Differentiation Of Osteoblastic Cells Research Articles

Development of nanoparticles for delivering recombinant human secretory leukocyte protease inhibitor (rhSLPI) for enhancing human osteoblast differentiation

The global number of elderly people has been increasing and the world can now be considered as an aging society. One of the most concerning health problems in the elderly is a musculoskeletal disease, in which fractures and osteoporosis are the most common problems. The deteriorating bone quality in the elderly is a limitation for treatment and for patient recovery. Several drugs and biomolecules have been studied for enhancing osteoblast properties and differentiation. The secretory leukocyte protease inhibitor (SLPI), which is a serine protease inhibitory protein, has been reported to enhance osteoblast cell adhesion, proliferation, and differentiation. However, the application of SLPI in real clinical settings is limited due to its short half-life in circulation and the fact that it can be destroyed by circulating protease enzymes. Therefore, the application of nanoparticle encapsulation might be of benefit for SLPI delivery. This study aims to fabricate the liposome nanoparticles encapsulating recombinant human SLPI or rhSLPI (rhSLPI-LNPs) for augmenting the half-life and enhancing human osteoblast differentiation. The liposome nanoparticles (LNPs) were fabricated by the thin film hydration method. Encapsulation of rhSLPI was performed by adding 0.33 µg/mL rhSLPI in ultrapure water to the thin film lipid and then rhSLPI-LNPs formed into vesicles. The size and zeta potential of blank-LNPs or rhSLPI-LNPs were measured by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The results showed that the size of both blank-LNPs and rhSLPI-LNPs was approximately 200 nm, and the zeta potentials were −55.6 ± 16.90 mV and −64 ± 7.86 mV, respectively. The rhSLPI encapsulation was confirmed by ELISA. The results showed that liposome nanoparticles could encapsulate rhSLPI. For cytotoxicity, rhSLPI-LNPs treatment did not cause any toxicity to the human osteoblast cell line (hFOB 1.19). In addition, pre-incubation of hFOB 1.19 with rhSLPI-LNPs could significantly enhance osteoblast adhesion when compared with the untreated group.

Activating Wnt/β-catenin signaling by autophagic degradation of APC contributes to the osteoblast differentiation effect of soy isoflavone on osteoporotic mesenchymal stem cells.

The functional role of autophagy in regulating differentiation of bone marrow mesenchymal stem cells (MSCs) has been studied extensively, but the underlying mechanism remains largely unknown. The Wnt/β-catenin signaling pathway plays apivotal role in the initiation of osteoblast differentiation of mesenchymal progenitor cells, and the stability of core protein β-catenin is tightly controlled by the APC/Axin/GSK-3β/Ck1α complex. Here we showed that genistein, a predominant soy isoflavone, stimulated osteoblast differentiation of MSCs in vivo and in vitro. Female rats were subjected to bilateral ovariectomy (OVX); four weeks after surgery the rats were orally administered genistein (50 mg·kg-1·d-1) for 8 weeks. The results showed that genistein administration significantly suppressed the bone loss and bone-fat imbalance, and stimulated bone formation in OVX rats. In vitro, genistein (10 nM) markedly activated autophagy and Wnt/β-catenin signaling pathway, and stimulated osteoblast differentiation in OVX-MSCs. Furthermore, we found that genistein promoted autophagic degradation of adenomatous polyposis coli (APC), thus initiated β-catenin-driven osteoblast differentiation. Notably, genistein activated autophagy through transcription factor EB (TFEB) rather than mammalian target of rapamycin (mTOR). These findings unveil the mechanism of how autophagy regulates osteogenesis in OVX-MSCs, which expands our understanding that such interplay could be employed as a useful therapeutic strategy for treating postmenopausal osteoporosis.

