Micromass Cultures Of Cells Research Articles

Geometric analysis of chondrogenic self-organisation of embryonic limb bud cells in micromass culture

Spatial and temporal control of chondrogenesis generates precise, species-specific patterns of skeletal structures in the developing vertebrate limb. The pattern-template is laid down when mesenchymal cells at the core of the early limb bud condense and undergo chondrogenic differentiation. Although the mechanisms involved in organising such complex patterns are not fully understood, the interplay between BMP and Wnt signalling pathways is fundamental. Primary embryonic limb bud cells grown under high-density micromass culture conditions spontaneously create a simple cartilage nodule pattern, presenting a model to investigate pattern generation. We describe a novel analytical approach to quantify geometric properties and spatial relationships between chondrogenic condensations, utilizing the micromass model. We follow the emergence of pattern in live cultures with nodules forming at regular distances, growing and changing shape over time. Gene expression profiling supports rapid chondrogenesis and transition to hypertrophy, mimicking the process of endochondral ossification within the limb bud. Manipulating the signalling environment through addition of BMP or Wnt ligands, as well as the BMP pathway antagonist Noggin, altered the differentiation profile and nodule pattern. BMP2 addition increased chondrogenesis while WNT3A or Noggin had the opposite effect, but with distinct pattern outcomes. Titrating these pro- and anti-chondrogenic factors and examining the resulting patterns support the hypothesis that regularly spaced cartilage nodules formed by primary limb bud cells in micromass culture are influenced by the balance of Wnt and BMP signalling under a Turing-like mechanism. This study demonstrates an approach for investigating the mechanisms governing chondrogenic spatial organization using simple micromass culture.

Comparative analysis of chondrogenic and epigenetic differences in fore or hind limb bud-derived chicken micromass cell cultures

Purpose: Chicken embryos are often-used experimental models in developmental biology researches. Micromass cell culture system derived from mesenchymal cells of the embryo limb buds can serve as a reliable in vitro model for cartilage differentiation. In order to harvest the required high number of cells, these cell cultures are established from a mixture of fore and hind limb buds. However, the epigenetic regulation of the two limb bud types may vary. Our objective was to investigate the different chondrogenic and epigenetic regulation of fore or hind limb bud-derived high density cultures. Methods: Chicken embryos of Hamburger-Hamilton 22-24 stages were used to establish primary micromass cell cultures of chondrifying mesenchymal cells. Fore or hind limb buds were collected separately. Cell cultures established from a mixture of fore and hind limb buds served as control groups. Cultures were harvested on the 3rd and 6th day of culturing (according to the major steps of in vitro chondrogenesis). Changes in the quantity of cartilage-specific extracellular matrix were examined by dimethyl-methylene blue and toluidine blue metachromatic staining methods. Real-time quantitative PCR reactions were carried out to study the alterations of cartilage-specific (Sox9: SRY-box 9, Col2a1: collagen type II alpha 1 chain, Col1a1: collagen type I alpha 1 chain, Acan: aggrecan, Has2: hyaluronan synthase 2) and epigenetic marker gene expression levels (Dnmt3a: DNA methyltransferase 3 alpha, Tet1: tet methylcytosine dioxygenase 1, Ogt: O-linked N-acetylglucosamine (GlcNAc) transferase, Hdac3: histone deacetylase 3, Hdac4: histone deacetylase 4). Protein expressions of specific chondrogenic and epigenetic markers were examined by Western Blot technique. Measurements of mitochondrial activity with MTT-assay were also carried out. Results: Fore limb bud-derived micromass cell cultures showed a significantly pronounced and more advanced cartilage formation in comparison with hind limb bud- as well as mixed limb bud-derived micromass cell cultures. This phenomenon was verified not only with specific histological staining methods, but also with modern molecular biological techniques on mRNA and protein levels. Interestingly, our results indicated that the epigenetic regulatory pathway was altered in a negative way in the two examined cell culture types compared to the control cell culture. Results of the MTT-assay showed that the mitochondrial activity of hind limb bud-derived high density cultures was significantly lower compared to the other two experimental groups. Conclusions: Chicken limb bud-derived micromass cell culture system is a popular experimental model for chondrogenic researches. However, no modern scientific results can be found on specific molecular characteristics of high density cultures established from fore or hind limb buds separately. Our research aims to optimise this cell culture system for later scientific investigations, with a special regard to epigenetic analysis. Our experiments were supported by the EFOP-3.6.1-16-2016-00022, Debrecen Venture Catapult program.

