Tuesday, December 28, 2010

Height Gaining with Hyaluronic Acid

In the article on BMP-2, we learned that Hyaluronic Acid was very helpful in promoting bone formation in bone autographs.  Hyaluronic Acid helps with cell migration and proliferation.  How can Hyaluronic Acid help with height growth?

Hyaluronan is essential for the expansion of the cranial base growth plates. 

"Exquisite control of chondrocyte function in the zone of hypertrophy results in expansive growth of cartilaginous growth plates[The hypertrophic phase is essential for height growth]. We hypothesize that hyaluronan-mediated hydrostatic pressure causes lacunae expansion in the zone of hypertrophy[hyaluronan induces hydrostatic pressure like LSJL which causes the expansion of lacunae, normally lacunae is associated with osteocytes but they can contain chondrocytes as well]; an important mechanism in cartilaginous growth plate and associated skeletal expansion. The role of hyaluronan and CD44 in this mechanism was studied using organ culture of the bipolar cranial base synchondroses. Hyaluronan was present in the hypertrophic zones, pericellular to the hypertrophic chondrocytes, while no hyaluronan was detected in the resting, proliferating and maturing zones. This localization of hyaluronan was associated with increased lacunae size, suggesting that chondrocytes deposit and retain pericellular hyaluronan as they mature. In comparison, Toluidine Blue staining was associated with the territorial matrix. Hyaluronidase, the hyaluronan-degrading enzyme, and CD44, the receptor for hyaluronan which also participates in the uptake and degradation of hyaluronan, were co-localized within the zone of ossification[There are ways to degrade hyaluronic Acid in the zone of ossification]. This pattern of expression suggests that cells in the early zone of ossification internalize and degrade hyaluronan through a CD44-mediated mechanism. Treatment of the cultured segments with either Streptomyces hyaluronidase or hyaluronan hexasaccharides inhibited lacunae expansion. These observations demonstrate that hyaluronan-mediated mechanisms play an important role in controlling normal skeletal lengthening." 

So maybe a CD44 inhibitor(which inhibits Hyaluronan by being a receptor for it) can increase height by extending the hypertrophic phase.  Hyaluronan induces hydrostatic pressure which is what we're trying to do with LSJL via lateral compression however there was no hyaluronan in the resting zone(the stem cell zone).  We'll have to study to see what the effects of hyaluronan are on Mesenchymal Stem Cells. 

Type II collagen-hyaluronan hydrogel--a step towards a scaffold for intervertebral disc tissue engineering. 

"The specific aim of this study was to optimise a composite hydrogel composed of type II collagen[cartilage] and hyaluronic acid (HA) as a carrier for mesenchymal stem cells. Hydrogel stabilisation was achieved by means of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking. Optimal hydrogel properties were determined by investigating different concentrations of EDC (8 mM, 24 mM and 48 mM). Stable hydrogels were obtained independent of the concentration of carbodiimide used. The hydrogels cross-linked by the lowest concentration of EDC (8 mM) demonstrated high swelling properties. Additionally, improved proliferation of seeded rat mesenchymal stem cells (rMSCs) and hydrogel stability levels in culture were observed with this 8 mM cross-linked hydrogel. Results from this study indicate that EDC/NHS (8 mM) cross-linked type II collagen/HA hydrogel was capable of supporting viability of rMSCs, and furthermore their differentiation into a chondrogenic lineage." 

Unfortunately this study involved Type II collagen which is hyaline cartilage which isn't available in post-pubertal growth plates.  Injection of stem cells with HA acid into active growth plates may be a way to increase height though.  It seems like hyaluronic acid may be one of the differences between AC and growth plate chondrocytes as HA is absorbed before the ossification phase.

Chondrocytes from patients with osteoarthritis express typical extracellular matrix molecules once grown onto a three-dimensional hyaluronan-based scaffold. 

"Chondrocytes from cartilage of [osteoarthritic] patients and from healthy donors were used to evaluate the expression of some extracellular matrix molecules once these cells were grown onto a hyaluronan-based scaffold already used in clinical practice. Constructs were analyzed by immunohistochemical and real-time PCR analyses. Chondrocytes from control and patients with OA cartilages expressed the same extracellular matrix molecules even if at different amount. These differences, which were appreciable both at protein and molecular levels, were not evident once the cells were grown onto Hyaff-11 scaffold. In this experimental culture condition, the cells derived from control and patients with OA showed a significant increase of collagen type II, Sox-9, and aggrecan and a decrease of collagen type I[so chondrogenic genes increased whereas osteogenic genes decreased(Collagen Type I which is bone)] compared with chondrocytes grown in monolayer. On the other hand, MMPs were downregulated in both the cell types evaluated by the specific action of TIMP-1 which was highly expressed at molecular and protein levels in the two groups. The growth of chondrocytes onto Hyaff-11 membrane seems to erase the differences between the cells derived from normal and OA cartilages. The cells seem to benefit of the "hyaluronan" presence which is able to create an ideal environment for the expression of cartilage genes even in absence of specific growth factors. This is of particular relevance hypothesizing the use of tissue engineering therapeutical approach also in patients with OA." 

