Browsing by Subject "Osteogenesis"
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Item Open AccessDesigning topographically textured microparticles for induction and modulation of osteogenesis in mesenchymal stem cell engineering.(Biomaterials, 2021-01) Amer, Mahetab H; Alvarez-Paino, Marta; McLaren, Jane; Pappalardo, Francesco; Trujillo, Sara; Wong, Jing Qian; Shrestha, Sumana; Abdelrazig, Salah; Stevens, Lee A; Lee, Jong Bong; Kim, Dong-Hyun; González-García, Cristina; Needham, David; Salmerón-Sánchez, Manuel; Shakesheff, Kevin M; Alexander, Morgan R; Alexander, Cameron; Rose, Felicity RajMesenchymal stem cells are the focus of intense research in bone development and regeneration. The potential of microparticles as modulating moieties of osteogenic response by utilizing their architectural features is demonstrated herein. Topographically textured microparticles of varying microscale features are produced by exploiting phase-separation of a readily soluble sacrificial component from polylactic acid. The influence of varying topographical features on primary human mesenchymal stem cell attachment, proliferation and markers of osteogenesis is investigated. In the absence of osteoinductive supplements, cells cultured on textured microparticles exhibit notably increased expression of osteogenic markers relative to conventional smooth microparticles. They also exhibit varying morphological, attachment and proliferation responses. Significantly altered gene expression and metabolic profiles are observed, with varying histological characteristics in vivo. This study highlights how tailoring topographical design offers cell-instructive 3D microenvironments which allow manipulation of stem cell fate by eliciting the desired downstream response without use of exogenous osteoinductive factors. Item Open AccessEnhanced osteogenic potential of mesenchymal stem cells from cortical bone: a comparative analysis.(Stem cell research & therapy, 2015-10) Fernandez-Moure, Joseph S; Corradetti, Bruna; Chan, Paige; Van Eps, Jeffrey L; Janecek, Trevor; Rameshwar, Pranela; Weiner, Bradley K; Tasciotti, Ennio
IntroductionMesenchymal stem cells (MSCs) hold great promise for regenerative therapies in the musculoskeletal system. Although MSCs from bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) have been extensively characterized, there is still debate as to the ideal source of MSCs for tissue-engineering applications in bone repair.
MethodsMSCs were isolated from cortical bone fragments (CBF-MSCs) obtained from patients undergoing laminectomy, selected by fluorescence-activated cell sorting analysis, and tested for their potential to undergo mesodermic differentiation. CBF-MSCs were then compared with BM-MSCs and AD-MSCs for their colony-forming unit capability and osteogenic potential in both normoxia and hypoxia. After 2 and 4 weeks in inducing media, differentiation was assessed qualitatively and quantitatively by the evaluation of alkaline phosphatase (ALP) expression and mineral deposition (Von Kossa staining). Transcriptional activity of osteoblastogenesis-associated genes (Alp, RUNX2, Spp1, and Bglap) was also analyzed.
ResultsThe cortical fraction of the bone contains a subset of cells positive for MSC-associated markers and capable of tri-lineage differentiation. The hypoxic conditions were generally more effective in inducing osteogenesis for the three cell lines. However, at 2 and 4 weeks, greater calcium deposition and ALP expression were observed in both hypoxic and normoxic conditions in CBF-MSCs compared with AD- and BM-MSCs. These functional observations were further corroborated by gene expression analysis, which showed a significant upregulation of Bglap, Alp, and Spp1, with a 22.50 (±4.55)-, 46.56 (±7.4)-, 71.46 (±4.16)-fold increase compared with their uninduced counterparts.
ConclusionsThis novel population of MSCs retains a greater biosynthetic activity in vitro, which was found increased in hypoxic conditions. The present study demonstrates that quantitative differences between MSCs retrieved from bone marrow, adipose, and the cortical portion of the bone with respect to their osteogenic potential exist and suggests the cortical bone as suitable candidate to use for orthopedic tissue engineering and regenerative medicine.
Item Open AccessG protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation.(Stem cell research & therapy, 2022-01-29) Brozowski, Jaime M; Timoshchenko, Roman G; Serafin, D Stephen; Allyn, Brittney; Koontz, Jessica; Rabjohns, Emily M; Rampersad, Rishi R; Ren, Yinshi; Eudy, Amanda M; Harris, Taylor F; Abraham, David; Mattox, Daniel; Rubin, Clinton T; Hilton, Matthew J; Rubin, Janet; Allbritton, Nancy L; Billard, Matthew J; Tarrant, Teresa K
BackgroundThe bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling.
MethodsMurine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures.
ResultsGRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of β-arrestin-2 in sphingosine-1-phosphate receptor internalization.
ConclusionsOur work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting β-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.
