In our discussion on water and height, we went over the differences between chondrocytes and osteoblasts and tried to determine why chondrocytes can cause interstitial growth whereas osteoblasts cannot. Both chondrocytes and osteoblasts secret ECM(osteoblasts secret Type I collagen) but cartilage is far more hydrophillic(water loving). Since cartilage is more hydrophillic it is more prone to hypertrophy thus that could be a key to why chondrocytes are a key to growing taller. Do osteoblasts undergo hypertrophy and if they do then why don't they make you taller like chondrocytes? Osteoblasts also undergo apoptosis but maybe water release from apoptotic chondrocytes can help you grow taller.
Chronic hyperglycemia modulates osteoblast gene expression through osmotic and non-osmotic pathways.
"Insulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with osteoporosis and increased fracture rates. The mechanisms underlying IDDM associated bone loss are not known. Previously we demonstrated that osteoblasts exhibit a response to acute (1 and 24 h) hyperglycemia and hyperosmolality[so there is a high number of solute in the body so osteoblasts release water and shrink]. Here we examined the influence of chronic hyperglycemia (30 mM) and its associated hyperosmolality on osteoblast phenotype. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions[MMP-13 degrades extracellular matrix, so hyperosmotic conditions suppress degradation of the extracellular matrix] (mannitol treatment). Acute hyperglycemia for a 48-h period was also capable of inducing alkaline phosphatase and suppressing osteocalcin, MMP-13, VEGF, and GAPDH expression in differentiated osteoblasts. This suggests that acute responses in differentiated cells are maintained chronically. In addition, hyperglycemic and hyperosmotic conditions increased PPARgamma2 expression[PPARgamma is usually associated with increased adipocyte differentiation and reduced osteoblast differentiation], although this increase reached significance only in 21 days chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARgamma2 expression is increased in type I diabetic mouse model bones, these findings suggest that diabetes-associated hyperglycemia may modulate osteoblast gene expression, function and bone formation and thereby contribute to type I diabetic bone loss."
So, hyperosmotic conditions are catabolic to bone cells. So osteoblasts do respond to water much like chondrocytes.
"increased expression of PPARγ2, aP2 and resistin in streptozotocin-induced diabetic mice corresponded with increased adipocyte maturation and suggested the possibility that IDDM may also affect lineage selection of mesenchymal stem cells, leading to adipocyte rather than osteoblast maturation."
"cells can also respond to hyperglycemia through an osmotic response. Because osteoblasts express glucose transporters, GLUT-1 and -3, with low Km (1–2 mM and <1 mM, respectively), glucose transport is maximal at euglycemic[normal blood glucose levels] state (glucose concentration of 3–5.5 mM) so an increase in extracellular glucose could be an osmotic stress." Since cells are transporting less glucose there is more glucose outside a cell therefore water leaves the cell to restore concentration to normal.
"During osmoadaptation to extracellular hyperosmotic conditions, virtually all cells undergo a volume change and shrink"<-therefore osteoblasts should be capable of undergoing hypertrophy as well.
"Osteoblast morphology and number does not change under chronic hyperglycemia"<-remember hyperglycemia causes hyperosmolarity as well. So hyperosmolarity does not cause osteoblast hypertrophy(which would be a part of morphology). So even though water is leaving the cell, osteoblasts do not stay shrunken in size.
"During this period osteoblasts undergo immediate volumetric changes (cell shrinking) induced by hyperglycemia-associated hyperosmolality"<-Hypertonic means water leaving the cell so it makes sense for cells to shrink
So osteoblasts are osmotically sensitive like chondrocytes. Do osteoblasts swell(hypertrophy) in response to hyposmotic(water enters the cell) solutions?
Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells.
"The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of approximately 30% in suspended (i.e., round) cells and approximately 60% in attached (i.e., spread) cells that was independent of subpassage number[so osteoblast cells did cell and the swelling did not return to normal after time as shown by the incomplete regulatory volume decrease]. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with BAPTA-AM and by depletion of Cl-, indicating the role of calcium-regulated K+ and Cl- efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (approximately 0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na(+)-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl(-)-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl- that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi."
It could be a location issue that chondrocytes are in a better position to increase height than osteoblasts. However, there are osteoblasts at the surface of the bone with potential for hypertrophy and the ability to secrete extracellular matrix. However, key components to chondrocyte hypertrophy may not be due to osmotic swelling and may be due to other factors. It could be these other forms of hypertrophy that are responsible for height growth.
Since cartilage is hydrophillic, chondrocytes are a lot better at manipulating water levels since it can store water in it's ECM. Osteoblasts do not have water stored in it's ECM. Osmotic lysis is more likely to occur in osteoblasts than chondrocytes as chondrocytes have the water storage ability of the cartilage. Since chondrocytes do have water stored in the ECM, they can better orchestrate the osmotic lysis of the cells thus resulting in an explosion pushing the bone apart.
Hyperosmotic conditions were found to result in more chondrocyte apoptosis. Water leaving the cell resulted in apoptosis rather than water flooding into the chondrocyte. During terminal differentiation, cartilage is absorbed leading to less water outside the cell therefore chondrocytes release water to result in osmotic balance this results in chondrocyte apoptosis. This response did not seem to occur in osteoblast cells which only exprienced decreased expression of MMP-13 and increased levels of PPARgamma2 in response to a hyperosmotic environment like those experienced by chondrocytes who just had their ECM degraded.
There is evidence that there is an active role of chondrocyte apoptosis in endochondral ossification and that active role may play a role in how physically growth plates make you taller. Other cells do have the ability to influence body shape for example you could have swollen skin. Osteoblasts have the ability to influence shape too but only by secreting new matrix beneath the periosteum. This however does not involve hypertrophy or apoptosis but is only the result of matrix secretion.
Therefore, matrix secretion may only be able to cause apopsitional growth but hypertrophy and apoptosis may be needed for interstitial growth.
Growth plate chondrocytes have to do something else besides proliferate, divide and secrete ECM to make us taller. Lots of cells perform those functions and don't make us taller. There needs to be a force generated pushing the bone apart from within to make room for new bone. Choreographed chondrocyte apoptosis has the ability to do that like a string of dynamite. Osteoclasts can degrade ECM all at once like triggers resulting in chondrocytes going off at once resulting in a big "explosive" force.
If the force isn't big enough like isolated osteoblast apoptosis you won't grow taller.
Tuesday, September 27, 2011
Tuesday, September 20, 2011
Growth Plate transplants not so far fetched
Note that I believe that hydrostatic pressure induced chondrogenic differentiation of stem cells is currently the best way to grow taller. That doesn't mean the science of stem cells in growth plate repair can't help us in our quest for height.
Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy.
"The potentials of mesenchymal stem cells (MSCs) in cartilage repair include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling[if they identify some carriers that are already in the body we could take advantage of that to gain height]. Gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells [can be used to regenerate cartilage]. [What is] the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes? As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone."
What we're doing with LSJL is mobilizing endogenous MSCs from the bone marrow.
Let's look at the three criteria for successful growth plate cartilage repair(and by extension successful formation of new growth plate cartilage):
1) MSCs capable of chondrogenic capacity. This should be true of bone marrow MSCs as they are capable of mesenchymal chondrosarcoma(which involves chondrogenic differentiation). Telomere Length and Methylation Status play a role also.
2) The second factor is a way of encouraging chondrogenic differentiation. This is hydrostatic pressure. Which we induce by laterally compressing the bone to increase the fluid pressure within the bone marrow. There are alternatives to this such as LIPUS plus TGF-Beta.
3)The scaffolds for transplantation or direct filling. Well, the MSCs are already in the body so there's no need for transplantation or filling. They are already in a position to increase height.
In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes.
"A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP])[MSCs were co-cultured with chondrocytes]. Committed MSCs were seeded into a collagen scaffold[bone is technically already a collagen scaffold Type II collagen] and analyzed for their neocartilage-forming ability.
Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules[studying these chondrogenic adhesion molecules could be a way to help us gain height] and stimulated cartilage developmental growth factors, including members of the transforming growth factor beta superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold.
Human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes."
Now, LSJL can't co-culture MSCs with growth plate chondrocytes as there are no growth plate chondrocytes left after fusion. However, LSJL can induce chondrogenesis by other means. This does mean that maybe pre-fusion that something like LIPUS can increase height on it's own as LIPUS causes shear strain in the bone disrupting the actin cytoskeleton and could allow for adhesion of MSCs to chondrocytes.
Application of autologous bone marrow derived mesenchymal stem cells to an ovine model of growth plate cartilage injury.
"Injury to growth plate cartilage in children can lead to bone bridge formation and result in bone growth deformities. Mesenchymal stem/stromal cells (MSC) offer a promising therapeutic option for regeneration of damaged cartilage, due to their self renewing and multi-lineage differentiation attributes. Our laboratory has recently characterised MSCs derived from ovine bone marrow, and demonstrated these cells form cartilage-like tissue when transplanted within the gelatin sponge, Gelfoam[Will have to explore height increase applications of Gelfoam], in vivo. In the current study, autologous bone marrow MSC were seeded into Gelfoam scaffold containing TGF-beta1, and transplanted into a surgically created defect of the proximal ovine tibial growth plate. Examination of implants at 5 week post-operatively revealed transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Importantly, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated due to transplantation of MSCs or the gelatin scaffold."
So the transplant failed to induce chondrogenesis and instead underwent fibrogenesis. Note that they didn't use shear strain at all and from the previous research shear strain plus TGF-Beta is needed for chondrogenesis. Also the gelatin scaffold may have been insufficient.
"Analysis of growth plate defects treated with Gelfoam scaffold and autologous MSC revealed a tissue composition consisting of dense fibrous (38.2 ± 6.1%), fibrous (29.6 ± 10.0%), fat (24.2 ± 6.6%) and bone (7.9 ± 1.8%)."
Also they inserted the growth plate scaffold directly into the growth plate defect that may be a part of the problem and they would've had better luck with a scaffold in a bone fracture.
But at least the technology is there. They just need better ways to induce chondrogenesis. Of course, you could just do LSJL.
Here's a growth plate transplant experiment in action:
Assessment of epiphyseal plate allograft viability and function after ex vivo storage in university of wisconsin solution.
"Compromised epiphyseal plate function can result in limb deformities. Microvascular transplantation of an epiphyseal plate allograft is a potentially effective approach to reestablish longitudinal limb growth. The goal of this study was to determine a time frame for which proximal tibial epiphyseal plate allografts could be stored in University of Wisconsin Preservation Solution (UWPS) and remain functional in vivo after microvascular transplantation.
Proximal tibial epiphyseal plate allografts from skeletally immature female New Zealand White rabbits (10 to 12 wk of age) were used. Allografts (isolated on the popliteal arteriovenous pedicle) were stored ex vivo in cold UWPS for periods of up to 21 days. Chondrocyte viability, phenotype, and extracellular matrix composition of growth plate cartilage was assessed. Microvascular transplantations of nonstored or prestored (3 d) allografts were performed and analysis of bromodeoxyuridine and calcein incorporation was done to determine chondrocyte proliferation and new bone growth, respectively.
In vitro analysis showed that, compared with control tissue, epiphyseal plate chondrocyte viability, organization, and collagen extracellular matrix was preserved up to 4 days in cold UWPS. Microvascular transplantation of nonstored epiphyseal plate allografts was successful[scientists can transfer growth plates that have not been stored]. Despite care being taken to ensure vascular patency during the microvascular procedure, transplantation of prestored allografts failed due to absent flow in the larger vessels and in the allograft based upon the visualization of organized thrombus within the vascular pedicle, and absent flow within the composite graft itself. However, growth plate viability and function was detected in a peripheral region of a single allograft where partial blood flow had been maintained during the transplantation period.