So CD44 may be the key difference between growth plate chondrocytes and articular cartilage chondrocytes. 

Intra-articular injection of hyaluronan restores the aberrant expression of matrix metalloproteinase-13 in osteoarthritic subchondral bone. 

"We investigated the effects of intra-articular injection of hyaluronan (IAI-HA) on subchondral bone in rabbit OA model. OA was induced by anterior cruciate ligament transection, with some rabbits receiving IAI-HA. OA was graded morphologically, and expression of mRNA was assessed by real-time RT-PCR. Tissue sections were stained with hyaluronan-binding protein, and penetration of fluorescent hyaluronan was assessed. The in vitro inhibitory effect of hyaluronan on MMP-13 was analyzed in human osteoarthritic subchondral bone osteoblasts (OA Ob) by real-time RT-PCR and ELISA. Binding of hyaluronan to OA Ob via CD44 was assessed by immunofluorescence cytochemistry. Expression of MMP-13 and IL-6 mRNA in cartilage and subchondral bone, and morphological OA grade, increased over time. IAI-HA ameliorated the OA grade and selectively suppressed MMP-13 mRNA in subchondral bone. IAI-HA[Hyaluronan] enhanced the hyaluronan staining of subchondral bone marrow cells and osteocyte lacunae. Fluorescence was observed in the subchondral bone marrow space. In OA Ob, hyaluronan reduced the expression and production of MMP-13, and anti-CD44 antibody blocked hyaluronan binding to OA Ob. These findings indicate that regulation of MMP-13 in subchondral bone may be a critical mechanism during IAI-HA." 

Hyaluronic Acid reduces MMP-13 expression which means that HA can increase height gaining.  Lack of CD44 in articular cartilage is what likely blocks ossification of articular cartilage as shown by the anti-CD44 antibodies.  So if you inject HA+Type II collagen you may just form new growth plates or you can just perform LSJL and get the hydrostatic pressure that way.  

Hyaluronic Acid at a high Molecular Weight may counteract cAMP expression which inhibits osteoblast adhesion which could affect an osteoblast based height increase method. 

Hyaluronic acid reverses the abnormal synthetic activity of human osteoarthritic subchondral bone osteoblasts.

"It is becoming recognized that the extracellular matrix influences the metabolism of cells both in vivo and in vitro and can modify their responses to external stimuli[growth plate chondrocytes have this matrix and thus manipulating this matrix can affect your height]. Indeed, the glycosaminoglycan/proteoglycan matrix is of major importance for the proliferation and/or differentiation of a number of cells[Altering this matrix can alter growth plate chondrocyte proliferation and differentiation]. Here, we determined the potential role of hyaluronic acid (HA) of increasing molecular weight (MW) to alter the expression of metabolic markers and cytokine production by human osteoarthritic (OA) subchondral osteoblasts (Ob). Both 1,25(OH)(2)D(3)-induced alkaline phosphatase activity (ALPase) and osteocalcin release were increased in OA Ob when compared to normal. HA reduced osteocalcin release in OA Ob at MW of 300 and above, whereas HA failed to significantly modify ALPase. Parathyroid hormone (PTH) stimulated cyclic AMP (cAMP) formation by OA Ob. HA had a biphasic effect on this PTH-dependent activity, totally inhibiting cAMP formation at MW of 300 and 800[This could help height growth by preventing osteoblast adhesion to the growth plate and preventing terminal differentiation but osteoblast adhesion and terminal differentiation is likely an important part of the process so this may reduce height growth]. HA of increasing MW progressively reduced the levels of Prostaglandin E(2) (PGE(2)) and interleukin-6 (IL-6) produced by OA Ob. Interestingly, urokinase plasminogen activator (uPA) and and PA inhibitor-1 (PAI-1) levels were not significantly affected by HA of increasing MW; however, the PAI-1 to uPA ratio showed a slight, yet nonsignificant increase. Surprisingly, uPA activity was increased in OA Ob under the same conditions. Last, HA had no effect on the production of insulin-like growth factor-1 by these cells. Our data suggest that high MW HA can modify cellular parameters in OA Ob that are increased when compared to normal. The effect of HA on inflammatory mediators, such as PGE(2) and IL-6, and on uPA activity is more striking at higher MW as well. Taken together, these results could suggest that HA of increasing MW has positive effects on OA Ob by modifying their biological synthetic capacities[and possibly chondrocytes and stem cells]."

