Thursday, October 8, 2009

Comparison of gene expression changes to hydrostatic pressure in cartilage to LSJL.

By studying the effects of hydrostatic pressure to already existing cartilage we can see what changes in gene expression may be due to chondrogenic induction in MSCs by LSJL.

Influence of extracellular matrix on the expression of inflammatory cytokines, proteases, and apoptosis-related genes induced by hydrostatic pressure in three-dimensionally cultured chondrocytes.

"Chondrocytes were isolated from rabbit joints and cultured in alginate beads. Immediately after embedding (0W group) or after 2 weeks culture (2W group), the amounts of glycosaminoglycan (GAG) in the alginate beads were quantified. Both groups were exposed to continuous HP of 10 or 50 MPa for 12 h. The expression of inflammatory cytokines, proteases, and apoptosis-related factors were examined by reverse transcription-polymerase chain reaction (RT-PCR). The expression of proteoglycan core protein (PG) and collagen type II were quantified by real-time RT-PCR.
All of the GAG components in alginate beads markedly increased in the 2W group. The expression of PG and collagen type II increased after exposure to 10 MPa in both groups. In the 0W group, these levels decreased after exposure to 50 MPa of HP. The expression of interleukins IL-6 and IL-8 increased after exposure to HP in the 0W group. HP at 50 MPa induced mRNA expression of ADAMTS-5 in the 0W group but not in the 2W group. The expression of Fas increased after exposure to HP in the 0W group.
These findings suggested that nonphysiological, excessive HP on chondrocytes with the ECM in poor condition reduced matrix gene expression and increased expression of the genes associated with apoptosis and catabolism of the cartilage matrix."

"HP influenced the expression of cytokines, including interleukins IL-6{up in LSJL} and IL-8 and tumor necrosis factor-α (TNFα), from the chondrocytes in in vitro two-dimensional cultures"

The study found that 10MPa of hydrostatic pressure could induce increased proteoglycan levels but no changes in Sox9 were observed.

Modulation of gene expression of rabbit chondrocytes by dynamic compression in polyurethane scaffolds with collagen gel encapsulation.

"The aim of this study was to investigate the effects of dynamic compression on gene expression of rabbit chondrocytes which were seeded in elastic polyurethane scaffolds with or without collagen gel encapsulation. Dynamic compression of 20% or 30% strain with 0.1 Hz frequency was applied to the cell-seeded scaffolds for 4, 8, 12, or 24 h, and then the expression of the three genes related to chondrogenic phenotype, type I and II collagens and aggrecan, was analyzed by RT-PCR. We also investigated the gene expression of the compressed chondrocytes, which had experienced 12-h 30% strain dynamic loading, during the post-compression resting period. We found that the expression of type II collagen did not seem to respond to cyclic compression. On the other hand,aggrecan gene was stimulated by dynamic compression. The stimulatory effect disappeared gradually after the dynamic compression was ceased. Furthermore, the mechano-response of the chondrocytes to aggrecan expression was delayed by collagen gel encapsulation. The expression of type I collagen was enhanced by collagen gel. We found that collagen gel encapsulation prolonged the expression of aggrecan and type I collagen during post-compression resting period. We demonstrated that mechanical and biochemical stimuli modulate the gene expression of chondrocytes."

"The expression of COL II gene was not stimulated, but slightly depressed by 20% or 30% strain of dynamic compression, regardless of collagen gel encapsulation. After cyclic compression was lifted, the expression of COL II gene remained at a similar level during the resting period."

"stimulation of aggrecan synthesis in cartilage explants by dynamic compression is co-localized to the regions with high interstitial fluid flow.  Fluid flow generates shear stress, streaming potentials and currents, and induces physicochemical changes including mobile ion concentrations and osmotic pressure, which may modulate chondrocyte gene expression."

cDNA array reveals mechanosensitive genes in chondrocytic cells under hydrostatic pressure.

"we examined the effect of continuous HP on the gene expression profile of a chondrocytic cell line (HCS-2/8) using a cDNA array containing 588 well-characterized human genes under tight transcriptional control."

"Increases in cyclic AMP and hyperpolarization of chondrocyte plasma membrane occur under intermittent HP, due to opening of small conductance Ca2+-dependent K+ channel. This hyperpolarization response was dependent on α5β1 integrins, leading to release of interleukin 4"

" Myo-inositol 1,4,5,-triphosphate and Ca2+/calmodulin system were shown to mediate increased aggrecan mRNA levels in compressed chondrocytes"

"Continuous 30-MPa pressure induces a specific stress response by increasing cellular hsp70 mRNA and protein levels in chondrocytic cells. The accumulation of hsp70 mRNA occurs due to stabilization of hsp70 mRNA, without induction of the gene itself, and protein synthesis is required for the stabilization"

"High pressure also increased mRNA levels of interleukin 6 and tumour necrosis factor α. Interleukin 6 may be connected with the increased synthesis of Hsp90 observed in human chondrocytic T/C28a4 cell line, since interleukin 6 is known to activate hsp90β gene"

"Cells were exposed to continuous 30 MPa HP for 3, 6 and 12 h."

Genes upregulated by LSJL and had fold change at 3, 6, and 12:
egr1-up, peak, up
Hbegf-up, up, up

Genes downregulated by LSJL:
Id2-up, down, down

C-fos levels were raised but not as significantly as by LSJL.