Ex vivo storage in cold UWPS for 3 days maintains growth plate chondrocyte viability and function in vivo. However, future studies must be directed toward investigating the direct effect of ex vivo storage on the integrity and function of the vascular pedicles."
So growth plate transplantation is possible along as the growth plate is not in storage for more than 3 days. Although I don't see why you wouldn't just differentiate a new growth plate in the bone marrow.
Here's a study related to formation of new growth plates so they can be available for transplant:
Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage.
Engineering osteochondral constructs through spatial regulation of endochondral ossification.
Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy.
"The potentials of mesenchymal stem cells (MSCs) in cartilage repair include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling[if they identify some carriers that are already in the body we could take advantage of that to gain height]. Gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells [can be used to regenerate cartilage]. [What is] the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes? As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone."
What we're doing with LSJL is mobilizing endogenous MSCs from the bone marrow.
Let's look at the three criteria for successful growth plate cartilage repair(and by extension successful formation of new growth plate cartilage):
1) MSCs capable of chondrogenic capacity. This should be true of bone marrow MSCs as they are capable of mesenchymal chondrosarcoma(which involves chondrogenic differentiation). Telomere Length and Methylation Status play a role also.
2) The second factor is a way of encouraging chondrogenic differentiation. This is hydrostatic pressure. Which we induce by laterally compressing the bone to increase the fluid pressure within the bone marrow. There are alternatives to this such as LIPUS plus TGF-Beta.
3)The scaffolds for transplantation or direct filling. Well, the MSCs are already in the body so there's no need for transplantation or filling. They are already in a position to increase height.
In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes.
"A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP])[MSCs were co-cultured with chondrocytes]. Committed MSCs were seeded into a collagen scaffold[bone is technically already a collagen scaffold Type II collagen] and analyzed for their neocartilage-forming ability.
Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules[studying these chondrogenic adhesion molecules could be a way to help us gain height] and stimulated cartilage developmental growth factors, including members of the transforming growth factor beta superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold.
Human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes."
Now, LSJL can't co-culture MSCs with growth plate chondrocytes as there are no growth plate chondrocytes left after fusion. However, LSJL can induce chondrogenesis by other means. This does mean that maybe pre-fusion that something like LIPUS can increase height on it's own as LIPUS causes shear strain in the bone disrupting the actin cytoskeleton and could allow for adhesion of MSCs to chondrocytes.
Application of autologous bone marrow derived mesenchymal stem cells to an ovine model of growth plate cartilage injury.
"Injury to growth plate cartilage in children can lead to bone bridge formation and result in bone growth deformities. Mesenchymal stem/stromal cells (MSC) offer a promising therapeutic option for regeneration of damaged cartilage, due to their self renewing and multi-lineage differentiation attributes. Our laboratory has recently characterised MSCs derived from ovine bone marrow, and demonstrated these cells form cartilage-like tissue when transplanted within the gelatin sponge, Gelfoam[Will have to explore height increase applications of Gelfoam], in vivo. In the current study, autologous bone marrow MSC were seeded into Gelfoam scaffold containing TGF-beta1, and transplanted into a surgically created defect of the proximal ovine tibial growth plate. Examination of implants at 5 week post-operatively revealed transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Importantly, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated due to transplantation of MSCs or the gelatin scaffold."
So the transplant failed to induce chondrogenesis and instead underwent fibrogenesis. Note that they didn't use shear strain at all and from the previous research shear strain plus TGF-Beta is needed for chondrogenesis. Also the gelatin scaffold may have been insufficient.
"Analysis of growth plate defects treated with Gelfoam scaffold and autologous MSC revealed a tissue composition consisting of dense fibrous (38.2 ± 6.1%), fibrous (29.6 ± 10.0%), fat (24.2 ± 6.6%) and bone (7.9 ± 1.8%)."
Also they inserted the growth plate scaffold directly into the growth plate defect that may be a part of the problem and they would've had better luck with a scaffold in a bone fracture.
But at least the technology is there. They just need better ways to induce chondrogenesis. Of course, you could just do LSJL.
Here's a growth plate transplant experiment in action:
Assessment of epiphyseal plate allograft viability and function after ex vivo storage in university of wisconsin solution.
"Compromised epiphyseal plate function can result in limb deformities. Microvascular transplantation of an epiphyseal plate allograft is a potentially effective approach to reestablish longitudinal limb growth. The goal of this study was to determine a time frame for which proximal tibial epiphyseal plate allografts could be stored in University of Wisconsin Preservation Solution (UWPS) and remain functional in vivo after microvascular transplantation.
Proximal tibial epiphyseal plate allografts from skeletally immature female New Zealand White rabbits (10 to 12 wk of age) were used. Allografts (isolated on the popliteal arteriovenous pedicle) were stored ex vivo in cold UWPS for periods of up to 21 days. Chondrocyte viability, phenotype, and extracellular matrix composition of growth plate cartilage was assessed. Microvascular transplantations of nonstored or prestored (3 d) allografts were performed and analysis of bromodeoxyuridine and calcein incorporation was done to determine chondrocyte proliferation and new bone growth, respectively.
In vitro analysis showed that, compared with control tissue, epiphyseal plate chondrocyte viability, organization, and collagen extracellular matrix was preserved up to 4 days in cold UWPS. Microvascular transplantation of nonstored epiphyseal plate allografts was successful[scientists can transfer growth plates that have not been stored]. Despite care being taken to ensure vascular patency during the microvascular procedure, transplantation of prestored allografts failed due to absent flow in the larger vessels and in the allograft based upon the visualization of organized thrombus within the vascular pedicle, and absent flow within the composite graft itself. However, growth plate viability and function was detected in a peripheral region of a single allograft where partial blood flow had been maintained during the transplantation period.
Ex vivo storage in cold UWPS for 3 days maintains growth plate chondrocyte viability and function in vivo. However, future studies must be directed toward investigating the direct effect of ex vivo storage on the integrity and function of the vascular pedicles."
So growth plate transplantation is possible along as the growth plate is not in storage for more than 3 days. Although I don't see why you wouldn't just differentiate a new growth plate in the bone marrow.
Here's a study related to formation of new growth plates so they can be available for transplant:
Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage.
"We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). [Are] chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP? hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. We compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development."
"Pellet cultures were used to induce chondrogenic differentiation of hfMSCs"
"The mean diameter of the pellets increased with time, as well as the amount of glycosaminoglycans, a major constituent of the cartilaginous extracellular matrix. Immunofluorescent staining for collagen type II demonstrated the presence of chondrocytes after 1 week of pellet culture. The expression of collagen type II increased over time. Hypertrophic chondrocytes were first detected after 3 weeks, as evidenced by immunohistochemical staining for collagen type X. These collagen type X positive cells were located in a discrete ring-like zone surrounded by collagen type II positive chondrocytes. In all stages of differentiation, the chondrogenic core of the pellets was surrounded by a thin layer of two to three undifferentiated cells"
"Global gene expression microarray analysis showed that the Wnt antagonist DKK1 and FRZB and the BMP antagonist GREM1 are highly expressed in articular cartilage as compared to growth plate cartilage."
"PANX3, EPYC{up 6 fold in LSJL}, WNT11 and LEF1 are highly expressed in growth plate cartilage as compared to articular cartilage"
KEGG Pathways expressed in growth plates:
Focal Adhesion
Cytokine and Cytokine Receptor Interaction
Wnt and IHH signaling
Complement and coagulation
TGFBeta Signaling
Cell Communication and Extracellular Matrix Interaction
B-cell receptor signaling
Click on the image to see it enlarged.
"Analysis of protein interactions of all genes that were ≥3.29-fold changed after 5 weeks of chondrogenic differentiation as compared to undifferentiated hfMSC"
Genes changed that were not in highlighted clusters in diagram also altered in LSJL:
CAPN6{up}
LRRC1{down}
CADM1{down}
ITGBL1{up}
ARL6ip1{down}
Acta2{up}
Angptl1{up}
Tmem100{up}
Scn3a{up}
Slc38a4{up}
Dpt{up}
Cluster A(all genes listed genes that were altered by LSJL are noted as these genes may not have been altered by LSJL immediately but may have been altered by LSJL at a later time point or at under 2 fold. Bolded means that the genes are centrally located in the cluster thus if they are altered by LSJL it's more likely that the other genes are too):
ADAMTS1{up}
OMD
GDF5
Sp7
Acan{up}
Epyc{up}
Col10a1{up}
Adamts5
Col9a2
Ptprz1
Cntnap1
Ctsb
Fap
Comp
Col9a3{up}
KIAA1199
Slc24a2
CHAD
PTH1R
Spp1
Col2a1{up}
Chi3l1
Fmod
Col11a1{up}
29.2%
Cluster B:
S100P
TGFBR3
HSPA8
UQCRFS1
CAV1
CRLF1{up}
LRP4
Basp1{down}
Grem1
Has2
FST
Bambi
Wif1
15.4%
Cluster C:
ID3
Hey1
Slc14a1
F13a1
Wnt11
Vcam1{down}
Nqo1
Akr1c3
Serpina3
MMP1
Plau
Fos{up}
Jun{up}
Sox8
21.4%
Cluster D:
Ccnb1
Prc1
Ndc80
Pbk
Ube2c
Plk2
Ccnc
Orc6l
Dlgap5
Melk{down}
10%
Cluster E:
Pcolce2
Ogn{down}
Smoc2{up}
66%
LSJL likely alters expression of clusters A, C, and E.
Now here's human fetal MSCs gene expression versus growth plate gene expression:
"Analysis of protein interactions of genes that are differentially expressed in undifferentiated hfMSC (week 0) compared to average expression profiles of growth plate cartilage of 3 prepubertal donors "
No Cluster Genes that were altered in growth plate cartilage and LSJL:
Gldn{up}
Fxyd6{up}
BSP{up}
Tagln{up}
Postn{up}
Acta2{up}
Col16a1{up}
Vgll3{up}
Scn3a{up}
Fzd2{up}
Capn6{up}
Arl4c{down}
Tardbp{down}
Ankrd29{up}
Slc5a1{up}
Spp1{up}
Cluster A:
Acan{up}
Omd
Matn2{up}
Adamts1{up}
Frzb
MMP9
Ptprz1
Col9a2
Col10a1{up}
Matn3{up}
Epyc{up}
Csgalnact1
Cntnap1
Phlda2
Col2a1{up}
Sox9{up}
Comp
Matn1
Col11a1{up}
Col11a2
Fmod
MMP13
Ctsk
Chi3l1
Lect1
Itga10
Pth1r
FGFR3
CHAD
Prelp
Gprasp1{down}
Wisp3
Col9a1{up}
33.3%
Cluster B:
Grem1
Rgmb
Sost
Gasp1
Has2
Col15a1{up}
glipr1
bambi
fat3
daam1
Wif1
Tmemff2
C4orf49
7.7%
Cluster C:
MMP1
IGFBP3
GBP1
Fap
Tnfaip8{down}
Fos{up}
Fosb{up}
Serpina3
37.5%
Cluster D:
Clu
Serpine1{up}
Gas6
F13a1
Lpl
Lif
Loxl1
TNIK
Vegfc
Serpina1
PF4
Neto2
CH25H{up}
15.4%
Cluster E:
S100B
Myo5c
Prss23
Man1a1
Boc
Ptx3
Serpina5
CTCFL
Clgn
Rbp4
Ifit1
Cdh13{up}
Adipoq
Gpnmb
Sox8
Cxcl14
Smoc2{up}
Ogn{down}
Pcolce2
Hbb
Hbd
Hba2
F3
TF
IGFBP4
MYO5C
11.5%
Cluster F:
BDNF
Eno4
Uchl1
Scg2
Scrg1
Cnih3
Spry2
Stmn2
0%
LSJL likely alters clusters A and C.
