Wednesday, February 24, 2010

Ways to increase Chondrocyte Proliferation

I don't think trying to increase chondrocyte proliferation in pre-fused growth plate teens is the way to go(Chondrocytes have to have their proliferative capacity adjusted first).  However, the faster chondrocytes proliferate the faster you will grow taller.  

Catch-up growth after dexamethasone withdrawal occurs in cultured postnatal rat metatarsal bones. 

"The growth of fetal and postnatal bones [was] stimulated by IGF1 (100 ng/ml) and inhibited by dexamethasone (Dexa; 1 microM). We found that the capacity to undergo catch-up growth was restricted to postnatal bones. Catch-up growth occurred after postnatal bones had been exposed to Dexa for 7 or 12 days but not after a more prolonged exposure (19 days). Incomplete catch-up growth resulted in compromised bone length when assessed at the end of the 4-month period of culture. While exposure to Dexa was associated with decreased chondrocyte proliferation and differentiation, catch-up growth was only associated with increased cell proliferation. The phenomenon of catch-up growth after Dexa treatment is intrinsic to the growth plate and primarily mediated by an upregulation of chondrocyte proliferation."

"The direct effects of Dexa are believed to be partly caused by downregulation of the GH receptor and/or the IGF1 receptor at the growth plate level"

GHR and IGF1R are very important as they determine the growth plate response to GH and IGF-1.  GHR and IGF1R are more important than GH and IGF-1.  Dexamethasone can induce chondrogenic differentiation in stem cells which is why it is so appealing.  But it seems that the growth plate can not recover from the loss of GH and IGF-1 receptors.  Therefore, a way to increase chondrocyte proliferation is to not take Dexamethasone. 

The growth plate has the ability to downregulate and upregulate chondrocyte proliferation.  Any method that increases chondrocyte proliferation has to get around this positive and negative feedback mechanism. 

Partial rescue of postnatal growth plate abnormalities in Ihh mutants by expression of a constitutively active PTH/PTHrP receptor.

Postnatal refers to phenomenon occurring after birth. 

"Ihh expression in postnatal chondrocytes has a non-redundant role in maintaining a growth plate and sustaining trabecular bone after birth. Loss of Ihh in postnatal chondrocytes results in fusion of the growth plate and a decrease in trabecular bone....Expression of a Jansen receptor in chondrocytes was able to rescue abnormal chondrocyte differentiation but not impaired chondrocyte proliferation and the bone anomalies in mice lacking the Ihh gene in chondrocytes after birth. Taken together, our findings suggest that Ihh has both PTHrP-dependent and -independent functions during postnatal endochondral bone development." 

"The Ihh signal is transduced through smoothened (Smo), a putative G protein-coupled seven-transmembrane domain protein. In the absence of Ihh protein, Smo is repressed by the Ihh target gene patched (Ptch), another cell surface receptor for Ihh."

"overexpression of either Ihh or a constitutively active Smo allele specifically in cartilage results in increased activity of the Ihh signaling pathway to promote chondrocyte proliferation".  Ihh also induces Cyclin D1.

"collagen type 2 is also expressed in the kidney"<-maybe we can recruit COL2A1 from there?

Chondrocyte differentiation refers to mesenchymal stem cells turning into chondrocytes usually in cartilage.  Osteoblast differentiation refers to the ability for mesenchymal stem cells to turn into osteoblasts usually in bone.  

If you're a teenager and your indian hedgehog isn't working then that's some 'free growth' that has potentially been lost as Ihh is an important factor in chondrocyte proliferation. 

Chondrocyte p21(WAF1/CIP1) expression is increased by dexamethasone but does not contribute to dexamethasone-induced growth retardation in vivo. 

"The inhibitory effects of glucocorticoids (GCs) on chondrocyte proliferation are consistent with GCs disrupting cell cycle progression and promoting cell cycle exit. Cyclin-dependent kinase inhibitors (CDKIs) force cells to exit the cell cycle and differentiate, and studies have shown that expression of the CDKI p21(CIP1/WAF1) is increased in terminally differentiated cells. p21 mRNA and protein expression was increased during chondrocyte differentiation and after exposure to dexamethasone (Dex, 10(-6 )M) in murine chondrogenic ATDC5 cells... In addition, p21 ablation had no effect on the reduction in width of the growth plate or reduced mineral apposition rate in Dex-treated mice. However, an alteration in growth rate and epiphyseal structure is evident when comparing p21(-/-) and wild-type mice. p21 is involved in the maintenance of the growth plate." 

