In previous articles, I've talked about factors that affect chondrocyte proliferation. There are tons of genes whose expression we can alter by mechanical loading and cells we can transport to areas that could increase growth(like stem cells or IGF-1) by increasing fluid flow in the bone. Long bone grows mainly as a result of chondrogenesis. Are there any other stages of chondrogenesis aside from chondrocyte proliferation or differentiation that we can manipulate to make ourselves taller?
Identification of Five Developmental Processes during Chondrogenic Differentiation of Embryonic Stem Cells.
"ESCs were differentiated into the chondrocyte lineage, forming small cartilaginous aggregates in suspension. Differentiated ESCs showed that chondrogenesis was typically characterized by five overlapping stages. During the first stage, cell condensation and aggregate formation was observed. The second stage was characterized by differentiation into chondrocytes and fibril scaffold formation within spherical aggregates. Deposition of cartilaginous extracellular matrix and cartilage formation were hallmarks of the third stage[Chondrocytes probably deposit this matrix] Apoptosis of chondrocytes, hypertrophy and/or degradation of cartilage occurred during the fourth stage. Finally, during the fifth stage, bone replacement with membranous calcified tissues took place.
ESCs show the chondrogenic differentiation pathway from the pluripotent stem cell to terminal skeletogenesis through these five stages in vitro. During each stage, morphological changes acquired in preceding stages played an important role in further development as a scaffold or template in subsequent stages."
Stage four and five of chondrogenic development are well known as part of endochondral ossifcation. Could the deposition of extracellular matrix and cartilage formation be important in making yourself taller? Chondrocytes have to die to be invaded by bone cells and extracellular matrix would inhibit that. Although a larger hyaline cartilage layer(growth plate resting zone) could be beneficial for growth. Stem cells tend to take up the characteristics of the surrounding tissues. Stem cells within the hyaline cartilage growth plate line tend to differentiate into chondrocytes so stages one and two should automatically occur once the stem cells arrive in the hyaline cartilage growth plate line.
The balance of WNT and FGF signaling influences mesenchymal stem cell fate during skeletal development
"Craniosynostosis, a developmental disorder resulting from premature closure of the gaps (sutures) between skull bones, can be caused by excessive intramembranous ossification, a type of bone formation that does not involve formation of a cartilage template (chondrogenesis). Endochondral ossification, a type of bone formation that proceeds through a cartilage intermediate, caused by switching the fate of mesenchymal stem cells to chondrocytes, can also result in craniosynostosis. Simultaneous knockout of Axin2, a negative regulator of the WNT-beta-catenin pathway, and decreased activity of fibroblast growth factor (FGF) receptor 1 (FGFR1) in mice induced ectopic chondrogenesis[upregulating Beta-Catenin and downregulating FGFR1 is an interesting way to induce chondrogenesis], leading to abnormal suture morphogenesis and fusion. Genetic analyses revealed that activation of beta-catenin cooperated with FGFR1 to alter the lineage commitment of mesenchymal stem cells to differentiate into chondrocytes, from which cartilage is formed. We showed that the WNT-beta-catenin pathway directly controlled the stem cell population by regulating its renewal and proliferation, and indirectly modulated lineage specification by setting the balance of the FGF and bone morphogenetic protein pathways. This study identifies endochondral ossification as a mechanism of suture closure during development and implicates this process in craniosynostosis."
You know what this study shows? It shows that endochondral ossification(long bone growth) can occur at the fibrocartilage sutures between the bones of the skull. There are fibroblast cells within your synovial joints as well.
Since articular cartilage is like hyaline cartilage, and like this study shows endochondral ossifcation is possible at a lot of joint spaces. Maybe LSJL could work within the articular cartilage and not just the hyaline cartilage but one difference between the articular cartilage and the sutures in the skull is the synovial fluid. So even if articular cartilage began proliferating and differentiating chondrocytes the synovial fluid should provide enough nutrients to prevent the chondrocytes from dying and being invaded by bone cells.
"WNT–β-catenin pathway directly controls the skeletal precursor population by stimulating its renewal and proliferation, and indirectly influences lineage specification by setting the balance of the FGF and bone morphogenetic protein (BMP) pathways[The WNT/Beta-Catenin pathway determines the lineage of mesenchymal stem cells]. Switching the fate of mesenchymal stem cells to chondrocytes is an etiologic mechanism for craniosynostosis in mice[switching the fate of MSCs to chondrocytes causes the fusion of the sutures in the brain, manipulating the WNT/Beta-Catenin can be used to induce chondrogenesis]."
"loss of FGFR1 activity tips the balance toward chondrogenesis."<-inhibiting FGFR1 activity in the bone may be a way to encourage height growth.
