Wednesday, December 29, 2010

Height Growth and Osteosarcoma

An interesting tidbit about Chondrosarcoma, was that it tended not to occur in the long bones of adults possibly due to the shortened telomeres of mesenchymal stem cells there.  If we were to know that Osteosarcoma was possible to occur in the femur and tibia and other long bones than it is likely that telomere length is a likely limiting factor to increasing height after puberty.  It could be the reason why some individuals have reported growing taller from the GHenerate and IGH-1 combo due to the content of IGH-1(putting aside the lack of photographic evidence of this height increase for a moment).  Short telomeres inhibit stem cells from differentiating to chondrocytes so if you are older in age and are not growing taller with Lateral Synovial Joint Loading then consider supplementing with Astragalus Membranaceous.(Astragalus Membranaceous  ) What can Osteosarcoma tell us about height growth?

Epidemiological data for common bone sarcomas. 

"At the largest center for histopathology in Pakistan, we looked at three common bone sarcomas in our practice i.e. osteosarcoma, Ewing's sarcoma and chondrosarcoma. An overwhelming majority of osteosarcomas in our study occurred in the second and third decades of life; they were common in males; and femur, tibia and hip bone were the commonest bones involved accounting for an overwhelming majority of cases. The large majority of Ewing's Sarcomas in our study occurred in the first three decades of life; they were more common in males; vertebrae, tibia, femur and hip bone were the commonest sites. In our study, chondrosarcomas showed a wide range of age distribution and occurred quite commonly in the young[so maybe chondorsarcomas can occur in the long bones in later ages]. Except for the greater occurrence of chondrosarcoma in young patients; and comparatively less frequent involvement of upper limb bones, most of the bone sarcoma trends in our population appear to be similar to published western data." 

In this study there was the odd distribution that showed that osteosarcoma more than chondrosarcoma decreased in occurrence in the old versus the young.    Which is odd considering the telomere shortening that occurs with age as chondrogenic differentiation requires longer telomeres than does osteogenic differentiation. 

The epidemiology of osteosarcoma. 

"Osteosarcoma derives from primitive bone-forming mesenchymal cells[why we are interested in osteosarcoma; to study mesenchymal stem cell osteogenic differentiation] and is the most common primary bone malignancy. The incidence rates and 95% confidence intervals of osteosarcoma for all races and both sexes are 4.0 (3.5-4.6) for the range 0-14 years and 5.0 (4.6-5.6) for the range 0-19 years per year per million persons. Among childhood cancers, osteosarcoma occurs eighth in general incidence and in the following order: leukemia (30%), brain and other nervous system cancers (22.3%), neuroblastoma (7.3%), Wilms tumor (5.6%), Non-Hodgkin lymphoma (4.5%), rhabdomyosarcoma (3.1%), retinoblastoma (2.8%), osteosarcoma (2.4%), and Ewing sarcoma (1.4%). The incidence rates of childhood and adolescent osteosarcoma with 95% confidence intervals areas follows: Blacks, 6.8/year/million; Hispanics, 6.5/year/million; and Caucasians, 4.6/year/million. Osteosarcoma has a bimodal age distribution, having the first peak during adolescence and the second peak in older adulthood. The first peak is in the 10-14-year-old age group, coinciding with the pubertal growth spurt. This suggests a close relationship between the adolescent growth spurt and osteosarcoma. The second osteosarcoma peak is in adults older than 65 years of age; it is more likely to represent a second malignancy, frequently related to Paget's disease. The incidence of osteosarcoma has always been considered to be higher in males than in females, occurring at a rate of 5.4 per million persons per year in males vs. 4.0 per million in females, with a higher incidence in blacks (6.8 per million persons per year) and Hispanics (6.5 per million), than in whites (4.6 per million). Osteosarcoma commonly occurs in the long bones of the extremities near the metaphyseal growth plates[That's where the Mesenchymal Stem cells are]. The most common sites are the femur (42%, with 75% of tumors in the distal femur), the tibia (19%, with 80% of tumors in the proximal tibia), and the humerus (10%, with 90% of tumors in the proximal humerus). Other likely locations are the skull or jaw (8%) and the pelvis (8%).." 

The reason that osteosarcoma coincides with the pubertal growth spurt is due to the increase in cellular proliferation rates.  The faster cells are proliferating the more likely you are to be able to detect an abnormality.  So an increase in cellular proliferation may not cause the osteosarcoma but rather merely make it more prevalent. 

Characterization of human multicentric osteosarcoma using newly established cells derived from multicentric osteosarcoma.

"Human multicentric osteosarcoma (HMOS) is a rare, aggressive variant of osteosarcoma. We used newly established HMOS cells, which were derived from primary (HMOS-A) and secondary (HMOS-P) lesions, respectively, to perform a basic study analyzing the cellular biology and gene expression of HMOS.
We performed a cell growth assay, an invasion assay, DNA microarray analysis, quantitative real-time RT-PCR (Qrt-PCR), and a telomerase assay and compared the results between HMOS-A, HMOS-P, and human osteosarcoma (HOS) cell lines (MNNG-HOS and Saos-2).
The cell biological analysis revealed that HMOS-A and HMOS-P had similar characteristics to Saos-2, and the invasion assay showed that they had similar characteristics to MNNG-HOS. The DNA microarray study showed that the gene expression profiles of HMOS-A and HMOS-P were similar to that of MNNG-HOS, but the overexpression of MMP-2, MMP-9, and MT1-MMP was observed in HMOS-A and HMOS-P[so these genes are very powerful in changing the spread of bone], which was correlated with the invasiveness of the extracellular matrix[increase mmp-2, mmp-9, and MT1-MMP to increase extracellular matrix invasiveness which can and cannot be a good thing for height growth], and collagen type-4 (COL-4) and VEGF were also detected. HMOS-A and HMOS-P showed low telomerase activity similar to Saos-2, which are known to be telomerase negative, but a similar telomere length and telomerase protein to MNNG-HOS.
HMOS-A and HMOS-P demonstrated strong invasive ability, and their gene expression profiles correlated with the invasiveness of the extracellular matrix. Their telomerase activity was low, but they did not shown the typical features of alternative lengthening of telomeres (ALT)."

