Monday, October 24, 2011

Growing Taller by Increasing the Periosteal Width

We know that it's possible to increase the size of the periosteum.  Flat bones are not completely covered by periosteum but if the flat bone is in the right location it is people to use an increase in the size of the periosteum to increase height.  Some irregular bones and short bones are also partially covered by periosteum.  For example, the tips of the fingers may have some periosteum allowing you to get some increase in wing span there.

To date most studies have shown increases in periosteal width to be almost insignificant as a result of exercises. And, periosteal width increases tend to need more stimulus to increase than trabecular or cortical bone size. Sprinting increases the size of the periosteum of the tibia by putting lots of shearing and compression forces on the bone(shearing forces in a way cause microfractures on the periosteum).

The most likely way to effectively increase periosteal width is by the usage of heavy weights(such as deadlifts+deadlift variations) which means that growing taller via periosteal width is not the right method if per say a girl wants to maintain her secondary sex characteristics(her femininity).

A workable method to increase torso length can likely be found in some sort of LIPUS method by increasing hydrostatic pressure in a method similar to LSJL.  The vertebral bones do not have periosteum on the top and bottom.

Increases in periosteal width may play a role however by making you taller via the flat bone of the skull and the calcaneus. And, perhaps, if we optimize the exercises we perform we might be able to increase periosteal width by a lot more than 4%. Although, admittedly this is unlikely as sprinting is already incredibly effective at causing shearing forces on the periosteum of the tibia although the calcaneus may benefit from sprinting.

Growth Hormone, however, may be minorly effective on increasing body height via increasing periosteal width(on the flat bone of the skull) even if it cannot increase height on the long bones without a mutation.

This study shows that the periosteum can induce TGF-Beta which is a boon for chondrogenesis so the periosteum may have a secondary effect on height if not a primary one.

Coculture between periosteal explants and articular chondrocytes induces expression of TGF-beta1 and collagen I.

"Micromass pellets of human articular chondrocytes were cocultured for up to 28 days with human periosteal explants either with physical contact or separated by a membrane allowing paracrine interactions only. Quantitative reverse transcription (RT)-PCR, ELISA, immunohistochemistry and collagen isolation were used to analyse the expression and secretion of TGF-beta1, collagens I and II and chondrogenic differentiation markers such as MIA (CD-RAP) and aggrecan.
TGF-beta1 gene expression was induced significantly in paracrine cocultures in periosteum, whereas it was repressed in physical contact cocultures. However, a higher TGF-beta1 secretion rate was observed in physical contact cocultures compared with periosteal monocultures. The expression of COL2A1, melanoma inhibitory activity (cartilage-derived retinoic acid-sensitive protein) [MIA (CD-RAP)] and aggrecan was mainly unaffected by culture conditions, whereas COL1A1 gene expression was increased in periosteal paracrine cocultures. Collagen I staining was induced in micromass pellets from paracrine cocultures, whereas it was repressed in chondrocytes from physical contact cocultures.
We found evidence for a bidirectional regulating system with paracrine signalling pathways between periosteum and articular chondrocytes[extremely likely that this signaling pathway is shared by epiphyseal chondrocytes as well]. Stimulation of TGF-beta1 and COL1A1 gene expression in periosteal paracrine cocultures and the increased release of TGF-beta1 protein in physical contact conditions indicate an anabolic, and not merely chondrogenic micro-environment in this in vitro model for periosteal-based ACI."

"periosteum carries a thin proliferative cambium layer containing mesenchymal cells with chondrogenic and osteogenic potential which contribute to repair tissue formation"<-Periosteum is attached to the bone with sharpey's fibers too so it's possible that the periosteum contributes some mesenchymal cells for usage during LSJL and it's also possible that deformation of the periosteum during LSJL could lead to some of the height growth.

"While BMPs promote hypertrophy, signalling by TGF-βs favours a stable chondrogenic phenotype and inhibits or delays hypertrophy"<-TGF-Beta helps get stem cells to chondrocytes.  Chondrocytes formed in bone make you taller.

Influence of cyclic bending loading on in vivo skeletal tissue regeneration from periosteal origin.

"Periosteum osteogenic and chondrogenic properties stimulate the proliferation then differentiation of mesenchymal precursor cells originating from its deeper layers and from neighboring host tissues[controlling the periosteum is key to controlling the chondrocyte differentiation that we're trying to induce with LSJL]. The local mechanical environment plays a role in regulating this differentiation of cells into lineages involved in the skeletal regeneration process[we can alter this local mechanical environment with stimuli like LSJL].
The aim of this experimental animal study is to explore the influence of cyclic high amplitude bending-loading on skeletal tissue regeneration[LSJL likely applies some degree of bending and bending may generate some hydrostatic pressure so it may have similar effects to LSJL]. The hypothesis is that this mechanical loading modality can orient the skeletogenesis process towards the development of anatomical and histological articular structures.
A vascularised periosteal flap was transferred in close proximity to each knee joint line in 17 rabbits[so they're moving the periosteum to a location not seen in normal development]. On one side, the tibiofemoral joint space was bridged and loading occurred when the animal bent its knee during spontaneous locomotion. On the other side, the flap was placed 12 mm distal to the joint line producing no loading during bending. Tissue regeneration was chronologically analyzed on histologic samples taken from the 4th day to the 6th month.
The structure and mechanical behavior of regenerating tissue evolved over time. As a result of the cyclic bending-loading regimen, cartilage tissue was maintained in specific areas of the regenerating tissue. When loading was discontinued, final osteogenic and fibrogenic differentiation occurred in the neoformed cartilage[the periosteum resulted in new cartilage formation and the cartilage underwent osteogenic differentiation]. Fissures developed in the cartilage aggregates resulting in pseudo-gaps suggesting similar processes to embryonic articular development. Ongoing mesenchymal stem cells stimulation was identified in the host tissues contiguous to the periosteal transfer[the periosteum stimulated MSC development as well]."

Since the periosteum is so important to chondrogenic differentiation and mesenchymal stem cell stimulation, it's likely that increasing periosteal width has benefits as well.

"mechanotransduction modulates the metabolism and synthesis of immature cells as well as their differentiation into different cell lineages."<-LSJL involves mechanotransduction

"High local strain directs precursor cell differentiation into fibrous tissue. On the other hand, mild stress directs precursor cell differentiation into osteochondrogenic cells with direct ossification associated with weak hydrostatic stresses while cartilage growth is favored by higher compressive stresses"<-With LSJL we are going for highly compressive stresses with the clamp.  Direct osteochondrogenic growth likely results in no height growth as there is probably no chondrocyte hypertrophy or apoptosis which is likely what is responsible for the actual change in bone size.

"The mesenchymal precursor cells brought to the surgical bed by the periosteum and the host tissues proliferate before differentiating"<-the wider the periosteum likely the more mesenchymal precursor cells that are available.

"Significant proliferation of precursor cells constituting an undifferentiated blastema in the area of flap, and the first step in cell differentiation was found in both groups on the 4th day. In the “control” group, the development of neotissue was observed along the medial gastrocnemius. In the “loaded” group, it developed on the medial side of the knee, and remained separate from the intact joint capsule"

"After the 4th day, chondrogenic differentiation of mesenchymal precursor cells, which is a key step in enchondral ossification, was similar in both experimental groups. In the “control” group, a process of ossification of the neotissue matrix gradually replaced all of the cartilage with bone. Between the 15th and 30th day, all the cartilage had disappeared and was replaced either with bone or fibrous tissue. After the 30th day, a segment of long bone, whose mean length was identical to that of the flap (27–32 mm), had formed in the posterior compartment of the muscle[a new segment of long bone formed identical to the length of the periosteum, stretch the periosteum to grow taller?  Limb lengthening surgery does involve stretching the periosteum]. A medullary cavity had developed and usually included bone marrow. Osteoclasts were identified on the surfaces of newly formed bone. At 6 months, the regenerated tissue was composed of 90% bone and 10% fibrous tissue.
In the “loaded” group cartilage and fibrocartilage, differentiation continued until the 3rd month. The presence of cartilage was gradually limited to the ends and to the middle of the newly formed tissue[just like in endochondral ossification with the primary ossification center in the middle and the secondary ossification centers in the end at the epiphysis]. These areas extended to the initial junction with the support bone and to the tibiofemoral joint space, respectively. After the 3rd month, the newly formed skeletal tissue was detached from at least one of its points of attachment to the support bone. Knee bending no longer caused the regenerated tissue to bend. The cartilage had completely disappeared from the newly formed tissues. At 6 months, a bone segment with a medullary cavity had finally developed on the medial side of the knee. It barely interfered with articular range of motion because it was structurally separate from its initial support bone."<-so the formation of skeletal tissue adapts to movement which means that a method like LSJL which alters skeletal formation will not cause problems as the body adapts.  There were no knee bending problems despite the formation of new bone.

