Acacia arabica (Lam.) Willd. On osteoblastogenesis, osteoblast proliferation, osteoclastic activity, and bone calcium mineralization.

OBJECTIVES: Since ancient times Acacia arabica (Lam.) Willd. (AA) consumed for the bone and muscle related disorder like the bone fracture, rheumatoid arthritis, and bone loss. To study the effects of the aqueous (AAA) and ethanolic extract (AAE) of AA on osteoblast proliferation and differentiation, osteoclastic activity and bone matrix mineralization using in vitro primary bone-marrow cultures. METHODS: Effect of AAA and AAE was estimated using four in vitro assays. Primary bone marrow cell culture, isolated from rat femur bone, was used for all the assays. Cell growth and viability were assessed by standard colorimetric assays like MTT assay. The differentiation of mesenchymal stem cells into osteoblastic lineage was evaluated by the measuring the levels of the osteoblast-specific marker, alkaline phosphatase. Antiosteoclastic action and matrix mineralization were measured using TRAP assay and Alizarin red-s staining assay, respectively. RESULTS: It indicates that AAA causes more increase in osteoblast differentiation and a reduction in osteoclast activity as compared to AAE. In osteoblast proliferation assay, AAA was found to promote more cell proliferation as compared to AAE. Higher concentrations of AAA significantly increased mineralization of bone-like matrix. CONCLUSIONS: The extracts of AA have a significant positive influence on osteogenesis and they inhibit osteoclastogenesis. Hence, these extracts have the potential to be developed as a therapy for osteoporosis.

Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

A Neuroprotective Bovine Colostrum Attenuates Apoptosis in Dexamethasone-Treated MC3T3-E1 Osteoblastic Cells.

Glucocorticoid-induced osteoporosis (GIO) is one of the most common secondary forms of osteoporosis. GIO is partially due to the apoptosis of osteoblasts and osteocytes. In addition, high doses of dexamethasone (DEX), a synthetic glucocorticoid receptor agonist, induces neurodegeneration by initiating inflammatory processes leading to neural apoptosis. Here, a neuroprotective bovine colostrum against glucocorticoid-induced neuronal damage was investigated for its anti-apoptotic activity in glucocorticoid-treated MC3T3-E1 osteoblastic cells. A model of apoptotic osteoblastic cells was developed by exposing MC3T3-E1 cells to DEX (0-700 µM). Colostrum co-treated with DEX was executed at 0.1-5.0 mg/mL. Cell viability was measured for all treatment schedules. Caspase-3 activation was assessed to determine both osteoblast apoptosis under DEX exposure and its potential prevention by colostrum co-treatment. Glutathione reduced (GSH) was measured to determine whether DEX-mediated oxidative stress-driven apoptosis is alleviated by colostrum co-treatment. Western blot was performed to determine the levels of p-ERK1/2, Bcl-XL, Bax, and Hsp70 proteins upon DEX or DEX plus colostrum exposure. Colostrum prevented the decrease in cell viability and the increase in caspase-3 activation and oxidative stress caused by DEX exposure. Cells, upon colostrum co-treated with DEX, exhibited higher levels of p-ERK1/2 and lower levels of Bcl-XL, Bax, and Hsp70. Our data support the notion that colostrum may be able to reduce DEX-induced apoptosis possibly via the activation of the ERK pathway and modulation of the Hsp70 system. We provided preliminary evidence on how bovine colostrum, as a complex and multi-component dairy product, in addition to its neuroprotective action, may affect osteoblastic cell survival undergoing apoptosis.

Electromagnetic stimulation increases mitochondrial function in osteogenic cells and promotes bone fracture repair.

