Differential osteoblastic activity in primary metaphyseal trabecular and secondary trabeculae of c-fos deficient mice.

OBJECTIVES: It has been highlighted that osteoblastic activities in remodeling-based bone formation are coupled with osteoclastic bone resorption while those in modeling-based bone formation are independent of osteoclasts. This study aimed to verify whether modeling-based bone formation can occur in the absence of osteoclasts. METHODS: We performed histochemical analyses on the bone of eight-week-old male wild-type and c-fos-/- mice. Histochemical analyses were conducted on primary trabeculae near the chondro-osseous junction (COJ), sites of modeling-based bone formation, and secondary trabeculae, sites of remodeling-based bone formation, in the femora and tibiae of mice. RESULTS: Alkaline phosphatase (ALP) immunoreactivity, a marker of osteoblastic lineages, was observed in the metaphyseal trabeculae of wild-type mice, while ALP was scattered throughout the femora of c-fos-/- mice. PHOSPHO1, an enzyme involved in matrix vesicle-mediated mineralization, was predominantly detected in primary trabeculae and also within short lines of osteoblasts in secondary trabeculae of wild-type mice. In contrast, femora of c-fos-/- mice showed several patches of PHOSPHO1 positivity in the primary trabeculae, but there were hardly any patches of PHOSPHO1 in secondary trabeculae. Calcein labeling was consistently observed in primary trabeculae close to the COJ in both wild-type and c-fos-/- mice; however, calcein labeling in the secondary trabeculae was only detected in wild-type mice. Transmission electron microscopic examination demonstrated abundant rough endoplasmic reticulum in the osteoblasts in secondary trabeculae of wild-type mice, but not in those of c-fos-/- mice. CONCLUSIONS: Osteoblastic activities at the sites of modeling-based bone formation may be maintained in the absence of osteoclasts.

Aucubin prevents steroid-induced osteoblast apoptosis by enhancing autophagy via AMPK activation.

Steroid-induced osteoblast apoptosis is a crucial pathological process in steroid-induced osteonecrosis of the femoral head (SONFH). Autophagy can resist apoptosis and AMPK plays an important role in autophagy regulation. Aucubin from the small tree Eucommia ulmoides Oliv., which has a long history of use in orthopaedics and traumatology in Asian medicine, can promote bone formation, but whether it can slow or prevent steroid-osteoblast apoptosis is unclear. Therefore, we investigated the pathogenesis of SONFH and how the osteoblast responds to aucubin under the dexamethasone stimulation. In human femoral head osteonecrosis specimens, we found that the autophage and apoptosis level were increased, and the AMPK signalling was crucial to autophagy. We observed that aucubin could prevent dexamethasone-induced apoptosis in osteoblasts by enhancing the level of autophagy. Further, we confirmed that the regulatory effect of aucubin on autophagy and apoptosis was achieved by activating AMPK signalling. We have demonstrated a mechanism of disease progression and shown that aucubin could enhance autophagy through AMPK signalling to prevent osteoblast apoptosis. These findings provide a basis for the further investigation of the potential therapeutic role of aucubin in the SONFH.

Vitamin D3/vitamin K2/magnesium-loaded polylactic acid/tricalcium phosphate/polycaprolactone composite nanofibers demonstrated osteoinductive effect by increasing Runx2 via Wnt/ß-catenin pathway.

Vitamin D3, vitamin K2, and Mg (10%, 1.25%, and 5%, w/w, respectively)-loaded PLA (12%, w/v) (TCP (5%, w/v))/PCL (12%, w/v) 1:1 (v/v) composite nanofibers (DKMF) were produced by electrospinning method (ES) and their osteoinductive effects were investigated in cell culture test. Neither pure nanofibers nor DKMF caused a significant cytotoxic effect in fibroblasts. The induction of the stem cell differentiation into osteogenic cells was observed in the cell culture with both DKMF and pure nanofibers, separately. Vitamin D3, vitamin K2, and magnesium demonstrated to support the osteogenic differentiation of mesenchymal stem cells by expressing Runx2, BMP2, and osteopontin and suppressing PPAR-γ and Sox9. Therefore, the Wnt/ß-catenin signaling pathway was activated by DKMF. DKMF promoted large axonal sprouting and needle-like elongation of osteoblast cells and enhanced cellular functions such as migration, infiltration, proliferation, and differentiation after seven days of incubation using confocal laser scanning microscopy. The results showed that DKMF demonstrated sustained drug release for 144 h, tougher and stronger structure, higher tensile strength, increased water up-take capacity, decreased degradation ratio, and slightly lower Tm and Tg values compared to pure nanofibers. Consequently, DKMF is a promising treatment approach in bone tissue engineering due to its osteoinductive effects.