" In the earliest stage (4th day), lytic activity was observed in the tissues in contact with the transfer. This corresponded to necrosis of the superficial layers of muscle in immediate contact with the periosteum. This first stage was followed by a process of muscular regeneration which systematically resulted in complete repair without scar tissue in less than 14 days."<-muscle will adapt to the formation of new bone by remodeling.

"Variations in hydrostatic pressure influence the mechanisms that regulate the proliferation and differentiation of mesenchymal precursor cells. They stimulate proliferation in vitro, while in vivo, they redirect differentiation of precursors of bone tissue towards a cartilage phenotype[<-Why LSJL makes you taller].
Differentiation into chondrogenic cell lines is favored by a local mechanical environment associating high hydrostatic pressures and mild strains[In the LSJL rat study they used relatively low microstrain, maybe it's ideal for LSJL's effectiveness to minimize the microstrain while maximizing the hydrostatic pressure]. High amplitude strain inhibits angiogenesis thus influencing enchondral ossification"

"In our experimental protocol, loading of neotissue by cyclic bending generated a complex mechanical environment which could be described by numerous physical variables such as strain, variations in pressure or fluid as well as shear stress or movements at the cell/matrix and cell/cell interfaces"<-Cyclic bending generated changes in hydrostatic pressure just like LSJL.

"The structure and mechanical response of regenerating tissue evolves over time. As it matures, the regenerating tissue ossifies and mineralisation occurs so that it gradually becomes rigid. This process, which is incompatible with high amplitude knee movements, caused the regenerating tissue to break off from the anchor points of its support bone so that bending-loading no longer occurred[this shouldn't be a problem for us as we are striving for new cartilage formation in the epiphysis not the knee]. We then observed the disappearance of neocartilage, although it had been maintained until this event at the 3rd month. Thus, the process of enchondral ossification was interrupted, and the cells did not finish their differentiation into cartilage. Nevertheless the deep layer of the periosteum contains cell precursors which are engaged in chondrogenic differentiation, and which form cartilage during monoclonal cell cultures"

"the segments of new cartilage sandwiched between two ossifying structures were not in a physiochemical environment that favored the stability of the cartilage phenotype. The molecular constituents of the extracellular matrix send signals of differentiation to its mesenchymal precursor cells. Thus, although the environment of the articular cavity and the new cartilaginous tissue are chondrogenic, contact with the extracellular bone matrix directs precursors towards osteogenic differentiation"<-This is a problem with LSJL as the stem cells are in an extracellular bone matrix.

" the maintenance of the cartilage phenotype became dependent upon continued cyclic mechanical loading"<-so the frequency of LSJL may have to increase to maintain cartilage phenotype.

Conclusion:  The periosteum is a key source of mesenchymal precursor cells thus increasing periosteal width may help you indirectly grow taller.  The stem cells being activated in LSJL are in an extracellular bone matrix thus it may be necessary to load more frequently to maintain a cartilage phenotype.

Here's an article about direct hypertrophy of periosteal cells which would likely increase both periosteal width and length:

Remodeling of Actin Cytoskeleton in Mouse Periosteal Cells under Mechanical Loading Induces Periosteal Cell Proliferation during Bone Formation.

"The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo.
We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2[remember BMP-2 can help with chondrogenic differentiation as well], resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells[so altering the actin cytoskeleton of the periosteum affects the hypertrophy of the periosteum].
These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation."

"The periosteum is a membrane that lines the outer surface of all bones, except at the joints of long bones. This membrane, which consists of dense irregular connective tissue, is divided into an outer fibrous layer and an inner osteogenic layer. The fibrous layer contains fibroblasts, whereas the osteogenic layer contains the progenitor cells that develop into osteoblasts. In the observation of molecular and cellular phenomena acted by mechanical stress in vitro, the mechanical stress causes remodeling of cell-matrix adhesions, in which the cytoskeleton rapidly responds to external force by actin assembly"

" Upon detailed analysis, we observed that the mechanical loading rapidly decreased the quantity of stress fibers of the actin cytoskeleton and changed the nuclear shapes in the periosteal cells, and then disorganized actin cytoskeleton was remodeled in a time-dependent manner."<-Since mechanical loading decreases the number of stress fibers this could indicate a need for a deconditioning period to allow for new stress fibers to form.

"In addition, to identify the character of the hypertrophic periosteum, we performed anti-periostin antibody staining, since periostin is a typical marker of periosteum"

"At day 3, we found periostin to be expressed throughout the side opposite to the loading point in hypertrophic periosteum"<-So periosteum hypertrophies at the side opposite of the loading point.  So if we want periosteum to increase in length we need to load the ends of the periosteum.

"and this signal had decreased in intensity at day 7 because of the reduced area of periosteum by woven bone formation"<-Periosteum reduces itself when it forms new woven bone, this could explain why adults stop growing taller.  Periosteum reduces it's own area when it secretes new woven bone eventually periosteum is no longer next to the growth plate and growth plates need to be next to periosteum thus growth stops.

"The nucleus itself has been proposed to act a cellular mechanosensor, with alterations in nuclear shape causing conformational changes in chromatin structure and organization and directly affecting transcriptional regulation. By this machinery, extracellular forces can be transmitted across the cytoskeleton to the nucleus, resulting in intranuclear deformations; and the actin cytoskeleton is thought to provide protrusive and contractile forces and compressive bearing microtubules to from a polarized network allowing organelle and protein movement throughout the cell. In fact, compressive stress induces shape changes in chondrocyte nuclei; and collagen synthesis is strongly correlated with nuclear shape"<-So if we induce stress in a certain way we can change nuclei to be more chondrogenic.  This is done by causing hydrostatic pressure like with LSJL.

Direct transformation from quiescence to bone formation in the adult periosteum following a single brief period of bone loading.

"This experiment documents the direct transformation of the normal, quiescent, adult periosteum to active bone formation. The osteogenic stimulus was provided by a single short period of dynamic loading. Periosteal activation and the production of new bone within 5 days of loading was unaccompanied by resorption or the presence of osteoclasts. We therefore conclude that an adult resting periosteum can become directly converted to formation as a physiologic response to an appropriate osteogenic stimulus without the need for resorption. To distinguish this process from remodeling we suggest it be called renewed modeling. It is notable that a single short exposure to an "osteogenic" loading regime can influence the full cascade of cellular events between quiescence and active bone formation."

Aging changes mechanical loading thresholds for bone formation in rats

"The effect of aging on the mechanical loading thresholds for osteogenesis was investigated in rats. We applied mechanical loads varying from 30 to 64 N to the tibiae of 43 19-month-old rats using a four-point bending apparatus. Bone formation rates were measured on the periosteal and endocortical surfaces of the tibial midshaft using double-label histomorphometry. Bone formation rates from the old rats were compared with results from adult (9-month-old) rats that we reported earlier. Bone formation on the periosteal surface of the old rats was predominantly woven-fibered. Periosteal bone formation was observed in a lower percentage of the old rats compared with the younger adult rats for applied loads of 40 N and greater (59% old, 100% adult). However, in the old rats that formed woven bone there were no significant differences in woven bone area (p = 0.1) or surface (p = 0.24) compared with younger adult rats. Therefore, the periosteum of old rats had a higher threshold for activation by mechanical loading, but after activation occurred, the cells had the same capacity to form woven bone as younger adult animals. On the endocortical surface, relative bone formation rates in old rats showed a marginal (p = 0.06) increase in response to an applied load of 64 N but was not increased at lower loads. The relative bone formation rate in the old rats was over 16-fold less than that reported for the younger adult rats at an applied load of 64 N and the relative bone forming surface in old rats in this study was 5-fold less than it was in younger rats under similar loading conditions. In the younger adult rats, a mechanical threshold for lamellar bone formation of 1050 μstrain was calculated for the endocortical bone surface. The old rats required over 1700 μstrain on the endocortical surface before bone formation was increased. The data suggest that both the periosteal and endocortical surfaces of the tibiae of older rats are less responsive to mechanical stimuli."

Friday, October 21, 2011


Site-1 Protease Is Essential to Growth Plate Maintenance and Is a Critical Regulator of Chondrocyte Hypertrophic Differentiation in Postnatal Mice

"Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice. "

"During unfolded protein response, S1P is involved in the maturation of the endoplasmic reticulum membrane-bound transcription factors ATF6, old astrocyte specifically induced substance, and cAMP-responsive element-binding protein H"

"Arrows in H and K point to the SOC (also see P) that is pushed aside to accommodate the additional growth plate shown in P7–P28 mutants (P) with normal hypertrophic (h) cells (R). The arrow in R points to the dead, inactive primary growth plate. In the older P7–P42 mutant mice, this new growth plate allows for development of new trabecular bone (arrowheads in I, L, and T), which is now aligned with the cortical bone (arrow in I and T) due to bending of the distal femur."

