Alkaline Phosphatase Activity Of Osteoblasts Research Articles

Identification of Common Pathogenic Pathways Involved in Hemochromatosis Arthritis and Calcium Pyrophosphate Deposition Disease: a Review.

Arthritis is a common clinical manifestation of hereditary hemochromatosis (HH), and HH is one of a handful of conditions linked to calcium pyrophosphate deposition (CPPD) in joints. The connection between these two types of arthritis has not yet been fully elucidated. In light of new pathogenic pathways recently implicated in CPPD involving bone, we reviewed the literature on the etiology of hemochromatosis arthropathy (HHA) seeking shared pathogenic mechanisms. Clinical observations reinforce striking similarities between HHA and CPPD even in the absence of CPP crystals. They share a similar joint distribution, low grade synovial inflammation, and generalized bone loss. Excess iron damages chondrocytes and bone cells in vitro. While direct effects of iron on cartilage are not consistently seen in animal models of HH, there is decreased osteoblast alkaline phosphatase activity, and increased osteoclastogenesis. These abnormalities are also seen in CPPD. Joint repair processes may also be impaired in both CPPD and HHA. Possible shared pathogenic pathways relate more to bone and abnormal damage/repair mechanisms than direct damage to articular cartilage. While additional work is necessary to fully understand the pathogenesis of arthritis in HH and to firmly establish causal links with CPPD, this review provides some plausible hypotheses explaining the overlap of these two forms of arthritis.

Role of tissue factor in delayed bone repair induced by diabetic state in mice.

BackgroundTissue factor (TF) is the primary activator of the extrinsic coagulation protease cascade. Although TF plays roles in various pathological states, such as thrombosis, inflammatory diseases, cancer, and atherosclerosis, its involvement in bone metabolism remains unknown.Materials and methodsThe present study examined the roles of TF in delayed bone repair induced by a diabetic state in mice using wild-type (WT) and low TF-expressing (LTF) male mice. A diabetic state was induced by intraperitoneal injections of streptozotocin (STZ).ResultsA prolonged diabetic state significantly reduced total and trabecular bone mineral densities (BMD) as well as cortical bone thickness in WT and LTF mice; these BMD parameters were similar between WT and LTF mice treated with or without STZ. The diabetic state induced in WT mice delayed the repair of the femur following injury. The diabetic state induced in LTF mice was associated with further delays in bone repair. In in vitro experiments, TF significantly decreased receptor activator of nuclear factor-κB ligand-induced osteoclast formation and osteoclastogenic gene expression in RAW264.7 cells. However, it did not affect the gene expression levels of runt-related transcription factor 2 and osterix as well as alkaline phosphatase activity in mouse primary osteoblasts.ConclusionLow TF state was associated with enhanced bone repair delay induced by diabetic state in mice. The TF-induced suppression of bone remodeling may be a contributing factor to the protective effects of TF against delayed bone repair in a diabetic state.

Biologically Augmented Suture for Ligament Bracing Procedures Positively Affects Human Ligamentocytes and Osteoblasts In Vitro

The purpose was to evaluate the response of human ligamentocytes and osteoblasts after biological augmentation with thrombin, concentrated bone marrow aspirate (cBMA), or platelet-rich plasma (PRP) on two different types of nonresorbable flat braided suture used for ligament bracing. Uncoated (U) and collagen-coated (C) flat braided suture material was augmented with either thrombin (T), cBMA (B), PRP (P), or a combination of these three (A), while platelet-poor plasma was used as a source for fibrin (F) in each assay. Previously cultured ligamentocytes and osteoblasts were added with a defined density and assayed after the required time period for adhesion, proliferation, and alkaline phosphatase activity. Biological augmentation of uncoated [(UFT, UFBT, UFA; P < .001), (UFPT; P= .017)] and collagen-coated suture (CFT, CFPT, CFBT, CFA; P < .001) led to a significantly higher ligamentocyte adhesion. Significantly higher adhesion was also observed for osteoblasts (UFT, UFPT, UFBT, UFA; P < .001; CFT, CFPT, CFBT, CFA; P < .001). Similarly, ligamentocyte proliferation was significantly higher [(UFT, UFPT, UFA; P= .009), (UFBT; P= .001), (CFT; P=.009), (CFBT; P= .001), and (CFA; P= .01)]. Osteoblasts showed significantly higher proliferation as well [(UFT, UFPT,UFA; P= .002), (UFBT; P= .001); (CFT: P= .003), and (CFPT, CFBT, CFA; P= .001)]. Augmentation with thrombin, PRP, and BMA for uncoated (UFT; P= .006, UFPT; P= .035, UFBT; P= .001) and BMA for coated suture (CFBT; P= .027) led to significantly higher alkaline phosphatase activity. Biological enhancement of suture used for ligament bracing significantly increased ligamentocyte and osteoblast adhesion and proliferation, as well as alkaline phosphatase activity of osteoblasts in an invitro model. After biological augmentation, cellular adhesion, proliferation, and alkaline phosphatase activity changed up to 1,077%, 190%, and 78%, respectively. Furthermore, no overall superiority between uncoated or collagen-coated suture material was observed for cellular adhesion, proliferation, or alkaline phosphatase activity. This study provides invitro data on a new treatment concept of biologic augmentation for acute ligamentous lesions treated with ligament bracing that has not been widely described. This concept may improve the healing of injured ligaments, in addition to providing immediate biomechanical stabilization.