Bone fracture is a growing public health burden and there is a clinical need for non-invasive therapies to aid in the fracture healing process. Previous studies have demonstrated the utility of electromagnetic (EM) fields in promoting bone repair; however, its underlying mechanism of action is unclear. Interestingly, there is a growing body of literature describing positive effects of an EM field on mitochondria. In our own work, we have previously demonstrated that differentiation of osteoprogenitors into osteoblasts involves activation of mitochondrial oxidative phosphorylation (OxPhos). Therefore, it was reasonable to propose that EM field therapy exerts bone anabolic effects via stimulation of mitochondrial OxPhos. In this study, we show that application of a low intensity constant EM field source on osteogenic cells in vitro resulted in increased mitochondrial membrane potential and respiratory complex I activity and induced osteogenic differentiation. In the presence of mitochondrial inhibitor antimycin A, the osteoinductive effect was reversed, confirming that this effect was mediated via increased OxPhos activity. Using a mouse tibial bone fracture model in vivo, we show that application of a low intensity constant EM field source enhanced fracture repair via improved biomechanical properties and increased callus bone mineralization. Overall, this study provides supporting evidence that EM field therapy promotes bone fracture repair through mitochondrial OxPhos activation.

Skeleton interoception regulates bone and fat metabolism through hypothalamic neuroendocrine NPY.

The central nervous system regulates activity of peripheral organs through interoception. In our previous study, we have demonstrated that PGE2/EP4 skeleton interception regulate bone homeostasis. Here, we show that ascending skeleton interoceptive signaling downregulates expression of hypothalamic neuropeptide Y (NPY) and induce lipolysis of adipose tissue for osteoblastic bone formation. Specifically, the ascending skeleton interoceptive signaling induces expression of small heterodimer partner-interacting leucine zipper protein (SMILE) in the hypothalamus. SMILE binds to pCREB as a transcriptional heterodimer on Npy promoters to inhibit NPY expression. Knockout of EP4 in sensory nerve increases expression of NPY causing bone catabolism and fat anabolism. Importantly, inhibition of NPY Y1 receptor (Y1R) accelerated oxidation of free fatty acids in osteoblasts and rescued bone loss in AvilCre:Ptger4fl/fl mice. Thus, downregulation of hypothalamic NPY expression lipolyzes free fatty acids for anabolic bone formation through a neuroendocrine descending interoceptive regulation.

Ginkgonitroside, a new nitrophenyl glycoside and bioactive compounds from Ginkgo biloba leaves controlling adipocyte and osteoblast differentiation.

Eight compounds (1-8) including one novel nitrophenyl glycoside, ginkgonitroside (1) were isolated from the leaves of Ginkgo biloba, a popular medicinal plant. The structure of the new compound was characterized using extensive spectroscopic analyses via 1D and 2D NMR data interpretations, HR-ESIMS, and chemical transformation. To the best of our knowledge, the present study is the first to report the presence of nitrophenyl glycosides, which are relatively unique phytochemicals in natural products, in G. biloba. The isolated compounds (1-8) were examined for their effects on the regulation of mesenchymal stem cell (MSC) differentiation. Compounds 1-3 and 8 were able to suppress MSC differentiation toward adipocytes. In contrast, compounds 5 and 8 showed activity promoting osteogenic differentiation of MSCs. These findings demonstrate that the active compounds showed regulatory activity on MSC differentiation between adipocytes and osteocytes.

In Situ Coatings of Silver Nanoparticles for Biofilm Treatment in Implant-Retention Surgeries: Antimicrobial Activities in Monoculture and Coculture.

Bacterial biofilms are indicated in most medical device-associated infections. Treating these biofilms is challenging yet critically important for applications such as in device-retention surgeries, which can have reinfection rates of up to 80%. This in vitro study centered around our new method of treating biofilm and preventing reinfection. Ionic silver (Ag, in the form of silver nitrate) combined with dopamine and a biofilm-lysing enzyme (α-amylase) were applied to model 4-day-old Staphylococcus aureus biofilms on titanium substrates to degrade the extracellular matrix of the biofilm and kill the biofilm bacteria. In this process, the oxidative self-polymerization of dopamine converted Ag ions into Ag nanoparticles that, together with the resultant self-adhering polydopamine (PDA), formed coatings that strongly bound to the treated substrates. Surprisingly, although these Ag/PDA coatings significantly reduced S. aureus growth in standard bacterial monoculture, they showed much lower antimicrobial activity in coculture of the bacteria and osteoblastic MC3T3-E1 cells in which the bacteria were also found attached to the osteoblasts. This S. aureus- osteoblast interaction was also linked to bacterial survival against gentamicin treatment observed in coculture. Our study thus provided clear evidence suggesting that bacteria's interactions with tissue cells surrounding implants may significantly contribute to their resistance to antimicrobial treatment.

