Monday, June 14, 2010

Height Increasement with Collagen?

The collagen in bone gives bone it's elasticity.  Collagen allows bone to temporarily lengthen(microstrain) and if microfractures occur while the bone is in this stretched state it stands to reason that the bone will maintain some of this state.  If we can increase the collagen content of bone, then we should be able to put more of a distraction force on the cortical bone.  Microfractures will maintain some of this distracted state.  This cortical bone diaphysis stretching is what is employed by Sky in his limb center.  And if it works(we're waiting on his before/after pictures), it's a viable alternative/supplement to lateral synovial joint loading.  How do we increase the collagen content in bone(for it's height increasement applications)?  Alternatively, is there anyway to enhance expression of the gene COL2A1 which controls the formation of hyaline cartilage(which is the resting zone of the growth plate)?

Collagen is formed by amino acids and peptides. 

Large-scale gene expression in bone marrow mesenchymal stem cells: a putative role for COL10A1 in osteoarthritis.

"To elucidate disease-specific molecular changes occurring in osteoarthritis (OA) by analysing the differential gene expression profiles of bone marrow mesenchymal stem cells (BM-MSCs) from patients with OA compared with those without OA. Expression profiles of BM-MSCs from eight paired patients with OA and patients with hip fracture without signs of OA were compared by DNA microarray expression analysis and significant differences were evaluated by computational Gene Set Enrichment Analysis. To validate the involvement of COL10A1 as part of the most downregulated gene set in OA, three tagging single nucleotide polymorphisms were genotyped in 191 patients with OA and 283 controls. COL10A1 expression was also assessed by quantitative RT-PCR in additional subjects. Expression levels in 9% (1967) of the overall transcripts were significantly different (p<0.05) between MSCs from patients with OA and controls (532 genes reached twofold differences: 240 were upregulated and 292 were downregulated). Cell development and differentiation were the functional categories accounting for most genes with altered expression. Interestingly, several genes related to the Wnt/-catenin pathway and collagen genes were downregulated in MSCs from patients with OA. The collagen gene set was clearly downregulated in OA. Furthermore, the expression of COL10A1 was significantly reduced in patients with OA. A genetic association between the COL10A1 rs11965969 polymorphism and OA was also found.  COL10A1 downregulation seems to have a role in the establishment of a defective and/or unstable subchondral cartilage matrix in OA disease. It is proposed that OA may be linked to the intrinsic defective regenerative potential of BM-MSCs resulting from its reduced expression of fate commitment-related genes."

Conclusion:  Collagen is increased by Mesenchymal Stem Cells.  To increase collagen content in bone or the hyaline cartilage growth plate line then one needs to increase expression of the genes that control mesenchymal stem cell lineage(such as COL2A1 for hyaline cartilage). 

The study states that it has supplement data detailing gene expression but I could not find it.

Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system. 

"Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen{up}, aggrecan{up}, Sry-related HMG box (SOX-9){up}, and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen{up} and bone sialoprotein (BSP){Up} genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3{up} and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. Alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA." 

Subchondral osteoblasts expressed type I collagen at a 5:1 ratio to cartilage chondrocytes whereas cartilage chondrocytes expressed type II collagen at a 5:1 ratio to subchondral osteoblasts.

Mechanical stress upregulated TGF-B1 in mechanically stressed osteoblasts.

Mechanical stress studies are always interesting because they are something that we can perform on our own.  This study shows that chondrocytes co-cultured with osteoblasts are more likely to hypertrophy(one of the stages of growth).  Osteoblasts are prevalent in bone so there should be plenty of opportunities to co-culture near the hyaline cartilage.  In this case, loading is bad but it is very high magnitude loading.  This may have applications to lateral synovial joint loading as if you are not getting results it may be worth it to lighten the weight in case the load is "overstressing" the osteoblasts.  

So... 

Collagen is produced by Mesenchymal Stem Cells.  If we want to produce more collagen we need to enhance proliferation of MSCs but enhance the expression of genes that make MSCs undergo a collagenic lineage.  If you are not getting results with LSJL, try lowering the weights you use(10lbs is too light.  I think between 50-70lbs may be the sweet spot to maximize the positive benefits of mechanical loading whereas not overstressing say the osteoblasts). 

Type I collagen may help as well:

The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite.