Although the fact that this took place in embryos could mean that the mechanisms of this neo growth plate formation are not the same as those present in an adult.

Thursday, October 20, 2011

Increase Bone Length by Stretching?

I'm not talking about ordinary stretching as in the Grow Taller 4 Idiot's Program, Yoga, or Pilates.  I'm talking more about Sky from Easy Height's new limb center.  Before my research stumbled upon the lateral synovial joint loading system, I believed that the best way to gain height was by stretching the bones.  You see bone is elastic, by the very definition of microstrain theory the bone is constantly changing in length. If the bone did not change in length then over time bone density would decrease.  One thousand units of microstrain is equivalent to 0.1% change in length.  If our bones did not lengthen or compress by 0.1% every day they would fall into disuse according to mechanostat theory

The problem with stretches like the medieval rack or sitting/sleeping with ankle weights is that they stretch more the cartilage and ligaments than the bones themselves.  Sky of easy height was working on a program that stretched the bone.  Unfortunately, Sky disappeared before sharing his data.

If your tie a rope around the top of your ankle and then load the bottom of the rope with iron plates, your leg is being stretched down and out(being lengthened).  If you then tie a rope around the bottom of the ankle and then tie the middle of the rope around a bar; and then load that rope with iron plates your leg is being stretched upwards and out.  If you do both of these at the same time with equal amounts of force then the up and down forces cancel out and your bone is only being lengthened outwards.

You could also alternate between pulling your ankle up and down which would proceed to lengthen your bone in a zig-zag motion.  We know that bone is elastic and that bone has the ability to microfracture.  If you stretch your bone and microfractures occur in a stretched state then the bone should maintain some of that elasticity.  Pull a pencil apart, when you do microscopic damage occurs in the length of the pencil.  The pencil is now longer than before.  Unlike a pencil however, bone has the ability to heal those microfractures.  So you distract the bone cause microfractures, the microfractures heal, and then you distract the bone again.  Gradually, becoming taller and taller over time.

One element of Sky's multitude of experiments that he has kept in his new Shinbone Version 2011 is cycling with ankle weights(with a raised saddle).  The hypothesis of why cycling with ankle weights would work is that you are stretching your leg bone forcing your leg bone to reach lower and lower. Now if Sky does have a method of performing cycling to properly put a stretching force on the leg bone then it could work.

There are a couple of problems involved with the heavy iron plates method however.  You are putting load on your tendons and ligaments.  You have to find some way to nullify the tendons and ligaments.  If you perform this method say around the ankle then you are probably only going to stretch the tibia and not the fibula.  The iron plates method would probably best be used in parts of the limb where there is only one bone such as the femur and humerus.

Essentially, a bone stretching method could work.  In contrast to LSJL, you would want the load slightly below the growth plate(you only want to stretch your cortical bone in contrast to LSJL where you are trying to deliver red bone marrow stem cells into your growth plate).  You would then have to find a way to perform it without putting all the strain on your tendons/ligaments.  You would then have to find a way to equally push the bone down from the top and the bottom.  You would have to make sure that you were causing microfractures during the process.  If you read the last study, you can see why it may be worth it to perform bone stretching while doing LSJL.

Osteodistraction of the maxilla in transverse deficiency in adults: Analysis of the literature and clinical case.

"Osteogenic distraction is a bone regeneration and reconstruction technique. [Osteogenic distraction is] "the process of creating new bone by stretching"[bone stretching can best be analyzed by tensile strain, however this seems to be a different form of stretching]. Disjunction entails separating two anatomical structures at their junction system and, therefore, at a suture[so they're not stretching the bone they're separating the junctions between the bone]. Usually, it involves separating two semi-maxillae in the transverse dimension by means of an osteotomy[osteotomy refers to bone cutting so they are cutting the bone but now it seems like they are separating two separate bones and not cutting the bones itself]. Transverse maxillary distraction appears to offer an alternative of choice to orthognathic surgery alone, which is frequently prone to relapse. The greatest benefit of osteogenic distraction lies in its greater potential for expansion and concurrent growth of the soft tissues. Among other things, this technique increases arch length, thus precluding tooth extractions in cases of maxillary crowding, and appears to provide more stable results than conventional surgical intermaxillary disjunction."

If osteodistraction does work by stretching between the bones then perhaps something that stretches the cartilagenous area of the knee could help you to grow taller.

Effects of osteoinduction on bone regeneration in distraction: results of a pilot study.

"Rate and frequency of distraction as well as stimulatory effects transmitted by growth factors and local gene therapy have a decisive influence on bone regeneration. In a pilot study we tested the effect of four different morphogenetic and mitotic proteins and a genetically transferred vector system on bone healing in continuous osteodistraction in a large animal experiment on 24 Goettingen mini-pigs. For this purpose bone morphogenetic protein (BMP-2), BMP-7, TGF-beta, IGF-1 and a liposome vector were instilled into the distraction gap. The animals were killed after 1-4 weeks of consolidation. Histological and radiological evaluations showed maximum bone formation after the application of BMP-2/7, whereas the application of TGF-beta, IGF-1 and the liposomal vector had only a limited effect on bone regeneration. The quantitative analysis demonstrated an average amount of bone in the distraction gap of 50% and 61% after instillation of BMP-2 and 7, respectively. The BMP-2 expression, however, was maximal after induction with the non-viral vector. Only after BMP-2/7 application could physical, radiographic and histological evidence of bone union be detected. In bone distraction with a short observation period the application of morphogenetic proteins seems to enhance bone regeneration significantly. Before application in humans further studies are necessary to measure the dose-effect relationship, the mode of application and the efficacy of different inductive proteins. The combination of osteodistraction with osteoinduction, however, could shorten treatment times dramatically."

If you look in this picture you can see that the bone grew back without any compounds like BMP-2/7 or TGF-Beta1

A1 is one week after distraction A2 is two weeks.  So the bone does grow without chemicals and it also looks like there was no periosteum involved in contrast to distraction osteogenesis.  However the periosteal progenitor cells are capable of migration.

"Within the context of ossification, cellular elements with increased BMP-2 expression were found both in the distraction zone, and in the consolidated osseous area close to the osteotomy region. A reduced BMP-2 expression was found in the central distraction zones of those animals, where induction did not stimulate bone regeneration in the distraction region"<-so bone does not seem to form without BMP-2 in the distraction region in this study

"Labelled bone marrow stem cells are systematically mobilized and attracted to fracture sites from remote cell depots"<-Stem cells are still involved.  Also remember that a callus might generate hydrostatic pressure.

In the article on limb lengthening surgery, we learned that stretching Type I Collagen might cause a mechanotransduction based signal that results in the increase of the size of the micronuclei of bone cells thus causing bone hypertrophy.   This would mean that there would be no need for the fracture and that all you'd need is for tensile strain on Type I Collagen.

Limb bud mesenchyme cultured under tensile strain remodel collagen type I tubes to produce fibrillar collagen type II.

"In this work, we studied the effects of tensile strain on limb bud mesenchymal cells (MSC) cultured on a collagen type I tubular scaffold. A novel bioreactor was designed to culture the cells while subjecting the tubular scaffold to tensile stress and strain. Control samples included unseeded and MSC-seeded tubes cultured for 2 weeks under unloaded, no-strain conditions, and unseeded tubes subjected to prolonged tensile stress and strain. Mechanical properties of tube specimens were measured under oscillatory compressive stress. Following mechanical testing, scaffolds were fixed for immunohistochemistry or frozen for mRNA extraction. The storage modulii of both seeded/unstrained and seeded/strained tubes were significantly less than that of unseeded tubes, suggesting that MSC disrupted the structure and elasticity of the tubes' collagen type I. At a frequency of 1.0 Hz, the loss tangent of seeded/strained tubes was more than 2.5 times greater than that of seeded/unstrained tubes, and almost 6 times greater than that of unseeded tubes. Confocal microscopy and qRT-PCR results demonstrated that collagen type II and aggrecan expression was upregulated in the seeded/strained tubes.  Culture under tensile strain induces MSC to remodel the collagen type I tube with collagen type II and aggrecan expression into fibrils dispersed throughout the matrix[so basically tensile strain on the mesenchyme encourages removal of bone and the creation of cartilagenous, possibly growth plate like structures.  Note there is mesenchymal tissue in the bone marrow]. The seeded/unstrained tubes manifested less collagen type II with a more random expression pattern. Compared to seeded/unstrained tubes, qRT-PCR for collagen type II in the seeded/strained tubes showed a 4-fold increase in the message for collagen type II and a 13-fold increase in the message for aggrecan. These results demonstrate that MSC cultured for at least some period under tensile strain are able to remodel collagen type I scaffolds to produce a more viscous construct having many of the mechanical and biological features of engineered cartilage."