Proximity proteomics identifies PAK4 as a component of Afadin-Nectin junctions.

Human PAK4 is an ubiquitously expressed p21-activated kinase which acts downstream of Cdc42. Since PAK4 is enriched in cell-cell junctions, we probed the local protein environment around the kinase with a view to understanding its location and substrates. We report that U2OS cells expressing PAK4-BirA-GFP identify a subset of 27 PAK4-proximal proteins that are primarily cell-cell junction components. Afadin/AF6 showed the highest relative biotin labelling and links to the nectin family of homophilic junctional proteins. Reciprocally >50% of the PAK4-proximal proteins were identified by Afadin BioID. Co-precipitation experiments failed to identify junctional proteins, emphasizing the advantage of the BioID method. Mechanistically PAK4 depended on Afadin for its junctional localization, which is similar to the situation in Drosophila. A highly ranked PAK4-proximal protein LZTS2 was immuno-localized with Afadin at cell-cell junctions. Though PAK4 and Cdc42 are junctional, BioID analysis did not yield conventional cadherins, indicating their spatial segregation. To identify cellular PAK4 substrates we then assessed rapid changes (12') in phospho-proteome after treatment with two PAK inhibitors. Among the PAK4-proximal junctional proteins seventeen PAK4 sites were identified. We anticipate mammalian group II PAKs are selective for the Afadin/nectin sub-compartment, with a demonstrably distinct localization from tight and cadherin junctions.

Osteoblasts-derived exosomes regulate osteoclast differentiation through miR-503-3p/Hpse axis.

MicroRNAs (miRNAs) are involved in bone remodeling by regulating the balance of bone formation and resorption. Increasing evidence has confirmed that the communication between osteoclast and osteoblast through secreting exosomes and transferring miRNAs. It has been reported that mineralized osteoblasts release exosomes containing more miR-503-3p. However, the roles and molecular mechanisms of osteoblast exosomes-derived miR-503-3p in osteoclast differentiation remain elusive. Here, we isolated exosomes from the supernatant of osteoblasts and identified the exosome characterization through transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot assay. In addition, we found that exosomes and miR-503-3p secreted by osteoblasts inhibited the differentiation of osteoclast progenitor cells. Meanwhile, we found that Hpse (heparanase gene) was a target gene of miR-503-3p and miR-503-3p inhibited the osteoclast differentiation through downregulating the expression of Hpse. In summary, our results demonstrated the roles and the mechanism of osteoblast-derived exosomes inhibited the osteoclast differentiation via miR-503-3p/Hpse axis.

Label-retention expansion microscopy.

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

Genetically Modified Ferritin Nanoparticles with Bone-Targeting Peptides for Bone Imaging.

Bone homeostasis plays a major role in supporting and protecting various organs as well as a body structure by maintaining the balance of activities of the osteoblasts and osteoclasts. Unbalanced differentiation and functions of these cells result in various skeletal diseases, such as osteoporosis, osteopetrosis, and Paget's disease. Although various synthetic nanomaterials have been developed for bone imaging and therapy through the chemical conjugation, they are associated with serious drawbacks, including heterogeneity and random orientation, in turn resulting in low efficiency. Here, we report the synthesis of bone-targeting ferritin nanoparticles for bone imaging. Ferritin, which is a globular protein composed of 24 subunits, was employed as a carrier molecule. Bone-targeting peptides that have been reported to specifically bind to osteoblast and hydroxyapatite were genetically fused to the N-terminus of the heavy subunit of human ferritin in such a way that the peptides faced outwards. Ferritin nanoparticles with fused bone-targeting peptides were also conjugated with fluorescent dyes to assess their binding ability using osteoblast imaging and a hydroxyapatite binding assay; the results showed their specific binding with osteoblasts and hydroxyapatite. Using in vivo analysis, a specific fluorescent signal from the lower limb was observed, demonstrating a highly selective affinity of the modified nanoparticles for the bone tissue. These promising results indicate a specific binding ability of the nanoscale targeting system to the bone tissue, which might potentially be used for bone disease therapy in future clinical applications.