"Type I collagen in vitro can initiate and orientate the growth of carbonated apatite mineral in the absence of any other vertebrate extracellular matrix molecules of calcifying tissues. Collagen matrix influences the structural characteristics on the atomic scale, and controls the size and the three-dimensional distribution of apatite at larger length scales."

"Circulating fluids continuously bathe the calcified connective tissue over both collagen inter- and intrafibrillar spaces"

"The 3D organization of collagen, similar to that described in human compact bone, has been reproduced in vitro by exploiting the capacity of collagen molecules to self-assemble spontaneously under high concentrations (at least 80 mg ml−1)"

"Collagen has a further impact on the hydration environment and local structure of phosphate in apatite"<-can we manipulate this to grow taller?

"Collagen can sequestrate large quantities of calcium, phosphate and carbonate ions, which precipitate spontaneously in apatite without additives. The concentration of calcium ions strongly influences the distribution of apatite crystals within the collagen matrix. Hence, the Ca-rich proteins in the ECF may play an important role as inhibitors of apatite precipitation whereas those in bone ECM may concentrate the ions locally in the gap regions described as the first site of nucleation for apatite crystals"

Other collagen types may help as well:

Minor cartilage collagens type IX and XI are expressed during embryonic stem cell-derived in vitro chondrogenesis.

"In vitro differentiation of murine embryonic stem (ES) cells via embryoid bodies (EBs) recapitulates the cellular differentiation steps of chondrogenesis. Differentiated chondrocytes lose their characteristic phenotype when they are kept in monolayer culture. The aim of this study was to further characterize the chondrogenic nodules derived by in vitro-differentiation of murine ES cells for the distribution of collagen types II, IX and XI{all three types of collagen are upregulated by LSJL} in comparison to in vitro dedifferentiating primary chondrocytes from murine embryonic ribs. Expression of cartilage collagens and other extracellular matrix proteins was analyzed. ES cell-derived chondrocyte differentiation starts with mesenchymal condensations synthesizing high amounts of fibronectin {Possible fibronectin related proteins like FNDC4 and FNDC6 are upregulated by LSJL}. Later, the matrix of the mature cartilage nodules consists of type II collagen, proteoglycans and the minor collagens type IX and XI {all of these proteins are upregulated by LSJL providing evidence that LSJL can form cartilage nodules that could potentially undergo endochondral ossification and make you grow taller}. The nodules show a three-dimensional structure with multiple layers of collagen type II-positive cells. At late differentiation stages these chondrocytes were located at lateral regions of the nodules. Similar to the distribution pattern of collagen type II positive cells, the cells staining positive for collagen type IX and XI were present in the surface regions, but not in the central areas of the chondrogenic nodules. During cultivation of the primary murine rib chondrocytes expression of chondrogenic marker genes such as collagen type II and aggrecan declined and many chondrocytes lost characteristic cartilage matrix proteins and converted to an elongated, fibroblastoid shape with prominent actin stress fibers."

"Early mesenchymal progenitor cells are characterized by the production of hyaluronan {hyaluronan synthase1 is upregulated 4.5-fold by LSJL}, fibronectin and collagen type I {upregulated by LSJL}. Condensations resulting from the aggregation of mesenchymal precursor cells can be visualized by their affinity to the lectin peanut binding agglutinin (PNA), binding to the cell surface of condensing cells"

"Collagen type IX was first described as a proteoglycan since it possesses a large glycosaminoglycan domain. The isoform mainly found in cartilage contains a large N-terminal globular domain (NC4) and is located on the surface of heterotypic type II/XI fibrils mediating cross-links either with collagen type ÍI fibrils or other collagen type IX molecules and interaction with glycosaminoglycans. Collagen type XI is a heterotrimeric protein located at the interior of collagen II/XI fibrils. A lack of the Col11a1 gene encoding the alpha1(XI) chain in mice results in autosomal recessive chondrodysplasia (cho) showing a shortened spine and thoracic skeleton with flared metaphyses"

"Collagen type XI has also been implied to have a function in the regulation of the diameter of collagen fibrils. Besides cartilage tissue, collagen type XI a1 chain mRNA expression was found in trabecular bone"<-so some COL11A1 expression can be explained by trabecular bone expression.

With the exception of Sox9 no proteins associated with the development of early mesenchymal condensations like PNA, N-cadherin, and Fibronectin were expressed over threshold in LSJL.