So the telomerase activity correlated with different types of Osteosarcoma and the invasiveness of the extracellular matrix.  However, low telomerase activity may mean that they already had sufficiently long telomeres but how would they keep telomere length long with no telomerase and no alternate lengthening of telomeres(the type of telomere lengthening that causes cancer, not astragalus).  So shorter telomere length increases the extracellular matrix invasiviness of bone cell types which could mean that shortening of the telomeres in the bone cells along the growth plate can result in fusion(invasion of bone cell matrix against the cartilage).  Increasing telomere length of bone cells can slow down fusion.

So there doesn't seem to be a correlation between age and chondrosarcoma versus osteosarcoma which is what we would hope for in stating that telomere length correlates with the chondrogenic potential of mesenchymal stem cells.  However, cancer usually involves Alternate Lengthening of Telomeres which is a confounding variable in terms of telomere length.  We do know that telomere length is extremely important though.

Astragalus and Puerarin(via the PI3K pathway) both upregulate telomerase activity.  Whether this is sufficient to achieve chondrogenic differentiation is unclear given that there are probably telomere binding proteins involved as well at a cellular level.

Osteosarcoma may contribute to height growth(of course the osteosarcoma and height growth can be caused by the same factor):

Relationship between height at diagnosis and bone tumours in young people: a meta-analysis.

"Some evidence exists that patients with osteosarcoma and Ewing sarcoma are taller than the general population[Ewing sarcoma and osteosarcoma differ by origin, Ewing sarcoma forms new bone by round cell origin whereas osteosarcoma by mesenchymal origin, neither type implies a chondrogenic intermediary which means that the tumors are not likely to increase height by endochondral ossification]. Relevant studies linking osteosarcoma and Ewing sarcoma with height at diagnosis were identified. 14 studies examined the height of patients with osteosarcoma or Ewing sarcoma. Meta-analyses on SD scores found patients with osteosarcoma were 0.260 SD (95% CI: 0.088-0.432) taller than the reference population (five studies). A meta-analysis on percentages found 62% (95% CI: 57%-67%) of patients were estimated to have a height above the median (six studies). Patients with Ewing sarcoma were 0.096 SD (95% CI 0.004 0.188) taller (four studies). Only one study reported the percentage of Ewing sarcoma patients with height above the median.The average height of patients with osteosarcoma, but not Ewing sarcoma, was significantly above the average height of the reference population by 2-3 centimetres. The observed differences indicate the involvement of pubertal longitudinal bone growth in osteosarcoma development while different biological pathways could be relevant for Ewing sarcoma."

Do the biological processes in osteosarcoma make you taller?  Or are there common causes between height gain and osteosarcoma?

Since Ewing sarcoma and Osteosarcoma have the same effect which is an increase in bone formation but have different origins mesenchymal versus round cell, it is likely that the changes in properties of the mesenchymal cell are causing the increase in height rather than the increase in bone formation.

"In dogs, where osteosarcoma is 40 to 50 times more common than in humans, an increasing risk of osteosarcoma has been seen with increasing weight and increasing height of the dog which persists even after adjusting for breed size"<-so increase in height may cause the osteosarcoma.  Since there are more cell divisions the taller you are the more likely for an abnormality to occur causing osteosarcoma.

If the height increase displayed by children with osteosarcoma is caused by changes in the properties of mesenchymal stem cells then it would be worthwhile to analyze the properties of MSCs in individuals with osteosarcoma to see if any of these can be applied to healthy individuals to increase height.

"An interesting link seems to exist between growth, particularly in adolescence, and both osteosarcoma and Ewing sarcoma. A particular bone tumour of a given cell type usually arose in a field where the homologous cells were most active and so osteosarcoma arose in the metaphysis which had abundant osteoclasts whereas round cell tumours (Ewing sarcoma) develop in the bone-free marrow cavity of the mid-shaft (diaphysis). Further studies on human and canine osteosarcoma suggested a link between growth and occurrence of these tumours. Price observed that there was an overall preponderance of osteosarcoma in males, with the mean age of occurrence later than in females. There was also a predilection of osteosarcoma for long bones of the lower limb with the mean age of occurrence later than osteosarcoma in the upper arm."<-Osteosarcoma develops in the bone whereas ewing sarcoma develops in the bone marrow thus osteosarcoma has more ability to increase height. More osteoclasts indicate increased likelihood of Osteosarcoma. It could just be that more cells equals a greater chance for a cellular process to go wrong.

The study also suggests that osteosarcoma could be linked to height growth than Ewing Sarcoma due to the fact that osteosarcoma is more likely to occur in the lower limbs than ewing sarcoma which incidence is distributed more evenly.  Lower limb growth is more associated with height.

This does not rule out the possibility that the pathology of Osteosarcoma can in some way contribute to height growth and some of the properties of Osteosarcoma can be used to safely induce adult height growth.

Mesenchymal chondrosarcoma: clinicopathologic study of 20 cases.

Mesenchymal chondrosarcomas managed to grow within the bone.  The problem is that they began to invade the soft tissues outside the bone.

Figure 20 is kind of what we're looking to induce with LSJL with chondrogenesis surrounding the bony trabaculae(pink region).  It doesn't look like endochondral ossification is being induced however.

"The histology of mesenchymal chondrosarcoma is typically characterized by a unique, biphasic pattern, composed of mesenchymal and chondrocytic components.The background of undifferentiated mesenchymal, small, round or spindled cells is interrupted by highly differentiated islands of hyaline cartilage. The primitive, round cell component often contains a hemangiopericytomatous vascular pattern, and the cartilage component may include sites of osteoid production, ossification, or calcification"

c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis.