Bioactivity and biocompatibility of porous gradient bioceramic coating prepared via laser cladding process

In this work, porous gradient bioceramic coating was fabricated via laser cladding process, and its bioactivity and biocompatibility were evaluated by various tests such as simulated body fluid (SBF) soaking, cell proliferation, cell fixation, osteoblast staining, osteoblast alkaline phosphatase (ALP) activity, and hydroxyproline (HYP) activity. Various porous bioceramic coatings were prepared with 0, 2.5, 5, 7.5, and 10wt% forming agents, and they were denoted as 0 wt% PFA, 2.5 wt% PFA, 5 wt% PFA, 7.5 wt% PFA, and 10 wt% PFA coating, respectively. Among these coatings, 2.5 wt% PFA and 5 wt% PFA coatings after immersing in SBF showed good ability towards mineralization and deposition of bioactive phase components, i.e., HA and β-TCP. In contrast to other coatings prepared in this work, the presence of pore structure on the surfaces of 2.5 wt% PFA and 5 wt% PFA coatings favored the deposition of bone-like apatite. The results obtained from the cell proliferation experiment revealed that 2.5–5 wt% PFA porous coatings possessed superior cell proliferation ability when compared to the other coatings. After investigating the cell fixation morphology, the pore structures of 2.5–5 wt% PFA coatings were shown to be more beneficial for the attachment and growth of MG63 cells on the surface or within the pore structure. Furthermore, the osteoblast staining test proved an extensive wide-ranged MG63 cell growth on the surfaces of 2.5–5 wt% PFA porous coatings. The ALP activity result revealed the increase in ALP contents for 2.5–10 wt% PFA porous coatings. This was especially noticeable for 2.5 wt% PFA coating as it presented the highest ALP content after 6 days. The HYP activity result indicated a continuous HYP content increment for 0–10 wt% PFA porous coatings, with only a slight difference observed between 2 days and 6 days. Therefore, a porous gradient coating with a certain pore structure, i.e., large pore size, even pore distribution, and high pore number, could significantly influence its bioactivity and biocompatibility. Based on the collective results, 2.5 wt% PFA coating with a uniform pore distribution and numerous large-sized pores exhibited the best comprehensive bioactivity and biocompatibility.

Effect of Erxian Decoction on proteomics and PI3K signaling pathway of osteoblasts stimulated by H2O2