A new strategy for discovering effective substances and mechanisms of traditional Chinese medicine based on standardized drug containing plasma and the absorbed ingredients composition, a case study of Xian-Ling-Gu-Bao capsules.

ETHNOPHARMACOLOGICAL RELEVANCE: The overall therapeutic effect of traditional Chinese medicine formulae (TCMF) was achieved by the interactions of multiple components with multiple targets. However, current pharmacology research strategies have struggled to identify effective substance groups and encountered challenges in elucidating the underlying mechanisms of TCMF. AIM: In this study, a comprehensive strategy was proposed and applied to elucidate the interactions of the multiple components that underlie the functions of the famous TCMF: Xian-Ling-Gu-Bao (XLGB) capsule on bone metabolism in vivo and to elucidate the molecular mechanisms underlying the effects of XLGB on bone cells, especially on osteoblasts. METHODS: The efficacy of XLGB in the protection against bones loss in ovariectomized (OVX) rats was confirmed by Micro-CT analysis. The anti-osteoporosis mechanism involved in the systemic regulatory actions of XLGB was elucidated by transcriptome sequencing analysis on bone marrow mesenchymal stem cells isolated from OVX rats. Moreover, the components absorbed in XLGB-treated plasma were characterized by mass spectrometry analysis, and subsequently, a standardized preparation process of drug-containing plasma was established. The synergistic osteogenic effect of the multiple components in plasma was investigated by a combination and then knockout of components using pre-osteoblast MC3T3-E1 cells. In order to decipher the underlying mechanism of XLGB, the targets of the absorbed components on bone were predicted by target prediction and network pharmacology analysis, then several interactions were validated by biochemical and cell-based assay. RESULTS: A total of 18 genes, including HDC, CXCL1/2, TNF, IL6 and Il1b, were newly found to be the major target genes regulated by XLGB. Interestingly, we found that a combination of the three absorbed components, i.e. MSP, rather than their single form at the same concentration, stimulated the formation of calcified nodules in MC3T3-E1 cells, suggesting a synergistic effect of these components. Besides, target prediction and experimental validation confirmed the binding affinity of corylin and icaritin for estrogen receptor α and ß, the inhibitory activity of isobavachin and isobavachalcone on glycogen synthase kinase-3ß, and the inhibitory activity of isobavachalcone on cathepsin K. The cell-based assay further confirmed the result of the biochemical assay. A network that integrated absorbed components of XLGB-targets-perturbation genes-pathways against osteoporosis was established. CONCLUSION: Our current study provides a new systemic strategy for discovering active ingredient groups of TCM formulae and understanding their underlying mechanisms.

Magnesium in joint health and osteoarthritis.

Osteoarthritis (OA) is a prevalent debilitating age-related skeletal disease. The hallmark of OA is the degradation of articular cartilage that cushions the joint during movement. It is characterized by chronic pain and disability. Magnesium, a critical trace element in the human body, plays a pivotal role in metabolism homeostasis and the energy balance. Humans obtain magnesium mainly from the diet. However, inadequate magnesium intake is not uncommon. Moreover, the magnesium status deteriorates with ageing. There has been a growing body of clinical studies pointing to an intimate relationship between dietary magnesium and OA although the conclusion remains controversial. As reported, the magnesium ion concentration is essential to determine cell fate. Firstly, the low-concentration magnesium ions induced human fibroblasts senescence. Magnesium supplementation was also able to mitigate chondrocyte apoptosis, and to facilitate chondrocyte proliferation and differentiation. In this literature review, we will outline the existing evidence in animals and humans. We will also discuss the controversies on plasma or intracellular level of magnesium as the indicator of magnesium status. In addition, we put forward the interplay between dietary magnesium intake and intestinal microbiome to modulate the inflammatory milieu in the conjecture of OA pathogenesis. This leads to an emerging hypothesis that the synergistic effect of magnesium and probiotics may open a new avenue for the prevention and treatment of OA.

