Wednesday, August 15, 2012

Comparison of cyclic compressive loading gene expression to LSJL gene expression

Here's the genes expressed during LSJL.  Not by chondrocytes but in adult bone marrow there are no chondrocytes to express the chondrogenic genes.

Note this study had successful chondroinduction so if the genes are similar that bodes well for LSJL.

Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading.

"Cyclic compressive loading may promote chondrogenesis of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose cultures through the transforming growth factor (TGF)-beta signaling pathway. The activating protein 1 (AP-1) (Jun-Fos) complex mediated autoinduction of TGF-beta1 and its binding activity [are] essential for promoting chondrogenesis of mesenchymal cells[overexpression of c-Fos has been shown to inhibit chondrogenesis, maybe it depends on the cell line ATDC5 cells are already pro-chondrogenic and c-Fos may inhibit chondrogenesis in this cell type but not other cell types, LSJL has never used this cell line], whereas Sox9 was identified as an essential transcription factor for chondrogenesis of embryonic mesenchymal cells. The objective of this study was to examine temporal expression patterns of early responsive genes (Sox9, c-Fos, c-Jun, and TGF-beta type I and II receptors)[LSJL increases c-Fos and TGF-beta RI) and induction of their corresponding proteins in agarose culture of rabbit BM-MSCs subjected to cyclic compressive loading. The rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell-agarose constructs were made by suspending BM-MSCs in 2% agarose gel (10(7) cells/ml) for cyclic, unconfined compression tests performed in a custom-made bioreactor. In the loading experiment, specimens were subjected to sinusoidal loading with a magnitude of 15% strain at a frequency of 1 hertz for 4 hours per day. Experiments were conducted for 2 consecutive days. This study showed that cyclic compressive loading promoted gene expressions of Sox9, c-Jun, and both TGF-beta receptors and productions of their corresponding proteins, whereas those gene expressions exhibited different temporal expression patterns among genes and between 2 days of testing. The gene expression of c-Fos was detected only in the samples subjected to 1-hour dynamic compressive loading.  The TGF-beta signal transduction and activities of AP-1 and Sox9 are involved in the early stage of BM-MSC chondrogenesis promoted by dynamic compressive loading."

"specimens of the loading group were placed into the testing chamber of the bioreactor developed previously  and then preloaded with 5% static strain for 300 seconds. Following preloading, sinusoidal compressive loading was applied with a magnitude of 15% at a frequency of 1 hertz for 4 hours. After the unconfined compression test, specimens were unloaded and incubated for 20 hours to allow constructs to recover. Experiments were conducted for 2 consecutive days. Specimens of the control group were cultured in serum-free medium that was also used in the compression tests. The culture medium was changed every day after the compression test. For each day of testing, gene expressions of the loading group were analyzed at three time points (after 1, 2, and 4 hours of loading) during the 4-hour compression test and three time points (after 4, 8, and 20 hours of rest) during the recovery period. To examine protein induction, the samples, which were subjected to 2 and 4 hours of loading and recovered for 2 and 4 hours after 4-hour loading at the first day of testing, were used for protein analyses. Samples of the control group were analyzed at the beginning of the compression test and the recovery period."

"The intracellular TGF-β signal transduction is initiated by type I receptor (TβR-I) after its phosphorylation by ligand-bound type II receptor (TβR-II)"

"Activating protein 1 (AP-1), a transcription factor complex of the Jun and Fos nuclear oncoproteins, is one of the downstream targets for mitogen-activated protein kinase (MAPK) signaling pathway. MAPKs regulated chondrogenesis of chick embryo limb bud cells at postprecartilage condensation stages"

"overexpression of c-Fos gene inhibited chondrocyte differentiation of ATDC5 chondrogenic cells"

"During the 4-hour compression test, gene expressions of c-Jun, Sox9, type II collagen, TGF-β1, TβR-I, and TβR-II for the loading group gradually increased and reached the peak after 2 hours of loading and then decreased from that peak after 4 hours of loading. Only the loading group exhibited a weak expression of c-Fos gene after 1 hour of loading . After the 4-hour compression test, all gene expressions of the loading group decreased to a level similar to (i.e., c-Jun, type II collagen, TGF-β1, and TβR-II) or slightly higher than (i.e., Sox9) those of the control group except that the expression of the TβR-I gene exhibited another peak after 8 hours of rest "

"the significant upregulation of TβR-I and TβR-II gene expressions occurred earlier than that of type II collagen gene expression during the compression test"<-which may explain why type II collagen wasn't observed in the LSJL gene expression study.

