Tuesday, February 15, 2011

Organic Development of chondrocytes

One of the thoughts as to why humans cannot grow post fusion is due to the belief that chondrocytes cannot grow organically.  There are lots of stem cells present in the bone marrow, as can be seen by the large amount of red dots present in the bone marrow such as in the link shown above.  If you look at these diagrams of epiphyseal distraction, you can see that everything else ossifies aside from the hyaline cartilage growth plate line(there is ossification within the hyaline cartilage as a result of microfracture; growth plate injuries often create a bony bridge in the fracture site; even in the very gradual epiphyseal distraction method there was still some ossification).

The goal of lateral synovial joint loading is to increase interstitial fluid flow by using methods such as the table clamp to send mesenchymal stem cells to the hyaline cartilage growth plate line where they will undergo a chondrogenic lineage.  If you look at the rat's under LSJL slide(right click and click on view image to see it in it's entirety) you can see stem cells being captured and taken into the hyaline cartilage growth plate line in slide B.  Can chondrogenesis be induced organically despite growth plate fusion?

Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium. 

Interstitial fluid flow such as that created by using the table clamp or pressing on the epiphysis of the bone with the dumbell increases hydrostatic pressure. 

"This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells[LSJL encourages chondrogenic differentiation of stromal cells, stem cells are a subset of stromal cells] (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days[1 Hz means load for a duration of 1 second, 1 MPa is the type of pressure used in steam locomotives, standard atmospheric pressure is 0.1 MPa at sea level], or remained in identical culture conditions but without exposure to pressure. we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1)[Most of these genes are associated with chondrogenic differentiation with the exception of Runx2]. With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo." 

"MSCs can differentiate along the chondrogenic lineage in three-dimensional culture conditions in the presence of soluble regulatory factors such as members of the TGF-β family. Other chondrogenic influences on the differentiation of mesenchymal cells are certain types of mechanical loading. Hydrostatic pressure and compressive loading[lateral joint loading results in compressive forces against the chondrocytes as well as well] increased cartilage matrix accumulation in cultured MSCs and in embryonic limb bud cells in vitro."

"Without the addition of TGF-β, the effects of mechanical stimuli are unclear; in some studies mechanical stimuli alone was sufficient to induce skeletal progenitor cells to adopt the cartilage phenotype"

"Loaded scaffolds were exposed to 1 MPa of pressure at a loading frequency of 1 Hz for 4 h per day, as this loading regime had previously been found to be effective in inducing chondrogenesis."

"The duration of the experiment was 10 days, as the results of a pilot study indicated that chondrogenic matrix accumulation could be visualized at this timepoint."

It's unclear whether the stem cells remained in an hypoxic environment.

"the hydrostatic pressure protocol increased the mRNA expression of both collagen I and II"

This mixture could lead to fibrocartilage rather than hyaline cartilage the authors speculate.

This was the medium used in the study: "1.0 mg/mL insulin, 0.55 mg/mL transferrin, 0.5 μg/mL sodium selenite, 50 mg/mL bovine serum albumin, 470 μg/mL linoleic acid), 10 nM dexamethasone, 50 μg/mL l-ascorbic acid, 1 mM sodium pyruvate, 40 μg/mL l-proline, 0.584 g/L l-glutamine, and 100 IU each penicillin and streptomycin."

Most of this aside for example the dexamethasone is available in the human body.

Previously, we found that periosteal loading increased expression of TGF-Beta whereas dynamic loading of chondrocytes increased expression of aggrecan and type II Collagen.  Dynamic loading of chondrocytes did not increase expression of Sox9 which is very important in endochondral ossification.  In the LSJL, studies Sox9 was not upregulated, however, the scientists only studied gene expression by bone and not by cartilage.  This study found that TGF Beta1 was not upregulated in contrast to LSJL which found that TGF Beta 1 was upregulated.  A possible explanation for this is that the collagen sponges contained no periosteum.   

