Promotion effect of FGF23 on osteopenia in congenital scoliosis through FGFr3/TNAP/OPN pathway / 中华医学杂志(英文版)

BACKGROUND@#Congenital scoliosis (CS) is a complex spinal malformation of unknown etiology with abnormal bone metabolism. Fibroblast growth factor 23 (FGF23), secreted by osteoblasts and osteocytes, can inhibit bone formation and mineralization. This research aims to investigate the relationship between CS and FGF23.@*METHODS@#We collected peripheral blood from two pairs of identical twins for methylation sequencing of the target region. FGF23 mRNA levels in the peripheral blood of CS patients and age-matched controls were measured. Receiver operator characteristic (ROC) curve analyses were conducted to evaluate the specificity and sensitivity of FGF23. The expression levels of FGF23 and its downstream factors fibroblast growth factor receptor 3 (FGFr3)/tissue non-specific alkaline phosphatase (TNAP)/osteopontin (OPN) in primary osteoblasts from CS patients (CS-Ob) and controls (CT-Ob) were detected. In addition, the osteogenic abilities of FGF23-knockdown or FGF23-overexpressing Ob were examined.@*RESULTS@#DNA methylation of the FGF23 gene in CS patients was decreased compared to that of their identical twins, accompanied by increased mRNA levels. CS patients had increased peripheral blood FGF23 mRNA levels and decreased computed tomography (CT) values compared with controls. The FGF23 mRNA levels were negatively correlated with the CT value of the spine, and ROCs of FGF23 mRNA levels showed high sensitivity and specificity for CS. Additionally, significantly increased levels of FGF23, FGFr3, OPN, impaired osteogenic mineralization and lower TNAP levels were observed in CS-Ob. Moreover, FGF23 overexpression in CT-Ob increased FGFr3 and OPN levels and decreased TNAP levels, while FGF23 knockdown induced downregulation of FGFr3 and OPN but upregulation of TNAP in CS-Ob. Mineralization of CS-Ob was rescued after FGF23 knockdown.@*CONCLUSIONS@#Our results suggested increased peripheral blood FGF23 levels, decreased bone mineral density in CS patients, and a good predictive ability of CS by peripheral blood FGF23 levels. FGF23 may contribute to osteopenia in CS patients through FGFr3/TNAP / OPN pathway.

Role of osteopontin in diet-induced brown gallstone formation in rats / 中华医学杂志(英文版)

BACKGROUND@#Although osteopontin (OPN) is expressed in the liver and pigment gallstones of patients with hepatolithiasis, its role in pigment gallstone formation remains unclear. This study aimed to explore the function of OPN in pigment gallstone formation.@*METHODS@#Rats were fed a chow diet (CD) or lithogenic diet (LD) for 10 consecutive weeks; blocking tests were then performed using an OPN antibody (OPN-Ab). Incidence of gallstones and levels of several bile components, OPN, tumor necrosis factor alpha (TNF-α), and cholesterol 7 alpha-hydroxylase (CYP7A1) were analyzed. To determine TNF-α expression in hepatic macrophages and both CYP7A1 and bile acid (BA) expression in liver cells, recombinant rat OPN and recombinant rat TNF-α were used to treat rat hepatic macrophages and rat liver cells, respectively. Chi-square or Fisher exact tests were used to analyze qualitative data, Student t-test or one-way analysis of variance were used to analyze qualitative data.@*RESULTS@#Incidence of gallstones was higher in LD-fed rats than in CD-fed rats (80% vs. 10%, P < 0.05). BA content significantly decreased in bile (t = -36.08, P < 0.01) and liver tissue (t = -16.16, P < 0.01) of LD-fed rats. Both hepatic OPN protein expression (t = 9.78, P < 0.01) and TNF-α level (t = 8.83, P < 0.01) distinctly increased in the LD group; what's more, CYP7A1 mRNA and protein levels (t = -12.35, P < 0.01) were markedly down-regulated in the LD group. Following OPN-Ab pretreatment, gallstone formation decreased (85% vs. 25%, χ2 = 14.55, P < 0.01), liver TNF-α expression (F = 20.36, P < 0.01) was down-regulated in the LD group, and CYP7A1 expression (F = 17.51, P < 0.01) was up-regulated. Through CD44 and integrin receptors, OPN promoted TNF-α production in macrophage (F = 1041, P < 0.01), which suppressed CYP7A1 expression (F = 48.08, P < 0.01) and reduced liver BA synthesis (F = 119.4, P < 0.01).@*CONCLUSIONS@#We provide novel evidence of OPN involvement in pigmented gallstone pathogenesis in rats.

