Photobiomodulation Modulates Proliferation and Gene Expression Related to Calcium Signaling in Human Osteoblast Cells.

Introduction: Photobiomodulation with low-level laser treatment can enhance bone formation by stimulating the cell division of osteoblasts and increasing the amount of protein deposition, thus encouraging the formation of new bone. The aim of this study was to evaluate the effects of photobiomodulation with a low-level laser on proliferation and gene expression related to calcium signaling in human osteoblasts. Methods: Osteoblastic cell lines of the hFOB1.19 lineage, human osteoblasts, were grown and assigned into two groups, control (C; n=78 cultured wells) and photobiomodulation (L; n=78 cultured wells) with n=6 per day of the experimental period. Cells were cultured (immature at 34 ºC), and after maturation at 37 ºC, group L cells were exposed to laser irradiation with a low-level laser device (gallium and aluminum arsenide), at a wavelength of 808 nm, a power output of 200 mW, and a power density of 200 mW/cm2. The energy delivered to the cells was 37 J/cm2, with a beam area of 0.02 mm2 and an exposure time of 5 seconds. This treatment was applied daily for a period of 13 days. Following this, the number of cells was counted, and RNA was isolated, measured, and then converted into cDNA for further quantification using a comparative Ct method with real-time polymerase chain reaction. The results were then subjected to statistical analysis through a Mann-Whitney test, with a significance level of P<0.05. Results: The cell count in the L group (37.25x10±4±22.02) was statistically higher compared to the control group (22.75x10±4±7.660) with a P value of 0.0259. The values of 2-ΔΔCt for S100A6, plasma membrane calcium ATPase (PMCA), and calmodulin genes indicated hyper-expression on the thirteenth day, while the osteocalcin gene showed hypo-expression. Conclusion: The study suggests that the photobiomodulation mechanism with a low-level laser may regulate gene expression in human osteoblasts in a dose-dependent and cumulative manner.

Osteoblasts-derived exosomes as potential novel communicators in particle-induced periprosthetic osteolysis.

The inflammatory response to wear particles derived from hip prothesis is considered a hallmark of periprosthetic osteolysis, which can ultimately lead to the need for revision surgery. Exosomes (Exos) have been associated with various bone pathologies, and there is increasing recognition in the literature that they actively transport molecules throughout the body. The role of wear particles in osteoblast-derived Exos is unknown, and the potential contribution of Exos to osteoimmune communication and periprosthetic osteolysis niche is still in its infancy. Given this, we investigate how titanium dioxide nanoparticles (TiO2 NPs), similar in size and composition to prosthetic wear particles, affect Exos biogenesis. Two osteoblastic cell models commonly used to study the response of osteoblasts to wear particles were selected as a proof of concept. The contribution of Exos to periprosthetic osteolysis was assessed by functional assays in which primary human macrophages were stimulated with bone-derived Exos. We demonstrated that TiO2 NPs enter multivesicular bodies, the nascent of Exos, altering osteoblast-derived Exos secretion and molecular cargo. No significant differences were observed in Exos morphology and size. However, functional assays reveal that Exos cargo enriched in uPA stimulates macrophages to a mixed M1 and M2 phenotype, inducing the release of pro- and anti-inflammatory signals characteristic of periprosthetic osteolysis. In addition, we demonstrated the expression of uPA in exosomes derived from the urine of patients with osteolysis. These results suggest that uPA can be a potential biomarker of osteolysis. In the future, uPa may serve as a possible non-invasive biomarker to identify patients at risk for peri-implant osteolysis.

HIV Modulates Osteoblast Differentiation via Upregulation of RANKL and Vitronectin.

Bone loss is a prevalent characteristic among people with HIV (PWH). We focused on mesenchymal stem cells (MSCs) and osteoblasts, examining their susceptibility to different HIV strains (R5- and X4-tropic) and the subsequent effects on bone tissue homeostasis. Our findings suggest that MSCs and osteoblasts are susceptible to R5- and X4-tropic HIV but do not support productive HIV replication. HIV exposure during the osteoblast differentiation process revealed that the virus could not alter mineral and organic matrix deposition. However, the reduction in runt-related transcription factor 2 (RUNX2) transcription, the increase in the transcription of nuclear receptor activator ligand kappa B (RANKL), and the augmentation of vitronectin deposition strongly suggested that X4- and R5-HIV could affect bone homeostasis. This study highlights the HIV ability to alter MSCs' differentiation into osteoblasts, critical for maintaining bone and adipose tissue homeostasis and function.

In Vitro Evaluation of Cellular Interactions with Nanostructured Spheres of Alginate and Zinc-Substituted Carbonated Hydroxyapatite.