The effects of titanium topography and chemical composition on human osteoblast cell.

The objective of this study was to evaluate and compare titanium surfaces: machined (MA); sintered ceramic-blasted (HAS); sintered ceramic-blasted and acid-etched (HAS DE) and to determine the effects of surface topography, roughness and chemical composition on human osteoblast cell reaction. Titanium surface samples were analyzed with respect to surface chemical composition, topography, and roughness. The effects of material surface characteristics on osteoblasts was examined by analyzing osteoblast morphology, viability and differentiation. Osteoblasts cultured on these materials had attached, spread and proliferated on every sample. The viability of osteoblasts cultured on HAS and HAS DE samples increased more intensively in time comparing to MA sample. The viability of osteoblast cultured on HAS samples increased more intensively in the early phases of culture while for cells cultured on HAS DE the cells viability increased later in time. Alkaline phosphate activity was the highest for the cells cultured on HAS sample and statistically higher than for the MA sample. The least activity occurred on the smooth MA sample along with the rougher HAS DE samples. All the examined samples were found to be biocompatible, as indicated by cell attachment, proliferation, and differentiation. Titanium surfaces modification improved the dynamics of osteoblast viability increase. Osteoblast differentiation was found to be affected by the etching procedure and presence of Ca and P on the surface.

Omega-3 fatty acid-rich fish oil supplementation prevents rosiglitazone-induced osteopenia in aging C57BL/6 mice and in vitro studies.

Rosiglitazone is an effective insulin-sensitizer, however associated with bone loss mainly due to increased bone resorption and bone marrow adiposity. We investigated the effect of the co-administration of fish oil rich in omega-3 fatty acids (FAs) on rosiglitazone-induced bone loss in C57BL/6 mice and the mechanisms underlying potential preventive effect. Mice fed the iso-caloric diet supplemented with fish oil exhibited significantly higher levels of bone density in different regions compared to the other groups. In the same cohort of mice, reduced activity of COX-2, enhanced activity of alkaline phosphatase, lower levels of cathepsin k, PPAR-γ, and pro-inflammatory cytokines, and a higher level of anti-inflammatory cytokines were observed. Moreover, fish oil restored rosiglitazone-induced down-regulation of osteoblast differentiation and up-regulation of adipocyte differentiation in C3H10T1/2 cells and inhibited the up-regulation of osteoclast differentiation of RANKL-treated RAW264.7 cells. We finally tested our hypothesis on human Mesenchymal Stromal Cells differentiated to osteocytes and adipocytes confirming the beneficial effect of docosahexaenoic acid (DHA) omega-3 FA during treatment with rosiglitazone, through the down-regulation of adipogenic genes, such as adipsin and FABP4 along the PPARγ/FABP4 axis, and reducing the capability of osteocytes to switch toward adipogenesis. Fish oil may prevent rosiglitazone-induced bone loss by inhibiting inflammation, osteoclastogenesis, and adipogenesis and by enhancing osteogenesis in the bone microenvironment.

Limonoid Triterpene, Obacunone Increases Runt-Related Transcription Factor 2 to Promote Osteoblast Differentiation and Function.

