Enhanced osteogenic differentiation potential of Arnica montana and Bellis perennis in C3H10T1/2 multipotent mesenchymal stem cells.

BACKGROUND: Arnica montana and Bellis perennis are two medicinal plants that are thought to accelerate bone repair in homoeopathic literature. Mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to differentiate and regenerate bone or osteogenesis. Hence, we aimed to determine the role of Arnica montana and Bellis perennis on the osteogenic differentiation of the C3H10T1/2 stem cell line. METHODS AND RESULTS: The cell proliferation of Arnica montana and Bellis perennis was evaluated by MTT assay. Osteogenic differentiation of C3H10T1/2 was induced by the addition of ß-glycerophosphate, ascorbic acid and dexamethasone in the differentiation medium over 3 weeks. Cells were treated with Arnica montana and Bellis perennis individually as well as in combination. The osteogenic differentiation potential of Arnica montana and Bellis perennis to differentiate C3H10T1/2 into osteoblasts was measured by alkaline phosphatase activity, alizarin red staining and the expression of Osteocalcin using immunostaining and qRT-PCR. Arnica montana and Bellis perennis could enhance C3H10T1/2 cell proliferation at 1600 µg. Further, the compound showed the ability to augment osteogenesis as confirmed by increased expression of alkaline phosphatase and enhanced calcium accumulation as seen by the Alizarin Red staining and quantification. Enhanced osteogenesis was further supported by the increased expression of osteocalcin in the treated cells with individual and combined doses of Arnica montana and Bellis perennis. Therefore, the findings provide additional support for the positive impact of Arnica montana and Bellis perennis on bone formation. CONCLUSIONS: Our findings suggest that homoeopathic compounds Arnica montana and Bellis perennis can augment osteogenesis individually as well as in combination.

Osteocalcin improves glucose tolerance, insulin sensitivity and secretion in older male mice.

Osteocalcin deficient mice (OC-/-), on a mixed 129/BL6J background, were reported to show glucose intolerance, insulin insensitivity and reduced insulin secretion at 1-6 mos of age. This is controversial as two studies in OC-/- mice on different backgrounds (C3H/BL6 (5-6 mos.) and C57BL/6N (5 and 9 mos.)) found no effect on glucose metabolism. To determine the role of OC in glucose metabolism we conducted glucose tolerance tests (GTT), insulin tolerances tests (ITT) and glucose stimulated insulin secretion (GSIS) on 6 and 9.5 month-old male OC-/- and OC+/+ mice on a pure C57BL/6J background and fed a normal chow diet. All results were analyzed with a two-way repeated measures ANOVA. The GTT results showed no effect on males at 6 months of age but glucose intolerance was significantly increased (p < 0.05) in male OC-/- mice at 9.5 months of age. The ITT results indicated significantly increased insulin resistance in male OC-/- mice. Glucose stimulated insulin secretion (GSIS) showed insulin significantly (p < 0.05) reduced in OC-/- at several time points. Mouse Osteocalcin injected into OC-/- mice decreased the glucose level. Our results confirm the role of OC in glucose metabolism and insulin sensitivity and demonstrate a role in insulin secretion in older male mice on a C57BL/6J background. Differences in background, age, or experimental procedures could explain controversial results. A delayed onset of the effect of OC on glucose metabolism at 9.5 months in male C57BL/6J mice highlights the importance of background on phenotype. Consideration of genetic background and age may be beneficial for human studies on osteocalcin and glucose homeostasis and may be relevant to the elderly where osteocalcin is reduced.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

Osteocalcin of maternal and embryonic origins synergize to establish homeostasis in offspring.