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

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

Large-Pore Platelet-Rich Fibrin with a Mg Ring to Allow MC3T3-E1 Preosteoblast Migration and to Improve Osteogenic Ability for Bone Defect Repair.

Platelet-rich fibrin (PRF) is a natural fibrin meshwork material with multiple functions that are suitable for tissue engineering applications. PRF provides a suitable scaffold for critical-size bone defect treatment due to its platelet cytokines and rich growth factors. However, the structure of PRF not only promotes cell attachment but also, due to its density, provides a pool for cell migration into the PRF to facilitate regeneration. In our study, we used repeated freeze drying to enlarge the pores of PRF to engineer large-pore PRF (LPPRF), a type of PRF that has expanded pores for cell migration. Moreover, a biodegradable Mg ring was used to provide stability to bone defects and the release of Mg ions during degradation may enhance osteoconduction and osteoinduction. Our results revealed that cell migration was more extensive when LPPRF was used rather than when PRF was used and that LPPRF retained the growth factors present in PRF. Moreover, the Mg ions released from the Mg ring during degradation significantly enhanced the calcium deposition of MC3T3-E1 preosteoblasts. In the present study, a bone substitute comprising LPPRF combined with a Mg ring was demonstrated to have much potential for critical-size bone defect repair.

Injectable tricalcium phosphate/calcium sulfate granule enhances bone repair by reversible setting reaction.

Towards repairing bone defects, calcium sulfate and calcium phosphate cement have been recognized as promising bone grafts. However, the current bone cements are generally lack of proper porosity for cell migration and new tissue formation. On the other hand, porous scaffold cannot be delivered by injection, which limits its use its clinical use. Herein, we develop a novel tricalcium phosphate/calcium sulfate granule to overcome the limitations of injectable cements and traditional scaffolds. The biocompatible granule underwent in situ self-setting to form scaffold with porous structure after injection. It contributes to calcium deposition and upregulation of osteogenic genes of mesenchymal stem cells in a time-dependent manner. Within three months, cavitary bone defects of distal rabbit femurs implanted the granules exhibited better bone formation than those with those implanted with autologous bone.

Effect of surface topography on in vitro osteoblast function and mechanical performance of 3D printed titanium.

Critical-sized defects remain a significant challenge in orthopaedics. 3D printed scaffolds are a promising treatment but are still limited due to inconsistent osseous integration. The goal of the study is to understand how changing the surface roughness of 3D printed titanium either by surface treatment or artificially printing rough topography impacts the mechanical and biological properties of 3D printed titanium. Titanium tensile samples and discs were printed via laser powder bed fusion. Roughness was manipulated by post-processing printed samples or by directly printing rough features. Experimental groups in order of increasing surface roughness were Polished, Blasted, As Built, Sprouts, and Rough Sprouts. Tensile behavior of samples showed reduced strength with increasing surface roughness. MC3T3 pre-osteoblasts were seeded on discs and analyzed for cellular proliferation, differentiation, and matrix deposition at 0, 2, and 4 weeks. Printing roughness diminished mechanical properties such as tensile strength and ductility without clear benefit to cell growth. Roughness features were printed on mesoscale, unlike samples in literature in which roughness on microscale demonstrated an increase in cell activity. The data suggest that printing artificial roughness on titanium scaffold is not an effective strategy to promote osseous integration.

Atomic Layer Deposition of ZrO2 on Titanium Inhibits Bacterial Adhesion and Enhances Osteoblast Viability.

PURPOSE: The study was intended to create a uniform zirconia layer even on the surface of complex structures via atomic layer deposition (ALD). The impact of crystalline zirconia deposited by ALD on bacterial adhesion and osteoblast viability was assessed via surface treatment of dental implants. METHODS: Amorphous zirconia was deposited using an atomic layer deposition reactor (Atomic Classic, CN1, Hwaseong, Korea) on titanium discs. Heating the samples at 400°C resulted in crystallization. Samples were divided into three groups: the control group, the group carrying amorphous ALD-zirconia (Z group), and the heat-treated group following zirconia ALD deposition (ZH group).The surface of each sample was analyzed, followed by the assessment of adhesion of Streptococcus mutans and Porphyromonas gingivalis, and viability and differentiation of MC3T3-E1 cells. RESULTS: The adhesion of S. mutans and P. gingivalis was significantly reduced in the Z and ZH groups compared with the control group (P < 0.05). The viability of MC3T3-E1 cells was significantly increased in the ZH group compared with the control group (P < 0.001), while no significant differences were observed in the Z group (P > 0.05). Differentiation of MC3T3-E1 cells showed a marginally significant increase in the ZH group compared with the control group (P < 0.1), while no significant differences were found in the Z group (P > 0.1). CONCLUSION: Compared with the pure titanium group, the groups that were coated with zirconia via ALD showed a decreased adhesion of S. mutans during the early stages of biofilm formation and P. gingivalis adhesion inducing peri-implantitis, and an increase in MC3T3-E1 cell viability and differentiation. The findings indicate the possibility of treating the implant surface to reduce peri-implantitis and improve osseointegration.