Root bark of Dictamnus dasycarpus Turcz. has been widely used as a traditional medicine and is a well-known anti-inflammatory agent. We isolated limonoid triterpene, obacunone (Obac) from the dried root bark of D. dasycarpus. Obac has been reported to exhibit varieties of biological activities including anti-inflammatory, anti-cancer, and anti-oxidant effects. This study aimed to investigate the beneficial effects and biological mechanisms of Obac in osteoblast differentiation and bone matrix mineralization. In the present study, Obac at concentrations ranging from 1 to 100 µM showed no proliferation effects in MC3T3-E1. The treatment of Obac (1 and 10 µM) increased wound healing and migration rates in a dose-dependent manner. Alkaline phosphatase (ALP) staining and activity showed that Obac (1 and 10 µM) enhanced early osteoblast differentiation in a dose-dependent manner. Obac also increased late osteoblast differentiation in a dose-dependent manner, as indicated by the mineralized nodule formation of ARS staining. The effects of Obac on osteoblast differentiation was validated by the levels of mRNAs encoding the bone differentiation markers, including Alp, bone sialoprotein (Bsp), osteopontin (Opn), and osteocalcin (Ocn). Obac increased the expression of bone morphogenetic protein (BMP), and the phosphorylation of smad1/5/8, and the expression of runt-related transcription factor 2 (RUNX2); Obac also inhibited GSK3ß and upregulated the protein level of ß-catenin in a dose-dependent manner during osteoblast differentiation. Obac-mediated osteoblast differentiation was attenuated by a BMP2 inhibitor, Noggin and a Wnt/ß-catenin inhibitor, Dickkopf-1 (Dkk1) with the abolishment of RUNX2 expression and nuclear accumulation by Obac. Taken together, the findings of this study demonstrate that Obac has pharmacological and biological activates to promote osteoblast differentiation and bone mineralization through BMP2, ß-catenin, and RUNX2 pathways, and suggest that Obac might be a therapeutic effect for the treatment and prevention of bone diseases such as osteoporosis and periodontitis.

Pinus roxburghii alleviates bone porosity and loss in postmenopausal osteoporosis by regulating estrogen, calcium homeostasis and receptor activator of nuclear factor-κB, osteoprotegerin, cathepsin bone markers.

OBJECTIVES: The study was aimed to evaluate the potential of hydroalcoholic extract of Pinus roxburghii (PRE) stem bark in post-menopausal osteoporosis and its underlying mechanisms. METHODS: In silico docking of the markers was done using AutoDock version 4.2. for molecular targets: receptor activator of nuclear factor-κB (RANK), osteoprotegerin (OPG) and Cathepsin. Female Wistar rats of bodyweight 200-250 g were employed and surgical ovariectomy (OVX) was performed. PRE was administered at a dose of 100 and 200 mg/kg whereas standard drug, raloxifene given at 1 mg/kg orally for eight weeks. KEY FINDINGS: PRE (20 and 40 µg/mL) significantly increased the cellular proliferation in osteoblastic UMR cell lines 11.58 and 15.09% respectively. Eight weeks after surgical removal of ovaries, a significant bone porosity was confirmed by modulation in bone breaking strength of tibia, lumber, and femur; bone mineral density (BMD), calcium, phosphorus, hydroxyproline levels in OVX group. Treatment with PRE 100 and 200 mg/kg significantly restored the bone loss. Real-time polymerase chain reaction (RT-PCR) analysis of molecular markers RANK, OPG and cathepsin and histology also confirmed the attenuation of bone loss. The quantification of quercetin, gallic acid, caffeic acid, catechin, tannic acid and ascorbic acid was done by high-performance liquid chromatography (HPLC) and high performance thin layer chromatography. CONCLUSIONS: P. roxburghii produced anti-osteoporotic effect possibly due to estrogenic modulation, and improved bone remodeling.

Agastache rugosa ethanol extract suppresses bone loss via induction of osteoblast differentiation with alteration of gut microbiota.