Many physiological osteocalcin-regulated functions are affected in adult offspring of mothers experiencing unhealthy pregnancy. Furthermore, osteocalcin signaling during gestation influences cognition and adrenal steroidogenesis in adult mice. Together these observations suggest that osteocalcin may broadly function during pregnancy to determine organismal homeostasis in adult mammals. To test this hypothesis, we analyzed in unchallenged wildtype and Osteocalcin-deficient, newborn and adult mice of various genotypes and origin maintained on different genetic backgrounds, the functions of osteocalcin in the pancreas, liver and testes and their molecular underpinnings. This analysis revealed that providing mothers are Osteocalcin-deficient, Osteocalcin haploinsufficiency in embryos hampers insulin secretion, liver gluconeogenesis, glucose homeostasis, testes steroidogenesis in adult offspring; inhibits cell proliferation in developing pancreatic islets and testes; and disrupts distinct programs of gene expression in these organs and in the brain. This study indicates that osteocalcin exerts dominant functions in most organs it influences. Furthermore, through their synergistic regulation of multiple physiological functions, osteocalcin of maternal and embryonic origins contributes to the establishment and maintenance of organismal homeostasis in newborn and adult offspring.

Osteocalcin: Promoter or Inhibitor of Hydroxyapatite Growth?

Osteocalcin is the most abundant noncollagenous bone protein and the functions in bone remineralization as well as in inhibition of bone growth have remained unclear. In this contribution, we explain the dual role of osteocalcin in the nucleation of new calcium phosphate during bone remodeling and in the inhibition of hydroxyapatite crystal growth at the molecular scale. The mechanism was derived using pH-resolved all-atom models for the protein, phosphate species, and hydroxyapatite, along with molecular dynamics simulations and experimental and clinical observations. Osteocalcin binds to (hkl) hydroxyapatite surfaces through multiple residues, identified in this work, and the fingerprint of binding residues varies as a function of the (hkl) crystal facet and pH value. On balance, the affinity of osteocalcin to hydroxyapatite slows down crystal growth. The unique tricalcium γ-carboxylglutamic acid (Gla) domain hereby rarely adsorbs to hydroxyapatite surfaces and faces instead toward the solution. The Gla domain enables prenucleation of calcium phosphate for new bone formation at a slightly acidic pH of 5. The growth of prenucleation clusters of calcium phosphate continues upon increase in pH value from 5 to 7 and is much less favorable, or not observed, on the native osteocalcin structure at and above neutral pH values of 7. The results provide mechanistic insight into the early stages of bone remodeling from the molecular scale, help inform mutations of osteocalcin to modify binding to apatites, support drug design, and guide toward potential cures for osteoporosis and hyperosteogeny.

Platelet-Rich Plasma Lysate Enhances the Osteogenic Differentiation of Adipose-Derived Stem Cells.

ABSTRACT: Adipose-derived stem cells (ADSCs) have become an accepted source of cells in bone tissue engineering. This study aimed to investigate whether platelet-rich plasma (PRP) lysate can replace traditional fetal bovine serum as a culture medium with the enhanced proliferation and osteogenic potential of ADSCs. We divided the experiment into 5 groups where the ADSCs were cultured in an osteogenic medium containing 2.5%, 5%, 7.5%, and 10% PRP lysate with 10% fetal bovine serum as the control group. The cell proliferation, alkaline phosphatase (ALP) activity, ALP stain, alizarin red stain, osteocalcin (OCN) protein expression, and osteogenic-specific gene expression were analyzed and compared among these groups. The outcome showed that all PRP lysate-treated groups had good ALP stain and ALP activity performance. Better alizarin red stains were found in the 2.5%, 5%, and 7.5% PRP lysate groups. The 2.5% and 5% PRP lysate groups showed superior results in OCN quantitative polymerase chain reaction, whereas the 5% and 7.5% PRP lysate groups showed higher OCN protein expressions. Early RUNX2 (Runt-related transcription factor 2 () genes were the most expressed in the 5% PRP lysate group, followed by the 2.5% PRP lysate group, and then the 7.5% PRP lysate group. Thus, we concluded that 5% PRP lysate seemed to provide the optimal effect on enhancing the osteogenic potential of ADSCs. Platelet-rich plasma lysate-treated ADSCs were considered to be a good cell source for application in treating nonunion or bone defects in the future.