Stromal cell-derived factor-1α promotes recruitment and differentiation of nucleus pulposus-derived stem cells.

BACKGROUNDIntervertebral disc (IVD) degeneration is a condition characterized by a reduction in the water and extracellular matrix content of the nucleus pulposus (NP) and is considered as one of the dominating contributing factors to low back pain. Recent evidence suggests that stromal cell-derived factor 1α (SDF-1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) direct the migration of stem cells associated with injury repair in different musculoskeletal tissues.AIMTo investigate the effects of SDF-1α on recruitment and chondrogenic differentiation of nucleus pulposus-derived stem cells (NPSCs).METHODSWe performed real-time RT-PCR and enzyme-linked immunosorbent assay to examine the expression of SDF-1α in nucleus pulposus cells after treatment with pro-inflammatory cytokines in vitro. An animal model of IVD degeneration was established using annular fibrosus puncture in rat coccygeal discs. Tissue samples were collected from normal control and degeneration groups. Differences in the expression of SDF-1α between the normal and degenerative IVDs were analyzed by immunohistochemistry. The migration capacity of NPSCs induced by SDF-1α was evaluated using wound healing and transwell migration assays. To determine the effect of SDF-1α on chondrogenic differentiation of NPSCs, we conducted cell micromass culture and examined the expression levels of Sox-9, aggrecan, and collagen II. Moreover, the roles of SDF-1/CXCR4 axis in the migration and chondrogenesis differentiation of NPSCs were analyzed by immunofluorescence, immunoblotting, and real-time RT-PCR.RESULTSSDF-1α was significantly upregulated in the native IVD cells cultured in vitro with pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mimicking the degenerative settings. Immunohistochemical staining showed that the level of SDF-1α was also significantly higher in the degenerative group than in the normal group. SDF-1α enhanced the migration capacity of NPSCs in a dose-dependent manner. In addition, SDF-1α induced chondrogenic differentiation of NPSCs, as evidenced by the increased expression of chondrogenic markers using histological and immunoblotting analyses. Real-time RT-PCR, immunoblotting, and immunofluorescence showed that SDF-1α not only increased CXCR4 expression but also stimulated translocation of CXCR4 from the cytoplasm to membrane, accompanied by cytoskeletal rearrangement. Furthermore, blocking CXCR4 with AMD3100 effectively suppressed the SDF-1α-induced migration and differentiation capacities of NPSCs.CONCLUSIONThese findings demonstrate that SDF-1α has the potential to enhance recruitment and chondrogenic differentiation of NPSCs via SDF-1/CXCR4 chemotaxis signals that contribute to IVD regeneration.

Gelatin microspheres releasing transforming growth factor drive in vitro chondrogenesis of human periosteum derived cells in micromass culture