Hyaluronic Acid can alter growth plate chondrocytes by modifying their molecular environment.

"It is known that HA interacts with HA receptors (CD44 receptors) on a number of cells, yet it remained to be demonstrated that HA could act upon chondrocytes or other cell types within the joint"<-the effectiveness of Hyaluronic Acid is based on the number and location of CD44 receptors and other receptors for Hyaluronic Acid.

Here's a study that shows that HA receptors exist in the growth plate.  However, CD44 receptors are not present within these growth plate chondrocytes.  CD44 does not modulate Hyaluronic Acid effectiveness on GP chondrocytes.

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

"CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle."

"HA mediates hydrostatic pressure by adsorbing water, indicating that HA is associated with increase in lacunae size of chondrocytes."<-this is why Hyaluronic Acid can help you grow taller.
Here's another study that shows that the potential height increasing effects of both Hyaluronic Acid and Chondroitin Sulfate:

Matrix molecule influence on chondrocyte phenotype and proteoglycan 4 expression by alginate-embedded zonal chondrocytes and mesenchymal stem cells.

"Articular cartilage resists load and provides frictionless movement at joint surfaces. The tissue is organized into the superficial, middle, deep, and calcified zones throughout its depth, each which serve distinct functions. Proteoglycan 4 (PRG4)[also known as lubricin], found in the superficial zone, is a critical component of the joint's lubricating mechanisms. Maintenance of both the chondrocyte and zonal chondrocyte phenotype remain challenges for in vitro culture and tissue engineering. Here we investigate the expression of PRG4 mRNA and protein by primary bovine superficial zone chondrocytes, middle/deep zone chondrocytes, and mesenchymal stem cells encapsulated in alginate hydrogels with hyaluronic acid (HA) and chondroitin sulfate (CS) additives. Chondrogenic phenotype and differentiation markers are evaluated by mRNA expression, histochemical, and immunohistochemical staining. Results show middle/deep cells express no measurable PRG4 mRNA by day 7. In contrast, superficial zone cells express elevated PRG4 mRNA throughout culture time. This expression can be significantly enhanced up to 15-fold by addition of both HA and CS to scaffolds. Conversely, PRG4 mRNA expression is downregulated (up to 5-fold) by CS and HA in differentiating MSCs, possibly due to build up of entrapped protein. HA and CS demonstrate favorable effects on chondrogenesis by upregulating transcription factor Sox9 mRNA (up to 4.6-fold) and downregulating type I collagen mRNA (up to 18-fold). Results highlight the important relationship between matrix components and expression of critical lubricating proteins in an engineered cartilage scaffold."

So both Chondroitin Sulfate and Hylauronic Acid may enhance chondrogenesis by enhancing Sox9.

"Proteoglycan 4 (PRG4), a large glycoprotein encoded by the PRG4 gene, is the critical boundary-lubrication mechanism in articular cartilage.1 Superficial cells secrete elevated levels of PRG4 to provide lubrication at the articulating surface."<-Maybe PRG4 can play a role in height.  "Bone marrow MSCs were shown to secret up to 10 times as much PRG4 in alginate culture than mixed zone chondrocytes or mensical fibrochondrocytes. However, MSC hydrogel constructs had poor ability to localize PRG4 to improve lubrication."

"PRG4 is a marker for superficial zone chondrocytes"

"bone marrow derived MSCs express PRG4 mRNA during chondrogenic differentiation, and matrix molecules can aid in retention of the protein within the scaffold."

Chondrogenic differentiation of bone marrow concentrate grown onto a hylauronan scaffold: Rationale for its use in the treatment of cartilage lesions.

"The use of Bone Marrow Concentrate (BMC) enables the implant of a cell population surrounded by its microenvironment. Moreover, the cells within the bone marrow niche are able to regulate stem cell behavior through direct physical contact and by secreting paracrine factors. The aim of this study was to investigate the phenotype of cells within BMC and their ability to differentiate into chondrogenic lineage once seeded onto a hyaluronan-based scaffold (Hyaff-11) already used in clinic. The chondrogenic potential of BMC has been evaluated by means of morphological, histological, immunohistochemical and molecular analyses. Cells within BMC grown onto HYAFF-11 are able to differentiate into chondrogenic sense by the expression and production of specific extracellular molecules."

