Monday, August 8, 2011

Growing Taller with Synovium Derived Stem Cells

Earlier, we learned that osteophytes can form from synoium derived stem cells.  This is why it is speculated that LSJL makes your bones grow wider more easily than it does make your bones grow longer.  The synovial cavities are more easily compressible than bone and thus it is easier to increase hydrostatic pressure.  At any point where the hydrostatic pressure is sufficient to induce chondrogenic differentiation in the bone marrow it should also be sufficient to induce chondrogenic differentiation in the synovium.

However, articular cartilage has long been regarded as difficult and slow to heal due to the lack of vascularity of cartilage.  The microfracture technique is used to allow bone marrow to seep into the articular cartilage.  So if there are stem cells in the synovium why doesn't the articular cartilage heal more efficiently?  How can we use the stem cells in the synovium to help us grow taller?

Extracellular matrix deposited by synovium-derived stem cells delays replicative senescent chondrocyte dedifferentiation and enhances redifferentiation.

"[We] assess the effect of extracellular matrix (ECM) deposited by synovium-derived stem cells (SDSCs) on articular chondrocyte expansion and maintenance of differentiation status and redifferentiation capacity. Passage 0 (P0) pig articular chondrocytes were expanded for six passages on plastic flasks (Plastic), SDSC-derived ECM (ECM), or substrate switching from either Plastic to ECM (PtoE) or ECM to Plastic (EtoP). Cell morphology, gene expression profiles, and immunophenotypes at each passage were used to characterize differentiation status of expanded cells. Chondrocytes at P0, P2, and P6 were assessed for redifferentiation capacity in a pellet culture system treated with either TGF-β1- or serum-containing medium for 14 days. ECM not only greatly enhanced chondrocyte expansion but also delayed dedifferentiation of expanded chondrocytes. Intriguingly, compared to a dramatic decrease in CD90 + /CD105+ cells and CD90+ cells, CD105+ cells dramatically increased when chondrocytes were plated on Plastic; on the contrary, ECM expansion dramatically increased CD90+ cells and delayed the decrease of CD90 + /CD105+ cells. Interestingly, expanded chondrocytes on ECM also acquired a strong redifferentiation capacity, particularly in the pellets treated with TGF-β1[ECM levels may affect chondrocyte proliferative capacity]. In conclusion, the ratio of CD90 to CD105 may serve as a marker indicative of proliferation and redifferentiation capacity of dedifferentiated chondrocytes{may be possible to manipulate the ratio of cells in the growth plate to grow taller}. ECM deposited by SDSCs provides a tissue-specific three-dimensional microenvironment for ex vivo expansion of articular chondrocytes while retaining redifferentiation capacity."

"Articular cartilage is a unique, hypocellular[contains less than the normal number of cells] and avascular tissue, mostly made of extracellular collagens and proteoglycans (PGs); it has a limited ability to self heal after trauma and degenerative disease"

"Plastic dishes coated with collagen II favored expanded chondrocytes’ chondrogenic potential"<-the more Type II collagen the easier it is to cause chondrogenic differentiation.  Thus it is easier to induce chondrogenic differentiation in already functional growth plates.

"Superficial zone protein (SZP) synthesized by both chondrocytes and synovial cells bordering the joint cavity  provides a protective microenvironment for cartilage progenitor cells at the surface of articular cartilage"<-so the location of cartilage mesenchymal stem cells is at the joint cavity

"A certain subpopulation of multipotent MSCs or progenitor cells in the cartilage tissue [exists] besides terminally differentiated chondrocytes"

"A potential mechanism underlying ECM enhancing chondrocyte proliferation and redifferentiation capacity may be explained by ECM acting through the integrin-mediated extracellular signal-activated kinase (ERK) signal transduction pathway. ECM would interact with chondrocytes through integrins and other signaling receptors, in which [some] integrins were proven to be highly expressed in the cells attaching on 3D ECM. Afterwards, the phosphorylation of ERK1/2 is upregulated by the activated [an] integrin via the Src/Ras/Raf signaling pathway; then the high level of phosphorylated ERK1/2 would enhance TGF-Beta1-mediated redifferentiation of expanded chondrocytes on ECM."<-this is why hyaluronic acid supplementation may make you taller by increasing the ECM proteins in the bone marrow.

