRNA levels. Furthermore, COL1 kDa also inhibited bone density loss in osteoporotic mice. Taken together, CP may reduce RANKL-induced osteoclast activity while promoting osteoblast synthesis, and therefore may act as a potential therapeutic agent for the prevention and control of osteoporosis.

Strontium chloride improves bone mass by affecting the gut microbiota in young male rats.

Bone mass accumulated in early adulthood is an important determinant of bone mass throughout the lifespan, and inadequate bone deposition may lead to associated skeletal diseases. Recent studies suggest that gut bacteria may be potential factors in boosting bone mass. Strontium (Sr) as a key bioactive element has been shown to improve bone quality, but the precise way that maintains the equilibrium of the gut microbiome and bone health is still not well understood. We explored the capacity of SrCl2 solutions of varying concentrations (0, 100, 200 and 400 mg/kg BW) on bone quality in 7-week-old male Wistar rats and attempted to elucidate the mechanism through gut microbes. The results showed that in a Wistar rat model under normal growth conditions, serum Ca levels increased after Sr-treatment and showed a dose-dependent increase with Sr concentration. Three-point mechanics and Micro-CT results showed that Sr exposure enhanced bone biomechanical properties and improved bone microarchitecture. In addition, the osteoblast gene markers BMP, BGP, RUNX2, OPG and ALP mRNA levels were significantly increased to varying degrees after Sr treatment, and the osteoclast markers RANKL and TRAP were accompanied by varying degrees of reduction. These experimental results show that Sr improves bones from multiple angles. Further investigation of the microbial population revealed that the composition of the gut microbiome was changed due to Sr, with the abundance of 6 of the bacteria showing a different dose dependence with Sr concentration than the control group. To investigate whether alterations in bacterial flora were responsible for the effects of Sr on bone remodeling, a further pearson correlation analysis was done, 4 types of bacteria (Ruminococcaceae_UCG-014, Lachnospiraceae_NK4A136_group, Alistipes and Weissella) were deduced to be the primary contributors to Sr-relieved bone loss. Of these, we focused our analysis on the most firmly associated Ruminococcaceae_UCG-014. To summarize, our current research explores changes in bone mass following Sr intervention in young individuals, and the connection between Sr-altered intestinal flora and potentially beneficial bacteria in the attenuation of bone loss. These discoveries underscore the importance of the "gut-bone" axis, contributing to an understanding of how Sr affects bone quality, and providing a fresh idea for bone mass accumulation in young individuals and thereby preventing disease due to acquired bone mass deficiency.

Sema3A Modified PDLSCs Exhibited Enhanced Osteogenic Capabilities and Stimulated Differentiation of Pre-Osteoblasts.

Genetically engineered stem cells, not only acting as vector delivering growth factors or cytokines but also exhibiting improved cell properties, are promising cells for periodontal tissue regeneration. Sema3A is a power secretory osteoprotective factor. In this study, we aimed to construct Sema3A modified periodontal ligament stem cells (PDLSCs) and evaluated their osteogenic capability and crosstalk with pre-osteoblasts MC3T3-E1. First, Sema3A modified PDLSCs was constructed using lentivirus infection system carrying Sema3A gene and the transduction efficiency was analyzed. The osteogenic differentiation and proliferation of Sema3A-PDLSCs was evaluated. Then, MC3T3-E1 was directly co-cultured with Sema3A-PDLSCs or cultured in condition medium of Sema3A-PDLSCs and the osteogenic ability of MC3T3-E1 was assessed. The results showed that Sema3A-PDLSCs expressed and secreted upregulated Sema3A protein, which confirmed successful construction of Sema3A modified PDLSCs. After osteogenic induction, Sema3A-PDLSCs expressed upregulated ALP, OCN, RUNX2, and SP7 mRNA, expressed higher ALP activity, and produced more mineralization nodes, compared with Vector-PDLSCs. Whereas, there was no obvious differences in proliferation between Sema3A-PDLSCs and Vector-PDLSCs. MC3T3-E1 expressed upregulated mRNA of ALP, OCN, RUNX2, and SP7 when directly co-cultured with Sema3A-PDLSCs than Vector-PDLSCs. MC3T3-E1 also expressed upregulated osteogenic markers, showed higher ALP activity, and produced more mineralization nodes when cultured using condition medium of Sema3A-PDLSCs instead of Vector-PDLSCs. In conclusion, our results indicated that Sema3A modified PDLSCs showed enhanced osteogenic capability, and also facilitated differentiation of pre-osteoblasts.