"Growth and differentiation factor 5 (GDF5), previously reported as stimulator of chondrocyte proliferation, was highly expressed at the earliest time point observed and down regulated thereafter."
"Cartilage matrix analysis is often limited to examining collagen type 2 and aggrecan expression. These markers are characteristic for hyaline cartilage and cannot distinguish growth plate cartilage from articular cartilage. Indeed, Huang et al. performed global microarray analysis of adult bovine MSCs at time 0 and after 28 days of differentiation in agarose constructs and compared the gene expression profile to that of chondrocytes isolated from articular cartilage. They showed that chondrogenically differentiating MSC do not form articular cartilage at 28 days in the presence of TGFβ3"
Engineering osteochondral constructs through spatial regulation of endochondral ossification.
"Chondrogenically primed bone marrow derived mesenchymal stem cells (MSCs) have been shown to become hypertrophic and undergo endochondral ossification when implanted in vivo. Modulating this endochondral phenotype may be an attractive approach to engineering the osseous phase of an osteochondral implant. [We engineered] an osteochondral tissue by promoting endochondral ossification in one layer of a bi-layered construct and stable cartilage in the other. The top-half of bi-layered agarose hydrogels were seeded with culture expanded chondrocytes (termed chondral layer) and the bottom half of the bi-layered agarose hydrogels with MSCs (termed osseous layer). Constructs were cultured in a chondrogenic medium for 21 days and thereafter were either maintained in a chondrogenic medium, transferred to a hypertrophic medium, or implanted subcutaneously into nude mice. This structured chondrogenic bi-layered co-culture was found to enhance chondrogenesis in the chondral layer, appearing to help re-establish the chondrogenic phenotype that is lost in chondrocytes during monolayer expansion. The bi-layered co-culture appeared to suppress hypertrophy and mineralisation in the osseous layer. The addition of hypertrophic factors to the media was found to induce mineralisation of the osseous layer in vitro. A similar result was observed in vivo where endochondral ossification was restricted to the osseous layer of the construct leading to the development of an osteochondral tissue."
Fully differentiated chondrocytes do not undergo endochondral ossification upon implantation whereas MSCs derived into chondrocytes do.
"A structured co-culture of chondrocytes and MSCs significantly enhanced collagen synthesis in the top chondral layer of bi-layered engineered constructs compared to single layer constructs that only contained chondrocytes (133.32 ± 21.8 vs. 72.45 ± 18.63 ng/ng). MSCs in single layer constructs accumulated significantly more collagen compared to MSCs in the bottom osseous layer of bi-layered constructs (154.65 ± 14.53 vs. 83.57 ± 21.38 ng/ng)."
"No evidence of mineralisation was observed in bi-layered constructs maintained in a hypertrophic medium without additional β-glycerophosphate supplementation (HM-). When β-glycerophosphate was added to the hypertophic medium (HM+), mineralisation of the osseous layer was observed"
"Both hypertrophic media formulations resulted in apparent elongation of the interface between the osseous and chondral layer of bi-layered constructs. sGAG accumulation in the chondral layer of the engineered tissue was significantly reduced for constructs maintained in HM+ compared to CM"
"Mineralisation of the osseous layer correlated with significant cell death as evidenced by a reduction in the DNA content in this layer of bi-layered constructs when cultured in a hypertrophic medium with additional β-glycerophosphate supplementation (HM+)"
"Mineral volume, was significantly greater for single layer MSC constructs compared to bi-layered constructs (6.09± 0.59 vs. 1.36 ± 0.42 mm3; n=3)"
"[There was] reduced type X collagen accumulation in the osseous layer of bi-layered constructs while type II collagen accumulation increased in the chondral layer."
"bi-layered coculture suppresses hypertrophy of MSCs and enhances chondrogenesis of chondrocytes"
"Single layer chondrocyte seeded constructs stained weakly for collagen type II, indicating that a certain degree of de-differentiation had occurred prior to hydrogel encapsulation."
"chondrogenically primed MSCs release growth factors and cytokines such as TGF-β3, BMP-2, IGF-1 and FGF-2"
"In hypertrophic media formulations, both with and without β-glycerophosphate supplementation, elongation of the interface between the two cell types was observed, suggesting perhaps that aspects of long bone growth are being mimicked in this culture system."
Monday, September 19, 2011
juvenile versus adult articular cartilage gene expression
What genes are expressed differently in the articular cartilage and how can that impact height growth?
Enhanced Tissue Regeneration Potential of Juvenile Articular Cartilage.
"Articular cartilage harvested from juvenile (age, 4 months) and adult (age, 6-8 years) bovine femoral condyles was cultured for 4 weeks to monitor chondrocyte migration, glycosaminoglycan content conservation, and new tissue formation. The cartilage cell density and proliferative activity were also compared. Compared with adult cartilage, juvenile bovine cartilage demonstrated a significantly greater cell density, higher cell proliferation rate, increased cell outgrowth, elevated glycosaminoglycan content, and enhanced matrix metallopeptidase 2{up} activity. During 4 weeks in culture, only juvenile cartilage was able to generate new cartilaginous tissues, which exhibited pronounced labeling for proteoglycan and type II collagen but not type I collagen. With over 19,000 genes analyzed, distinctive gene expression profiles were identified. The genes mostly involved in cartilage growth and expansion, such as COL2A1{up}, COL9A1{up}, MMP2{up}, MMP14{up}, and TGFB3, were upregulated in juvenile cartilage, whereas the genes primarily responsible for structural integrity, such as COMP, FN1, TIMP2, TIMP3, and BMP2{up}, were upregulated in adult cartilage. As the first comprehensive comparison between juvenile and adult bovine articular cartilage at the tissue, cellular, and molecular levels, the results strongly suggest that juvenile cartilage possesses superior chondrogenic activity and enhanced regenerative potential over its adult counterpart. Additionally, the differential gene expression profiles of juvenile and adult cartilage suggest possible mechanisms underlying cartilage age-related changes in their regeneration capabilities, structural components, and biological properties."
"It appeared that numerous juvenile chondrocytes were able to break down the matrix entrapment in their migrating front, forming migration channels. Through these channels, the juvenile chondrocytes that resided far away from the cut margins were able to migrate out. However, these channels were not observed from adult cartilage, as the migrating adult chondrocytes mostly resided adjacent to the cut edges of the cartilage discs"
Another gene upregulated in juvenile articular cartilage is IGF2.
"breakdown of the collagen network by extensive cutting has been shown to promote chondrocyte migration"<-Maybe we can cause migration of the chondrocytes to form new growth plates?
"abundant open surfaces of the cartilage discs may provide a permissive environment, allowing the “passive” migration of the chondrocytes residing adjacent to the cut margins in both age groups. Juvenile chondrocytes, however, appeared to migrate in a more active manner by breaking down the local ECM network at their migrating front. These different migration activities could be explained by our GeneChip array findings. As a cell membrane–anchored enzyme, MMP14 has been reported to localize at the migration front and promote cell migration by cleaving the cellular adhesion molecule CD44 and triggering MMP activation cascades. Because MMP2, MMP13, MMP14, and MMP16 were all upregulated in juvenile cartilage and the overexpression of MMP2 was further confirmed by both qPCR and MMP2 activity assays, we hypothesize that the migration of juvenile chondrocytes is facilitated by a MMP activation cascade wherein MMP14 activates pro-MMP2, which in turn activates pro-MMP13. As a broad-spectrum ECM-destructing enzyme, MMP13, whose expression was 1.6-fold higher in juvenile cartilage, cleaves local type II collagen, aggrecan, and pericellular ECM components. As a result, activation of these juvenile cartilage upregulated MMPs not only increases chondrocyte mobility by detaching them from the surrounding ECM but also opens up a migration pathway by digesting their pericellular ECM barriers."
" adult cartilage expresses elevated levels of TIMP2 and TIMP3, which block the MMP activation cascade, leading to the inhibition of “active” chondrocyte migration."
Enhanced Tissue Regeneration Potential of Juvenile Articular Cartilage.
"Articular cartilage harvested from juvenile (age, 4 months) and adult (age, 6-8 years) bovine femoral condyles was cultured for 4 weeks to monitor chondrocyte migration, glycosaminoglycan content conservation, and new tissue formation. The cartilage cell density and proliferative activity were also compared. Compared with adult cartilage, juvenile bovine cartilage demonstrated a significantly greater cell density, higher cell proliferation rate, increased cell outgrowth, elevated glycosaminoglycan content, and enhanced matrix metallopeptidase 2{up} activity. During 4 weeks in culture, only juvenile cartilage was able to generate new cartilaginous tissues, which exhibited pronounced labeling for proteoglycan and type II collagen but not type I collagen. With over 19,000 genes analyzed, distinctive gene expression profiles were identified. The genes mostly involved in cartilage growth and expansion, such as COL2A1{up}, COL9A1{up}, MMP2{up}, MMP14{up}, and TGFB3, were upregulated in juvenile cartilage, whereas the genes primarily responsible for structural integrity, such as COMP, FN1, TIMP2, TIMP3, and BMP2{up}, were upregulated in adult cartilage. As the first comprehensive comparison between juvenile and adult bovine articular cartilage at the tissue, cellular, and molecular levels, the results strongly suggest that juvenile cartilage possesses superior chondrogenic activity and enhanced regenerative potential over its adult counterpart. Additionally, the differential gene expression profiles of juvenile and adult cartilage suggest possible mechanisms underlying cartilage age-related changes in their regeneration capabilities, structural components, and biological properties."
"It appeared that numerous juvenile chondrocytes were able to break down the matrix entrapment in their migrating front, forming migration channels. Through these channels, the juvenile chondrocytes that resided far away from the cut margins were able to migrate out. However, these channels were not observed from adult cartilage, as the migrating adult chondrocytes mostly resided adjacent to the cut edges of the cartilage discs"
Another gene upregulated in juvenile articular cartilage is IGF2.
"breakdown of the collagen network by extensive cutting has been shown to promote chondrocyte migration"<-Maybe we can cause migration of the chondrocytes to form new growth plates?
"abundant open surfaces of the cartilage discs may provide a permissive environment, allowing the “passive” migration of the chondrocytes residing adjacent to the cut margins in both age groups. Juvenile chondrocytes, however, appeared to migrate in a more active manner by breaking down the local ECM network at their migrating front. These different migration activities could be explained by our GeneChip array findings. As a cell membrane–anchored enzyme, MMP14 has been reported to localize at the migration front and promote cell migration by cleaving the cellular adhesion molecule CD44 and triggering MMP activation cascades. Because MMP2, MMP13, MMP14, and MMP16 were all upregulated in juvenile cartilage and the overexpression of MMP2 was further confirmed by both qPCR and MMP2 activity assays, we hypothesize that the migration of juvenile chondrocytes is facilitated by a MMP activation cascade wherein MMP14 activates pro-MMP2, which in turn activates pro-MMP13. As a broad-spectrum ECM-destructing enzyme, MMP13, whose expression was 1.6-fold higher in juvenile cartilage, cleaves local type II collagen, aggrecan, and pericellular ECM components. As a result, activation of these juvenile cartilage upregulated MMPs not only increases chondrocyte mobility by detaching them from the surrounding ECM but also opens up a migration pathway by digesting their pericellular ECM barriers."
" adult cartilage expresses elevated levels of TIMP2 and TIMP3, which block the MMP activation cascade, leading to the inhibition of “active” chondrocyte migration."
