Proteasome inhibition-enhanced fracture repair is associated with increased mesenchymal progenitor cells in mice.

The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.

Ataxia-televangelist mutated (ATM)/ ATR serine/threonine kinase (ATR)-mediated RAD51 recombinase (RAD51) promotes osteogenic differentiation and inhibits osteoclastogenesis in osteoporosis.

Osteoporosis is a metabolic bone disease that significantly affects the quality of life and can even lead to death. In this study, we aimed to investigate the role of RAD51 recombinase (RAD51) in osteoblast and osteoclast differentiation. We analyzed differentially expressed genes using microarray analysis. The osteogenic differentiation capability was analyzed by alkaline phosphatase (ALP) staining and alizarin red staining assays. Osteogenesis and osteoclast related genes expression was detected using quantitative real-time PCR (qPCR) and Western blotting. The phosphorylation of Ataxia-telangiectasia mutated (ATM) and ATR serine/threonine kinase (ATR) was tested using Western blotting. The effect of RAD51 on osteoporosis was also explored in vivo. The results showed that RAD51 was downregulated in osteoporosis, but upregulated in differentiated osteoblasts. Overexpression of RAD51 enhanced the differentiation of osteoblasts and suppressed the formation of osteoclasts. Furthermore, p-ATM and p-ATR levels were upregulated in osteoblasts and downregulated in osteoclasts. RAD51 expression was reduced by the ATM/ATR pathway inhibitor AZ20. AZ20 treatment inhibited osteoblastogenesis and promoted osteoclastogenesis, whereas RAD51 reversed the effects induced by AZ20. Moreover, RAD51 improved bone microarchitecture in vivo. Taken together, ATM/ATR signaling-mediated RAD51 promoted osteogenic differentiation and suppressed osteoclastogenesis. These findings reveal a critical role for RAD51 in osteoporosis.

Incretins Enhance PGF2α-Induced Synthesis of IL-6 and Osteoprotegerin in Osteoblasts.

Incretins including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), which are secreted from the small intestine after oral food ingestion, are currently well-known to stimulate insulin secretion from pancreatic ß-cells and used for the treatment of type 2 diabetes mellitus. We have previously reported that prostaglandin F2α (PGF2α) stimulates the synthesis of interleukin-6 (IL-6) and osteoprotegerin in osteoblast-like MC3T3-E1 cells, and that IL-6 and osteoprotegerin release are mediated through the p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways. In the present study, we investigated the effects of incretins including GLP-1 and GIP, on the PGF2α-induced synthesis of IL-6 and osteoprotegerin and examined the detailed mechanism in osteoblast-like MC3T3-E1 cells. We found that GIP and GLP-1 significantly stimulated the PGF2α-induced synthesis of IL-6 in osteoblast-like MC3T3-E1 cells. In addition, GIP and GLP-1 significantly enhanced the PGF2α-induced mRNA expression levels of IL-6. On the other hand, GIP and GLP-1 markedly stimulated the PGF2α-induced synthesis of osteoprotegerin. However, the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or JNK induced by PGF2α was not affected by GIP or GLP-1. Therefore, these results strongly suggest that incretins enhance the PGF2α-induced synthesis of IL-6 and osteoprotegerin in osteoblast-like MC3T3-E1 cells. However, these syntheses are not mediated through p44/p42 MAP kinase, p38 MAP kinase, or JNK pathways.

Loss of phosphatidylinositol-4-phosphate 5-kinase type-1 gamma (Pip5k1c) in mesenchymal stem cells leads to osteopenia by impairing bone remodeling.

