Indiana University-Purdue University Indianapolis (IUPUI) / The hormonal and cellular mechanisms controlling bone formation are not completely understood. The proline-rich tyrosine kinase 2 (Pyk2) is important for osteoblast (OB) activity and bone formation. However, female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone volume/total volume. Previously, our laboratory found ovariectomized Pyk2-KO mice supplemented with 17β-estradiol (E2) exhibited a greater increase in bone volume than WT mice treated with E2. The overall hypotheses of our studies are that Pyk2 regulates OB activity by modulating the E2-signaling cascade and that a Pyk2-inhibitor will promote OB activity and be suitable for bone regeneration applications. In Aim1, we determined the mechanism of action of Pyk2 and E2 in OBs. Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In the presence of E2 or raloxifene, a selective estrogen receptor (ER) modulator, both matrix formation and mineralization were further increased in Pyk2-KO OBs, but not WT OBs. Consistent with a role of Pyk2 in E2 signaling, Pyk2-depletion led to the proteasome-mediated degradation of ERα, but not ERβ. Finally, we found Pyk2-depletion and E2 have an additive effect on ERK phosphorylation, known to increase cell differentiation and survival. In Aim2, we developed a Pyk2-inhibitor loaded hydrogel and evaluated its viscosity, gelation time, swelling, degradation, and release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin exhibited viscosity and shear-thinning behavior suitable for use as an injectable-carrier. Importantly, the Pyk2-inhibitor-hydrogel was cytocompatible, retained its inhibitory activity against Pyk2 leading to an increase in OB activity. In conclusion, therapeutic strategies targeting Pyk2 may improve systemic bone formation, while Pyk2-inhibitor loaded hydrogels may be suitable for targeted bone regeneration in craniofacial and/or the other skeletal defects.

<p>Bone loss diseases, including osteoporosis affect millions of people worldwide. Understanding the underlying mechanisms behind bone homeostasis and adaptation is essential to uncovering new therapeutic targets for the prevention and treatment of bone loss diseases. Primary cilia have been implicated in the development and mechanosensation of various tissue types, including bone. The goal of the studies outlined in this thesis is to determine the mechanosensory role of primary cilia in bone cell function, bone growth, and adaptation. This goal was achieved by exploring two specific scenarios. In the first study, mice models with conditional knockouts of MKS5, a ciliary protein, in osteocytes were utilized to demonstrate that dysfunctional primary cilia in those cells result in impaired loading-induced bone formation. The hypothesis tested is that the existence of functioning primary cilia on osteocytes is crucial for proper bone adaptation following stress. The results of this study support the hypothesis, with the conditional knockout mice showing significantly lower loading-induced bone formation compared to controls. The second study highlighted the importance of the osteoblast primary cilia in bone growth by using mice models with osteoblast-specific deletion of the cilia. The hypothesis tested is that the presence of the primary cilia is crucial for proper bone growth. The results show that conditional knockout mice have lower body weights, decreased femur length, and a significantly lower rate of bone formation, confirming that the primary cilia play a great role in bone growth and development. This study has highlighted the role of primary cilia in bone health and this topic merits further investigation. </p>

This project involves the design and development of a cell stretching bioreactor device that can work in conjunction with a Random Positioning Machine (RPM) apparatus. Microgravity environments, such as in space, have been shown to induce alterations in cellular development due to inadequate mechanical loading of biological tissue. Because of this, long-term spaceflight has led to many health concerns, including osteoporosis and muscle atrophy. Space travel is rare and costly, making this research difficult to conduct, however; techniques to simulate microgravity on Earth can be achieved by using a Random Positioning Machine. This device has been a beneficial tool used to study the effect gravity has on cellular growth, yet certain tissues in the body, such as bone and muscle, require mechanical stress, strain, and mechanical loading to develop properly. Because of this, a device that can induce strain on cells while subjected to microgravity conditions is needed to further improve cellular research for space exploration. The constructed bioreactor consists of 3D printed and custom-made components that can induce uniaxial cyclic strain on cells adhered to an elastic membrane. Validation and testing of the device have shown that this bioreactor is suitable for cellular experimentation to work in conjunction with an RPM to deliver a controlled amount of strain while under microgravity conditions. / Bioengineering

No description available.