Tuesday, September 13, 2011
How much can supplements help with LSJL
Many people look to chemical methods in terms of increasing height. LSJL is in many ways a chemical method increasing the hydrostatic pressure(thus increasing the amount of water, a chemical, per square inch). Hydrostatic Pressure has been shown to induce chondrogenic differentiation. Hydrostatic pressure has been shown to induce chondrogenic differentiation in Type I Collagen(bone). No other substances were used, however, a hydrostatic pressure of 1 MPa was used. The peak pressure observed in the LSJL rat studies was about 0.0013 MPa. Now there are already other chemicals present in the body that encourage chondrogenic differentiation like IGF-1. The more chondrogenic the medium, the lower the threshold of hydrostatic pressure required and the more likely to grow taller.
By supplementing we can recreate the medium in the bone marrow in which scientists have had success in inducing chondrogenic differentiation. Since we don't directly have access to TGF-Beta1, IGF-1, and BMP-2, we have to find ways to indirectly increase them. Since we can't directly inject them, their anabolic effects will occur at a systematic level. So, if you're Justin Bieber, and don't want a deeper voice don't take anything. If you want to be tall but not bigger otherwise then don't take anything.
Dexamethasone can induce chondrogenic differentiation but it also decreases cellular proliferation. It may or may not have permanent affects on height but for now we'll avoid it.
Melatonin indirectly increases TGF-Beta1 but there's an enzyme that is upregulated in response to exogenous Melatonin so it is necessary to cycle Melatonin. Both TGF-Beta and BMP-2 are helpful for early induction of chondrogenesis. However, during active growth plates TGF-Beta is preferable to BMP-2 as BMP-2 encourages mineralization.
Cnidium can increase BMP-2 levels as well as IGF-1 levels. Horny goat weed increases both TGF-Beta and BMP-2. IGF-1 has been shown to increase chondrocyte differentiation levels.
Hyaluronic Acid has been shown to enhance development into a chondrogenic lineage. And High Molecular Weight Hyaluronic Acid has been shown to increase serum levels of HA(divided among all the body tissues of course). Chondroitin has been shown to help stem cell proliferation but I have not directly found something stated it's influence in stem cell differentiation into chondrocytes.
Myostatin has been to shown to inhibit chondrocyte differentiation. So that means creatine can help you grow taller as it inhibits myostatin. Testosterone boosters lower myostatin as well so they can help you grow taller too.
FGF-2 also helps induce chondrogenic differentiation. I don't know of any supplements that can increase FGF-2 levels however.
It's hard to gauge the safety and efficacy of all the supplements. How much of a chondrogenic medium is needed in conjunction with LSJL to induce chondrogenic differentiation? Observe all supplement instructions. Also, when you add a new supplement ask yourself if you notice any new phenotype differences. If you notice differences after supplementation that means that the supplement is likely proving to be effective in making a more chondrogenic medium in your bone marrow. Although we cannot say for sure whether the converse is true, in that even if you don't notice any changes in phenotype it can still be helping make a more chondrogenic medium in your bone marrow.
By supplementing we can recreate the medium in the bone marrow in which scientists have had success in inducing chondrogenic differentiation. Since we don't directly have access to TGF-Beta1, IGF-1, and BMP-2, we have to find ways to indirectly increase them. Since we can't directly inject them, their anabolic effects will occur at a systematic level. So, if you're Justin Bieber, and don't want a deeper voice don't take anything. If you want to be tall but not bigger otherwise then don't take anything.
Dexamethasone can induce chondrogenic differentiation but it also decreases cellular proliferation. It may or may not have permanent affects on height but for now we'll avoid it.
Melatonin indirectly increases TGF-Beta1 but there's an enzyme that is upregulated in response to exogenous Melatonin so it is necessary to cycle Melatonin. Both TGF-Beta and BMP-2 are helpful for early induction of chondrogenesis. However, during active growth plates TGF-Beta is preferable to BMP-2 as BMP-2 encourages mineralization.
Cnidium can increase BMP-2 levels as well as IGF-1 levels. Horny goat weed increases both TGF-Beta and BMP-2. IGF-1 has been shown to increase chondrocyte differentiation levels.
Hyaluronic Acid has been shown to enhance development into a chondrogenic lineage. And High Molecular Weight Hyaluronic Acid has been shown to increase serum levels of HA(divided among all the body tissues of course). Chondroitin has been shown to help stem cell proliferation but I have not directly found something stated it's influence in stem cell differentiation into chondrocytes.
Myostatin has been to shown to inhibit chondrocyte differentiation. So that means creatine can help you grow taller as it inhibits myostatin. Testosterone boosters lower myostatin as well so they can help you grow taller too.
FGF-2 also helps induce chondrogenic differentiation. I don't know of any supplements that can increase FGF-2 levels however.
It's hard to gauge the safety and efficacy of all the supplements. How much of a chondrogenic medium is needed in conjunction with LSJL to induce chondrogenic differentiation? Observe all supplement instructions. Also, when you add a new supplement ask yourself if you notice any new phenotype differences. If you notice differences after supplementation that means that the supplement is likely proving to be effective in making a more chondrogenic medium in your bone marrow. Although we cannot say for sure whether the converse is true, in that even if you don't notice any changes in phenotype it can still be helping make a more chondrogenic medium in your bone marrow.
Monday, September 12, 2011
Grow Taller without Thrombospondin-1?
Thrombospondin-1 prevents excessive ossification in cartilage repair tissue induced by osteogenic protein-1.
"thrombospondin-1 (TSP-1) osteogenic protein-1 (OP-1). In miniature pigs, articular cartilage lesions in the femoral trochlea were treated by the microfracture technique and either received no further treatment (MFX), or were treated by additional application of recombinant osteogenic protein-1 (MFX+OP-1), recombinant TSP-1 (MFX+TSP-1), or a combination of both proteins (MFX+TSP-1+OP-1). Six and 26 weeks after surgery, the repair tissue and the degree of endochondral ossification were assessed by histochemical and immunohistochemical methods detecting collagen types I, II, X, TSP-1, and CD31. Microfracture treatment merely induced the formation of inferior fibrocartilaginous repair tissue. OP-1 stimulated chondrogenesis, but also induced chondrocyte hypertrophy, characterized by synthesis of collagen type X, and excessive bone formation. Application of TSP-1 inhibited inadvertant endochondral ossification, but failed to induce chondrogenesis. In contrast, the simultaneous application of both TSP-1 and OP-1 induced and maintained a permanent, nonhypertrophic chondrocyte-like phenotype within cartilage repair tissue. OP-1 and TSP-1 complement each other in a functional manner. While OP-1 induces chondrogenesis of the ingrowing cells, TSP-1 prevents their further hypertrophic differentiation and prevents excessive endochondral ossification within the lesions."
So THBS1 doesn't inhibit chondrogenesis.
"Stimulation with TSP-1 for 24 or 48 h significantly inhibited the mRNA expression of GADD45β, a factor that was recently shown to be involved in chondrocyte hypertrophy. On the contrary, single stimulation with OP-1 for 24 h significantly induced the expression of GADD45β, an effect that could be neutralized by simultaneous application of TSP-1"
BMP-7 induces both Sox9 and Runx2.
Tuesday, September 6, 2011
Increasing Height by Manipulating the Cell Cycle
Stem Cells and Chondrocytes like all cells engage in a cell cycle. A cell that has left the cycle is senescent and has stopped dividing. Once all the cells stop dividing the left over extracellular matrix like collagen type II is degraded. Thus, forcing us to try to induce chondrogenic differentiation with no cartilagenous template. The longer cells spend in the cell cycle, the more time before senescence and the taller you will grow.
Cell cycle analysis of proliferative zone chondrocytes in growth plates elongating at different rates.
"Regulation of postnatal growth of long bones occurs in multiple levels of chondrocytic activity, including stem cell proliferation, proliferative zone cycling, and regulation of changes in chondrocytic shape during hypertrophy[thus altering all those things can help us grow taller during ordinary development and when trying to induce chondrogenesis once more with LSJL]. The differentiation sequence of chondrocytes is the same in all growth plates, but rates of elongation at a single point in time and over a period of time differ widely among individual growth plates, which suggests that the rates of sequential gene activation and suppression in this phenotypic pattern can vary. The purpose of this study was to investigate, directly and in vivo, parameters of the cell cycle of proliferative chondrocytes in growth plates growing at widely different rates at a single point in time in order to analyze the relationship between cell cycle time, including the duration of each phase of the cell cycle (G1, S, G2, and M), and the rate of growth[In G1 cells grow but there is no DNA replication, in stage S DNA replication begins, in G2 cells resume growing and continue DNA replication, in stage M growth and DNA replication ceases and division occurs]. The experimental design used repeated pulse labeling with bromodeoxyuridine and was analyzed using a regression model of time of pulse label with increasing labeling index. Total cell cycle time was calculated as the inverse of the slope of the relationship of the labeling index and the time between labels. The y intercept was the calculated labeling index at time zero. Multiple comparison contrasts were used to test for individual differences among four growth plates with growth rates ranging from approximately 50 to 400 microns per 24 hours from 28-day-old rats. The estimate of total cell cycle time for the proximal tibial growth plate was 30.9 hours[So if we were to induce one stem cell to differentiate into a chondrocyte with LSJL we would have about 31 hours to try to induce more stem cell chondrogenesis before mitosis occurs followed by senescence]. Cell cycle times for the other three growth plates were 34.0, 48.7, and 76.3 hours for the distal radius, distal tibia and proximal radius, respectively[the proximal radius is by the elbow, it's the bump on the side. It may be worthwhile to target that region if it does spend so much time in the cell cycle]. Although the times for the proximal tibia and distal radius did not differ significantly, all other times were significantly different (p < 0.05). Almost all differences in total cell cycle time were attributable to significant differences in the length of the G1 phase[thus we likely(but not necessarily) want to stimulate the G1 phase of the cell cycle, note that the distal radius has a long cell cycle and most people have relatively longer arms than their height this could be correlated to G1 cell cycle length]. The S phase was estimated at 3.4-6.1 hours; the G2 phase, at 3.0 hours; and the M phase, at 0.5-0.6 hours. The current study suggests that regulation through cell cycle parameters, specifically in the G1 phase, may be involved in overall regulation of differential postnatal long bone growth. It has previously been established that increase and shape change of cellular volume during hypertrophy may be regulated at the level of individual growth plates and that both are significant in understanding differential growth of long bone at this level. By demonstrating that chondrocytes in the proliferating zone have different cell cycle times that are regulated primarily through differences in the duration of G1, this study suggests that, in addition to systemic controls of chondrocyte proliferation, local controls may modulate rates of proliferation of individual growth plates and thus may be another locally mediated regulator of differential growth."
"The increase in length of a bone achieved by a specific growth plate in any one 24-hour period is determined by a complex interplay of proliferative kinetics, matrix synthesis throughout the growth plate with controlled matrix degradation, and chondrocytic enlargement during hypertrophy that is accompanied by a disproportionate increase in height (in the direction of growth) compared with width as the volume of the cell expands"
According to the chart in the study, growth rate is actually inverse to the time spent in G1. Of course growth rate does not correlate to final adult height. But this indicates that it may be worthwhile to actually diminish time spent in G1. Though by definition of having a longer cell cycle it makes sense to have a slower growth rate because everything takes a longer time to do.
"Circadian rhythms associated with mitosis of growth plate chondrocytes also have been reported"<-thus light, absence of light, and melatonin may be able to manipulate mitosis and the bodies height increasing ability.
"when chondrocytic performance has been studied in mammalian growth plates growing at different rates, it has been demonstrated repeatedly that the growth plates elongating at the fastest rate have the largest hypertrophic chondrocytes with respect to final volume"<-thus it may be worthwhile to try to increase growth rate.