Phosphatidylinositol-4-phosphate 5-kinase type-1 gamma (Pip5k1c) is a lipid kinase that plays a pivotal role in the regulation of receptor-mediated calcium signaling in multiple tissues; however, its role in the skeleton is not clear. Here, we show that while deleting Pip5k1c expression in the mesenchymal stem cells using Prx1-Cre transgenic mice does not impair the intramembranous and endochondral ossification during skeletal development, it does cause osteopenia in adult mice, but not rapidly growing young mice. We found Pip5k1c loss dramatically decreases osteoblast formation and osteoid and mineral deposition, leading to reduced bone formation. Furthermore, Pip5k1c loss inhibits osteoblastic, but promotes adipogenic, differentiation of bone marrow stromal cells. Pip5k1c deficiency also impairs cytoplasmic calcium influx and inactivates the calcium/calmodulin-dependent protein kinase, which regulates levels of transcription factor Runx2 by modulating its stability and subsequent osteoblast and bone formation. In addition, Pip5k1c loss reduces levels of the receptor activator of nuclear factor-κB ligand, but not that of osteoprotegerin, its decoy receptor, in osteoblasts in bone and in sera. Finally, we found Pip5k1c loss impairs the ability of bone marrow stromal cells to support osteoclast formation of bone marrow monocytes and reduces the osteoclast precursor population in bone marrow, resulting in reduced osteoclast formation and bone resorption. We conclude Pip5k1c deficiency causes a low-turnover osteopenia in mice, with impairment of bone formation being greater than that of bone resorption. Collectively, we uncover a novel function and mechanism of Pip5k1c in the control of bone mass and identify a potential therapeutic target for osteoporosis.

Overexpression of PIK3R1 Promotes Bone Formation by Regulating Osteoblast Differentiation and Osteoclast Formation.

In an effort to bolster our understanding of regulation of bone formation in the context of osteoporosis, we screened out differentially expressed genes in osteoporosis patients with high and low bone mineral density by bioinformatics analysis. PIK3R1 is increasingly being nominated as a pivotal mediator in the differentiation of osteoblasts and osteoclasts that is closely related to bone formation. However, the specific mechanisms underlying the way that PIK3R1 affects bone metabolism are not fully elucidated. We intended to examine the potential mechanism by which PIK3R1 regulates osteoblast differentiation. Enrichment analysis was therefore carried out for differentially expressed genes. We noted that the estrogen signaling pathway, TNF signaling pathway, and osteoclast differentiation were markedly associated with ossification, and they displayed enrichment in PIK3R1. Based on western blot, qRT-PCR, and differentiation analysis in vitro, we found that upregulation of PIK3R1 enhanced osteoblastic differentiation, as evidenced by increased levels of investigated osteoblast-related genes as well as activities of ALP and ARS, while it notably decreased levels of investigated osteoclast-related genes. On the contrary, downregulation of PIK3R1 decreased levels of osteoblast-related genes and increased levels of osteoclast-related genes. Besides, in vitro experiments revealed that PIK3R1 facilitated proliferation and repressed apoptosis of osteoblasts but had an opposite impact on osteoclasts. In summary, PIK3R1 exhibits an osteoprotective effect via regulating osteoblast differentiation, which can be represented as a promising therapeutic target for osteoporosis.

Therapeutic potential of phosphodiesterase inhibitors in the treatment of osteoporosis: Scopes for therapeutic repurposing and discovery of new oral osteoanabolic drugs.

Cyclic nucleotide phosphodiesterases (PDEs) are ubiquitously expressed enzymes that hydrolyze phosphodiester bond in the second messenger molecules including cAMP and cGMP. A wide range of drugs blocks one or more PDEs thereby preventing the inactivation of cAMP/cGMP. PDEs are differentially expressed in bone cells including osteoblasts, osteoclasts and chondrocytes. Intracellular increases in cAMP/cGMP levels in osteoblasts result in osteogenic response. Acting via the type 1 PTH receptor, teriparatide and abaloparatide increase intracellular cAMP and induce osteoanabolic effect, and many PDE inhibitors mimic this effect in preclinical studies. Since all osteoanabolic drugs are injectable and that oral drugs are considered to improve the treatment adherence and persistence, osteogenic PDE inhibitors could be a promising alternative to the currently available osteogenic therapies and directly assessed clinically in drug repurposing mode. Similar to teriparatide/abaloparatide, PDE inhibitors while stimulating osteoblast function also promote osteoclast function through stimulation of receptor activator of nuclear factor kappa-B ligand production from osteoblasts. In this review, we critically discussed the effects of PDE inhibitors in bone cells from cellular signalling to a variety of preclinical models that evaluated the bone formation mechanisms. We identified pentoxifylline (a non-selective PDE inhibitor) and rolipram (a PDE4 selective inhibitor) being the most studied inhibitors with osteogenic effect in preclinical models of bone loss at ≤ human equivalent doses, which suggest their potential for post-menopausal osteoporosis treatment through therapeutic repurposing. Subsequently, we treated pentoxifylline and rolipram as prototypical osteogenic PDEs to predict new chemotypes via the computer-aided design strategies for new drugs, based on the structural biology of PDEs.