"In the knee, static compression of the joint creates hydrostatic stress, and movement of  the joint creates shear stresses. However, because the knee is a non-conformal surface, the cartilage will also experience a directional tensile stress and strain during use"<-We use static compression in LSJL to create hydrostatic pressure on the bone marrow of the epiphysis.

So tensile strain involved in limb lengthening surgery may stimulate cartilage formation by tensile strain on the mesenchymal tissue itself.  Here's the effects of tensile strain directly on the Type I Collagen.
Deformation-dependent enzyme mechanokinetic cleavage of type I collagen.

"Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%[but is inhibition of collagen cleavage good or bad for height growth?]. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsilon<3%), (2) a rapid decrease ( approximately 50%) in the transition of the toe-to-heel region (epsilon congruent with3%) followed by (3) a constant value throughout the heel (epsilon=3-5%) and linear (epsilon=5-10%) regions. This observation suggests that the mechanism for the strain-dependent inhibition of enzyme cleavage of the collagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule."

"Collagen degradation is a mechanism for extracellular matrix (ECM) remodeling and maintenance[the possibility for remodeling Type I Collagen into Type II Collagen which is the cartilage of the growth plate], and in response to trauma, disease and inflammation. Collagenases-1, 2 and 3 are the primary enzymes that act to degrade interstitial collagens (types I, II and III) in humans and animals. These collagenases are part of a larger family of enzymes (matrix metalloproteinases or MMPs) characterized by a zinc dependency for catalytic activity. MMPs are secreted by the cell as inert zymogens in response to the cell being activated by inflammatory cytokines, such as growth factors (interleukin-1) and mechanical loads. In order for collagen cleavage to occur, the collagenase (MMPs-1, 8 and 13, respectively) gains access to the collagen triple helix by binding to the enzyme’s attachment domain along the α-chains, followed by separation (unwinding) of the α-chains to expose the cleavage site, and then cleavage of the α-chain by the enzyme’s catalytic domain[tensile strain of 11% or greater(which means that the bone is stretched to 11% of it's original length) results in the in ability for collagen cleavage]. Collagenases contain two protein domains joined by a linker (hinge), a hemopexin C domain to which the collagen molecule attaches, and a catalytic domain responsible for the α-chain cleavage. MMP-1, 8 and 13 will cleave all three α-chains of interstitial collagens by a single scission at a specific site, located 3/4 from the N terminal and 1/4 from the C terminal, which is characterized by a Gly775-Ile776 or Gly775-Leu776 peptide bond, resulting in two fragments of the collagen molecule. Following this initial cleavage other MMPs (mainly gelatinases and stromelysins) can collectively further degrade the collagen fragments. However, the mechanism of the initial cleavage of the collagen molecule must originate with collagenase binding, triple helix unfolding and ¾-¼ scissoring."

"increasing tensile strain up to 4% (grip-to-grip) resulted in a decrease in the rate of enzymatic degradation, while strains above this (to 7%) caused an increase in the rate"<-Thus perhaps why limb lengthening surgery only stretches by 1mm a day which is well below 4%.

"mechanical deformation of type I collagen fibers caused by an axial strain (elongation) applied to the fiber will result in a significant decrease in the rate of collagen degradation by bacterial collagenase"<-This could cause height growth, if production of Type I Collagen outweighs degradation of Type I Collagen then bone could become longer.

It's possible that hydrostatic pressure is involved and that decreased cleavage results in increased hydrostatic pressure.

"Due to the self assembly nature of collagen, there appears to be a long range attraction which prevents molecules from coming too far apart and which induces the self assembly where hydrogen water bridges surrounding the molecule act as specific recognition sites for attracting other collagen molecules[So collagen attracts water to form water bridges]. More important, however, is that an exponential increase in the interaction energy (forces between triple helices) occurs as the α-chain separation distance decreases[as the Type I collagen fibers get farther apart the interaction cost increases]. Heightened hydration interaction forces are observed nearing the last 10-20 angstroms of α-chain separation (osmotic stress as high as 1000 MPa have been measured)[Increased hydration interaction forces due to increased distrace results in increased hydrostatic pressure]. This increased interaction force is [possibly] due to the energy required to rearrange the hydrogen bonding network near the molecular surfaces of macromolecules, such as might occur as the collagen molecule’s diameter is reduced as the molecule is stretched in response to an axial tensile load[tensile strain lowers the collagen molecule's diameter thus increasing the energy for hydrogen bond interaction]. Alterations in the ionic strength will also effect electrostatic interactions (<1 MPa at 15-60 Å). When the collagen molecules come into close proximity the van der Waals forces (<1 MPa at 10-25Å separation) result in an attraction or repulsion dynamic. Thus, a high concentration of repulsion ionic character will position the molecules further away from neighboring molecules creating a greater separation distance and ultimately a larger diameter. Conversely, attractive ionic character would link the molecules with a greater affinity, resulting in smaller diameters."

Stretching Type I Collagen lowers the diameter resulting in an increased energy required to rearrange the hydrogen bonding network.  This increases hydrostatic pressure.  So again it all leads to hydrostatic pressure.

So bone stretching may help during LSJL by lowering Type I Collagen fibril diameter thereby increasing hydrostatic pressure.

An exponential law for stretching–relaxation properties of bone piezovoltages

"Bone can change its mass, shape and density to adapt its external environment"

Ways to alter bone modeling include pizeoelectric potential, streaming potential, and fluid-generated shear stress.

"the piezoelectricity of bone arises from the organic components (mainly collagen)"

"Collagen molecules are filled and coated by platelet like tiny mineral crystals, which form the mineralized collagen fibril. A group of collagen fibrils embedded in the mineral crystals form a hierarchical structure of collagen fiber"

" Based on the hypothesis the deduced main reason for the stretching-exponential behavior is the triple helices structure of collagen fibrils distributed randomly in bone, which suffer relatively large deformation, under external loads, including self-deformations and relative slipping between molecule chains. The relative slipping movements may change the dielectric constants and resistances of bone, which leads to multiple relaxation time behaviors during deformation of bone."

Body height changes with hyperextension.

"The purpose of this study was to determine if the overall body height, as measured by a stadiometer, could be increased by brief episodes of hyperextension rather like a stretch that people frequently employ when arising. The subjects were loaded with 10 kg and the recovery with quiet sitting was compared to hyperextension 'stretches'. 15 s of hyperextension caused a significant temporary height increase [due to disc rehydration]."

"lifts up to 32 kg, and lateral bending with lifts up to 10 kg [were performed], the following parameters were estimated: lumbosacral angle and elongation, contribution of each lumbar segment to the lordosis reduction, relative pelvic/spine motion, and trunk velocity. "

-6.33mm was lost after 5 minutes of sitting.

"unloaded sitting with five hyperextensions resulted in an average height gain of +5.05 mm"

Exercise and the height of horses.

"The heights of 89 horses were measured at the withers before and after half a furlong of trotting exercise. The mean (+/- sd) height increase after exercise was 1.75 +/- 0.86 cm and the horses returned to their resting height within seven minutes. There was no linear relationship between gain in height and pre-exercise height."

Couldn't get this full study and it would be important before drawing conclusions.

Tuesday, October 18, 2011

Gain in Height by decreasing bone marrow fat content

It was once speculated that bone marrow turned to fat post epiphyseal fusion.  Later it was learned that that is based on nutrition and that if the bone marrow turns to fat then bone marrow can be restored with proper nutrition again.  Stem cell count decreases with age.  Since our goal with Lateral Synovial Joint Loading is to get stem cells to differentiate into chondrocytes(via hydrostatic pressure, which we induce by compression by a table clamp or dumbell) it behooves us to try to increase our stem cell count as much as possible.  How do we increase our stem cell count so we have more stem cells available for differentiation?  We already know that mechanical stimulation increases mesenchymal stem cell count.  One way is to discourage stem cells from differentiating into fat leaving more to differentiate into chondrocytes and osteoblasts. 

Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: Correlation with cytokines. 

"Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N=100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean=30.7, SEM=2) decreased and IL-6 levels (mean=4.4, SEM=1) increased with age as did marrow fat (mean=1.2mmfat/g, SEM=0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive." 