Melatonin has a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/secretion / Melatonina tem efeito estimulador em osteoblastos, pela regulação positiva da expressão/secreção de COL-I e OPN

ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.

RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.

Expressão da Osteopontina após utilização de matriz dentinária humana desmineralizada em alvéolos de ratos: análise imunohistoquímica / Osteopontin expression after use of demineralized human dentin matrix in rats alveoli: immunohistochemistry analysis

Apesar de sua capacidade de reparo, o tecido ósseo pode ser submetido a alguns tipos de fraturas, cirurgias ou patologias que podem levar a grandes defeitos ósseos. As principais estratégias de tratamento de defeitos ósseos são baseados em osteoindução ou osteocondução. Matriz dentinária desmineralizada humana (MDDH) é uma alternativa biocompatível para preencher defeitos ósseos, melhorando a qualidade e quantidade de osso produzido. 24 Ratos Wistar foram selecionados, submetidos à extração de ambos os segundos molares superiores (direito e esquerdo). Os alvéolos foram separados em dois grupos: controle (direita) preenchido com coágulo sanguíneo e experimental (esquerda) preenchido com MDDH. Os animais foram sacrificados aos 5, 10 e 21 dias. Foram realizadas análises histológicas, histomorfométricas (análise de variância - ANOVA e teste de Tukey) e imunohistoquímica para osteopontina (OPN) como indicador de osteogênese. Aos 5 dias MDDH foi incorporada pelas novas trabéculas ósseas. Aos 10 dias observou-se organização do tecido conjuntivo e trabéculas no grupo experimental. Detectou-se coloração intensa para OPN em área adjacente à MDDH no grupo experimental. Aos 21 dias no grupo experimental verificou-se trabéculas maduras. Houve diferença estatísticamente significatva (p <0,05). Maior número de trabéculas em grupos experimentais que nos grupos controle em todos os períodos de análise. MDDH implantadas em alvéolos de ratos, induz a aceleração da osteogênese. Presença OPN observada mais intensamente aos 10 dias próximo à MDDH

BACKGROUND - Despite its good capacity for regeneration, bone tissue subjected to some types of fractures or surgery that can lead to large bone defects. The major bone defects treatment strategies are based in osteoinduction or osteoconduction. Demineralized human dentin matrix (DHDM) is a biocompatible alternative to fill bone defect, improving the quantity and quality of bone produced. METHODS - Wistar rats were selected, submitted to the extraction of both second molars (right and left). Alveoli were separated into two groups: control (right) filled with blood clot and experimental (left) filled with DHDM. Animals were sacrificed at 5, 10 and 21 days. Histological and histoquantitative analyzes (analysis of variance - ANOVA, and Tukey's test) were performed and immunostaining for osteopontin (OPN) as osteogenesis indicator. RESULTS - 5 days - DHDM incorporated by new trabeculae. 10 days - connective tissue organization and new trabeculae in the experimental group. Intense staining for OPN close to DHDM in the experimental group. 21 days - experimental group showing mature trabeculae. Statistical difference observed (p<0.05). Higher number of trabeculae in experimental groups in all periods of analysis. CONCLUSIONS - DHDM implanted in alveoli induces the acceleration of osteogenesis. Presence of OPN observed more intensely at 10 days close to DHDM