Development of orthophosphosilicate glass/poly(lactic acid) composite anisotropic scaffolds for simultaneous reconstruction of bone quality and quantity.

Reconstruction of organ-specific architecture is necessary to recover the original organ function. The anisotropic structure of bone tissue is strongly related to the collagen fibril alignment and bone apatite crystal direction. Bone regeneration indicates following two main process; first, restoration of bone mineral density (BMD; bone quantity), and second, restoring bone apatite c-axis orientation (bone quality). In addition to BMD, bone quality is the most important factor among bone mechanical properties. Recovery of the original bone function requires development of novel scaffolds with simultaneous reconstruction of bone quality and quantity. Herein, novel orthophosphosilicate glass (PSG)/poly(lactic acid) composite anisotropic scaffolds were developed to control cell alignment and enhance bone formation, which are important for the simultaneous reconstruction of bone quality and quantity. The strategy to control cell alignment and bone formation involved designing anisotropic scaffolds in combination with the release of therapeutic ions by PSGs. The morphology of fibrous scaffolds containing PSGs was quantitatively designed using electrospinning. This successfully modulated cell alignment and subsequent bone apatite c-axis orientation along the fiber-oriented direction. The released silicate and Mg2+ ions from PSGs in scaffolds improved cell adhesion, proliferation, and calcification. To best of our knowledge, this is the first report demonstrating that the anisotropic scaffolds containing bioactive glasses regenerate bone tissues with simultaneous reconstruction of bone quality and quantity via stimulating osteoblasts by inorganic ions and designing morphology of scaffolds.

Evaluation of osteogenic and antibacterial properties of strontium/silver-containing porous TiO2 coatings prepared by micro-arc oxidation.

Ti and Ti alloys are bioinert materials and two frequent problems associated with them are bacterial infection and lack of osteogenic potential for rapid bone integration. To overcome the problems, the present study incorporated strontium (Sr) and silver (Ag) simultaneously into porous TiO2 coatings through a single-step technique, micro-arc oxidation (MAO). Incorporation of Sr and Ag brought no significant changes to coating micromorphology and physicochemical properties, but endowed TiO2 coatings with both strong antibacterial activity and osteogenic ability. Antibacterial activity increased with Ag contents in the coatings. When Ag content reached 0.58 wt%, the coating showed both excellent short-term (100.0%) and long-term (77.6%) antibacterial activities. Sr/Ag-containing coatings with 18.23 wt% Sr and 0.58 wt% Ag also presented good cytocompatibility for preosteoblast adhesion and proliferation, and promoted preosteoblast osteogenic differentiation both short-termly and long-termly. However, higher Ag content (1.29 wt%) showed toxic effects to preosteoblasts. In summary, MAO is a simple and effective way to incorporate Sr and Ag into porous TiO2 coatings and Sr/Ag-containing TiO2 coating with 18.5 wt% Sr and 0.58 wt% Ag has both good osteogenic activity and strong antibacterial capability short-termly and long-termly. Therefore, such coatings are valuable for clinical application to strengthen osseointegration and long-term high quality use of titanum implants.

Multiparametric Analysis of Focal Adhesions in Bidimensional Substrates.

Focal adhesions in planar substrates constitute an excellent cellular resource to evaluate different parameters related to cell morphology, cytoskeletal organization, and adhesive strength. However, their intrinsic heterogeneity in terms of size, molecular composition, orientation, and so on complicates their analysis. Here, we describe a simple and straightforward ImageJ/Fiji-based method to quantify several parameters that describe the morphology and relative composition of focal adhesions. This type of analysis can be implemented in various ways and become useful for drug and shRNA screenings.

Preparation of Thin Frozen Sections from Nonfixed and Undecalcified Hard Tissues Using Kawamoto's Film Method (2020).

