Height Increase Pages

Wednesday, December 28, 2011

Grow Taller with Ascorbic Acid(Vitamin C)

Previously, we talked about the potential of vitamins augmenting potential height growth methods.  Now, we find a study that shows the ability of Ascorbic Acid to directly induce chondrogenic differentation.

The cell line used in the study is ATDC5 which is a rat cell line so human chondrocyte cells might have different results.

The mechanism of ascorbic acid-induced differentiation of ATDC5 chondrogenic cells.

"The ATDC5 cell line exhibits a multistep process of chondrogenic differentiation analogous to that observed during endochondral bone formation.  Insulin at high concentrations [can induce ATDC differentiation]. spontaneous differentiation of ATDC5 cells maintained in ascorbic acid-containing α-MEM [occurred]{so it has to be Vitamin C containing MEM alpha, MEM stands for Minimum Essentail Medium a listing of what the contains can be found here, none of the compounds would be anything particularly unusual to find in the body except for something like phenol red which is used more as a measurement tool}. A comparison of the differentiation events in response to high-dose insulin vs. ascorbic acid showed similar expression patterns of key genes, including collagen II, Runx2, Sox9, Indian hedgehog, and collagen X{Col2, Sox9, and Col10 are upregulated by LSJL}.  In contrast to high-dose insulin, which downregulates both IGF-I and insulin receptors, there were only minimal changes in the abundance of these receptors during ascorbic acid-induced differentiation[so ascorbic acid is better than Insulin as it doesn't impact IGF-1 and insulin receptors]. ascorbic acid exposure was associated with ERK activation[so this leads us to believe that activating ERK increases chondrogenic differentiation and may help us to grow taller], and ERK inhibition attenuated ascorbic acid-induced differentiation{so ERK enhances Vitamin C-induced chondrogenic differentiation}. ERK activation [has an inhibitory effect] during IGF-I-induced differentiation{ERK impairs IGF-1 induced chondrogenic differentiation}. Inhibition of collagen formation with a proline analog markedly attenuated the differentiating effect of ascorbic acid on ATDC5 cells. When plates were conditioned with ATDC5 cells exposed to ascorbic acid, ATDC5 cells were able to differentiate in the absence of ascorbic acid. matrix formation early in the differentiation process is essential for ascorbic acid-induced ATDC5 differentiation[so maybe ascorbic acid plays a role in ECM interaction?]. ascorbic acid can promote the differentiation of ATDC5 cells by promoting the formation of collagenous matrix and that matrix formation mediates activation of the ERK signaling pathway{so forming the cartilage matrix is key to early height growth}, which promotes the differentiation program."

"At the growth plate, ascorbic acid deficiency can result in decreased chondrocyte proliferation, impaired matrix synthesis, and a reduction in osteoblast cell number"

"ERK activation can be mediated by the cross-linking of extracellular matrix with integrins. In human chondrocytes, the interaction of collagen II with the β1-integrin receptor has been shown to activate ERK" <-So injecting type II collagen into the bone and increasing serum levels of the Beta1-integrin receptor may be one way to grow taller.

"Given that ascorbic acid is required for the formation of collagen triple helices, the ability of ascorbic acid to induce ATDC5 cell differentiation [may depend] on its ability to promote synthesis of collagen matrix."<-So amounts of ascorbic acid greater than that required to form collagen triple helices would be useless.

"Ascorbic acid stimulates procollagen hydroxylation and processing and is required for collagen fibril assembly and collagen secretion"<-collagen hydroxylation is the addition of hydroxyl groups to the amino acids proline or lysine.  Supranormal levels of ascorbic acid, proline, and lysine could stimulation supranormal collagen fibril assembly and collagen secretion.

"The rate-limiting step in this overall process is the hydroxylation and secretion of unprocessed procollagen chains that accumulate in the endoplasmic reticulum of ascorbic acid-deficient cells"<-so you have to have enough Vitamin C such that no cell is definition to maximize height growth.