Secreted Phosphoprotein 1 Promoter Genetic Variants Are Associated with the Response to Pegylated Interferon alpha Plus Ribavirin Combination Therapy in Egyptian Patients with Chronic Hepatitis C Virus Infection

BACKGROUND/AIMS: The T-helper 1 (TH1) immune reaction is essential for the eradication of hepatitis C virus (HCV) during pegylated interferon alpha (PEG-IFN-alpha)- and ribavirin (RBV)-based therapy in chronic HCV patients. Secreted phosphoprotein 1 (SPP1) was shown to be a crucial cytokine for the initiation of a TH1 immune response. We aimed to investigate whether SPP1 single nucleotide polymorphisms (SNPs) may influence sustained virological response (SVR) rates. METHODS: Two SNPs in the promoter region of SPP1 at the -443 C>T and -1748 G>A loci were genotyped in 100 patients with chronic HCV genotype 4 infection using a TaqMan SNP genotyping assay. RESULTS: Sixty-seven patients achieved a SVR, and 33 patients showed no SVR. Patients carrying the T/T genotype at the -443 locus showed a significantly higher SVR rate than those carrying the C/T or C/C genotype (83.67% vs 50.98%, pT and -1748 G>A loci may be useful markers for predicting the response to PEG-IFN-alpha-2b plus RBV therapy in Egyptian patients with chronic HCV genotype 4 infection.

Myxovirus resistance, osteopontin and suppressor of cytokine signaling 3 polymorphisms predict hepatitis C virus therapy response in an admixed patient population: comparison with IL28B

OBJECTIVES: Suppressor of cytokine signaling 3, myxovirus resistance protein and osteopontin gene polymorphisms may influence the therapeutic response in patients with chronic hepatitis C, and an association with IL28 might increase the power to predict sustained virologic response. Our aims were to evaluate the association between myxovirus resistance protein, osteopontin and suppressor of cytokine signaling 3 gene polymorphisms in combination with IL28B and to assess the therapy response in hepatitis C patients treated with pegylated-interferon plus ribavirin. METHOD: Myxovirus resistance protein, osteopontin, suppressor of cytokine signaling 3 and IL28B polymorphisms were analyzed by PCR-restriction fragment length polymorphism, direct sequencing and real-time PCR. Ancestry was determined using genetic markers. RESULTS: We analyzed 181 individuals, including 52 who were sustained virologic responders. The protective genotype frequencies among the sustained virologic response group were as follows: the G/G suppressor of cytokine signaling 3 (rs4969170) (62.2%); T/T osteopontin (rs2853744) (60%); T/T osteopontin (rs11730582) (64.3%); and the G/T myxovirus resistance protein (rs2071430) genotype (54%). The patients who had ≥3 of the protective genotypes from the myxovirus resistance protein, the suppressor of cytokine signaling 3 and osteopontin had a greater than 90% probability of achieving a sustained response (p<0.0001). The C/C IL28B genotype was present in 58.8% of the subjects in this group. The sustained virological response rates increased to 85.7% and 91.7% by analyzing C/C IL28B with the T/T osteopontin genotype at rs11730582 and the G/G suppressor of cytokine signaling 3 genotype, respectively. Genetic ancestry analysis revealed an admixed population. CONCLUSION: Hepatitis C genotype 1 patients who were responders to interferon-based therapy had a high frequency of multiple protective polymorphisms in the myxovirus ...

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin, and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.