"Id proteins down-regulate the expression of immediate-early genes c-fos and egr-1 by antagonizing the ETS domain proteins SAP-1 and Elk-1 responsible for induction of these genes"

High pressure effects on cellular expression profile and mRNA stability. A cDNA array analysis.

"Induction of stress response in immortalized chondrocytes exposed to high static hydrostatic pressure increases the stability of heat shock protein 70 mRNA.  For this purpose a cDNA array analysis was used to compare mRNA expression profile in pressurized vs. non-pressurized human chondrosarcoma cells (HCS 2/8). mRNA stability was analyzed using actinomycin-treated[inhibits transcription thus measuring how much of a fold change is due to increased stability and not transcription] and nontreated samples collected after pressure treatment. A number of immediate-early genes, and genes regulating cell cycle and growth were up-regulated due to high pressure. Decrease in osteonectin and fibronectin mRNAs was observed. Also bikunin, cdc37 homologue and Tiam1, genes linked with hyaluronan metabolism, were down-regulated. In general, stability of down-regulated mRNA species appeared to increase. However, no increase in mRNA above control level due to stabilization was noticed in the genes available in the array. On the other hand, mRNAs of certain immediate-early genes, like c-jun, jun-B and c-myc, became destabilized under pressure treatment. Increased accumulation of mRNA on account of stabilization under high pressure conditions seems to be a tightly regulated, specific phenomenon."

"The pressure level of the test chamber was selected to be 30 MPa and static mode of pressure loading was used."

Genes up by 6 hour hydrostatic pressure also up(or -down) in LSJL:
Genes downregulated in actomyosin D treated pressure cells versus regular pressure cells are in bold.


Genes down by 6 hour hydrostatic pressure:
Genes upregulated in actomyosin D treated pressure cells versus regular pressure cells are in bold.


Transcriptional activation in chondrocytes submitted to hydrostatic pressure.

"we have observed several differentially expressed cDNA bands under continuous 30 MPa hydrostatic pressure, while 30 MPa cyclic pressure at 1 Hz produced much fewer changes. In the first phase of our studies, we have focused on the effects of 30 MPa hydrostatic pressure because it causes a unique hsp70-mediated stress response in immortalized chondrocytes."

"Under 30 MPa continuous hydrostatic pressure, four different time points were chosen (0, 3, 6 and 24 h) for the experiments."

No similarities were noted between gene expression here and LSJL gene expression.

Cyclic Hydrostatic Pressure Stimulates Enhanced Bone Development in the Foetal Chick Femur in Vitro.

"Hydrostatic pressure regimes of 0-279 kPa at 0.005 - 2Hz were applied to organotypically cultured ex vivo chick foetal femurs (e11) for 1hour per day in a custom designed bioreactor for 14days. The mineralised portion of the developing femur cultured under any cyclic hydrostatic pressure regime was significantly larger and/or denser than unstimulated controls but that constant (non-cycling) hydrostatic pressure had no effect on bone growth. The increase in bone formation was directly proportional to stimulation frequency (R(2)=0.917), but independent of the magnitude of the pressure applied, whilst even very low frequencies of stimulation (0.005Hz) had significant effects on bone growth. Expression of Type-II collagen in both epiphyses and diaphysis was significantly upregulated (1.48-fold and 1.95-fold respectively){possibly indicative of ectopic cartilage formation}, together with osteogenic genes (osteonectin and osteopontin) and the osteocyte maturation marker CD44."<-all these genes other than CD44 were upregulated by LSJL.

"[Day 11 chick femur tissue] is primarily composed of osteoblasts and progenitor cells with a number of encased osteoblasts maturing into osteocytes and negligible numbers of osteoclasts. At this stage of development, the onset of primary mineralisation is underway in the region surrounding the diaphysial shaft (bone collar) allowing the progress of bone maturation to be observed by X-ray microtomography in response to experimental treatments. Vascular invasion has not yet occurred in the organ, although pores in the mineralising collar exist to admit ingrowth of blood vessels to the endosteum."

"Sections through femurs cultured for two weeks in osteogenic medium with or without stimulation and stained for calcium (alizarin red) showed that bone formation under both control conditions (A–C) and under hydrostatic stimulation (D–F) was similar, with the periosteum and mineralising layer (A and D), bone collar (B and E) and epiphyseal tissues (C and F) forming organotypically"<-No visible cartilage formation but it wasn't stained for that.  F is more blue than C is both are epiphyseal tissues so the hydrostatic pressure did likely enhance chondrogenesis in the femoral epiphysis.

"[In the epiphysis] chondrogenic transcription factor SOX9 was downregulated to 0.70-fold — despite observations of increased sulphated glycosaminoglycan content in this region."  This is contrary to LSJL where Sox9 was upregulated.  No significant difference in Runx2 expression was observed in the epiphysis.  CD44 was not statistically different in the epiphysis as well.

"the mineralising region is maturing more rapidly in femurs that received hydrostatic stimulation."

Sox9 and Runx2 were non-significant downregulated in the diaphysis.

According to the author James Henstock: "We didn't see any definite changes in whole bone length but the bone collar does extend further towards both ends of the femur. The mechanically stimulated bones grow straighter than the unstimulated ones which we attribute to the increased physical stiffness of the more densely mineralised collar as the whole bone increases in size which resists it being contorted by contact with the underlying membrane."

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