Diamond-like carbon (DLC)-coated titanium surface inhibits bacterial growth and modulates human alveolar bone cell responses in vitro

Modifying the titanium alloy (Ti-6Al-4V) surface plays a key role in improving biological responses. The present work aimed to determine the response of human alveolar bone cells to the diamond-like carbon (DLC) film and to evaluate the antibacterial effect of DLC-coated titanium. The alloys were deposited with different compositions, including C2H2 (DLC), carbon tetrafluoride (CF4:C2H2; F-DLC), and tetramethylsilane (Si(CH3)4:C2H2; Si-DLC). The surface morphology and wettability were measured using an atomic force microscopy (AFM) and a contact angle measurement. Titanium coated with DLC, F-DLC, or Si-DLC increased surface roughness and hydrophobicity. The ratio of spreading cells to initial rounded attaching cells at 20 min of all modified surfaces was higher than that of the control surfaces. F-DLC showed better cell spreading at 20 min compared to other conditions, as observed by scanning electron microscopy (SEM). The DLC, F-DLC, and Si-DLC coating significantly promoted cell proliferation compared to the Ti-6Al-4V control surfaces. ALP mRNA expression was significantly reduced on DLC surfaces compared to the control on day 14. Additionally, ALP and BMP2 mRNA expression increased on F-DLC-coated surfaces, but there were no statistically significant differences. However, DLC surfaces promoted higher mineral deposition than the control. The lower Staphylococcus aureus colony was observed on the surface of the DLC and F-DLC. In conclusion, the DLC-modified Ti-4Al-6V surface modulates cell attachment, proliferation, osteogenic marker gene expression, and bacterial growth. Herein, DLC-coated titanium alloys are still needed for continued development as an alternative biomaterial in dental applications.

Metabolic shift and the effect of mitochondrial respiration on the osteogenic differentiation of dental pulp stem cells.

Metabolism shifts from glycolysis to mitochondrial oxidative phosphorylation are vital during the differentiation of stem cells. Mitochondria have a direct function in differentiation. However, the metabolic shift and the effect of mitochondria in regulating the osteogenic differentiation of human dental pulp stem cells (hDPSCs) remain unclear. Human dental pulp stem cells were collected from five healthy donors. Osteogenic differentiation was induced by osteogenic induction medium. The activities of alkaline phosphatase, hexokinase, pyruvate kinase, and lactate dehydrogenase were analyzed by enzymatic activity kits. The extracellular acidification rate and the mitochondrial oxygen consumption rate were measured. The mRNA levels of COL-1, ALP, TFAM, and NRF1 were analyzed. The protein levels of p-AMPK and AMPK were detected by western blotting. Glycolysis decreased after a slight increase, while mitochondrial oxidative phosphorylation continued to increase when cells grew in osteogenic induction medium. Therefore, the metabolism of differentiating cells switched to mitochondrial respiration. Next, inhibiting mitochondrial respiration with carbonyl cyanide-chlorophenylhydrazone, a mitochondrial uncoupler inhibited hDPSCs differentiation with less ALP activity and decreased ALP and COL-1 mRNA expression. Furthermore, mitochondrial uncoupling led to AMPK activation. 5-Aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, simulated the effect of mitochondrial uncoupling by inhibiting osteogenic differentiation, mitochondrial biogenesis, and mitochondrial morphology. Mitochondrial uncoupling and activation of AMPK depressed mitochondrial oxidative phosphorylation and inhibited differentiation, suggesting that they may serve as regulators to halt osteogenic differentiation from impaired mitochondrial oxidative phosphorylation.