"it has been demonstrated that shape change accompanying volume increase during hypertrophy
also is significant, and that cumulative differential height increases in the direction of growth from
proliferative to hypertrophic chondrocytes correlate positively with rate of growth"<-So the size of hypertrophic chondrocytes plays a large role in bone growth. Although the height growth could be caused be a secondary factor related to hypertrophy like osmotic pressure changes that occur with the degradation of proteoglycans.
Chondrocyte p21(WAF1/CIP1) expression is increased by dexamethasone but does not contribute to dexamethasone-induced growth retardation in vivo.
p21 is a regulator of the G1 stage of the cell cycle and can cause senescence. However, just be the title you can see that this surprisingly does not induce growth retardation.
"It has been shown that cell cycle genes play an important role in the coordination of chondrocyte proliferation and differentiation. 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[thus to grow taller you may want to inhibit CDKIs]. In this study, 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 4-week-old mice lacking a functional p21 gene, Dex caused a reduction in body weight compared to saline control null mice, but this was consistent with the reduction in body weight observed in Dex-treated wild-type littermates. 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[the reduction in growth plate width occur regardless of removal of the p21 gene in Dex-treated mice]. However, an alteration in growth rate and epiphyseal structure is evident when comparing p21(-/-) and wild-type mice[so removal of p21 in normal mice does affect the growth plate, maybe Dexamethasone and p21 work via similar inhibitors]. These findings suggest that p21 does not directly contribute to GC-induced growth retardation in vivo but is involved in the maintenance of the growth plate."
"Disruption of the p57 gene has been shown to cause delayed chondrocyte differentiation, resulting in skeletal deformations and shortened limbs"<-thus you may not want to delay chondrocyte differentiation
"mice deficient in p27 do not show any obvious skeletal phenotypes, though they are larger than wild-type mice"<-inhibiting p27 may be a way to increase height. Both p21 and p27 play similar roles. If you inhibit p27 but maintain fully functional p21 you should be able to maintain full functionality without uncontrolled cellular proliferation and result in being taller. PI3K-Akt inactivates p27 but inhibiting p27 does increase risk of cancer(because now you have to rely solely on p21 to regulate the cell cycle). PI3K is stimulated by Insulin, IGF-1, and exercise. The PI3K pathway is also stimulated by Puerarin(which is contained in Ghenerate).
Here's a study that explains what happens to articular chondrocytes in relation to senescence and aging, we can use this information about apply it to growth plate chondrocytes:
Events in Articular Chondrocytes with Aging.
"One of the most pronounced age-related changes in chondrocytes is the exhibition of a senescent phenotype, which is the result of several factors including the accumulation of reactive oxygen species[so could anti-oxidants help stave off senescence?] and advanced glycation end products. Compared with a normal chondrocyte, senescent chondrocytes exhibit an impaired ability to respond to many mechanical and inflammatory insults to the articular cartilage[so could mechanical and inflammatory signals be key to growth in growth plate chondrocytes]. Furthermore, protein secretion is altered in aging chondrocytes, demonstrated by a decrease in anabolic activity and increased production of proinflammatory cytokines and matrix-degrading enzymes."
This could very well apply to growth plate chondrocytes as well as the final stage is apoptosis. You can perhaps slow down this senescence somewhat with anti-oxidants and telomere lengtheners like astragalus.
In addition, to telomere length and reactive oxygen species number being possible preventable ways of slowing down cellular senescence, so too is the amount of advanced glycogen end-products.
"AGEs are produced through a nonenzymatic reaction between reducing sugars and free amino groups of proteins, lipids, or nucleic acids. AGEs are formed within the body, and are also derived from cooking techniques that involve “browning” foods. Excessive levels of AGEs in the body are pathogenic, and its effects include increased production of oxidative stress and inflammation. In chondrocytes, AGEs increase production of inflammatory cytokine tumor necrosis factor-α (TNF-α) and inflammatory mediators prostaglandin E2 and nitric oxide, suppress collagen II production, and stimulate expression of degradative enzymes matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)"<-so AGE number can be reduced by avoiding cooking techniques that brown foods.
Remember that osteoarthritis is very similar to endochondral ossification except that osteoarthritis does not seem to increase height. Thus insights into delaying senescence in osteoarthritis can help us delay senescence in the growth plate.
Cell-cycle control and the cartilage growth plate.
"Progression through the eukaryotic cell-cycle is controlled by cyclin-dependent kinases (CDKs)"
"The activity of CDKs is highly regulated by a number of mechanisms: (a) the level of their respective partner proteins, the cyclins, (b) the levels of inhibitory proteins of the Cip/Kip (p21, p27, p57) and Ink (p15, p16, p18, p19) families (CDK inhibitors or CKIs), and (c) inhibitory and stimulatory phosphorylation of various CDK residues "
"High levels of cyclins therefore generally stimulate cell-cycle progression and proliferation through activation of CDKs, whereas high levels of CKIs antagonize these processes. The most prominent targets of CDKs are the retinoblastoma protein (pRb) and the closely related p107 and p130 proteins. In their hypophosphorylated forms, these proteins (commonly referred to as pocket proteins) form complexes with transcription factors of the E2F family. These complexes, in association with histone deacetylases, repress transcription of E2F target genes. Upon stepwise phosphorylation of pocket proteins by CDKs the complexes dissociate, and free E2F factors can now activate the transcription of genes required for cell-cycle progression and DNA replication."
"Within the growth plate, cyclin D1 expression is specific for the proliferative zone at the mRNA"
"cyclin D1 gene [is] a target of the transcription factor ATF-2 in chondrocytes"
Both Wnt5a over- and under- expression reduce Cyclin D1 activity.
"intracellular signaling molecules such as integrin-linked kinase, the small GTPase RhoA and the transcription factor c-Fos also stimulate cyclin D1 expression in cartilage."<-LSJL upregulates c-Fos. LSJL affects Cyclin D1 given the effects of LSJL on c-Fos it is likely to increase Cyclin D1 expression.
"p21 expression in chondrocytes is induced or enhanced by FGF signaling through the transcription factor STAT1 "<-increased p21 expression may be a part of FGFR3 dwarfism.
Cell cycle analysis of proliferative zone chondrocytes in growth plates elongating at different rates.
"Regulation of postnatal growth of long bones occurs in multiple levels of chondrocytic activity, including stem cell proliferation, proliferative zone cycling, and regulation of changes in chondrocytic shape during hypertrophy[thus altering all those things can help us grow taller during ordinary development and when trying to induce chondrogenesis once more with LSJL]. The differentiation sequence of chondrocytes is the same in all growth plates, but rates of elongation at a single point in time and over a period of time differ widely among individual growth plates, which suggests that the rates of sequential gene activation and suppression in this phenotypic pattern can vary. The purpose of this study was to investigate, directly and in vivo, parameters of the cell cycle of proliferative chondrocytes in growth plates growing at widely different rates at a single point in time in order to analyze the relationship between cell cycle time, including the duration of each phase of the cell cycle (G1, S, G2, and M), and the rate of growth[In G1 cells grow but there is no DNA replication, in stage S DNA replication begins, in G2 cells resume growing and continue DNA replication, in stage M growth and DNA replication ceases and division occurs]. The experimental design used repeated pulse labeling with bromodeoxyuridine and was analyzed using a regression model of time of pulse label with increasing labeling index. Total cell cycle time was calculated as the inverse of the slope of the relationship of the labeling index and the time between labels. The y intercept was the calculated labeling index at time zero. Multiple comparison contrasts were used to test for individual differences among four growth plates with growth rates ranging from approximately 50 to 400 microns per 24 hours from 28-day-old rats. The estimate of total cell cycle time for the proximal tibial growth plate was 30.9 hours[So if we were to induce one stem cell to differentiate into a chondrocyte with LSJL we would have about 31 hours to try to induce more stem cell chondrogenesis before mitosis occurs followed by senescence]. Cell cycle times for the other three growth plates were 34.0, 48.7, and 76.3 hours for the distal radius, distal tibia and proximal radius, respectively[the proximal radius is by the elbow, it's the bump on the side. It may be worthwhile to target that region if it does spend so much time in the cell cycle]. Although the times for the proximal tibia and distal radius did not differ significantly, all other times were significantly different (p < 0.05). Almost all differences in total cell cycle time were attributable to significant differences in the length of the G1 phase[thus we likely(but not necessarily) want to stimulate the G1 phase of the cell cycle, note that the distal radius has a long cell cycle and most people have relatively longer arms than their height this could be correlated to G1 cell cycle length]. The S phase was estimated at 3.4-6.1 hours; the G2 phase, at 3.0 hours; and the M phase, at 0.5-0.6 hours. The current study suggests that regulation through cell cycle parameters, specifically in the G1 phase, may be involved in overall regulation of differential postnatal long bone growth. It has previously been established that increase and shape change of cellular volume during hypertrophy may be regulated at the level of individual growth plates and that both are significant in understanding differential growth of long bone at this level. By demonstrating that chondrocytes in the proliferating zone have different cell cycle times that are regulated primarily through differences in the duration of G1, this study suggests that, in addition to systemic controls of chondrocyte proliferation, local controls may modulate rates of proliferation of individual growth plates and thus may be another locally mediated regulator of differential growth."
"The increase in length of a bone achieved by a specific growth plate in any one 24-hour period is determined by a complex interplay of proliferative kinetics, matrix synthesis throughout the growth plate with controlled matrix degradation, and chondrocytic enlargement during hypertrophy that is accompanied by a disproportionate increase in height (in the direction of growth) compared with width as the volume of the cell expands"
According to the chart in the study, growth rate is actually inverse to the time spent in G1. Of course growth rate does not correlate to final adult height. But this indicates that it may be worthwhile to actually diminish time spent in G1. Though by definition of having a longer cell cycle it makes sense to have a slower growth rate because everything takes a longer time to do.
"Circadian rhythms associated with mitosis of growth plate chondrocytes also have been reported"<-thus light, absence of light, and melatonin may be able to manipulate mitosis and the bodies height increasing ability.
"when chondrocytic performance has been studied in mammalian growth plates growing at different rates, it has been demonstrated repeatedly that the growth plates elongating at the fastest rate have the largest hypertrophic chondrocytes with respect to final volume"<-thus it may be worthwhile to try to increase growth rate.
"it has been demonstrated that shape change accompanying volume increase during hypertrophy
also is significant, and that cumulative differential height increases in the direction of growth from
proliferative to hypertrophic chondrocytes correlate positively with rate of growth"<-So the size of hypertrophic chondrocytes plays a large role in bone growth. Although the height growth could be caused be a secondary factor related to hypertrophy like osmotic pressure changes that occur with the degradation of proteoglycans.
Chondrocyte p21(WAF1/CIP1) expression is increased by dexamethasone but does not contribute to dexamethasone-induced growth retardation in vivo.
p21 is a regulator of the G1 stage of the cell cycle and can cause senescence. However, just be the title you can see that this surprisingly does not induce growth retardation.
"It has been shown that cell cycle genes play an important role in the coordination of chondrocyte proliferation and differentiation. 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[thus to grow taller you may want to inhibit CDKIs]. In this study, 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 4-week-old mice lacking a functional p21 gene, Dex caused a reduction in body weight compared to saline control null mice, but this was consistent with the reduction in body weight observed in Dex-treated wild-type littermates. 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[the reduction in growth plate width occur regardless of removal of the p21 gene in Dex-treated mice]. However, an alteration in growth rate and epiphyseal structure is evident when comparing p21(-/-) and wild-type mice[so removal of p21 in normal mice does affect the growth plate, maybe Dexamethasone and p21 work via similar inhibitors]. These findings suggest that p21 does not directly contribute to GC-induced growth retardation in vivo but is involved in the maintenance of the growth plate."