Effects of Cocoa Pod Husk Extract (Theobroma Cacao L.) on Alveolar Bone in Experimental Periodontitis Rats

Periodontitis is a destructive inflammatory disease of the periodontal tissues with a high prevalence in the world. Phorphyromonas gingivalis as one of the main bacteria causing periodontitis produces virulence factors that trigger immune cells to produce proinflammatory cytokines that can reduce the number of osteoblasts, thereby triggering alveolar bone resorption. Osteoblast cell differentiation is mediated by a number of growth factors such as cytokines and bone morphogenic proteins (BMPs). SRP mechanical therapy is sometimes not optimal and Metronidazole as an effective antibiotic against periodontitis has been reported to have several adverse effects. Utilization of cocoa pod waste (Theobroma cacao L) with its polyphenol content has antibacterial, antioxidant and anti-inflammatory effects. This study aims to determine the effect of ethanol extract gel from cocoa pod husk extract (Theobroma cacao L.) on the number of osteoblasts and osteoclasts, as well as the expression of BMP-2 in the alveolar bone of periodontitis-model rats. This laboratory experimental study used 24 rats which were divided into negative control group (CMC-Na gel), positive control group (metronidazole gel), and treatment group (100 mg/mL cocoa pod husk extract gel). 24 Wistar rats were divided into 3 groups which were observed on days 7 and 14. The number of osteoblasts and osteoclasts of alveolar bone were observed by hematoxillin eosin staining and BMP-2 expression by immunohistochemistry staining. One-way ANOVA test showed that there was a significant difference (p < 0.05) between groups. The LSD test average of BMP-2 expression in the treatment group on the day 7 and day 14 was higher than the negative control group (p < 0.05). The conclusion of the study was that giving 100 mg/mL of cacao pod husk extract gel (Theobroma cacao L.) increased the number of osteoblasts and increased the expression of BMP-2 in the alveolar bone of periodontitis rats. HIGHLIGHTS Cocoa pod husk extract gel (Theobroma cacao) 100 mg/mL increased the ratio of osteoblasts to osteoclasts and increased BMP-2 expression in the alveolar bone of periodontitis rats The histological staining with IHC was analyzed using Quantitative Pathology 0.3.2 software The cocoa pod husk (Theobroma Cacao L.) extract gel effectively triggers and increased BMP-2 which stimulates bone formation in periodontitis rat models GRAPHICAL ABSTRACT

Litsea glutinosa extract promotes fracture healing and prevents bone loss via BMP2/SMAD1 signaling.

Estrogen deficiency is one of the main causes for postmenopausal osteoporosis. Current osteoporotic therapies are of high cost and associated with serious side effects. So there is an urgent need for cost-effective anti-osteoporotic agents. Anti-osteoporotic activity of Litsea glutinosa extract (LGE) is less explored. Moreover, its role in fracture healing and mechanism of action is still unknown. In the present study we explore the osteoprotective potential of LGE in osteoblast cells and fractured and ovariectomized (Ovx) mice models. Alkaline phosphatase (ALP), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and mineralization assays revealed that LGE treatment increased osteoblast cell differentiation, viability and mineralization. LGE treatment at 0.01 μg increased the expression of BMP2, PSMAD, RUNX2 and type 1 col. LGE also mitigated RANKL-induced osteoclastogenesis. Next, drill hole injury Balb/C mice model was treated with LGE for 12 days. Micro-CT analysis and Calcein labeling at the fracture site showed that LGE (20 mg/kg) enhanced new bone formation and bone regeneration, also increased expression of BMP2/SMAD1 signaling genes at fracture site. Ovx mice were treated with LGE for 1 month. μCT analysis indicated that the treatment of LGE at 20 mg/kg dose prevented the alteration in bone microarchitecture and maintained bone mineral density and bone mineral content. Treatment also increased bone strength and restored the bone turnover markers. Furthermore, in bone samples, LGE increased osteogenesis by enhancing the expression of BMP2/SMAD1 signaling components and decreased osteoclast number and surface. We conclude that LGE promotes osteogenesis via modulating the BMP2/SMAD1 signaling pathway. The study advocates the therapeutic potential of LGE in osteoporosis treatment.