Synthesis of amide derivatives of 3-aryl-3H-benzopyrans as osteogenic agent concomitant with anticancer activity

The present study reports a series of 3-aryl-3H-benzopyran-based amide derivatives as osteogenic agents concomitant with anticancer activity. Six target compounds viz 22e, 22f, 23i, and 24b-d showed good osteogenic activity at 1 pM and 100 pM concentrations. One of the potential molecules, 24b, effectively induced ALP activity and mRNA expression of osteogenic marker genes at 1 pM and bone mineralization at 100 pM concentrations. These molecules also presented significant growth inhibition of osteosarcoma (MG63) and estrogen-dependent and -independent (MCF-7 and MDA-MB-231) breast cancer cells. The most active compound, 24b, inhibited the growth of all the cancer cells within the IC50 10.45–12.66 µM. The mechanistic studies about 24b showed that 24b induced apoptosis via activation of the Caspase-3 enzyme and inhibited cancer cell migration. In silico molecular docking performed for 24b revealed its interaction with estrogen receptor-β (ER-β) preferentially.

Effects of a Functional Ice Cream Enriched with Milk Proteins on Bone Metabolism: A Feasibility Clinical Study and In Vitro Investigation.

Background: Milk proteins (MPs) and their derivative whey proteins (WPs) are important components of human diet that might prevent bone loss. We aimed to investigate the effects of MP on the bones of postmenopausal women, along with the effects of WP on osteoblast cells. Methods: We conducted a feasibility controlled clinical study with 62 postmenopausal women who were asked to consume an MP-enriched ice cream. We also investigated the effect of WP on the ERK1/2 and AKT pathways, RUNX2, alkaline phosphatase, RANKL/OPG ratio, and COL1A of Saos-2. Results: After 12 weeks, we found a greater bone mineral density and bone alkaline phosphatase reduction in women who consumed the MP-enriched ice cream compared to the control group (p = 0.03 and p = 0.02, respectively). In Saos-2 cells, WP upregulated ERK1/2 and AKT pathways (p = 0.002 and p = 0.016), cell proliferation (p = 0.03), and osteoblast differentiation markers, along with downregulating RANKL/OPG (p &lt; 0.001). Moreover, the inhibition of ERK1/2 by PD184253 reverted the effects on both the RUNX2 and ALP mRNA expression and cells proliferation (p = 0.028, p = 0.004, and p = 0.003, respectively) when treated with WP. Conclusions: WP upregulates cell proliferation, RUNX2, and alkaline phosphatase through the activation of the ERK1/2 pathways on Saos-2. These mechanisms probably contribute to preventing bone loss in postmenopausal women.

Comprehensive analysis of M2 macrophage-derived exosomes facilitating osteogenic differentiation of human periodontal ligament stem cells

BackgroundThe role of periodontal ligament stem cells (PDLSCs) and macrophage polarization in periodontal tissue regeneration and bone remodeling during orthodontic tooth movement (OTM) has been well documented. Nevertheless, the interactions between macrophages and PDLSCs in OTM remain to be investigated. Consequently, the present study was proposed to explore the effect of different polarization states of macrophages on the osteogenic differentiation of PDLSCs.MethodsAfter M0, M1 and M2 macrophage-derived exosomes (M0-exo, M1-exo and M2-exo) treatment of primary cultured human PDLSCs, respectively, mineralized nodules were observed by Alizarin red S staining, and the expression of ALP and OCN mRNA and protein were detected by RT-qPCR and Western blotting, correspondingly. Identification of differentially expressed microRNAs (DE-miRNA) in M0-exo and M2-exo by miRNA microarray, and GO and KEGG enrichment analysis of DE-miRNA targets, and construction of protein–protein interaction networks.ResultsM2-exo augmented mineralized nodule formation and upregulated ALP and OCN expression in PDLSCs, while M0-exo had no significant effect. Compared to M0-exo, a total of 52 DE-miRNAs were identified in M2-exo. The expression of hsa-miR-6507-3p, hsa-miR-4731-3p, hsa-miR-4728-3p, hsa-miR-3614-5p and hsa-miR-6785-3p was significantly down-regulated, and the expression of hsa-miR-6085, hsa-miR-4800-5p, hsa-miR-4778-5p, hsa-miR-6780b-5p and hsa-miR-1227-5p was significantly up-regulated in M2-exo compared to M0-exo. GO and KEGG enrichment analysis revealed that the downstream targets of DE-miRNAs were mainly involved in the differentiation and migration of multiple cells.ConclusionsIn summary, we have indicated for the first time that M2-exo can promote osteogenic differentiation of human PDLSCs, and have revealed the functions and pathways involved in the DE-miRNAs of M0-exo and M2-exo and their downstream targets.