Osteogenic gene expression in osteoblastic differentiation of human mesenchymal stem cells

During differentiation of mesenchymal stem cells [MSCs] into various cells, the expression of a variety of genes undergoes some changes; in this study we decided to investigate the expression rate of some genes like osteopontin [OPN] and osteocalcin [OCN] during this process in order to find a better and faster way for these cells to be differentiated into osteoblasts. In this experimental study, the mononuclear cells of bone marrow were separated and then cultured in DMEM-LG culture media with 10% FBS. During some definite days, the RNA of differentiating cells was extracted. Then, the effective genes in osteogenesis like OPN and OCN were amplified by speciefic primers. The mesenchymal cells were cultured on 3D calcium phosphate scaffolds, and finally the activity rate of the alkaline phosphatase was examined. This research has demonstrated that in the process of differentiation, the expression of the two genes of OPN and OCN changed orderly with the maximum expression of OPN in the 6th day and the maximum expression of OCN in the 7th and 8th days of differentiation. The osteogenic differentiation of MSCs was not confirmed by the coloration of mineral sediments. The activity rate of alkaline phosphatase revealed the preference of 3D calcium phosphate scaffold to 2D environment in this differentiation. The calcium phosphate scaffold positively affects the differentiation process. The expression of OPN and OCN genes changes during differentiation and can be used as away to a better and faster differentiation of these cells into osteoblast

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9 percent) for 15 days. Thyroidectomy increased OPG (~40 percent) and OPN (~75 percent) mRNA expression, while T3 treatment reduced OPG (~40 percent) and OPN (~75 percent) in Tx, and both (~50 percent) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

Osteopontin expression and its possible functions in the aortic disorders and coronary artery disease / A expressão da osteopontina e as suas funções possíveis nas desordens aórticas e doença arterial coronariana

BACKGROUND: Osteopontin (OPN) has been verified to be closely associated with oncogenesis and remodeling processes. But this cytokine was rarely assessed in the presence of aortopathies, especially acute aortic dissection. The aim of the present study was to evaluate the expressions of OPN by way of molecular biological approaches so as to offer a better understanding of the possible mechanisms of the aortopathies. METHODS: Consecutive patients with type A acute aortic dissection (20 patients), aortic aneurysm (nine patients) or coronary artery disease (21 patients) referred to this hospital for surgical operations were enrolled into this study. Blood samples of the surgical patients after systematic heparinization, and control fast morning blood samples drawn from 21 young healthy volunteers who had no evidence of any healthy problems were investigated for enzyme linked immunosorbent assay (ELISA). The surgical specimens of the aortic tissues collected from the surgical patients during the operations were obtained for quantitative realtime reverse transcription polymerase chain reaction (RT-PCR) for OPN mRNA, western blot assay for OPN protein, and for immunohistochemical staining of OPN. Ascending aortic tissues from the autopsies of the healthy individuals dying of accident were obtained as controls of immunohistochemistry. RESULTS: By quantitative RT-PCR, the expressions of OPN mRNA were all upregulated in all three surgical groups. The quantitative results did not reveal any intergroup differences. Western blot assay revealed that OPN was positive with similar intensities of expressions in all three surgical groups. Quantitative western blot analyses of OPN expressions did not show any significance between groups. The OPN expressions by ELISA in the aortic tissue were 3.09311 ± 1.65737, 3.40414 ± 1.15095, and 1.68243 ± 0.31119 pg/mg protein in the aortic dissection, aortic aneurysm, and coronary artery disease groups, respectively. The OPN level of the patients with coronary artery disease was much lower than those with aortic dissection (P = 0.033) or with aortic aneurysm (P = 0.019). By unparametric tests, there were significant differences in the aortic OPN contents among aortic dissection, aortic aneurysm and coronary artery disease groups (P < 0.01). A significant direct correlation was present between plasma OPN concentration and the time interval from the onset to surgery of aortic dissection (Y = 0.1420X + 2.4838, r² = 0.5623, r = 0.750, P = 0.032). By immunohistochemistry, OPN was expressed in the aortic cells: in the intima, it was weaker in all three surgical groups in comparison with the healthy control; in the media, it was weak in the aortic dissection, intense positive in aortic aneurysm, focal positive in the coronary artery disease, but evenly positive in the healthy control groups; and in the adventitia, it was positive in the aortic dissection, coronary artery disease and healthy control groups, but weak positive in the aortic aneurysm group. CONCLUSION: These data may provide evidences that OPN may play a role in the pathogenesis of aortopathies including aortic dissection, aortic aneurysm, and coronary artery disease. OPN might be of potential perspective as a clinically diagnostic tool in the evaluations of the complex remodeling process incorporating vascular injury and repair.