"we established a mouse osteosarcoma (OS) model through overexpression of c-MYC in bone marrow stromal cells (BMSCs) derived from Ink4a/Arf (-/-) mice. Single-cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly different clones: highly tumorigenic cells, similar to bipotent-committed osteochondral progenitor cells, and low-tumorigenic tripotent cells, similar to mesenchymal stem cells (MSCs). It is noteworthy that both bipotent and tripotent cells were capable of generating histologically similar, lethal OS, suggesting that both committed progenitor cells and MSCs can become OS cells of origin. Shifting mesenchymal differentiation by depleting PPARγ in tripotent MSC-like cells and overexpressing PPARγ in bipotent cells affected cell proliferation and tumorigenic activity.  Differentiation potential has a key role in OS tumorigenic activity, and that the suppression of adipogenic ability is a critical factor for the development of OS."

"both WT and Ink4aKO BMSCs were positive for CD44, Sca-1 and Thy-1 (CD90.2), and were negative for CD45, CD31 and c-Kit. Under optimal differentiation conditions, both types of BMSCs were able to undergo adipogenesis, osteogenesis or chondrogenesis"

"The nuclei of the tumor cells located in these mature bone and cartilage formations were strongly positive for Runx2 and Sox9 immunoreactivity, respectively. In contrast, nuclei from immature parts of the tumor were negative for these genes. Moreover, immature tumor cells were enriched at the interface between normal tissue and OS, whereas osteogenesis and chondrogenesis were apparent mainly in the non-interfacial regions of the tumor"

Tuesday, December 28, 2010

Height Gaining with Hyaluronic Acid

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

"PRG4 is a marker for superficial zone chondrocytes"

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Saturday, December 25, 2010

Get a Taller Stature with TGF-Beta1

Getting a taller stature involves stem cells differentiating into chondrocytes.  Two ways to achieve that are:  Hydrostatic Pressure(LSJL) and LIPUS(Ultrasound)+TGF-Beta1.  LIPUS however does increase TGF-Beta synthesis.  Although one study of LIPUS, studied the effects of LIPUS on the Mandibular Condyle.  It reported that LIPUS on it's own(without TGF-Beta1) did not significantly affect cartilaginous layer thickness.  However, specific parts of that layer did change in thickness relative to before but the overall layer did not increase.  So it's unclear whether the increase in TGF-Beta1 induced by LIPUS is sufficient to induce mesenchymal stem cell differentiation.  You also need available mesenchymal stem cells that have sufficiently long telomeres also.

How does TGF-Beta work in endochondral ossification?  What are some ways we can manipulate TGF-Beta in order to get a taller stature? 

Smad signaling determines chondrogenic differentiation of bone-marrow derived mesenchymal stem cells: Inhibition of Smad 1/5/8P prevents terminal differentiation and calcification. 

"The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in TGF-β induced chondrogenic differentiation of bone-marrow derived mesenchymal stem cells (BMSCs) in order to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs[i.e. can you grow taller(preventing terminal differentiation) by manipulating Smad signaling pathways(which affect TGF-Beta)]. Terminally differentiated chondrocytes produced in-vitro by chondrogenic differentiation of BMSCs or studied ex-vivo during murine embryonic limb formation, stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex-vivo articular cartilage samples that lacked expression for MMP13 and collagen X only expressed Smad2/3P[So if there's only Smad2/3P there's no expression of MMP13 and Collagen X which are involved in terminal differentiation, this perhaps inhibiting Smad1/5/8Phosphorylation may help you grow taller]. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis[however both pathways are needed in early chondrogenesis, so for LSJL for example you would need both pathways but for a case like an active growth plate you would only want Smad2/3P]. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of MMP13, collagen X and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization." 

Many of the Smad pathways are involved in the phosphorylation of TGF-Beta.  The Smad2/3P is critical for proper bone deposition whereas Smad 1/5/8P is not essential and result in terminal differentiation.   Therefore, ways of inhibiting Smad 1/5/8 phosphorylation may be a mechanism of increasing height.   So you want to allow for Smad2/3P but not Smad 1/5/8P.  You want Smad 1/5/8 just not for it to be phosphorylated(deactivated).   However, with something like LSJL which involves early chondrogenesis you would want Smad 1/5/8P.

"This terminal differentiation of BMSCs may be advantageous for tissue engineering of bone through the endochondral route"<-And terminal differentiation may be needed for endochondral ossification.  But prolonging Smad 1/5/8P may help you grow taller longer.

"TGF-β signaling requires binding to complexes of type II and type I serine/threonine kinase receptors, followed by receptor-Smad phosphorylation at their C-terminus. The canonical TGF-β pathway is the Smad2/3 pathway. However, by use of an alternate type I receptor (ALK1 instead of ALK5) Smad1/5/8 is phosphorylated instead of Smad2/3. The Smad1/5/8 route is commonly known as the route activated by bone morphogenetic proteins (BMPs), also a member of the TGF-β superfamily and a very potent inducer of bone formation"  TGF-Beta normally is involved with Smad 2/3P whereas BMP-2 is involved with Smad1/5/8P.

"Although chondrogenesis and cartilage formation are achieved, it eventually leads to terminal differentiation of chondrocytes instead of the production of stable hyaline cartilage."<-So scientists have a way for endochondral ossification(which causes height growth) just not stable hyaline cartilage for the joint.

"both TGF-β and BMP are important for early chondrogenesis"<-TGF-Beta is upregulated by LSJL whereas there aren't definitive signs that BMP-2 is.  This could be a problem with LSJL effectivity.  Ways of upregulating BMP-2 may be needed to enhance LSJL effectiveness.

Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes.

"TGF-β isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-β receptors. Endoglin is a TGF-β co-receptor that binds TGF-β with high affinity in the presence of the type II TGF-β receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-β signalling receptors.
Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-β-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples.
Endoglin enhances TGF-β1-induced Smad1/5 phosphorylation and inhibits TGF-β1-induced Smad2 phosphorylation[Remember Smad1/5 phosphorylation is bad for height growth, and Smad2 phosphorylation is necessary for height growth therefore Endoglin is bad for height growth], Smad3-driven transcriptional activity and ECM production in human chondrocytes. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-β1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage.
Endoglin regulates the balance between TGF-β/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes." 