Activation of the CB1 receptor is modulated by aspartate residue D2.63176 in transmembrane helix (TMH) II. Interestingly, D2.63 does not affect the affinity for ligand binding at the CB1 receptor. Studies in class A GPCRs have suggested an ionic interaction between residues of TMHII and VII. In this report, modeling studies identified residue K373, in the extracellular (EC)-3 loop, in charged interactions with D2.63. We investigated this possibility by performing reciprocal mutations and biochemical studies. D2.63176A, K373A, D2.63176A-K373A, and the reciprocal mutant with the interacting residues juxtaposed, D2.63176K-K373D were characterized using radioligand binding and guanosine 5'-3-O-(thio)triphosphate functional assays. None of the mutations resulted in a significant change in the binding affinity of CP55,940 or SR141716A. Computational results indicate that the D2.63176-K373 ionic interaction strongly influences the conformation(s) of the EC-3 loop, providing a structure-based rationale for the importance of the EC-3 loop to signal transduction in CB1. Specifically, the putative ionic interaction results in the EC-3 loop pulling over the top (extracellular side) of the receptor; this EC-3 loop conformation may serve protective and mechanistic roles. These results suggest that the ionic interaction between D2.63176 and K373 is crucial for CB1 signal transduction. This work may help to aide drug design efforts for the effective treatment of different diseases. The cannabinoid receptors of osteoblasts may represent a target for the treatment of bone disorders such as osteoporosis. Our research demonstrates that cannabinoids can affect important signaling molecules in osteoblasts. In MC3T3-E1 osteoblastic cells, the CB1 antagonist, AM251, has been reported to induce increases in Runx2 mRNA, mineralized bone nodule formation, and activation of signaling molecules such as ERK and AKT (Wu et al., 2011). Studies from our lab characterizing mice in which both CB1 and CB2 receptors were inactivated by homologous recombination have demonstrated increased bone mass coupled with enhanced osteoblast differentiation of bone marrow stromal cells in culture (manuscript in preparation). We explored the effect of antagonizing CB1 and CB2 cannabinoid receptors in osteoblastic cells to gain insights into molecular pathways that may help to explain the effects of the endocannabinoid system (ECS) in bone development. Our data was generated by running time course experiments with MC3T3-E1 cells under the influence of SR141716A, SR144528 or both in combination. The cells were harvested with a lysis buffer at specific time points and analyzed by western blot analysis. Quantification of protein activation was calculated using LiCor imaging equipment and software. Within 15 minutes, treatment with the CB1 receptor antagonist SR141716A resulted in several fold increases in pERK, pSMAD158, and pAKT. SR144528, a CB2 receptor antagonist, caused increases in pERK and pSMAD158, but not pAKT. When both antagonists were applied together, pERK and pSMAD158 levels increased, while pAKT signaling was diminished compared to SR141716A alone. The finding that cannabinoid receptor antagonists alter the activity of the SMAD158 complex is a novel finding, which suggests that cannabinoids can influence bone morphogenic signaling pathways, and therefore play a significant role in osteoblast differentiation and function. / Cell Biology

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a dynamic organ that responds to its external environment. Cell signaling cascades are initiated within bone cells when changes in mechanical loading occur. To describe these molecular signaling networks that sense a mechanical signal and convert it into a transcriptional response, we proposed the mechanosome model. “GO” and “STOP” mechansomes contain an adhesion-associated protein and a nucleocytoplasmic shuttling transcription factor. “GO” mechanosomes functions to promote the anabolic response of bone to mechanical loading, while “STOP” mechanosomes function to suppress the anabolic response of bone to mechanical loading. While much work has been done to describe the molecular mechanisms that enhance the anabolic response of bone to loading, less is known about the signaling mechanisms that suppress bone’s response to loading. We studied two adhesion-associated proteins, Src and Pyk2, which may function as “STOP” mechanosomes. Src kinase is involved in a number of signaling pathways that respond to changes in external loads on