"collagen II hydrogels could stimulate the differentiation of bone marrow mesenchymal stem cells in the absence of growth factors."<-Thus injecting type II collagen hydrogels into your bone marrow could make you taller.

Since the compounds of Vitamin C and the other compounds of the medium are so readily available in the body it's likely to be a problem with the stem cells themselves that's inhibiting chondrogenic differentiation.

Thus study was done on adult stem cells:

Effects of osteogenic differentiation inducers on in vitro expanded adult mesenchymal stromalcells.

"MSC were isolated from the bone marrow of donors (46-73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: a-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and ß-glycerophosphate (C4).
MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation[we don't want Runx-2 and type I collagen as that is osteogenic while we want chondrogenic so we would not want Beta-glyerophosphate(dexamethasone has been found to have potential height decreasing effects)], i.e. from C1 to C4, while ALP and osteocalcin were differently regulated."

Chondrogenic markers were not measured in this study.  However, using a different cell line produced very different results in regards to Vitamin C.

Here's a study that shows that age doesn't affect stem cell differentiation however:

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent.

"Ex vivo cultures of primary human MSCs [were taken] from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, [levels of] the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, [and] Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH gene [levels] characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed. Progenitor cells could be expanded in the long term from all bone marrow donations. MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels.  The highest ALP activity was found in probands of the age group >65 years. we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected [and] no significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype[This page lists the micromass protocol for MSCs to generate chondrocytes].  Cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Donor age does not affect osteogenic differentiation potential."

"Expanded MSCs were released by trypsin treatment, counted and resuspended at a density of 1 × 106. The medium was changed to 500 μl DMEM with 10% FCS, 1% antibiotic mix (penicillin/streptomycin), 37.5 μg/ml (100 μmol/l) ascorbate-2 phosphate, and 10–7 mol/l dexamethasone. Pellet cultures were incubated with 10 ng/ml recombinant human transforming growth factor-β3 during chondrogenesis. After 3 weeks the samples were used."

"Pellet cultures of MSCs resulted in the formation of dense nodules consistent with chondrogenic differentiation."<-So forming a pellet culture is most important in inducing chondrogenesis. Can hydrostatic pressure like that induced by LSJL result in the MSC formation of pellet cultures?

"CD9{down in LSJL}, a mediator of tetraspanin protein-driven cellular adhesion, invasion, and fusion, was verified in mesenchymal cells during expansion "

So Vitamin C enhances both chondrogenic and osteogenic differentiation. Age does not seem to affect whether differentiation is headed towards an osteogenic or chondrogenic path but rather how the stem cells are cultured. Hydrostatic pressure may change the culture within the bone marrow to be more chondrogenic.

Ascorbate-enhanced chondrogenesis of ATDC5 cells.

"The ATDC5 cell line exhibits the multistep chondrogenic differentiation observed during endochondral bone formation. However, it takes up to two months to complete the process of cell expansion, insulin addition to promote differentiation and further changes in culture conditions effectively to induce hypertrophy.  By adding ascorbate, the prechondrogenic proliferation phase was shortened from 21 to 7 days, with production of cartilaginous nodules during the chondrogenic phase, after insulin addition, that were greater in number and larger in size.  [There was] much greater matrix elaboration and the mRNA expression of sox9, aggrecan and collagen type II were all significantly increased earlier and to a much higher degree when compared with controls. [Hypertrophic indicators] Col10a1, Runx2 and Mmp13 were all induced within 7-10 days. addition of ascorbate to ATDC5 cultures shortened the prechondrogenic proliferation phase, produced earlier chondrogenic differentiation, heightened gene expression and robust hypertrophic differentiation."

Increasing serum levels of Vitamin C, proline, and lysine may increase height but there may be negative feed mechanisms and points of diminishing returns.

Effects of both vitamin C and mechanical stimulation on improving the mechanical characteristics of regenerated cartilage.