Endoglin is a receptor for TGF-Beta. Finding a way to inhibit Endoglin may be a way to make people taller.

"Accompanying [the] morphological changes [to a more fibroblastic morphology] is the loss of the chondrogenic (differentiated) phenotype or ‘chondrocyte dedifferentiation’ which is characterized by a decrease in type II collagen expression"<-So type II collagen expression is key to maintaining chondrogenesis.  Col2A1 can induce CNP as well.

E-selectin ligand-1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-beta. 

"TGF-beta signaling is a critical determinant of growth plate homeostasis.  TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus.  Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation."

Surprising, ESL knockout mice had shorter growth plates and height growth.  So ESL-1 is not something that can be specifically targeted for inhibition as TGF-Beta serves different functions at different stages of differentiation and ESL-1 function is needed to inhibit TGF-Beta signaling at certain growth plate stages.

"ADAMTSL2 mutations in geleophysic dysplasia patients have recently been reported to cause elevated TGF-β secretion and activity, leading to disproportionate short stature and brachydactyly in humans. In contrast, fibrillin1 mutations in Marfan syndrome exhibit increased TGF-β activity but result in tall stature. In earlier skeletal developmental stages, Esl1 is highly expressed in the perichondrium but at low levels in the cartilage. Similarly, fibrillin-1 and ADMTSL2 also exhibit strong expression in the perichondrium, where TGF-βs is abundantly synthesized, suggesting that the perichondrium is particularly important for production and regulation of TGF-β activity and regulation of the growth plate"-The perichondrium is a developing periosteum meaning that shear strain on the periosteum whether through LIPUS or LSJL may increase TGF-Beta activity. 

A role for age-related changes in TGFbeta signaling in aberrant chondrocyte differentiation and osteoarthritis. 

"We postulate that the dual effects of TGFbeta on chondrocytes can be explained by the fact that TGFbeta can signal via different receptors and related Smad signaling routes. On chondrocytes, TGFbeta not only signals via the canonical type I receptor ALK5 but also via the ALK1 receptor. Notably, signaling via ALK5 (Smad2/3 route) results in markedly different chondrocyte responses than ALK1 signaling (Smad1/5/8), and we postulate that the balance between ALK5 and ALK1 expression on chondrocytes will determine the overall effect of TGFbeta on these cells. Importantly, signaling via ALK1, but not ALK5, stimulates MMP-13 expression by chondrocytes. In cartilage of ageing mice and in experimental OA models we have found that the ALK1/ALK5 ratio is significantly increased, favoring TGFbeta signaling via the Smad1/5/8 route, changes in chondrocyte differentiation and MMP-13 expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. In this paper we summarize concepts in OA, its link with ageing and disturbed growth factor responses, and a potential role of TGFbeta signaling in OA development." 

Smad 2/3 phosphorylation is good whereas Smad 1/5/8P is bad for height growth.  ALK1 stimulates MMP-13 chondrogenic expression.  Remember, F-spondin reduced height by as much as 30% and F-spondin increased levels of MMP-13. 

Inhibiting MMP-13, Smad 1/5/8P is a way to get a taller stature. Stimulating Smad's 2/3P is also a way to get a taller stature.

TGF-β but not BMP signaling induces prechondrogenic condensation through ATP oscillations during chondrogenesis.

"Although both TGF-β and BMP signaling enhance expression of adhesion molecules during chondrogenesis, TGF-β but not BMP signaling can initiate condensation of uncondensed mesenchymal cells. ATP oscillations play a critical role in prechondrogenic condensation. Thus, the current study examined whether ATP oscillations are associated with the differential actions of TGF-β and BMP signaling on prechondrogenic condensation. The result revealed that while both TGF-β1 and BMP2 stimulated chondrogenic differentiation, TGF-β1 but not BMP2 induced prechondrogenic condensation. It was also found that TGF-β1 but not BMP2 induced ATP oscillations and inhibition of TGF-β but not BMP signaling prevented insulin-induced ATP oscillations. Moreover, blockage of ATP oscillations inhibited TGF-β1-induced prechondrogenic condensation[prechondrogenic condensation may be dependent on ATP oscillations]. TGF-β1-driven ATP oscillations and prechondrogenic condensation depended on Ca(2+) influx via voltage-dependent calcium channels."

"BMP signaling is more effective in condensed cells and has little effect on mesenchymal cells at low density"<-So BMP would likely have no effect on adult epiphyseal MSC's.

"TGF-β signaling drives Ca2+ oscillations by stimulating extracellular ATP signaling and modulating Ca2+ influx via VDCC and then TGF-β-driven Ca2+ oscillations subsequently generate ATP oscillations."<-This we can stimulate extracellular ATP signaling by other means to drive Ca2+ oscillations.

TGF-Beta may be able to induce significant height on it's own as shown by this mutation the resulted in a seven foot tall person.

Camurati-Engelmann disease: unique variant featuring a novel mutation in TGFβ1 encoding transforming growth factor beta 1 and a missense change in TNFSF11 encoding RANK ligand.