Coupling BCP Ceramics with Micro‐Vibration Stimulation Field for Cascade Amplification from Immune Activation to Bone Regeneration

AbstractThe osteoimmunology has revealed that immune system plays an important role in maintaining bone metabolism and remodeling. As long‐term physiological factor in bone, mechanical stimulation such as micro‐vibration stimulation (MVS) exerts effects on regulating osteogenesis and immune response. In this study, the osteo‐immunodulatory effects of bicalcium phosphate (BCP) ceramics coupled with MVS are investigated. This results find that the combination of BCP ceramics and MVS may exert synergistic effects on the polarization and functional status of macrophages through activating plasma membrance Ca2+ ATPase (PMCA) channel, reducing the intracellular calcium ion concentration, and inhibiting downstream extracellular signal‐regulated kinase (ERK)1/2 signaling pathway. BCP ceramics coupled MVS could drive the macrophage polarization to wound‐healing M2 phenotype to decrease the production of pro‐inflammatory factors, enhance the secretion of anti‐inflammatory cytokines and growth factors such as transforming growth factor (TGF)‐β1 and bone morphogenetic protein (BMP)‐2. Moreover, BCP and MVS‐modulated macrophage secretion pattern can trigger the BMP/TGF‐Smad signaling pathways to induce osteoblastic differentiation of bone marrow mesenchymal stromal cells (BM‐MSCs) in vitro, and maintain cellular viability and promote the formation of collagen‐rich osteoid like tissues and mature blood vessels in vivo. This study demonstrates that the introduction of mechanical stimuli like non‐invasive MVS is an effective strategy to improve bone repair effects of biomaterials through endowing them with superior osteo‐immunodulatory capacity.

Mangiferin-Enriched Mn-Hydroxyapatite Coupled with β-TCP Scaffolds Simultaneously Exhibit Osteogenicity and Anti-Bacterial Efficacy.

Biphasic calcium phosphate (BCP) containing β-tricalcium phosphate and manganese (Mn)-substituted hydroxyapatite (HAP) was synthesized. Biomedical scaffolds were prepared using this synthesized powder on a sacrificial polyurethane sponge template after the incorporation of mangiferin (MAN). Mn was substituted at a concentration of 5% and 10% in HAP to examine the efficacy of Mn at various concentrations. The phase analysis of the as-formed BCP scaffold was carried out by X-ray diffraction analysis, while the qualitative observation of morphology and the osteoblast cell differentiation were carried out by scanning electron microscopy and confocal laser scanning microscopy techniques. Gene expressions of osteocalcin, collagen 1, and RUNX2 were carried out using qRT-PCR analyses. Significantly higher (p < 0.05) levels of ALP activity were observed with extended osteoblast induction on the mangiferin-incorporated BCP scaffolds. After characterization of the specimens, it was found that the scaffolds with 10% Mn-incorporated BCP with mangiferin showed better osteogenicity and simultaneously the same scaffolds exhibited higher anti-bacterial properties as observed from the bacterial viability test. This study was carried out to evaluate the efficacy of Mn and MAN in BCP for osteogenicity and antibacterial action.

Bone marrow stromal cells generate an osteoinductive microenvironment when cultured on titanium–aluminum–vanadium substrates with biomimetic multiscale surface roughness