Bone contains a lot of Type I collagen which was the medium used for the stem cells in the study.  So that part of the study is easy to recreate.  Hydrostatic pressure is pretty synonymous with interstitial fluid flow so that part of the study can be recreated by LSJL.  One thing you'll notice is that the stem cells were not in the hyaline cartilage growth plate line.  This means that chondrogenesis can occur organically even without an open growth plate.

Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro.

"[What are] the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro? Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa[the amount of pressure used by a pressure washer] and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis." 

"Normal hMSCs (positive for CD105, CD166, CD29, and CD44 and negative for CD14, CD34, and CD45) from normal human bone marrow"

"Hip joint contact pressures up to 18 MPa exist during some activities, such as rising from a chair[but note this pressure is not applied within the epiphysis of the bone]"

"The effect of IHP on SOX9 expression may involve changes in cytoskeletal organization, G-protein activation, and transcription factor translocation into the nucleus."

Here's an example of what a stem cell looks like with intermittent hydrostatic pressure applied:

The HE and Saf-O refers to a form of staining.  TGF-Beta3 +IHP is better but IHP is good enough to induce chondrogenic differentiation.  "IHP applied with and without addition of TGF-Beta3 also increased gene expression for all three chondrogenic proteins[Aggrecan, Sox9, Type II Collagen]."

I can't really think of a good way to measure pressure for LSJL as all the pressure gauges are for tires and water level.

Stem cells are sensitive to mechanotransduction which makes their mechanosensitivity important.  Hydrostatic Pressure induces dynamic compression as it's the flow of the fluid that makes the compression dynamic.

Dose- and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro.

"MSCs were exposed to 0.1, 1, and 10 MPa of IHP at a frequency of 1 Hz for 4 h/day for 3, 7, and 14 days in the presence of transforming growth factor (TGF-beta3). Chondrogenesis was characterized by gene expression, macromolecule production, and extracellular matrix deposition. Exposure of hMSCs to 0.1 MPa of IHP increased SOX9 and aggrecan mRNA expression by 2.2- and 5.6-fold, respectively, whereas type II collagen mRNA expression responded maximally at 10 MPa. Production of sulfated glycosaminoglycan responded to IHP of 1 MPa and 10 MPa, whereas collagen levels increased only at 10 MPa. Morphologically, matrix condensation occurred with increased IHP, concomitant with collagen expression. Different levels of IHP differentially modulate hMSC chondrogenesis in the presence of TGF-beta3."

"hydrostatic pressure (50 MPa) [induces] heat shock proteins[HSP70]."  5MPa to cartilage increases TGFB3 levels.

"[Stem] Cells were positive for CD105, CD166, CD29, and CD44 and negative for CD14, CD34, and CD45."

Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells.

"[We] investigate the mechanotransduction of bovine bone marrow stromal cells (BMSCs) through the interactions between transforming growth factor beta1 (TGF-beta1), dexamethasone, and dynamic compressive loading. The addition of TGF-beta1 increased cell viability, extracellular matrix (ECM) gene expression, matrix synthesis, and sulfated glycosaminoglycan content over basal construct medium[Stimulating the other bone cells increases TGF-Beta1 which induces all these pro-chondrogenic effects]. The addition of dexamethasone further enhanced extracellular matrix gene expression and protein synthesis[dexamethasone is not needed]. There was little stimulation of ECM gene expression or matrix synthesis in any medium group by mechanical loading introduced on day 8. There was significant stimulation of ECM gene expression and matrix synthesis in chondrogenic media by dynamic loading introduced on day 16. The level of stimulation was also dependent on the medium supplements, with the samples treated with basal medium being the least responsive and the samples treated with TGF-beta1 and dexamethasone being the most responsive at day 16. Collagen I and collagen II gene expressions were more responsive to dynamic loading than aggrecan gene expression. Dynamic compression upregulated Smad2/3 phosphorylation in samples treated with basal and TGF-beta1 media. Interactions between mechanical stimuli and TGF-beta signaling may be an important mechanotransduction pathway for BMSCs, and they indicate that mechanosensitivity may vary during the process of chondrogenesis."

TGF-Beta1 affects mechanical sensitivity of bone marrow mesenchymal stem cells.