For cartilage tissue engineering, several in vitro culture methodologies have displayed potential for the chondrogenic differentiation of mesenchymal stem cells (MSCs). Micromasses, cell aggregates or pellets, and cell sheets are all structures with high cell density that provides for abundant cell-cell interactions, which have been demonstrated to be important for chondrogenesis. Recently, these culture systems have been improved via the incorporation of growth factor releasing components such as degradable microspheres within the structures, further enhancing chondrogenesis. Herein, we incorporated different amounts of gelatin microspheres releasing transforming growth factor β1 (TGF-β1) into micromasses composed of human periosteum derived cells (hPDCs), an MSC-like cell population. The aim of this research was to investigate chondrogenic stimulation by TGF-β1 delivery from these degradable microspheres in comparison to exogenous supplementation with TGF-β1 in the culture medium. Microscopy showed that the gelatin microspheres could be successfully incorporated within hPDC micromasses without interfering with the formation of the structure, while biochemical analysis and histology demonstrated increasing DNA content at week 2 and accumulation of glycosaminoglycan and collagen at weeks 2 and 4. Importantly, similar chondrogenesis was achieved when TGF-β1 was delivered from the microspheres compared to controls with TGF-β1 in the medium. Increasing the amount of growth factor within the micromasses by increasing the amount of microspheres added did not further improve chondrogenesis of the hPDCs. These findings demonstrate the potential of using cytokine releasing, gelatin microspheres to enhance the chondrogenic capabilities of hPDC micromasses as an alternative to supplementation of the culture medium with growth factors. Statement of significanceGelatin microspheres are utilized for growth factor delivery to enhance chondrogenesis of mesenchymal stem cells (MSCs) in high cell density culture systems. Herein, we employ a new combination of these microspheres with micromasses of human periosteum-derived cells, which possess ease of isolation, excellent expansion potential, and MSC-like differentiation capabilities. The resulting localized delivery of transforming growth factor β1 increases glycosaminoglycan and collagen production within the micromasses without exogenous stimulation in the medium. This unique combination is able to drive chondrogenesis up to similar levels as seen in micromasses that do receive exogenous stimulation. The addition of growth factor releasing microspheres to high cell density micromasses has the potential to reduce costs associated with this strategy for cartilage tissue engineering.

Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation

C3H10T1/2, a mouse mesenchymal stem cell line, is a well-known in vitro model of chondrogenesis that can be easily employed to recapitulate some of the mechanisms intervening in this process. Moreover, these cells can be used to validate the effect of candidate molecules identified by high throughput screening approaches applied to the development of targeted therapy for human disorders in which chondrogenic differentiation may be involved, as in conditions characterized by heterotopic endochondral bone formation. Chondrogenic differentiation of C3H10T1/2 cells can be monitored by applying quantitative polymerase chain reaction (qPCR), one of the most sensitive methods that allows detection of small dynamic changes in gene expression between samples obtained under different experimental conditions. In this work, we have used qPCR to monitor the expression of specific markers during chondrogenic differentiation of C3H10T1/2 cells in micromass cultures. Then we have applied the geNorm approach to identify the most stable reference genes suitable to get a robust normalization of the obtained expression data. Among 12 candidate reference genes (Ap3d1, Csnk2a2, Cdc40, Fbxw2, Fbxo38, Htatsf1, Mon2, Pak1ip1, Zfp91, 18S, ActB, GAPDH) we identified Mon2 and Ap3d1 as the most stable ones during chondrogenesis. ActB, GAPDH and 18S, the most commonly used in the literature, resulted to have an expression level too high compared to the differentiation markers (Sox9, Collagen type 2a1, Collagen type 10a1 and Collagen type 1a1), therefore are actually less recommended for these experimental conditions. In conclusion, we identified nine reference genes that can be equally used to obtain a robust normalization of the gene expression variation during the C3H10T1/2 chondrogenic differentiation.

Effect of the PTHrP(1-34) analog abaloparatide on inducing chondrogenesis involves inhibition of intracellular reactive oxygen species production