"stem cell activity may be considered not only the expression of one or more intrinsic cell capabilities, but also the result of interaction between cells with specific abilities and their niche, represented by extracellular matrix, adhesion molecules, growth factors, cytokines, and chemokines. Bone marrow contains not only stem cells and precursors cells as a source of regeneration tissue but also accessory cells that support angiogenesis and vasculogenesis by producing several growth factors."

"Bone marrow was obtained from the iliac crest of 10 patients (mean age 30.35 ± 12.63; 6 females and 4 males) surgically treated with autologous cell transplantation for chondral defects."

"The harvested bone marrow was reduced in volume directly in the operating room, by removing most of the red cells and plasma."

"CFU-F assays demonstrated that BMC was able to generate new fibroblast colonies from single cells. Few and small new colonies where observed at day 7. A significant increase of the number of colonies was noticed at day 14"<-maybe this can occur in LSJL at which point new growth plates can be formed.

"In particular, SOX-9, Collagen type II, Aggrecan, COMP, and Fibromodulin which were not expressed at day 0, became detectable at day 40 but with a significant increase at day 52"<-maybe at those stages more primitive chondrocyte markers could be observed like FGF2.  Unfortunately they were not measured.

"Decorin mRNA which is not detectable at day 0, showed a high level of expression at day 40 with a tendency to decrease at day 52"

"ALP was already expressed at day 0 and showed a tendency to increase until day 52"<-maybe this native ALP in human adult stem cells is inhibitory to chondrogenic differentiation and needs to be addressed.

"At day 40 ultrastructural analysis of samples, allowed the identification of extracellular matrix surroundings the chondrocyte-like cells. The nuclei showed an ovoidal or plurilobated appearance. The cytoplasm of the cells contained a variable amount of glycogen particles and lipid droplets"

"the bond of these cells with hyaluronan molecule is able to enhance their chondrogenic differentiation potential via stimulated expression of specific target genes"<-The increase of hyaluronic synthase by LSJL may prime stem cells for chondrogenic differentiation.

"Light and electron microscopy performed highlighted the interaction of the cells with Haff-11 scaffold, demonstrating the colonization of this last by nucleated cells which entered in contact with its fibers acquiring a fibroblastic appearance. These cells, that with the passing of time express adult chodrocytes phenotype, produce an extracellular matrix mainly composed by repetitive collagen fibrils."

Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells via biomimetic and bioactive poly-ε-caprolactone scaffolds.

"Nanofibrous (N: ∼400 nm) and microfibrous (M: ∼10 μm) poly-ε-caprolactone (PCL) scaffolds were combined with 1% high-molecular-weight sodium hyaluronate (NHA/MHA), 1% hyaluronan (HA) and 200 ng transforming growth factor-beta 1 (TGF-β1; NTGF/MTGF), or 0.1% bovine serum albumin (N/M). Scaffolds were seeded with MSCs from bone marrow and cultured without growth factors in vitro. Cultures with chondrogenic medium supplemented with TGF-β1 served as controls. Proliferation, migration, and release of TGF-β1 were investigated.  NTGF and MTGF exhibited primarily an initial release of TGF-β1. None of the factors released by the scaffolds recruited MSCs. The expression of aggrecan was dependent on the scaffold ultrastructure with nanofibers promoting increasing and microfibers decreasing expression levels. Composites containing HA demonstrated elevated seeding efficiency and lower type I collagen expression. Expression of type II collagen was dependent on continuous or late supply of TGF-β1, which was not provided by our scaffold design. The initial release of TGF-β1 induced an expression of type I collagen and osteogenic marker genes. Initial release of HA is sufficient in terms of directing the implanted MSCs toward a chondrogenic end, whereas a late release of TGF-β1 is preferred to foster type II and avoid type I collagen expression."

"TGF-β1 is a peptide homodimer that is naturally found in human platelets and bone in highest concentrations"

"HA is a linear polysaccharide, which naturally occurs in articular cartilage and synovial fluid. It is unique among cartilage glycosaminoglycans and fundamental for hyaline matrix homeostasis and the cellular microenvironment."<-So HA can alter the cellular microenvironment and favor chondrogenesis.

"human serum derived from whole blood recruits bone marrow-derived MSC. MSC migration was stimulated by a variety of chemokines and growth factors, such as bone morphogenetic proteins or platelet-derived growth factors.  Hyaluronic acid and synovial fluid, showing high amounts of endogenous HA, stimulated the migration of human subchondral progenitor cells."