So increasing IGF-1 levels and decreasing IL-6 levels may increase stem cell count.  IL-6 is an inflammatory cytokine so it likely "kills" stem cells.  However, the last sentence of the study indicates that there is no correlation between IL-6 and IGF-1 and stem cell count and rather that it's some other process related to aging.  This could be something like Methylation Status or Telomere length[although the effectiveness of both is in question].  Supplementing with B-6, B-12, or Folic Acid will work for the former unless for some reason you are non responsive and then it would be best to take S-Adenosyl Methionine.  Telomere length involves supplements like Astragalus and you may increase telomere length with weight lifting also.  None of those will work unless your body is deficient as you can't make your body methylate cells it doesn't want to by supplementing with SAM-e[and SAM-e is an expensive experiment]. 

It's very likely that increased IL-6 and decreased IGF-1 levels are just the by-product of some pathway that happens to decrease MSC number rather than increased IL-6 reducing MSCs.

The pathophysiology of the aging skeleton. 

"In recent decades the population of both elderly men and women has grown substantially worldwide. Aging is associated with a number of pathologies involving various organs including the skeleton. Age-related bone loss and resultant osteoporosis put the elderly population at an increased risk for fractures and morbidity. Fortunately, in parallel our understanding of this malady has also grown substantially in recent years. A number of clinical as well as translational studies have been pivotal in providing us with an understanding of the pathophysiology of this condition. This article discusses the current concepts of age-related modulation of the skeleton involving intrinsic factors such as genetics, hormonal changes, levels of oxidative stress[IL-6, TNF-alpha], and changes in telomere length, as well as extrinsic factors such as nutritional and lifestyle choices. It also briefly outlines recent studies on the relationship between bone and fat in the marrow as well as the periphery." 

"There has been much recent interest in the relationship between fat and the aging skeleton. This is very important because both bone-forming osteoblastic cells and fat-forming adipocytic cells arise from a common progenitor in the marrow. Another exciting and recent area of investigation is the central regulation of bone mass and how this might be affected by age. Finally, a number of recent studies point to telomere shortening and its association with age-related bone loss."<-So osteoblastic cells compete with adipocytic cells for progenitors more than chondrogenic cells although adipocytic cells may still leave a smaller pool of progenitors for potential chondrocytes.

"With aging, hematopoietic tissue is replaced by fatty bone marrow, with consequent reduction in osteoblast number and function. Further, there appears to be a predominant differentiation of MSCs into adipocytes at the expense of osteoblasts"<-Again this likely applies to chondrocytes as well but the effect is not being reported.

"More recent studies found that a higher percentage of body fat was associated with a higher risk for osteoporosis, osteopenia, and non-spine fractures, as well as an inverse relationship between fat mass and bone mass after adjusting for the mechanical loading effects of body weight has been reported"<-so overall fat mass does reduce the available pool of bone marrow progenitors for chondrogenic and osteoblastic activities.  This can be reversed by mechanical loading somewhat but it's better to maximize mechanical loading and ensure that the amount of fat is not so great as to strikingly inhibit osteoblastic and chondrogenic stem cell differentiation.

PPARγ: a circadian transcription factor in adipogenesis and osteogenesis. 

"Peroxisome proliferator-activated receptor γ (PPARγ) is a critical factor for adipogenesis and glucose metabolism, but accumulating evidence demonstrates the involvement of PPARγ in skeletal metabolism as well. PPARγ agonists[agonist means that it activates], the thiazolidinediones, have been widely used for the treatment of type 2 diabetes mellitus owing to their effectiveness in lowering blood glucose levels. However, the use of thiazolidinediones has been associated with bone loss and fractures. Thiazolidinedione-induced alterations in the bone marrow milieu-that is, increased bone marrow adiposity with suppression of osteogenesis-could partially explain the pathogenesis of drug-induced bone loss. Furthermore, several lines of evidence place PPARγ at the center of a regulatory loop between circadian networks and metabolic output. PPARγ exhibits a circadian expression pattern that is magnified by consumption of a high-fat diet. One gene with circadian regulation in peripheral tissues, nocturnin, has been shown to enhance PPARγ activity. Importantly, mice deficient in nocturnin are protected from diet-induced obesity, exhibit impaired circadian expression of PPARγ and have increased bone mass. This Review focuses on new findings regarding the role of PPARγ in adipose tissue and skeletal metabolism and summarizes the emerging role of PPARγ as an integral part of a complex circadian regulatory system that modulates food storage, energy consumption and skeletal metabolism." 

PPAR-lambda may be what's involved in "turning" bone marrow into fat.  PPAR-lambda's effects are augmented by a high-fat diet and a gene called nocturnin.  So, you can inhibit PPAR-lambda by not eating a high fat diet.  Also, ways of decreasing the expression of nocturnin will decrease the number of MSC's turning into fat. 

The Effects of Native and Synthetic Estrogenic Compounds as well as Vitamin D Less-Calcemic Analogs on Adipocytes Content in Rat Bone Marrow. 

"We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17betaf0 (E2) modulate bone morphology in rat female model. Aim: We now analyze the effects of phytoestrogens, E2, SERMs and the lesscalcemic analogs of vitamin D: JKF1624F2-2 (JKF) or QW1624F2-2 (QW) on fat content in bone marrow (BM) from long bones in ovariectomized female rats (OVX). Materials and Methods: OVX rats were injected with treatments known to affect bone formation, 5 days per week for 2.5 month for analysis of fat content in BM. Results: In OVX young adults there is a decreased bone formation and a 10 folds increase in fat cells content in BM. Treatment with E2, raloxifene (Ral) or Femarelle (DT56a) resulted in almost completely abolishment of fat cells content. Daidzein (D) decreased fat cells content by 80%, genistein (G) or biochainin A (BA) did not change fat cells content and carboxy BA (cBA) had a small but significant effect. JKF or QW did not affect fat cells content, whereas combined treatment of JKF or QW with E2 resulted in complete abolishment of fat cells content. These changes in fat cells content are inversely correlated with changes in bone formation." 

So, Vitamin D will help prevent bone marrow from turning into fat. 

One way to increase stem cell count is to reduce bone marrow fat content.  To do that you can lower the amount of fat in your diet and make sure you have enough Vitamin D.  You can also increase stem cell count by mechanical stimulation and various supplements.

Bone marrow fat content may not be the only thing that changes but also the sub-populations of the bone marrow itself.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging.

"Life-long hematopoiesis depends on the support by mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment[if we can alter the composition of the stromal cell microenvironment than we can reverse some of the changes that occur during development and aging]. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271brightCD146- and CD271brightCD146+ subsets correlates with donor age. The main subset in adults was CD271brightCD146-, whereas the CD271brightCD146+ population was dominant in pediatric and fetal bone marrow[increasing the amount of CD271brightCD146+ population may help increase the differentiative potential of stem cells into chondrocytes]. A third subpopulation of CD271-CD146+ cells contained colony-forming-unit-fibroblasts in fetal samples only[Thus fetal marrow has even better differentiation potential with CFUFs in the subpopulation of CD271-CD146+ cells]. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which the subpopulations may have different functions."

"Mesenchymal stromal cells (MSC) cultured from adult and fetal tissues constitute a heterogeneous cell population. Although a panel of markers, including CD105 (Endoglin) and CD90 (Thy-1), was introduced to define cultured MSC, the cells initiating the culture remain unidentified. Recently, the low-affinity nerve growth factor receptor CD271 and melanoma cell adhesion molecule CD146 were described for prospective isolation of MSC with colony forming unit-fibroblast capacity."<-The presence of CD271 and CD146 means that the MSC population has good differentiation capabilities.

"These subsets had a similar capacity to differentiate and to support hematopoiesis, but their localization in human BM was different. CD271+/CD146-lo cells were bone-lining, while CD271+CD146+ had a perivascular localization."<-perivascular means surrounding a blood vessel.  Adults have more of the bone-lining stem cells which would explain why there's more apopsitional growth.  Whereas youthful individuals have perivascular stem cells which will enable more interstitial(height gaining) growth.

"CD271+CD146-/lo and CD271+CD146+ respectively localize to endosteal or perivascular niches in vivo"<-Thus even if there's growth stimulation for a perivascular region a bone lining cell will still localize to a bone-lining(appositional thereby non-height gaining region).  Thus, perivascular localizating bone marrow may be essential for height growth.

Alternatively, the number of subpopulations may be a function of need.  There is far perivascular growth without growth plates thus it makes sense that there are less mesenchymal stem cells localized to perform perivascular growth.

Microarchitectural Changes in the Aging Skeleton

"The main cortical age-related change is increased porosity due to negatively balanced osteonal remodeling and expansion of Haversian canals, which occasionally merge with endosteal and periosteal resorption bays, thus leading to rapid cortical thinning and cortical weakening."