The present study aimed to explore the effect of Erxian Decoction on proteomics of osteoblasts stimulated by hydrogen peroxide(H_2O_2) and its protective mechanism with the H_2O_2-induced cell model of oxidative stress. The primary osteoblasts were cultured from the skulls of newborn rats(within 24 hours) and divided into a control group, a model group, a Fosamax group, and an Erxian Decoction group. Blank serum was added in the control group and model group, and the drug-containing serum was added correspondingly to the remaining two groups. After 45 hours, H_2O_(2 )stimulation was conducted for three hours except for the control group, followed by protein extraction. Nano-LC-LTQ-Orbitrap system was used for protein detection, Protein Discovery for protein identification, and SIEVE for quantitative and qualitative analysis. Furthermore, following the blocking of PI3 K signaling pathway by LY294002(10 μmol·L~(-1)), a control group, a model group, an LY294002 group, an Erxian Decoction group, and an Erxian Decoction + LY294002 group were set up to observe the effect of Erxian Decoction on cell proliferation, alkaline phosphatase(ALP) activity, and the relative expression of BMP-2, OPG, p-Akt, p-FoxO1 of osteoblasts stimulated by H_2O_2 under LY294002 intervention. The results revealed that 78 differential proteins were discovered between the Erxian Decoction group and model group, which were involved in the regulation of PI3 K/Akt, glucagon, estrogen, insulin, and other signaling pathways. LY294002 blunted the promoting effect of Erxian Decoction on osteoblast proliferation and significantly down-regulated the expression of OPG and p-FoxO1, whereas its down-regulation on the expression of BMP-2 and p-Akt was not significant. Both LY294002 and Erxian Decoction increased the ALP activity of osteoblasts, which may be related to the cell state and the cell differentiation. The above results suggest that Erxian Decoction can protect osteoblasts stimulated by H_2O_2, with the PI3 K/Akt signaling pathway as one of the internal mechanisms.

Zeolite Socony Mobil-Five Coating on Ti-24 Nb-4 Zr-7.9 Sn Promotes Biocompatibility and Osteogenesis In Vitro and In Vivo.

The aim of this study was to evaluate the biocompatibility and osteogenic potential of a Zeolite Socony Mobil-5 (ZSM-5) coating on a Ti-24 Nb-4 Zr-7.9 Sn (Ti-2448) surface. ZSM-5-modified Ti-2448 (ZSM-5/Ti-2448) and Ti-2448 (control) groups were employed. The physical and chemical properties of the two types of samples were evaluated by scanning electron microscopy, Fourier-transform infrared spectroscopy, nitrogen adsorption/desorption, and contact angle methods. The surface of the ZSM-5/Ti-2448 was rougher than that of the original Ti-2448, while the contact angle of the ZSM-5/Ti-2448 was smaller than that of Ti-2448. In addition, the ZSM-5/Ti-2448 largely increased the specific surface area and introduced silanol groups. A bone-like apatite layer could be formed on the surface of ZSM-5/Ti-2448 after 14 days of incubation in a simulated body fluid. ZSM-5/Ti-2448 was not cytotoxic. The number and alkaline phosphatase (ALP) activity of osteoblasts on ZSM-5/Ti-2448 were significantly higher than those on Ti-2448 surfaces, obtained in vitro using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and ALP activity assays. Few inflammatory cells were observed around ZSM-5/Ti-2448 after insertion into the femurs of Japanese white rabbits after 4, 12, and 26 weeks through hematoxylin–eosin staining. The average gray scale of transforming growth factor-β1 (TGF-β1) on ZSM-5/Ti-2448 peaked earlier than that on Ti-2448, according to immunohistochemical staining. These results indicate that ZSM-5/Ti-2448 has a good biocompatibility and improved early osteogenic potential compared to a noncoated Ti-2448.

Short carboxyl terminal parathyroid hormone peptides modulate human parathyroid hormone signaling in mouse osteoblasts

BackgroundNovel human parathyroid hormone (hPTH) peptides of unknown biological activity have recently been identified in the serum of subjects with normal renal function, chronic renal failure, and end-stage renal disease through the application of liquid chromatography-high resolution mass spectrometry. Purposeof experiments: To determine the bioactivity of these peptides, we synthesized hPTH28-84, hPTH38-84, and hPTH45-84 peptides by solid phase peptide synthesis and tested their bioactivity in MC3T3-E1 mouse osteoblasts, either individually or together with the native hormone, hPTH1-84, by assessing the accumulation of 3´,5´-cyclic adenosine monophosphate (cAMP) and the induction of alkaline phosphatase activity. ResultsIncreasing doses of hPTH1-84 (1–100 nM) increased the accumulation of cAMP and alkaline phosphatase activity in osteoblasts. hPTH28-84, hPTH38-84, and hPTH45-84 in concentrations of 1–100 nM were biologically inert. Surprisingly, 100 nM hPTH38-84 and hPTH45-84 increased the accumulation of cAMP in osteoblasts treated with increasing amounts of hPTH1-84. Human PTH28-84 had no effects on cAMP activity alone or in combination with hPTH1-84. Conversely, 100 nM hPTH38-84, hPTH45-84, and hPTH28-84 blocked the activation of alkaline phosphatase activity by hPTH1-84. ConclusionsThe data show that the short carboxyl-terminal hPTH peptides, hPTH38-84 and hPTH45-84, increase the amount of cellular cAMP generated in cultured osteoblasts in response to treatment with full-length hPTH1-84 when compared to full-length hPTH1-84 alone. Human PTH28-84 had no effect on cAMP activity alone or in combination with hPTH1-84. Human PTH28-84, hPTH38-84 and hPTH45-84 reduced the effects of hPTH1-84 in osteoblasts with respect to the induction of alkaline phosphatase activity compared to hPTH1-84 alone. Short carboxyl peptides of human PTH are biologically inert but when administered together with full-length hPTH1-84 modulate the bioactivity of hPTH1-84 in osteoblasts.