Osteoarthritis (OA) is a degenerative joint disease characterized by a progressive loss of articular cartilage. Mesenchymal stem cells transplanted to damaged tissues are promising for OA cartilage repair. However, these cells are poor survival after transplantation and acquire hypertrophic properties during chondrogenic induction. Parathyroid hormone-related protein (PTHrP) promotes chondrogenesis and suppresses chondrocyte hypertrophic differentiation. Additionally, PTHrP was reported to have anti-oxidant effects. The synthetic PTHrP(1-34) analog abaloparatide (ABL) is a newly approved drug for osteoporosis therapy. It is unknown whether ABL stimulates chondrogenesis and affects intracellular reactive oxygen species (ROS) production. By using mouse embryonic limb bud mesenchymal stem cells in micromass culture as an in vitro model of chondrogenic differentiation, we found that mesenchymal stem cells in micromass cultures spontaneously produced ROS, and N-acetyl-l-cysteine, a potent antioxidant, enhanced chondrogenesis. The effect of ABL on stimulation of chondrogenesis is involved in its inhibition of intracellular ROS generation. These novel findings support the use of ABL for the damaged cartilage regeneration.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Reduces Oxidative and Mechanical Stress-Evoked Matrix Degradation in Chondrifying Cell Cultures.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide also secreted by non-neural cells, including chondrocytes. PACAP signaling is involved in the regulation of chondrogenesis, but little is known about its connection to matrix turnover during cartilage formation and under cellular stress in developing cartilage. We found that the expression and activity of hyaluronidases (Hyals), matrix metalloproteinases (MMP), and aggrecanase were permanent during the course of chondrogenesis in primary chicken micromass cell cultures, although protein levels changed daily, along with moderate and relatively constant enzymatic activity. Next, we investigated whether PACAP influences matrix destructing enzyme activity during oxidative and mechanical stress in chondrogenic cells. Exogenous PACAP lowered Hyals and aggrecanase expression and activity during cellular stress. Expression and activation of the majority of cartilage matrix specific MMPs such as MMP1, MMP7, MMP8, and MMP13, were also decreased by PACAP addition upon oxidative and mechanical stress, while the activity of MMP9 seemed not to be influenced by the neuropeptide. These results suggest that application of PACAP can help to preserve the integrity of the newly synthetized cartilage matrix via signaling mechanisms, which ultimately inhibit the activity of matrix destroying enzymes under cellular stress. It implies the prospect that application of PACAP can ameliorate articular cartilage destruction in joint diseases.

Embryonic Midfacial Palatal Organ Culture Methods in Developmental Toxicology.

The morphogenesis of the secondary palate provides an interesting model for many of the processes involved in embryonic development. A number of in vitro models have been used to study craniofacial development, including whole embryo culture, palatal mesenchymal and micromass cell cultures, and Trowell-like palatal cultures in which dissected palates are cultured individually or as pairs in contact on a support above medium. This chapter presents a detailed protocol for the culture of maxillary midfacial tissues, including the palatal shelves, in suspension culture. This method involves isolation of the midfacial tissues (maxillary arch and palatal shelves) and suspension of the tissues in medium in flasks. On a rocker in an incubator, the palatal shelves elevate, grow, make contact, and fuse in a time span analogous to that occurring in the intact embryo in utero.

Improved Protocol for Chondrogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells -Effect of PTHrP and FGF-2 on TGFβ1/BMP2-Induced Chondrocytes Hypertrophy.

Growth factors have a pivotal role in chondrogenic differentiation of stem cells. The differential effects of known growth factors involved in the maintenance and homeostasis of cartilage tissue have been previously studied in vitro. However, there are few reported researches about the interactional effects of growth factors on chondrogenic differentiation of stem cells. The aim of this study is to examine the combined effects of four key growth factors on chondrogenic differentiation of mesenchymal stem cells (MSCs). Isolated and expanded rabbit bone marrow-derived MSCs underwent chondrogenic differentiation in a micromass cell culture system that used a combination of the following growth factors: transforming growth factor beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP2), parathyroid hormone related protein (PTHrP), and fibroblast growth factor 2 (FGF2) according to a defined program. The chondrogenic differentiation program was analyzed by histochemistry methods, quantitative RT-PCR (qRT-PCR), and measurement of matrix deposition of sulfated glycosaminoglycan (sGAG) and collagen content at days 16, 23, and 30. The results showed that the short-term combination of TGF-β1 and BMP-2 increased sGAG and collagen content, Alkaline phosphates (ALP) activity, and type X collagen (COL X) expression. Application of either PTHrP or FGF2 simultaneously decreased TGF-β1/BMP-2 induced hypertrophy and chondrogenic markers (at least for FGF2). However, successive application of PTHrP and FGF2 dramatically maintained the synergistic effects of TGF-β1/BMP-2 on the chondrogenic differentiation potential of MSCs and decreased unwanted hypertrophic markers. This new method can be used effectively in chondrogenic differentiation programs.

Dynamic behavior of different quantities of osteoblasts during formation of micromass cultures.