So, there are definitely stem cells in the articular cartilage and ECM levels increase chondrogenic potential.  In osteoarthritic or other form of degenerated cartilage there is likely to be very low levels of ECM which is likely why the cartilage does not regenerate properly in those circumstances as the ECM is too low for proper chondrogenic differentiation.

Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.

"We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (HMSC). We exposed chondrogenically differentiated HMSC pellets, to IL-1α, TNF-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel respectively{we don't have access to things like Enbrel but many anti-oxidants inhibit TNF-alpha and interleukins}. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis{which is why anti-oxidants are important to meet with increased doses of TNF-alpha and IL-1alpha due to mechanical stimuli} when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. hMSC chondrogenesis is blocked by either IL-1α or TNFalpha alone."

Another reason for the lack of regeneration in articular cartilage could be the presence of inflammatory cytokines.  We can use this in our pursuits for height increase by trying to inhibit these cytokinies.

"Chondrocytes are the only residing cells in cartilage (less than 10% of total cartilage volume in humans) and they function to construct, remodel and maintain the abundant extracellular matrix"<-the cartilagenous growth plate is pure.  Thus when performing something like LSJL which attempts to induce new growth plates by means of hydrostatic pressure we want to try to induce a cartilagenous environment as possible.

"Addition of TGFβ to the chondrogenic differentiation medium was sufficient to induce chondrogenesis"<-we know that LSJL upregulates genes in the TGF-Beta pathway.

"Blocking the inhibitory effect of IL-1 on chondrogenesis was dependent on NF-kappaB translocation to the nucleus. When this was prevented, so was the inhibitory effect. TNF-α inhibition of chondrocytic differentiation was dependent on NF-kappaB mediated posttranscriptional Sox9 and MyoD down-regulation"

"Unfortunately the NF-kappaB pathway is not a suitable blocking candidate as this is crucial for chondrogenesis itself"<-so it's best inhibit IL-1 and TNF-alpha.

Synovial Fluid Progenitors Expressing CD90+ from Normal but Not Osteoarthritic Joints Undergo Chondrogenic Differentiation without Micro-Mass Culture.

"Mesenchymal progenitor cells (MPCs) can differentiate into chondrocytes. Recently, a progenitor cell population has been found within the synovial fluid that shares many similarities with bone marrow MPCs. These synovial fluid MPCs (sfMPCs) [have] a bias for cartilage differentiation [and increased expression of CD44 and UDPGD(an enzyme related to hyaluronan synthesis)]. sfMPCs were isolated from human and canine synovial fluid collected from normal individuals and those with osteoarthritis (human: clinician-diagnosed, canine: experimental) to compare the differentiation potential of CD90+ vs. CD90- sfMPCs, and to determine if CD90 (Thy-1) is a predictive marker of synovial fluid progenitors with chondrogenic capacity in vitro.
sfMPCs were derived from synovial fluid from normal and OA knee joints. These cells were induced to differentiate into chondrocytes.
The CD90+ subpopulation of sfMPCs had increased chondrogenic potential compared to the CD90- population. sfMPCs derived from healthy joints did not require a micro-mass step for efficient chondrogenesis. Whereas sfMPCs from OA synovial fluid retain the ability to undergo chondrogenic differentiation, they require micro-mass culture conditions {So we would want to inject CD90+ stem cells into our epiphysis}."

"The CD90-negative fraction displayed reduced levels of Sox9, Collagen 2 and Aggrecan mRNA"

"whereas whole populations of sfMPCs from normal individuals aggregate and express high levels of chondrogenic markers, whole populations of sfMPCs from OA joints do not aggregate spontaneously, grow only as a monolayer, and express low levels of chondrogenic markers during differentiation"<-does LSJL induce spontaneous chondrogenic aggregation?