"Disruption of the p57 gene has been shown to cause delayed chondrocyte differentiation, resulting in skeletal deformations and shortened limbs"<-thus you may not want to delay chondrocyte differentiation
"mice deficient in p27 do not show any obvious skeletal phenotypes, though they are larger than wild-type mice"<-inhibiting p27 may be a way to increase height. Both p21 and p27 play similar roles. If you inhibit p27 but maintain fully functional p21 you should be able to maintain full functionality without uncontrolled cellular proliferation and result in being taller. PI3K-Akt inactivates p27 but inhibiting p27 does increase risk of cancer(because now you have to rely solely on p21 to regulate the cell cycle). PI3K is stimulated by Insulin, IGF-1, and exercise. The PI3K pathway is also stimulated by Puerarin(which is contained in Ghenerate).
Here's a study that explains what happens to articular chondrocytes in relation to senescence and aging, we can use this information about apply it to growth plate chondrocytes:
Events in Articular Chondrocytes with Aging.
"One of the most pronounced age-related changes in chondrocytes is the exhibition of a senescent phenotype, which is the result of several factors including the accumulation of reactive oxygen species[so could anti-oxidants help stave off senescence?] and advanced glycation end products. Compared with a normal chondrocyte, senescent chondrocytes exhibit an impaired ability to respond to many mechanical and inflammatory insults to the articular cartilage[so could mechanical and inflammatory signals be key to growth in growth plate chondrocytes]. Furthermore, protein secretion is altered in aging chondrocytes, demonstrated by a decrease in anabolic activity and increased production of proinflammatory cytokines and matrix-degrading enzymes."
Phenotype of chondrocyte aging
|
Molecular events
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|---|---|
Altered gene expression related to senescence
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• ↑ GADD45β and C/EBPβ → ↑ p21 transcription
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• ↓ SIRT1 → ↑ p53, ↑ p21
| |
• ↑ Caveolin 1 → ↑ p53, ↑ p21
| |
• ↑ β-Galactosidase
| |
DNA and telomere dysfunction
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• ↓ TRF → telomere shortening
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• ↓ XRCC5 → ↑ DNA damage
| |
• Mitochondrial DNA degradation
| |
Altered protein secretion
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• ↑ Proinflammatory cytokines (ie, IL-1β, TNF-α) and proinflammatory mediators (PGE2, NO)
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• ↑ MMPs (−1, −3, −13) and ADAMTS (−4, −5)
| |
Oxidative damage
|
• ↑ ROS production
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• ↓ Antioxidant enzyme activity
| |
• Mitochondrial dysfunction
| |
↓ Growth factor response
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• Impaired responsiveness to IGF-1 , OP-1/BMP-7 , TGF-β
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Cell death
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• ↓ IGF-1 and OP-1 → reduced cellularity
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• ↓ CK2 → apoptosis
| |
• ↓ HMGB2 → apoptosis
|
This could very well apply to growth plate chondrocytes as well as the final stage is apoptosis. You can perhaps slow down this senescence somewhat with anti-oxidants and telomere lengtheners like astragalus.
In addition, to telomere length and reactive oxygen species number being possible preventable ways of slowing down cellular senescence, so too is the amount of advanced glycogen end-products.
"AGEs are produced through a nonenzymatic reaction between reducing sugars and free amino groups of proteins, lipids, or nucleic acids. AGEs are formed within the body, and are also derived from cooking techniques that involve “browning” foods. Excessive levels of AGEs in the body are pathogenic, and its effects include increased production of oxidative stress and inflammation. In chondrocytes, AGEs increase production of inflammatory cytokine tumor necrosis factor-α (TNF-α) and inflammatory mediators prostaglandin E2 and nitric oxide, suppress collagen II production, and stimulate expression of degradative enzymes matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)"<-so AGE number can be reduced by avoiding cooking techniques that brown foods.
Remember that osteoarthritis is very similar to endochondral ossification except that osteoarthritis does not seem to increase height. Thus insights into delaying senescence in osteoarthritis can help us delay senescence in the growth plate.
Cell-cycle control and the cartilage growth plate.
"Progression through the eukaryotic cell-cycle is controlled by cyclin-dependent kinases (CDKs)"
"The activity of CDKs is highly regulated by a number of mechanisms: (a) the level of their respective partner proteins, the cyclins, (b) the levels of inhibitory proteins of the Cip/Kip (p21, p27, p57) and Ink (p15, p16, p18, p19) families (CDK inhibitors or CKIs), and (c) inhibitory and stimulatory phosphorylation of various CDK residues "
"High levels of cyclins therefore generally stimulate cell-cycle progression and proliferation through activation of CDKs, whereas high levels of CKIs antagonize these processes. The most prominent targets of CDKs are the retinoblastoma protein (pRb) and the closely related p107 and p130 proteins. In their hypophosphorylated forms, these proteins (commonly referred to as pocket proteins) form complexes with transcription factors of the E2F family. These complexes, in association with histone deacetylases, repress transcription of E2F target genes. Upon stepwise phosphorylation of pocket proteins by CDKs the complexes dissociate, and free E2F factors can now activate the transcription of genes required for cell-cycle progression and DNA replication."
"Within the growth plate, cyclin D1 expression is specific for the proliferative zone at the mRNA"
"cyclin D1 gene [is] a target of the transcription factor ATF-2 in chondrocytes"
Both Wnt5a over- and under- expression reduce Cyclin D1 activity.
"intracellular signaling molecules such as integrin-linked kinase, the small GTPase RhoA and the transcription factor c-Fos also stimulate cyclin D1 expression in cartilage."<-LSJL upregulates c-Fos. LSJL affects Cyclin D1 given the effects of LSJL on c-Fos it is likely to increase Cyclin D1 expression.
"p21 expression in chondrocytes is induced or enhanced by FGF signaling through the transcription factor STAT1 "<-increased p21 expression may be a part of FGFR3 dwarfism.
" overexpression of an activated FGFR3 gene in transgenic mice also induces expression of the p16, p18, and p19 genes, CDK inhibitors of the INK family. In addition, FGF1 induces expression of p27 and p57 in RCS cells"
"Expression of p21 and the related p27 protein in chondrocytes is also enhanced by thyroid hormone, a well-characterized inducer of chondrocyte hypertrophy, and by bone morphogenetic protein (BMP)-2. Finally, the chondrogenic transcription factor Sox9 and signaling through the c-Raf/MEK/ERK MAP kinase cascade have also been shown to be important positive regulators of p21 expression, whereas parathyroid hormone represses p21 expression in the chondrogenic cell line ATDC5. In addition, chondrocyte proliferation is enhanced and p57 expression decreased in mice with cartilage-specific inactivation of the gene encoding HIF-1α, a transcription factor involved in the cellular response to hypoxia"
"expression of p21 and p16 is increased in mice deficient for β1 integrin, suggesting that integrin signaling suppresses expression of these cell-cycle inhibitors"
" both β1 integrin deficiency and loss of integrin-linked kinase, a crucial mediator of integrin signaling, result in reduced chondrocyte proliferation through modulation of cell-cycle gene expression [through] different target genes (p16 and p21 vs. cyclinD1)"
"Disruption of the p27 gene results in generalized overgrowth, indicating effects on the growth plates"
"Overexpression of E2F1 in ATDC5 cells resulted in a [dwarfism] phenotype of delayed early and late differentiation. Interestingly, overexpression of the similar E2F2, E2F3, or E2F4 proteins caused much milder phenotypes and did not block late (hypertrophic) chondrocyte differentiation, although high levels of E2F4 in proliferating chondrocytes have been reported "
Mechanotransduction and Chondrogenesis
Dynamic compressive strain influences chondrogenic gene expression in human periosteal cells: a case study.
"Dynamic compression [selectively enhances] chondrogenic [and] osteogenic differentiation in human periosteal cells from two donors. Donor derived human periosteal cells were expanded in monolayer culture before seeding in 3% (w/v) agarose constructs. Intermittent dynamic compression (1 Hz, 15% strain) was applied to constructs, in the presence or absence of 10 ng/ml TGF-β3, for up to 4 days. The combined effect of TGF-β3 and compressive loading on the expression levels of the Sox-9, Runx-2, ALP, Collagen X, and collagen type I genes was donor dependent. A synergistic effect was noted only in donor two, with peak mRNA expression levels at 24 h, particularly Sox-9 which increased 59.0-fold."
Sox9 increased without any TGF-Beta.
"Runx-2 and Sox-9 gene expressions were highest in constructs treated with TGF-β3 in chondrogenic media and subjected to intermittent compression over 24 h"
"In periosteal cell-constructs derived from donor 1, loading did not have a positive effect on Sox-9 or Runx-2 mRNA levels. In the absence of TGF-β3, periosteal cells responded to mechanical compression through a significant down-regulation of Sox-9 expression at 12 h (1.6-fold ↓) and 48 h (1.7-fold ↓). A similar decrease (1.6-fold) was found in Runx-2 expression after 48 h of loading in constructs treated with TGF-β3."
"stimulated TGF-β signalling pathways [modulates] mechanotransduction, directly or indirectly, by increasing the sensitivity of periosteal cells to loading through the activation of mechanosensitive proteins, such as focal adhesion kinase and paxillin"
"Dynamic compressive loading promoted gene expression and protein production of both TGF-β receptors (TGF--I and II) in rabbit-MSCs in agarose. TGF--1, phosphorylated by TGF-β, initiates intracellular signal transduction, which mediates chondrogenic differentiation of chondroprogenitor cells and mesenchymal stem cells"
"As the tissue undergoes a compressive load, the pressurization of the fluid phase initially supports the applied load, because water is trapped within the solid matrix of the tissue because of its low permeability"
"In the joint, cartilage is typically exposed to stresses between 3 and 10MPa, with stress as high as 18MPa having been reported in the hip joint. These stresses [are] translated to HP due to fluid phase pressurization"<-MPa's above these levels have been reported to have anti-chondrogenic effects.
"HP has direct effects on cell membrane ion channels. Static HP on isolated bovine chondrocytes for 20s or 10min [resulted in the] sodium–potassium (Na/K) pump [being] substantially inhibited when going from 2.5 to 5MPa" "[HP] activates Na/hydrogen and stretch-activated calcium (Ca) channels, and triggers release of intracellular Ca stores."<-And this release according to our hypothesis induces chondrogenic differentiation.
"detrimental effects are apparent with loading times exceeding 2h [for loads as low as 15 MPa]."
Induction of chondrogenic phenotype in synovium-derived progenitor cells by intermittent hydrostatic pressure.
"SPCs[synovium derived progenitor cells], bone marrow-derived progenitor cells and skin fibroblasts from rabbits were subjected to IHP ranging from 1.0 to 5.0 MPa. The mRNA expression of proteoglycan core protein (PG), collagen type II and SOX-9 was examined The production of SOX-9 protein and glycosaminoglycan (GAG) by SPCs was analyzed . Mitogen-activated protein (MAP) kinase inhibitors for c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and the p38 pathway were used.
mRNA expression of PG, collagen type II and SOX-9 was significantly enhanced only in SPCs receiving 5.0 MPa of IHP. The production of SOX-9 protein and GAG by SPCs was also increased by exposure to 5.0 MPa of IHP. These up-regulated expressions were suppressed by pretreatment with an inhibitor of JNK, but not with inhibitors of ERK or p38."