Polymorphisms in the Runx2 and osteocalcin genes affect BMD in postmenopausal women: a systematic review and meta-analysis.

PURPOSE: Runx2 and osteocalcin have pivotal roles in bone homeostasis. Polymorphism of these two genes could alter the function of osteoblasts and consequently bone mineral density (BMD). Attempts to understand the relationship between these polymorphisms and BMD in postmenopausal women across a variety of populations have yielded inconsistent results. This meta-analysis seeks to define the relationship between these polymorphisms with BMD in postmenopausal women. METHODS: Eligible studies were identified from three electronic databases. Data were extracted from the eligible studies (4 studies on Runx2 and 6 studies on osteocalcin), and associations of Runx2 T > C and osteocalcin HindIII polymorphisms with BMD in postmenopausal women were assessed using standard difference in means (SDM) and 95% confidence intervals (CI) as statistical measures. RESULTS: A significant difference in the lumbar spine (LS) BMD in postmenopausal women was observed between the TT and CC homozygotes for the Runx2 T > C (SDM = -0.445, p-value = 0.034). The mutant genotypes (CC) showed significantly lower LS BMD in comparison to wild type genotypes under recessive model of genetic analysis (TC + TT vs. CC: SDM = -0.451, p-value = 0.032). For osteocalcin, HindIII polymorphism, the mutant genotypes (HH) was associated with significantly higher BMD for both LS and femoral neck (FN) than the wild type (hh) homozygotes (SDM = 0.152, p-value = 0.008 and SDM = 0.139, p-value = 0.016 for LS and FN, respectively). There was no association between total hip (TH) BMD and the osteocalcin HindIII polymorphism. CONCLUSIONS: Runx2 T > C and osteocalcin HindIII polymorphisms influence the level of BMD in postmenopausal women and may be used as predictive markers of osteoporosis.

In Vitro and In Vivo Bone-Forming Effect of a Low-Molecular-Weight Collagen Peptide.

This study reveals that low-molecular-weight collagen peptide (LMWCP) can stimulate the differentiation and the mineralization of MC3T3-E1 cells in vitro and attenuate the bone remodeling process in ovariectomized (OVX) Sprague-Dawley rats in vivo. Moreover, the assessed LMWCP increased the activity of alkaline phosphatase (ALP), synthesis of collagen, and mineralization in MC3T3-E1 cells. Additionally, mRNA levels of bone metabolism-related factors such as the collagen type I alpha 1 chain, osteocalcin (OCN), osterix, bone sialoprotein, and the Runt family-associated transcription factor 2 were increased in cells treated with 1,000 µg/ml of LMWCP. Furthermore, we demonstrated that critical bone morphometric parameters exhibited significant differences between the LMWCP (400 mg/kg)-receiving and vehicle-treated rat groups. Moreover, the expression of type I collagen and the activity of ALP were found to be higher in both the femur and lumbar vertebrae of OVX rats treated with LMWCP. Finally, the administration of LMWCP managed to alleviate osteogenic parameters such as the ALP activity and the levels of the bone alkaline phosphatase, the OCN, and the procollagen type 1 N-terminal propeptide in OVX rats. Thus, our findings suggest that LMWCP is a promising candidate for the development of food-based prevention strategies against osteoporosis.

Roles of vitamin K­dependent protein in biomineralization (Review).