"Periosteal porosity contributes to age-related weakening of the skeleton. It results from negatively balanced osteonal remodeling, which leads to a progressive increase in the volume fraction of osteonal (Haversian) canals (cortical porosity) and thus to significant deficits in cortical mineralized matrix and decreased resistance to fracturing, as intracortical porosity accounts for about 70% of elastic modulus and 55% of yield stress . Neighboring canals progressively increase in size and merge to become super-osteons."

Note the large amount of adipose tissue.

Spontaneous chondrogenic differentiation

Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system.

"Bovine bone marrow mesenchymal stem cells (MSCs) cultured in condensate culture, spontaneous and independent for any external biostimulants, undergo chondrogenic differentiation. In the present study, the bovine MSC chondrogenesis pathway was studied by analyzing stage-specific gene expression using quantitative "Real Time" reverse transcriptase polymerase chain reaction (qRT-PCR). Results showed that bovine MSCs underwent complete chondrogenesis; the initial stage was characterized by expression. of sox9 messenger ribonucleic acid (mRNA), followed by high transcription of chondrocyte specific genes, collagen type II and IX, biglycan and cartilage oligomeric matrix protein, and the final prehypertrophic and/or hypertrophic stage was distinguished by increased expression of collagen type X[LSJL induces expression of all of these except COMP]. From day 7 to day 14 of differentiation increased mRNA expression of the transforming growth factors beta1{upregulation is predicted} and beta2, basic fibroblast growth factor (FGF 2){up in LSJL}, bone morphogenic protein 6 (BMP 6), insulin-like growth factors 1, parathyroid hormone related peptide and indian hedgehog (Ihh) were detected. These results suggest that these well know chondrogenic growth factors may play a role in bovine chondrogenesis in autocrine and/or paracrine manner. On day 21 of the culture, FGF 2, BMP 6 and Ihh were highly expressed, compared to cells cultured in monolayer manner, which suggests a possible function in maintaining the terminal stage of differentiation. This data extends our knowledge about the unusual species-specific bovine MSC chondrogenesis, allowing us to define the phenotype of the differentiated cells."

"For inducing chondrogenesis in vitro, strong cell to cell interaction, growth factors or cytokines possessing chondrogenic potentiality and structure which support three dimensional cell orientations were reported to be necessary"

"Strong cell to cell interaction mediated by cell adhesion molecules such as N-cadherin and integrins allowed MSC conversion to prechondroblasts at the precartilage mesenchymal condensation stage during limb development.  This condition in vitro can be obtained by pellet or micromass culture systems."

"Bone marrow was aspirated from three calves months old.  The bone marrow
sample was washed twice with phosphate-buffered saline and twice with Dulbecco‘ s Modified Eagle Medium DMEM...Non-adherent cells were removed by changing the culture medium."

"MSCs from the first or second passage were used in the experiments. There were not notable differences on chondrogenic potential among cells from different passages, or reconstructed cells after deep freeze in liquid nitrogen. Within one or two days of samples preparations, cells formed compact pellet, which increased in size during culturing"

"Cells cultured in monolayer manner with serum free-chemically defined medium had bipolar to polygonal fibroblastic cell-shape and grew in uniform monolayer"

"[Cells grown in pellet culture] plump, round cell-shape, located in lacunae and surrounded with notable newly synthesized ECM"

"Cell shape and structure of the newly generated tissue had typical characteristics of chondrocytes and cartilage.  Only cells in the few periphery layer of the pellet had elongated , fibroblast-like shape ."

"Sox9 was firstly detected on day 7 and its expression increased in the following 7 days of culture, when reaches the maximum [expression], and then decreased at the terminal stage"

"over expression of [Sox9] in mouse MSC can enhance chondrogenesis, and that cell mediated sox9 gene therapy could be treatment for articular cartilage regeneration"

Monday, October 17, 2011

Tensile strain versus LSJL genes

LSJL causes dynamic tensile strain on the growth plates.  How much does dynamic tensile strain explain the effects of LSJL?

Cyclic tensile strain facilitates the ossification of ligamentum flavum through β-catenin signaling pathway: in vitro analysis.

"Histological, immunohistochemical, and real-time reverse transcription-polymerase chain reaction analyses of the expression of cell signaling and transcriptional factors in human ossification of ligamentum flavum (OLF).
To test the hypothesis that β-catenin plays a role in the ossification of OLF cells in response to cyclic tensile strain.
Several studies have investigated the roles of biomechanical and metabolic factors in the development and progression of OLF, based on the importance of genetic and biological factors. The process of ossification includes enchondral ossification.
Using real-time reverse transcription-polymerase chain reaction, we analyzed the mRNA expression levels of signaling factors known to be involved in the ossification process (β-catenin, Runx2, Sox9, and osteopontin) in cultured OLF cells subjected to cyclic tensile strain. Cyclic tensile strain was produced by Flexercell FX-3000 (Flexercell International, Hillsborough, NC), applied for 0, 6, 12, or 24 hours. The localization of these factors was examined in decalcified paraffin OLF sections by immunohistochemistry. Controlled samples were harvested from nonossified ligamentum flavum of patients who underwent thoracic posterior surgical procedures.
Under resting conditions (no tensile strain), the mRNA levels of β-catenin, Runx2, Sox9, and osteopontin in cultured OLF cells were significantly higher than in the control non-OLF cells. Application of cyclic tensile strain to OLF cells resulted in significant increases in mRNA expression levels of β-catenin, Runx2, Sox9, and osteopontin at 24 hours. Hypertrophic chondrocytes present around the calcification front were immunopositive for Runx2 and osteopontin. Immunoreactivity of β-catenin and Sox9 was strongly present in premature chondrocytes in the fibrocartilage area.
Our results indicated that cyclic tensile strain applied to OLF cells activated their ossification through a process mediated by the β-catenin signaling pathway."

"From a histological point of view, the progression of OLF correlates with enchondral ossification. Structurally, pathological specimens exhibit an ossification front, including a calcified cartilage layer, calcification front, and fibrocartilage layer, between the bone formation and the ligamentous fiber area."

Sox9 increased to 8 fold at 6 to 24 hour time points.

"non-OLF cells showed no immunoreactivity for [beta]-catenin, Runx2, Sox9, and osteopontin without strain but became positive for Sox9 after tensile strain application"<-Thus tensile strain may be able to induce ectopic chondrogenesis.

Effects of tensile strain and fluid flow on osteoarthritic human chondrocyte metabolism in vitro.

"Primary high-density monolayer chondrocytes cultures were exposed to varying magnitudes of tensile strain and fluid-flow using a four-point bending system. Metabolic changes were quantified by real-time PCR measurement of aggrecan, IL-6, SOX-9, and type II collagen gene expression, and by determination of nitric oxide levels in the culture medium. A linear regression model was used to investigate the roles of strain, fluid flow, and their interaction on metabolic activity. Aggrecan, type II collagen, and SOX9 mRNA expression were negatively correlated to increases in applied strain and fluid flow. An effect of the strain on the induction of nitric oxide release and IL-6 gene expression varied by level of fluid flow (and visa versa). This interaction between strain and fluid flow was negative for nitric oxide and positive for IL-6. These results confirm that articular chondrocyte metabolism is responsive to tensile strain and fluid flow under in vitro loading conditions. Although the articular chondrocytes reacted to the mechanically applied stress, it was notable that there was a differential effect of tensile strain and fluid flow on anabolic and catabolic markers."

" In porcine articular cartilage, cyclic tensile strain upregulates the catabolic mediators, nitric oxide (NO), prostaglandin E2, and MMP-1. Aggrecan and type II collagen were also upregulated at 3 h, but this effect was muted at all later time points (up to 24 h). One confounding variable of previous tensile strain loading systems is the unintended effect of fluid motion of the culture medium on the cells."

"The three anabolic markers (aggrecan, type II collagen, SOX9) were independently responsive to strain and fluid flow. The two catabolic markers (NO and IL-6) were responsive to the interaction of strain and fluid flow. NO production was also affected independently by strain and fluid flow."

The minimum microstrain here is 850 which is much higher than that generated in LSJL which was 20.

"For mechanical loading, each chondrocyte-seeded substrate was placed in a loading chamber containing 15 mL of serum-free medium. Homogenous cyclic tensile strain was applied to the chondrocyte-seeded substrate by using a custom-made four-point bending device, which was driven by computer-controlled linear actuator assembly with an interface controller to regulate vertical displacement and displacement rate"

Enhancement of nitric oxide and proteoglycan synthesis due to cyclic tensile strain loaded on chondrocytes attached to fibronectin.