So maybe chondroinduction occurs along the JNK pathway. "the JNK pathway is involved in signal transduction for IHP and in increased expression of c-Jun"
Mesenchymal Stem Cells: Role of Mechanical Strain in Promoting Apoptosis and Differentiation
"In contrast to necrotic cell death, apoptosis is an ordered process that does not trigger a pronounced inflammatory response in the surrounding tissue, due in part to the maintenance of an intact plasma membrane"
"the apoptotic process occurred as an essential step in normal ontogenesis indicating that apoptosis is an inherently programmed pathway responsible for determining cellular fate and acts as a counterbalance to mitosis thus maintaining homeostasis in the organism"
"Stretch-activated cation channels (SACCs) are understood to be involved in mechanotransduction and the presence of these receptors has been identified on osteocytes where they act as mechanical transducers. Blocking SACCs with gadolinium chloride does not protect against strain-induced apoptosis, indicating that these channels are not involved in the upregulation of apoptotic pathways in MSCs. However, in a separate study investigating the effect of 2.5% continuous strain on MSC differentiation, upregulation of collagen type I is observed. This induction of collagen type I is decreased when SACCs are blocked, highlighting a role for these receptors in the strain-induced expression of bone-related proteins"
"Mechanical stress is also thought to elongate the cell membrane which can result in activation of p38 MAPKs and production of ROS. Both of these signals converge at the level of p53. The p38 MAPK pathway directly phosphorylates p53, while ROS production causes oxidative DNA damage. Indirect activation of p53 by p38 MAPKs may occur by regulation of oxidase activation whereby p53 might be involved in a positive feedback loop by stimulating free radical generation itself. Expression of the pro-apoptotic Bax protein and mitochondrial dysfunction also mediate apoptosis following mechanical stress. Strain-induced apoptosis has also been observed in MSCs as a result of signalling via L-type calcium channels, calpain activity and JNK activation. Application of a continuous 10% strain results in L-type calcium channel activity leading to increased intracellular calcium concentrations which may be responsible for inducing conformational changes in the cysteine protease calpain, leading to its activation. Strain-mediated apoptosis is prevented when cells are strained in the presence of a calpain inhibitor, implicating these proteases in the apoptotic response of MSCs to tensile strain. Calpain has also been associated with JNK activity in response to 10% tensile strain"
"Mechanotransduction signalling pathways and MSC fate decisions in response to mechanical strain. Integrin receptors link the cell membrane to the ECM. Upon receptor ligation, integrins cluster within the membrane and form focal adhesion complexes leading to actin polymerisation via talin, vinculin (VCL) and paxilin (PXN) recruitment. Downstream of these events the key integrin signal mediator, FAK, is activated leading to GTPase Rho, MAPK and ERK 1/2 activation. Stretch-activated cation channels stimulated by mechanical strain activate PI3K and p38 leading to phosphorylation of p53. Application of 2.5% mechanical strain induces MSC differentiation via these pathways. Conversely, apoptotic pathways are upregulated in response to ≥7.5% mechanical strain. Upregulation of p38 and subsequent phosphorylation of p53, in conjunction with cytochrome-c release from the mitochondria, leads to caspase-3 activation via holoenzyme formation of Apaf-1 and caspase-9. Activation of voltage-activated calcium channels increases intracellular calcium concentration, which alters calpain conformation. This calpain activation is associated with p38 phosphorylation leading to cellular apoptosis"
The external mechanical environment can override the influence of local substrate in determining stem cell fate.
Mesenchymal Stem Cells: Role of Mechanical Strain in Promoting Apoptosis and Differentiation
"In contrast to necrotic cell death, apoptosis is an ordered process that does not trigger a pronounced inflammatory response in the surrounding tissue, due in part to the maintenance of an intact plasma membrane"
"the apoptotic process occurred as an essential step in normal ontogenesis indicating that apoptosis is an inherently programmed pathway responsible for determining cellular fate and acts as a counterbalance to mitosis thus maintaining homeostasis in the organism"
"Stretch-activated cation channels (SACCs) are understood to be involved in mechanotransduction and the presence of these receptors has been identified on osteocytes where they act as mechanical transducers. Blocking SACCs with gadolinium chloride does not protect against strain-induced apoptosis, indicating that these channels are not involved in the upregulation of apoptotic pathways in MSCs. However, in a separate study investigating the effect of 2.5% continuous strain on MSC differentiation, upregulation of collagen type I is observed. This induction of collagen type I is decreased when SACCs are blocked, highlighting a role for these receptors in the strain-induced expression of bone-related proteins"
"Mechanical stress is also thought to elongate the cell membrane which can result in activation of p38 MAPKs and production of ROS. Both of these signals converge at the level of p53. The p38 MAPK pathway directly phosphorylates p53, while ROS production causes oxidative DNA damage. Indirect activation of p53 by p38 MAPKs may occur by regulation of oxidase activation whereby p53 might be involved in a positive feedback loop by stimulating free radical generation itself. Expression of the pro-apoptotic Bax protein and mitochondrial dysfunction also mediate apoptosis following mechanical stress. Strain-induced apoptosis has also been observed in MSCs as a result of signalling via L-type calcium channels, calpain activity and JNK activation. Application of a continuous 10% strain results in L-type calcium channel activity leading to increased intracellular calcium concentrations which may be responsible for inducing conformational changes in the cysteine protease calpain, leading to its activation. Strain-mediated apoptosis is prevented when cells are strained in the presence of a calpain inhibitor, implicating these proteases in the apoptotic response of MSCs to tensile strain. Calpain has also been associated with JNK activity in response to 10% tensile strain"
"Mechanotransduction signalling pathways and MSC fate decisions in response to mechanical strain. Integrin receptors link the cell membrane to the ECM. Upon receptor ligation, integrins cluster within the membrane and form focal adhesion complexes leading to actin polymerisation via talin, vinculin (VCL) and paxilin (PXN) recruitment. Downstream of these events the key integrin signal mediator, FAK, is activated leading to GTPase Rho, MAPK and ERK 1/2 activation. Stretch-activated cation channels stimulated by mechanical strain activate PI3K and p38 leading to phosphorylation of p53. Application of 2.5% mechanical strain induces MSC differentiation via these pathways. Conversely, apoptotic pathways are upregulated in response to ≥7.5% mechanical strain. Upregulation of p38 and subsequent phosphorylation of p53, in conjunction with cytochrome-c release from the mitochondria, leads to caspase-3 activation via holoenzyme formation of Apaf-1 and caspase-9. Activation of voltage-activated calcium channels increases intracellular calcium concentration, which alters calpain conformation. This calpain activation is associated with p38 phosphorylation leading to cellular apoptosis"
The external mechanical environment can override the influence of local substrate in determining stem cell fate.
"Bone marrow derived mesenchymal stem cells (MSCs) were seeded in agarose and fibrin hydrogels and subjected to dynamic compression in the presence of different concentrations of TGF-β3. Markers of chondrogenic, myogenic and endochondral differentiation were assessed. MSCs embedded within agarose hydrogels adopted a spherical cell morphology, while cells directly adhered to the fibrin matrix and took on a spread morphology. Free-swelling agarose constructs stained positively for chondrogenic markers, with MSCs appearing to progress towards terminal differentiation as indicated by mineral staining. MSC seeded fibrin constructs progressed along an alternative myogenic pathway in long-term free-swelling culture. Dynamic compression suppressed differentiation towards any investigated lineage in both fibrin and agarose hydrogels in the short-term. Given that fibrin clots have been shown to support a chondrogenic phenotype in vivo within mechanically loaded joint defect environments, we next explored the influence of long term (42 days) dynamic compression on MSC differentiation{so you might not get results with LSJL for 42 days}. Mechanical signals generated by this extrinsic loading ultimately governed MSC fate, directing MSCs along a chondrogenic pathway as opposed to the default myogenic phenotype supported within unloaded fibrin clots. In conclusion, this study demonstrates that external cues such as the mechanical environment can override the influence specific substrates, scaffolds or hydrogels have on determining mesenchymal stem cell fate."
The emergence of mechanoregulated endochondral ossification in evolution.
The emergence of mechanoregulated endochondral ossification in evolution.
"The emergence of mechanosensitive genes that trigger endochondral ossification in evolution will stabilise in the population and create a variable mechanoregulated response, if the endochondral ossification process enhances fitness for survival. The fitness of animals in a population is determined by their ability to heal their bones. With the emergence of mechanosensitive genes through evolution enabling skeletal cells to modulate their synthetic activities, novel differentiation pathways such as endochondral ossification could have emerged, which when favoured by natural selection is maintained in a population. Evolutionary forces do not lead to a single optimal mechanoregulated response but that the capacity of endochondral ossification exists with variability in a population."
"low mechanical stimuli promote bone differentiation and high stimuli inhibits differentiation of mesenchymal stem cells to bone cells"
"the fracture callus [is] initially filled with granulation tissue and replaced with bone through endochondral ossification."
"thinner bones have been associated with higher mechanosensitivity and thicker bones with low mechanoresponsiveness"
"Thicker bones would be able to better resist daily loads, but be heavy to carry and less mechanosensitive if subjected to injury. "
Mechanical regulation of chondrogenesis.
"the effects of dynamic compressive loading alone (that is, in the absence of exogenous growth factors) on MSC chondrogenesis appear to be minimal and transient" Referring to the study mentioned, growth factors decrease dramatically at the third week. Supplementary tables with gene expression data are provided that will have to be analyzed. The study does state however "Despite
the absence of TGF-β3, by week 6, a subset of MSCs in CM- had undergone chondrogenesis to a limited extent, depositing a small amount of type II collagen in the immediate pericellular space" And gene expression decreases in both the chondrogenic plus and minus mediums at the third week.
"The chondrogenic effects of loading on MSCs in the absence of growth factors also appear to be transient. For example, while loading in the absence of growth factors increased Col2α1 and aggrecan gene expression after 1 and 2 weeks of loading, expression of these chondrogenic markers returned to baseline levels after an additional week of continued loading"
"Joint loading leads to complex tissue strains, including components of compression, tension, and shear, producing direct cellular and nuclear deformation. indirect biophysical factors are also generated as a result of the exudation of interstitial water and ions from cartilage, including streaming potentials, changes in local pH and osmolarity, and hydrostatic pressure"
" the MSC response to hydrostatic loading does not require a preculture period"
"with TGFβ supplementation, while 0.1 MPa was sufficient to increase Sox9 expression, upregulation of Col2α1 expression only occurred with loading at 10 MPa"
" in the absence of exogenous TGFβ, hydrostatic pressure increased expression and secretion of TGFβ1, as well as the phosphorylation of Smad2/3 and p38 mitogen-activated protein kinase"
"Mechanical loading of the cartilage layer results in large gradients in hydrostatic pressure, which subsequently induce flow of the interstitial fluid within the extracellular matrix. One way that mechanical loading is predicted to enhance tissue maturation is through this flow-mediated nutrient and growth factor exchange, as well as through physical activation of growth factors. Loading may also influence tissue maturation through direct transduction of fluid shear stress across the cellular membrane."
"the TRPV4 ion channel has been identified as the major sensor of osmolarity in chondrocytes, and activation of this channel leads to an influx of calcium ions."
Mechanical regulation of chondrogenesis.
"the effects of dynamic compressive loading alone (that is, in the absence of exogenous growth factors) on MSC chondrogenesis appear to be minimal and transient" Referring to the study mentioned, growth factors decrease dramatically at the third week. Supplementary tables with gene expression data are provided that will have to be analyzed. The study does state however "Despite
the absence of TGF-β3, by week 6, a subset of MSCs in CM- had undergone chondrogenesis to a limited extent, depositing a small amount of type II collagen in the immediate pericellular space" And gene expression decreases in both the chondrogenic plus and minus mediums at the third week.