bone. An inhibition of Src causes an increase in the expression of the anabolic bone gene osteocalcin. Additionally, mechanical stimulation of osteoblasts and osteocytes by fluid shear stress further enhanced expression of osteocalcin when Src activity was inhibited. Importantly, fluid shear stress stimulated an increase in nuclear Src activation and activity. The mechanism by which Src participates in attenuating anabolic gene transcription remains unknown. The studies described here suggest Src and Pyk2 increase their association in response to fluid shear stress. Pyk2, a protein-tyrosine kinase, exhibits nucleocytoplasmic shuttling, increased association with methyl-CpG-binding protein 2 (MBD2), and suppression of osteopontin expression in response to fluid shear stress. MBD2, known to be involved in DNA methylation and interpretation of DNA methylation patterns, may aid in fluid shear stress-induced suppression of anabolic bone genes. We conclude that both Src and Pyk2 play a role in regulating bone mass, possibly through a complex with MBD2, and function to limit the anabolic response of bone cells to fluid shear stress through the suppression of anabolic bone gene expression. Taken together, these data support the hypothesis that “STOP” mechanosomes exist and their activity is simulated in response to fluid shear stress.

Os eventos celulares e extracelulares que ocorrem durante o processo de osseointegração do titânio (Ti) são bastante influenciados por suas propriedades de superfície, como morfologia, topografia e composição química. A modificação bioquímica da superfície do Ti consiste em imobilizar proteínas ou peptídeos nessa superfície com a finalidade de induzir respostas celulares e teciduais específicas na interface osso-implante que acelerem ou aumentem a osseointegração. O objetivo deste estudo foi avaliar o desenvolvimento do fenótipo osteoblástico em culturas de células crescidas sobre Ti revestido com colágeno tipo I. Para tanto, células osteoblásticas derivadas de fragmentos ósseos do processo alveolar de humanos foram cultivadas sobre discos de Ti usinados revestidos (Ti-col) ou não (Ti-usinado) com colágeno tipo I e foram avaliados os seguintes parâmetros: adesão, morfologia e proliferação celulares, síntese de proteína total, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada, e expressão de genes marcadores do fenótipo osteoblástico por reação em cadeia da polimerase em tempo real (PCR em tempo real). O Ti-col alterou o crescimento e a expressão gênica das culturas e não teve efeito na adesão e morfologia celulares, síntese de proteína total, atividade de ALP e formação de matriz mineralizada comparado ao Ti-usinado. Esses resultados indicam que a superfície Ti-col pode favorecer um maior crescimento da cultura durante a fase proliferativa e um aumento e/ou aceleração da diferenciação, como indicado por alterações na expressão gênica de marcadores do fenótipo osteoblástico. Portanto, essa modificação de superfície pode ter um impacto nos processos de reparo e remodelação do tecido ósseo adjacente a implantes, favorecendo a ocorrência de maior formação óssea. / Cellular and extracellular events that occur during titanium (Ti) osseointegration process are highly influenced by its surface properties, such as morphology, topography and chemical composition. The objective of biochemical modification of Ti is to immobilize proteins or peptides on its surface in order to induce specific cellular and tissue responses at the boneimplant interface in order to accelerate or enhance osseointegration. The aim of this study was to evaluate the osteoblastic phenotype development in cells grown on collagen type I-coated Ti surface. Osteoblastic cells from human alveolar bone fragments were cultured on turned Ti either coated with collagen type I (col-Ti) or not (turned-Ti) and the following parameters were assessed: cell adhesion, morphology, and proliferation, total protein content, alkaline phosphatase (ALP) activity, bone-like formation and gene expression of osteoblastic markers by real-time polymerase chain reaction (real-time PCR). Col-Ti altered culture growth and gene expression of osteoblastic markers without affecting cell adhesion, morphology, protein synthesis, ALP activity, and matrix mineralization. These results demonstrated that col-Ti favours cell growth during the proliferative phase and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase, suggesting that this Ti surface modification may affect the processes of bone healing and remodelling.