PURPOSE: Osteoporosis is a metabolic skeletal disease characterized by bone loss and an increased risk of fractures. This study aimed to investigate the therapeutic effect of Agastache rugosa on postmenopausal osteoporosis and elucidate its mechanisms in modulating the bone status. METHODS AND RESULTS: In the osteoblast differentiation process with MC3T3-E1 pre-osteoblasts, ethanol extract of Agastache rugosa (EEAR) and its compounds increased the expression of the proteins and genes of the osteoblast differentiation-related markers such as Runt-related transcription factor 2 (RUNX2) and ß-catenin along with the elevation of calcium deposits. An ovariectomized mouse model was utilized to determine the impact of EEAR extract on postmenopausal osteoporosis. Twelve weeks of AR treatment suppressed the loss of bone strength, which was observed through micro-computed tomography. AR elevated osteogenic markers in the bone marrow cells, and collagen type 1 alpha 1 in the distal femoral bone. The results of the 16S rRNA gene sequencing analysis of cecal gut microbiomes demonstrated that AR reversed the ovariectomy-induced changes in the gut microbiomes. CONCLUSION: Ethanol extract of Agastache rugosa has a therapeutic effect on postmenopausal osteoporosis via bone morphogenic protein, transforming growth factor ß, and Wnt signaling pathway. It also increases the diversity of gut microbiota. Therefore, these data suggest that EEAR could be a potential candidate to treat postmenopausal osteoporosis.

In vivo performance of Al2O3-Ti bone implants in the rat femur.

BACKGROUND: Alumina-titanium (Al2O3-Ti) biocomposites have been recently developed with improved mechanical properties for use in heavily loaded orthopedic sites. Their biological performance, however, has not been investigated yet. METHODS: The aim of the present study was to evaluate the in vivo biological interaction of Al2O3-Ti. Spark plasma sintering (SPS) was used to fabricate Al2O3-Ti composites with 25 vol.%, 50 vol.%, and 75 vol.% Ti content. Pure alumina and titanium were also fabricated by the same procedure for comparison. The fabricated composite disks were cut into small bars and implanted into medullary canals of rat femurs. The histological analysis and scanning electron microscopy (SEM) observation were carried out to determine the bone formation ability of these materials and to evaluate the bone-implant interfaces. RESULTS: The histological observation showed the formation of osteoblast, osteocytes with lacuna, bone with lamellar structures, and blood vessels indicating that the healing and remodeling of the bone, and vasculature reconstruction occurred after 4 and 8 weeks of implantation. However, superior bone formation and maturation were obtained after 8 weeks. SEM images also showed stronger interfaces at week 8. There were differences between the composites in percentages of bone area (TB%) and the number of osteocytes. The 50Ti composite showed higher TB% at week 4, while 25Ti and 75Ti represented higher TB% at week 8. All the composites showed a higher number of osteocytes compared to 100Ti, particularly 75Ti. CONCLUSIONS: The fabricated composites have the potential to be used in load-bearing orthopedic applications.

Bioactive effects of nonthermal argon-oxygen plasma on inorganic bovine bone surface.

As a commonly used bone substitute material in the clinic, inorganic bovine bone has the characteristics of osteoconduction but not osteoinduction. This study aimed to treat inorganic bovine bone using nonthermal argon-oxygen plasma (NTAOP) to obtain greater bioreactivity for enhancing adhesion, proliferation and differentiation of mouse preosteoblast MC3T3-E1 cells. In this study, inorganic bovine bone was activated by NTAOP, and the surface characteristics were analyzed. MC3T3-E1 cells were then seeded onto the surface of inorganic bovine bone. Cell morphology, proliferation and osteogenic differentiation were examined. There was no obvious change in the surface morphology of specimens between the two groups. Regarding the elemental composition of the material, the amount of surface carbon was reduced, whereas oxygen, phosphorus and calcium levels were increased in the NTAOP group. Further studies showed that the NTAOP groups performed better than their untreated counterparts in terms of supporting cell proliferation and differentiation. Inorganic bovine bone treated with NTAOP can promote preosteoblast adhesion, proliferation and differentiation.

Effects of the amide alkaloid piperyline on apoptosis, autophagy, and differentiation of pre-osteoblasts.