Compounds Isolated from Wikstroemia taiwanensis Regulate Bone Remodeling by Modulating Osteoblast and Osteoclast Activities.

Bone remodeling, a dynamic process in which bone formation by osteoblast is preceded by bone resorption by osteoclast, is a vital physiological process for maintaining bone mass and strength, imbalances in which could precipitate osteoporosis. Due to the unilateral mechanism of the existing bone remodeling drugs, identifying compounds that could regulate the balance between osteoclast and osteoblast could improve the treatment of osteoporosis. Here, we show that compounds isolated from Wikstroemia taiwanensis modulate osteoclast and osteoblast activities. Specifically, astragalin (1) and kaempferol 3-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (2), besides increasing mineral deposition, increased alkaline phosphatase activity (137.2% for 1 and 115.8% for 2) and ESR-α expression (112.8% for 1 and 122.5% for 2) in primary human osteoblasts. In contrast, compounds 1, 2, 3, and 5 inhibited tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor-κB ligand-induced osteoclasts by 40.8, 17.1, 25.9, and 14.5% and also decreased the number of TRAP-positive cells by 51.6, 26.8, 20.5, and 18.6%, respectively. Our findings, therefore, showed that compounds isolated from W. taiwanensis could increase osteoblast activity while simultaneously decreasing osteoclast activity, and hence, warrant further evaluation for development as anti-osteoporosis agents.

Epigenetic reprogramming enhances the therapeutic efficacy of osteoblast-derived extracellular vesicles to promote human bone marrow stem cell osteogenic differentiation.

Extracellular vesicles (EVs) are emerging in tissue engineering as promising acellular tools, circumventing many of the limitations associated with cell‐based therapies. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Therefore, this study aimed to investigate the potential of augmenting osteoblast epigenetic functionality using the HDAC inhibitor Trichostatin A (TSA) to enhance the therapeutic efficacy of osteoblast‐derived EVs for bone regeneration. TSA was found to substantially alter osteoblast epigenetic function through reduced HDAC activity and increased histone acetylation. Treatment with TSA also significantly enhanced osteoblast alkaline phosphatase activity (1.35‐fold), collagen production (2.8‐fold) and calcium deposition (1.55‐fold) during osteogenic culture (P ≤ 0.001). EVs derived from TSA‐treated osteoblasts (TSA‐EVs) exhibited reduced particle size (1‐05‐fold) (P > 0.05), concentration (1.4‐fold) (P > 0.05) and protein content (1.16‐fold) (P ≤ 0.001) when compared to untreated EVs. TSA‐EVs significantly enhanced the proliferation (1.13‐fold) and migration (1.3‐fold) of human bone marrow stem cells (hBMSCs) when compared to untreated EVs (P ≤ 0.05). Moreover, TSA‐EVs upregulated hBMSCs osteoblast‐related gene and protein expression (ALP, Col1a, BSP1 and OCN) when compared to cells cultured with untreated EVs. Importantly, TSA‐EVs elicited a time‐dose dependent increase in hBMSCs extracellular matrix mineralisation. MicroRNA profiling revealed a set of differentially expressed microRNAs from TSA‐EVs, which were osteogenic‐related. Target prediction demonstrated these microRNAs were involved in regulating pathways such as ‘endocytosis’ and ‘Wnt signalling pathway’. Moreover, proteomics analysis identified the enrichment of proteins involved in transcriptional regulation within TSA‐EVs. Taken together, our findings suggest that altering osteoblasts’ epigenome accelerates their mineralisation and promotes the osteoinductive potency of secreted EVs partly due to the delivery of pro‐osteogenic microRNAs and transcriptional regulating proteins. As such, for the first time we demonstrate the potential to harness epigenetic regulation as a novel engineering approach to enhance EVs therapeutic efficacy for bone repair.