"HA-containing scaffolds fostered proliferation, initial neomatrix retention, and increasing glycosaminoglycan deposition. They prevented fibroblastic dedifferentiation, stress fiber formation and resulted in the expression of the adhesion molecule CD44."

"if one wants to maintain the chondrogenic phenotype of human bone marrow-derived MSCs on fibrous PCL scaffolds, TGF-β1 may be detrimental at least during the early stages of differentiation."

Hyaluronan initiates chondrogenesis mainly via CD44 in human adipose derived stem cells.

"a microenvironment enriched with hyaluronan (HA) initiated and enhanced chondrogenesis in human adipose derived stem cells (hADSCs). We further hypothesize that HA-induced chondrogenesis in hADSCs is mainly due to the interaction of HA and CD44 (HA-CD44), a cell surface receptor of HA. The HA-CD44 interaction was tested by examining the mRNA expression of hyaluronidase-1 (Hyal-1) and chondrogenic marker genes (SOX-9, collagen type II, and aggrecan) in hADSCs cultured on HA-coated wells. Cartilaginous matrix formation, sulfated glycosaminoglycan (sGAG) and collagen productions by hADSCs affected by HA-CD44 interaction were tested in a 3D fibrin hydrogel. About 99.9% of hADSCs possess CD44. The mRNA expressions of Hyal-1 and chondrogenic marker genes were up-regulated by HA in hADSCs on HA-coated wells. Blocking HA-CD44 interaction by anti-CD44 antibody completely inhibited Hyal-1 expression and reduced chondrogenic marker gene expression, which indicates that HA induced chondrogenesis in hADSCs mainly acts through HA-CD44 interaction. A two-hour pre-incubation and co-culture of cells with HA in hydrogel (HA/fibrin hydrogel) not only assisted in hADSC survival but also enhanced expression of Hyal-1 and chondrogenic marker genes. Higher levels of sGAG and total collagen were also found in HA/fibrin hydrogel group. Immunocytochemistry showed more collagen type II but less collagen type X in HA/fibrin than in fibrin hydrogels."

Hyaluronan likely enhances chondrogenesis in mesenchymal stem cells as well and there is adipose tissue within bone.

Stem cells from ages 18-65 were used.
Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis.

"Methacrylated hyaluronic acid (HA) hydrogels provide a backbone polymer with which mesenchymal stem cells (MSCs) can interact through several cell surface receptors that are expressed by MSCs, including CD44 and CD168. This 3D hydrogel environment supports the chondrogenesis of MSCs, through functional blockade{so blocking expression of certain genes could be more important in inducing chondrogenesis than activating genes} that these specific cell-material interactions play a role in this process. Beyond matrix interactions, cadherin molecules, a family of transmembrane glycoproteins, play a critical role in tissue development during embryogenesis, and N-cadherin is a key factor in mediating cell-cell interactions during mesenchymal condensation and chondrogenesis.  We functionalized HA hydrogels with N-cadherin mimetic peptides and evaluated their role in regulating chondrogenesis and cartilage matrix deposition by encapsulated MSCs. Conjugation of cadherin peptides onto HA hydrogels promotes both early chondrogenesis of MSCs and cartilage-specific matrix production with culture, compared with unmodified controls or those with inclusion of a scrambled peptide domain. This enhanced chondrogenesis was abolished via treatment with N-cadherin-specific antibodies, confirming the contribution of these N-cadherin peptides to chondrogenesis. Subcutaneous implantation of MSC-seeded constructs also showed superior neocartilage formation in implants functionalized with N-cadherin mimetic peptides compared with controls."

"expression of the deletion mutant form of N-cadherin, which lacks either the extracellular homotypic interaction domains or the intracellular β-catenin binding site, results in decreased cellular condensation and impaired chondrogenesis"  Cadherin interactions stimulated chondrogenesis from days 1-6 of the experiment but did not have an effect after that.

Too high expression of N-Cadherin 4-fold had the effect of inhibiting chondrogenesis.
Hyaluronic Acid will definitely help during the chondrocyte hypertrophy phase by increasing peak chondrocyte hypertrophy.  Surprisingly, Hyaluronic Acid is available for sale: NOW Foods Hyaluronic Acid 100mg 2X Plus, 60 Vcaps.   The Molecular Weight of the Hyaluronic Acid is not listed.  I can't speak on the delivery effectiveness of Hyaluronic Acid of actually getting into the bone(it's a lot easier to get into the articular cartilage) but if it can get inside the bone it should help you grow taller by increasing hydrostatic pressure one..  Supplementing with HA may be a way to increase height during development barring various negative feed back mechanisms.

1 comment:

  1. It does have benefits but this can only be possible when started from younger years to see results.