Apesar da ampla aplicação clínica de plasma rico em plaquetas (PRP), a sua eficácia no reparo de defeitos ósseos e na osseointegração de implantes metálicos continua sendo questionada. Em vista disso, objetivo do presente estudo foi avaliar os efeitos de um coquetel contendo os principais fatores de crescimento (GFs) e proteínas de PRP no desenvolvimento do fenótipo osteogênico in vitro sobre titânio (Ti). O coquetel referido continha PDGF-BB, TGF-&beta;1, TGF- &beta;2, albumina, fibronectina e trombospondina. Células da linhagem osteoblástica foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sob condições osteogênicas convencionais até a subconfluência, sendo, em seguida, subcultivadas sobre superfície de Ti. As subculturas foram expostas durante os 7 primeiros dias a meio osteogênico, suplementado com GFs e proteínas, e apenas ao meio osteogênico nos 7 dias subseqüentes. Os grupos controles foram expostos apenas ao meio osteogênico. Nos experimentos dose-resposta foram utilizadas culturas primárias de calvária de ratos, as quais foram expostas ao coquetel de GFs e proteínas e às suas diluições de 1:10 e 1:100. Culturas derivadas de osso alveolar humano expostas ao coquetel de GFs e proteínas apresentaram: aumento significativo do número de células a partir do dia 4 e da proliferação celular em 1 e 4 dias; redução significativa nos níveis de atividade de fosfatase alcalina (ALP) em 4, 7 e 10 dias e ausência de marcação com vermelho de Alizarina em 14 dias. Apesar de as diluições 1:10 e 1:100 restaurarem a atividade proliferativa das culturas aos níveis controles, formações de matriz calcificada foram observadas apenas na diluição 1:100. Os resultados do presente trabalho mostram que o coquetel de GFs e proteínas inibe o desenvolvimento do fenótipo osteogênico de culturas de células osteoblásticas humanas e de ratos crescidas sobre Ti. / Background: Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. The objective of the present study was to evaluate the effects of a well-defined mixture of growth factors (GFs) and proteins (GFs+proteins) on the development of the osteogenic phenotype on titanium (Ti) in vitro. The composition of the mixture was based on the major components found in PRP preparations. Methods: The PRP-like mixture contained PDGF-BB, TGF-&beta;1, TGF-&beta;2, albumin, fibronectin, and thrombospondin. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic condition until subconfluence. They were then subcultured on Ti discs up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with GFs+proteins and to osteogenic medium alone thereafter. Control cultures were exposed to only osteogenic medium throughout the culture interval. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs+proteins and 1:10 or 1:100 dilutions of the mixture. Results: Treated human-derived cell cultures exhibited a significantly higher number of cells from day 4 on and of cycling cells at days 1 and 4, significantly reduced levels for alkaline phosphatase (ALP) activity, and no Alizarin red stained areas at day 14. Although the 1:10 and 1:100 dilutions restored the proliferative activity of rat calvaria-derived osteogenic cells to control levels, mineralized bone-like nodule formation was only observed with the 1:100 dilution. Conclusions: The present results demonstrated that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti.

