Understanding the recruitment of multipotent skeletal progenitor cells and the factors that influence their differentiation would be helpful in providing a means for harnessing the regenerative capacity of skeletal progenitor cells in bone tissue engineering. In order to track the recruitment of skeletal stem cells in fracture healing, transgenic mice containing a Tamoxifen-inducible Cre recombinase that had been placed under the control of a 2.4 kb Prx1 promotor were used to induce conditional expression in periosteal skeletal stem cells that express the Prx1 gene. In order to initially see the cells expressing Prx1, a green fluorescent protein gene (GFP) had also been put downstream to the Prx1 promotor. We then crossed these Prx1CreER-GFP transgenic mice with a second strain containing the Beta-galactosidase gene that becomes constitutively expressed after recombination by the Cre recombinase. The enzymatic activity of Beta-galactosidase was then used to generate a colormetric staining reaction that was used to visualize the cells in which recombination had occurred based on a blue staining product. The recombination activity should only be present in Prx1 expressing cells and their progeny. The goal of the present study was to assess several different approaches to optimize the Beta-galactosidase enzymatic staining protocol and to visualize the Prx1-expressing cells during fracture healing. These studies further examined those populations of cells in the fracture calluses that became labeled and arose from the stem cell populations that had expressed Prx1 at post-operative day 7 and 14. The optimization of a staining method for histology will allow this study to track Prx1 cell fates in a fracture model both in response to specific drug treatments, mechanical loading of the fracture during healing and under pathological conditions that effect healing.

Tem sido sugerido que um adequado reparo ósseo pode ser obtido por biomateriais híbridos, produzidos pela combinação de células e materiais substitutos ósseos macroporosos. O objetivo geral do presente estudo foi avaliar a aplicação do biomaterial híbrido formado pelo arcabouço de PLGA/CaP e células-tronco mesenquimais e osteoblastos derivados de medula óssea na engenharia de tecido ósseo. Para verificar qual o tamanho de poro deste arcabouço é mais adequado para este fim, foram realizados experimentos in vitro, que avaliaram a proliferação celular, atividade de fosfatase alcalina (ALP) e expressão quantitativa de genes marcadores do fenótipo osteoblástico em células cultivadas sobre os arcabouços; e in vivo, que avaliaram a formação óssea após implantação do arcabouço em defeitos ósseos críticos de calvária de ratos. Também foi avaliado o efeito do tamanho dos poros sobre o carreamento celular através da força centrifuga. Para avaliar o efeito do soro fetal bovino, utilizado na suplementação do meio de cultura celular, na resposta tecidual in vivo, arcabouços expostos ao soro foram implantados em defeitos ósseos críticos de calvária de ratos. O efeito da retirada do soro fetal bovino do meio de cultura sobre osteoblastos foi analisado através da proliferação celular, atividade de ALP, conteúdo de proteína total e mineralização. Para avaliar o efeito do estágio de diferenciação celular sobre a formação óssea, células em diferentes estágios de diferenciação osteoblástica associadas ao arcabouço de PLGA/CaP foram implantadas de forma autóloga em defeitos ósseos críticos de calvária de ratos. Arcabouços com tamanhos de poros de aproximadamente 1000 µm promovem maior diferenciação osteoblástica e melhor carreamento celular, enquanto arcabouços com poros de aproximadamente 500 µm promovem maior formação óssea e vascular in vivo. O soro fetal bovino influenciou negativamente a resposta tecidual e a formação óssea. A retirada do soro fetal bovino do meio de cultura reduziu a proliferação celular e a atividade de ALP sem afetar o conteúdo de proteína total e a formação de matriz mineralizada. Células-tronco mesenquimais indiferenciadas e em fase inicial de diferenciação osteoblástica (7 dias) promoveram maior formação óssea, portanto permitiriam a obtenção de um biomaterial híbrido com maior potencial osteogênico. / It has been suggested that an adequate bone repair can be obtained by hybrid biomaterials, produced by combining osteoprogenitor cells and macroporous bone substitutes. The aim of this study was to evaluate the application of hybrid biomaterial formed by PLGA/CaP scaffold and bone marrow-derived mesenchymal stem cells and osteoblasts in bone tissue engineering. The effect of scaffold pore size was evaluated in vitro assessing cell growth, alkaline phosphatase (ALP) activity, and quantitative gene expression of osteoblastic phenotype markers in osteoblasts cultured on scaffolds; and in vivo assesseing bone formation after implantation of scaffolds in critical size rat calvaria defects. It was also evaluated the effect of pore size on cell seeding by centrifugal force. To evaluate the effect of fetal calf serum used to supplement the cell culture medium on in vivo tissue response, scaffolds exposed to serum were implanted in critical size rat calvaria defects. The effect of withdrawal of fetal calf serum in the culture medium on osteoblasts was analyzed by cell growth, ALP activity, total protein content and mineralization. To evaluate the effect of cell differentiation stage on bone repair, cells either undifferentiated or in different stages of osteoblast differentiation associated with the PLGA/CaP were implanted autologously in critical size rat calvaria. Scaffolds with pore sizes of around 1000 µm would favor the osteoblast differentiation and display a better cell seeding, while the scaffolds with pore sizes of around 500 µm would favor increased bone and vascular formation. The fetal calf serum influenced negatively the in vivo tissue response and bone formation. The withdrawal of fetal calf serum in the culture medium reduced cell growth and ALP activity without affecting the total protein content and the formation of mineralized matrix Mesenchymal stem cells and osteoblats at the early stage of differentiation (7 days) promoted greater bone formation, therefore they would allow the obtention of a hybrid biomaterial with higher osteogenic potential.