BACKGROUND: Amide alkaloidsare typical constituents in plants of the Piperaceae family. Most of the pharmacological properties of Piper nigrum L. are attributed to the major amide alkaloid, piperine. Piperyline (PIPE) is a further amide alkaloid that has been isolated from P. nigrum. HYPOTHESIS/PURPOSE: This study was performed to examine the biological effects of PIPE on pre-osteoblasts and elucidate the underlying mechanisms. STUDY DESIGN: We investigated the effects of PIPE in MC3T3E-1 cells, which are widely used for studying osteoblast behavior in in vitro cell systems. METHODS: We evaluated cell viability based on the MTT assay, apoptosis by TUNEL staining, adhesion and migration by cell adhesion and migration assays, and osteoblast differentiation by alkaline phosphatase activity and staining. Western blot and immunocytochemical analyses were used to investigate cell signaling pathways. RESULTS: We found that at concentrations ranging from 1 to 30 µM, PIPE inhibited cell growth and induced apoptosis in pre-osteoblasts, which was accompanied by the upregulation of apoptotic proteins but downregulation of anti-apoptotic proteins. In contrast, PIPE had no appreciable effect on the autophagy pathway. Nevertheless, PIPE reduced cell adhesion and migration via the inactivation of non-receptor tyrosine kinase (Src)/focal adhesion kinase (FAK) and mitogen-activated protein kinases, and also promoted the downregulation of matrix metalloproteinase 2 and 9 levels. Furthermore, at concentrations of 10 and 30 µM, PIPE suppressed osteoblast differentiation, as indicated by reductions in alkaline phosphatase staining and activity. In addition, PIPE reduced the protein levels of phospho-Smad1/5/8 and runt-related transcription factor 2, and the mRNA levels of osteopontin, alkaline phosphatase, and osteocalcin. CONCLUSION: The findings of this study indicate that PIPE has biological effects associated with cell adhesion, migration, proliferation, and osteoblast differentiation, and suggest a potential role for this alkaloid in the treatment of bone diseases.

Collagen Extract Derived from Yeonsan Ogye Chicken Increases Bone Microarchitecture by Suppressing the RANKL/OPG Ratio via the JNK Signaling Pathway.

Yeonsan Ogye is a traditional Korean chicken breed (Gallus domesticus, GD), with a dominant gene for fibromelanosis, showing entirely black fluffy head feathers, ear lobes, and pupils. GD collagen extract (78.6 g per 100 g total protein) was derived from the flesh of Yeonsan Ogye. The effects of GD collagen on bone mass, microarchitecture, osteogenic, osteoclastogenic differentiations, and function factor expression were investigated in ovariectomized (OVX) rats. GD collagen stimulated osteogenesis in OVX rats and increased tibial bone strength and calcium content. Micro-computed tomography analysis of tibia cross-sections revealed that GD collagen attenuated the OVX-induced changes in trabecular thickness, spacing, and number. GD collagen stimulated alkaline phosphatase activity, bone-specific matrix proteins (alkaline phosphatase (ALP), osteocalcin, collagen type I (COL-I)) and mineralization by activating bone morphogenetic protein 2 (BMP-2)/mothers against decapentaplegic homolog 5 (SMAD5)/runt-related transcription factor 2 (Runx2). GD collagen inhibited osteoclast differentiation and function gene markers (TRAP, cathepsin K) by interfering with the Wnt signaling, increasing OPG production, and reducing the expression of RANKL, TRAP, and cathepsin K. GD collagen promoted osteogenesis by activating the p38 signal pathway and prevented osteoclastogenesis by lowering the RANKL/OPG ratio and blocking the JNK signaling pathway. Dietary supplementation with GD collagen might inhibit osteoclastogenesis, stimulate osteoblastogenesis, and regulate bone metabolism.

Bone defect reconstruction with a novel biomaterial containing calcium phosphate and aluminum oxide reinforcement.