A method for preparing frozen sections with an adhesive film is described. In order to observe fine structures and weak fluorescence of samples, new types of adhesive films [Cryofilm type 3C(16UF) and 4D(16UF)] are used. The adhesive film is made with very clear and very low autofluorescence. For gene analysis, a very thin adhesive film (LMD film) is used to cut by means of the laser microdissection (LMD). For MALDI mass spectrometry imaging (MALDI-MSI), a conductive adhesive film (Cryofilm type MS) is used to avoid electric charge of the sample. A biological sample is frozen quickly and freeze-embedded. The frozen sample is cut with a very sharp disposable blade made from fine tungsten carbide. The combination of the adhesive films and the blade can generate 3 micrometer thick sections from samples including bone, while it is also possible to generate 1 µm thick sections. The morphology of bone and soft tissues are preserved using this method. Cells such as osteoblasts, fibroblasts, and osteoclasts are clearly observed with an oil immersion lens at high magnification. Sections generated using the Cryofilm type 3C(16UF) shows weak fluorescent signals more clearly than sections generated with the previously reported adhesive films [Cryofilm type 2C(9) and 2C(10)]. Furthermore fluorescence of the fine structures in cells is clearly shown using a super-high-resolution microscope. Several staining and experimental methods such as histology, histochemistry, enzyme histochemistry, immunohistochemistry, and in situ hybridization can be performed on these sections. This method is also useful for preparing frozen sections of large sample such as a whole-body mouse and rat. In gene analysis, gene quality of sample collected from the section made with the LMD film is superior to that of sample made by a conventional method. The Cryofilm type MS makes almost complete section from tissues including hard tissues and large samples. The satisfactory signals are detected from the section with MALDI-MSI.

Comparative analysis of titanium coating on cobalt-chrome alloy in vitro and in vivo direct metal fabrication vs. plasma spraying.

BACKGROUND: Titanium surface coating on cobalt-chromium (CoCr) alloy has characteristics desirable for an orthopedic implant as follows: strength, osteointegrative capability, and biocompatibility. Creating such a coated surface takes a challenging process and two dissimilar metals are not easily welded. In our study, we utilized additive manufacturing with a 3D printing called direct metal fabrication (DMF) and compared it to the plasma spraying method (TPS), to coat titanium onto CoCr alloy. We hypothesized that this would yield a coated surface quality as acceptable or better than the already established method of plasma spraying. For this, we compared characteristics of titanium-coated surfaces created by direct metal fabrication method (DMF) and titanium plasma spraying (TPS), both in vitro and in vivo, for (1) cell morphology, (2) confocal microscopy images of immunofluorescent assay of RUNX2 and fibronectin, (3) quantification of cell proliferation rate, (4) push-out biomechanical test, and (5) bone histomorphometry. METHOD: For in vitro study, human osteoblast cells were seeded onto the coated surfaces. Cellular morphology was observed with a scanning electron microscope. Cellular proliferation was validated with ELISA, immunofluorescent assay. For in vivo study, coated rods were inserted into the distal femur of the rabbit and then harvested. The rods were biomechanically tested with a push-out test and observed for histomorphometry to evaluate the microscopic bone to implant ratio. RESULT: For cell morphology observation, lamellipodia and filopodia, a cytoplasmic projection extending into porous structure, formed on both surfaces created by DMF and TPS. The proliferation of the osteoblasts, the DMF group showed a better result at different optic density levels (p = 0.035, 0.005, 0.001). Expression and distribution of fibronectin and Runx-2 genes showed similar degrees of expressions. The biomechanical push-out test yielded a similar result (p = 0.714). Histomorphometry analysis also showed a similar result (p = 0.657). CONCLUSION: In conclusion, DMF is a method which can reliably create a proper titanium surface on CoCr alloy. The resulting product of the surface shows a similar quality to that of the plasma spraying method, both in vivo and in vitro, in terms of biological and mechanical property.

Chitosan Coating of TiO2 Nanotube Arrays for Improved Metformin Release and Osteoblast Differentiation.