"[We studied] vitamin C (VC) and mechanical stimulation on development of the extracellular matrix (ECM) and improvement in mechanical properties of a chondrocyte-agarose construct in a regenerating tissue disease model of hyaline cartilage. We used primary bovine chondrocytes and two types of VC, ascorbic acid (AsA) as an acidic form and ascorbic acid 2-phosphate (A2P) as a non-acidic form, and applied uniaxial compressive strain to the tissue model using a purpose-built bioreactor. When added to the medium in free-swelling culture conditions, A2P downregulated development of ECM and suppressed improvement of the tangent modulus more than AsA.  Application of mechanical stimulation to the construct both increased the tangent modulus more than the free-swelling group containing A2P and enhanced the ECM network of inner tissue to levels nearly as high as the free-swelling group containing AsA. Mechanical stimulation and strain appears to enhance the supply of nutrients and improve the synthesis of ECM via mechanotransduction pathways of chondrocytes. Mechanical stimulation is necessary for homogenous development of ECM in a cell-associated construct with a view to implantation of a large-sized articular cartilage defect."

"Even though A2P has no physiological activity, it can nevertheless reproduce the effect of VC activity after dephosphorylation by an alkaline phosphatase (ALP). Dephosphorylated A2P, or AsA, penetrates chondrocytes through a VC transporter, chiefly the sodium-dependent VC transporter 2 (SVCT2), and supports collagen synthesis as a cofactor in the rough endoplasmic reticulum. In free-swelling culture conditions, we only observed a small number of collagen molecules distributed in the construct. We also observed that the tangent moduli of both A2P dose groups were lower than the moduli of AsA(2.2). We must also consider the reaction rates of both dephosphorylation of A2P by ALP and the transport of AsA into the cytosol via SVCT2."

Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures.

"Most strategies [for articular cartilage regeneration] require ascorbate supplementation to promote matrix formation by isolated chondrocytes. We evaluate ascorbate forms and concentrations on in vitro cartilage formation in porcine chondrocyte high-density pellet cultures. l-ascorbate[AsA], sodium l-ascorbate[NaAs], and l-ascorbate-2-phosphate[AsAP] were administered in 100 microM, 200 microM, and 400 microM in the culture medium over 16 days. Pellet thickness increased independently from the supplemented ascorbate form and concentration. Hydroxyproline content increased as well, but here, medium concentration of AsAP and low concentration of AsA showed a more pronounced effect. Proteoglycan and collagen formation could be proven in all supplemented cultures. Non-supplemented cultures, however, showed no stable matrix formation at all. Effects on the gene expression pattern of cartilage marker genes (type I and type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP)) were studied compared to non-supplemented control cultures. Expression level of cartilage marker genes was elevated in all cultures showing that dedifferentiation of chondrocytes could be prevented. All supplementations caused a similar effect except for low concentration of AsA, which resulted in an even higher expression level of all marker genes.  We could not detect a pronounced difference between ascorbate and its derivates as well as between the different concentrations."

"Ascorbate has a transcriptional effect on cartilage formation promoting type II collagen and prolyl-4-hydroxylase gene expression. At the post-transcriptional level, ascorbate is responsible for the reduction of iron to its ferrous state. This reaction is important for the hydroxylation of proline and lysine. Latter hydroxy amino acids are specific for type II collagens. Their non-covalent bonds support collagen triple helix and fibril formation. Collagen secretion from the cells is promoted by ascorbate-dependent hydroxylation within the endoplasmatic reticulum"

"Standard concentration of [Vitamin C is] about 280 μM"

"NaAs has the advantage of being non-acidic, while AsA is acidic in aqueous solutions such as culture medium. AsAP is not only non-acidic, but also characterized by a longer half-life in aqueous solutions compared to the other derivatives. It is internalized, dephosphorylated, and highly concentrated in the aqueous phase of the cells. AsA, NaAs and AsAP were administered in 100 μM, 200 μM, and 400 μM over 16 days. "

"The best results of 5 μg/mg were achieved by supplementation with 100 μM of AsA"

1 comment:

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