"TGF-β signals from the cell surface via a transmembrane serine/threonine kinase receptor complex. Upon ligand binding, the type II receptor subunit engages and transphosphorylates a type I receptor subunit (TβRI), which in turn phosphorylates the receptor-activated Smad proteins (R-Smads) Smad2 and Smad3[So we want TGF-Beta to be released from the osteoblast cell surface and then engage a type I receptor subunit in a mesenchymal stem cell]. A protein complex with Smad4 forms with the activated R-Smads and translocates into the nucleus, where the complex interacts with additional transcription factors, binding to the promoters of responsive genes and regulating their expression by cooperating with other activators or repressors. In addition to the Smad pathway, TGF-β  [activates] signaling pathways, including p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC). TGF-β signaling [activates] the p38 MAPK pathway through activation of mitogen-activated protein kinase kinase 1 and subsequent ERK/ELK signaling. G-protein-dependent activation of PKC results from TGF-β stimulation of growth plate chondrocytes. TGF-β responsiveness may require the activation of the R-Smad2/4 complexes[lack of response to LSJL may be a failure of the R-Smad2/4 complex], as well as other signaling pathways"

"dexamethasone induced Sox9 upregulation in the pluripotent mesoblastic C1 line"

TGF-Beta1 plus Dynamic Compression(hydrostatic pressure) = chondrogenic differentiation = height growth

Evidence that hydrostatic pressure can induce differentiation in vitro:

The mechanical environment of bone marrow: a review.

"Bone marrow is a viscous tissue that resides in the confines of bones and houses pluripotent stem cells. Due to its confinement by bones, the marrow has a unique mechanical environment which has been shown to be affected from external factors, such as physiological activity and disuse. The mechanical environment of bone marrow can be defined by determining hydrostatic pressure, fluid flow induced shear stress, and viscosity. The hydrostatic pressure values of bone marrow reported in the literature vary in the range of 10.7-120 mmHg for mammals[This is the value we are trying to increase with LSJL], which is generally accepted to be around one fourth of the systemic blood pressure. Viscosity values of bone marrow have been reported to be between 37.5 and 400 cP for mammals, which is dependent on the marrow composition and temperature. Marrow's mechanical and compositional properties have been implicated to be changing during common bone diseases, aging or disuse. In vitro experiments have demonstrated that the resident mesenchymal stem and progenitor cells in adult marrow are responsive to hydrostatic pressure[Lateral Loading can affect mesenchymal stem cells], fluid shear or to local compositional factors such as medium viscosity. Therefore, the changes in the mechanical and compositional microenvironment of marrow may affect the fate of resident stem cells in vivo as well, which in turn may alter the homeostasis of bone."

"The close proximity of marrow within medullary cavities of bones subject the marrow to physiological loads as well"<-Lateral loading can affect the bone marrow.

"When the marrow hematopoietic activity increases, adipose tissue undergoes resorption to provide more space, or vice versa."<-If Adipose Tissue is inversly related to bone marrow activity then fat gain may be bad for height growth.

"Bone nutrient vessels enter the marrow cavity to make connections with marrow vessels. Small arteries of marrow also enter the bone, make a loop and return back to where they originated from"<-So it could be possible for chondrogenic stem cells to travel to where they are needed.

"The fixed femurs of rats in vivo [had loads applied] ranging from 0 to 12.25 kg for 1 min. Pressure values [rised] two-fold (12–14 mmHg increase) upon loading."12.25 kg is about 25 lbs so 25lbs increases hydrostatic pressure by about 12-144 mmHG, a very useful measurement for performing LSJL.  Note that larger animals like dogs tend to have larger hydrostatic pressure values than mice.

"Contraction of the quadriceps muscles with electrical stimulation [resulted] in a pressure increase of 60 mmHg"<-So if you contract your muscles at the same as performing LSJL you can get a larger pressure gain.