PARP Activity Fine-tunes the DNA Replication Choreography of Chk1-depleted Cells.

Checkpoint Kinase 1 (Chk1) prevents DNA damage by adjusting the replication choreography in the face of replication stress. Chk1 depletion provokes slow and asymmetrical fork movement, yet the signals governing such changes remain unclear. We sought to investigate whether poly(ADP-ribose) polymerases (PARPs), key players of the DNA damage response, intervene in the DNA replication of Chk1-depleted cells. We demonstrate that PARP inhibition selectively alleviates the reduced fork elongation rates, without relieving fork asymmetry in Chk1-depleted cells. While the contribution of PARPs to fork elongation is not unprecedented, we found that their role in Chk1-depleted cells extends beyond fork movement. PARP-dependent fork deceleration induced mild dormant origin firing upon Chk1 depletion, augmenting the global rates of DNA synthesis. Thus, we have identified PARPs as novel regulators of replication fork dynamics in Chk1-depleted cells.

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation.

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.

The virulence factor GroEL promotes gelatinase secretion from cells in the osteoblast lineage: Implication for direct crosstalk between bacteria and adult cells.

OBJECTIVE: The aim of this study was to demonstrate the influence of the virulence factor GroEL on osteoblast behavior by characterizing the changes of secreted gelatinases. DESIGN: ELISA was performed to detect GroEL from samples from patients with or without apical periodontitis. An apical periodontitis model was established in rats and the expression of MMP-2, MMP-9 and NF-κB was evaluated by immunofluorescence staining. The primary osteoblasts and osteoblast-like MC3T3 cells were stimulated with recombinant GroEL, and gelatin zymography was used to determine the activity and expression of MMP-2 and MMP-9. Western blot was used to screen signaling pathways, and immunofluorescence staining was performed to confirm the activated signaling. RESULTS: First, we found expression of GroEL to be higher in oral saliva, gingival crevicular fluid and periradicular granulation tissue of patients with apical periodontitis than it was in healthy control patients. We next found that recombinant GroEL could increase the activity of the gelatinases, MMP-2 and MMP-9, which were secreted by both primary osteoblasts and MC3T3 cells. In a rat apical periodontitis model, strong expression of gelatinases was confirmed. Then, we found that GroEL-enhanced gelatinase activity was mediated through activation of NF-κB signaling. Acetylated NF-κB accumulated in the cell nucleus and bound to the promoter of MMP-2 and MMP-9 genes, thus initiating their high expression. CONCLUSION: This study reveals a direct interaction between oral bacteria and adult cells by demonstrating that gelatinase secretion is induced by GroEL, which partially explains bone resorption through gelatinase activation.

Effects of inhibiting PDK­1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation in vitro.

Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3­phosphoinositide­dependent protein kinase­1 (PDK­1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK­1 in the process of mouse osteoblast differentiation in vitro. In the BX­912 group, BX­912, a specific inhibitor of PDK­1, was added to osteoblast induction medium (OBM) to treat bone marrow mesenchymal stem cells (BMSCs), whereas the control group was treated with OBM alone. Homozygote PDK1flox/flox mice were designed and generated, and were used to obtain BMSCsPDK1flox/flox. Subsequently, an adenovirus containing Cre recombinase enzyme (pHBAd­cre­EGFP) was used to disrupt the PDK­1 gene in BMSCsPDK1flox/flox; this served as the pHBAd­cre­EGFP group and the efficiency of the disruption was verified. Western blot analysis demonstrated that the protein expression levels of phosphorylated (p)­PDK1 and p­AKT were gradually increased during the osteoblast differentiation process. Notably, BX­912 treatment and disruption of the PDK­1 gene with pHBAd­cre­EGFP effectively reduced the number of alkaline phosphatase (ALP)­positive cells and the optical density value of ALP activity, as well as the formation of cell mineralization. The mRNA expression levels of PDK­1 in the pHBAd­cre­EGFP group were significantly downregulated compared with those in the empty vector virus group on days 3­7. The mRNA expression levels of the osteoblast­related genes RUNX2, osteocalcin and collagen I were significantly decreased in the BX­912 and pHBAd­cre­EGFP groups on days 7 and 21 compared with those in the control and empty vector virus groups. Overall, the results indicated that BX­912 and disruption of the PDK­1 gene in vitro significantly inhibited the differentiation and maturation of osteoblasts. These experimental results provided an experimental and theoretical basis for the role of PDK­1 in osteoblasts.

Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice.

Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.

Crosstalk between the COX2-PGE2-EP4 signaling pathway and primary cilia in osteoblasts after mechanical stimulation.

Primary cilia have been found to function as mechanosensors in low-magnitude high-frequency vibration (LMHFV)-induced osteogenesis. The PGE2 also regulates bone homeostasis and mechanical osteogenesis through its receptor EP4 signaling, but its involvement in LMHFV-induced or in primary cilia-induced osteogenesis has not been investigated. We hypothesized that LMHFV stimulates osteoblast (OB) differentiation by activating the COX2-PGE2-EP pathway in a manner dependent on primary cilia and that primary cilia are also affected by the PGE2 pathway. In this study, through western blot analysis, RNA interference, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and cytochemical staining, we observed that COX2, mPGES-1, and PGE2 levels were markedly elevated in cells treated with LMHFV and were greatly decreased in LMHFV-treated cells following IFT88 silencing. EP4 expression was significantly increased in OBs following LMHFV treatment, but IFT88 silencing significantly blocked this increase. EP4 localized to the bases of primary cilia. LMHFV reduced the length and abundance of primary cilia, but the cells could self-repair their primary cilia after mechanical damage. EP4 antagonism significantly blocked the LMHFV-induced increase in IFT88 expression and blocked the recovery of primary cilia length and the proportion of cells with primary cilia. In addition, COX2 or EP4 antagonism disrupted LMHFV-induced osteogenesis. These results demonstrate the integration of and crosstalk between primary cilia and the COX2-PGE2-EP4 signaling pathway under mechanical stimulation.

Pentraxin 3 promotes the osteoblastic differentiation of MC3T3-E1 cells through the PI3K/Akt signaling pathway.

Osteoblast cells are responsible for synthesizing new bone tissue, and determining how to control osteoblastic differentiation is vital to the treatment of osteoporosis. In the present study, we show that pentraxin 3 (PTX3) signaling is involved in the regulation of osteoblastic differentiation in MC3T3-E1 cells. Our data reveal that PTX3 is abundantly expressed in MC3T3-E1 cells and that its expression is inducible by the introduction of osteogenic induction medium (OIM). Overexpression of PTX3 was observed to significantly increase the expression of four osteoblast signature genes, including Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN) and osterix (OSX), suggesting that the overexpression of PTX3 promotes osteoblastic differentiation. The relative level of gene expression between OIM and OIM plus overexpressed PTX3 was evaluated using the Affymetrix Gene Chip® mouse gene microarray. PTX3-related differentially expressed genes (DEGs) were screened. Gene ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway enrichment analyses were performed, and the PI3K/Akt signaling pathway was primarily involved in the osteogenic differentiation of PTX3. Protein-protein interactions (PPIs) were also constructed, and the molecular complex detection (MCODE) plugin calculated modules of PPI networks. Moreover, we show that the effect of PTX3 is mediated by its induction of the PI3K/Akt signaling pathway. Mechanistically, we show that the action of PTX3 requires the activation of PI3K and Akt, and deactivation of PI3K by its inhibitor LY294002 weakens the PTX3-mediated induction of osteoblast signature genes, ALP and matrix mineralization. The present study revealed a new role played by PTX3 and suggest a potential mechanism governing the osteoblastic differentiation of MC3T3-E1 cells.

Carbohydrate responsive element binding protein (ChREBP) negatively regulates osteoblast differentiation via protein phosphatase 2A Cα dependent manner.

Carbohydrate responsive element binding protein (ChREBP) is a major transcription factor of lipogenesis regulated by glucose status in the liver. However, the function of ChREBP in osteogenic differentiation is unclear. The present study examined the role of ChREBP in osteoblast differentiation in MC3T3-E1 preosteoblast cell line. The mRNA expression of ChREBP, protein phosphatase 2A catalytic subunit-α (PP2A Cα) and the osteogenic genes such as, DNA-binding protein inhibitor (Id1), runt-related transcription factor-2 (Runx2), and alkaline phosphatase (ALP) was measured by qPCR and RT-PCR. Runx2, ChREBP, and PP2A Cα, protein levels were evaluated by Western blotting. ALP staining experiment was carried out to evaluate ALP enzyme activity, and a luciferase reporter assay was performed to analyze Runx2 transcriptional activity. Expression of ChREBP and PP2A Cα did not change during bone morphogenetic protein-2 (BMP2)-induced osteoblast differentiation. Overexpression of ChREBP reduced the osteogenic genes (Runx2 and ALP) expression and ALP activity, while knockdown of ChREBP had the opposite effects. Overexpression of PP2A Cα increased ChREBP expression, while inhibition of PP2A Cα using okadaic acid not only inhibited the expression of ChREBP, but also restored the mRNA and protein expression of Runx2 and activity of ALP enzyme. These results demonstrate that ChREBP inhibits BMP2-induced osteoblast differentiation in a PP2A Cα- dependent manner.