"Only some single cells expressed the extracellular matrix protein collagen type X characteristic for hypertrophic chondrocytes and peanut agglutinin, a marker for early mesenchymal condensations at three days after cell isolation. The same was true for N-cadherin"<-LSJL gene expression was taken at 49 hours which is below three days however unlike this study COL10A1 was upregulated above threshold.

"Collagen type IX seems to be preferentially expressed by cells isolated from “permanent” cartilage tissues, which do not undergo endochondral ossification"

Autologous Collagen-induced Chondrogenesis: Single-stage Arthroscopic Cartilage Repair Technique.

"Autologous collagen-induced chondrogenesis is a novel, single-staged arthroscopic cartilage repair technique using microdrilling and atelocollagen or fibrin gel application under carbon dioxide insufflation. Atelocollagen is a highly purified type I collagen obtained following the treatment of skin dermis with pepsin and telopeptide removal, making it nonimmunogenic. In this procedure, atelocollagen mixed with fibrinogen and thrombin in a 2-way syringe can maintain the shape of the articular surface approximately 5 minutes after application due to the reaction between the thrombin and fibrinogen. Carbon dioxide insufflation facilitates the application of the gel under dry conditions. Ten patients (mean age, 38 years) with symptomatic chondral defects in the knee who were treated arthroscopically with microdrilling and atelocollagen application were retrospectively analyzed. All defects were International Cartilage Repair Society grade III or IV and were 2 to 8 cm(2) in size intraoperatively. For the clinical assessment, Lysholm score was assessed preoperatively and at 2-year follow-up. All patients underwent morphological magnetic resonance imaging at 1.5-Tesla at 1-year follow-up. Mean Magnetic Resonance Imaging Observation of Cartilage Repair Tissue score at 1-year follow-up was 70.4±20.2 (range, 15-95). The Magnetic Resonance Imaging Observation of Cartilage Repair Tissue score for patellar lesions was similar to that of lesions in other locations: 73.3±11.7 vs 68.1±25.5, respectively. This technique had encouraging clinical results at 2-year follow-up. Morphological magnetic resonance imaging shows good cartilage defect filling, and the biochemical magnetic resonance imaging suggests hyaline-like repair tissue."

Can this technique be used to produce growth plate cartilage?

The issue is that it actually applies a gel so it might be complicated to do within the bone marrow and it's unclear whether there was any cartilage overgrowth within the knee joint.

Nanomechanics of collagen microfibrils.

"collagen-rich tissues are built with the collagen fibril as fundamental building block. These fibrils have a diameter in the range of 30 to 500 nm, a length up to the millimeter range and are assembled in complex hierarchical assemblies, whose structure depend on the particular tissue"

"In bone, the organic collagen protein matrix alone is not sufficient to provide the stiffness and resistance to compression required for this tissue (which has to carry considerable loads) and additional stiffening is provided by the inclusion of mineral hydroxyapatite crystals into collagen fibrils and particularly in the gap region"

"When subjected to mechanical load, collagen microfibrils feature two distinct deformation regimes. In the small-strain regime (<10%) the predicted Young’s modulus is ≈300 MPa, while in the large-strain regime (>10%) the microfibril shows a severely increased tangent stiffness, with a Young’s modulus of ≈1.2 GPa "

6 comments:

  1. Is anyone having any success with LSJL, at all?

    I tried it a little bit for the first time today, just with 20 lbs on the ankles. It seems like thats where the growth is really coming from, so i'll think i'll just try it there for now. That should be effective, right?

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  2. I've been doing it for about 2 weeks now but not going to measure myself until I get back to school. (2 More Months) that way I don't stress/obsess about it. I used 40 lbs but going up to 50 tonight.

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  3. How do you tollerate that kind of weight, 20 is uncomfortable enough but 50?

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  4. Would supplementing or topical use of hyaluronic acid have any benefit or how could one cope with it in regimen?

    Or am I way off with my questions?

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  5. Hey Tyler, now that you've taken a few days off from LSJL are you still feeling tenderness in the areas you are performing it on? Or any pains at all in your ankles/shins/legs?

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  6. There's lots of things that can aid in the increase of height, I don't know about hyaluronic acid off hand. I'm not feeling tenderness in any of the areas I'm performing. In fact the tenderness usually goes completely away a couple of minutes after I perform the exercise.

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