"Cyclic tensile strain was applied to bovine articular chondrocytes. PG and NO synthesis were determined by [35S] sulfate incorporation and chemiluminescence analysis, respectively. To determine the expression of inducible NO synthase (iNOS), quantitative RT-PCR was used.
Enhanced PG and NO synthesis were evident when cyclic tensile strain was applied to chondrocytes seeded on fibronectin-coated plates. When NO production was inhibited, PG synthesis was further enhanced.
Cyclic tensile strain loaded on the chondrocytes enhanced NO synthesis and this enhanced NO inhibited PG synthesis."

"a low frequency and magnitude of cyclic tensile strain applied to chondrocytes increased proteoglycan (PG) synthesis, a high frequency and magnitude of strain decreased its synthesis"

"In articular cartilage, NO production is increased in chondrocytes exposed to fluid shear stress. Static and intermittent compression loaded on articular cartilage explants enhanced NO production"

a5b1 is the major fibronectin receptor in chondrocytes and the major mechanoreceptor.

"there is a linear relationship between the vacuum level (kPa) and maximal percent elongation of cells. In this study, the frequency (10 cycles per minute, i. e. three seconds of strain followed by three seconds of relaxation) and magnitude (10 kPa, 7%) of strain"

NO production was low until 12 hours.

According to Mechanical Stress inhibits chondrogenesis through ERK-1/2 phosphorylation in micromass culture, mechanical stress can inhibit chondrogenesis on embryonic like stem cells.

Mechanical Stretch Induces ERK1/2 Phosphorylation in Micromass Culture

"Our previous study demonstrated that tension inhibited chondrocyte differentiation through integrin mediated cell-extracellular matrix adhesion in micromass culture derived from embryonic rat limb bud cells. In the present study, we investigated the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in micromass cultures under a hypothesis that stretch induces ERK-1/2 phosphorylation in short term immediately after force loading. 
Embryonic limb bud cells were isolated from embryonic day 12 Sprague-Dawley rats. Dissociated cells by 0.25mg/ml collagenase and 0.25mg/ml trypsin EDTA were assembled to micromass culture on a Flexcer cell plate. After two drops of 50μl of 4.66x106 cells/ml suspension were plated around the center of wells of the dish and maintained for 5 hours in a CO2 incubator at 37°C, 2ml of 10% fetal bovine serum supplemented DMEM was added. After 4 days, micromass cultures were stretched for 0.5, 1.0, 1.5, 3.0, 6.0 and 12.0 hours prior to the isolation of protein samples. Western blot analysis for phosphorylated and non-phosphorylated ERK-1/2 was performed on samples from non-stretched control and stretched cultures. 
The expression level of ERK-1/2 was maintained to be constant level throughout the experimental period in rat limb bud derived micromass cultures. Phosphorylation of ERK-1/2 increased and peaked at 1.0 hour after stretch loaded, while basal level of the phosphorylarion of ERK-1/2 was maintained in the control non-stretched culture. 
Signaling through ERK-1/2 was activated by stretch-based shearing stress in micromass cultures and was suggested to be involved in the inhibition of chondrogenesis by shearing stress generated by stretching."

Molecular mechanisms of the response to mechanical stimulation during chondrocyte differentiation

"During embryonic development, mesenchymal stem cells congregate along with fibronectin and tenascin in the location where future long bone will be formed in the immature tissue-specific ECM. Once cellular condensation begins using these ECM molecules as a scaffold, the cells in the aggregation begin to express cell–cell adhesion molecules such as N-cadherin and N-CAM. After cellular condensation, mesenchymal cells start to differentiate into chondrocytes by firstly expressing the transcription factors Sox-5, -6, and -9, which regulate the gene expression of the phenotypic genes, Col2a1 and aggrecan. By producing these cartilage-specific ECM macromolecules, cell shape changes from ovoid to round, and polarity is obtained by arranging the positions of the nucleus and a lipid storage area in the cytosol, and the intercellular space is enlarged through the progression of chondrocyte differentiation. Further hypertrophic differentiation continues as endochondral bone formation progresses during growth. Chondrocytes terminally differentiate into hypertrophic chondrocytes expressing the Col10a1 gene and induce calcification of the cartilaginous matrix, which is later replaced by bone."

" a recent study revealed the structural changes that occur in integrin-related molecules after mechanical stimulation lead to the activation of downstream signaling mediated by the MAPK pathway and/or small GTPases such as Cdc42, Ras, and Rho"

"Mechanical stretching directly activates ERK-1/2, but not JNK or p38 MAPK. The phosphorylation of ERK-1/2 even increased under the inhibition of protein production, which can be interpreted as showing that the signal was transferred to the nucleus after the cell had sensed it."<-LSJL increases p38 MAPK phosphorylation which could be why ERK1/2 was not chondroinhibitory in LSJL.

Intermittent Cyclic Mechanical Tension-Induced Calcification and downregulation of ankh gene expression of end plate chondrocytes.

"Intermittent Cyclic Mechanical Tension (ICMT) was applied to end plate chondrocytes by using an FX-4000T Flexercell Tension Plus unit (Flexcell International Corporation, Hillsborough, NC). 
Rat end plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) was applied for 25 days, 4 hours a day and continued to culture for 5 days. End plate chondrocytes were incubated for 12 hours in the presence or absence of 10 ng/mL of transforming growth factor-β1 (TGF-β1) (prepared from a stock solution at 10 μg/mL in 2 mM citric acid containing 2 mg/mL bovine serum albumin) in MEM/F-12 containing a final concentration of 1% FCS. End plate chondrocytes calcification was stained by alizarin red S (AR-S). End plate chondrocytes viability was examined by LIVE/DEAD viability/cytotoxicity kit.
LIVE/DEAD assay verified that the nonloading (NC) group and the ICMT group end plate chondrocytes remained adherent, with no change in viability after the application of ICMT. Alizarin red staining showed that ICMT induced the calcification of end plate chondrocytes. Real-time reverse transcription-polymerase chain reaction showed that mRNA expression of endogenous TGF-β1 decreased and mRNA expression of type I, type X, osteocalcin and osteopontin increased after ICMT. The ankh gene expression of both mRNA and protein levels decreased in the ICMT stimulation. The ankh gene expression of both mRNA and protein levels increased in TGF-β1 stimulation. Compared with NC group, the alkaline phosphatase activities significantly increased in ICMT group.
Our results directly showed that ICMT induced the calcification and downregulation of ankh gene expression of end plate chondrocytes, which may be caused by the endogenous TGF-β1."

"One of the main pathways for nutrients to reach the avascular nucleus pulpous is by diffusion from the blood supply of the vertebral body through the end plate cartilage. The end plate cartilage is a layer of hyaline cartilage lying between the vertebral body and the intervertebral disc. End plate calcification could impede the passage of nutrients from the blood to the intervertebral disc and lead to the alterations in mechanical material properties of disc and make the end plate fail to maintain the nucleus pulposus and could also accelerate degenerative process of intervertebral disc."

"Many genes associated with cartilage calcification have been confirmed, such as COL9A2, COL9A3{up in LSJL}, AGCI, CLIP, TNAP, ANK, and transforming growth factor-[beta]1 (TGF-[beta]1)"

"A multipass transmembrane protein, ANK, seemed to function as an inorganic pyrophosphate (PPi) transporter or regulator of PPi transport, and PPi is a potent inhibitor of basic calcium phosphate (BCP) crystals formation. The ANK protein is a transporter able to export iPPi from the cells and is known to be upregulated in osteoarthritis."

Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells.

"We investigated mechanical stretch-regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry-type HMG box (SOX) 9 and α1(II) collagen (COL2A1) were assessed by real-time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch-related protein-DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA-binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretch-induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression."

"the total amounts of endogenous SOX9 were similar between non-stretched and 4-h-stretched cells"

"5% uniaxial CTS (0.5 Hz, 4 h) induced the highest expression of COL2A1 and SOX9 genes in human inner meniscus cells"

Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes.

"Chondrocyte-seeded alginate constructs were exposed to one of the two loading modes for a period of 3 h per day for 3 days. Gene expression was analyzed using real-time RT-PCR. Cyclic tension upregulated the expression of Cbfa1, MMP-13, CTGF, type X collagen and VEGF and downregulated the expression of TIMP-1. Cyclic tension also upregulated the expression of type 2 collagen, COMP and lubricin, but did not change the expression of SOX9 and aggrecan. Cyclic hydrostatic pressure downregulated the expression of MMP-13 and type I collagen and upregulated expression of TIMP-1 compared to the unloaded controls. Hydrostatic pressure may slow chondrocyte differentiation and have a chondroprotective, anti-angiogenic influence on cartilage tissue. Our results suggest that cyclic tension activates the Cbfa1/MMP-13 pathway and increases the expression of terminal differentiation hypertrophic markers."