"In a tubular bone, the nutrient artery enters the marrow cavity, runs longitudinally in the center, then branches out toward the endosteum of the surrounding bone, leading to specialized vascular structures known as sinuses or sinusoids. Several of these sinuses may then combine to form collecting sinuses which lead to the central sinus or vein. This vein runs longitudinally next to the nutrient artery. Blood in marrow flows from the center toward the bone and then returns back to the center. This structural configuration yields high numbers of vessels and sinuses in the periphery (resulting in a slower flow rate of blood and higher surface area) where most of the exchange occurs. Therefore, hematopoiesis is maximal in the closer proximities to the bone surface leaving the central parts with relatively little hematopoietic activity. Due to this fact, it is possible to observe a transition region between red marrow and fatty marrow radially (red marrow being closer to the endosteal surfaces. Similarly, a longitudinal macroscopic distribution is observed as red marrow in the proximal half and fatty marrow in the distal half of the bones. Bone and marrow are connected by vasculature. Bone nutrient vessels enter the marrow cavity to make connections with marrow vessels. Small arteries of marrow also enter the bone, make a loop and return back to where they originated from.."

"There are numerous in vitro studies subjecting marrow-derived mesenchymal stem cells to high hydrostatic pressures (750–75,000 mmHg) in an effort to induce chondrogenic phenotype. Even though the chondrogenic differentiation of the progenitor cells does not take place in marrow cavity[meaning they eliminate the variable of blood loss via veins], those studies are related in indicating the sensitivity of the progenitors to the magnitude and the mode (cyclic, intermittent or static) of loading. In general, higher pressure (∼75,000 mmHg) over lower (∼750 mmHg) and intermittent loading over static have proved to be more effective in chondrogenesis"<-That's quite a lot of pressure.

Here's what the author(Ozan Akkus) has to say chondrogenic differentiation not taking place in the marrow cavity:

"It is more of an opinion and the statement of an observation. We believe that this may have something to do, in part, with the mechanical environment of the marrow, that it is not conducive to chondrogenesis. Otherwise, there may be other factors in play, such as suppression by neighboring hematopoietic cells. As we know, marrow is highly vascular and cartilage is avascular. So the vascularity is suppressive to chondrogenesis."

"Layout of bone marrow in a cross-sectional view of a tubular bone. Bone (B) is surrounding the bone marrow (BM). Central artery (CA) and central vein (CV) are running parallel to each other and longitudinally along the long bone (perpendicular to the plane of the page). The central artery and central vein branch toward the periphery to form arterioles (A) and sinusoids (S) which then combine and join with the central vein. Hematopoietic space (H) is interspersed by the sinuses. Developing red blood cells and granulocytic cells appear in the hematopoietic space. Megakaryocytes develop subjacent to the endothelium of marrow sinuses. It is possible to observe the radial distribution of marrow as the yellow marrow in the central regions and the red marrow in the periphery. (b) A toluidine-blue stained section taken transversely to the longer axis of a tubular bone. The micrograph displays the endosteal junction between bone and marrow (125×). The distribution of abundant number of red blood cells indicates that the bone marrow is hematopoietic"<-this is the cortical bone however and we are looking to induce new growth plates in the epiphysis.

Dynamic compression stimulates proteoglycan synthesis by mesenchymal stem cells in the absence of chondrogenic cytokines.

"Dynamic compression was applied to agarose hydrogels seeded with bone marrow-derived adult equine MSCs. In the absence of the chondrogenic cytokine transforming growth factor beta (TGFbeta), dynamic compression applied for 12 h per day led to significantly greater proteoglycan synthesis than in unloaded TGFbeta-free cultures, although at a rate that was approximately 20% to 35% of unloaded TGFbeta cultures. These data suggest that the emergence of aggrecan dominated a chondrogenic response to loading as increases in proteoglycan synthesis. Cross-sectional analyses were conducted to subjectively identify potential spatial distributions of heterogeneous differentiation. In loaded samples, cell viability and metachromatic staining was low near the porous compression platen interface but increased with depth[the increase in depth relates to hydrostatic pressure as the more depth the larger the HP], reaching levels in the lower portion of the hydrogel that resembled unloaded TGFbeta cultures. These results suggest that the combination of high hydrostatic pressure and low dynamic strain and fluid flow had a stronger effect on chondrogenesis than did low hydrostatic pressure coupled with high dynamic strain and fluid flow. Next, the 12-h per day loading protocol was applied in the presence of TGFbeta. Biosynthesis in loaded cultures was less than in unloaded TGFbeta samples[so perhaps you don't want to increase TGF-Beta levels when joint loading]. Taken together, these data suggest that the duration of loading necessary to stimulate mechanoinduction of MSCs may not be optimal for neo-tissue accumulation in the presence of chondrogenic cytokines[but maybe higher levels of TGF-Beta just mean loading has to be applied for a shorter period of time]."