Vitamin K (VK), a fat­soluble vitamin, is well known as an anticoagulant in the clinic. It is essential for the post­translational activation of VK­dependent proteins (VKDPs) because hydroquinone VK is a cofactor of glutamine carboxylase. At present, 17 VKDPs are known, which are mainly involved in coagulation and calcification. When Glu residues are carboxylated to Gla residues, these proteins gain a higher calcium­binding ability, which explains why VK has an important role in blood coagulation and biomineralization. However, the current view on the role of VK and several VKDPs in biomineralization remains inconsistent. For instance, conflicting results have been reported regarding the effect of osteocalcin gene knockout on the bone of mice; matrix Gla protein (MGP) promotes osteoblasts mineralization but inhibits vascular smooth muscle cell mineralization. The present review aimed to summarize the existing evidence that several VKDPs, including osteocalcin, MGP, Gla­rich protein and growth arrest specific 6 are closely related to calcification, including bone health, vascular calcification and lithiasis. The current review discussed these controversies and provided suggestions for future studies on VKDPs, i.e. taking into account dietary habits, geographical environments and genetic backgrounds.

Mechanosignaling-related angiocrine factors drive osteoblastic phenotype in response to zirconia.

BACKGROUND: The growing use of zirconia as a ceramic material in dentistry is attributed to its biocompatibility, mechanical properties, esthetic appearance, and reduced bacterial adhesion. These favorable properties make ceramic materials a viable alternative to commonly used titanium alloys. Mimicking the physiological properties of blood flow, particularly the mechanosignaling in endothelial cells (ECs), is crucial for enhancing our understanding of their role in the response to zirconia exposure. METHODS: In this study, EC cultures were subjected to shear stress while being exposed to zirconia for up to 3 days. The conditioned medium obtained from these cultures was then used to expose osteoblasts for a duration of 7 days. To investigate the effects of zirconia on osteoblasts, we examined the expression of genes associated with osteoblast differentiation, including Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Additionally, we assessed the impact of mechanosignaling-related angiocrine factors on extracellular matrix (ECM) remodeling by measuring the activities of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) during the acquisition of the osteogenic phenotype, which precedes mineralization. RESULTS: Our data revealed that mechanosignaling-related angiocrine factors play a crucial role in promoting an osteoblastic phenotype in response to zirconia exposure. Specifically, exposed osteoblasts exhibited significantly higher expression levels of genes associated with osteoblast differentiation, such as Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Furthermore, the activities of MMP2 and MMP9, which are involved in ECM remodeling, were modulated by mechanosignaling-related angiocrine factors. This modulation is likely an initial event preceding the mineralization phase. CONCLUSION: Based on our findings, we propose that mechanosignaling drives the release of angiocrine factors capable of modulating the osteogenic phenotype at the biointerface with zirconia. This process creates a microenvironment that promotes wound healing and osseointegration. Moreover, these results highlight the importance of considering the mechanosignaling of endothelial cells in the modulation of bone healing and osseointegration in the context of blood vessel effects. Our data provide new insights and open avenues for further investigation into the influence of mechanosignaling on bone healing and the osseointegration of dental devices.

Lactoferrin-Anchored Tannylated Mesoporous Silica Nanomaterials-Induced Bone Fusion in a Rat Model of Lumbar Spinal Fusion.

Lactoferrin (LF) is a potent antiviral, anti-inflammatory, and antibacterial agent found in cow and human colostrum which acts as an osteogenic growth factor. This study aimed to investigate whether LF-anchored tannylated mesoporous silica nanomaterials (TA-MSN-LF) function as a bone fusion material in a rat model. In this study, we created TA-MSN-LF and measured the effects of low (1 µg) and high (100 µg) TA-MSN-LF concentrations in a spinal fusion animal model. Rats were assigned to four groups in this study: defect, MSN, TA-MSN-LF-low (1 µg/mL), and TA-MSN-LF-high (100 µg/mL). Eight weeks after surgery, a greater amount of radiological fusion was identified in the TA-MSN-LF groups than in the other groups. Hematoxylin and eosin staining showed that new bone fusion was induced in the TA-MSN-LF groups. Additionally, osteocalcin, a marker of bone formation, was detected by immunohistochemistry, and its intensity was induced in the TA-MSN-LF groups. The formation of new vessels was induced in the TA-MSN-LF-high group. We also confirmed an increase in the serum osteocalcin level and the mRNA expression of osteocalcin and osteopontin in the TA-MSN-LF groups. TA-MSN-LF showed effective bone fusion and angiogenesis in rats. We suggest that TA-MSN-LF is a potent material for spinal bone fusion.