The increasing demand for effective bone regeneration materials drives the exploration of biomaterials with enhanced bioactivity and biocompatibility, such as zinc-substituted compounds. This study investigates the in vitro cellular interactions with nanostructured spheres composed of alginate/carbonated hydroxyapatite (CHA), compared to zinc-substituted CHA (ZnCHA). This work aimed to compare the physicochemical properties and biological effects of ZnCHA and CHA on osteoblasts. ZnCHA was synthesized using a wet chemical method, followed by characterization through X-ray diffraction, Fourier transform infrared spectroscopy, total organic carbon analysis, Wavelength-dispersive X-ray spectroscopy, and BET surface area analysis to assess ion release and structural changes. Biological evaluation was conducted using cell viability, proliferation, and biomineralization assays on osteoblasts. Results showed successful incorporation of zinc and carbonate, leading to reduced crystallinity and increased surface area. Cell viability and proliferation assays indicated ZnCHA's cytocompatibility and enhanced osteoblastic activity, with increased mineralization nodules compared to CHA samples. The study concludes that ZnCHA composites are promising candidates for bone tissue engineering, demonstrating improved cytocompatibility and potential for further preclinical evaluations.

Exploring the osteogenic potential of semisynthetic triterpenes from Combretum leprosum: An in vitro and in silico study.

Combretum leprosum Mart. is a plant of the Combretaceae family, widely distributed in the Northeast region of Brazil, popularly used as an anti-inflammatory agent, and rich in triterpenes. This study evaluated in vitro and in silico potential osteogenic of two semisynthetic triterpenes (CL-P2 and CL-P2A) obtained from the pentacyclic triterpene 3ß,6ß,16ß-trihydroxylup-20(29)-ene (CL-1) isolated from C. leprosum. Assays were carried out in cultured murine osteoblasts (OFCOL II), first investigating the possible toxicity of the compounds on these cells through viability assays (MTT). Cell proliferation and activation were investigated by immunohistochemical evaluation of Ki-67, bone alkaline phosphatase (ALP) activity, and mineralization test by Von Kossa. Molecular docking analysis was performed to predict the binding affinity of CL-P2 and CL-P2A to target proteins involved in the regulation of osteogenesis, including: bone morphogenetic protein 2 (BMP-2), proteins related to Wingless-related integration (WNT) pathway (Low-density lipoprotein receptor-related protein 6-LRP6 and sclerostin-SOST), and receptor activator of nuclear factor (NF)-kB-ligand (RANK-L). Next, Western Blot and immunofluorescence investigated BMP-2, WNT, RANK-L, and OPG protein expressions in cultured murine osteoblasts (OFCOL II). None of the CL-P2 and CL-P2A concentrations were toxic to osteoblasts. Increased cell proliferation, ALP activity, and bone mineralization were observed. Molecular docking assays demonstrated interactions with BMP-2, LRP6, SOST, and RANK-L/OPG. There was observed increased expression of BMP-2, WNT, and RANK-L/OPG proteins. These results suggest, for the first time, the osteogenic potential of CL-P2 and CL-P2A.

The Involvement of microRNAs in Bone Remodeling Signaling Pathways and Their Role in the Development of Osteoporosis.

Bone remodeling, crucial for maintaining the balance between bone resorption and formation, relies on the coordinated activity of osteoclasts and osteoblasts. During osteoclastogenesis, hematopoietic stem cells (HSCs) differentiate into the osteoclast lineage through the signaling pathways OPG/RANK/RANKL. On the other hand, during osteoblastogenesis, mesenchymal stem cells (MSCs) differentiate into the osteoblast lineage through activation of the signaling pathways TGF-ß/BMP/Wnt. Recent studies have shown that bone remodeling is regulated by post-transcriptional mechanisms including microRNAs (miRNAs). miRNAs are small, single-stranded, noncoding RNAs approximately 22 nucleotides in length. miRNAs can regulate virtually all cellular processes through binding to miRNA-response elements (MRE) at the 3' untranslated region (3'UTR) of the target mRNA. miRNAs are involved in controlling gene expression during osteogenic differentiation through the regulation of key signaling cascades during bone formation and resorption. Alterations of miRNA expression could favor the development of bone disorders, including osteoporosis. This review provides a general description of the miRNAs involved in bone remodeling and their significance in osteoporosis development.

The Incorporation of Zinc into Hydroxyapatite and Its Influence on the Cellular Response to Biomaterials: A Systematic Review.