Osseointegration of titanium-based implants possessing complex macroscale/microscale/mesoscale/nanoscale (multiscale) topographies support a direct and functional connection with native bone tissue by promoting recruitment, attachment and osteoblastic differentiation of bone marrow stromal cells (MSCs). Recent studies show that the MSCs on these surfaces produce factors, including bone morphogenetic protein 2 (BMP2) that can cause MSCs not on the surface to undergo osteoblast differentiation, suggesting they may produce an osteogenic environment in vivo. This study examined if soluble factors produced by MSCs in contact with titanium–aluminum–vanadium (Ti6Al4V) implants possessing a complex multiscale biomimetic topography are able to induce osteogenesis ectopically. Ti6Al4V disks were grit-blasted and acid-etched to create surfaces possessing macroscale and microscale roughness (MM), micro/meso/nanoscale topography (MN), and macro/micro/meso/nanoscale topography (MMNTM). Polyether-ether-ketone (PEEK) disks were also fabricated by machining to medical-grade specifications. Surface properties were assessed by scanning electron microscopy, contact angle, optical profilometry, and x-ray photoelectron spectroscopy. MSCs were cultured in growth media (GM). Proteins and local factors in their conditioned media (CM) were measured on days 4, 8, 10 and 14: osteocalcin, osteopontin, osteoprotegerin, BMP2, BMP4, and cytokines interleukins 6, 4 and 10 (IL6, IL4, and IL10). CM was collected from D14 MSCs on MMNTM and tissue culture polystyrene (TCPS) and lyophilized. Gel capsules containing active demineralized bone matrix (DBM), heat-inactivated DBM (iDBM), and iDBM + MMN-GM were implanted bilaterally in the gastrocnemius of athymic nude mice (N = 8 capsules/group). Controls included iDBM + GM; iDBM + TCPS-CM from D5 to D10 MSCs; iDBM + MMN-CM from D5 to D10; and iDBM + rhBMP2 (R&D Systems) at a concentration similar to D5–D10 production of MSCs on MMNTM surfaces. Legs were harvested at 35D. Bone formation was assessed by micro computed tomography and histomorphometry (hematoxylin and eosin staining) with the histology scored according to ASTM 2529–13. DNA was greatest on PEEK at all time points; DNA was lowest on MN at early time points, but increased with time. Cells on PEEK exhibited small changes in differentiation with reduced production of BMP2. Osteoblast differentiation was greatest on the MN and MMNTM, reflecting increased production of BMP2 and BMP4. Pro-regenerative cytokines IL4 and IL10 were increased on Ti-based surfaces; IL6 was reduced compared to PEEK. None of the media from TCPS cultures was osteoinductive. However, MMN-CM exhibited increased bone formation compared to iDBM and iDBM + rhBMP2. Furthermore, exogenous rhBMP2 alone, at the concentration found in MMN-CM collected from D5 to D10 cultures, failed to induce new bone, indicating that other factors in the CM play a critical role in that osteoinductive microenvironment. MSCs cultured on MMNTM Ti6Al4V surfaces differentiate and produce an increase in local factors, including BMP2, and the CM from these cultures can induce ectopic bone formation compared to control groups, indicating that the increased bone formation arises from the local response by MSCs to a biomimetic, multiscale surface topography.

Atf7ip Inhibits Osteoblast Differentiation via Negative Regulation of the Sp7 Transcription Factor

Epigenetic modifications are critical for cell differentiation and growth. As a regulator of H3K9 methylation, Setdb1 is implicated in osteoblast proliferation and differentiation. The activity and nucleus localization of Setdb1 are regulated by its binding partner, Atf7ip. However, whether Atf7ip is involved in the regulation of osteoblast differentiation remains largely unclear. In the present study, we found that Atf7ip expression was upregulated during the osteogenesis of primary bone marrow stromal cells and MC3T3-E1 cells, and was induced in PTH-treated cells. The overexpression of Atf7ip impaired osteoblast differentiation in MC3T3-E1 cells regardless of PTH treatment, as measured by the expression of osteoblast differentiation markers, Alp-positive cells, Alp activity, and calcium deposition. Conversely, the depletion of Atf7ip in MC3T3-E1 cells promoted osteoblast differentiation. Compared with the control mice, animals with Atf7ip deletion in the osteoblasts (Oc-Cre;Atf7ipf/f) showed more bone formation and a significant increase in the bone trabeculae microarchitecture, as reflected by μ-CT and bone histomorphometry. Mechanistically, Atf7ip contributed to the nucleus localization of Setdb1 in MC3T3-E1, but did not affect Setdb1 expression. Atf7ip negatively regulated Sp7 expression, and through specific siRNA, Sp7 knockdown attenuated the enhancing role of Atf7ip deletion in osteoblast differentiation. Through these data, we identified Atf7ip as a novel negative regulator of osteogenesis, possibly via its epigenetic regulation of Sp7 expression, and demonstrated that Atf7ip inhibition is a potential therapeutic measure for enhancing bone formation.

