Abstract: Despite the crucial role of osteoclasts in the physiological process of bone repair, most bone tissue engineering strategies have focused on osteoblast-biomaterial interactions. Although Biosilicate® with two crystalline phases (BioS-2P) exhibits osteogenic properties and significant bone formation, its effects on osteoclasts are unknown. This study aimed to investigate the in vitro and in vivo effects of BioS-2P on osteoclast differentiation and activity. RAW 264.7 cells were cultured in osteoclastogenic medium (OCM) or OCM conditioned with BioS-2P (OCM-BioS-2P), and the cell morphology, viability, and osteoclast differentiation were evaluated. BioS-2P scaffolds were implanted into rat calvarial defects, and the bone tissue was evaluated using tartrate-resistant acid phosphatase (TRAP) staining and RT-polymerase chain reaction (PCR) after 2 and 4 weeks to determine the gene expressions of osteoclast markers and compare them with those of the bone grown in empty defects (Control). OCM-BioS-2P favored osteoclast viability and activity, as evidenced by an increase in the TRAP-positive cells and matrix resorption. The bone tissue grown on BioS-2P scaffolds exhibited higher expression of the osteoclast marker genes (Ctsk, Mmp 9, Rank) after 2 and 4 weeks and the RankL/Opg ratio after 2 weeks. Trap gene expression was lower at 2 weeks, and a higher number of TRAP-stained areas were observed in the newly formed bone on BioS-2P scaffolds at both 2 and 4 weeks compared to the Controls. These results enhanced our understanding of the role of bioactive glass-ceramics in bone repair, and highlighted their role in the modulation of osteoclastic activities and promotion of interactions between bone tissues and biomaterials.

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Abstract Cell therapy associated with guided bone regeneration (GBR) can be used to treat bone defects under challenging conditions such as osteoporosis. This study aimed to evaluate the effect of mesenchymal stem cells (MSCs) in combination with a poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT) membrane on bone repair in osteoporotic rats. Osteoporosis was induced in female rats by bilateral removal of the ovaries (OVX) or sham surgery (SHAM), and the osteoporotic condition was characterized after 5 months by microtomographic and morphometric analyses. Calvarial defects were created in osteoporotic rats that immediately received the PVDF-TrFE/BT membrane. After 2 weeks, bone marrow-derived MSCs from healthy rats, characterized by the expression of surface markers using flow cytometry, or phosphate-buffered saline (PBS) (Control) were injected into the defects and bone formation was evaluated 4 weeks post-injection by microtomographic, morphometric, and histological analyses. A reduction in the amount of bone tissue in the femurs of OVX compared with SHAM rats confirmed the osteoporotic condition of the experimental model. More bone formation was observed when the defects were injected with MSCs compared to that with PBS. The modification that we are proposing in this study for the classical GBR approach where cells are locally injected after a membrane implantation may be a promising therapeutic strategy to increase bone formation under osteoporotic condition.

ABSTRACT Aging negatively affects bone/titanium implant interactions. Our hypothesis is that the unbalance between osteogenesis and adipogenesis induced by aging may be involved in this phenomenon. Objective We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. Material and Methods Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. Results Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. Conclusions These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population.

ABSTRACT The ability of hemostatic agents to promote bone repair has been investigated using in vitro and in vivo models but, up to now, the results are inconclusive. Objective In this context, the aim of this study was to compare the potential of bone repair of collagen sponge with fibrin glue in a rat calvarial defect model. Material and Methods Defects of 5 mm in diameter were created in rat calvariae and treated with either collagen sponge or fibrin glue; untreated defects were used as control. At 4 and 8 weeks, histological analysis and micro-CT-based histomorphometry were carried out and data were compared by two-way ANOVA followed by Student-Newman-Keuls test when appropriated (p≤0.05). Results Three-dimensional reconstructions showed increased bone formation in defects treated with either collagen sponge or fibrin glue compared with untreated defects, which was confirmed by the histological analysis. Morphometric parameters indicated the progression of bone formation from 4 to 8 weeks. Additionally, fibrin glue displayed slightly higher bone formation rate when compared with collagen sponge. Conclusion Our results have shown the benefits of using collagen sponge and fibrin glue to promote new bone formation in rat calvarial bone defects, the latter being discreetly more advantageous.