"chondrocyte aggregation is mediated by β1-Integrin, while Cadherins seem to play a dominant role in the aggregation of MSCs during chondrogenesis"<-LSJL upregulates ITGBL1 2.436 fold.  Which is integrin-beta like1.

Rejuvenation of chondrogenic potential in a young stem cell microenvironment.

"Autologous cells suffer from limited cell number and senescence during ex vivo expansion for cartilage repair. Here we found that expansion on extracellular matrix (ECM) deposited by fetal synovium-derived stem cells (SDSCs) (FE) was superior to ECM deposited by adult SDSCs (AE) in promoting cell proliferation and chondrogenic potential. Unique proteins in FE might be responsible for the rejuvenation effect of FE while advantageous proteins in AE might contribute to differentiation more than to proliferation. Compared to AE, the lower elasticity of FE yielded expanded adult SDSCs with lower elasticity which could be responsible for the enhancement of chondrogenic and adipogenic differentiation. MAPK and noncanonical Wnt signals were actively involved in ECM-mediated adult SDSC rejuvenation."

"Adult MSCs lack telomerase activity resulting in telomere shortening after serial passaging in vitro"

"decellularized ECM from human adult stem cells, such as SDSCs or BMSCs, exhibited a limited capacity to rejuvenate expanded stem cells' chondrogenic potential."

"compared to up-regulation of p-p38 and p-Jnk, ECM expansion dramatically down-regulated p-Erk; despite the fact that p-Erk bounced back in the cell condensation phase, the lowest level was seen in FE4 pellets; different from the change of p-Erk, ECM expansion yielded ASDSC pellets with a relatively lower level of p-Jnk in the cell condensation phase though no significant difference was found among groups after 10-day chondrogenic induction. Our data also showed that ECM pretreatment down-regulated Wnt3a but up-regulated Wnt5a and Wnt11 in expanded SDSCs; these effects were relayed through cell condensation and chondrogenic differentiation, especially for the Wnt11-mediated non-canonical pathway"

"FE had more fibrillin-2 (76:2), tenascin C{downregulated in LSJL} (259:120), and clusterin (5:0) than AE."<-these proteins may be responsible for the enhanced chondrogenesis.

"enhanced expression of fibrillin-2 and tenascin C has been observed in adults with fibroproliferative conditions, such as wound healing and sclerosis"

"AE had more biglycan{up} (95:37), decorin (95:40), dermatopontin{up} (35:16), elastin (18:2), periostin{up} (400:125), thrombospondin-1{up} (79:6), and TGFβ1 (258:164) than FE."

" AE has more matrix components favoring cell differentiation and apoptosis rather than cell proliferation, which is consistent with the observed differences in elasticity and ECM/collagen architecture, i.e. fibrillar collagens were shifted into the “insoluble” fraction in AE but not in FE."

"in human BMSCs showed that ECM expansion dramatically increased the level of integrin β5 but decreased integrin β1"

" AE expansion yielded higher cell viability (DNA% by day 0) during chondrogenic induction; FE expansion yielded the highest GAG amount per pellet and ratio of GAG to DNA (chondrogenic index) followed by those from AE expansion with PL expansion having the least"

The matrix alterations of LSJL are more consistant to adult ECM than fetal ECM.

So there are stem cells in the synovial joint.  The reason why joints regenerate poorly is likely due to a depleted ECM and the presence of inflammatory cytokines.  We can use these facts to grow taller by taking anti-oxidants to decrease serum levels of inflammatory cytokines.  An adult bone has no Type II collagen in the growth plate and thus dedifferentiation of stem cells into chondrocytes is a potential factor.  To avoid this more frequent application of LSJL throughout the day may be needed to give time for freshly differentiated chondrocytes to secrete ECM before dedifferentiation occurs.  Frequent bursts of hydrostatic pressure plays the role that Type II collagen plays in a unfused growth plate.  In a younger growth plate this may not be necessary as there is already ECM encouraging a chondrogenic micro environment.

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