Kisspeptin (Kp-10) inhibits in vitro osteogenic differentiation of multipotent mesenchymal stromal cells extracted from the bone marrow of adult rats.

Kisspeptin (Kp-10) is a neuropeptide that binds to GPR54 receptors, exerting several functions mainly in the nervous and reproductive systems of the body. However, its effects and mechanisms of action on the skeletal system remain poorly understood. This study evaluated the effects of different concentrations of Kp-10 on in vitro osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) extracted from the bone marrow (BM) of adult Wistar rats. Two-month-old female rats were euthanized to extract BM from long bones to obtain MSCs. Four experimental groups were established in vitro: a control and Kp-10 at concentrations of 0.01, 0.05 and, 0.1 µg/mL. After induction of osteogenic differentiation, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase activity, collagen synthesis, percentage of area covered by MSCs/field and mineralized nodules/field, and immunocytochemistry of the GPR54 receptor tests. Furthermore, evaluation of gene transcripts for type I collagen, Runx-2, Bmp-2, bone sialoprotein, osteocalcin and osteopontin was performed using real-time RT-qPCR. It was observed that MSCs expressed GPR54 receptor to which Kp-10 binds during osteogenic differentiation, promoting a negative effect on osteogenic differentiation. This effect was observed at all the Kp-10 concentrations in a concentration-dependent manner, characterized by a decrease in the activity of alkaline phosphatase, collagen synthesis, mineralized nodules, and decreased expression of gene transcripts for type I collagen, osteocalcin, osteopontin, and Runx-2. Thus, Kp-10 inhibits in vitro osteogenic differentiation of MSCs extracted from the BM of adult Wistar rats.

Osteonectin bidirectionally regulates osteoblast mineralization.

OBJECTIVE: The aim of this study was to investigate whether Osteonectin/Secreted protein acidic and rich in cysteine (ON/SPARC) had a two-way dose-dependent regulatory effect on osteoblast mineralization and its molecular mechanism. METHODS: Initially, different concentrations of ON were added in osteoblasts, and the gene of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) were detected using reverse-transcription quantitative polymerase chain reaction (RT-PCR). Secondly, based on the above results, the Optima and inhibitory concentration of ON for osteoblast mineralization were determined and regrouped, the Control group was also set up, and the gene detections of Collagen 1 (Col 1), Discoidin domain receptor 2 (DDR2) and p38 mitogen­activated protein kinase were added using RT-PCR. In the third stage of the experiment, osteoblasts were pretreated with 0.4Mm ethyl-3,4-dihydroxybenzoate (DHB) (a specific inhibitor of collagen synthesis) for 3 h before adding the optima SPARC, the gene and protein expressions of OCN, OPN, BSP, ALP, DDR2, ALP, Col 1, DDR2 and P38 were detected by RT­qPCR and western blot analysis, and the mineralized nodules were observed by alizarin red staining. RESULTS: The results showed that the expression of OCN, OPN, BSP, ALP, DDR2, ALP, Col 1, DDR2 and P38 genes and proteins in osteoblasts were significantly enhanced by 1 ug/ml ON, 100 ug/ml ON or 1 ug/ml ON added with 3,4 DHB significantly inhibited the expressions of DDR2, P38 and the above-mentioned mineralization indexes, and significantly reduced the formation of mineralized nodules. CONCLUSION: This study suggested that ON had a bidirectional dose-dependent regulatory effect on osteoblast mineralization, and the activation of P38 pathway by collagen binding to DDR2 was also an important molecular mechanism.