Effects of fluid flow shear stress to mouse muscle cells on the bone actions of muscle cell-derived extracellular vesicless.

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.

The value of the hedgehog signal in osteoblasts in fluoride-induced bone-tissue injury

ObjectiveThis study was designed to observe the expression of important hedgehog (Hh) signal factors in the bone tissue of rats with chronic fluorosis and cultured osteoblasts in order to investigate the role and significance of the Hh signal in fluoride-induced bone injury.MethodsHealthy Sprague-Dawley (SD) rats were randomly divided into four groups: the control group, the fluorosis group (F Group), the fluoride + blocker group (F + Cycl group: rats were treated with fluoride + cyclopamine), and the fluoride + blocker control group (F + DMSO group). After 6 months of intervention, the urinary fluoride content of rats in each group was detected. The primary osteoblasts of rats were selected for cell experiment, and the experiment was carried out after the cells were passaged from the second to the fourth generation.ResultsThe proliferation rate of primary rat osteoblasts presented time-affected and dose-affected relationships in a short time under treatment with a low dose of sodium fluoride (NaF), but the proliferation of osteoblasts was inhibited by long-term and high-dose NaF exposure. In the F group, the alkaline phosphatase (ALP) activity of osteoblasts increased gradually. The ALP activity was lower in the F + Cycl group than in the F group, and there was no significant difference between the F + DMSO group and F group. With the increase in fluoride exposure, the expression of Hh signal factors and osteogenic-related factor proteins increased gradually. The expressions of Indian hedgehog (Ihh), smoothened (Smo), Glioma-associated oncogene homolog (Gli) 2, and Runt-related transcription factor 2 (Runx2)in the F + Cycl group increased with the dose of fluoride but they were significantly inhibited compared with the F group. Compared with the control group, the content of urinary fluoride in the F group was significantly higher (P < 0.05), but there was no significant change in urinary fluoride content in the F + Cycl group and the F + DMSO group. Compared with the control group, the serum bone alkaline phosphatase (BALP) contents of rats in the other groups increased after 6 months’ intake of fluoride water (P < 0.05). After drug blocking, the serum BALP content in the F + Cycl group was lower than that in the F + DMSO group (P < 0.05). The BALP content in the F + DMSO group was similar to that in the F group: it did not decrease. The mRNA expressions of Ihh, Smo, Gli2, and Runx2 in bone tissue of the F group were significantly higher than those in the control group (P < 0.05). After cyclopamine blocking, the expressions decreased (P < 0.05), but the differences between the F + DMSO group and F group were not statistically significant.ConclusionHh signal plays an important role in fluoride-induced bone injury. The effective inhibition of cyclopamine is expected to be a new target for the treatment of skeletal damage caused by fluorosis.

Expression of miR-182 in patients with fracture of tibial plateau and its regulative effects on the fracture healing.

To investigate the expression of miR-182 in patients with fracture of tibial plateau (FTP) and its effects on osteoblasts and fracture healing. The patients with fracture of tibial plateau treated in our hospital and healthy subjects who received physical examination from January 2017 to January 2018 were collected. The expression of miR-182 in the serum was detected. The osteoblasts from SD rats were cultured and transfected with miR-182, anti-miR-182, miR-NC or anti-miR-NC using transfection reagent LipofectamineTM 2000. The proliferation, alkaline phosphatase (ALP) activity, calcification and osteogenic gene expression of osteoblasts were detected. The rat models with fracture of tibial plateau were divided into control group, fracture group, fracture+miR-182 group, and fracture+anti-miR-182 group. The levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and transforming growth factor β (TGFβ) in serum were detected by enzyme-linked immunosorbent assay (ELISA). Compared with the controls, the expression of miR-182 in serum was significantly elevated in patients with fracture of tibial plateau. Overexpression of miR-182 inhibited the proliferation of osteoblasts, while the knockdown of miR-182 increased the proliferation. MiR-182 could decrease the ALP activity of osteoblasts, while anti-miR-182 increased the ALP activity. Osteoblast calcification ability was significantly decreased by overexpression of miR-182. Knockdown of miR-182 increased the calcification ability of osteoblasts and the expression of osteogenic genes. MiR-182 could inhibit the expression of osteogenic genes. The levels of VEGF, EGF and TGFβ in the fracture group were higher than those in the control group, while the levels in the fracture+miR-182 group were higher than those in the fracture group. The levels of VEGF, EGF and TGFβ in the anti-miR-182 group were lower than those in the fracture group. MiR-182 is elevated in patients with fracture of tibial plateau, which can inhibit the proliferation and differentiation of osteoblasts and affect the fracture healing. The knockdown of miR-182 might be a new method for treating fracture healing.