Myeloma cells shift osteoblastogenesis to adipogenesis by inhibiting the ubiquitin ligase MURF1 in mesenchymal stem cells.

The suppression of bone formation is a hallmark of multiple myeloma. Myeloma cells inhibit osteoblastogenesis from mesenchymal stem cells (MSCs), which can also differentiate into adipocytes. We investigated myeloma-MSC interactions and the effects of such interactions on the differentiation of MSCs into adipocytes or osteoblasts using single-cell RNA sequencing, in vitro coculture, and subcutaneous injection of MSCs and myeloma cells into mice. Our results revealed that the α4 integrin subunit on myeloma cells stimulated vascular cell adhesion molecule-1 (VCAM1) on MSCs, leading to the activation of protein kinase C ß1 (PKCß1) signaling and repression of the muscle ring-finger protein-1 (MURF1)-mediated ubiquitylation of peroxisome proliferator-activated receptor γ2 (PPARγ2). Stabilized PPARγ2 proteins enhanced adipogenesis and consequently reduced osteoblastogenesis from MSCs, thus suppressing bone formation in vitro and in vivo. These findings reveal that suppressed bone formation is a direct consequence of myeloma-MSC contact that promotes the differentiation of MSCs into adipocytes at the expense of osteoblasts. Thus, this study provides a potential strategy for treating bone resorption in patients with myeloma by counteracting tumor-MSC interactions.

Excessive production of mitochondrion­derived reactive oxygen species induced by titanium ions leads to autophagic cell death of osteoblasts via the SIRT3/SOD2 pathway.

The incidence of peri-implant bone loss is high, and is a difficult condition to treat. Previous studies have shown that titanium (Ti) ions released from implants can lead to osteoblast cell damage, but the specific mechanisms have not been elucidated. The present study established a Ti ion damage osteoblast cell model. The levels of mitochondrion­derived reactive oxygen species (mROS) and autophagy, cell viability and the sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) pathway were examined in this model. It was found that Ti ions decreased osteoblast viability. Moreover, with increased Ti ion concentration, the expression levels of microtubule associated protein 1 light chain 3α (LC3) progressively increased, P62 decreased, autophagic flow increased and mROS levels increased. After the addition of an autophagy inhibitor Bafilomycin A1 and Mito­TEMPO, a mitochondrial antioxidant, the production of mROS was inhibited, the level of autophagy was decreased and cell activity was improved. In addition, with increased Ti ion concentration, the activity of SOD2 decreased, the acetylation level of SOD2 increased, the SIRT3 mRNA and protein expression levels decreased, and the activity of SIRT3 was significantly decreased. Furthermore, it was demonstrated that SIRT3 overexpression reduced the acetylation of SOD2 and increased the activity of SOD2, as well as reducing the production of mROS and the expression level of LC3, thus increasing cell viability. Therefore, the present results suggested that excessive production of mROS induced by Ti ions led to autophagic cell death of osteoblasts, which is dependent on the SIRT3/SOD2 pathway.

KMN-159, a novel EP4 receptor selective agonist, stimulates osteoblastic differentiation in cultured whole rat bone marrow.