Higher weight in partially leptin-resistant db/+ mice is associated with positive effects on bone.

Absence of leptin confers metabolic dysfunction resulting in morbid obesity. Bone growth and maturation are also impaired. Partial leptin resistance is more common than leptin deficiency and, when induced by feeding mice a high fat diet, often has a negative effect on bone. Here, we used a genetic model to investigate the skeletal effects of partial and total leptin resistance in mice. This was accomplished by comparing the skeletal phenotypes of 17-week-old female C57Bl6/J wild-type (WT) mice, partial leptin receptor-deficient (db/+) mice and leptin receptor-deficient (db/db) mice (n = 7-8/group), all fed a standard diet. Compared to WT mice, db/db mice were dramatically heavier and hyperleptinemic. These mice were also hypogonadal, hyperglycemic, osteopenic and had lower serum levels of bone turnover markers, osteocalcin and C-terminal telopeptide of type I collagen (CTX). Compared to WT mice, db/+ mice were 14% heavier, had 149% more abdominal white adipose tissue, and were mildly hyperglycemic. db/+ mice did not differ from WT mice in uterine weight or serum levels of markers of bone turnover, although there was a trend for lower osteocalcin. At the bone microarchitectural level, db/+ mice differed from WT mice in having more massive femurs and a trend (P = 0.072) for larger vertebrae. These findings suggest that db/+ mice fed a normal mouse diet compensate for partial leptin resistance by increasing white adipose tissue mass which results in higher leptin levels. Our findings suggest that db/+ mice are a useful diet-independent model for studying the effects of partial leptin resistance on the skeleton.

Genetic polymorphisms of rs73620203 in the transforming growth interacting factor gene associated with rheumatoid arthritis in a Chinese population.

OBJECTIVE: To explore the association of single nucleotide polymorphisms (SNPs) in the transforming growth interacting factor (TGIF) gene with bone metabolism markers and rheumatoid arthritis (RA) susceptibility. METHODS: Three SNPs were genotyped in 155 RA patients and 168 healthy controls using high-resolution melting (HRM) analysis. The serum levels of osteocalcin, bone alkaline phosphatase (BALP), and ß type I collagen-crosslinked C telopeptide (ß-CTX) were detected using electrochemical luminescence in 108 patients randomly selected from the RA group. RESULTS: Genotype and allele frequency analysis showed that rs73620203 was associated with bone erosion in RA (P = 0.012 and P = 0.003, respectively), and individuals carrying the T allele for rs73620203 showed a decreased RA risk (OR = 0.59, 95% CI = 0.42-0.84; P = 0.003). In sex-specific analysis, the rs73620203 polymorphism was associated with susceptibility to RA in women (P = 0.022 and P = 0.006, respectively). In addition, RA patients with three genotypes at the rs73620203 locus showed significant differences in serum osteocalcin and BALP (P = 0.006 and P = 0.037, respectively). Haplotype analysis revealed that the haploid ATG and GCA frequencies were significantly lower in the RA group (P = 0.036, OR = 0.693; P = 0.002, OR = 0.189, respectively), while the haploid ACA frequency of the RA group was enhanced (P < 0.01, OR = 5.058). CONCLUSION: Our study provides the first evidence that rs73620203 is associated with RA susceptibility and the relationship between TGIF gene SNPs and the regulation of bone metabolism in RA patients.

INFLUENCE OF CHOLESTEROL ENRICHED DIET ON GENES EXPRESSION ENCODING BONE MORPHOGENETIC PROTEIN-2 AND OSTEOCALCIN IN MOUSE MANDIBLE.