High phosphate content in bioactive glasses promotes osteogenesis in vitro and in vivo

ObjectivesHigh phosphate content was found to significantly increase apatite formation of bioactive glasses (BGs) in vitro. However, there is very limited understanding of the effect of phosphate contents on osteogenesis which is important for clinical applications. The aims of this study were to investigate how phosphate content influences apatite formation ability of bioactive glasses in α-MEM culture medium and whether high phosphate content in bioactive glasses promotes osteogenesis in vitro and in vivo. MethodsFour phosphate containing bioactive glasses were synthesized via a melt-quench method and characterized using X-ray powder Diffraction (XRD), TGA-DSC and Fourier transform infra-red spectroscopy (FTIR). The apatite formation ability in α-MEM culture medium and the in vitro and in vivo osteogenic potential of these bioactive glass were explored. ResultsFTIR spectra confirmed faster apatite formation with an increase in phosphate content. The culture media containing ions released from the BGs showed enhanced cell viability and alkaline phosphatase activity of osteoblasts. Osteoblasts cultured with extracted BGs culture media generally showed increased proliferation, mineralized nodule formation, osteogenic and angiogenic genes expression with an increase in phosphate content in the glass compositions. An in vivo study demonstrated a larger amount of new bone formation in the calvarial defects implanted with high phosphate containing BG granules compared with that of BG without the presence of phosphate at 8 weeks post-surgery. SignificantThe presence of higher phosphate content accelerates apatite formation and promotes osteogenesis, indicating that both apatite formation and osteogenesis of bioactive glasses can be tailored by varying phosphate content for specific clinical needs and personalized treatments.

The Influence of Sodium Citrate on the Characteristics and Biological Activity of Plasma Rich in Growth Factors

Aim: This study investigated the effect of sodium citrate on the properties and biological activity of plasma rich in growth factors (PRGF). Methods: PRGF was obtained from trisodium citrate and plain extraction tubes. Hematological parameters, growth factors' release kinetics from both PRGF clots and their releasates' biological effect on human bone cells were evaluated. Results: The platelet enrichment factor, the growth factors' content and the release kinetic of PRGF were similar for both groups. The proliferation, collagen type I synthesis and tissue-nonspecific alkaline phosphatase activity of human osteoblasts showed no statistically significant differences. Conclusion: The use of sodium citrate does not influence the composition, the growth factors' release kinetics or the biological effect of PRGF, but it increases its clinical versatility.

Genistein Improves Bone Healing via Triggering Estrogen Receptor Alpha-Mediated Expressions of Osteogenesis-Associated Genes and Consequent Maturation of Osteoblasts.

Osteoporosis-associated fractures may cause higher morbidity and mortality. Our previous study showed the effects of genistein, a phytoestrogen, on the induction of estrogen receptor alpha (ERα) gene expression and stimulation of osteoblast mineralization. In this study, rat calvarial osteoblasts and an animal bone defect model were used to investigate the effects of genistein on bone healing. Treatment with genistein caused a time-dependent increase in alkaline phosphatase (ALP) activity in rat osteoblasts. Levels of cytosolic and nuclear ERα significantly augmented following exposure to genistein. Subsequently, genistein elevated levels of ALP mRNA and protein in rat osteoblasts. Moreover, genistein induced other osteogenesis-associated osteocalcin and Runx2 mRNA and protein expressions. Knocking-down ERα using RNA interference concurrently inhibited genistein-induced Runx2, osteocalcin, and ALP mRNA expression. Attractively, administration of ICR mice suffering bone defects with genistein caused significant increases in the callus width, chondrocyte proliferation, and ALP synthesis. Results of microcomputed tomography revealed that administration of genistein increased trabecular bone numbers and improved the bone thickness and volume. This study showed that genistein can improve bone healing via triggering ERα-mediated osteogenesis-associated gene expressions and subsequent osteoblast maturation.