A current goal of dental implant research is the development of titanium (Ti) surfaces to improve osseointegration. Plasma nitriding treatments generate surfaces that favor osteoblast differentiation, a key event to the process of osteogenesis. Based on this, it is possible to hypothesize that plasma-nitrided Ti implants may positively impact osseointegration. Objective The aim of this study was to evaluate the in vivo bone response to Ti surfaces modified by plasma-nitriding treatments. Material and Methods Surface treatments consisted of 20% N2 and 80% H2, 450°C and 1.5 mbar during 1 h for planar and 3 h for hollow cathode. Untreated surface was used as control. Ten implants of each surface were placed into rabbit tibiae and 6 weeks post-implantation they were harvested for histological and histomorphometric analyses. Results Bone formation was observed in contact with all implants without statistically significant differences among the evaluated surfaces in terms of bone-to-implant contact, bone area between threads, and bone area within the mirror area. Conclusion Our results indicate that plasma nitriding treatments generate Ti implants that induce similar bone response to the untreated ones. Thus, as these treatments improve the physico-chemical properties of Ti without affecting its biocompatibility, they could be combined with modifications that favor bone formation in order to develop new implant surfaces.

O objetivo deste estudo foi caracterizar a microestrutura de uma superfície de titânio comercialmente puro (cpTi) condicionada com HCl/H2SO4 (acid etched) (AE-cpTi) e investigar sua citocompatibilidade in vitro, comparada à do cpTi usinado (turned) (T-cpTi). O T-cpTi apresentou uma superfície com sulcos e o AE-cpTi exibiu uma superfície caracterizada pela presença de micro-vales. Os parâmetros de superfície indicaram que a superfície AE-cpTi é mais isotrópica e apresenta uma área maior quando comparada à superfície T-cpTi. A espessura da camada de óxido foi similar para as duas superfícies; no entanto, a AE-cpTi apresentou maiores quantidades de Ti e O e menor, de C. A proliferação de células osteoblásticas, a atividade de fosfatase alcalina e a formação de matriz mineralizada foram maiores na superfície T-cpTi que na AE-cpTi. Esses resultados mostram que o condicionamento ácido produziu uma superfície com características topográficas e químicas diferentes quando comparadas às da superfície usinada. Além disso, observou-se que essas modificações de superfície afetaram de forma negativa a citocompatibilidade in vitro do cpTi como demonstrado pela inibição da proliferação celular e da expressão do fenótipo osteoblástico.

The aims of this study were to characterize the microstructure of a commercially pure titanium (cpTi) surface etched with HCl/H2SO4 (AE-cpTi) and to investigate its in vitro cytocompatibility compared to turned cpTi (T-cpTi). T-cpTi showed a grooved surface and AE-cpTi revealed a surface characterized by the presence of micropits. Surface parameters indicated that the AE-cpTi surface is more isotropic and present a greater area compared to T-cpTi. The oxide film thickness was similar between both surfaces; however, AE-cpTi presented more Ti and O and less C. Osteoblastic cell proliferation, alkaline phosphatase activity, and bone-like nodule formation were greater on T-cpTi than on AE-cpTi. These results show that acid etching treatment produced a surface with different topographical and chemical features compared to the turned one, and such surface modification affected negatively the in vitro cytocompatibility of cpTi as demonstrated by decreasing culture growth and expression of osteoblastic phenotype.