OBJECTIVE: The aim: To evaluate the mRNA expression of the key regulators of osteogenesis - osteocalcin and BMP-2 in the mouse embryos mandible (17th day of pregnancy) which were borne by females on high-cholesterol diet for 30 days before fertilization and throughout pregnancy. PATIENTS AND METHODS: Materials and methods: Experimental hypercholesterolemia (2%) was simulated by adding Cholesterol to the diet for 60 days. In experiment were used 40 mature female white mice that were randomly divided to control and experimental groups. The control group were fed with standard chow diet, the experimental group with diet with cholesterol enriched diet (with addition of 2 grams of Cholesterol per 100 grams of standard chow). The mandibles of mouse embryos (E-17) were examined by using molecular genetic methods. RESULTS: Results: In control group the relative level of BMP-2 mRNA / actin mRNA was 27.0«2.82, the relative level of and osteocalcin mRNA / actin mRNA was 30.5«6,28. In the jaws of animals in the experimental group with cholesterol enriched diet, the expression relative level of BMP-2 was 30.9«5.81 that is by 14,4% higher than in control group. Therefore, the expression level of оsteocalcin, on the contrary, decreased by 22.3% and was 23.7+5.31. CONCLUSION: Conclusions: Our study report influence of the cholesterol enriched diet (2%) on mRNA expression of BMP-2 and osteocalcin encoding genes. The embryos from mouse on cholesterol enriched diet (2%) had increased level of BMP-2 gene expression, however significantly decreased level of osteocalcin gene expression.

Osteogenic stimulation of osteoprogenitors by putamen ovi peptides and hyaluronic acid.

Eggshell peptides (EP) majorly contribute to rapid bone building in chicks, wherefore this paper investigated their potential for stimulating osteogenesis in vitro. In this study, the effects of EP, also called putamen ovi peptides and a combination of hyaluronic acid with EP in cell culture medium were tested towards proliferation, differentiation, gene expression and mineralization of bovine osteoprogenitors and primary human osteoblasts. The influence of EP at concentrations of 0.005 g/L, 0.5 g/L and 0.5 g/L with 0.25% hyaluronic acid was analyzed using immunocytochemical staining of bone-specific matrix proteins, namely collagen type I, osteonectin, osteopontin and osteocalcin, to prove osteoblastic differentiation. Additionally, Richardson-staining was performed. All tests revealed a superior osteoblastic differentiation with EP at 0.5 g/L after 5 days of cultivation. Hyaluronic acid alone showed controversial results and partially constrained osteoblastic differentiation in combination with EP to a level as low as for pure EP at 0.005 g/L. Of particular interest is the osteoblast-typical mineralization, as an important indicator of bone formation, which was measured indirectly via the calcium concentration after cultivation over 4 weeks. The mineralization showed an increase by a factor of 286 during the cultivation of primary human osteoblasts with hyaluronic acid and EP. Meanwhile, cell cultures treated with EP (0.5 g/L) only showed an 80-fold increase in calcium concentration.The influence of EP (0.5 g/L) on primary human osteoblasts was investigated by gene expression after 2 weeks of cultivation. Microarray and qRT-PCR analysis showed a strongly increased expression of main important genes in bone formation, bone regeneration and the physiological bone remodelling processes. Namely, BMP 2, osteopontin and the matrix metalloproteinases 1 and 9, were present during in vitro osteoprogenitor culture with EP. By explicitly underlining the potential of eggshell peptides for stimulating osteogenesis, as well as emphasizing complex and controversial interaction with hyaluronan, this manuscript is relevant for developing new functionalized biomaterials for bone regeneration.

Considering the Value of 3D Cultures for Enhancing the Understanding of Adhesion, Proliferation, and Osteogenesis on Titanium Dental Implants.