BACKGROUND: Reconstruction of metaphyseal fractures represents a clinical challenge for orthopedic surgeons. Especially in osteoporotic bone, these fractures are frequently accompanied by osseous substance defects. In order to ensure rapid mobilization of patients, high stability requirements must be met by osteosynthesis. Various bone graft materials have been introduced in the past, such as autologous bone or exogenous bone substitute materials. These are used as bone void fillers or as augmentation techniques to ensure safe fixation of osteosynthesis. New calcium phosphate-based bone void-filling materials could be a promising alternative to autologous bone or to the currently and widely used polymethylmethacrylate (PMMA)-based cement. The aim of this study was to evaluate a novel paste-like bone void filler in vivo and in vitro with regard to biocompatibility and osteoconductivity. METHODS: In addition to in vitro testing of cell compatibility using pre-osteoblasts (MC3T3-E1), 35 Wistar rats were treated in vivo with implantation of various material mixtures based on calcium phosphate and aluminum oxide reinforcement in a metaphyseal drill hole defect. After 4 weeks, an examination by micro-computed tomography (µCT) and histology was performed. RESULTS: The in vitro analysis showed good biocompatibility with a high cell survival of osteoblasts. In the in vivo experiments, a significantly higher bone ingrowth compared to the empty defect was shown by µCT and histological analysis. Here, the group receiving material reinforced with aluminum oxide (Al2O3) showed a bone volume/tissue volume (BV/TV) of 89.19% compared to a BV/TV of 83.14% for the empty defect (p = 0.0013). In the group treated with a polysaccharide matrix, no increase in BV/TV was observed given a mean ratio of 80.14%. Scoring of histological sections did not reveal a significant difference between CaP and CaP that was substituted with Al2O3. CONCLUSION: The results of this study show an encouraging first step towards the development of new pasty, bone void-filling materials. We demonstrated that a new paste-like bone-filling material, based on calcium phosphate granulates and aluminum oxide to provide strength, exhibits good biocompatibility and osteoconductivity. Further biomechanical test in an osteoporotic animal model will have to be performed, to prove feasibility in metaphyseal defects.

Effects and Mechanisms of Exogenous Electromagnetic Field on Bone Cells: A Review.

Osteoporosis, fractures, and other bone diseases or injuries represent serious health problems in modern society. A variety of treatments including drugs, surgeries, physical therapies, etc. have been used to prevent or delay the progression of these diseases/injuries with limited effects. Electromagnetic field (EMF) has been used to non-invasively treat bone diseases, such as fracture and osteoporosis, for many years. However, because a variety of cellular and molecular events can be affected by EMF with various parameters, the precise bioeffects and underlying mechanisms of specific EMF on bone cells are still obscure. Here, we summarize the common therapeutic parameters (frequency and intensity) of major types of EMF used to treat bone cells taken from 32 papers we selected from the PubMed database published in English from 1991 to 2018. Briefly, pulse EMF promotes the proliferation of osteoblasts when its frequency is 7.5-15 Hz or 50-75 Hz and the intensity is 0.40-1.55 mT or 3.8-4 mT. Sinusoidal EMF, with 0.9-4.8 mT and 45-60 Hz, and static magnetic field with 0.1-0.4 mT or 400 mT, can promote osteoblast differentiation and maturation. Finally, we summarize the latest advances on the molecular signaling pathways influenced by EMF in osteoblasts and osteoclasts. A variety of molecules such as adenosine receptors, calcium channels, BMP2, Notch, Wnt1, etc., can be influenced by EMF in osteoblasts. For osteoclasts, EMF affects RANK, NF-κB, MAPK, etc. We speculate that EMF with different frequencies and intensities exert distinct bioeffects on specific bone cells. More high-quality work is required to explore the detailed effects and underlying mechanisms of EMF on bone cells/skeleton to optimize the application of EMF on bone diseases/injuries. Bioelectromagnetics. 2020;41:263-278 © 2020 Bioelectromagnetics Society.