"In mammals, there are two families of CDKIs: the CIP/KIP family, composed of p21WAF1/CIP1, p27KIP1, and p57KIP1, which bind to and inhibit G1 CDKs, and the INK4 proteins p16INK4A, p15INK4B, p18INK4C and p19INK4D, which inhibit CDK4 and, to a lesser extent, CDK6. The Cip/Kip family acts on most cyclin/CDK complexes and is essential to inhibit G1 progression and S1 entry"

"Disruption of the p57 gene has been shown to cause delayed chondrocyte differentiation, resulting in skeletal deformations and shortened limbs"<-maybe increasing p57 expression can increase height?

"mice deficient in p27 [are larger]"

"In chondrocytes, p21 expression is p38/MAPK-dependent"

"p21 blocks the formation of cyclin/CDK complexes, which promote cell cycle progression through the phosphorylation of pocket proteins pRb (retinoblastoma protein), p107, and p130. Once phosphorylated, these pocket proteins form complexes with E2F transcription factors, leading to the transcription of genes necessary for cell cycle progression and DNA replication."

p21 could be very valuable.  P21 could be vital in the formation of new growth plates. 

Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification. 

"Chondrogenesis and subsequent endochondral ossification are processes tightly regulated by the transcription factor Sox9 (SRY-related high mobility group-Box gene 9). coactivator-associated arginine methyltransferase 1 (CARM1) regulates chondrocyte proliferation via arginine methylation of Sox9. CARM1-null mice display delayed endochondral ossification and decreased chondrocyte proliferation. Conversely, cartilage development of CARM1 transgenic mice was accelerated. CARM1 specifically methylates Sox9 at its HMG domain in vivo and in vitro. Arg-methylation of Sox9 by CARM1 disrupts interaction of Sox9 with beta-catenin, regulating Cyclin D1 expression and cell cycle progression of chondrocytes. CARM1 [is] an important regulator of chondrocyte proliferation during embryogenesis." 

"CARM1 has been shown to synergistically activate transcription with nuclear receptors in combination with other coactivators, such as p160 family, p300/CBP and SRC-2/TIF2/GRIP1. After recruitment to promoters of steroid-responsive genes, CARM1 methylates specific residues (Arg17 and Arg26) at the N-terminus of histone H3 resulting in transcriptional activation"

CARM1 null mice had reduced height.  CARM1 transgenic mice had increased height.

"Sox9 could be methylated by CARM1/PRMT4"

"Decreased chondrocyte proliferation phenotypes seen in Sox9 transgenic mice are partly explained by the model that Sox9 competes with Tcf/Lef for binding to beta-catenin and regulates chondocyte proliferation via Cyclin D1 expression. The beta-catenin/Tcf complex binds the Tcf/Lef consensus site in the Cyclin D1 promoter, transactivating Cyclin D1" Sox9 methylation by CARM1 inhibits Sox9 degradation of Beta-Catenin.

"CARM1 and Sox9 may cooperatively regulate CyclinD1 promoter activity."

So CARM1 can increase height and allow for more Sox9 by inhibiting the likely mechanism where too much Sox9 decreases height by inhibiting Cyclin D1.

Growth plate mechanics and mechanobiology. A survey of present understanding. 

"The longitudinal growth of bones is apparently controlled by modifying the numbers of growth plate chondrocytes in the proliferative zone, their rate of proliferation, the amount of chondrocytic hypertrophy and the controlled synthesis and degradation of matrix throughout the growth plate. These variables may be modulated to produce a change in growth rate in the presence of sustained or cyclic mechanical load. Tissue and cellular deformations involved in the transduction of mechanical stimuli depend on the growth plate tissue material properties that are highly anisotropic, time-dependent, and that differ in different zones of the growth plate and with developmental stages. There is little information about the effects of time-varying changes in volume, water content, osmolarity of matrix, etc. on differentiation, maturation and metabolic activity of chondrocytes. Also, the effects of shear forces and torsion on the growth plate are incompletely characterized. Future work on growth plate mechanobiology should distinguish between changes in the regulation of bone growth resulting from different processes, such as direct stimulation of the cell nuclei, physico-chemical stimuli, mechanical degradation of matrix or cellular components and possible alterations of local blood supply."