"c-Jun and c-Fos proteins can form a stable heterodimer (AP-1 complex) with a high affinity for the DNA target sequence. It has been shown that the AP-1 complex can positively regulate c-Jun, whereas the binding of AP-1 complex to the promoter regions of TGF-β1 gene mediated autoinduction of TGF-β1. This study found that the induction of c-Fos gene expression was detected earlier than the significant upregulation of c-Jun and TGF-β1 gene expressions during the dynamic compression test. It may suggest that AP-1 complex was formed by dimerizing the newly synthesized c-Fos protein with the pre-existing c-Jun protein and then upregulated the expression of c-Jun and TGF-β1 genes[so maybe if LSJL doesn't upregulate c-Fos it doesn't matter?]. TGF-β1 treatment [is] able to activate gene expressions of c-Jun and c-Fos, TGF-β1 upregulation by dynamic compression loading may be able to mediate transcription of c-Fos and c-Jun through feedback mechanism. Activity of the AP-1 complex may play an important role in regulating chondrocyte differentiation of chondrogenic cell lines and limb mesenchymal cells as well as TGF-β–induced type II collagen expression in chondrocytes. Dynamic compressive loading may promote chondrogenic gene expressions of BM-MSCs through activation of the AP-1 complex."

Development of methods for studying the differentiation of human mesenchymal stem cells under cyclic compressive strain.

"hMSC were successfully isolated, purified using D7-FIB antibody, cloned, and characterized. The cells were subsequently analyzed using fluorescence-activated cell sorting using a panel of antibodies and differentiation into multiple cell lineages. D7FIB-positive cells were then seeded into collagen-alginate scaffolds and subjected to 10% or 15% cyclic compressive strain for 4 out of 24 hours for up to 21 days in a bespoke servo-assisted displacement-controlled device. Cells were analyzed using adenosine triphosphate assay to determine cell number, live-dead cell assay, and quantitative real-time polymerase chain reaction at 7 and 21 days. Cloned D7-FIB-positive hMSCs showed evidence of differentiation to an osteogenic lineage under 10% cyclic compressive strain alone (core binding factor alpha 1 (CBFA-1) was significantly upregulated at 7 and 21 days by a factor of 18.3 and 32.2, respectively) and to an osteo-chondrogenic lineage under 15% cyclic compressive strain alone (increased expression of CBFA-1, Sox9, and aggrecan). A combination of a composite viscoelastic scaffold and controlled cyclic compressive strain may be useful for study of the differentiation of MSC."

So heavier loads may be more effective at inducing chondrogenesis.

"Cell-seeded scaffolds were then subjected to cyclic compressive strain of 10% or 15% (in separate experiments) using a sinusoidal waveform with a frequency of 1Hz for 4 hours per day for 21 days. The strain amplitude was controlled to within 0.5% across all stations, and the 0 position was controlled to within 1% across all wells. After 7 and 21 days of culture, six statically cultured and six dynamically cultured scaffolds were harvested"

"Sox9 was significantly upregulated at 7 and 21 days by factors of 1.3 and 1.6, respectively, under static culture. Under dynamic conditions, Sox9 at 7 and 21 days was significantly upregulated by factors of 2.6 and 1.9, respectively. Aggrecan was significantly upregulated at 7 and 21 days by factors of 1.4 and 1.6, respectively, under static culture. Under dynamic conditions, aggrecan was significantly upregulated at 7 and 21 days by factors of 1.3 and 2.0, respectively."<-LJSL gene expression was done after 49 hours so it may not have been long enough to induce Aggrecan and Sox9 expression.

So maybe the loads weren't heavy enough to induce chondrogenic gene expression and an increase in load by 50% so 0.75N for rats may be more chondrogenic.

Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.

"Unconfined cyclic compression [can] induce chondrogenic differentiation of human mesenchymal stem cell (hMSC) in agarose[non-biogradeable material] culture. Of the possible biocompatible materials available for tissue engineering, fibrin is a natural regenerative scaffold, which possesses several desired characteristics including a controllable degradation rate and low immunogenicity. The objective of the present study was to determine the capability of fibrin gels for supporting chondrogenesis of hMSCs under cyclic compression. To optimize the system, three concentrations of fibrin gel (40, 60, and 80 mg/mL) and three different stimulus frequencies (0.1, 0.5, and 1.0 Hz) were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Cyclic compression (10% strain) at frequencies >0.5 Hz and gel concentration of 40 mg/mL fibrinogen appears to maintain cellular viability within scaffolds. Similarly, variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by hMSC in fibrin constructs after 8 h of compression spread out over 2 days."

"Aggrecan expression was upregulated in all compressed samples compared to controls regardless of gel concentration or stimulus frequency applied"

"only the lowest concentration gels (1.0× gel concentration) present a significant upregulation of this gene’s expression when compared to its control."

"Only the 1 Hz[versus 0.5 and 0.1Hz] stimulated cells present an increased expression of the collagen II gene when compared to the control samples"

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