"We report a 32-year-old man and his 59-year-old mother with a unique and extensive variant of Camurati-Engelmann disease (CED) featuring histopathological changes of osteomalacia and alterations within TGFβ1 and TNFSF11 encoding TGFβ1 and RANKL, respectively. He suffered leg pain and weakness since childhood and reportedly grew until his late 20s, reaching 7 feet in height. He had deafness, perforated nasal septum, torus palatinus, disproportionately long limbs with knock-knees, low muscle mass, and pseudoclubbing. Radiographs revealed generalized skeletal abnormalities, including wide bones and cortical and trabecular bone thickening in keeping with CED, except that long bone ends were also affected. Lumbar spine and hip BMD Z-scores were + 7.7 and + 4.4, respectively. Biochemical markers of bone turnover were elevated. Hypocalciuria accompanied low serum 25-hydroxyvitamin D (25[OH]D) levels. Pituitary hypogonadism and low serum insulin-like growth factor (IGF)-1 were present. Karyotype was normal. Despite vitamin D repletion, iliac crest histology revealed severe osteomalacia. Exon 1 of TNFRSF11A (RANK), exons 2, 3, and 4 of LRP5, and all coding exons and adjacent mRNA splice junctions of TNFRSF11B (OPG), SQSTM1 (sequestosome 1), and TNSALP (tissue nonspecific alkaline phosphatase) were intact. His asymptomatic and less dysmorphic 5'11″ mother, also with low serum 25(OH)D, had milder clinical, radiological, biochemical, and histopathological findings. Both individuals were heterozygous for a novel 12-bp duplication (c.27_38dup, p.L10_L13dup) in exon 1 of TGFβ1, predicting four additional leucine residues in the latency-associated-peptide segment of TGFβ1, consistent with CED. The son was also homozygous for a single base transversion in TNFSF11, predicting a nonconservative amino acid change (c.107C > G, p.Pro36Arg) in the intracellular domain of RANKL that was heterozygous in his nonconsanguineous parents. This TNFSF11 variant was not found in the SNP Database, nor in published TNFSF11 association studies, but it occurred in four of the 134 TNFSF11 alleles (3.0%) we tested randomly among individuals without CED. Perhaps the unique phenotype of this CED family is conditioned by altered RANKL activity."

"[The] mutations in TGFβ1 seem to enhance TGFβ1 anabolic effects within bone by freeing TGFβ1 from the latency-associated-peptide"<-maybe we can do the same thing?

"His feet were remarkably small for his extreme height (shoe size 11)"

"His subnormal serum creatinine of 0.5 mg/dL (0.7–1.5 Nl) perhaps reflected low muscle mass. Plasma protein was slightly elevated at 8.7 g/dL (6.5–8.5 Nl). Serum total bilirubin was low at 0.2 mg/dL (0.3–1.1 Nl). Serum testosterone was deficient at 152 ng/dL (241–827 Nl) while follicle stimulating hormone was 1.6 IU/L (1.4–18 Nl for men), prolactin 5.2 ng/mL (2.1–18.0 Nl), and 17β-estradiol < 40 pg/mL (< 52 Nl for men). Serum random growth hormone was elevated at 2.17 ng/mL (0.01–0.97 Nl), but IGF-1 was low at 69 ng/mL (115–307 Nl), suggesting undernutrition. However, serum ferritin was 66 ng/mL (22–322 Nl)."

"Markers of bone turnover, including serum ALP, indicated rapid skeletal remodeling"

"A novel heterozygous TGFβ1 duplication in keeping with CED was identified in both individuals, but the son is also homozygous and his mother heterozygous for a missense change in TNFSF11 encoding RANKL."<-So maybe you need both mutations in RANKL and TGF-Beta to get the height gain.

"The pro-TGFβ1 precursor molecule dimerizes and undergoes proteolytic cleavage to separate the LAP and TGFβ1 moieties. However, the LAP(Latency-associated peptide) remains associated in the bone matrix with mature TGFβ1 until subjected to specific activation conditions. Release of LAP activates circulating TGFβ1 and presumably also TGFβ1 within bone."

"All 10 TGFβ1 mutations causing CED involve LAP; none are within TGFβ1 itself"<-So maybe you need to cause the release of LAP to get TGF-Beta1 to increase height.

The influence of delayed compressive stress on TGF-β1-induced chondrogenic differentiation of rat BMSCs through Smad-dependent and Smad-independent pathways.

"Col2α1 mRNA was increased by delayed dynamic compressive stress initiated at the 8th day of chondrogenic culture. The current work is to further study the possibility of using delayed mechanical stress to relay chondrogenesis initiated by exogenous TGF-β1. Mechanical stimulation was delivered from day 8 to day 14 of chondrogenic culture. It showed that delayed compressive stress not only stimulated gene expression and protein synthesis of chondrocyte-specific markers, but also stimulated the endogenous TGF-β1 gene transcription, protein expression and the subsequent activation even when exogenous TGF-β1 was discontinued. Furthermore, mechanical stress also promoted protein phosphorylation and nuclear translocation of Smad2/3, the TGF-β1 downstream effectors. Inhibition TGF-β with SB431542 significantly affected the stress-induced chondrogenic gene expression. In addition, phosphorylated-p38 {phosphorylated p38 is pro-chondrogenic} and RhoB{Rhobtb1 was downregulated by LSJL} were upregulated by delayed loading in a TGF-β-related manner. Phosphorylated-ERK1/2{p-ERK1 is anti chondrogenic} and Wnt7a were also increased, but in a TGF-β-independent way."

"TGF-β activates ERK, p38, RhoB, Wnt and PKC"

"p38 MAPK was activated by cyclic compressive force in a rapid and transient manner to mediate subsequent transcriptional regulation in chondrogenic differentiation of rat BMSCs"

"ompressive stress promoted gene expression of chondrogenic markers even in the absence of exogenous TGF-β1, and the stress-induced gene expression was affected by the TβRI{downregulated by LSJL} inhibitor, SB431542, in different degrees. The stress-induced mRNA level of Col2α1, Sox9 and Runx2 was respectively repressed to 72.4%, 61.6%, and 74.1% after being treated with SB431542 for 3 days. In contrast, the stress-induced gene expression of Aggrecan and Ihh was unaffected by SB431542"

" Inactivation of TGF-β1 by the specific inhibitor SB431542 abrogated the up-regulation of p-p38 and RhoB by dynamic stress at varying degrees. Mechanical stress also increased the levels of p-ERK1/2 and Wnt7a to certain extents, whereas p-ERK1/2 and Wnt7a were unaffected by SB431542 treatment"<-although downregulation of TGFBetaR1 could be part of the negative feedback mechanism to load and expression could be restored over time.