Zinc is known for its role in enhancing bone metabolism, cell proliferation, and tissue regeneration. Several studies proposed the incorporation of zinc into hydroxyapatite (HA) to produce biomaterials (ZnHA) that stimulate and accelerate bone healing. This systematic review aimed to understand the physicochemical characteristics of zinc-doped HA-based biomaterials and the evidence of their biological effects on osteoblastic cells. A comprehensive literature search was conducted from 2022 to 2024, covering all years of publications, in three databases (Web of Science, PUBMED, Scopus), retrieving 609 entries, with 36 articles included in the analysis according to the selection criteria. The selected studies provided data on the material's physicochemical properties, the methods of zinc incorporation, and the biological effects of ZnHA on bone cells. The production of ZnHA typically involves the wet chemical synthesis of HA and ZnHA precursors, followed by deposition on substrates using processes such as liquid precursor plasma spraying (LPPS). Characterization techniques confirmed the successful incorporation of zinc into the HA lattice. The findings indicated that zinc incorporation into HA at low concentrations is non-cytotoxic and beneficial for bone cells. ZnHA was found to stimulate cell proliferation, adhesion, and the production of osteogenic factors, thereby promoting in vitro mineralization. However, the optimal zinc concentration for the desired effects varied across studies, making it challenging to establish a standardized concentration. ZnHA materials are biocompatible and enhance osteoblast proliferation and differentiation. However, the mechanisms of zinc release and the ideal concentrations for optimal tissue regeneration require further investigation. Standardizing these parameters is essential for the effective clinical application of ZnHA.

Comparison of Radiographic Outcomes Assessed via the Radiographic Union Scale for Tibial Fractures and Alkaline Phosphatase Levels during the Tibial Healing Process: A Series of Case Reports.

BACKGROUND/OBJECTIVES: Tibial diaphysis fractures are common injuries resulting from high-to-low-energy traumas in patients of all age groups, but few reports currently provide complementary parameters for the assessment of bone healing processes in the postoperative period. Serum alkaline phosphatase (ALP) and the scores from the Radiographic Union Scale for Tibial Fractures (RUST) can promote new horizons in this context. Therefore, the aim of this study was to assess the behavior of ALP and RUST through within-subject comparisons from immediately post-surgery to 49 days after tibial diaphysis fracture repair. METHODS: This article included four case studies where patients underwent the same procedures. Adults of both sexes aged 18 to 60 years with tibial fractures requiring surgery were included. After surgical intervention (T1), the patients were followed for 49 days after surgery, returning for follow-up appointments on the 21st (T2) and 49th (T3) days. At the follow-up appointments, new X-ray images were obtained, and blood samples were collected for ALP measurement. RESULTS: Serum ALP levels increased by T2 following tibial reamed intramedullary nailing surgery. While this increase persisted into T3 for two patients, a decline was observed during the same period for the other two patients. Both events are indicative of the bone consolidation process, and RUST scores at the T3 corroborate this perspective for all patients included in this study. Considering that delta ALP (T3-T1 value) was lower in patients who exhibited the highest RUST score, we suggest that a synchronized analysis between ALP and RUST allows medics to diagnose bone consolidation. CONCLUSIONS: Therefore, it can be concluded that the analysis of ALP alongside RUST may be complementary for evaluating bone consolidation following tibial reamed intramedullary nailing surgery, but future studies are needed to confirm this assertion.

Novel PVA-Hyaluronan-Siloxane Hybrid Nanofiber Mats for Bone Tissue Engineering.

Hyaluronan (HA) is a natural biodegradable biopolymer; its biological functions include cell adhesion, cell proliferation, and differentiation as well as decreasing inflammation, angiogenesis, and regeneration of damaged tissue. This makes it a suitable candidate for fabricating nanomaterials with potential use in tissue engineering. However, HA nanofiber production is restricted due to the high viscosity, low evaporation rate, and high surface tension of HA solutions. Here, hybrids in the form of continuous and randomly aligned polyvinyl alcohol (PVA)-(HA)-siloxane nanofibers were obtained using an electrospinning process. PVA-HA fibers were crosslinked by a 3D siloxane organic-inorganic matrix via sol-gel that restricts natural hydrophilicity and stiffens the structure. The hybrid nanofiber mats were characterized by FT-IR, micro-Raman spectroscopy, SEM, and biological properties. The PVA/HA ratio influenced the morphology of the hybrid nanofibers. Nanofibers with high PVA content (10PVA-8 and 10PVA-10) form mats with few beaded nanofibers, while those with high HA content (5PVA-8 and 5PVA-10) exhibit mats with mound patterns formed by "ribbon-like" nanofibers. The hybrid nanofibers were used as mats to support osteoblast growth, and they showed outstanding biological properties supporting cell adhesion, cell proliferation, and cell differentiation. Importantly, the 5PVA-8 mats show 3D spherical osteoblast morphology; this suggests the formation of tissue growth. These novel HA-based nanomaterials represent a relevant advance in designing nanofibers with unique properties for potential tissue regeneration.

Analysis of the cytotoxicity and bioactivity of CeraSeal, BioRoot™ and AH Plus® sealers in pre-osteoblast lineage cells.