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

O objetivo desse estudo foi avaliar a resposta do tecido ósseo à superfície de titânio (Ti) enriquecida com Ca e P obtida por anodização (BSP-AK). Três cilindros de Ti (4,1 x 12 mm) BSP-AK ou usinado (controle) foram implantados bilateralmente nos úmeros de dois cães de raça indefinida. Oito semanas após a implantação, os fragmentos ósseos contendo os implantes foram removidos e processados para análises histológica e histomorfométrica. A formação óssea foi observada na região cortical e no canal medular até aproximadamente um terço da extensão do implante. Na maioria dos casos, feixes de fibras colágenas dispostos paralelamente à superfície do implante foram observados na região medular. Nessa região observaram-se também áreas focais de formação de matriz mineralizada e osteoblastos ativos. Os implantes do grupo BSP-AK apresentaram média de contato osso-implante 2,3%, com medidas variando de 0,0 a 7,2% e os do grupo controle tiveram média 0,4%, com medidas variando de 0,0 a 1,3%. Apesar do teste de Mann-Whitney não mostrar diferença estatisticamente significante entre os grupos, nossos resultados indicaram uma tendência para a ocorrência de osteogênese de contato na superfície BSP-AK em um modelo experimental que resulta em baixos valores de contato osso-implante.

This study evaluated bone response to a Ca- and P- enriched titanium (Ti) surface treated by a multiphase anodic spark deposition coating (BSP-AK). Two mongrel dogs received bilateral implantation of 3 Ti cylinders (4.1 x 12 mm) in the humerus, being either BSP-AK treated or untreated (machined - control). At 8 weeks postimplantation, bone fragments containing the implants were harvested and processed for histologic and histomorphometric analyses. Bone formation was observed in cortical area and towards the medullary canal associated to approximately 1/3 of implant extension. In most cases, in the medullary area, collagen fiber bundles were detected adjacent and oriented parallel to Ti surfaces. Such connective tissue formation exhibited focal areas of mineralized matrix lined by active osteoblasts. The mean percentages of bone-to-implant contact were 2.3 (0.0-7.2 range) for BSP-AK and 0.4 (0.0-1.3 range) for control. Although the Mann-Whitney test did not detect statistically significant differences between groups, these results indicate a trend of BSP-AK treated surfaces to support contact osteogenesis in an experimental model that produces low bone-to-implant contact values.

Culturas de células de medula óssea têm sido utilizadas para avaliar a biocompatibilidade de substitutos ósseos que poderiam ser empregados em cirurgias maxilofaciais e ortopédicas. No entanto, ainda não está claro se células em subculturas mantêm a habilidade para se diferenciarem em osteoblastos. O objetivo deste estudo foi comparar o desenvolvimento do fenótipo osteoblástico em subculturas seriadas de células de medula óssea humana. Células da primeira à terceira passagem foram cultivadas (2x10(4) células/poço) em alfa-MEM suplementado. As células foram incubadas a 37ºC e 5% CO2 / 95% ar atmosférico. A adesão celular foi avaliada em 4 h e 24 h. Aos 7, 14 e 21 dias, a proliferação e viabilidade celulares, conteúdo de proteína total e atividade de fosfatase alcalina (ALP) foram avaliadas. A formação de matriz mineralizada foi avaliada aos 14 e 21 dias. Os dados foram comparados por análise de variância a dois critérios e teste de Duncan. Adesão e viabilidade celular e conteúdo de proteína total não foram afetados pela subcultura seriada. Entretanto, a subcultura seriada interferiu negativamente na diferenciação osteoblástica, como demonstrado pelos parâmetros osteoblásticos da segunda e terceira passagens, tais como a proliferação celular contínua, a baixa atividade de ALP e a baixa quantidade de matriz mineralizada formada, quando comparados à primeira passagem. Portanto, é importante avaliar a capacidade das células para se diferenciarem em osteoblastos antes de selecionar a população de células adequada para testar a biocompatibilidade de materiais para substituir tecido ósseo.

