Thursday, September 20, 2012

LSJL gene expression versus dynamic chondrocyte compression

LSJL involves a lateral compressive force.  Since in the 14 week old rats there were growth plates in the bone, it's important to see how much of the upregulation was due to stimulus of the growth plate itself and how much was stimulation of stem cells to differentiate into chondrocytes to form new growth plates.

"We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium."

"The main collagen-binding integrin on chondrocytes in cartilage is α10β1 integrin, whereas α11β1 integrin is more characteristic of mesenchymal tissues."

Upregulated Genes:

Fos(up in LSJL)
Egr1(up in LSJL)
Nr4a1(up in LSJL)
Fosb(up in LSJL)
Junb(up in LSJL)
Atf3(up in LSJL)
Cyr61(up in LSJL)
Zfp36(up in LSJL, may be a limiting agent against growth)
Bgn(up in LSJL)

Downregulated Genes:
Sdpr(down in LSJL)
Htra1(up in LSJL, inhibits TGFbeta signaling)
MMP3(up in LSJL)
Col11a1(up in LSJL)

Of note is that the expression of Sox9, COL2A1, Aggrecan did not appear to be altered.  Thus the induction of new growth plates by LSJL may not require existing chondrocytes.

The effects of intermittent dynamic loading on chondrogenic and osteogenic differentiation of human marrow stromal cells encapsulated in RGD-modified poly(ethylene glycol) hydrogels.

"Poly(ethylene glycol) hydrogels were fabricated with tethered cell adhesion moieties, RGD. Cell-laden hydrogels were subjected to 4 h daily intermittent dynamic compressive loading (0.3Hz, 15% amplitude strain) for up to 14 days and the cell response evaluated by gene expression and matrix deposition for chondrogenic and osteogenic markers. The three-dimensional hydrogel supported chondrogenesis and osteogenesis under free swelling conditions, as shown by the up-regulation of cartilage-related markers (SOX9, Col II, Col X, and aggrecan) and staining for type II collagen and aggrecan and osteogenically by up-regulation of ALP and staining for type I collagen and for mineralization. Under dynamic loading the expression of cartilage-related markers SOX9, Col II, Col X, and aggrecan were down-regulated, along with reduced aggrecan staining and no positive staining for type II collagen. The bone-related markers RUNX2, Col I, and ALP were down-regulated and positive staining for type I collagen and mineralization was reduced.  The selected loading regime appears to have an inhibitory effect on chondrogenesis and osteogenesis of hMSC encapsulated in PEG-RGD hydrogels after 14 days in culture, potentially due to overloading of the differentiating hMSC before sufficient pericellular matrix is produced and/or due to large strains, particularly for osteogenically differentiating hMSC."

"dynamic compressive loading at 0.1 Hz and 5% amplitude strains led to a 79% increase in glycosaminoglycan content compared with statically compressed explants"

"Intermittent loading (0.5 h on, 1.5 h off) was applied for 16 h, followed by 8 h of rest. During the loading period a dynamic compressive strain was applied from 0% to 15% with a sinusoidal waveform and at a frequency of 0.3 Hz, while the resting period experienced no strain"

"SOX9 and Col X were upregulated by day 14. Col II increased 1900-fold by day 7 and remained high (2200-fold) at day 14. ACAN increased 70-fold by day 7 and dropped to levels similar to the basal condition by day 14 [in CDM(chondrogenic differentiation medium) with no load]."

"The application of loading to CDM constructs downregulated the expression of SOX9, ACAN, and Col I by day 7"

"either reduced loading (e.g. lower strains) and/or delaying the application of loading may be important in the differentiation potential of MSC."

Mechanical signals control SOX-9, VEGF, and c-Myc expression and cell proliferation during inflammation via integrin-linked kinase, B-Raf, and ERK1/2-dependent signaling in articular chondrocytes.

"Cytokines like interleukin (IL)-1beta suppress homeostatic mechanisms and inhibit cartilage repair and cell proliferation. Matrix synthesis and chondrocyte (AC) proliferation are upregulated by the physiological levels of mechanical forces.
ACs isolated from articular cartilage were exposed to low/physiologic levels of dynamic strain in the presence of IL-1beta.
Mechanoactivation, but not IL-1beta treatment, of ACs initiated integrin-linked kinase activation. Mechanical signals induced activation and subsequent C-Raf-mediated activation of MAP kinases (MEK1/2). IL-1beta activated B-Raf kinase activity. Dynamic strain did not induce B-Raf activation but instead inhibited IL-1beta-induced B-Raf activation. Both mechanical signals and IL-1beta induced ERK1/2 phosphorylation but discrete gene expression. ERK1/2 activation by mechanical forces induced SRY-related protein-9 (SOX-9), vascular endothelial cell growth factor (VEGF), and c-Myc mRNA expression and AC proliferation. IL-1beta did not induce SOX-9, VEGF, and c-Myc gene expression and inhibited AC cell proliferation. SOX-9, VEGF, and Myc gene transcription and AC proliferation induced by mechanical signals were sustained in the presence of IL-1beta."

ERK1/2 has two phosphorylation sites and mechanical loading and IL-1B can phosphorylation different sites.

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