BACKGROUND: The objective of the present study was to evaluate in vitro the cytotoxicity and bioactivity of various endodontic sealers (CeraSeal, BioRoot™ and AH Plus®) in pre-osteoblast mouse cells (MC3T3 cells). METHODS: MC3T3 cells (ATCC CRL-2594) were plated in 1 × 104 cells/well in 96-well plates in contact with endodontic sealers at concentrations of 1:10 and 1:100. Cell viability was evaluated by MTT assay after 24 and 48 h. In addition, sealer bioactivity was measured by RT-PCR for mediator of inflammation (Tnf, Ptgs2) and mineralization (Runx2, Msx1, Ssp1 and Dmp1) after 24 h and by Alizarin Red S Assay of mineralization after 28 days. Data were analyzed using one-way ANOVA followed by the Tukey's post-test at a significance level of 5%. RESULTS: BioRoot™ presented 24-hour cytotoxicity (p < 0.05) at 1:10 concentration. In the period of 48 h, no endodontic cement was cytotoxic to the cells compared to the control (p > 0.05). TNF-α gene expression was induced by AH Plus® (p < 0.05), while Ptgs2 was induced by the CeraSeal and BioRoot™ (p < 0.05). The expression of Runx2 was stimulated by BioRoot™ and AH Plus® (p < 0.05). In contrast, the expression of Dmp-1 Dmp1 was higher for the CeraSeal and BioRoot™ (p < 0.05). Nonetheless, the sealers did not impact the formation of mineralization nodules (p > 0.05). CONCLUSION: CeraSeal, BioRoot™ and AH Plus® sealers were not cytotoxic to MC3T3 cells within 48 h, but differentially induced the expression of genes related to inflammation and mineralization without impacting biomineralization by the cells.

Fibroblasts and osteoblasts behavior after contact with different titanium surfaces used as implant abutment: An in vitro experimental study.

Background: The goal of this in vitro study was to compare three different surfaces: two types of implant surfaces commercially available ([a] smooth/machined and [b] acid-treated surface) versus (c) anodized surface. Discs were manufactured with commercially pure titanium (CP) grade IV, which were subsequently analyzed by scanning microscopy and fibroblastic and osteoblastic cell cultures. Methods: Ninety-nine discs (5 × 2 mm) were manufactured in titanium grade IV and received different surface treatments: (i) Mach group: machined; (ii) AA group: double acid etch; and (iii) AN group: anodizing treatment. Three discs from each group were analyzed by Scanning Electron Microscopy (SEM) to obtain surface topography images and qualitatively analyzed by EDS. Balb/c 3T3 fibroblasts and pre-osteoblastic cells (MC3T3-E1 lineage) were used to investigate each group's biological response (n = 10/cellular type). The data were compared statistically using the ANOVA one-way test, considered as a statistically significant difference p < 0.05. Results: The AA group had numerous micropores with diameters between 5 and 10 µm, while nanopores between 1 and 5 nm were measured in the AN group. The EDX spectrum showed a high titanium concentration in all the analyzed samples. The contact angle and wetting tension were higher in the AA, whereas similar results were observed for the other groups. A lower result was observed for base width in the AA, which was higher in the other two groups. The AN showed the best values in the fibroblast cells, followed by Mach and AA; whereas, in the culture of the MC3T3 cells, the result was precisely the opposite (AA > Mach > AN). There was similar behavior for cell adhesion for the test groups (Mach and AN), with greater adhesion of Balb/c 3T3 fibroblasts compared to MC3T3 cells; in the AA group, there was greater adherence for MC3T3 cells compared to Balb/c 3T3 fibroblasts. Conclusions: The findings suggest that different surface characteristics can produce different biological responses, possibly cell-line dependent. These findings have important implications for the design of implantable medical devices, where the surface characteristics can significantly impact its biocompatibility.

The Association of Nanostructured Carbonated Hydroxyapatite with Denatured Albumin and Platelet-Rich Fibrin: Impacts on Growth Factors Release and Osteoblast Behavior.

Platelet-rich Fibrin (PRF), a second-generation blood concentrate, offers a versatile structure for bone regeneration due to its composition of fibrin, growth factors, and cytokines, with adaptations like denatured albumin-enriched with liquid PRF (Alb-PRF), showing potential for enhanced stability and growth factor dynamics. Researchers have also explored the combination of PRF with other biomaterials, aiming to create a three-dimensional framework for enhanced cell recruitment, proliferation, and differentiation in bone repair studies. This study aimed to evaluate a combination of Alb-PRF with nanostructured carbonated hydroxyapatite microspheres (Alb-ncHA-PRF), and how this association affects the release capacity of growth factors and immunomodulatory molecules, and its impact on the behavior of MG63 human osteoblast-like cells. Alb-PRF membranes were prepared and associated with nanocarboapatite (ncHA) microspheres during polymerization. MG63 cells were exposed to eluates of both membranes to assess cell viability, proliferation, mineralization, and alkaline phosphatase (ALP) activity. The ultrastructural analysis has shown that the spheres were shattered, and fragments were incorporated into both the fibrin mesh and the albumin gel of Alb-PRF. Alb-ncHA-PRF presented a reduced release of growth factors and cytokines when compared to Alb-PRF (p < 0.05). Alb-ncHA-PRF was able to stimulate osteoblast proliferation and ALP activity at lower levels than those observed by Alb-PRF and was unable to positively affect in vitro mineralization by MG63 cells. These findings indicate that the addition of ncHA spheres reduces the biological activity of Alb-PRF, impairing its initial effects on osteoblast behavior.