Bone marrow cells have been used for testing biocompatibility of bone substitute materials that would be applied in maxillofacial and orthopedic surgeries. However, it remains unclear whether cells in serial subcultures retain the ability to differentiate into osteoblasts. The purpose of this study was to compare the development of osteoblast phenotype of serially passaged cells from human bone marrow. Cells from first to third passage were cultured (2x10(4) cells/well) in supplemented culture medium. Cells were incubated at 37ºC in a humidified atmosphere of 5% CO2 and 95% air. Cell attachment was assessed at 4 and 24 h. At 7, 14 and 21 days, cell proliferation, cell viability, total protein content and alkaline phosphatase (ALP) activity were evaluated. Bone-like formation was evaluated at 14 and 21 days. Data were compared by two-way ANOVA and Duncan's multiple range test. Cell attachment, cell viability and total protein content were not affected by serial subcultures. However, serial subcultures did interfered negatively with osteoblast differentiation as shown by osteoblast parameters observed in second and third subcultures, such as continuous cell proliferation, lower ALP activity and bone-like formation in comparison to first subculture. Therefore, it is important to evaluate cell ability to growth and differentiate before selecting the cell population for studies that investigate the biocompatibility of materials to replace bone tissue.

A dexametasona (Dex) induz diferenciação osteoblástica em diversos modelos de cultura de células. Este estudo investigou o efeito do tratamento contínuo e descontínuo com Dex sobre a diferenciação de células de medula óssea humana (BMSC). Células da cultura primária e da primeira passagem foram cultivadas em meio de cultura com e sem Dex 10-7 M (37ºC e 5% CO2 / 95% ar atmosférico). Aos 7, 14 e 21 dias, os seguintes parâmetros foram avaliados: proliferação e viabilidade celulares, conteúdo de proteína total, atividade de fosfatase alcalina (ALP) e formação de matriz mineralizada. Os dados foram comparados por análise de variância a dois critérios. A Dex não afetou a viabilidade celular e o conteúdo de proteína total, mas reduziu o número de células. A atividade de ALP e a formação de matriz mineralizada foram aumentadas quando apenas a primeira passagem ou cultura primária e primeira passagem foram tratadas com Dex, em comparação aos grupos que não tiveram contato com Dex após a primeira passagem. Estes resultados indicam que, para BMSC humanas, a presença contínua de Dex não parece ser necessária para o desenvolvimento do fenótipo osteoblástico. Contudo, a Dex deve estar presente após a primeira passagem para permitir a diferenciação osteoblástica expressa por proliferação celular reduzida e aumento da atividade de ALP e da formação de matriz mineralizada.

Dexamethasone (Dex) has been shown to induce osteoblast differentiation in several cell culture systems. This study investigated the effect of continuous and discontinuous treatment with Dex on osteoblast differentiation of human bone marrow stromal cells (BMSC). Primary culture and first passage were cultured in media with or without Dex 10-7 M. During the culture period, cells were incubated at 37ºC in humidified atmosphere of 5% CO2 and 95% air. At 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity and bone-like formation were evaluated. Data were compared by two-way analysis of variance. Dex did not affect cell viability and total protein content, but reduced cell number. ALP activity and bone-like formation increased when only first passage or both primary culture and first passage were treated with Dex, in comparison to the groups that did not have contact with Dex after first passage. The results of this study indicate that, for human BMSC, continuous presence of Dex did not appear to be required for development of the osteoblast phenotype, but Dex must be present after first passage to allow osteoblast differentiation expressed by reduced cell proliferation and increased ALP activity and bone-like formation.

Sabe-se que superfícies rugosas melhoram as respostas biológica e biomecânica dos implantes de titânio (Ti). O objetivo desse estudo foi investigar o efeito da rugosidade de superfície do Ti sobre a resposta de células de medula óssea humana, avaliando: adesão e proliferação celulares, síntese de proteína total, atividade de fosfatase alcalina (ALP), e a formação de nódulos de matriz mineralizada. Células foram cultivadas sobre discos de titânio comercialmente puro (cpTi) com quatro diferentes rugosidades de superfície (Ra). A adesão foi avaliada após 4 horas. Após 21 dias, a proliferação, a quantidade de proteína total e a atividade de ALP foram avaliadas. A formação de nódulos de matriz mineralizada foi avaliada após 28 dias em cultura. Os dados foram comparados por ANOVA e teste de Duncan. A adesão não foi afetada pela Ra. Para as células cultivadas sobre Ti com Ra variando entre 0,80 µm e 1,90 µm a proliferação foi reduzida enquanto o conteúdo de proteína total e atividade de ALP foram aumentadas. Houve um aumento estatisticamente não-significante na formação de nódulos de matriz mineralizada sobre superfícies com Ra próxima de 0,80 µm. Esses resultados sugerem que, para o Ti, uma Ra variando entre 0,80 µm e 1,90 µm otimizaria respostas celulares intermediárias e finais, mas não afetaria respostas iniciais, e superfíces lisas não favoreceria nenhuma das respostas avaliadas.