Different roles of TGF-β in the multi-lineage differentiation of stem cells

"Supplementation with TGF-β1 could initiate and promote chondrogenesis of synovium-derived stem cell (SDSCs), but TGF-β1 alone was insufficient to fully differentiate SDSCs into chondrocytes. However, HDAC4 overexpression can promote TGF-β1-induced SDSC chondrogenesis but inhibit chondrogenically differentiated stem cell hypertrophy"

"C-type natriuretic peptide/NPR-B signaling pathway was activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may be involved in glycosaminoglycan synthesis during this process in a dose-dependent manner"

" The chondrogenesis of trabecular bone-derived MPCs was initiated and maintained by TGF-β1 through the differential chondro-stimulatory activities of p38, ERK-1, and JNK. TGF-β1-mediated MAPK activation also controlled wnt-7a gene expression and WNT-mediated signaling through the intracellular β-catenin-TCF pathway, which probably regulated the expression of cell adhesion protein, N-cadherin"

Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells.

"This study investigated the role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10(-8) and 10(-7) M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10(-6) M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect."

So this study confirms that not only is TGF-Beta capable of inducing chondrogenic differentiation of epiphyseal bone marrow MSCs but that TGF-Beta stimulates CNP.

"CNP increases the number of chondrogenic condensations, induces the expression of N-cadherin, stimulates GAG synthesis, but does not alter the expression of the chondrogenic transcription factors Sox9, -5, and -6, or of the main ECM genes encoding collagen type II and aggrecan of mouse embryonic limb bud cells in micromass culture. "

Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway.

"This study investigated the effect of mechanical load on human mesenchymal stem cell (hMSC) differentiation under different exogenous transforming growth factor-beta1 (TGF-beta(1)) concentrations (0, 1 or 10 ng/ml).  Human MSCs were seeded into fibrin-biodegradable polyurethane scaffolds at a cell density of 5 x 10(6) cells per scaffold and stimulated using our bioreactor. One hour of surface motion superimposed on cyclic compression was applied once a day over seven consecutive days. Scaffolds were analysed for gene expression, DNA content and glycosaminoglycan amount. Addition of TGF-beta(1) in the culture medium was sufficient to induce chondrogenesis of hMSCs. Depending on the TGF-beta(1) concentration of the culture medium, mechanical load stimulated chondrogenesis of hMSCs compared to the unloaded scaffolds, with a much stronger effect on gene expression at lower TGF-beta(1) concentrations. With TGF-beta(1) absent in the culture medium, mechanical load stimulated gene transcripts and protein synthesis of TGF-beta(1) and TGF-beta(3). TGF-beta type I receptor inhibitor LY364947 blocked the up-regulation on TGF-beta(1) and TGF-beta(3) production stimulated by mechanical load, and also blocked the chondrogenesis of hMSCs. Mechanical load promotes chondrogenesis of hMSCs through TGF-beta pathway by up-regulating TGF-beta gene expression and protein synthesis."

LSJL stimulates the TGF-Beta pathway.

"Dimeric ligands of the TGF-β superfamily signal across cell membranes by assembling heterotetrameric complexes of structurally related serine/threonine–kinase receptor pairs, designated types I and II. TGF-β complexes assemble cooperatively through recruitment of the low-affinity (type I) receptor by the ligand-bound high-affinity (type II) pair. The type II receptor phosphorylates the type I receptor, which in turn activates type I receptor kinase activity"

"The loaded group was exposed to ball oscillation of ±25° at 1 Hz. Simultaneously, dynamic compression was applied at 1 Hz with 10% sinusoidal strain, superimposed on a 10% static offset strain, resulting in an actual strain amplitude of 10–20%. Mechanical loading was performed 1 hr a day over 7 consecutive days. The group of unloaded constructs served as controls. After 7 days in pre-culture and 7 days of loading the top 10% of the construct was used for gene expression analysis."

TGFB1 increased TGFB3, Sp7, Col1, Aggrecan, and Col2.  It decreased PRG4 expression.  Mechanical loading also increased TGFB3.

"The standard chondrogenic TGF-β1 level (10 ng/ml) [used to induce chondrogenesis] is non-physiological and artificially high. In this situation the effects of mechanical load on the chondrogenesis of hMSCs are masked, as seen by very small increase in the type II collagen (1.2-fold) and aggrecan (1.5-fold) gene level between control and loaded samples in our study"

"Dexamethasone is necessary in the process of chondrogenic differentiation stimulated by mechanical load. We performed an experiment with neither dexamethasone nor TGF-β1 in the culture medium. Mechanical load did not affect the gene expression profile except for 7.45-fold increase of PRG4 mRNA expression"

Shearing of synovial fluid activates latent TGF-β.

"TGF-β is synthesized in an inactive latent complex that is unable to bind to membrane receptors, thus unable to induce a cellular biological response until it has been activated. In addition to activation by chemical mediators, mechanical forces may activate latent TGF-β via integrin-mediated cellular contractions, or mechanical shearing of blood serum. Since TGF-β is present in synovial fluid in latent form, and since normal diarthrodial joint function produces fluid shear, this study tested the hypothesis that the native latent TGF-β1 of synovial fluid can be activated by shearing.
Synovial fluid from 26 bovine joints and three adult human joints was sheared at mean shear rates up to 4000 s(-1) for up to 15 h.
Unsheared synovial fluid was found to contain high levels of latent TGF-β1 (4.35 ± 2.02 ng/mL bovine, 1.84 ± 0.89 ng/mL human; mean ± radius of 95% confidence interval) and low amounts (<0.05 ng/mL) of the active peptide. Synovial fluid concentrations of active TGF-β1 increased monotonically with shear rate and shearing duration, reaching levels of 2.64 ± 1.22 ng/mL for bovine and 0.60 ± 0.39 ng/mL for human synovial fluid. Following termination of shearing, there was no statistical change in these active levels over the next 8 h for either species, demonstrating long-term stability of the activated peptide. The unsheared control group continued to exhibit negligible levels of active TGF-β1 at all times."

Will shear have the same impact in the bone marrow?