Bioactive Composite Methacrylated Gellan Gum for 3D-Printed Bone Tissue-Engineered Scaffolds

Gellan gum (GG) was chemically modified with methacrylic moieties to produce a photocrosslinkable biomaterial ink, hereinafter called methacrylated GG (GGMA), with improved physico-chemical properties, mechanical behavior and stability under physiological conditions. Afterwards, GGMA was functionalized by incorporating two different bioactive compounds, a naturally derived eumelanin extracted from the black soldier fly (BSF-Eumel), or hydroxyapatite nanoparticles (HAp), synthesized by the sol-gel method. Different ink formulations based on GGMA (2 and 4% (w/v)), BSF-Eumel, at a selected concentration (0.3125 mg/mL), or HAp (10 and 30% wHAp/wGGMA) were developed and processed by three-dimensional (3D) printing. All the functionalized GGMA-based ink formulations allowed obtaining 3D-printed GGMA-based scaffolds with a well-organized structure. For both bioactive signals, the scaffolds with the highest GGMA concentration (4% (w/v)) and the highest percentage of infill (45%) showed the best performances in terms of morphological and mechanical properties. Indeed, these scaffolds showed a good structural integrity over 28 days. Given the presence of negatively charged groups along the eumelanin backbone, scaffolds consisting of GGMA/BSF-Eumel demonstrated a higher stability. From a mechanical point of view, GGMA/BSF-Eumel scaffolds exhibited values of storage modulus similar to those of GGMA ones, while the inclusion of HAp at 30% (wHAp/wGGMA) led to a storage modulus of 32.5 kPa, 3.5-fold greater than neat GGMA. In vitro studies proved the capability of the bioactivated 3D-printed scaffolds to support 7F2 osteoblast cell growth and differentiation. BSF-Eumel and HAp triggered a different time-dependent physiological response in the osteoblasts. Specifically, while the ink with BSF-Eumel acted as a stimulus towards cell proliferation, reaching the highest value at 14 days, a higher expression of alkaline phosphatase activity was detected for scaffolds consisting of GGMA and HAp. The overall findings demonstrated the possible use of these biomaterial inks for 3D-printed bone tissue-engineered scaffolds.

Conditioned medium of PC‑3 prostate cancer cells affects microRNA and mRNA profiles in mechanically strained osteoblasts.

Bone is the main site of metastasis from prostate cancer; therefore, it is important to investigate the microRNAs (miRNAs) and mRNA associated with bone metastases from prostate cancer. Since an appropriate mechanical environment is important in the growth of bone, in the present study, the miRNA, mRNA, and long non-coding RNA (lncRNA) profiles of mechanically strained osteoblasts treated with conditioned medium (CM) from PC-3 prostate cancer cells were studied. MC3T3-E1 osteoblastic cells were treated with the CM of PC-3 prostate cancer cells and were simultaneously stimulated with a mechanical tensile strain of 2,500 µε at 0.5 Hz; the osteoblastic differentiation of the MC3T3-E1 cells was then assessed. In addition, the differential expression levels of mRNA, miRNA and lncRNA in MC3T3-E1 cells treated with the CM of PC-3 cells were screened, and some of the miRNAs and mRNAs were verified by reverse transcription-quantitative PCR (RT-qPCR). The signal molecules and signaling pathways associated with osteogenic differentiation were predicted by bioinformatics analysis. The CM of PC-3 prostate cancer cells suppressed osteoblastic differentiation of MC3T3-E1 cells. A total of seven upregulated miRNAs and 12 downregulated miRNAs were selected by sequencing and further verified using RT-qPCR, and related differentially expressed genes (11 upregulated and 12 downregulated genes) were also selected by sequencing and further verified using RT-qPCR; subsequently, according to the enrichment of differentially expressed genes in signaling pathways, nine signaling pathways involved in osteogenic differentiation were screened out. Furthermore, a functional mRNA-miRNA-lncRNA regulatory network was constructed. The differentially expressed miRNAs, mRNAs and lncRNAs may provide a novel signature in bone metastases of prostate cancer. Notably, some of the signaling pathways and related genes may be associated with pathological osteogenic differentiation caused by bone metastasis of prostate cancer.

Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease

Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves.Lower levels of bioavailable H2S and higher levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine β-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1β and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1β and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation.We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.

Hyaluronan and Proteoglycan Link Protein 1 Activates the BMP4/Smad1/5/8 Signaling Pathway to Promote Osteogenic Differentiation: an Implication in Fracture Healing.

Osteoblast regeneration, characterized by osteoblast differentiation, is the basis of fracture healing and accelerates fracture repair. It has been reported that hyaluronan and proteoglycan link protein 1 (HAPLN1) is overexpressed during osteoblast differentiation and regulates cartilage regeneration, but its function in fracture healing remains unclear. To elucidate this issue, we collected clinical blood samples of fracture healing, established a femoral fracture rat model, and induced an osteoblast differentiation cell model. We found that HAPLN1 was overexpressed in the serum of patients with fracture healing and the bone tissues of rats with fracture healing. Furthermore, the expression of HAPLN1 was increased time dependently during the osteogenic differentiation of MC3T3-E1 cells. HAPLN1 silencing prevented osteoblast differentiation and mineralization in MC3T3-E1 cells as evidenced by decreased osteoblast differentiation-related factors, suppressed alkaline phosphatase activities, and reduced alizarin red positive staining. Mechanically, the bone morphogenic protein 4 (BMP4)/Smad1/5/8 pathway, a facilitator of osteoblastic differentiation, was found to be inhibited by HAPLN1 knockdown, and inhibition of BMP4/Smad1/5/8 signaling enhanced the effects caused by HAPLN1 silencing. These findings demonstrated that HAPLN1 might promote fracture healing by facilitating osteogenic differentiation through the BMP4/Smad1/5/8 pathway, indicating that targeting HAPLN1 may be a feasible therapeutic candidate for fracture repair.

BMSC-Derived Exosomal CircHIPK3 Promotes Osteogenic Differentiation of MC3T3-E1 Cells via Mitophagy

Exosome-based therapy is emerging as a promising strategy to promote bone regeneration due to exosomal bioactive cargos, among which circular RNA (circRNA) has recently been recognized as the key effector. The role of exosomal circRNA derived from bone marrow mesenchymal stem cells (BMSCs) has not been well-defined. The present study aimed to clarify the regulatory function and molecular mechanism of BMSC-derived exosomal circRNA in osteogenesis. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) were isolated and identified. BMSC-Exos' pro-osteogenic effect on MC3T3-E1 cells was validated by alkaline phosphatase (ALP) activity and Alizarin Red staining. Through bioinformatic analysis and molecular experiments, circHIPK3 was selected and verified as the key circRNA of BMSC-Exos to promote osteoblast differentiation of MC3T3-E1 cells. Mechanistically, circHIPK3 acted as an miR-29a-5p sponge and functioned in mitophagy via targeting miR-29a-5p and PINK1. Additionally, we showed that the mitophagy level of MC3T3-E1 cells were mediated by BMSC-Exos, which promoted the osteogenic differentiation. Collectively, our results revealed an important role for BMSC-derived exosomal circHIPK3 in osteogenesis. These findings provide a potentially effective therapeutic strategy for bone regeneration.

The biological responses of osteoblasts on titanium: Effect of oxygen level and surface roughness

Due to the general application of invitro test, cell culture is generally selected to evaluate the cytocompatibility of devices and materials. The choice of test condition should depend on the probable site and clinical application. The oxygen content of human body could be estimated around 5%∼12%, and the oxygen level of healing bone fracture range from 0.8%∼3.8%%. However, materials for bone implant are traditionally evaluated under laboratory normoxia condition (21% O2) invitro. The aim was to study the effect of oxygen level on osteoblast upon high stiffness titanium with different roughness. After sandblasted and acid-etched (SLA) process, we create titanium surfaces with four different roughness. The differentiation and proliferation of MC3T3-E1 osteoblast cultured on SLA-treated specimens were evaluated in designed chamber with oxygen level of 1%, 5%, 10%, 21%. By scanning electron microscopy, all samples had sub-micro pit inside the micro-holes upon SLA-treated Ti disk surface. The decrease of oxygen level from 21% to 5% promoted osteoblast growth of SLA-treated specimens, but 1% O2 delayed cell proliferation. The surface roughness of specimens influenced osteoblast cell differentiation. The differentiation and proliferation ability of the cells upon SLA-treated specimens is proportional to oxygen level. Our results demonstrated that 5% O2 will easily discriminate osteoblasts responses on different SLA-treated specimens. These results suggest that hypoxia (5% O2) environment is better model for biological evaluation of bone-related materials.

Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway

To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism. MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells. The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05). Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.

SPARC is a decoy counterpart for c‑Fos and is associated with osteoblastic differentiation of bone marrow stromal cells by inhibiting adipogenesis.

Secreted protein acidic and rich in cysteine (SPARC), also called basement‑membrane protein 40 or osteonectin, is a matricellular protein that is abundant not only in bone tissue as a non‑collagenous protein but is also ubiquitously expressed in non‑calcified tissue. SPARC is located intracellularly and disruption of the Sparc gene has been reported to reduce bone formation and increase fat tissue; however, the mechanism by which SPARC inhibits adipogenesis remains unclear. The present study evaluated the intracellular function of SPARC in adipogenesis using the bone marrow stromal cell line ST2. When ST2 cells with low SPARC production were cloned, intrinsic activator protein‑1 (AP‑1) activity was markedly higher, mineralized nodule formation was significantly lower and lipid accumulation was significantly increased compared with in the parental ST2 cells. Forced expression of secreted SPARC with the signal peptide‑coding sequences of wild‑type Sparc or preprotrypsin in SPARC‑low ST2 cells significantly reduced AP‑1 transcription activity; however, these reductions were not observed in the absence of signal peptide sequences. Recombinant SPARC, produced using Brevibacillusbrevis, specifically bound to c‑Fos but not c‑Jun and inhibited the binding of c‑Fos/c‑Jun to a TPA‑response element sequence. These data suggested that SPARC was incorporated into the cells from the extracellular spaces and serves an intracellular role as a decoy counterpart for c‑Fos, as well as being associated with osteoblastogenesis through the inhibition of adipogenesis. These findings may provide new insights into regenerative medicine.

Effects of areca nut consumption on cell differentiation of osteoblasts, myoblasts, and fibroblasts

Areca nut is used worldwide as a hallucinogenic addicting drug along the tropical belt. Arecoline, a toxic compound, is the most important alkaloid in areca nuts. The adverse effects of oral uptake and chewing of areca nut are well known. For example, the possibility of cancer caused by chewing areca nuts is widely discussed. Chewing areca nut has other adverse effects on other organs, including abnormal cell differentiation, oral cancer, and several other diseases. The use of areca nut is also associated with low birthweight. Skeletal musculature is the largest organ in the body and is attached to the bones. During embryo development, the differentiation of bone and muscle cells is critical. In this article, we reviewed the effects of areca nut and arecoline on embryonic cell differentiation, particularly osteoblasts, myoblasts, and fibroblasts.

Chebulinic acid alleviates LPS-induced inflammatory bone loss by targeting the crosstalk between reactive oxygen species/NFκB signaling in osteoblast cells.

Chebulinic acid (CA), a plant ellagitannin derived from Triphala, is reported to exhibit both anti-inflammatory & anti-oxidant activity apart from anti-tumour property. However, its role in inflammatory bone loss conditions was unexplored. We hypothesized that CA may prevent the bone loss under inflammatory conditions induced by lipopolysaccharide (LPS) in 10-week-old male C57BL/6J mice. Micro-CT analysis and histomorphometric evaluations were carried out where it was found that CA significantly improved the bone micro-architectures by enhancing trabecular connectivity and strength of the bone. CA also increased the bone regeneration as examined by calcein labelling and ex-vivo mineralisation along with maintaining the bone serum markers. Further, CA ameliorated the reduction in osteoblast cell differentiation, proliferation and viability after LPS stimulation. DCFDA and Mitosox staining revealed that CA presented remarkable protective effects against LPS treatment by attenuating oxidative stress, both at cellular & mitochondrial levels. In addition, CA significantly decreased the production of pro-inflammatory cytokines, and down-regulated the phosphorylation of NFκB and IκBα, indicating that CA could attenuate the inflammatory impairment to primary osteoblast cells by suppressing the NFkB signalling pathway. Taken together, the protective role of CA against LPS-induced bone loss & inhibitory effect on total ROS levels hold promise as a potential novel therapeutic strategy for the inflammatory diseases in bones.

