Osteogenic potential of mesenchymal cells derived from canine umbilical cord matrix co-cultured with platelet-rich plasma and demineralized bone matrix

Canine mesenchymal cells (MSCs) derived from Wharton's jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.

Expression of non-neuronal cholinergic system in maxilla of rat in vivo

BACKGROUND: Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, which is also produced in a variety of non-neuronal tissues and cell. The existence of ACh in maxilla in vivo and potential regulation role for osteogenesis need further study. RESULTS: Components of the cholinergic system (ACh, esterase, choline acetyltransferase, high-affinity choline uptake, n- and mAChRs) were determined in maxilla of rat in vivo, by means of Real-Time PCR and immunohistochemistry. Results showed RNA for CarAT, carnitine/acylcarnitine translocase member 20 (Slc25a20), VAChT, OCTN2, OCT1, OCT3, organic cation transporter member 4 (Slc22a4), AChE, BChE, nAChR subunits α1, α2, α3, α5, α7, α10, ß1, ß2, ß4, γ and mAChR subunits M1, M2, M3, M4, M5 were detected in rat's maxilla. RNA of VAChT, AChE, nAChR subunits α2, ß1, ß4 and mAChR subunits M4 had abundant expression (2(-ΔCt) > 0.03). Immunohistochemical staining was conducted for ACh, VAChT, nAChRα7 and AChE. ACh was expressed in mesenchymal cells, chondroblast, bone and cartilage matrix and bone marrow cells, The VAChT expression was very extensively while ACh receptor α7 was strongly expressed in newly formed bone matrix of endochondral and bone marrow ossification, AchE was found only in mesenchymal stem cells, cartilage and bone marrow cells. CONCLUSIONS: ACh might exert its effect on the endochondral and bone marrow ossification, and bone matrix mineralization in maxilla.

Regulation of osteogenic differentiation of human adipose-derived stem cells by controlling electromagnetic field conditions

Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation.

Efeito da triiodotironina na expressão das proteínas ósseas durante a diferenciação osteogênica de células-tronco mesenquimais / Effect of triiodothyronine on the bone proteins expression during osteogenic differentiation of mesenchymal stem cells

OBJETIVO: O objetivo deste estudo foi avaliar o efeito da T3 na expressão da osteocalcina, osteo­pontina e colágeno I durante a diferenciação osteogênica das células-tronco mesenquimais (CTM). MATERIAIS E MÉTODOS: As células da medula óssea de ratas Wistar jovens foram extraídas, cultivadas e separadas em cinco grupos: controle (indiferenciado), diferenciado (estímulo osteogênico) e diferenciado com T3 (10-3 nM, 10-2 nM e 100 nM). Para cada grupo, foram cultivadas quatro amostras que foram analisadas por RT-PCR tempo real aos 7, 14 e 21 dias, para quantificação dos transcritos gênicos para osteocalcina, osteopontina e colágeno I. RESULTADOS: Todos os grupos diferenciados sem T3 ou com T3 independentemente da concentração apresentaram expressão de colágeno I significativamente menor e expressão de osteocalcina e osteopontina significativamente maior em comparação a das CTM indiferenciadas. Mas o grupo T3 100 nM apresentou concentração de osteocalcina mais elevada e semelhante à da cultura de osteoblastos. CONCLUSÃO: Conclui-se que a triiodotironina não altera a expressão de osteopontina e de colágeno pelas CTM, mas aumenta a expressão da osteocalcina durante a diferenciação osteogênica in vitro, sendo esse efeito dose-dependente.

OBJECTIVE: The aim of this study was to evaluate the effect of T3 on the expression of osteocalcin, osteopontin and collagen I during osteogenic differentiation of mesenchymal stem cells (MSC). MATERIALS AND METHODS: The bone marrow cells of Wistar rats with 30 days of age were extracted, cultured and separated into five groups: control (undifferentiated), differentiated (osteogenic stimulus) and differentiated with T3 (10-3 nM, 10-2 nM and 100 nM). For each group, four samples were cultured and were analyzed by real time RT-PCR at 7, 14 and 21 days for quantification of gene transcripts for osteocalcin, osteopontin and collagen I. RESULTS: All the different groups without T3 or with T3 regardless of the concentration, showed the collagen I expression significantly lower expression, and osteocalcin and osteopontin expression significantly greater than that of undifferentiated MSC. Nevertheless, the group T3 100 nM showed higher expression of osteocalcin and a similar expression of the osteoblast culture. CONCLUSION: In conclusion, the triiodothyronine does not affect the expression of osteopontin and collagen I, but increases ostecalcin expression during osteogenic differentiation in vitro of the MSC, and this effect is dose-dependent.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Técnica histoquímica aplicada ao tecido ósseo desmineralizado e parafinado para o estudo do osteócito e suas conexões / Histochemical tecnique for osteocyte and its connections study in descalcified and paraffined bone tissue