Kefir peptides promote osteogenic differentiation to enhance bone fracture healing in rats

AimsFractures are the result of fragile bone structures after trauma caused by direct or indirect external impact or strong muscular contraction. Most fracture patients undergo surgical fixation to accelerate the healing process and restore the function of mutilated bone. Promoting the healing process remains an important issue for the treatment of bone fractures. Our previous studies demonstrated the remarkable bone-protective effects of kefir peptides (KPs) in ovariectomized rats and mice. In this study, we further evaluate the efficacy of KPs on fracture healing using a rat model of femoral fracture. Main methodsFifteen 8-week-old male Sprague Dawley (SD) rats were divided into the sham, mock, and KPs groups, in which the mock and the KPs groups underwent femur-fracture surgery with nail fixation, while the sham group underwent a sham operation. The next day, rats were orally administered with daily 400 mg/kg of KPs (KPs group) or distilled water (sham and mock groups) for four weeks. X-ray imaging, histochemical staining and serum osteogenic markers were applied for fracture healing evaluation. In vitro, mouse bone marrow mesenchymal stem cells (BMMSCs) and MC3T3-E1 line were subjected to osteoblast differentiation in the presence of KPs and compared with no KPs treatment. Key findingsThe results demonstrated that KPs treatment improved the progression of the fracture healing process (p < 0.05) and significantly increased the expressions of Col1a1, Alp, Spp1, Vegfa and Cox2 mRNA in the femurs of the KPs-treated fractured rats compared to those of the mock-treated fracture rats. In vitro, KPs treatment promoted bone regeneration factor (Col1a1, Alp, M-csf and Phospho1) expression in MC3T3-E1-derived osteoblast cultures (on Day 3) and enhanced osteogenic differentiation and mineralization in BMMSC-derived osteoblast cultures (on Day 17 and Day 21). SignificanceThis is the first study to show that KPs can help with fracture healing by promoting osteogenic differentiation, and it also suggests that KPs can be used as a nutritional supplement to accelerate fracture healing.

Effects of rmBMP-7 on Osteoblastic Cells Grown on a Nanostructured Titanium Surface

This study evaluates the effects of the availability of exogenous BMP-7 on osteoblastic cells’ differentiation on a nanotextured Ti surface obtained by chemical etching (Nano-Ti). The MC3T3-E1 and UMR-106 osteoblastic cell lines were cultured for 5 and 7 days, respectively, on a Nano-Ti surface and on a control surface (Control-Ti) in an osteogenic medium supplemented with either 40 or 200 ng/mL recombinant mouse (rm) BMP-7. The results showed that MC3T3-E1 cells exhibited distinct responsiveness when exposed to each of the two rmBMP-7 concentrations, irrespective of the surface. Even with 40 ng/mL rmBMP-7, important osteogenic effects were noticed for Control-Ti in terms of cell proliferation potential; Runx2, Osx, Alp, Bsp, Opn, and Smad1 mRNA expression; and in situ ALP activity. For Nano-Ti, the effects were limited to higher Alp, Bsp, and Opn mRNA expression and in situ ALP activity. On both surfaces, the osteogenic potential of UMR-106 cultures remained unaltered with 40 ng/mL rmBMP-7, but it was significantly reduced when the cultures were exposed to the 200 ng/mL concentration. The availability of rmBMP-7 to pre-osteoblastic cells at the concentrations used alters the expression profile of osteoblast markers, indicative of the acquisition of a more advanced stage of osteoblastic differentiation. This occurs less pronouncedly on the nanotextured Ti and without reflecting in higher mineralized matrix production by differentiated osteoblasts on both surfaces.