"In [the] latent complex, mature TGF-β 25 kDa peptide is linked non-covalently to a 70 kDa latency associated peptide (LAP), together forming the small latent complex (SLC). This complex may be disulfide-bonded to a latent TGF-β binding protein (LTBP, ∼180 kDa), constituting a configuration termed the large latent complex (LLC). In growth plate cartilage, both SLC and LLC are secreted by chondrocytes; the LLC can bind to the ECM via the LTBP "<-So shear strain can definitely activate the latent TGF-Beta in growth plate chondrocytes.

"matrix-bound latent TGF-β can be activated by stromelysin-1 (MMP-3){upregulated in LSJL} and collagenase-3 (MMP-13) released from cellular matrix vesicles during endochondral ossification"

In-vitro analysis of the expression of TGFbeta -superfamily-members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture.

"we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) and human mesenchymal stem cells (MSC) in cell culture"

"In dedifferentiating chondrocytes, the gene for TGFbeta1 was constantly expressed, while the gene for TGFbeta2 was never expressed. The genes for TGFalpha, TGFbeta4 and TGFbetai were activated with ongoing dedifferentiation. TGFbeta-receptor 3 was constantly expressed, while the genes for the TGFbeta-receptors 1 and 2 were never expressed."  TGFBeta3 was upregulated during dedifferentitation

"The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation [and were inactivated during chondrogenic differentiation], whereas the gene for LTBP3 was constantly expressed, and negative results were obtained for the gene for LTBP4. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation. During chondrogenic differentiation, the MSCs constantly expressed TGFbeta1, beta2, beta3 and beta4. LTBP1, LTBP2 and TGFbeta-R3 were downregulated. TGFbeta3, TGFbeta4, TGFbetai, LTBP1 and LTBP2 may assist the process of dedifferentiation, while TGFbeta1 and beta2 might not be involved in this process. Of the TGFbeta-receptors, only type 3 might be involved in dedifferentiation."

"TGFb3-production [during dedifferentiation] might be a signal to stimulate other cells or themselves in an autocrine way to redifferentiate."

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation.

"we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation. Transforming growth factor beta1 (TGF-beta1) served as the initiator of MC condensation. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-beta1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-beta1-induced condensation was CTGF dependent. Silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-beta1 was also found to be mediated by CTGF. CTGF, TGF-beta1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-beta1 induced MC condensation."

"when plated in micromass cultures under either TGF-β1 or BMP-2 stimulation, C3H10T1/2 cells condense and differentiate along the chondrocytic lineage"

"The addition of TGF-β1 and Col2α1 increased Sox9 expression in mesenchymal progenitor cells, while treatment with TGF-β1 and type I collagen (Col1α2) reduced Sox9 and Agc expression "

"CTGF stimulation alone or in conjunction with TGF-β1 cause a significant increase in Col1α2 expression both in vivo "

From tall to short: the role of TGFβ signaling in growth and its disorders.

"The acromelic dysplasia group is characterized by short stature, short hands and feet, stiff joint, and "muscular" build. Four disorders can now be ascribed to this group, namely Weill-Marchesani syndrome (WMS), geleophysic dysplasia (GD), acromicric dysplasia (AD), and Myhre syndrome (MS). Although closely similar, they can be distinguished by subtle clinical features and their pattern inheritance. WMS is characterized by the presence of dislocation of microspherophakia and has autosomal dominant or recessive mode of inheritance. GD is the more severe one, with a progressive cardiac valvular thickening, tracheal stenosis, bronchopulmonary insufficiency, often leading to an early death. AD has an autosomal dominant mode of inheritance, distinct facial and skeleton features (a hoarse voice and internal notch of the femoral head). Finally, MS is sporadic, characterized by prognathism, deafness, developmental delay, thickened calvarium, and large vertebrae with short and large pedicles. We first identified mutations in Fibrillin-1 (FBN1) in the dominant form of WMS and then mutations in A Disintegrin-like And Metalloproteinase domain with ThromboSpondin type 1 repeats 10 (ADAMTS10) in the recessive form of WMS.  ADAMTS10 [interacts with] FBN1. We then identified mutations in ADAMTSL2 in the recessive form of GD and a hotspot of mutations in FBN1 in the dominant form of GD and in AD (exon 41-42, encoding TGFβ binding protein-like domain 5 (TB5) of FBN1). Using a yeast double hybrid screen, we identified latent transforming growth factor-β (TGFβ) binding protein 1 as a partner of ADAMTSL2. We found an increased level of active TGFβ in the fibroblast medium from patients with FBN1 or ADAMTSL2 mutations and an enhanced phosphorylated SMAD2 level, allowing us to conclude at an enhanced TGFβ signaling in GD and AD. Finally, a direct interaction between ADAMTSL2 and FBN1 was demonstrated suggesting a dysregulation of FBN1/ADAMTSL2 interrelationship as the underlying mechanism of the short stature phenotypes. Using exome sequencing in MS probands, we identified de novo SMAD4 missense mutations, all involving isoleucine residue at position 500, in the MH2 domain. In MS fibroblasts, we found decreased ubiquitination level of SMAD4 and increased level of SMAD4 supporting a stabilization of SMAD4 protein. Functional SMAD4 is required for canonical signal transduction through the oligomerization with phosphorylated SMAD2/3 and SMAD1/5/8. We therefore studied the nuclear localization of mutant SMAD complexes and found that the complexes translocate to the nucleus. We finally observed a decreased expression of downstream TGFβ target genes supporting impaired TGFβ driven transcriptional control in MS. Short stature phenotypes [involve] TGFβ signaling. TGFβ signaling [is enhanced] in Marfan phenotypes[specifically via FBN1]."

Friday, December 24, 2010

The physics of growing taller via your growth plates

Height growth occurs primarily by chondrocyte hypertrophy.  Chondrocytes expand in size in both height and width making you taller.  Now the thing is that the growth plates are in between two parts of the bone:  the epiphysis and the diaphysis.  In growth plates under IGF-1, growth plate height became taller.  However, it doesn't really make sense that a hypertrophying chondrocyte could push apart two whole bones(the epiphysis and diaphysis) to make the whole bone longer.  In the weight loading of young chicks study, loading chicks inhibited bone growth.  However, the chicks were loaded 24/7 and thus there was no time for a distraction phase.  Compressive loading has been shown to result in catch up growth as long as there was a distraction phase afterwards(like sleep).