O osteócito vem sendo alvo de pesquisas recentes. A avaliação in situ de sua morfologia, da atividade e das características de suas conexões é difícil, portanto é realizada apenas com técnicas avançadas. Devido à importância desse tipo celular na manutenção da matriz óssea, este estudo propõe uma técnica de coloração pela prata como alternativa para o estudo do osteócito e suas conexões em tecido ósseo desmineralizado e parafinado. Cortes de 4æm do fêmur de ratas foram desmineralizados, desparafinados em xilol e hidratados em concentrações decrescentes de álcool etílico (ETOH) e água miliQ. Para a impregnação foram utilizadas soluções de nitrato de prata a 50 por cento e de ácido fórmico a 1 por cento com 2 por cento de gelatina microbiológica em estufa a 40ºC. Essa técnica permite visualizar facilmente as bordas lacunares dos osteócitos e suas conexões, proporcionando uma alternativa simples e eficaz para o estudo da morfologia desse tipo celular até então ainda não proposta com essa finalidade.

Hacia el conocimiento molecular del metabolismo óseo: una revisión del actual estado del arte / Toward the molecular understanding of bone metabolism: a review of the state-of-the-art

El tejido óseo posee características propias, tanto histológicas como fisológicas, que lo diferencian como un tejido altamente complejo, por sus propiedades de regulación mediante el mecanismo de formación-reabsorción; está comandado por hormonas como la paratiroidea, el péptido relacionado con la hormona paratiroidea, la vitamina D3, estrógenos, andrógenos, glucocorticoides, hormonas tiroideas y del crecimiento, entre otras. La matriz ósea está compuesta por dos constituyentes principales: la matriz orgánica y las sales inorgánicas. La matriz orgánica tiene como componente principal el colágeno tipo I, así como los tipos II y III, y por lo menos 12 proteínas no colágenas, a cuyas mutaciones se les atribuye la causa de algunos sindromes. Se han estudiado la glicoproteína acídica ósea, la osteocalcina, osteopontina, osteonectina, sialoproteína ósea, con propiedades y formaciones específicas para la síntesis y metabolismo del tejido óseo. La matriz inorgánica se compone de cantidades de citrato, carbonato e hidroxiapatita. La interacción molecular de estos componentes conduce a la mineralización del tejido con el concurso de componentes celulares como el osteoblasto y el osteoclasto. La proliferación y la maduración de la matriz preceden a la etapa de mineralización que se regula por actividades esencialmente hormonales. Los procesos de osteoclastogénesis y osteoblastogénesis se modulan molecularmente por la osteoprotegerina, que inhibe el desarrollo de osteoclastos bloqueando la molécula RANKL, factor de diferenciación osteoclástica con un importante papel en la reabsorción ósea. El aspecto molecular de la osteoblastogénesis es menos conocido, y hasta el momento sólo se conocen las proteínas morfogenéticas que participan en la diferenciación osteoblástica

Tumour induced osteomalacia: a report of two cases. A disease of defective matrix synthesis?

Tumour induced hypophosphataemic osteomalacia or rickets is a well delineated clinical entity. There is confusion, however, about the nomenclature and classification of the associated tumours. The tumour factor responsible for the biochemical abnormalities has also not been identified. We report here two cases: one, a 43 year old male with a soft tissue tumour in the left vastus medialis, and the other, a 25 year old female with a soft tissue tumour in the right anterior axillary fold. Reversal of biochemical abnormalities and clinical improvement occurred after removal of the tumour in both cases. Both tumours showed unusual morphology characterised by spindle cell component, large vascular spaces, osteoclast-like giant cells, calcification and ossification. The tumour in the second patient was benign, while the nature of the tumour in the first patient was debated. We speculate that defective matrix may be the cause of unusual histology of the tumours, and also the source of the phosphaturic factor.