Implantation of micromass cultures of osteoblastic cells offers the possibility of scaffold free tissue engineering for example, regeneration of bone defects. However, the details of cell dynamics during the formation of these micromasses are still not well understood. This study aims to investigate and clarify the extent to which cell quantity influences the dynamics of micromass formation of osteoblastic cell cultures. For this purpose, the migration and aggregation during this process are investigated by optical inspection employing image processing software that allows for automated tracking of cell groups using digital image correlation. An exponential time behavior is observed with respect to the velocity of the cells and the distance of the cells to their common center of gravity. Characteristic time constants are derived as quantitative measures of the cell dynamics. The results indicate that the time constants strongly depend on the quantity of cells, that is, will decrease with increasing cell quantity. © 2018 International Society for Advancement of Cytometry.

Roles of Ihh signaling in chondroprogenitor function in postnatal condylar cartilage

Condylar articular cartilage in mouse temporomandibular joint develops from progenitor cells near the articulating surface that proliferate, undergo chondrogenesis and mature into hypertrophic chondrocytes. However, it remains unclear how these processes are regulated, particularly postnatally. Here we focused on the apical polymorphic layer rich in progenitors and asked whether the phenotype and fate of the cells require signaling by Indian hedgehog (Ihh) previously studied in developing long bones. In condyles in newborn mice, the apical polymorphic/progenitor cell layer was ~10 cell layer-thick and expressed the articular matrix marker Tenascin-C (Tn-C), and the underlying thick cell layer expressed Tn-C as well as the chondrogenic master regulator Sox9. By 1 month, condylar cartilage had gained its full width, but became thinner along its main longitudinal axis and displayed hypertrophic chondrocytes. By 3 months, articular cartilage consisted of a 2–3 cell layer-thick zone of superficial cells and chondroprogenitors expressing both Tn-C and Sox9 and a bottom zone of chondrocytes displaying vertical matrix septa. EdU cell tracing in juvenile mice revealed that conversion of chondroprogenitors into chondrocytes and hypertrophic chondrocytes required about 48 and 72 h, respectively. Notably, EdU injection in 3 month-old mice labeled both progenitors and maturing chondrocytes by 96 h. Conditional ablation of Ihh in juvenile/early adult mice compromised chondroprogenitor organization and function and led to reduced chondroprogenitor and chondrocyte proliferation. The phenotype of mutant condyles worsened over time as indicated by apoptotic chondrocyte incidence, ectopic chondrocyte hypertrophy, chondrocyte column derangement and subchondral bone deterioration. In micromass cultures of condylar apical cells, hedgehog (Hh) treatment stimulated chondrogenesis and alkaline phosphatase (APase) activity, while treatment with HhAntag inhibited both. Our findings indicate that the chondroprogenitor layer is continuously engaged in condylar growth postnatally and its organization and functioning depend on hedgehog signaling.

Spatial control of chondrogenesis assessed through micromass cultures of embryonic limb bud cells

Spatial control of chondrogenesis assessed through micromass cultures of embryonic limb bud cells

New scaffolds encapsulating TGF-β3/BMP-7 combinations driving strong chondrogenic differentiation

The regeneration of articular cartilage remains an unresolved question despite the current access to a variety of tissue scaffolds activated with growth factors relevant to this application. Further advances might result from combining more than one of these factors; here, we propose a scaffold composition optimized for the dual delivery of BMP-7 and TGF-β3, two proteins with described chondrogenic activity. First, we tested in a mesenchymal stem cell micromass culture with TGF-β3 whether the exposure to microspheres loaded with BMP-7 would improve cartilage formation. Histology and qRT-PCR data confirmed that the sustained release of BMP-7 cooperates with TGF-β3 towards chondrogenic differentiation. Then, we optimized a scaffold prototype for tissue culture and dual encapsulation of BMP-7 and TGF-β3. The scaffolds were prepared from poly(lactic-co-glycolic acid), and BMP-7/TGF-β3 were loaded as nanocomplexes with heparin and Tetronic 1107. The scaffolds showed the sustained release of both proteins over four weeks, with minimal burst effect. We finally cultured human mesenchymal stem cells on these scaffolds, in the absence of exogenous chondrogenic factor supplementation. The cells cultured on the scaffolds loaded with BMP-7 and TGF-β3 showed clear signs of cartilage formation macroscopically and histologically. RT-PCR studies confirmed a clear upregulation of cartilage markers SOX9 and Aggrecan. In summary, scaffolds encapsulating BMP-7 and TGF-β3 can efficiently deliver a cooperative growth factor combination that drives efficient cartilage formation in human mesenchymal stem cell cultures. These results open attractive perspectives towards in vivo translation of this technology in cartilage regeneration experiments.

