Enabling bone formation in the aged skeleton via rest-inserted mechanical loading.

“The mild and moderate physical activity most successfully implemented in the elderly has proven ineffective in augmenting bone mass. We have recently reported that inserting 10 s of unloaded rest between load cycles transformed low-magnitude loading into a potent osteogenic regimen{but is it a chondrogenic regimen?} for both adolescent and adult animals. Here, we extended our observations and hypothesized that inserting rest between load cycles will initiate and enhance bone formation in the aged skeleton. Aged female C57BL/6 mice (21.5 months) were subject to 2-week mechanical loading protocols utilizing the noninvasive murine tibia loading device. We tested our hypothesis by examining whether (a) inserting 10 s of rest between low-magnitude load cycles can initiate bone formation in aged mice and (b) whether bone formation response in aged animals can be further enhanced by doubling strain magnitudes, inserting rest between these load cycles, and increasing the number of high-magnitude rest-inserted load cycles. We found that 50 cycles/day of low-magnitude cyclic loading (1200 microepsilon peak strain) did not influence bone formation rates in aged animals. In contrast, inserting 10 s of rest between each of these low-magnitude load cycles was sufficient to initiate and significantly increase periosteal bone formation (fivefold versus intact controls and twofold versus low-magnitude loading){we’re not looking for periosteal bone formation, we’re looking for neo-growth plate formation but the principles may be the same}. However, otherwise potent strategies of doubling induced strain magnitude (to 2400 microepsilon) and inserting rest (10 s, 20 s) and, lastly, utilizing fivefold the number of high-magnitude rest-inserted load cycles (2400 microepsilon, 250 cycles/day) were not effective in enhancing bone formation beyond that initiated via low-magnitude rest-inserted loading. We conclude that while rest-inserted loading was significantly more osteogenic in aged animals than the corresponding low-magnitude cyclic loading regimen, age-related osteoblastic deficits most likely diminished the ability to optimize this stimulus.”

“While the inability to perceive mild and moderate loading events as stimulatory may reflect potential deficits in numbers and/or viability of mechanosensory (e.g., osteocytic) cells, the inability to initiate and, especially, sustain bone formation more likely reflects potential for deficits in the numbers and/or function of osteoblastic cells. Additionally, the declining availability of biomolecules involved in coordinating and enhancing osteoblastic response to mechanical stimuli (e.g., TGF-β, IGF-1){these biomolecules are involved in chondrogenesis too so it’s important to monitor changes in these biomolecules due to aging} potentially compromises the ability of bone cells in aged tissue to perceive low and moderate magnitude loading events as being stimulatory. Last, the age-related decrease in the surface to volume ratio of bone mineral matrix and increased viscosity of interstitial fluids could decrease biophysical stimuli delivered to bone cells via standard exercise regimens{this could affect neo-growth plate formation too as the degree of biophysical stimuli delivered to cells would affect the ability to form new growth plates}”

“A total of 49 aged female C57BL/6 mice (mean ± SE; 21.5 ± 0.16 months)”

“The device fixes the proximal tibia (at the tuberosity) against motion and applies controlled loads to the distal tibia, thereby placing the tibia diaphysis under “cantilever” bending in the medial–lateral direction.”<-not quite like LSJL.

“a strain versus load calibration curve was determined and yielded peak strains in the range of 800–2400 με at the periosteal surface (and 600 to 1800 με peak strains at the endocortical surface) for loads of 0.4–1.2 N, respectively.”

“rest-inserted loading (particularly at low magnitudes) enhances rates of bone formation by primarily increasing mineral apposition rates compared to cyclic protocols”

“attempts at further enhancing the bone formation response to rest-inserted loading by doubling strain magnitude, inserting rest-intervals, and subjecting animals to five-fold the number of high-magnitude rest-inserted loading cycles were all ineffective in the aged skeleton.”

Here’s a diagram showing the zone of Ranvier in relation to the growth plate:

If we know what stresses are responsible for forming growth plates we can re-induce these mechanical stresses to form new growth plates.