Effect of energy level of photobiomodulation therapy on bone repair in rats / Efecto del nivel de energía de la terapia de fotobiomodulación sobre la reparación ósea en ratas

Abstract The aim of this experimental study was to determine the effect of photobiomodulation therapy on bone repair in a rat tibia osteotomy model at 15 and 30 days. The sample consisted of 36 male Holtzman rats that were randomized into 6 equal groups. Groups A1 and A2: osteotomy + 1 J laser energy. Groups B1 and B2: osteotomy + 3 J laser energy. Groups C1 and C2 (controls): osteotomy only. The bone repair was analyzed by histological evaluation of osteoblasts and osteocytes both at 15 days (groups A1, B1, and C1) and at 30 days (groups A2, B2, and C2). Within the results, in all groups a greater number of osteoblasts was found at 15 days vs 30 days (p<0.05), and a greater number of osteocytes in B1 and C2 vs B2 and C1, respectively (p<0.05). When evaluating the 3 groups worked up to 15 days, more osteoblasts were found in A1 and C1 vs B1 (p<0.001); and osteocytes predominated in A1 and B1 vs C1 (p<0.001). At 30 days there was a greater quantity of osteoblasts in C2 vs A2 and B2 (p<0.05) and of osteocytes in C2 vs B2 (p<0.05). It is concluded that 1 J photobiomodulation therapy improved bone repair at 15 days; however, this improvement was not observed at 30 days because there were no differences between the irradiated groups and the control.

Resumen El objetivo de este estudio experimental fue determinar el efecto de terapia de fotobiomodulación sobre la reparación ósea en un modelo de osteotomía de tibia de rata a los 15 y 30 días. La muestra estuvo compuesta por 36 ratas Holtzman macho que se aleatorizaron en 6 grupos iguales. Grupos A1 y A2: osteotomía + energía láser de 1 Joule. Grupos B1 y B2: osteotomía + energía láser 3 Joule. Grupos C1 y C2 (controles): solo osteotomía. La reparación ósea fue analizada por evaluación histológica de osteoblastos y osteocitos tanto a los 15 días (grupos A1, B1 y C1) como a los 30 días (grupos A2, B2 y C2). Como resultados se encontró que en todos los grupos hubo mayor número de osteoblastos a los 15 días vs. 30 días (p<0,05), y mayor número de osteocitos en B1 y C2 vs B2 y C1, respectivamente (p<0,05). Al evaluar a los animales a los 15 días, se observó mayor número de osteoblastos en A1 y C1 vs B1 (p<0.001); y mayor número de osteocitos en A1 y B1 vs C1 (p<0,001). Al evaluar a los ratones a los 30 días hubo mayor cantidad de osteoblastos en C2 vs A2 y B2 (p<0,05) y de osteocitos en C2 vs B2 (p<0,05). Se concluye que la terapia de fotobiomodulación con 1 Joule mejoró la reparación ósea a los 15 días; sin embargo, dicha mejora no se observó a los 30 días porque no hubo diferencias entre los grupos irradiados y el control.

Physical-chemical influences and cell behavior of natural compounds on titanium dental surfaces

Abstract The present study evaluated the influence of carvacrol, terpinene-4-ol, and chlorhexidine on the physical-chemical properties of titanium surfaces, cell viability, proliferation, adhesion, and spreading of fibroblasts and osteoblasts in vitro. Titanium surfaces (Ti) were treated with Carvacrol (Cvc), Terpinen-4-ol (T4ol), Chlorhexidine (CHX), DMSO, and ultrapure water (Control group). Physical-chemical modifications were evaluated by surface wettability, the surface free energy (SFE) calculated from the contact angle values using the Owens-Wendt-Rabel-Kaeble (OWRK) equation, scanning electron microscopy (SEM) and energy dispersive spectrometry probe (EDS) system. Cells were seeded onto Ti-treated surfaces and incubated for 24 h and 72 h, then evaluated by Alamar blue assay and fluorescence microscopy. Surfaces treated with Cvc and T4ol showed the presence of Na, O, and Cl. All surfaces showed hydrophilic characteristics and SFE values between 5.5 mN/m and 3.4 mN/m. On the other hand, EDS peaks demonstrated the presence of O and Cl after CHX treatment. A reduction of cell viability and adhesion was noted on titanium surfaces treated with CHX after 24 and 72h. In conclusion, the results indicate that the decontamination with Cvc and T4ol on Ti surfaces does not alter the surface proprieties and allows an adequate interaction with cells involved in the re-osseointegration process such as fibroblasts and osteoblasts.