Chondrogenesis of embryonic limb bud cells in micromass culture progresses rapidly to hypertrophy and is modulated by hydrostatic pressure.

Chondrogenesis in vivo is precisely controlled in time and space. The entire limb skeleton forms from cells at the core of the early limb bud that condense and undergo chondrogenic differentiation. Whether they form stable cartilage at the articular surface of the joint or transient cartilage that progresses to hypertrophy as endochondral bone, replacing the cartilage template of the skeletal rudiment, is spatially controlled over several days in the embryo. Here, we follow the differentiation of cells taken from the early limb bud (embryonic day 11.5), grown in high-density micromass culture and show that a self-organising pattern of evenly spaced cartilage nodules occurs spontaneously in growth medium. Although chondrogenesis is enhanced by addition of BMP6 to the medium, the spatial pattern of nodule formation is disrupted. We show rapid progression of the entire nodule to hypertrophy in culture and therefore loss of the local signals required to direct formation of stable cartilage. Dynamic hydrostatic pressure, which we have previously predicted to be a feature of the forming embryonic joint region, had a stabilising effect on chondrogenesis, reducing expression of hypertrophic marker genes. This demonstrates the use of micromass culture as a relatively simple assay to compare the effect of both biophysical and molecular signals on spatial and temporal control of chondrogenesis that could be used to examine the response of different types of progenitor cell, both adult- and embryo-derived.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

Novel role for cyclophilin A in regulation of chondrogenic commitment and endochondral ossification.

Recent studies showed that cyclophilin A (CypA) promotes NF-κB/p65 nuclear translocation, resulting in enhanced NF-κB activity and altered expression of its target genes, such as the Sox9 transcriptional factor, which plays a critical role in chondrogenic differentiation and endochondral ossification. In this report, we unveil the role of CypA in signal-induced chondrogenic differentiation and endochondral ossification. Expression levels of the chondrogenic differentiation markers and transcriptional regulators Sox9 and Runx2 were all significantly lower in CypA knockdown chondrogenic cells than in wild-type cells, indicating that CypA plays a functional role in chondrogenic differentiation. In vitro differentiation studies using micromass cultures of mouse limb bud cells further supported the conclusion that CypA is needed for chondrogenic differentiation. Newborn CypA-deficient pups double stained with alcian blue and alizarin red exhibited generalized, pronounced skeletal defects, while high-resolution micro-computed tomography (microCT) analyses of the femurs and lumbar vertebrae revealed delayed or incomplete endochondral ossification. Comparative histology and immunohistochemistry (IHC) analyses further verified the effects of CypA deficiency on chondrogenic differentiation. Our results provide evidence for the important contribution of CypA as a pertinent component acting through NF-κB-Sox9 in regulation of chondrogenesis signaling. These findings are important to better understand signal-induced chondrogenesis of chondrogenic progenitors in physiological and pathophysiological contexts.

The effect of growth-mimicking continuous strain on the early stages of skeletal development in micromass culture.

Embryonic skeletogenesis involves proliferation, condensation and subsequent chondrogenic differentiation of mesenchymal precursor cells, and the strains and stresses inherent to these processes have been hypothesized to influence skeletal development. The aim of this study was to determine the effect of growth-mimicking strain on the process of early skeletal development in vitro. To this end, we applied continuous uniaxial strain to embryonic skeletal precursor cells in micromass culture. Strain was applied at different times of culture to specifically address the effect of mechanical loading on the sequential stages of cellular proliferation, condensation and differentiation. We found that growth-mimicking strain at all three times did not affect proliferation or chondrogenic differentiation under the tested conditions. However, the timing of the applied strain did play a role in the density of mesenchymal condensations. This finding suggests that a mechanically dynamic environment, and specifically strain, can influence skeletal patterning. The growth-mimicking micromass model presented here may be a useful tool for further studies into the role of mechanical loading in early skeletal development.