This study would be more significant except it was published in 1988 before the scientific community began to focus more on genes and cellular epigenetics.  Just because the highly undifferentiated cells of a juvenile or infant may be induced to differentiate into the cells composing the Zone of Ranvier and in turn the growth plate does not mean that adult mesenchymal stem cells will do the same thing.

And I think going back to these old studies is a good thing as they focued more on mechanical factors rather than retrovirus genetic engineering.

One study found that interstitial fluid flow upregulates the mesenchymal condensation gene Msx2 but downregulates the chondrogenic gene Col2a1.  This is not necessarily a bad thing as mesenchymal condensation in a neo-Zone of Ranvier would be key to forming new growth plates and those genes would not be chondrogenic until they became a growth plate.  In fact in this study the Zone of Ranvier experienced more osteogenic than chondrogenic stimuli so it would be no surprise that a mesenchymal stem cell that may eventually become a growth plate chondrocyte may not express chondrogenic genes initially.

This study provides additional evidence for LSJL as LSJL compresses the bone and the joint thus altering the “Loading history” of the bone and joint.  Since the shear strain, caused by LSJL fluid flow, needs to be high this could explain why I’m getting more results for my fingers than my legs.

The role of mechanical loading histories in the development of diarthrodial joints.

“The role of mechanical loading history in chondroosseous development at the ends of long bones is explored using two-dimensional finite element models of chondroepiphyses. Loading histories are characterized in terms of discrete loading cases defined by joint contact pressure distributions and an associated number of loading cycles. An osteogenic stimulus throughout the chondroepiphyses is calculated following the theory that cyclic octahedral shear stresses promote endochondral ossification and cyclic compressive dilatational stresses inhibit ossification{Octahedral stresses are shear stresses that are acting on octahedral planes inside the bone.  The plane whose normal vector forms equal angles with the coordinate system is called octahedral plane.  But basically shear stress which is what LSJL can induce. A compressive dilational stress is the increase in volume per unit volume of a homogeneous substance.  So for example if you squeezed a stress ball and it got bigger}. The resulting distributions for the osteogenic stimulus predict the appearance of the secondary ossific nucleus and the shape of the developing bony epiphysis. The zone of Ranvier and the formation of articular cartilage and the growth plate are predicted by the models{Considering that the zone of Ranvier is the basis of the growth plate this is key}. Tissue stress histories constitute an important influence during skeletal morphogenesis {And we can expose the tissue to different stresses ala LSJL}.”

“Alterations in joint loading or motion can alter the pattern of ossification and growth in developing bone ends, and a reduction in joint forces can delay the appearance of the secondary ossific nuclei”

“The sequence of cartilage proliferation, maturation, degeneration, and ossification is the normal process for all cartilage in the appendicular skeleton. This process is (a) accelerated by intermittently applied deviatoric (shear) stresses (or strain energy) and (b) inhibited or prevented by intermittently applied compressive dilatational stresses (hydrostatic pressure).”

“The appearance of the secondary ossific nuclei in both chondroepiphyses was predicted. Increased osteogenic stimuli were also calculated at the edge of the advancing ossification front where the zone of Ranvier (ossification groove) forms. The areas where the osteogenic stimuli were low define those cartilaginous regions that become the growth plate and the articular cartilage.”

“the ossific nucleus appears in an area of high shear (deviatoric) stresses the edge of the advancing ossification front (zone of Ranvier or ossification groove) also experiences high shear stresses, and the joint surface, where articular cartilage forms, is exposed to high-magnitude hydrostatic compression”<-Since the zone of Ranvier is the key forming new growth plates, inducing high shear stress would be key to forming new growth plates and LSJL can induce shear stress via interstitial fluid flow.  According to the study Interstitial fluid flow: the mechanical environment of cells and foundation of meridians., interstitial fluid flow induces shear stresses.  Here’s Michael’s summary of LSJL.

“In both the convex and the concave chondroepiphysis, a state of high hydrostatic pressure is created directly beneath the loaded contact surface. In the interior regions of both chondroepiphyses, areas of high-magnitude octahedral shear stress are created. The location of these areas within the central zone of the chondroepiphyses shifts with the direction of the applied loading”<-If we mimic this state we can achieve chondroinduction.