Resumo O presente estudo avaliou a influência do carvacrol, terpineno-4-ol e clorexidina nas propriedades físico-químicas de superfícies de titânio, viabilidade celular, proliferação, adesão e esplhamento de fibroblastos e osteoblastos in vitro. Superfícies de titânio (Ti) foram tratadas com Carvacrol (Cvc), Terpinen-4-ol (T4ol), Clorexidina (CHX), DMSO e água ultrapura (Grupo Controle). As modificações físico-químicas foram avaliadas pela molhabilidade da superfície, a energia livre de superfície (ELS) calculada a partir dos valores do ângulo de contato usando a equação de Owens-Wendt-Rabel-Kaeble (OWRK), microscopia eletrônica de varredura (MEV) e espectroscopia de raios X por energia dispersiva (EDS). As células foram semeadas em superfícies tratadas com Ti e incubadas por 24 h e 72 h, e avaliadas pelo ensaio Alamar blue e microscopia de fluorescência. As superfícies tratadas com Cvc e T4ol mostraram a presença de Na, O e Cl. Todas as superfícies apresentaram características hidrofílicas e valores de ELS entre 5,5 mN/m e 3,4 mN/m. Por outro lado, os picos de EDS demonstraram a presença de O e Cl após o tratamento com CHX. Uma redução da viabilidade celular e adesão foi observada em superfícies de titânio tratadas com CHX após 24 e 72h. Em conclusão, os resultados indicam que a descontaminação com Cvc e T4ol em superfícies de Ti não altera as propriedades da superfície e permite uma interação adequada com células envolvidas no processo de reosseointegração como fibroblastos e osteoblastos.

The Action of Angiocrine Molecules Sourced from Mechanotransduction-Related Endothelial Cell Partially Explain the Successful of Titanium in Osseointegration.

Since Branemark's findings, titanium-based alloys have been widely used in implantology. However, their success in dental implants is not known when considering the heterogenicity of housing cells surrounding the peri-implant microenvironment. Additionally, they are expected to recapitulate the physiological coupling between endothelial cells and osteoblasts during appositional bone growth during osseointegration. To investigate whether this crosstalk was happening in this context, we considered the mechanotransduction-related endothelial cell signaling underlying laminar shear stress (up to 3 days), and this angiocrine factor-enriched medium was harvested further to use exposing pre-osteoblasts (pOb) for up to 7 days in vitro. Two titanium surfaces were considered, as follows: double acid etching treatment (w_DAE) and machined surfaces (wo_DAE). These surfaces were used to conditionate the cell culture medium as recommended by ISO10993-5:2016, and this titanium-enriched medium was later used to expose ECs. First, our data showed that there is a difference between the surfaces in releasing Ti molecules to the medium, providing very dynamic surfaces, where the w_DAE was around 25% higher (4 ng/mL) in comparison to the wo_DAE (3 ng/mL). Importantly, the ECs took up some of this titanium content for up to 3 days in culture. However, when this conditioned medium was used to expose pOb for up to 7 days, considering the angiocrine factors released from ECs, the concentration of Ti was lesser than previously reported, reaching around 1 ng/mL and 2 ng/mL, respectively. Thereafter, pOb exposed to this angiocrine factor-enriched medium presented a significant difference when considering the mechanosignaling subjected to the ECs. Shear-stressed ECs showed adequate crosstalk with osteoblasts, stimulating the higher expression of the Runx2 gene and driving higher expressions of Alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin. Mechanotransduction-related endothelial cell signaling as a source of angiocrine molecules also stimulated the higher expression of the Col3A1 gene in osteoblasts, which suggests it is a relevant protagonist during trabecular bone growth. In fact, we investigated ECM remodeling by first evaluating the expression of genes related to it, and our data showed a higher expression of matrix metalloproteinase (MMP) 2 and MMP9 in response to mechanosignaling-based angiocrine molecules, independent of considering w_DAE or the wo_DAE, and this profile reflected on the MMP2 and MMP9 activities evaluated via gelatin-based zymography. Complimentarily, the ECM remodeling seemed to be a very regulated mechanism in mature osteoblasts during the mineralization process once both TIMP metallopeptidase inhibitor 1 and 2 (TIMP1 and TIMP2, respectively) genes were significantly higher in response to mechanotransduction-related endothelial cell signaling as a source of angiocrine molecules. Altogether, our data show the relevance of mechanosignaling in favoring ECs' release of bioactive factors peri-implant, which is responsible for creating an osteogenic microenvironment able to drive osteoblast differentiation and modulate ECM remodeling. Taking this into account, it seems that mechanotransduction-based angiocrine molecules explain the successful use of titanium during osseointegration.