"altering β-catenin and FGF signaling still changed the fate of the bone marrow precursors, suggesting that both β-catenin and FGF signaling are important for chondrocyte development in multiple contexts."
There are mechanisms specific within the articular cartilage that prevent endochondral ossification from happening...
Articular chondrocytes secrete PTHrP and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis.
"[During cell-based cartilage regeneration the] unwanted upregulation of hypertrophic markers like alkaline phosphatase (ALP) and collagen-type X [occurs] during in vitro chondrogenesis and formation of instable calcifying cartilage at heterotopic sites[i.e. chondrocytes undergoing endochondral ossification]. In contrast, stable non-mineralizing cartilage is obtained from articular chondrocytes. Aim of this study was to address whether coculture with human articular chondrocytes (HAC) has the capacity to suppress undesired hypertrophy in differentiating MSC.
MSC were differentiated in chondrogenic medium which had or had not been conditioned by parallel chondrocyte pellet cultures, or were mixed in the same pellet with chondrocytes (1:1, 1:2) and cultured for six weeks. Following in vitro differentiation, pellets were transplanted into SCID mice.
The gene expression ratio of COL10A1/COL2A1 and IHH/COL2A1 was significantly reduced by HAC-conditioned medium and less collagen-type X protein was deposited relative to collagen-type II. ALP-activity was significantly lower (p<0.05) in the conditioned-medium-group and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of ALP was dose-dependent and in vivo calcification was fully inhibited. Chondrocytes secreted PTHrP throughout culture while PTHrP was downregulated in favour of IHH upregulation in control MSC after 2-3 weeks of chondrogenesis. Main inhibitory effects seen with HAC-conditioned medium could be reproduced by PTHrP supplementation of unconditioned medium."
So, in articular cartilage there is a method of inhibiting chondrocytes from undergoing endochondral ossification.
Conclusion: Endochondral ossification can occur at almost any cartilage layer(including fibrocartilage) however there are inhibitors of this occurring such as that within articular cartilage at the synovial joints. If you get stem cells to differentiate into chondrocytes, chondrogenesis and thereby endochondral ossifcation will occur.
Are these stem cell derived-chondrocytes different from "natural" chondrocytes?
Differences Between Chondrocytes and Bone Marrow-Derived Chondrogenic Cells.
"In this study, we analyzed the differences between the iMSCs[artificially induced Mesenchymal Stem Cells] and chondrocytes. Human bone marrow-derived MSCs were collected and induced to exhibit the chondrogenic phenotype by culturing the pelleted MSCs in a chemically defined culture medium supplemented with transforming growth factor-beta 1[note that maybe the marrow derived MSCs were not complete chondrocytes as the TGF-Beta 1 alone was not enough to induce full differentiation]. The molecular biological properties of iMSCs and culture-expanded chondrocytes, including their mRNA profiles and surface proteomics, were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry, respectively. The biomechanical properties of iMSCs and native chondrocytes, including their surface topology, adhesion force, and membrane stiffness, were analyzed using atomic force microscopy (AFM). Both iMSCs and chondrocytes presented type II collagen and glycosaminoglycan, whereas only chondrocytes presented type X collagen[Type X collagen is consistent with chondrocyte hypertrophy so derived chondrocytes don't hypertrophy. It's possible though for this to change with other stimulus and the stem cells just differentiated so they may begin expressing type X collagen over time]. Flow cytometric assays showed that the expression of type II collagen and integrin-1 was higher in the chondrocytes than in the iMSCs[integrin-1 mediates attachment so it can be very vital in forming growth plates]. AFM revealed that the MSCs, iMSCs, and chondrocytes greatly differed in their shape. The MSCs were spindle shaped and easily distinguishable from the spherical chondrocytes. The iMSCs appeared round and resembled the spherical chondrocytes; however, the iMSCs were flatter with a central hump of condensed mass and a surrounding thin and broad pleat[so again the iMSCs likely did not completely differentiate into chondrocytes and needed hydrostatic pressure or something to become more like spherical chondrocytes]. The mean adhesion force and mean surface stiffness were significantly lower for the iMSCs (4.54 nN and 0.109 N/m, respectively) than for the chondrocytes (6.86 nN and 0.134 N/m, respectively)."
"Although MSCs are found in various adult tissues, the MSCs from bone marrow have higher potential for chondrogenesis than the MSCs from other sources"<-this since heterotropic ossification can occur in other tissues it is very likely for heterotopic ossification to occur in bone marrow.
It's very likely that the low quality of iMSCs was due to the presence of only one differentiation factor(TGF-Beta1) rather than an inherent malfunction of MSC derived chondrocytes.
JAWS coordinates chondrogenesis and synovial joint positioning.