“the shape of the developing bony epiphysis will depend on the geometry of the bone end. In the model with a convex joint surface, the developing ossific nucleus is stimulated to produce a sphere-shaped bony epiphysis.  The model with a concave joint surface is stimulated to create a flatter, more disc-shaped bony epiphysis.”

“The stored energy in cartilage under mechanical loading is primarily in the form of deviatoric or shear energy owing to the nearly incompressible nature of this tissue. Some of this stored deviatoric energy is lost in hysteresis upon unloading. The energy dissipated during intermittent mechanical loading must be accounted for by a change in internal energy and/or a change in the temperature of the cartilage. It is possible that the direct mechanical alteration of cells or the increased temperature associated with energy dissipation caused by intermittent shear stresses could increase mitotic activity or activate specific biochemical pathways in the cells”

Here’s another study with some information on the Zone of Ranvier:

Stem Cells and Cartilage Development: Complexities of a Simple Tissue

“[The] biochemical composition [of cartilage] is uniquely suited to providing a combination of tensile strength with deformability, giving it mechanical properties that resemble those of a shock absorber, thereby dissipating forces across the bones, preventing them from fracturing during normal activity.”

“The balance between mechanical stiffness and flexibility is itself the result of interaction between the thin type II collagen fibrils, giving tensile strength, within which are trapped molecules of aggrecan, which are highly negatively charged and so bind water avidly”

“When unloaded, the water content of cartilage is about 70% of the wet weight. Under deforming load water flows out and when the load is reduced it flows back in, damping the effects of these forces. Damage to either the type II collagen or aggrecan may lead to loss of cartilage function”<-Maybe when LSJL loads the cartilage it results in water flowing out and possibly other nutrients and maybe even growth factors that can result in neo-growth plates.

“During cartilage development [cartilage transforms] from a relatively simple isotropic tissue with a high cell density and homogeneous distribution of collagen fibrils to an anisotropic tissue with a low density of chondrocytes growing in vertical columns and a unique arrangement of collagen fibrils.”

“Articular cartilage chondroprogenitor cells are derived from migration of mesenchymal cells out of the zone of Ranvier niche.  In early development these cells may accumulate in the surface zone and drive the process of appositional growth of cartilage but with maturity they become dissipated throughout the cartilage.”<-So maybe LSJL can drive these cells back into the zone of ranvier?

Since LSJL loads the articular cartilage which is part of the synovial joint this study may have implications on LSJL.

Dynamic mechanical compression of devitalized articular cartilage does not activate latent TGF-β.

“mechanical shearing of synovial fluid, induced during joint motion, rapidly activates a large fraction of its soluble latent TGF-β content. Based on this observation, the primary hypothesis of the current study is that the mechanical deformation of articular cartilage, induced by dynamic joint motion, can similarly activate the large stores of latent TGF-β bound to the tissue extracellular matrix (ECM). Here, devitalized deep zone articular cartilage cylindrical explants (n=84) were subjected to continuous dynamic mechanical loading (low strain: ±2% or high strain: ±7.5% at 0.5Hz) for up to 15h or maintained unloaded. TGF-β activation was measured in these samples over time while accounting for the active TGF-β that remains bound to the cartilage ECM. Results indicate that TGF-β1 is present in cartilage at high levels (68.5±20.6ng/mL) and resides predominantly in the latent form (>98% of total). Under dynamic loading, active TGF-β1 levels did not statistically increase from the initial value nor the corresponding unloaded control values for any test, indicating that physiologic dynamic compression of cartilage is unable to directly activate ECM-bound latent TGF-β via purely mechanical pathways and leading us to reject the hypothesis of this study. These results suggest that deep zone articular chondrocytes must alternatively obtain access to active TGF-β through chemical-mediated activation and further suggest that mechanical deformation is unlikely to directly activate the ECM-bound latent TGF-β of various other tissues, such as muscle, ligament, and tendon{or bones?}.”

If the TGF-Beta of the synovial joint is the only location can be activated it could explain why it is important to load there.