KMN-159 is the lead compound from a series of novel difluorolactam prostanoid EP4 receptor agonists aimed at inducing local bone formation while avoiding the inherent side effects of systemic EP4 activation. KMN-159 is a potent, selective small molecule possessing pharmacokinetic properties amenable to local administration. Unfractionated rat bone marrow cells (BMCs) were treated once at plating with escalating doses of KMN-159 (1 pM to 10 µM). The resulting elevated alkaline phosphatase (ALP) levels measured 9 days post-dose are consistent with increased osteoblastic differentiation and exposure to KMN-159 at low nanomolar concentrations for as little as 30 min was sufficient to induce complete osteoblast differentiation of the BMCs from both sexes and regardless of age. ALP induction was blocked by an EP4 receptor antagonist but not by EP1 or EP2 receptor antagonists and was not induced by EP2 or EP3 receptor agonists. Addition of BMCs to plates coated with KMN-159 24 days earlier resulted in ALP activation, highlighting the chemical stability of the compound. The expression of phenotype markers such as ALP, type I collagen, and osteocalcin was significantly elevated throughout the osteoblastic differentiation timecourse initiated by KMN-159 stimulation. An increased number of tartrate-resistant acid phosphatase-positive cells was observed KMN-159 or PGE2 treated BMCs but only in the presence of exogenous receptor activator of nuclear factor kappa-Β ligand (RANKL). No change in the number of adipocytes was observed. KMN-159 also increased bone healing in a rat calvarial defect model with a healing rate equivalent to recombinant human bone morphogenetic protein-2. Our studies show that KMN-159 is able to stimulate osteoblastic differentiation with a very short time of exposure, supporting its potential as a therapeutic candidate for augmenting bone mass.

Enhanced osteogenic differentiation of osteoblasts on CaTiO3 nanotube film.

Improved implant-bone interface interaction for rapid formation of strong and long-lasting bond is significantly important in orthopedic clinics. Herein, Ca-doped TiO2 nanotube film (M-CaNTs) with enhanced adhesion strength was fabricated on titanium (Ti) surface by an anodization-hydrothermal treatment. Results showed that TiO2 nanotube film (M-NTs) fabricated by modified anodization was amorphous, exhibiting 100-nm diameter and 12-nm tube wall thickness. After hydrothermal treatment, the nanotubular structure of M-CaNTs kept integrated, but was volume-expanded, exhibiting a decreased diameter (∼ 60 nm) and an increased wall thickness (∼ 30 nm). The formation of M-CaNTs proceeded preferentially at the interior surfaces of the closely aligned nanotubes, involving an in situ dissolution-recrystallization process. Though the adhesion strength of M-CaNTs was weakened by the volume-expansion derived internal stress, it was still higher than that of the traditionally obtained one. In the in vitro investigations, the combination of nanotubular structure and Ca2+ could expectedly enhance the attachment, spreading and proliferation of MC3T3-E1 cells, as well as promote the expressions of bone-specific genes, intracellular proteins and ALP activity, which in turn accelerated collagen secretion and ECM mineralization. This work provides an attractive potential for the surface modification of Ti-based implants in clinical application.

Rapid Fabrication of Anatomically-Shaped Bone Scaffolds Using Indirect 3D Printing and Perfusion Techniques.

Fused deposit modeling (FDM) 3D printing technology cannot generate scaffolds with high porosity while maintaining good integrity, anatomical-surface detail, or high surface area-to-volume ratio (S/V). Solvent casting and particulate leaching (SCPL) technique generates scaffolds with high porosity and high S/V. However, it is challenging to generate complex-shaped scaffolds; and solvent, particle and residual water removal are time consuming. Here we report techniques surmounting these problems, successfully generating a highly porous scaffold with the anatomical-shape characteristics of a human femur by polylactic acid polymer (PLA) and PLA-hydroxyapatite (HA) casting and salt leaching. The mold is water soluble and is easily removable. By perfusing with ethanol, water, and dry air sequentially, the solvent, salt, and residual water were removed 20 fold faster than utilizing conventional methods. The porosities are uniform throughout the femoral shaped scaffold generated with PLA or PLA-HA. Both scaffolds demonstrated good biocompatibility with the pre-osteoblasts (MC3T3-E1) fully attaching to the scaffold within 8 h. The cells demonstrated high viability and proliferation throughout the entire time course. The HA-incorporated scaffolds demonstrated significantly higher compressive strength, modulus and osteoinductivity as evidenced by higher levels of alkaline-phosphatase activity and calcium deposition. When 3D printing a 3D model at 95% porosity or above, our technology preserves integrity and surface detail when compared with FDM-generated scaffolds. Our technology can also generate scaffolds with a 31 fold larger S/V than FDM. We have developed a technology that is a versatile tool in creating personalized, patient-specific bone graft scaffolds efficiently with high porosity, good scaffold integrity, high anatomical-shaped surface detail and large S/V.

Tuning of Titanium Microfiber Scaffold with UV-Photofunctionalization for Enhanced Osteoblast Affinity and Function.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3-10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2-15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.