Therapeutic Potential of Liraglutide for Diabetes-Periodontitis Comorbidity: Killing Two Birds with One Stone.

Background The relationship between diabetes and periodontitis is bidirectional, and there is now consensus that periodontitis and diabetes are comorbid. There is a quest for a drug that can be used to treat both conditions simultaneously. This study evaluated the anti-inflammatory and osteoprotective effects of liraglutide (LIRA) on periodontitis in diabetic rats. Methods Male Wistar rats (n = 46) were randomly divided into four groups: control group (n = 8), LIRA group (n = 8), diabetes-associated periodontitis+0.9% saline group (diabetic periodontitis (DP)+NaCl group, n = 15), and diabetes-associated periodontitis+LIRA group (DP+LIRA group, n = 15). LIRA treatment lasted for 4 weeks (300 μg/kg/d) after establishment of a rat model of DP. The expression of IL-6, TNF-α, and IL-1β was detected by enzyme-linked immunosorbent assay. The morphological changes of periodontal tissues were observed by hematoxylin–eosin staining. The absorption of alveolar bone and its ultrastructural changes were observed by histomorphometry and microcomputed tomography. The expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in alveolar bone was detected by immunohistochemistry. The levels of Runx2 mRNA and ALP mRNA in the gingival epithelium were examined by quantitative real-time polymerase chain reaction. Results LIRA decreased alveolar bone resorption, improved the microstructure of alveolar bone, and reduced periodontal inflammation and damage (P < 0.05). LIRA also reduced blood glucose level and inhibited the secretion of serum IL-6, TNF-α, and IL-1β (P < 0.05). In addition, after treatment with LIRA, the ratio of RANKL/OPG was reduced, and the expression levels of ALP mRNA and Runx2 mRNA were upregulated (P < 0.05). Conclusions LIRA not only controls blood glucose level but also reduces inflammation and bone loss and enhances osteogenic differentiation in diabetes-associated periodontitis. Those indicate that LIRA may be used as a potential medicine for the adjunctive therapy of diabetes-periodontitis comorbidity.

EPLINβ Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINβ. In this study, we investigated the role of EPLINβ in osteoblasts using EPLINβ-deficient (EPLINβGT/GT) mice. The skeletal phenotype of EPLINβGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINβGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINβGT/GT mice, suggesting aberrant cell adhesion. In EPLINβGT/GT osteoblasts, α- and β-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINβ knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINβ knockdown cells, suggesting an important role for EPLINβ in osteoblast formation. In conclusion, we propose that EPLINβ is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

Effect of miR-34a on the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs) in Hyperlipidemia Rats

This study intends to explore miR-34a’s effect on BMSCs osteogenic differentiation of hyperlipidemia. 20 SD rats were equally assigned into control group (normal diet) and high-fat group (research diet). Cells were transfected with miR-34a mimic or negative control followed by meausring miR-34a, Dvl2, PPAR-γ, ALP, Runx2, and sp7, and ALP activity. The number of mineralized nodules, adipocytes, miR-34a and PPAR-γ expression in high-fat group were significantly increased and Dvl2, ALP, Runx2, and sp7 mRNA showed opposite profiles. Meanwhile, Runx2, ALP protein, cytoplasm and nuclear blue-black particles, Dvl2 protein and mRNA in miR-34a mimic group were significantly downregulated (P &lt; 0.05). Additionally, the luciferase activity of wild-type plasmid+miR-34a mimic group was significantly lower than mutant group, indicating that miR-34a targets Dvl2. In conclusion, miR-34a inhibits the osteogenic differentiation of hyperlipidemia BMSCs by inhibiting the expression of Dvl2, Runx2 and ALP activity, indicating that it might be target in the hyperlipidemia treatment.

Application of RUNX2 gene over expression vector modified exosomes from BMSC combined with calcium carbonate scaffold system in bone defect

To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect. Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining. The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05). Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.