Les protéoglycanes (PGs) sont des protéines présentes au niveau de la matrice extracellulaire et à la surface des cellules. Ils sont constitués d’une protéine sur laquelle sont attachées des chaînes de glycosaminoglycanes. Ils jouent un rôle essentiel dans plusieurs processus biologiques. Des mutations au niveau des gènes codant pour la protéine porteuse ou les enzymes impliquées dans la biosynthèse des GAGs sont associées à plusieurs syndromes et pathologies chez l’homme. L’initiation de la synthèse des GAGs est catalysée par la xylosyltransférase I (XT-I). La XT-I joue un rôle clé dans la régulation de la synthèse des PGs au niveau du cartilage et il a été montré récemment que les mutations hypomorphiques de la XT-I sont associées au syndrome du Desbuquois de type II (DBQD2) caractérisé par des anomalies squelettiques (ostéochondrodysplasie). Afin d’élucider le rôle de la XT-I dans le développement ostéoarticulaire, nous avons généré une souris transgénique conditionnelle Col2α1-CreERTM ;XylT1flox/flox permettant l’invalidation de la XT-I au niveau du cartilage. De façon intéressante, l’invalidation de la XT-I induit des anomalies du développement ostéoarticulaire caractérisées par un nanisme important et des défauts des éléments squelettiques. Des études histologiques et la microscopie SHG (génération de seconde harmonique) de la plaque de croissance ont permis de montrer l’importante de la XT-I dans la formation de la matrice extracellulaire (MEC), la fibrillation du collagène II, la maturation des chondrocytes et leur organisation en colonne dans la plaque de croissance. L’analyse des mécanismes moléculaires impliqués indique la perturbation de la voie de signalisation du TGF-β dans la plaque de croissance. D’autre part, des études histomorphométriques et histologiques des os ont révélé que la déficience en XT-I entraîne une accélération du processus d’ossification avec une stimulation de l’activité des ostéoclastes au niveau de l’os spongieux conduisant à une résorption osseuse importante et à une ossification accrue de l’os cortical. Ces travaux ont permis de révéler le rôle de la XT-I dans le développement ostéoarticulaire et dans le maintien de l’homéostasie du cartilage et du tissu osseux et ont mis en évidence le rôle de la voie du TGF-β dans les anomalies du développement. Ces travaux ouvrent également la voie pour le développement de thérapeutiques potentielles pour le traitement des patients atteints du syndrome de Desbuquois type II / Proteoglycans (PGs) are proteins present in the extracellular matrix and on the surface of cells. They consist of a protein to which chains of glycosaminoglycans (GAGs) are attached. PGs play an essential role in many biological processes and in the homeostasis of different tissues including cartilage, bone and skin. Mutations in the genes encoding PG core proteins or the enzymes involved in GAG biosynthesis are associated with several syndromes and pathologies in human. Initiation of GAG synthesis is catalyzed by xylosyltransferase I (XT-I). XT-I plays a key role in the regulation of the synthesis of PGs in cartilage and it has been shown recently that hypomorphic mutations of XT-I are associated with the Desbuquois syndrome type II (DBQD2), characterized by skeletal abnormalities (osteochondrodysplasia). To elucidate the role of XT-I in skeletal development, we generated a conditional transgenic mouse, Col2α1-CreERTM; XylT1flox/flox allowing the invalidation of XT-I gene in the cartilage. Interestingly, the invalidation of XT-I induces skeletal developmental abnormalities characterized by significant dwarfism, and defects in many skeletal elements. Histological studies and SHG microscopy (second harmonic generation) of the growth plate showed the importance of XT-I in extracellular matrix formation, fibrillation of collagen type II, maturation of chondrocytes and their organization in column in the growth plate. The analysis of the molecular mechanisms involved indicates the disruption of the TGF-β signaling pathway in the growth plate. On the other hand, histomorphometric and histological studies of the bones revealed that the XT-I deficiency causes an acceleration of the ossification process with a stimulation of the osteoclasts activity in spongy bone leading to bone resorption, and increased ossification of the cortical bone. This work revealed the role of XT-I in skeletal development and in the maintenance of cartilage and bone homeostasis and highlighted the role of the TGF-β pathway in developmental abnormalities. This work also paves the way for the development of potential therapeutics for the treatment of patients with Desbuquois syndrome type II