OBJETIVOS: A osteopontina (OPN) está estreitamente associada com os processos de oncogênese e remodelação. Entretanto, essa citocina era raramente avaliada na presença de aortopatias, especialmente na dissecção aórtica aguda. O objetivo do presente estudo foi avaliar a expressão de OPN por meio de abordagens moleculares biológicas, de modo a oferecer uma melhor compreensão dos possíveis mecanismos das aortopatias. MÉTODOS: Pacientes consecutivos com um tipo de dissecção aguda da aorta (20 pacientes), aneurisma da aorta (nove pacientes) ou doença arterial coronária (21 pacientes) foram incluídos neste estudo. As amostras de sangue depois da heparinização sistemática e de 21 voluntários jovens e saudáveis não apontaram nenhuma evidência de qualquer problema ao serem investigados por ensaio imunoenzimático (ELISA). Os espécimes cirúrgicos dos tecidos aórtica coletados dos pacientes durante as operações foram obtidos para a reação de transcrição reversa quantitativa em tempo real em cadeia da polimerase (RT-PCR) para OPN mRNA, técnica de Western blot para a proteína OPN, e imunohistoquímica de OPN. Amostras da aorta de indivíduos saudáveis que morreram de acidente foram obtidos para controle imunohistoquímico. RESULTADOS: Com uso do RT-PCR quantitativo, as expressões de OPN mRNA foram suprarreguladas em todos os três grupos cirúrgicos. Os resultados quantitativos não revelaram quaisquer diferenças intergrupais. Western blot revelou que OPN foi positiva com intensidade semelhante de expressões em todos os três grupos. As análises quantitativas Western blot de expressões OPN não apresentaram significâncias entre os grupos. As expressões OPN medidas pelo teste ELISA no tecido aórtico foram 3,09311 ± 1,65737, 3,40414 ± 1,15095 e 1,68243 ± 0,31119 pg/mg de proteína na dissecção de aorta, aneurisma da aorta, e grupos de doença arterial coronariana, respectivamente. O nível de OPN dos pacientes com doença arterial coronariana foi muito menor do que aqueles com dissecção aórtica (P = 0,033) ou com aneurisma da aorta (P = 0,019). Testes não-paramétricos apontaram diferenças significativas nos teores de aorta OPN entre dissecção aórtica, aneurisma da aorta e grupos com doença arterial coronariana (P <0,01). Uma correlação direta significativa estava presente entre a concentração plasmática OPN e o intervalo de tempo entre o início da cirurgia de dissecção de aorta (Y = 2,4838 + 0,1420X, r² = 0,5623, r = 0,750, P = 0,032). Pela imunohistoquímica, a OPN foi expressa em células aórticas: na íntima, foi fraca em todos os três grupos cirúrgicos em comparação ao grupo saudável; na média, era fraca na dissecção aórtica, positiva intensa no aneurisma de aorta, focal positivo na doença arterial coronariana, mas igualmente positiva no grupo controle; e na adventícia, positiva para a dissecção da aorta, doença arterial coronariana e grupos de controle saudáveis, mas fraca positiva no grupo de aneurisma da aorta. CONCLUSÃO: Estes dados fornecem evidências de que a OPN pode desempenhar um papel na patogênese da aortopatias, incluindo dissecção aórtica, aneurisma da aorta R e doença arterial coronariana. OPN tem perspectiva potencial como ferramenta de diagnóstico clínico nas avaliações do processo de remodelação complexa, incluindo lesão vascular e de reparação.