Tem sido sugerido que um adequado reparo ósseo pode ser obtido por biomateriais híbridos, produzidos pela combinação de células e materiais substitutos ósseos macroporosos. O objetivo geral do presente estudo foi avaliar a aplicação do biomaterial híbrido formado pelo arcabouço de PLGA/CaP e células-tronco mesenquimais e osteoblastos derivados de medula óssea na engenharia de tecido ósseo. Para verificar qual o tamanho de poro deste arcabouço é mais adequado para este fim, foram realizados experimentos in vitro, que avaliaram a proliferação celular, atividade de fosfatase alcalina (ALP) e expressão quantitativa de genes marcadores do fenótipo osteoblástico em células cultivadas sobre os arcabouços; e in vivo, que avaliaram a formação óssea após implantação do arcabouço em defeitos ósseos críticos de calvária de ratos. Também foi avaliado o efeito do tamanho dos poros sobre o carreamento celular através da força centrifuga. Para avaliar o efeito do soro fetal bovino, utilizado na suplementação do meio de cultura celular, na resposta tecidual in vivo, arcabouços expostos ao soro foram implantados em defeitos ósseos críticos de calvária de ratos. O efeito da retirada do soro fetal bovino do meio de cultura sobre osteoblastos foi analisado através da proliferação celular, atividade de ALP, conteúdo de proteína total e mineralização. Para avaliar o efeito do estágio de diferenciação celular sobre a formação óssea, células em diferentes estágios de diferenciação osteoblástica associadas ao arcabouço de PLGA/CaP foram implantadas de forma autóloga em defeitos ósseos críticos de calvária de ratos. Arcabouços com tamanhos de poros de aproximadamente 1000 µm promovem maior diferenciação osteoblástica e melhor carreamento celular, enquanto arcabouços com poros de aproximadamente 500 µm promovem maior formação óssea e vascular in vivo. O soro fetal bovino influenciou negativamente a resposta tecidual e a formação óssea. A retirada do soro fetal bovino do meio de cultura reduziu a proliferação celular e a atividade de ALP sem afetar o conteúdo de proteína total e a formação de matriz mineralizada. Células-tronco mesenquimais indiferenciadas e em fase inicial de diferenciação osteoblástica (7 dias) promoveram maior formação óssea, portanto permitiriam a obtenção de um biomaterial híbrido com maior potencial osteogênico. / It has been suggested that an adequate bone repair can be obtained by hybrid biomaterials, produced by combining osteoprogenitor cells and macroporous bone substitutes. The aim of this study was to evaluate the application of hybrid biomaterial formed by PLGA/CaP scaffold and bone marrow-derived mesenchymal stem cells and osteoblasts in bone tissue engineering. The effect of scaffold pore size was evaluated in vitro assessing cell growth, alkaline phosphatase (ALP) activity, and quantitative gene expression of osteoblastic phenotype markers in osteoblasts cultured on scaffolds; and in vivo assesseing bone formation after implantation of scaffolds in critical size rat calvaria defects. It was also evaluated the effect of pore size on cell seeding by centrifugal force. To evaluate the effect of fetal calf serum used to supplement the cell culture medium on in vivo tissue response, scaffolds exposed to serum were implanted in critical size rat calvaria defects. The effect of withdrawal of fetal calf serum in the culture medium on osteoblasts was analyzed by cell growth, ALP activity, total protein content and mineralization. To evaluate the effect of cell differentiation stage on bone repair, cells either undifferentiated or in different stages of osteoblast differentiation associated with the PLGA/CaP were implanted autologously in critical size rat calvaria. Scaffolds with pore sizes of around 1000 µm would favor the osteoblast differentiation and display a better cell seeding, while the scaffolds with pore sizes of around 500 µm would favor increased bone and vascular formation. The fetal calf serum influenced negatively the in vivo tissue response and bone formation. The withdrawal of fetal calf serum in the culture medium reduced cell growth and ALP activity without affecting the total protein content and the formation of mineralized matrix Mesenchymal stem cells and osteoblats at the early stage of differentiation (7 days) promoted greater bone formation, therefore they would allow the obtention of a hybrid biomaterial with higher osteogenic potential.