In vitro study of a novel multi-substituted hydroxyapatite nanopowder synthesized by an ultra-fast, efficient and green microwave-assisted method.

In order to improve the biological activity of hydroxyapatite (HA), a multi-substituted HA (SHA) nanopowder with the chemical composition of Ca9.5Mg0.25Sr0.25(PO4)5.5(SiO4)0.5(OH)1.2F0.8 was synthesized using the microwave-assisted method. X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) revealed that all ions were substituted in the HA crystal lattice. The HA and SHA nanoparticles had a semi-spherical morphology with the average size of 90 and 80nm, respectively. In-vitro bioactivity assessments showed that after the 28-day immersion of the samples in the simulated body fluid, the morphology of the precipitated apatites on the surface of the HA sample still consisted of spherical particles with a cauliflower-like structure. However, in the SHA sample, the morphology of the precipitated apatites was changed to a nanorod-like one similar to the bone-like apatite, which may be attributed the presence of Sr in the precipitated apatites. The results showed that the release of the substituted ions not only had no adverse effect on the cell viability and cell attachment, but also enhanced the alkaline phosphatase activity of MG63 osteoblast like cells in the SHA group, as compared to the HA and control groups. The results indicated that the simultaneous substitution of Si, Mg, Sr, and F in HA nanoparticles could effectively promote bioactivity, cell proliferation and differentiation. This novel HA composition could be, therefore, well used for implant coating, bone tissue engineering and other orthopedic applications.

Osteogenic activity of Na2Ti3O7/SrTiO3 hybrid coatings on titanium

Titanium (Ti) and its alloys have been widely adopted as implant materials due to their satisfactory biocompatibility and favorable mechanical property. However, they usually show poor osteogenic ability, hindering its clinical application. Herein, Na2Ti3O7/SrTiO3 hybrid coatings on Ti were fabricated via one-step alkali etching in a mixed solution of NaOH and Sr(OH)2 to enhance its osteogenic activity. Scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and transmission electron microscopy were used to characterize the samples. Biological experiments were conducted to evaluate their osteogenic activity. The results showed Na2Ti3O7 nanowire coatings could be grown on Ti surface and Sr could be incorporated into the coatings. Incorporation of Sr clustered the nanowires and high Sr content lead to the formation of spherical SrTiO3 with diameter of about 1 μm. Sr2+ could continuously release from the samples and the release amount increased with Sr content in the coatings. Although adhesion, spreading, viability and proliferation of osteoblasts on all the samples had no significant difference when compared with Sr-free one, they could significantly promote osteogenic differentiation as verified by enhanced alkaline phosphatase (ALP) activity, collagen secretion, and extracellular matrix mineralization of osteoblasts. Further studies depicted that the expression levels of osteogenic-related genes including ALP, type I collagen and transforming growth factor-β were significantly upregulated in Sr-containing samples. The desirable osteogenic capacity may be ascribed to the synergistic effect of continuous Sr2+ release and nanostructured morphology. The samples containing Sr of 5.65–11.07 at.% showed the best osteogenic capacity therefore possess great potential in hard-tissue implant field.

UV-Pre-Treated and Protein-Adsorbed Titanium Implants Exhibit Enhanced Osteoconductivity.