” During joint motion, opposing articular surfaces slide relative to one another, producing high levels of mechanical fluid shear (~ 104 s−1) in the synovial fluid”

” due to the presence of an overwhelming supply of non-specific binding sites in the cartilage ECM, active TGF-β from an external bathing solution predominantly binds to, and accumulates in the superficial zone (0–250 µm deep) and is unable to penetrate deeper into articular cartilage”

“TGF-β activated in synovial fluid can reach high concentrations in superficial articular cartilage, but it is unable to transport into the middle and deep zones of the tissue.”

” the shear rates of pressure-driven fluid flow through the interstitium of the tissue are far lower than those experienced in synovial fluid”

“mechanical loading in live tissue may alternatively modulate the secretion of various chemical activation mediators, such as MMPs and other proteases, and thus indirectly induce TGF-β activation.”

Here’s the previous update.  It’s hard to compare my two fingers against each other as the exact beginning of the finger is hidden by skin.

In the doctor’s office recently I measured 5’8 1/4″ versus 5’7 3/4″ previously.  Now I’ve stated I was 5’10” before and the reason for the discrepancy is that it’s a short nurse and I have a long skull with the hair it’s hard to tell where the peak of the skull is.  At other doctors I have measured higher 5’9 1/2″.  It still shows growth as the same nurse measured me 5’7 3/4″ two years in a row.

There’s a part of the door that’s 5’11” so if I got to there that would be definitive proof.  Maybe LSJL gains are just slow.  The finger gains are just more drastic.  Increasing the intensity on the finger I can see results in a week.  Clamping the finger has a much greater intensity.  Maybe more intensity on the leg is needed to generate faster results but the less intensity generates more quality height as there is no deformation of the leg in contrast to the finger.  The knuckle does not seem to be affected that much except it looks denser.  Thus emphasizing the importance of laterally loading the bone.

I’m going to start loading the thumb.

There’s the current picture of my finger:

You can see how much the joints have increased in width.  Comparing the right hand from the left to right hand you can see the difference more clearly.  But there’s the problem of getting the picture and it’s hard to align the two fingers correctly and see where the thumb ends and begins.  But that finger definitely increased in length and those bumps at the side of the finger are bone growth. There is no pain in the fingers.  Finger mobility is excellent.  Discoloration of the skin occurs during and following clamping but it heals in about a minute.  This discoloration does not occur on the leg. LSJL definitely works but you can clamp much more intensely on the finger than you can on the leg.  Maybe there’s a way to get more rapid growth on the legs but not the massive increase in width that occurs in the fingers.  Maybe there’s a happy medium between my current intensity on the fingers and on the legs as I’m basically applying the same intensity of clamping on the legs as I am on the fingers thus the slow growth.  When Michael returns to the US and we begin work on the LSJL that will be a key turning point or maybe there’s some device to get more intensity on the legs?

Here’s the before picture of the thumb:

The advantage of the thumb versus the finger is that I can laterally clamp the base joint of the thumb whereas I can’t do that on the finger due to being blocked by the hand so I have to clamp overhead.  I also want to see if my growth on the finger was a growth and how fast I can grow the thumb given that I’m much more experienced in clamping.

If you want to complain that this is no proof at all because it is just fingers.  I’m still working on leg proof and you should complain to people with more resources than me who are not devoted to height increase.  Millionaires like Ryan Seacrest who complain about their height all the time.  I’m doing the best I can with the resources I have available and have come further in the height increase arena than anyone else before me.

The prevalence of pseudoepiphyses in the metacarpals of the growing hand

“Normally the metacarpals have an epiphysis at one end — distally for the second to fifth and proximally for the first. Pseudoepiphyses are notches or clefts that occur at the non-epiphyseal ends of bones where an epiphyseal plate would be expected and are common incidental findings in the metacarpals of the growing hand. We aimed to identify the prevalence of pseudoepiphyses on serial radiographs of 610 healthy asymptomatic children. Pseudoepiphyses in the form of notches or clefts were common, identified most often in the second metacarpal (15.25%), fifth metacarpal (7.21%), and third metacarpal (0.49%). Complete pseudoepiphyses, in which the cleft extended across the full width of the metacarpal, were seen in the first metacarpal (1.97%) and the second metacarpal (1.31%). Pseudoepiphyses are a normal variant of metacarpal ossification and should not be confused with fractures in skeletally immature patients. ”

<-Is there any way to form psueodoepiphyses to grow taller?  Normally in long bones there are two epiphysis but in the finger bones there is only one epiphyseal plate except a psuedoepiphysis is a secondary growth plate like that of a normal long bone.