No description available.

Long-term exposure to pharmacological doses of glucocorticoids has been associated with the development of osteopenia and avascular necrosis. Bone loss may be partially attributed to a steroid-induced decrease in the osteoblastic differentiation of multipotent progenitor cells found in the bone marrow. In order to determine if there is a change in the osteogenic potential of the bone marrow stroma following glucocorticoid treatment, Sprague-Dawley rats were administered methylprednisolone for up to six weeks, then sacrificed at 0, 2, 4, or 6 weeks during treatment or 4 weeks after cessation of treatment. Femurs were collected and analyzed for evidence of steroid-induced osteopenia and bone marrow adipogenesis. Although glucocorticoid treatment did inhibit bone growth, differences in ultimate shear stress and mineral content were not detected. The volume of marrow fat increased with increasing duration of treatment, but returned to near control levels after cessation of treatment. Marrow stromal cells were isolated from tibias, cultured in the presence of osteogenic supplements, and analyzed for their capacity to differentiate into osteoblast-like cells in vitro. Glucocorticoid treatment diminished the absolute number of isolated stromal cells, but did not inhibit the relative levels of bone-like mineral deposition or osteocalcin expression and secretion. Although pharmacological glucocorticoid levels induce bone loss in vivo, physiologically equivalent concentrations have been shown to enhance the formation of bone-like tissue in vitro. However, glucocorticoids have also been reported to inhibit proliferation and type I collagen synthesis in marrow stromal cell cultures. In order to assess the effects of intermittent dexamethasone treatment on the progression of osteogenesis in rat marrow stromal cell culture, this synthetic glucocorticoid was removed from the culture medium after a variable period of initial supplementation. Cell layers were analyzed for total cell number, collagen synthesis, phenotypic marker expression, and matrix mineralization. Prolonged supplementation with dexamethasone decreased proliferation, but did not significantly affect collagen synthesis. Furthermore, increased treatment duration was found to increase bone sialoprotein expression and mineral deposition. The duration of glucocorticoid treatment may be a key factor for controlling the extent of differentiation in vitro. / Master of Science

We have shown that nuclear matrix protein 4 (Nmp4) attenuates the response to intermittent parathyroid hormone (PTH) in healthy and ovariectomized (OVX) female mice using a global knockout of the Nmp4 gene. Additionally, these mice have increased bone marrow osteoprogenitors and CD8+ T-cells which support osteoblast differentiation. The animals were not protected from bone loss following OVX, but retained the hypersensitivity seen in the intact mice. Mesenchymal stem/progenitor cells (osteoprogenitors) demonstrated increased growth rate in culture and showed more robust differentiation into mineralizing bone cells. Chromosome precipitation followed by next generation sequencing and bioinformatics analysis characterized Nmp4 as a negative regulator of synthetic processes and suggested the IGF1/Akt and BMP2/Smad biochemical pathways which are likely targets for Nmp4 regulation. We have experimentally verified these pathways in immortalized bone marrow mesenchymal cells from wild type and Nmp4-KO mice. Disabling Nmp4 in estrogen replete or depleted mice confers an enhanced bone formation from intermittent parathyroid hormone.