Titanium materials are essential treatment modalities in the medical field and serve as a tissue engineering scaffold and coating material for medical devices. Thus, there is a significant demand to improve the bioactivity of titanium for therapeutic and experimental purposes. We showed that ultraviolet light (UV)-pre-treatment changed the protein-adsorption ability and subsequent osteoconductivity of titanium. Fibronectin (FN) adsorption on UV-treated titanium was 20% and 30% greater after 1-min and 1-h incubation, respectively, than that of control titanium. After 3-h incubation, FN adsorption on UV-treated titanium remained 30% higher than that on the control. Osteoblasts were cultured on titanium disks after 1-h FN adsorption with or without UV-pre-treatment and on titanium disks without FN adsorption. The number of attached osteoblasts during the early stage of culture was 80% greater on UV-treated and FN-adsorbed (UV/FN) titanium than on FN-adsorbed (FN) titanium; osteoblasts attachment on UV/FN titanium was 2.6- and 2.1-fold greater than that on control- and UV-treated titanium, respectively. The alkaline phosphatase activity of osteoblasts on UV/FN titanium was increased 1.8-, 1.8-, and 2.4-fold compared with that on FN-adsorbed, UV-treated, and control titanium, respectively. The UV/FN implants exhibited 25% and 150% greater in vivo biomechanical strength of bone integration than the FN- and control implants, respectively. Bone morphogenetic protein-2 (BMP-2) adsorption on UV-treated titanium was 4.5-fold greater than that on control titanium after 1-min incubation, resulting in a 4-fold increase in osteoblast attachment. Thus, UV-pre-treatment of titanium accelerated its protein adsorptivity and osteoconductivity, providing a novel strategy for enhancing its bioactivity.

Myonectin inhibits the differentiation of osteoblasts and osteoclasts in mouse cells

Myonectin is a myokine, which is involved in the pathophysiology of diabetes and obesity, and various myokines are involved in the interactions between skeletal muscle and bone. However, roles of myonectin in bone have still remained unknown. We therefore examined the effects of myonectin on mouse osteoblast and osteoclast differentiation in vitro. Myonectin significantly suppressed the mRNA levels of osteogenic genes and alkaline phosphatase (ALP) activity in mouse osteoblasts. As for osteoclasts, myonectin significantly suppressed osteoclast formation as well as the mRNA levels of osteoclast-related genes enhanced by receptor activator nuclear factor κB ligand (RANKL) from mouse monocytic RAW264.7 cells. Moreover, myonectin significantly suppressed osteoclast formation from mouse bone marrow cells in the presence of macrophage-colony stimulating factor and RANKL. On the other hand, myonectin significantly suppressed RANKL-induced oxygen consumption rate and peroxisome proliferator-activated receptor γ coactivator-1β mRNA levels in RAW264.7 cells, although myonectin did not affect these mitochondrial biogenesis parameters in mouse osteoblasts. In conclusion, the present study demonstrated that myonectin suppresses the differentiation and ALP activity in mouse osteoblasts. Moreover, myonectin suppressed osteoclast differentiation from mouse bone marrow and RAW264.7 cells partly through an inhibition of mitochondrial biogenesis.

Effect of bisphosphonate treatment of titanium surfaces on alkaline phosphatase activity in osteoblasts: a systematic review and meta-analysis

BackgroundBisphosphonate coating of dental implants is a promising tool for surface modification aiming to improve the osseointegration process and clinical outcome. The biological effects of bisphosphonates are thought to be mainly associated with osteoclasts inhibition, whereas their effects on osteoblast function are unclear. A potential of bisphosphonate coated surfaces to stimulate osteoblast differentiation was investigated by several in vitro studies with contradictory results. The purpose of this systematic review and meta-analysis was to evaluate the effect of bisphosphonate coated implant surfaces on alkaline phosphatase activity in osteoblasts.MethodsIn vitro studies that assessed alkaline phosphatase activity in osteoblasts following cell culture on bisphosphonate coated titanium surfaces were searched in electronic databases PubMed/MEDLINE, Scopus and ISI Web of Science. Animal studies and clinical trials were excluded. The literature search was restricted to articles written in English and published up to August 2019. Publication bias was assessed by the construction of funnel plots.ResultsEleven studies met the inclusion criteria. Meta-analysis showed that coating of titanium surfaces with bisphosphonates increases alkaline phosphatase activity in osteoblasts after 3 days (n = 1), 7 (n = 7), 14 (n = 6) and 21 (n = 3) days. (7 days beta coefficient = 1.363, p-value = 0.001; 14 days beta coefficient = 1.325, p-value < 0.001; 21 days beta coefficient = 1.152, p-value = 0.159).ConclusionsThe meta-analysis suggests that bisphosphonate coatings of titanium implant surfaces may have beneficial effects on osteogenic behaviour of osteoblasts grown on titanium surfaces in vitro. Further studies are required to assess to which extent bisphosphonates coating might improve osseointegration in clinical situations.