Unveiling the role of osteoblastic micro RNAs in the skeleton: from biological functions to therapeutic potential / Rol de los micro-ARN osteoblásticos en el esqueleto: desde las funciones biológicas al potencial terapéutico

MicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene regulation. They function by binding to target messenger RNA (mRNA) molecules, leading to their degradation or inhibiting their translation into proteins. In the context of skeletal diseases, such as osteoporosis, osteoarthritis, and bone metastasis, there is growing evidence osteoblastic miRNAs, are involved in the regulation of bone formation and maintenance.Osteoblasts are bone-forming cells responsible for synthesizing and depositing the extracellular matrix, which ultimately mineralizes to form bone tissue. Osteoblastic miRNAs modulate various aspects of osteoblast function, including proliferation, differentiation, mineralization, and apoptosis. Dysregulation of these miRNAs can disrupt the balance between bone formation and resorption, leading to skeletal diseases.The therapeutic implications of targeting osteoblastic miRNAs in skeletal diseases are significant. Modulating the expression levels of specific miRNAs holds promise for developing novel therapeutic strategies to enhance bone formation, prevent bone loss, and promote bone regeneration. Potential therapeutic approaches include the use of synthetic miRNA mimics to restore miRNA expression in diseases associated with miRNA downregulation or the use of anti-miRNA oligonucleotides to inhibit miRNA function in diseases associated with miRNA upregulation.miRNA-based therapies are still in the early stages of development, and further research is needed to fully understand the complexity of miRNA networks. Additionally, the delivery of miRNAs to specific target tissues and cells remains a challenge that needs to be addressed for effective clinical translation. Nonetheless, targeting osteoblastic miRNAs represents a promising avenue for future therapeutic interventions in skeletal diseases. (AU)

Los micro-ARNs (miARNss) son pequeños ARN no codificantes que desempeñan un papel fundamental en la regulación génica postranscripcional. Ejercen su función al unir-se a moléculas de ARN mensajero (ARNm), promoviendo su degradación e inhibiendo su traducción en proteínas. En el contexto de las enfermedades esqueléticas, como la osteoporosis, la osteoartritis y la metástasis ósea existe evidencia de que los miARNs osteoblásticos están involucrados en la regulación de la formación y del mantenimiento óseo. Los osteoblastos son células formadoras de hueso responsables de sintetizar y depositar la matriz extracelular, que finalmente se mineraliza para formar el hueso. Los miARNs derivados de osteoblastos modulan varios aspectos de la función de estas células, incluida la proliferación, diferenciación, mineralización y la apoptosis. La desregulación de estos miARNs puede alterar el equilibrio entre la formación y la resorción ósea, lo que lleva a enfermedades óseas. Las implicaciones terapéuticas de los miARNs osteoblásticos en enfermedades esqueléticas son significativas. La modulación de los niveles de expresión de miARNs específicos es prometedora para desarrollar nuevas estrate-gias terapéuticas a fin de mejorar la formación, prevenir la pérdida y promover la regeneración ósea. Los enfoques terapéuticos potenciales incluyen el uso de miméticos de miARNs para restaurar la expresión de miARNs o el uso de oligonucleótidos anti-miARNs para inhibir su función. Las terapias basadas en miARNs aún se encuentran en las primeras etapas de desarrollo. La administración de miARNs a las células y los tejidos específicos sigue siendo un desafío para lograr una aplicación clínica eficaz. (AU)

Normoglycemia partially recovers the disrupted osteoblast differentiation of mesenchymal stem cells induced by type 1 but not type 2 diabetes mellitus.

Type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are characterized by changes in glucose metabolism and cause bone damage via a variety of mechanisms, including effects on osteoblasts. We aimed to evaluate the osteoblast differentiation of mesenchymal stem cells (MSCs) from rats with T1DM or T2DM and the effects of removing the hyperglycemic stimulus on the osteogenic potential of these cells. MSCs from healthy rats were cultured in normoglycemic medium, whereas MSCs from rats with T1DM or T2DM were cultured in hyperglycemic or normoglycemic medium. T1DM and T2DM reduced osteoblast differentiation of MSCs grown in hyperglycemic media, with T1DM having a more pronounced effect, as evidenced by alkaline phosphatase activity, RUNX2 protein expression, and extracellular matrix mineralization, and modulated the gene expression of several components of the bone morphogenetic protein signaling pathway. The restoration of the normoglycemic environment partially recovers the osteogenic potential of MSCs from rats with T1DM but not with T2DM. Our findings highlight the need for specific therapies to treat T1DM- or T2DM-induced bone loss, as both disrupt osteoblast differentiation at distinct levels and likely through different mechanisms.

Disruption of TNF-α signaling improves osteoblastic differentiation of adipose-derived mesenchymal stem cells and bone repair.