Item Open AccessGenetic engineering of mesenchymal stem cells and its application in human disease therapy.(Hum Gene Ther, 2010-11) Hodgkinson, Conrad P; Gomez, José A; Mirotsou, Maria; Dzau, Victor JThe use of stem cells for tissue regeneration and repair is advancing both at the bench and bedside. Stem cells isolated from bone marrow are currently being tested for their therapeutic potential in a variety of clinical conditions including cardiovascular injury, kidney failure, cancer, and neurological and bone disorders. Despite the advantages, stem cell therapy is still limited by low survival, engraftment, and homing to damage area as well as inefficiencies in differentiating into fully functional tissues. Genetic engineering of mesenchymal stem cells is being explored as a means to circumvent some of these problems. This review presents the current understanding of the use of genetically engineered mesenchymal stem cells in human disease therapy with emphasis on genetic modifications aimed to improve survival, homing, angiogenesis, and heart function after myocardial infarction. Advancements in other disease areas are also discussed. Item Open AccessBcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis.(International journal of molecular sciences, 2023-12) Moriishi, Takeshi; Kawai, Yosuke; Fukuyama, Ryo; Matsuo, Yuki; He, You-Wen; Akiyama, Haruhiko; Asahina, Izumi; Komori, ToshihisaBcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis. Item Open AccessMulti-Functional Small Molecule Alleviates Fracture Pain and Promotes Bone Healing.(Advanced science (Weinheim, Baden-Wurttemberg, Germany), 2023-12) Shih, Yu-Ru V; Kingsley, David; Newman, Hunter; Hoque, Jiaul; Gupta, Ankita; Lascelles, B Duncan X; Varghese, ShyniBone injuries such as fractures are one major cause of morbidities worldwide. A considerable number of fractures suffer from delayed healing, and the unresolved acute pain may transition to chronic and maladaptive pain. Current management of pain involves treatment with NSAIDs and opioids with substantial adverse effects. Herein, we tested the hypothesis that the purine molecule, adenosine, can simultaneously alleviate pain and promote healing in a mouse model of tibial fracture by targeting distinctive adenosine receptor subtypes in different cell populations. To achieve this, a biomaterial-assisted delivery of adenosine is utilized to localize and prolong its therapeutic effect at the injury site. The results demonstrate that local delivery of adenosine inhibited the nociceptive activity of peripheral neurons through activation of adenosine A1 receptor (ADORA1) and mitigated pain as demonstrated by weight bearing and open field movement tests. Concurrently, local delivery of adenosine at the fracture site promoted osteogenic differentiation of mesenchymal stromal cells through adenosine A2B receptor (ADORA2B) resulting in improved bone healing as shown by histological analyses and microCT imaging. This study demonstrates the dual role of adenosine and its material-assisted local delivery as a feasible therapeutic approach to treat bone trauma and associated pain. Item Open AccessPalmitoyl acyltransferase, Zdhhc13, facilitates bone mass acquisition by regulating postnatal epiphyseal development and endochondral ossification: a mouse model.(PLoS One, 2014) Song, I-Wen; Li, Wei-Ru; Chen, Li-Ying; Shen, Li-Fen; Liu, Kai-Ming; Yen, Jeffrey JY; Chen, Yi-Ju; Chen, Yu-Ju; Kraus, Virginia Byers; Wu, Jer-Yuarn; Lee, MT Michael; Chen, Yuan-TsongZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis. Item Open AccessPathogenesis of growth failure and partial reversal with gene therapy in murine and canine Glycogen Storage Disease type Ia.(Molecular Genetics and Metabolism, 2013-06) Brooks, Elizabeth Drake; Little, Dianne; Arumugam, Ramamani; Sun, Baodong; Curtis, Sarah; Demaster, Amanda; Maranzano, Michael; Jackson, Mark W; Kishnani, Priya; Freemark, Michael S; Koeberl, Dwight DGlycogen Storage Disease type Ia (GSD-Ia) in humans frequently causes delayed bone maturation, decrease in final adult height, and decreased growth velocity. This study evaluates the pathogenesis of growth failure and the effect of gene therapy on growth in GSD-Ia affected dogs and mice. Here we found that homozygous G6pase (-/-) mice with GSD-Ia have normal growth hormone (GH) levels in response to hypoglycemia, decreased insulin-like growth factor (IGF) 1 levels, and attenuated weight gain following administration of GH. Expression of hepatic GH receptor and IGF 1 mRNAs and hepatic STAT5 (phospho Y694) protein levels are reduced prior to and after GH administration, indicating GH resistance. However, restoration of G6Pase expression in the liver by treatment with adeno-associated virus 8 pseudotyped vector expressing G6Pase (AAV2/8-G6Pase) corrected body weight, but failed to normalize plasma IGF 1 in G6pase (-/-) mice. Untreated G6pase (-/-) mice also demonstrated severe delay of growth plate ossification at 12 days of age; those treated with AAV2/8-G6Pase at 14 days of age demonstrated skeletal dysplasia and limb shortening when analyzed radiographically at 6 months of age, in spite of apparent metabolic correction. Moreover, gene therapy with AAV2/9-G6Pase only partially corrected growth in GSD-Ia affected dogs as detected by weight and bone measurements and serum IGF 1 concentrations were persistently low in treated dogs. We also found that heterozygous GSD-Ia carrier dogs had decreased serum IGF 1, adult body weights and bone dimensions compared to wild-type littermates. In sum, these findings suggest that growth failure in GSD-Ia results, at least in part, from hepatic GH resistance. In addition, gene therapy improved growth in addition to promoting long-term survival in dogs and mice with GSD-Ia.