Exercise maintains bone homeostasis by promoting osteogenesis through STAT3

Bone exhibits changes in density, strength, and microarchitecture in relation to mechanical loading mediated by exercise. Appropriate exercise maintains bone homeostasis, while the absence of exercise leads to disuse bone loss. However, the acting mechanism of mechanotransduction in bone remains unclear. We performed the running-wheel exercise and tail suspension model to study the effects of exercise on bone metabolism, and found that osteoblastic Signal transducer and activator of transcription 3 (STAT3) activity was closely related to exercise-induced bone mass and metabolism changes. With the Flexcell tension-loading system in vitro, mechanical force promoted STAT3 activity, which was accompanied by increased osteoblastic differentiation of the bone marrow mesenchymal stem cells (BMSCs). In contrast, the inhibition of STAT3 phosphorylation blocked force-induced osteoblastic differentiation. Furthermore, pharmacological inactivation of STAT3 impaired the increase in exercise-induced bone mass and osteogenesis. With an inducible conditional deletion mouse model, we found that the osteoblast lineage-specific Stat3 deletion could also block force-induced osteoblastic differentiation in vitro and impair exercise-promoted bone mass and osteogenesis in vivo. This confirmed the crucial role of osteoblastic STAT3 in exercise-mediated bone metabolism. Finally, colivelin, a STAT3 agonist, promoted osteoblastic differentiation in vitro and partly rescued exercise loss-induced disuse bone loss by improving osteogenesis in the tail suspension model. Taken together, our study revealed the essential role of STAT3 in maintaining exercise-mediated bone homeostasis. In addition, STAT3 might act as a potential target for osteoporosis caused by exercise loss.

VCAM-1 and GFPT-2: Predictive markers of osteoblast differentiation in human dental pulp stem cells.

Dental pulp stem cells (DPSCs) have high proliferative and multilineage differentiation potential in mesenchymal stem cells. However, several studies have indicated that there are individual differences in the potential for osteogenic differentiation of DPSCs, and the factors determining these differences are unknown. To identify the genes responsible for the individual differences in the osteogenic differentiation ability of DPSCs. We divided DPSCs into high and low osteogenic differentiation ability groups (HG or LG) with ALP and von Kossa stain, and compared the gene expression patterns using RNA-seq. In addition, genes that may affect osteogenic differentiation were knocked down using small interfering RNA (siRNA) and their effects were investigated. The RNA-seq patterns revealed that VCAM1 and GFPT2 were significantly expressed at higher levels in the HG than in the LG. The results of siRNA analysis showed that VCAM1 and GFPT2 knockdown significantly reduced the expression of osteogenic markers. Furthermore, we analyzed the involvement of these two genes in cell signaling in DPSC differentiation. The results indicated that the VCAM1-mediated Ras-MEK-Erk and PI3K/Akt pathways are involved in the osteogenic differentiation of DPSCs, and that GFPT2-mediated HBP signaling influences the osteogenic differentiation of DPSCs. These findings indicate that DPSCs that highly express VCAM1 and GFPT2 have a high capacity for osteogenic differentiation. Evaluation of VCAM1 and GFPT2 expression in undifferentiated DPSCs may predict the outcome of bone regenerative therapy using DPSCs. Moreover, the expression levels of VCAM1 and GFPT2 in DPSCs may be useful in setting criteria for selecting donors for allogeneic cell transplantation for bone regeneration.