CXC chemokine ligand 12a enhances chondrocyte proliferation and maturation during endochondral bone formation

We investigated the roles of CXC chemokine ligand 12a (CXCL12a), also known as stromal cell-derived factor-1α (SDF-1α), in endochondral bone growth, which can give us important clues to understand the role of CXCL12a in osteoarthritis (OA). Primary chondrocytes and tibial explants from embryonic 15.5 day-old mice were cultured with recombinant mouse CXCL12a. To assess the role of CXCL12a in chondrogenic differentiation, we conducted mesenchymal cell micromass culture. In tibia organ cultures, CXCL12a increased total bone length in a dose-dependent manner through proportional effects on cartilage and bone. In accordance with increased length, CXCL12a increased the protein level of proliferation markers, such as cyclin D1 and proliferating cell nuclear antigen (PCNA), in primary chondrocytes as well as in tibia organ culture. In addition, CXCL12a increased the expression of Runx2, Col10 and MMP13 in primary chondrocytes and tibia organ culture system, implying a role of CXCL12a in chondrocyte maturation. Micromass cultures of limb-bud mesenchymal progenitor cells (MPCs) revealed that CXCL12a has a limited effect on early chondrogenesis, but significantly promoted maturation of chondrocytes. CXCL12a induced the phosphorylation of p38 and Erk1/2 MAP kinases and IκB. The increased expression of cyclin D1 by CXCL12a was significantly attenuated by inhibitors of MEK1 and NF-κB. On the other hand, p38 and Erk1/2 MAP kinase and NF-κB signaling were associated with CXCL12a-induced expression of Runx2 and MMP13, the marker of chondrocyte maturation. CXCL12a promoted the proliferation and maturation of chondrocytes, which strongly suggest that CXCL12a may have a negative effect on articular cartilage and contribute to OA progression.

Notch inhibits chondrogenic differentiation of mesenchymal progenitor cells by targeting Twist1

While Notch signaling plays a critical role in the regulation of cartilage formation, its downstream targets are unknown. To address this we performed gain and losses of function experiments and demonstrate that Notch inhibition of chondrogenesis acts via up-regulation of the transcription factor Twist1. Upon Notch activation, murine limb bud mesenchymal progenitor cells in micromass culture displayed an inhibition of chondrogenesis. Twist1 was found to be exclusively expressed in mesenchymal progenitor cells at the onset stage of chondrogenesis during Notch activation. Inhibition of Notch signaling in these cells significantly reduced protein expression of Twist1. Furthermore, the inhibition effect of NICD1 on MPC chondrogenesis was markedly reduced by knocking down of Twist1. Constitutively active Notch signaling significantly enhanced Twist1 promoter activity; whereas mutation studies indicated that a putative NICD/RBPjK binding element in the promoter region is required for the Notch-responsiveness of the Twist1 promoter. Finally, chromatin immunoprecipitation assays further confirmed that the Notch intracellular domain influences Twist1 by directly binding to the Twist1 promoter. These data provide a novel insight into understanding the molecular mechanisms behind Notch inhibition of the onset of chondrogenesis.

Effects of Teratogenic Drugs on CYP1A1 Activity in Differentiating Rat Embryo Cells

CYP1A1, a P450 isoenzyme, is involved in the phase I xenobiotic metabolism including teratogen drugs. In the present study, the ability of teratogens to elevate the embryonic expression of CYP1A1 was examined. Micromass cell cultures prepared from day 13 rat embryo limb buds (LB). LB cells were cultivated and exposed for 5 days to retinoic acid (RA), hydrocortisone (HC), caffeine (CA) and quinine (QN). CYP1A1 protein expression and activity were measured using immunofluorescence staining and ethoxyresorufin O-deethylation (EROD) assay, respectively. The EROD activity increased significantly following LB cells exposure to RA and HC (p<0.05) but the expression of CYP1A1 protein was reduced by these drugs, whereas the expression of CYP1A1 protein and EROD activity decreased significantly following the addition of CA and QN (p<0.05, p<0.01). Our findings show that studied teratogens have potency to increase CYP1A1 activity.