One of these finger bones has a second epiphyseal plate that’s a psuedoepiphysis.

“supernumerary epiphyses appear as a separate node of ossification in an island of hyaline cartilage.”

“three basic patterns of formation [of pseudoepiphysis formation]. In the first, a central osseous bridge extends outwards from the metaphysis and then expands into a ‘mushroom-like’ osseous structure. The circular notch proximal to this structure gives the bone the appearance of an epiphysis. If this notch is displaced to one side, it can give the appearance of a cleft or partial pseudo-epiphysis. The other two patterns recognized were of an abnormal peripheral osseous bridge, creating an eccentric notch in the bone, and of multiple abnormal osseous bridging points. In each situation, the area that appeared to be a ‘pseudo-physis’ lacked typical cell columns and these were incapable of significantly contributing to the postnatal growth of the involved bone.”<-Can we create these osseus bridges?

Complete pseudoepiphyses with associated enhanced growth in hands and feet: a report of 2 siblings-case report.

“We present 2 siblings with multiple complete pseudoepiphyses in their hands and feet with associated symptomatic enhanced growth. Physical examination of the 6-year-old boy revealed long slender fingers and hyperplastic great toes. Radiography showed complete pseudoepiphyses in the first metacarpals, proximal and middle phalanges of the hands, and proximal phalanges of the feet. The patient’s younger brother had a similar phenotype with slightly milder functional complaints. Genetic analysis did not reveal an underlying syndrome in these siblings. ”

Notice each finger bone has a second growth plate.  Psuedoepiphysis’ do not always increase growth.  Since many things do not show up in x-ray’s maybe what appeared to be psuedoepiphysis’ were actually something different entirely which would not show up on x-rays.

That these siblings both developed pseudoepiphysis’ makes a mechanical means of inducing psuedoepiphysis’ less likely but it’s still a possibility if they performed the same activities especially as they did not find a genetic link.  Although they could’ve missed it.  The two siblings were 5 and 6 year old boys.

Many of the supplements to encourage chondrogenic differentiation also encourage osteogenic differentiation.  Since growth plates are made of chondrocytes, it is much more advantageous to encourage chondrogenic over osteogenic differentiation.  Some of these factors include BMP2, TGFBeta1, etc.

This study suggests that Sox9 levels may be one factor affecting whether BMP2 encourages osteogenic or chondrogenic differentiation of mesenchymal stem cells:

Sox9 Potentiates BMP2-Induced Chondrogenic Differentiation and Inhibits BMP2-Induced Osteogenic Differentiation.

“Bone morphogenetic protein 2 (BMP2) is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification{but enchondral ossification is good for height growth, however without the growth base chondrocytes for the growth plate the stimuli for endochondral ossification is pointless}. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs) towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. Exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification.”

You’re not going to be able to genetically engineer your mesenchymal stem cells to be transgenic for Sox9 but with supplements and mechanical stimuli you could upregulate the MSC expression of Sox9.  Icariin increased Sox9 but only in cells that were already chondrocytes.  Electroacupencture increased Sox9 expression.  LSJL also upregulates Sox9.  Lactoferrin upregulated Sox9 in pluripotent stem cells.  Vitamin C increased Sox9 in pre-chondrogenic ATDC5 stem cells.  Quercetin increases Sox9 levels.  Kaempferol increases Sox9 also in ATDC5 cells.

Quercetin had the most prominent effect on increasing Sox9 in normal stem cells.

“BMP2 induced Sox9 expression was transient and relatively at a lower level during the early stages of MSCs differentiation.”

“Sox9 and BMP2 synergistically promoted chondrocytes condensation and proliferation. However, Sox9 inhibited BMP2 induced chondrocytes hypertrophy, and ossification.”<-So we want optimal levels of Sox9 to form neo growth plates as chondrocyte hypertrophy and ossification are vital stages in the growth plates mechanisms of increasing height.