BACKGROUND: Individuals with pathologic conditions and restorative deficiencies might benefit from a combinatorial approach encompassing stem cells and dental implants; however, due to the various surface textures and coatings, the influence of titanium dental implants on cells exhibits extensive, wide variations. Three-dimensional (3D) cultures of stem cells on whole dental implants are superior in testing implant properties and were used to examine their capabilities thoroughly. MATERIALS AND METHODS: The surface micro-topography of five titanium dental implants manufactured by sandblasting with titanium, aluminum, corundum, or laser sintered and laser machined was compared in this study. After characterization, including particle size distribution and roughness, the adhesion, proliferation, and viability of adipose-derived stem cells (ADSCs) cultured on the whole-body implants were tested at three time points (one to seven days). Finally, the capacity of the implant to induce ADSCs' spontaneous osteoblastic differentiation was examined at the same time points, assessing the gene expression of collagen type 1 (coll-I), osteonectin (osn), alkaline phosphatase (alp), and osteocalcin (osc). RESULTS: Laser-treated (Laser Mach and Laser Sint) implants exhibited the highest adhesion degree; however, limited proliferation was observed, except for Laser Sint implants, while viability differences were seen throughout the three time points, except for Ti Blast implants. Sandblasted surfaces (Al Blast, Cor Blast, and Ti Blast) outpaced the laser-treated ones, inducing higher amounts of coll-I, osn, and alp, but not osc. Among the sandblasted surfaces, Ti Blast showed moderate roughness and the highest superficial texture density, favoring the most significant spontaneous differentiation relative to all the other implant surfaces. CONCLUSIONS: The results indicate that 3D cultures of stem cells on whole-body titanium dental implants is a practical and physiologically appropriate way to test the biological characteristics of the implants, revealing peculiar differences in ADSCs' adhesion, proliferation, and activity toward osteogenic commitment in the absence of specific osteoinductive cues. In addition, the 3D method would allow researchers to test various implant surfaces more thoroughly. Integrating with preconditioned stem cells would inspire a more substantial combinatorial approach to promote a quicker recovery for patients with restorative impairments.

Effect of Insulin on Bone Formation Ability of Rat Alveolar Bone Marrow Mesenchymal Stem Cells.

The alveolar bone marrow mesenchymal stem cells (ABM-MSCs) play an important role in oral bone healing and regeneration. Insulin is considered to improve impaired oral bones due to local factors, systemic factors and pathological conditions. However, the effect of insulin on bone formation ability of ABM-MSCs still needs to be elucidated. The aim of this study was to determine the responsiveness of rat ABM-MSCs to insulin and to explore the underlying mechanism. We found that insulin promoted ABM-MSCs proliferation in a concentration-dependent manner, in which 10-6 M insulin exerted the most significant effect. 10-6 M insulin significantly promoted the type I collagen (COL-1) synthesis, alkaline phosphatase (ALP) activity, osteocalcin (OCN) expression, and mineralized matrix formation in ABM-MSCs, significantly enhanced the gene and protein expressions of intracellular COL-1, ALP, and OCN. Acute insulin stimulation significantly promoted insulin receptor (IR) phosphorylation, IR substrate-1 (IRS-1) protein expression, and mammalian target of rapamycin (mTOR) phosphorylation, but chronic insulin stimulation decreased these values, while inhibitor NT219 could attenuate these responses. When seeded on ß-tricalcium phosphate (ß-TCP), ABM-MSCs adhered and grew well, during the 28-day culture period, ABM-MSCs+ß-TCP +10-6 M insulin group showed significantly higher extracellular total COL-1 amino-terminus prolongation peptide content, ALP activity, OCN secretion, and Ca and P concentration. When implanted subcutaneously in severe combined immunodeficient mice for 1 month, the ABM-MSCs+ß-TCP +10-6 M insulin group obtained the most bone formation and blood vessels. These results showed that insulin promoted the proliferation and osteogenic differentiation of ABM-MSCs in vitro, and enhance osteogenesis and angiogenesis of ABM-MSCs in vivo. Inhibition studies demonstrated that the insulin-induced osteogenic differentiation of ABM-MSCs was dependent of insulin/mTOR signaling. It suggests that insulin has a direct anabolic effect on ABM-MSCs.

[Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells].

Objective: To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). Methods: The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. Results: The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05). Conclusion: Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.