Adipose tissue is an attractive source of mesenchymal stem cells (at-MSCs), but their low osteogenic potential limits their use in bone regeneration. Adipose tissue plays a role in pro-inflammatory diseases by releasing cytokines with a catabolic effect on bone, such as tumor necrosis factor-alpha (TNF-α). Thus, we hypothesized that endogenous TNF-α could have a negative effect on at-MSC differentiation into osteoblasts. Short interfering RNAs (siRNAs) targeting TNF-α receptors (siR1, siR2, and si1R/R2) were transfected into at-MSCs, and cell differentiation was assessed by measuring the expression of bone markers, ALP activity, and mineralized matrix. Scrambled was used as Control. Knockout at-MSCs (KOR1/R2) was injected in mice calvaria defects, and bone formation was evaluated by microtomography and histological analysis. Data were compared by Kruskal-Wallis or analysis of variance (5%). The expression of bone markers confirmed that at-MSCs differentiate less than bone marrow MSCs. In silenced cells, the expression of Alp, Runx2, and Opn was generally higher compared to Control. ALP, RUNX2, and OPN were expressed at elevated levels in silenced groups, most notably at-MSCs-siR1/R2. ALP was detected at high levels in at-MSCs-siR1/R2 and in-MSCs-siR1, followed by an increase in mineralized nodules in at-MSCs-siR1/R2. As the morphometric parameters increased, the groups treated with KOR1/R2 exhibited slight bone formation near the edges of the defects. Endogenous TNF-α inhibits osteoblast differentiation and activity in at-MSCs, and its disruption increases bone formation. While opening a path of investigation, that may lead to the development of new treatments for bone regeneration using at-MSC-based therapies.

JAK/STAT as a Potential Therapeutic Target for Osteolytic Diseases.

Several cytokines with major biological functions in inflammatory diseases exert their functions through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal transduction pathway. JAKs phosphorylate the cytoplasmic domain of the receptor, inducing the activation of its substrates, mainly the proteins known as STATs. STATs bind to these phosphorylated tyrosine residues and translocate from the cytoplasm to the nucleus, further regulating the transcription of several genes that regulate the inflammatory response. The JAK/STAT signaling pathway plays a critical role in the pathogenesis of inflammatory diseases. There is also increasing evidence indicating that the persistent activation of the JAK/STAT signaling pathway is related to several inflammatory bone (osteolytic) diseases. However, the specific mechanism remains to be clarified. JAK/STAT signaling pathway inhibitors have gained major scientific interest to explore their potential in the prevention of the destruction of mineralized tissues in osteolytic diseases. Here, our review highlights the importance of the JAK/STAT signaling pathway in inflammation-induced bone resorption and presents the results of clinical studies and experimental models of JAK inhibitors in osteolytic diseases.

The Multifarious Functions of Leukotrienes in Bone Metabolism.

Leukotrienes (LTs) are derived from arachidonic acid metabolism by the 5-lipoxygenase (5-LO) enzyme. The production of LTs is stimulated in the pathogenesis of rheumatoid arthritis (RA), osteoarthritis, and periodontitis, with a relevant contribution to bone resorption. However, its role in bone turnover, particularly the suppression of bone formation by modulating the function of osteoclasts and osteoblasts, remains unclear. We investigated the effects of LTs on bone metabolism and their impact on osteogenic differentiation and osteoclastogenesis using a 5-LO knockout (KO) mouse model. Results from micro-computed tomography (µCT) analysis of femur from 8-week-old 5-LO-deficient mice showed increased cortical bone and medullary region in females and males and decreased trabecular bone in females. In the vertebra, we observed increased marrow area in both females and males 5-LO KO and decreased trabecular bone only in females 5-LO KO. Immunohistochemistry (IHC) analysis showed higher levels of osteogenic markers tissue-nonspecific alkaline phosphatase (TNAP) and osteopontin (OPN) and lower expression of osteoclastogenic marker tartrate-resistant acid phosphatase (TRAP) in the femurs of 5-LO KO mice versus wild-type (WT). Alkaline phosphatase activity and mineralization assay results showed that the 5-LO absence enhances osteoblasts differentiation and mineralization but decreases the proliferation. Alkaline phosphatase (ALP), Bglap, and Sp7 gene expression were higher in 5-LO KO osteoblasts compared to WT cells. Eicosanoids production was higher in 5-LO KO osteoblasts except for thromboxane 2, which was lower in 5-LO-deficient mice. Proteomic analysis identified the downregulation of proteins related to adenosine triphosphate (ATP) metabolism in 5-LO KO osteoblasts, and the upregulation of transcription factors such as the adaptor-related protein complex 1 (AP-1 complex) in long bones from 5-LO KO mice leading to an increased bone formation pattern in 5-LO-deficient mice. We observed enormous differences in the morphology and function of osteoclasts with reduced bone resorption markers and impaired osteoclasts in 5-LO KO compared to WT osteoclasts. Altogether, these results demonstrate that the absence of 5-LO is related to the greater osteogenic profile. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).