"We show that mice carrying an insertional mutation in a previously uncharacterized gene, which we have named Jaws (joints abnormal with splitting), die perinatally with striking skeletal defects, including ectopic interphalangeal joints. These ectopic joints develop along the longitudinal axis and persist at birth, suggesting that JAWS is uniquely required for the orientation and consequent positioning of interphalangeal joints within the endochondral skeleton. Jaws mutant mice also exhibit severe chondrodysplasia characterized by delayed and disorganized maturation of growth plate chondrocytes, together with impaired chondroitin sulfation and abnormal metabolism of the chondroitin sulfate proteoglycan aggrecan. Our findings identify JAWS as a key regulator of chondrogenesis and synovial joint positioning required for the restriction of joint formation to discrete stereotyped locations in the embryonic skeleton."
"Jaws−/− chondrocytes lacked the pseudocolumnar organization of their WT counterparts"
"normal short-range but disrupted long-range Ihh signaling in the Jaws−/− growth plate"
Would overexpression of JAWS increase height or is JAWS only needed for proper endochondral ossification?
Effects of overexpression of membrane-bound transferrin-like protein (MTf) on chondrogenic differentiation in Vitro.
Effects of overexpression of membrane-bound transferrin-like protein (MTf) on chondrogenic differentiation in Vitro.
"Membrane-bound transferrin-like protein (MTf) is expressed in parallel with the expression of cartilage-characteristic genes during differentiation of chondrocytes, and the MTf level is much higher in cartilage than in other tissues. To investigate the role of MTf in cartilage, we examined the effects of growth factors on MTf expression in mouse prechondrogenic ATDC5 cells and the effect of MTf overexpression on differentiation of ATDC5 and mouse pluripotent mesenchymal C3H10T1/2 cells. In ATDC5 cultures, bone morphogenetic protein-2 and transforming growth factor-beta as well as insulin induced MTf mRNA expression when these peptides induced chondrogenic differentiation. Forced expression of rabbit MTf in ATDC5 cells induced aggrecan, type II collagen, matrilin-1, type X collagen mRNAs, and cell-shape changes from fibroblastic cells to spherical chondrocytes. Accordingly, the synthesis and accumulation of proteoglycans were higher in MTf-expressing cultures than in control cultures. These effects of MTf overexpression correlated with the MTf protein level on the cell surface and decreased in the presence of anti-MTf antibody. However, the aggrecan mRNA level in the ATDC5 cells overexpressing MTf was lower than that in wild type ATDC5 cells exposed to 10 microg/ml insulin. MTf overexpression in C3H10T1/2 cells also induced aggrecan and/or type II collagen mRNA but not the spherical phenotype[so Mtf could be chondroinductive]."
"BMP-2 elicited the greatest stimulation of MTf "
Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in self-gelling alginate discs reveals novel chondrogenic signature gene clusters.
Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in self-gelling alginate discs reveals novel chondrogenic signature gene clusters.
"We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced[LSJl gene expression was taken at 48 hours]. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. "
"A number of genes encoding proteins with a modifying effect on chondrogenesis were upregulated, including epiphycan (EPYC){upregulated over 6 fold in LSJL}, matrilin 3 (MATN3){upregulated in LSJL}, fibromodulin (FMOD), TIMP metalloproteinase inhibitor 4 (TIMP4), chondroadherin (CHAD), and dermatopontin (DPT){upregulated 3 fold by LSJL}"
Divide, accumulate, differentiate: cell condensation in skeletal development revisited.
"The pre-condensation phase is characterized by expression of Hox genes, growth factors (TGF-beta and BMP-2) and the cell surface proteoglycan receptor, syndecan-1"
"Other molecules, such as versican, syndecan-3 and tenascin, present in low concentrations before condensation, are up-regulated during condensation. Yet other molecules--Hox genes, transcription factors, growth factors (activin, BMP-4 and -5, GDF-5), cell adhesion molecules and proteoglycans--are only expressed during the condensation phase, while the transcription factor Pax-1, fibronectin, hyaluronan and hyaladherin are expressed both during and after condensation"
Divide, accumulate, differentiate: cell condensation in skeletal development revisited.
"The pre-condensation phase is characterized by expression of Hox genes, growth factors (TGF-beta and BMP-2) and the cell surface proteoglycan receptor, syndecan-1"
"Other molecules, such as versican, syndecan-3 and tenascin, present in low concentrations before condensation, are up-regulated during condensation. Yet other molecules--Hox genes, transcription factors, growth factors (activin, BMP-4 and -5, GDF-5), cell adhesion molecules and proteoglycans--are only expressed during the condensation phase, while the transcription factor Pax-1, fibronectin, hyaluronan and hyaladherin are expressed both during and after condensation"
So this furthers the hypothesis that LSJL induces pre-chondrogenic mesenchymal condensation.
