Cartilage morphogenesis in vitro
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ABSTRACT The morphogenetic capacity of prechondrogenic mesenchyme from two developmentally distinct sources was investigated in high density micromass cultures. We confirmed an earlier report (Weiss & Moscona, 1958) that scleral mesenchyme formed cartilage sheets whilst limb bud mesenchyme formed distinct cartilage nodules. It was thus suggested by these authors that this morphogenesis was tissue type specific. However, by varying cell density at inoculation (which controls cell configuration) and by varying the relative amount of prechondrogenic mesenchyme present in cultures we found that dramatic changes in morphogenesis could be brought about. Viewed in these terms we suggest that cartilage morphogenesis in vitro is dependent on cell configuration and the presence of non-chondrogenic cell types and hence is not necessarily a function of an intrinsic morphogenetic potential of the constituent cells.Keywords:
Mesenchyme
Chondrogenesis
ABSTRACT This study is a continuing investigation of the effect of the brachypod mouse mutation on cell interactions and chondrogenesis during early limb development. In this report, cell adhesiveness was assessed in fused fragments of brachypod and normal limb-bud mesenchyme. Examination of the interface of fused distal postaxial limb fragments show brachypod limb mesenchyme to be more adhesive than normal limb mesenchyme. Chondrogenesis within brachypod fragments is delayed and less extensive than in normal fragments. In addition, chondrogenesis within normal fragments is not affected by the juxtaposition of the brachypod fragment, and vice versa.
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Limb bud
Limb development
Ingression
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Recent evidence suggests that low oxygen tension (hypoxia) may control fetal development and differentiation. A crucial mediator of the adaptive response of cells to hypoxia is the transcription factor Hif-1α. In this study, we provide evidence that mesenchymal condensations that give origin to endochondral bones are hypoxic during fetal development, and we demonstrate that Hif-1α is expressed and transcriptionally active in limb bud mesenchyme and in mesenchymal condensations. To investigate the role of Hif-1α in mesenchymal condensations and in early chondrogenesis, we conditionally inactivated Hif-1α in limb bud mesenchyme using a Prx1 promoter-driven Cre transgenic mouse. Conditional knockout of Hif-1α in limb bud mesenchyme does not impair mesenchyme condensation, but alters the formation of the cartilaginous primordia. Late hypertrophic differentiation is also affected as a result of the delay in early chondrogenesis. In addition, mutant mice show a striking impairment of joint development. Our study demonstrates a crucial, and previously unrecognized, role of Hif-1α in early chondrogenesis and joint formation.
Mesenchyme
Chondrogenesis
Limb bud
Limb development
Endochondral ossification
Conditional gene knockout
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Primordium
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An increasing number of studies have detected mesenchymal stromal cells (MSCs) and mesenchymal progenitor cells (MPCs) in the peripheral blood (PB). This study aimed to systematically review the possibility of using the PB as a source for chondrogenic progenitors. PubMed, the Web of Science, and Embase were searched for relevant articles. The findings of the studies were reviewed to evaluate the biological characteristics of PB-derived MSCs, chondrogenic MPCs, and their applications in cartilage repair. Thirty-six articles were included in the final analysis, 29 of which indicated that PB is a potential source for chondrogenic progenitor cells. Thirty-two studies reporting in vitro data, including 79.2% (19/24) of studies on PB MSCs and 75% (6/8) of studies on chondrogenic PB MPCs, confirmed the existence of PB MSCs and PB MPCs, respectively; all in vivo investigations showed that using PB as a cell source enhanced cartilage repair. PB MSCs were found in most of the animal studies (12/13), whereas 7 of 11 human studies described the presence of PB MSCs. This systematic review strongly indicates the existence of MSCs in the PB of animals, whereas the presence of MSCs in human PB is less clear. Although the presence of both MSCs and chondrogenic MPCs in the PB, as well as a few favorable outcomes associated with the use of PB-derived progenitors for cartilage repair in vivo, suggests that the PB is a potential alternative source of chondrogenic progenitor cells for cartilage repair, the efficacy of these cells has not been compared to those from other sources, such as bone marrow or adipose tissue in controlled studies.
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�Cellshape isknown to influencethe chondrogenic differentiation ofculturedlimb bud mesenchyme cells(Solursh,M .,T.F.Linsenmayer,and K.L.Jensen,1982,Dev .Biol.,94: 259-264) .To testwhether specificcytoskeletalcomponents mediate thisinfluenceof cell shape, we examined differentcytoskeletondisruptingagentsfortheirabilityto affectchondrogenesis.Limb bud cellsculturedat subconfluentdensitieson plasticsubstratanormally become flattened,containnumerous cytoplasmicmicrotubulesand actinbundles,and do not undergo spontaneous chondrogenesis.Ifsuch culturesaretreatedwith 2 /Ag/mlcytochalasin D duringthe initial 3-24 h inculture,thecellsround up, losetheiractincables,and undergo chondrogenesis,asindicatedby theproductionofimmunologicallydetectabletype IIcollagen and a pericellular Alcianblue stainingmatrix.CytochalasinD alsoinduces cartilageformation by high-densityculturesof proximal limb bud cells,which normally become blocked ina protodifferentiated state.Inaddition,cytochalasinD was found toreversethenormal inhibition by fibronectinofchondrogenesis by proximal limb bud cellsculturedin hydrated Collagen gels.Agents thatdisruptmicrotubuleshave no apparent effecton the shape orchondrogenic differentiation of limb bud mesenchymal cells.These resultssuggestan involvement of the actincytoskeletonin controllingcellshape and chondrogenic differentiation of limb bud mesenchyme .Interactionsoftheactincytoskeletonand extracellular matrixcomponents may providea regulatorymechanism formesenchyme celldifferentiation intocartilageor fibrous connectivetissueinthedeveloping limb.
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Insoluble "biomatrix" of mesenchyme is a stimulator of mammary cell differentiation in vitro, but its effect in the morphogenesis is unknown. Fetal salivary mesenchyme induces intense local duct formation when implanted into adult mammary gland. We have therefore tested whether biomatrix prepared from fetal salivary mesenchyme retains this abillity to stimulate duct formation in vivo. Salivary mesenchyme isolated from mouse fetuses at 13.5-14.0 days of gestation, extracted sequentially with water and with 1 M NaCl, then digested with DNAse and RNAse was implanted into mammary glands of female mice and left for periods of 1-35 days. In approximately 40% of recipients, the local epithelium either formed cyst like structures, or else "spikes" of mammary epithelium penetrated the matrix forming a simplified ductwork inside it. Similar responses were elicited by salivary mesenchyme killed by freezing and also by biomatrix prepared from fetal mammary fat pad precursor tissue, mesenchyme of fetal lung, and fetal heart, liver, and brain. However when mesenchyme was either fixed with glutaraldehyde or sonicated and embedded in polymer blocks before implantation, no epithelial response was noted. These observations suggest that the biomatrix provides a passive scaffolding that contributes to morphogenesis of mammary ducts, is insufficient to support normal morphogenesis.
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Limb development
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