Use of calcium phosphate cement scaffolds for bone tissue engineering: in vitro study / A utilização do cimento de fosfato de cálcio como arcabouço para engenharia de tecido ósseo: estudo in vitro

Purpose: To evaluate the ability of macroporous tricalcium phosphate cement (CPC) scaffolds to enable the adhesion, proliferation, and differentiation of mesenchymal stem cells derived from human bone marrow. Methods: Cells from the iliac crest of an adult human donor were processed and cultured on macroporous CPC discs. Paraffin spheres sized between 100 and 250µm were used as porogens. Cells were cultured for 5, 10, and 15 days. Next, we assessed cells' behavior and morphology on the biomaterial by scanning electron microscopy. The expression levels of the BGLA and SSP1 genes and the alkaline phosphatase (ALP) activity were quantified by the quantitative real-time polymerase chain reaction technique (QT-PCR) using the fluorophore SYBR GREEN®. Results: QT-PCR detected the expression of the BGLA and SSP1 genes and the ALP activity in the periods of 10 and 15 days of culture. Thus, we found out that there was cell proliferation and differentiation in osteogenic cells. Conclusion: Macroporous CPC, with pore sized between 100 and 250µm and developed using paraffin spheres, enables adhesion, proliferation, and differentiation of mesenchymal stem cells in osteogenic cells and can be used as a scaffold for bone tissue engineering.

Objetivo: Avaliar a capacidade de suportes tridimensionais macroporosos de cimento de fosfato de cálcio (CFC), de permitir a adesão, proliferação e diferenciação de células-tronco mesenquimais derivadas da medula óssea humana. Métodos: células obtidas da crista ilíaca de um doador humano adulto foram processadas e cultivadas sobre suportes de CFC, macroporosos, que tiveram como corpo gerador de poros, microesferas de parafina, com tamanho entre 100 e 250µm. Os períodos de cultura estabelecidos foram de cinco, 10 e 15 dias. Após estes períodos, o comportamento e a morfologia das células junto ao biomaterial foram avaliados por meio de Microscopia Eletrônica de Varredura. Os níveis de expressão dos genes BGLA e SSP1 bem como a atividade da Fosfatase Alcalina (ALP) foram quantificados pela técnica de PCR em Tempo Real (QT-PCR) utilizando o fluoróforo SYBR Green®. Resultados: O QT-PCR detectou a expressão dos genes BGLA e SSP1 e a atividade da fosfatase alcalina nos períodos de 10 e 15 dias de cultura. No período de cinco dias, não foi observada a expressão de nenhum dos genes investigados. Conclusão: O CFC, macroporoso, com tamanho de poros entre 100 e 250µm, criados por meio da utilização de microesferas de parafina, permite a adesão, proliferação e diferenciação de células-tronco mesenquimais em células osteogênicas, podendo ser utilizado como arcabouço para engenharia de tecido ósseo.

Effects of low-level laser therapy on human osteoblastic cells grown on titanium

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.

Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumínio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possível sugerir possíveis benefícios do laser na osseointegração de implantes de Ti apesar do efeito deletério às células imediatamente após a irradiação.

Influence of the microenvironment on gene and protein expression of odontogenic-like and osteogenic-like cells

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC-23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cel ls, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.

Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development

Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology which is not fully elucidated yet. Epidemiological studies point to different etiologies in the cleft lip and palate subgroups, isolated cleft lip (CL), isolated cleft palate (CP) and combined cleft lip and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed. Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP and immunostaining in palatal shelves against SPP1, chemokine receptor 4 (CXCR4) and serglycin (PRG1) in human embryonic craniofacial tissue were positive, supporting a role for these genes in palatal development. However, gene expression profiles are subject to variations during growth and therefore we recommend that future gene expression in CL/P studies should use tissue from the correct embryonic time and place if possible, to overcome the biases in the presented study.