Os efeitos de preparações de plasma rico em plaquetas (PRP) sobre a atividade osteogênica in vitro e in vivo em contato com biomateriais são divergentes na literatura. O objetivo do presente estudo foi avaliar a expressão e/ou atividade de marcadores iniciais da diferenciação osteoblástica em culturas de células osteogênicas crescidas sobre titânio (Ti) e expostas a coquetel de fatores de crescimento e proteínas PRP-símile (coquetel de FCs). Células osteoblásticas primárias derivadas de calvárias de ratos recém-nascidos foram cultivadas em meio osteogênico e expostas, nos 7 primeiros dias, a coquetel de FCs nas diluições 1:1 (FCs), 1:10 (FCs/10), 1:100 (FCs/100) e 1:1000 (FCs/1000). Foram avaliados, nos tempos de 7, 10 e 14 dias: 1) os aspectos morfológicos e a imunomarcação de sialoproteína óssea (BSP) e osteopontina (OPN), por epifluorescência; 2) a proliferação celular, por ensaio de MTT; 3) a expressão de RNAm para o fator de transcrição Runx2, BSP e fosfatase alcalina (ALP), por PCR em tempo real; 4) a atividade de ALP, clivada da fração de membrana; 5) quantificação da mineralização, por extração do vermelho de Alizarina. Os resultados mostraram inibição da formação dos nódulos de matriz mineralizada em culturas FCs e atraso em seu desenvolvimento em FCs/10 e FCs/100, em comparação a FCs/1000 e controle. A expressão de Runx2, BSP e ALP era menor em todas as culturas expostas ao coquetel de FCs em 7 dias, sendo que para Runx2 e BSP notava-se o efeito concentração-dependente em 10 dias. Menores valores de atividade de ALP foram observados nas culturas FCs e FCs/10, com efeito concentração-dependente e correlação positiva com a mineralização em 7 dias, mas não em 10 e 14. Os resultados permitem concluir que a exposição ao coquetel de FCs inibe e/ou atrasa a diferenciação osteogênica de culturas primárias sobre Ti. Adicionalmente, a atividade de ALP de membrana pode ser considerada também um marcador inicial de diferenciação osteoblástica, indicativo do potencial osteogênico no modelo in vitro utilizado. / The effects of platelet-rich plasma (PRP) preparations on the in vitro and in vivo osteogenic activity in contact with biomaterials have been subject of debate and controversy in the literature. The aim of the present study was to evaluate the expression and/or activity of early markers of osteoblast differentiation in cultured osteogenic cells grown on titanium (Ti) and exposed to a PRP-like cocktail of growth factors and proteins (GFs cocktail). Primary osteoblastic cells derived from newborn rat calvarial bone were cultured in an osteogenic medium (control group) and exposed during the first 7 days of culture to the following dilutions of GFs cocktail: 1:1 (GFs), 1:10 (GFs/10), 1:100 (GFs/100) and 1:1000 (GFs/1000). At days 7, 10 and 14 of culture, the following parameters were assessed: 1) morphology and immunolabeling for bone sialoprotein (BSP) and osteopontin (OPN) by epifluorescence microscopy; 2) cell proliferation by MTT assay; 3) mRNA expression for the osteoblast markers Runx2, BSP and alkaline phosphatase (ALP) by real time PCR; 4) ALP activity following ALP cleavage from cell membrane; 5) mineralization by Alizarin red extraction. The results showed no mineralized nodules for the GFs group and a delayed nodule formation for GFs/10 and GFs/100 compared with GFs/1000 and control. Whereas Runx2, BSP and ALP mRNA levels were lower for all cultures exposed to the GFs cocktail at day 7, a concentration-dependent effect was noticed only for Runx2 and BSP at day 10. The GFs cocktail showed a concentration-dependent effect on ALP activity, with the lowest values for GFs and GFs/10 cultures and a positive correlation with mineralization at day 7 but not at day 10 or 14. In conclusion, inhibited and/or delayed osteogenic differentiation take place in primary cultures grown on Ti and exposed to the GFs cocktail. In addition, membrane ALP activity can also be considered an early marker of osteoblast differentiation, indicative of the in vitro osteogenic potential in the model used.