"The shapes of joint profiles may be determined in part by mechanical loading"

"Chondrocytic enlargement and matrix synthesis are strongly correlated since chondrocytes enlarge mostly in the growth direction, with little increase in their width. Hence matrix synthesis is required to fill the increased volume laterally surrounding each cell as it grows in the longitudinal direction"

"Growth plate mechanical behavior is comparable to articular cartilage"

"It is hypothesized that the more rigid reserve zone, which also constitutes a greater proportion of growth plate height in large animals, might provide mechanical support in growth plates of large species that are undergoing slower growth rates for long periods of time together with greater mechanical loading"

"There is a significant increase in the extracellular matrix elastic moduli from the reserve zone to the chondro-osseous junction"

"The length of the playing-side ulnar and 2nd metacarpal were respectively 3% and 3.7% longer than on the contralateral side [in adult tennis players]"

Altering the controlled synthesis or degradation of matrix may be a way to increase height growth.  It's interesting that the study mentions that the rate of chondrocyte proliferation may affect height growth.  Maybe the rate of chondrocyte proliferation could affect growth by upregulating certain hormones or by changing the structure of the bone(more proliferating chondrocytes = larger growth plate).

Mesenchymal Stem Cells may be another method of encouraging chondrocyte proliferation.

Trophic effects of mesenchymal stem cells increase chondrocyte proliferation and matrix formation.

"coculture of primary chondrocytes (PCs) with various sources of multipotent cells results in a higher relative amount of cartilage matrix formation than cultures containing only chondrocytes[if you add MSCs to a cartilagenous matrix then more ECM will be deposited]. We used coculture pellet models of human mesenchymal stem cells (hMSCs) and human PCs or bovine PCs (bPCs) and studied the fate and the contribution to cartilage formation of the individual cell populations during coculture. Enhanced cartilage matrix deposition was confirmed by histology and quantification of total glycosaminoglycan deposition. Species-specific quantitative polymerase chain reaction demonstrated that cartilage matrix gene expression was mainly from bovine origin when bPCs were used. Short tandem repeat analysis and species-specific quantitative polymerase chain reaction analysis of genomic DNA demonstrated the near-complete loss of MSCs in coculture pellets after 4 weeks of culture. In coculture pellets of immortalized MSCs and bPCs, chondrocyte proliferation was increased, which was partly mimicked using conditioned medium, and simultaneously preferential apoptosis of immortalized MSCs was induced. Taken together, our data clearly demonstrate that in pellet cocultures of MSCs and PCs, the former cells disappear over time. Increased cartilage formation in these cocultures is mainly due to a trophic role of the MSCs in stimulating chondrocyte proliferation and matrix deposition by chondrocytes rather than MSCs actively undergoing chondrogenic differentiation[this is very interesting, maybe the limit in chondrocyte proliferative capacity applies to MSCs too so injecting new MSCs into the growth plate would not exceed this capacity]."

If new growth plate MSCs don't exceed proliferative capacity by a feedback mechanism within the chondrocytes then LSJL may be less effective in open growth plates.  There is ample evidence that MSCs can differentiate into chondrocytes under hydrostatic pressure.  With active plates, LSJL may only increase matrix formation whereas with no cartilagenous growth plate LSJL doesn't have to worry about negative feedback from "Primary chondrocytes".

"evidence suggests that differentiation into tissue-specific cells cannot fully explain the benefits of transplanted MSCs in remodeling and recovery of damaged or lost tissue"<-So MSCs may mostly encourage existing cell activity.

"it has been suggested that increased cartilage formation in cocultures is due to chondrogenic differentiation of MSCs stimulated by factors secreted by chondrocytes. Indeed, chondrocyte-conditioned medium can induce chondrogenic differentiation of MSCs directly and in transwell cultures[a mechanism like LSJL]"

"DNA analysis of coculture pellets at 1, 2, and 3 weeks of culture demonstrated a steep drop in human DNA between 1 and 2 weeks with a further gradual decline at weeks 3 and 4"<-So the amount stem cell DNA decreased which you would not expect if stem cells were differentiating into chondrocytes.

"Even in cell pellets with an initial seeding of 80% iMSCs, hardly any human mRNA was detected"

"Cell death in iMSC-containing pellets started to increase significantly from day 5 onward."

"These data show that the change in ratio between MSC and PC during prolonged coculturing is in addition to apoptosis also due to increased proliferation of chondrocytes in pellet cultures."<-So maybe MSCs do help exceed proliferative capacity just not by proliferating themselves.

"By providing nutrients and growth factors, MSCs increase proliferation and differentiation of host-derived cells to help them to repair damaged tissues."<-So when there are already enough cells, MSCs provide resources.  However whether this increases proliferative capacity is unclear.

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