There is general agreement that rough surfaces improve both biologic and biomechanical responses to titanium (Ti) implants. The aim of this investigation was to study the effect of Ti surface roughness on the response of human bone marrow cell culture evaluating: cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on commercially pure titanium (cpTi) discs with four different average roughnesses (Ra). For attachment evaluation, cells were cultured for 4 h. After 21 days, cell proliferation, total protein content, and ALP activity were evaluated. For bone-like nodule formation, cells were cultured for 28 days. Data were compared by ANOVA and Duncan's multiple range test. Cell attachment was not affected by surface roughness. For cells cultured on Ti with Ra ranging from 0.80 µm to 1.90 µm, proliferation was reduced while total protein content, and ALP activity were increased. There was a non-statistically significant increase of bone-like nodule formation on a surface with Ra near 0.80 µm. These results suggest that for Ti an Ra ranging from 0.80 µm to 1.90 µm would optimize both intermediary and final cellular responses but not affect the initial response, and a smoother surface would not favor any evaluated response.

A hidroxiapatita (HA) tem sido utilizada como revestimento de implantes e para substituição de tecido ósseo. O objetivo deste estudo foi avaliar o efeito da topografia de superfície da HA, resultante da presença de microporosidade, sobre a adesão, a morfologia e proliferação celulares, a medida de proteína total e a atividade de fosfatase alcalina. Discos de HA com diferentes porcentagens de microporosidade (< 5%, 15% e 30%) foram fabricados por uma combinação das técnicas de pressão uniaxial e sinterização. Células ROS17/2.8 foram cultivadas sobre os discos de HA. Para a adesão, as células foram cultivadas por duas horas. A morfologia foi avaliada após sete dias. A proliferação, medida de proteína total e atividade de ALP foram avaliadas após sete e quatorze dias. Os dados foram comparados por ANOVA e teste de Duncan quando apropriado. A adesão (p = 0,11) e a medida de proteína total (p = 0,31) não foram afetadas pela topografia de superfície. A proliferação após sete e quatorze dias (p = 0,0007 e p = 0,003, respectivamente), e a atividade de ALP (p = 0,0007) foram significantemente menores na superfície irregular (HA30). Esses resultados sugerem que eventos iniciais não são afetados pela topografia, enquanto superfícies com topografias mais regulares (microporosidade de 15% ou menos) favoreceram eventos intermediários e finais, como proliferação e atividade de ALP.

Hydroxyapatite (HA) has been used in orthopedic, dental, and maxillofacial surgery as a bone substitute. The aim of this investigation was to study the effect of surface topography produced by the presence of microporosity on cell response, evaluating: cell attachment, cell morphology, cell proliferation, total protein content, and alkaline phosphatase (ALP) activity. HA discs with different percentages of microporosity (< 5%, 15%, and 30%) were confected by means of the combination of uniaxial powder pressing and different sintering conditions. ROS17/2.8 cells were cultured on HA discs. For the evaluation of attachment, cells were cultured for two hours. Cell morphology was evaluated after seven days. After seven and fourteen days, cell proliferation, total protein content, and ALP activity were measured. Data were compared by means of ANOVA and Duncan’s multiple range test, when appropriate. Cell attachment (p = 0.11) and total protein content (p = 0.31) were not affected by surface topography. Proliferation after 7 and 14 days (p = 0.0007 and p = 0.003, respectively), and ALP activity (p = 0.0007) were both significantly decreased by the most irregular surface (HA30). These results suggest that initial cell events were not affected by surface topography, while surfaces with more regular topography, as those present in HA with 15% or less of microporosity, favored intermediary and final events such as cell proliferation and ALP activity.