“Sox9 inhibits BMP2-induced early osteogenic differentiation.”<-So stem cells need to have high Sox9 expression to become chondrocytes but then levels of Sox9 need to increase to undergo endochondral ossification.

“we also explored the effect of Sox9 on skeletal development using the fetal limb culture assay. The skinned fetal limbs were isolated from mouse E18.5 perinatal embryos and cultured in the organ culture medium in presence of AdGFP, AdBMP2, and/or AdSox9 for 14 days. The limbs were infected with indicated recombinant adenoviruses effectively at day 5. On histological examination, both BMP2 and Sox9 induced chondrocytes proliferation and condensation. However, only BMP2 induced chondrocyte hypertrophy and ossification. When the limbs were co-infected with AdBMP2 and AdSox9, the proliferating chondrocyte zone was expanded with no obvious expansion of hypertrophic chondrocyte zone”

“combined treatment of BMP2 and Sox9 had the largest length of proliferating chondrocyte zone, while BMP2 alone exhibited the largest length of hypertrophic chondrocyte zone”<-So you’d be taller if you just had BMP2 and not Sox9.  This link is supported by genes such as Twist1 which supress Sox9 but which overexpression increases height.

” Sox9 alone was insufficient to induce MSCs chondrogenic differentiation, but required other growth factors, such as Sox5, Sox6, IGF1, FGF or TGF-β”

“transient overexpression of Sox9 using adenovirus vector was insufficient to induce chondrogenic differentiation of MSCs.”<-So you need sustained expression of Sox9 to induce the initial chondrogenesis.

“exogenous overexpression of Sox9 in BMP2-induced osteogenic differentiation of MSCs showed a significant decrease in the levels of Runx2 expression, sequentially with delayed osteogenic differentiation, and endochondral ossification. Apart from overexpression of Sox9, silencing or removing Runx2 might achieve a similar outcome in BMP2-induced MSCs differentiation.”<-Alternatively inducing Runx2 after chondrogenesis has been inducted by Sox9 might be the right way to get the chondrocytes back on track to endochondral ossification.

Given that you start with an initial pool of progenitor cells  to form a growth plate(although the idea with LSJL is to create new progenitors), to maximize height growth you want to maximize the growth per progenitor cell via increasing expression of genes like CNP and Twist1.  After this pool is exhausted how much growth you get per cell is less important as any growth is better than zero so it’s more important to induce this initial expression of Sox9 to get the right kind of cells in the first place.

Here’s a study about Notch inhibiting Twist1 which inhibits Sox9:

Notch inhibits chondrogenic differentiation of mesenchymal progenitor cells by targeting Twist1.

“Notch inhibition of chondrogenesis acts via up-regulation of the transcription factor Twist1. Upon Notch activation, murine limb bud mesenchymal progenitor cells in micromass culture displayed an inhibition of chondrogenesis. Twist1 was found to be exclusively expressed in mesenchymal progenitor cells at the onset stage of chondrogenesis during Notch activation. Inhibition of Notch signaling in these cells significantly reduced protein expression of Twist1. Furthermore, the inhibition effect of NICD1 on MPC chondrogenesis was markedly reduced by knocking down of Twist1. Constitutively active Notch signaling significantly enhanced Twist1 promoter activity; whereas mutation studies indicated that a putative NICD/RBPjK binding element in the promoter region is required for the Notch-responsiveness of the Twist1 promoter. Finally, chromatin immunoprecipitation assays further confirmed that the Notch intracellular domain influences Twist1 by directly binding to the Twist1 promoter.”

“Twist1 is developmentally expressed in mesoderm-derived embryonic tissues and postnatally in adult mesoderm-derived mesenchymal stem cells, where it functions as a major regulator of mesenchymal cell differentiation”

“mRNA expression of both Col2a1 and Agc1 in NICD1-expressing cells was down-regulated at time points 3–7 days, in which NICD1 protein expression was highly expressed, suggest an inhibition of cartilage matrix synthesis at that stage.”