"Sinusoidal strains of 9% peak-to-peak were applied to the alginate specimens"  HP was 5Mpa.  Both were loaded at 0.5Hz, 3 hrs per day for 3 days.

Effect of uniaxial stretching on rat bone mesenchymal stem cell: orientation and expressions of collagen types I and III and tenascin-C.

"Rat BMSCs were harvested from femoral and tibial bone marrow by density gradient centrifugation. Cells from passages 1-6 were characterized by flow cytometry using monoclonal antibodies. The recovered cells were stably positive for the markers CD90 and CD44 and negative for CD34 and CD45. A cyclic 10% uniaxial stretching at 1Hz was applied on rat BMSC for different time-courses. The length, width, and orientation of the cells were subsequently determined. Expression of collagen types I{up} and III{up} and tenascin-C mRNAs was measured by real-time RT-PCR, and the synthesis of these receptors was determined by radioimmunoassay. Results showed that uniaxial stretching lengthened and rearranged the cells. Compared with control groups, expression of collagen types I and III mRNAs was up-regulated after 12-h of stretching, while significant increase in synthesis of the two collagen protein types was not observed until after 24-h stretching. The expression of tenascin-C mRNA was significantly increased after a 24-h stretching. These data suggest that cyclic stretching promotes the synthesis of collagen types I and III and tenascin-C by the rat BMSC."

[Strain-induced tenogenic differentiation of bone marrow mesenchymal stem cells].

Both Scx and Tnmd were upregulated by LSJL.

"BMSCs were isolated by adherent culture from the bone marrow of 1-week-old SD rats. Inducing method of multiple differentiation and flow cytometry were applied to identify the cells. The stress-strain curve of SIS was measured with Instron machine. Purified BMSCs (2nd passage, 2.5 x 10(5) cells/cm2) were seeded on SIS (3 cm x 1 cm at size) and cultured for 2 days and then continued for another 5 days under strain stimulation (stretching frequency was 0.02 Hz, action time was 15 minutes/hour and 12 hours/day, strain amplitude was 5%) as experimental group, while the BMSCs-SIS composites were sustained static culture as control group. TCs were isolated from tail of 1-week-old SD rats. TCs-SIS composites were cultured under non-strained as positive control group. Scanning electron microscope (SEM) was used to examine the morphological changes of BMSCs after strain stimulation. The contents of Scleraxis and Tenomodulin in supernatant were tested by ELISA kit. Results The BMSCs could be induced to differentiate into osteoblasts and lipocytes, and showed the results of CD34-, CD45-, and CD90+, which were accorded with the biological characteristics of BMSCs. The failure test of SIS showed that the average elastic strain was 39.5%. SEM observation showed that the strain-stimulated BMSCs had the TCs-like morphological characteristics. The contents of Scleraxis and Tenomodulin in supernatant of experimental group, control group, and positive control group were (3.56 +/- 0.91) micromol/L and (4.27 +/- 1.10) micromol/L, (0.23 +/- 0.14) micromol/L and (0.16 +/- 0.10) micromol/L, and (14.73 +/- 2.30) micromol/L and (10.65 +/- 1.51) micromol/L, respectively. There were significant differences among 3 groups (P < 0.05)."

This validates that stretching plays a large role in LSJL response based on the effects of stretching on tendon related gene expression.

Bending loading produces tensile strain on the bone.

Mechanical stimulation alters tissue differentiation and molecular expression during bone healing.

"This study investigated the use of mechanical stimulation to promote cartilage rather than bone formation within an osteotomy. Retired breeder Sprague-Dawley rats (n = 85) underwent production of a mid-diaphyseal, transverse femoral osteotomy followed by external fixation. Beginning on postoperative day 10 and continuing for 1, 2, or 4 weeks, a cyclic bending motion (+35 degrees/-25 degrees at 1 Hz) was applied in the sagittal plane for 15 min/day for 5 consecutive days/week. Control animals experienced continuous rigid fixation. Histological and molecular analyses indicated that stimulation substantially altered normal bone healing. Stimulated specimens exhibited an increase in cartilage volume over time, while control specimens demonstrated bony bridging. Stimulation induced upregulation of cartilage-related genes (COL2A1 and COL10A1){both up in LSJL} and downregulation of bone morphogenetic proteins (BMPs) -4, -6 and -7. However, BMP-3 was upregulated with stimulation."

"a cyclic, axial, compressive displacement applied to a diaphyseal fracture or osteotomy enhances healing via formation of a larger cartilaginous callus and earlier bony bridging."

"in distraction osteogenesis, a series of tensile, step displacements is applied to the osteotomy gap to promote ossification and achieve bone lengthening."

"In fracture healing, less stable fixation induces expression of genes associated with chondrogenesis, cartilage extracellular matrix components, cell division, and inflammation."

"BMP-3 has been shown to antagonize the osteo-inductive effects of BMP-2."

Tensile strain may inhibit osteogenic genes like Runx2:

Continuous cyclic mechanical tension inhibited Runx2 expression in mesenchymal stem cells through RhoA-ERK1/2 pathway.

"CCMT[Continuous cyclic mechanical tension} [inhibits] the expression of osteogenic genes such as key transcription factor Runx2. RhoA regulates cell differentiation in response to mechanical stimuli. MAPK signaling acts as a downstream effector of RhoA. In MSCs, CCMT regulates the osteogenic master gene Runx2 through RhoA-ERK1/2 pathway. There is a decrease in RhoA activity after CCMT stimulation. Pre-treatment of CCMT-loaded MSCs with LPA, a RhoA activator, restores ALP activity and significantly rescues Runx2 expression, while pre-treatment with C3 toxin, a RhoA inhibitor, further decreases the activity of ALP and down-regulates the expression of Runx2. The inhibition of Runx2 expression after CCMT stimulation is mediated by RhoA-ERK1/2 pathway."

"Different from intermittent tensile loading, CCMT applies continuous stimulation throughout the study period, which could partially simulate mechanical overuse in vitro."  Intermittent tensile loading has been found to be pro-osteogenic.

CCMT also inhibited Osteopontin and Type I Collagen.  CCMT decreased ERK1/2 phosphorylation.

Tensile strain increases expression of CCN2 and COL2A1 by activating TGF-β-Smad2/3 pathway in chondrocytic cells.

"Physiologic mechanical stress stimulates expression of chondrogenic genes, such as multifunctional growth factor CYR61/CTGF/NOV (CCN) 2 and α1(II) collagen (COL2A1), and maintains cartilage homeostasis. Cyclic tensile strain (CTS) induces nuclear translocation of transforming growth factor (TGF)-β receptor-regulated Smad2/3 and the master chondrogenic transcription factor Sry-type HMG box (SOX) 9. CTS may induce TGF-β1 release and stimulate Smad-dependent chondrogenic gene expression in human chondrocytic SW1353 cells. Uni-axial CTS (0.5Hz, 5% strain) stimulated gene expression of CCN2 and COL2A1 in SW1353 cells, and induced TGF-β1 secretion. CCN2 synthesis and nuclear translocalization of Smad2/3 and SOX9 were stimulated by CTS. In addition, CTS increased the complex formation between phosphorylated Smad2/3 and SOX9. The CCN2 promoter activity was cooperatively enhanced by CTS and Smad3 in luciferase reporter assay. Chromatin immunoprecipitation revealed that CTS increased Smad2/3 interaction with the CCN2 promoter and the COL2A1 enhancer. CTS epigenetically stimulates CCN2 transcription via TGF-β1 release associated with Smad2/3 activation and enhances COL2A1 expression through the complex formation between SOX9 and Smad2/3."

The epigenetic stimulation of CCN2 may affect how applicable this could be to mesenchymal stem cells.

"immoderate cyclic tensile strain (CTS, 0.5 Hz, 10% strain) induces expression of catabolic factors, such as MMP-13 and ADAMTS-4/5/9, and inhibits expression of cartilage-specific ECM molecule α1(II) collagen (COL2A1) in human chondrocytic SW1353 cells. In addition, excessive CTS (10% strain) stimulates expression of MMP-3/13 and ADAMTS-5 in human articular chondrocytes. On the other hand, appropriate stretching force (5-6% strain) increases expression of anabolic factors, such as COL2A1 and multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, in human fibrochondrocytes derived from the inner meniscus and chondrocytic HCS-2/8 cells"

"CTS (0.5 Hz, 5% strain) induces the nuclear translocation of phosphorylated Smad2/3 and enhances Smad3-dependent CCN2 expression in inner meniscus cells"<-maybe this translocation can occur in MSCs.

Interesting that no CCN2 was detected above threshold in LSJL.

TGFB1 levels continued to increase with duration of tensile strain up to 120 minutes.

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