"The formation of a condensation is associated with reduction in hyaluronan (hyaluronate, hyaluronic acid) and increase in chondroitin sulphate. Hyaluronan blocks chondrogenesis; removal of hyaluronan permits chondrogenesis;"<-this is contrary to other evidence and LSJL increases both chondroitin and hyaluronan synthesis.
Identification and characterization of chondrogenic progenitor cells in the fascia of postnatal skeletal muscle.
Identification and characterization of chondrogenic progenitor cells in the fascia of postnatal skeletal muscle.
"Intramuscular injection of bone morphogenetic proteins (BMPs) has been shown to induce ectopic bone formation. A chondrogenic phase is typically observed in this process, which suggests that there may exist a chondrogenic subpopulation of cells residing in skeletal muscle. Two prospective cell populations were isolated from rat skeletal muscle: fascia-derived cells (FDCs), extracted from gluteus maximus muscle fascia (epimysium) and muscle-derived cells (MDCs) isolated from the muscle body. Both populations were investigated for their cell surface marker profiles (flowcytometry analysis), proliferation rates as well as their myogenic and chondrogenic potentials. The majority of FDCs expressed mesenchymal stromal cell markers but not endothelial cell markers. FDCs underwent chondrogenic differentiation after BMP4 treatment in vitro. Although MDCs showed chondrogenic potential, they expressed the myogenic cell marker desmin and readily underwent myogenic differentiation in vitro; however, the chondrogenic potential of the MDCs is confounded by the presence of FDC-like cells residing in the muscle perimysium and endomysium. To clarify the role of the muscle-derived myogenic cells in chondrogenesis, mixed pellets with varying ratios of FDCs and L6 myoblasts were formed and studied for chondrogenic potential. the chondrogenic potential of the mixed pellets decreased with the increased ratio of myogenic cells to FDCs supporting the role of FDCs in chondrogenesis."
" fascia tissue is also rich in blood vessels, which implies that these chondrogenic progenitor cells might reside in the ‘stem cell niche’ surrounding the blood vasculature. The blood cell walls may harbor a dormant reserve of chondrogenic progenitors that could be recruited when they receive a BMP4 stimulation signal."
"connective tissue-derived cells (epimysium, perimysium and endomysium) [may be] required for chondrogenesis"
High expression of vimentin(LSJL upregulates Vim) and low expression of desmin may be a more chondrogenic environment.
Metabolic labeling of human bone marrow mesenchymal stem cells for the quantitative analysis of their chondrogenic differentiation.
"The average age of the patients was 70 years (range 65-74 years). The study has been approved by the Ethics Committee of Galicia, and all patients gave their informed consent. Cells were isolated from trabecular bone marrow samples by washing the femoral heads"<-We're trying to induce chondrogenic differentiation in the trabecular bone marrow of the femoral epiphysis!
This study proves that adult epiphyseal bone marrow cells are capable of chondrogenesis.
"chondrogenesis [involves] protein synthesis and turnover, cytoskeleton organization and chaperones/stress response"
Metabolic labeling of human bone marrow mesenchymal stem cells for the quantitative analysis of their chondrogenic differentiation.
"The average age of the patients was 70 years (range 65-74 years). The study has been approved by the Ethics Committee of Galicia, and all patients gave their informed consent. Cells were isolated from trabecular bone marrow samples by washing the femoral heads"<-We're trying to induce chondrogenic differentiation in the trabecular bone marrow of the femoral epiphysis!
This study proves that adult epiphyseal bone marrow cells are capable of chondrogenesis.
"chondrogenesis [involves] protein synthesis and turnover, cytoskeleton organization and chaperones/stress response"
"the cell pellet was resuspended in DMEM medium with 20% FBS and then plated at a seeding density of 1x105 cells/cm2 and incubated at 37ºC in a 5% CO2 atmosphere. After 48 hours, the medium was replaced to remove non-adherent hematopoietic cells."<-Can LSJL induce a similar environment? It can increase the density by laterally compressing the epiphysis.
Genes upregulated at 14 days of chondrogenic differentiation versus day 2 of chondrogenic differentiation of HMBMSC's that are also altered during LSJL:
PTRF
ARPC4(down in LSJL)
VIM
COL6A2(partially up and down in LSJL)
COL6A3
LMNA
Tenascin(LSJL upregulates Tenascin W as TNN which is found in developing bone)
Downregulated:
It's possible to induce/stimulate through microfractures (using ultrasound) the proliferation/differentiation of the connective tissue of the cranial sutures even though it has formed a synostosis and flat bones are fused?
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