BACKGROUND: Ineffective integration has been recognized as one of the major causes of early orthopedic failure of titanium-based implants. One strategy to address this problem is to develop modified titanium surfaces that promote osteoblast differentiation. This study explored titanium surfaces modified with TiO2 nanotubes (TiO2 NTs) capable of localized drug delivery into bone and enhanced osteoblast cell differentiation. MATERIALS AND METHODS: Briefly, TiO2 NTs were subjected to anodic oxidation and loaded with Metformin, a widely used diabetes drug. To create surfaces with sustainable drug-eluting characteristics, TiO2 NTs were spin coated with a thin layer of chitosan. The surfaces were characterized via scanning electron microscopy, atomic force microscopy, and contact angle measurements. The surfaces were then exposed to mesenchymal bone marrow stem cells (MSCs) to evaluate cell adhesion, growth, differentiation, and morphology on the modified surfaces. RESULTS: A noticeable increase in drug release time (3 days vs 20 days) and a decrease in burst release characteristics (85% to 7%) was observed in coated samples as compared to uncoated samples, respectively. Chitosan-coated TiO2 NTs exhibited a considerable enhancement in cell adhesion, proliferation, and genetic expression of type I collagen, and alkaline phosphatase activity as compared to uncoated TiO2 NTs. CONCLUSION: TiO2 NT surfaces with a chitosan coating are capable of delivering Metformin to a bone site over a sustained period of time with the potential to enhance MSCs cell attachment, proliferation, and differentiation.

Epigenetic Regulation of Skeletal Tissue Integrity and Osteoporosis Development.

Bone turnover is sophisticatedly balanced by a dynamic coupling of bone formation and resorption at various rates. The orchestration of this continuous remodeling of the skeleton further affects other skeletal tissues through organ crosstalk. Chronic excessive bone resorption compromises bone mass and its porous microstructure as well as proper biomechanics. This accelerates the development of osteoporotic disorders, a leading cause of skeletal degeneration-associated disability and premature death. Bone-forming cells play important roles in maintaining bone deposit and osteoclastic resorption. A poor organelle machinery, such as mitochondrial dysfunction, endoplasmic reticulum stress, and defective autophagy, etc., dysregulates growth factor secretion, mineralization matrix production, or osteoclast-regulatory capacity in osteoblastic cells. A plethora of epigenetic pathways regulate bone formation, skeletal integrity, and the development of osteoporosis. MicroRNAs inhibit protein translation by binding the 3'-untranslated region of mRNAs or promote translation through post-transcriptional pathways. DNA methylation and post-translational modification of histones alter the chromatin structure, hindering histone enrichment in promoter regions. MicroRNA-processing enzymes and DNA as well as histone modification enzymes catalyze these modifying reactions. Gain and loss of these epigenetic modifiers in bone-forming cells affect their epigenetic landscapes, influencing bone homeostasis, microarchitectural integrity, and osteoporotic changes. This article conveys productive insights into biological roles of DNA methylation, microRNA, and histone modification and highlights their interactions during skeletal development and bone loss under physiological and pathological conditions.

Laser Nd:YAG patterning enhance human osteoblast behavior on zirconia implants.

Zirconia has been regarded as a promising material for dental implants, and Nd:YAG laser treatment has been proposed as a potential strategy to improve its bioactivity. The main aim of the present study was to evaluate the in vitro behavior of human fetal osteoblasts in contact with laser-textured zirconia implant surfaces assessing the effect of different texture patterns, spacing between laser passes and number of laser passes. Zirconia discs were produced and treated with Nd:YAG laser according to test group variables: texture (microgrooves and micropillar array), distance between surface features (25 µm, 30 µm and 35 µm), and laser passes [1, 2, 4, and 8]. Untextured sandblasted and acid-etched zirconia discs (SBAE) were used as controls. Human osteoblasts (hFOB 1.19) were cultured for 14 days on test and control samples. Morphology and cellular adhesion were observed using scanning electron microscopy (SEM). Cell viability and proliferation were evaluated at 1, 3, 7, and 14 days using a commercial resazurin-based method. Collagen type I was evaluated at 3 days using ELISA. Alkaline phosphatase (ALP) activity was evaluated at 7 days using a colorimetric enzymatic technique. Group comparisons were tested using ANOVA or Mann-Whitney test (Tukey's post hoc) using statistical software, and significance was set at p < 0.05. Cell viability and proliferation increased over time for all groups with statistically higher values for laser-textured groups when compared with control at 7 and 14 days in culture (p < 0.05). Collagen type I levels were higher for study groups (p < 0.05) when compared with control group. No statistically differences were detected for ALP activity levels between texture and control groups (p > 0.05). The results suggest that laser-machined zirconia implant surfaces may benefit biological osteoblast response. However, the type of texture, spacing at the range of 25-35 µm, and number of laser passes did not seem to be relevant variables.