"The chondrogenic effects of loading on MSCs in the absence of growth factors also appear to be transient. For example, while loading in the absence of growth factors increased Col2α1 and aggrecan gene expression after 1 and 2 weeks of loading, expression of these chondrogenic markers returned to baseline levels after an additional week of continued loading"
"Joint loading leads to complex tissue strains, including components of compression, tension, and shear, producing direct cellular and nuclear deformation. indirect biophysical factors are also generated as a result of the exudation of interstitial water and ions from cartilage, including streaming potentials, changes in local pH and osmolarity, and hydrostatic pressure"
" the MSC response to hydrostatic loading does not require a preculture period"
"with TGFβ supplementation, while 0.1 MPa was sufficient to increase Sox9 expression, upregulation of Col2α1 expression only occurred with loading at 10 MPa"
" in the absence of exogenous TGFβ, hydrostatic pressure increased expression and secretion of TGFβ1, as well as the phosphorylation of Smad2/3 and p38 mitogen-activated protein kinase"
"Mechanical loading of the cartilage layer results in large gradients in hydrostatic pressure, which subsequently induce flow of the interstitial fluid within the extracellular matrix. One way that mechanical loading is predicted to enhance tissue maturation is through this flow-mediated nutrient and growth factor exchange, as well as through physical activation of growth factors. Loading may also influence tissue maturation through direct transduction of fluid shear stress across the cellular membrane."
"the TRPV4 ion channel has been identified as the major sensor of osmolarity in chondrocytes, and activation of this channel leads to an influx of calcium ions."
Monday, September 5, 2011
Height Increase by inhibiting HSP90?
The effect of inhibition of heat-shock proteins on thiram-induced tibial dyschondroplasia.
"increased Col I was detected in the thiram-fed chicken growth plate on days 4, 9, 16 and 23 or around the TD lesion at the 16th and 23rd days."
"NADH DH, cytochrome C oxidase (COX) and ENO1 are important enzymes for transmitting electrons in the mitochondrial respiratory chain and in glucose metabolism. Thiram may oxidize cellular thiol-group-containing compounds, resulting in cellular injury and death by interfering with the –SH group(s) of numerous critical proteins and enzymes"
According to Genistein effects on growth and cell cycle of Candida albicans., Genistein increases HSP90 levels in plants.
"Tibial dyschondroplasia (TD)[growth plates that do not ossify] is a skeletal abnormality. Thiram-induced TD is characterized by enlarged, unvascularized growth plates, low levels of the vascular endothelial growth factor receptor Flk-1, abnormal chondrocyte differentiation, and lameness. Heat-shock protein 90 (Hsp90) [is inolved] in chondrocyte differentiation and growth-plate vascularization. Inhibition of Hsp90 activity in thiram-induced TD resulted in increased Flk-1 levels, re-instated normal growth-plate angiogenesis and morphology, and abrogated lameness. We evaluated the efficacy of various concentrations of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90 activity, in preventing growth-plate histopathology and lameness in TD-affected chicks. Low doses of 17-DMAG (2 injections, each of 100 or 300 μg) did not prevent TD development even though Flk-1 levels were restored, which suggests that Flk-1 is not the only rate-limiting factor in growth-plate angiogenesis. High doses of 17-DMAG (2 injections, each of 600 or 900 μg) prevented BW loss, decreased the TD score, reduced lesion width, restored proper chondrocyte differentiation, increased blood vessel invasion, and eliminated lameness. To assess the specificity of Hsp90, we evaluated the efficacy of the flavonoid quercetin, an inhibitor of Hsp70 synthesis, in preventing TD development; it decreased Hsp70 levels but not those of Hsp90 in the control growth plates and prevented upregulation of Hsp70 in the TD-affected growth plates. Dietary quercetin (at 100 or 500 ppm) did not prevent the hypoxia that is characteristic of the TD-affected growth plate or development of thiram-induced TD and lameness. In contrast to the anti-angiogenic effect of 17-DMAG observed in mammals, inhibition of Hsp90 activity in the unvascularized TD-affected growth plates resulted in activation of the angiogenic switch and restored normal growth-plate morphology."
Couldn't get full study which is unfortunate as it mentions quercetin as an HSP70.
Couldn't get full study which is unfortunate as it mentions quercetin as an HSP70.
"Hsp90β is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation.
Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1β or TNF-α) and nitric oxide donors (NOC-12 or SNP). For Hsp90β inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out.
Hsp90β was found to be increased by proinflammatory cytokines. Inhibition of Hsp90β by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1β in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90β. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes."
"the proinflammatory cytokine IL-1β acts as a positive modulator of Hsp90β abundance"
"Hsp90 [interacts with] glucocorticoid receptors, Akt/Protein kinase B and Raf-1, the tumor suppressor protein p53, and NOS family members"
"silencing Hsp90β significantly increased MMP-13"
"Hsp90 inhibition (of both α and β forms) blocked IL-1β-induced up-regulation of MMP-13 in equine articular chondrocytes"
"Thiram-induced tibial dyschondroplasia (TD) and vitamin-D deficiency rickets are avian bone disorders of different etiologies characterized by abnormal chondrocyte differentiation, enlarged and unvascularized growth plates, and lameness. Heat-shock protein 90 (Hsp90) is a proangiogenic factor in mammalian tissues and in tumors; therefore, Hsp90 inhibitors were developed as antiangiogenic factors. Administration of the Hsp90 inhibitor to TD- and rickets-afflicted chicks at the time of induction resulted in reduction in growth-plate size and, contrary to its antiangiogenic effect in tumors, a major invasion of blood vessels occurred in the growth plates. This was the result of upregulation of the VEGF receptor Flk-1, the major rate-limiting factor of vascularization in TD and rickets. In addition, the abnormal chondrocyte differentiation, as characterized by collagen type II expression and alkaline phosphatase activity, and the changes in hypoxia-inducible factor-1α (HIF-1α) in both disorders were restored. All these changes resulted in prevention of lameness. Inhibition of Hsp90 activity reduced growth-plate size, increased vascularization, and mitigated lameness also in TD chicks with established lesions. In contrast to the antiangiogenic effect of Hsp90 inhibitors observed in mammals, inhibition of Hsp90 activity in the unvascularized TD- and rickets-afflicted chicks resulted in activation of the angiogenic switch and reinstated normal growth-plate morphology."
HSP90 stimulation of NO pathway may be what increases growth plate size.
"HIF-1α is one of the major client proteins of heat-shock protein 90 (Hsp90), and it is required for the functioning and the rapid hypoxic stabilization of HIF-1α, which otherwise is degraded by the ubiquitin-proteasome protein system. Hsp90 is implicated in angiogenesis by affecting the VEGF/VEGF-receptor system at various levels"
HSP 90 is induced by high hydrostatic pressure.
Specific induction of heat shock protein 90beta by high hydrostatic pressure.
"In chondrocytes, a low-amplitude intermittent hydrostatic pressure induces production of extracellular matrix molecules, while high hydrostatic pressure inhibits it. High pressure increases cellular heat shock protein 70 level in a number of cell types on account of increased stabilisation of the heat shock protein 70 mRNA. In our experiments, only bovine primary chondrocytes, but not an immortalized chondrocytic cell line, could resist the induction of the stress response in the presence of continuous 30 MPa hydrostatic pressure. We have recently shown that protein synthesis is required for the stabilization. According to two-dimensional gel electrophoresis the synthesis of heat shock protein 90 was also increased in a chondrocytic cell line and in HeLa cells, and mass spectrometric analysis suggested that the induction was rather due to increase in heat shock protein 90beta than in heat shock protein 90alpha. The stress response was rather intense in HeLa cells, therefore, we investigated the effect of continuous 30 MPa hydrostatic pressure on the expression of the two heat shock protein 90 genes in HeLa cells using Northern and Western blot analyses. Heat shock protein 90beta mRNA level increased within 6 hours of exposure to 30 MPa hydrostatic pressure, while hsp90alpha level remained stable. At protein level there was a clear increase in the heat shock protein 90beta/heat shock protein 90alpha ratio, too."
The intracellular Ca(2+)-pump inhibitors thapsigargin and cyclopiazonic acid induce stress proteins in mammalian chondrocytes., states HSP90 can also be induced by inhitibion of the calcium pump.
Identification of differentially expressed genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia.
"Tibial dyschondroplasia (TD) is characterized by expansion of the proximal growth plates of the tibiotarsus that fail to form bone, lack blood vessels, and contain non-viable cells. Thiram (a carbamate pesticide), when fed to young broiler chicks, induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by selecting and identifying the genes differentially expressed in the TD growth plate of broiler chickens. Broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 96 h to induce TD (thiram-fed). mRNA was purified from the growth plates of control and thiram-fed broilers. Forward and reverse-subtracted cDNA libraries were generated by suppression subtractive hybridization technology. Ten selected genes from cDNA libraries were identified by real-time quantitative polymerase chain reaction. All were differentially expressed in TD growth plates. The levels of collagen type X (Col X){up}, pro-alpha-1 collagen type I (Col I alpha1){up}, collagen type IX (Col IX), NADH dehydrogenase (NADH DH), cytochrome C oxidase subunit III (COX III), enolase 1, alpha (ENO1){down}, carbonic anhydrase II (CA2) and heat shock protein 90 (Hsp90) mRNA transcripts were up-regulated, while the expression levels of Matrilin 3 (MATN3){up} and chondromodulin-I (ChM-I) were down-regulated. Col I and Hsp90 were detected by immunohistochemistry at different stages. Given that these genes are involved in matrix formation, endochondral ossification, developmental regulation, electron transport in the mitochondrial respiratory chain and vascularization."
Identification of differentially expressed genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia.
"Tibial dyschondroplasia (TD) is characterized by expansion of the proximal growth plates of the tibiotarsus that fail to form bone, lack blood vessels, and contain non-viable cells. Thiram (a carbamate pesticide), when fed to young broiler chicks, induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by selecting and identifying the genes differentially expressed in the TD growth plate of broiler chickens. Broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 96 h to induce TD (thiram-fed). mRNA was purified from the growth plates of control and thiram-fed broilers. Forward and reverse-subtracted cDNA libraries were generated by suppression subtractive hybridization technology. Ten selected genes from cDNA libraries were identified by real-time quantitative polymerase chain reaction. All were differentially expressed in TD growth plates. The levels of collagen type X (Col X){up}, pro-alpha-1 collagen type I (Col I alpha1){up}, collagen type IX (Col IX), NADH dehydrogenase (NADH DH), cytochrome C oxidase subunit III (COX III), enolase 1, alpha (ENO1){down}, carbonic anhydrase II (CA2) and heat shock protein 90 (Hsp90) mRNA transcripts were up-regulated, while the expression levels of Matrilin 3 (MATN3){up} and chondromodulin-I (ChM-I) were down-regulated. Col I and Hsp90 were detected by immunohistochemistry at different stages. Given that these genes are involved in matrix formation, endochondral ossification, developmental regulation, electron transport in the mitochondrial respiratory chain and vascularization."
"increased Col I was detected in the thiram-fed chicken growth plate on days 4, 9, 16 and 23 or around the TD lesion at the 16th and 23rd days."
"NADH DH, cytochrome C oxidase (COX) and ENO1 are important enzymes for transmitting electrons in the mitochondrial respiratory chain and in glucose metabolism. Thiram may oxidize cellular thiol-group-containing compounds, resulting in cellular injury and death by interfering with the –SH group(s) of numerous critical proteins and enzymes"
According to Genistein effects on growth and cell cycle of Candida albicans., Genistein increases HSP90 levels in plants.
I think the best thing is to inhibit HSP90 and to stimulate the CNP pathway over the NO pathway.