"quantitative extracellular matrix (ECM) synthesis studies have demonstrate that MSCs are capable of synthesizing neo-tissue on the order of that reported for chondrocyte culture."<-The MSCs in the bone marrow are capable of forming new growth plates(which is a chondrocyte culture).

"Dynamic hydrostatic loading[hydrostatic loading is performed by submerging a compound deeper under water], on the order of what occurs during routine joint functioning, has been proven to upregulate chondrogenic gene expression and cartilage-like protein synthesis in the presence of TGFβ."

"Experiments used a sinusoidal dynamic compression protocol of 2.5% strain amplitude superimposed on a 7.5% static offset strain at a frequency of 0.3Hz in displacement control; these loading parameters are within the physiological range of moderate, low-amplitude strain when applied to intact cartilage explants. At the initiation of loading, these parameters created maximum stress response of approximately 2kPa[a Kilopascal is 1000 of a MegaPascal, usually Hydrostatic Pressure is measured in MPa so the pressure range was well below typical Hydrostatic pressure].
For loading in the absence of TGFβ, two dynamic compression duty cycles were explored. The first protocol was defined by 6-h cycles consisting of 45-min periods of compression followed by 5h and 15min of free-swelling culture[I don't think we'd be able to mimic this cycle]. Each 6-h cycle was applied four times, followed by 24h of free-swelling culture such that loading was applied on alternate days. In the second protocol, 45min of dynamic compression was followed by 45min of free-swelling culture throughout the loading period, resulting in 12h of loading per day. In the presence of TGFβ, MSC-seeded agarose samples were loaded using the second, 12-h/d dynamic compression protocol."

"Given the low levels of biosynthesis in adult equine MSC cultures in the absence of TGFβ[In alternate day loading], these effects of dynamic compression were considered negligible."  So during LSJL, the loading likely has to be performed at least daily.

"H-proline and S-sulfate incorporation in dynamic compression samples were 20% of those of TGFβ+ samples (p < 0.05), whereas GAG accumulation in loaded cultures was 34% of that in TGFβ+ samples"<-this was twelve hours a day.

"Adjustment of the frequency of the applied loading from alternate day to daily loading, applied over 12 h/d, resulted in significantly greater proteoglycan synthesis than in TGFβ− cultures. These data suggest a chondrogenic response to loading because the emergence of aggrecan production, the major proteoglycan in cartilage, probably dominates S-sulfate incorporation and GAG accumulation. Therefore, the duration of the application of loading appeared to be a critical factor in stimulating proteoglycan synthesis, as occurs during chondrogenesis."<-So sustained loading may work in the presence of low pressure
"In the three dynamic compression samples, the zones near the chamber base contained the highest density of viable cells, suggesting that chondrogenesis was preferentially stimulated in deep zones of maximal hydrostatic pressure."

"The decrease in ECM synthesis and accumulation demonstrated an inhibitory effect of loading in TGFβ."

"Previously, finite element analyses have predicted compression-induced hydrostatic pressures of less than 1 kPa in agarose constructs compressed using near-physiological deformation levels. Although this value is significantly less than that measured in joints during normal activities, it is closer to the 0 to 3 kPa reported for continuous passive motion."<-Normal motion involves 0 to 3 kPa.  So, if you walked for 12 hours a day at 3 kPascals you might be able to grow taller?

We're not going to be able to perform loading for the periods of time in this study but hopefully we're performing our stimulus at a pressure much higher than 2 kPa so we can do the loading perhaps 1/500th of the time for 1 MPa.

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