En raison de l’augmentation de l'espérance de vie, le nombre de personnes âgées et par conséquent le taux de maladies chroniques augmente de plus en plus nécessitant le développement de nouveaux biomatériaux permettant la réparation osseuse. Le but de cette étude était d'évaluer le comportement in vitro de cellules ostéoprogénitrices cultivées sur des pastilles de phosphate tricalcique dopées (β-TCMP, Mg/Ca = 0,15) ou non (β-TCP) avec du magnésium dans un environnement statique et dynamique (débit de 0,3 mL/min), ainsi que la réponse de macrophages humains après contact avec des extraits des matériaux (ISO10993-12). Les pastilles de β-TCMP et β-TCP ont été obtenues par frittage respectivement d’une apatite calcium déficiente en calcium dopée au magnésium et de TCP (Merck). Les diffractogrammes et les spectres infrarouge ont confirmé la production de β-TCP et de β-TCMP, ratio Mg/Ca = 0,14. Des cellules osteoprogénitrices STRO+1A ont été cultivées sur des pastilles pendant 21 jours et leur comportement de prolifération et de différenciation cellulaire ont été vérifiés. Aucune différence entre les deux pastilles n’a été observée concernant le nombre de cellules après 21 jours de culture, ni sous condition statique ni dynamique. Cependant, il semble que l'environnement dynamique accélère la différenciation ostéoblastique. D'une façon générale, le β-TCMP n'a pas modifié la réponse des macrophages (le profil des cytokines) activés ou non par du lipopolysaccharide bactérien, cultivés pendant 72 heures avec des extraits de biomatériaux, par rapport au β-TCP. D’après ces résultats, les deux biomatériaux semblent être prometteurs pour le traitement des pertes osseuses. / Research on bone tissue regeneration is constantly expanding due to the improvement of life expectancy with a consequent increase in the rate of chronic diseases. The aim of this work was to evaluate the in vitro behavior of STRO+1A osteoprogenitor cells cultured onto dense tablets of tricalcium phosphate doped (β-TCMP, Mg/Ca = 0.15) or not (β-TCP) with magnesium under static and dynamic (flow rate of 0.3 mL/min), as well as the response of human macrophages after contact with the granules extracts of the materials (ISO10993-12: 2007). The β-TCMP and β-TCP tablets were obtained by sintering a calcium-deficient apatite doped with magnesium and commercial TCP (Merck), respectively. The diffractograms and infrared spectra confirmed the presence of β-TCP and β-TCMP with a Mg/Ca ratio of 0.14 (plasma spectrometry). A cell density of 5x103 was inoculated onto the tablets and then STRO+1A were cultured at 37°C/5% CO2 for up to 21 days, in which the proliferation and differentiation rate were assessed. There was no difference in cell density between the materials after 21 days of culture, neither under static nor dynamic conditions; however, it seems that the dynamic environment accelerated osteoblast differentiation. In a general way, β-TCMP did not change the response of macrophages (cytokine profile), activated or not, by bacterial lipopolysaccharide, cultured for up to 72 hours with the extracts of biomaterials, when compared to β-TCP. Based on these results, both materials appear to be adequate for bone loss therapy.

Multipotent mesenchymal stem cells (MSCs), found in many adult tissues, may be useful for regenerative medicine applications. Their identification and purification have been difficult due to their low frequency and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove contaminating hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOp) population that was also enriched for other mesenchymal precursors, including MSCs (Itoh and Aubin, 2009). To further enhance enrichment, we positively selected BMSCs and HipOps for CD73, a putative MSC marker, which resulted in no significant additional enrichment for osteoprogenitors when the population was tested in vitro. However, we also found that HipOps were enriched in vascular endothelial cells, and that removing these cells by further negative selection with CD31/PECAM resulted in a CD31(-) HipOp population with higher osteogenic capacity than HipOps in vitro and in vivo.

Multipotent mesenchymal stem cells (MSCs), found in many adult tissues, may be useful for regenerative medicine applications. Their identification and purification have been difficult due to their low frequency and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove contaminating hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOp) population that was also enriched for other mesenchymal precursors, including MSCs (Itoh and Aubin, 2009). To further enhance enrichment, we positively selected BMSCs and HipOps for CD73, a putative MSC marker, which resulted in no significant additional enrichment for osteoprogenitors when the population was tested in vitro. However, we also found that HipOps were enriched in vascular endothelial cells, and that removing these cells by further negative selection with CD31/PECAM resulted in a CD31(-) HipOp population with higher osteogenic capacity than HipOps in vitro and in vivo.