Hypertrophying chondrocytes cannot possibly push a very sturdy epiphysis away from a diaphysis.  However, chondrocytes are a lot more elastic than bones.  Imagine two pencils stuck together by glue.  If you pull the two pencils apart, you're not likely to increase the length of the pencils but you can increase the length of the glue.  Then this glue can ossify by endochondral ossification resulting in one really long pencil.  This distraction must be critical for growth as illustrated by the chicks where chicks could not grow much taller when distraction was prohibited by non-stop loading.

Take a look at the histology of rat growth plates under LSJL.  Be sure to click on view image to see the whole thing.  You can see stem cells acquiring a chondrogenic phenotype outside of the growth plate.  Especially in the middle at the top of slide B.  Meaning that LSJL can induce chondrogenic differentiation outside of the growth plate and telomere length(+other things) willing can induce endochondral ossification there.

Now what's the difference between a growth plate and an independent stem cell differentiating into a chondrocyte.  A gap in the bone?  If you look at the LSJL slides there really is no visible gap in the bone and the LSJL rats still managed to grow taller.  The issue is primarily the bone marrow which contains mesenchymal stem cells.  You can see that in the LSJL slides that the amount of bone marrow decreased at the bottom(but not in the center).  Also, if you look at the slides in epiphyseal distraction you can see a decrease in the number of stem cells and less bone marrow.

You can grow taller as a result of an independently differentiating chondrocyte as long as distraction is allowed.  The problem is bone marrow and number of mesenchymal stem cells.  So, the growth plate gap doesn't provide a physical limit it's the bone marrow.

Here's a study that illustrates what specific characteristics of the cartilage plate that may help in producing distraction forces:

Cartilage is held together by elastic glycan strings. Physiological and pathological implications 

"Animal shapes are maintained by connective tissue extracellular matrices (ECMs). ECM shapes depend on keeping collagen fibrils in the right places, held by regular frequent proteoglycan (PG) bridges attached at specific sites. The PGs carry anionic glycosaminoglycan (AGAG) ‘strings’ that span and determine interfibrillar distances, thus holding us together. I called these repeating structures ‘shape modules’. The strings are aggregated antiparallel chains of dermochondan, keratan and chondroitan sulphates (DS, KS and CS); stabilised by hydrophobic and H-bonds. Shape modules are elastic. AGAG/AGAG interactions break under stress and reform when the stress is removed and/or they contain an elastic sugar, L-iduronate (in DS).
Cartilage ECMs are also based on shape modules. Depots therein of aggrecan, the large PG which carries many chains of CS and KS, imbibe water, thereby exerting swelling pressure[Cartilage counteracts hydrostatic pressure]. External pressure forces this water into the elastic shape modules, from whence it is returned post compression. Cartilage anisotropic responses (along and at right angles to shape module axes) to compressive and tensile stresses are now interpretable. Inability to hold collagen fibrils together results in imbibition of excess water, fissuring and erosion, characteristic of this condition."

"Iduronate in DS[DS is a GAG] underwent a sudden extension of about 10% at a critical stress of about 200 pN. This change is reversible and can be repeated as long as the molecule remains stuck across the tensioners. Iduronate is thus an elastic unit, unique in this field although we observed similar behaviour by guluronate in alginate and galacturonate in pectates"<-Elastic sugars are able to extend thus helping to explain the height growth of endochondral ossification.

Endochondral ossification: how cartilage is converted into bone in the developing skeleton.

"Hypertrophic chondrocytes die, and as they do so, the transverse septa of cartilage matrix surrounding them are broken down, leaving vertical septa largely intact, but allowing entry of the invading cells of the ossification front: blood vessels, osteoclasts (multinucleate bone-resorbing cells), and precursors of osteoblasts (bone-forming cells) and bone marrow cells. The osteoclasts assist in the removal of cartilage matrix, and the differentiating osteoblasts use the remnants of cartilage matrix as a scaffold for the deposition of bone matrix."

"Light chondrocytes have sparse endoplasmic reticulum and an inconspicuous Golgi region. Dark chondrocytes, in contrast, have well developed endoplasmic reticulum and a prominent Golgi zone; they possess numerous cytoplasmic processes, with vesicles budding from the cell surface. We have recently provided evidence that light and dark chondrocytes represent two distinct post-proliferative chondrocyte populations, with different patterns of gene expression"

"Light cells appear to disintegrate within their cell membrane, whereas dark cells undergo progressive extrusion of cytoplasm into the extracellular space; for both cell types, nuclear condensation is late and irregular"

"The actin-binding protein adseverin has recently been shown to be expressed selectively by prehypertrophic chondrocytes as well as by hypertrophic chondrocytes. Overexpression of adseverin in non-hypertrophic chondrocytes induces changes in the actin cytoskeleton, a dramatic increase in volume and expression of molecular markers of hypertrophy."

"Tg737 gene encodes the primary cilium protein polaris. Kif3a [is] a subunit of the kinesin II motor complex which is required for intraflagellar transport and the formation of cilia"

"Carminerin is a transcriptional inhibitor of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which generates pyrophosphate by hydrolysing extracellular adenosine triphosphate analogues"

"Loss of Gli3{up in LSJL} in Ihh-null mice restores chondrocyte proliferation, allows reactivation of PTHrP expression and delays the accelerated onset of hypertrophic differentiation seen in Ihh-null mice."

"Mutations leading to constitutive activation of the PTH/PTHrP receptor are found in patients with Jansen-type metaphyseal dysplasia, which is characterised by short limbs associated with delayed chondrocyte hypertrophy"

"mice with targeted ablation of the gene for BMP receptor 1A in chondrocytes show an expanded domain of collagen type X expression"

"The C4st1 gene encodes chondroitin 4-sulphotransferase-1, which catalyses the sulphation at the 4-0 position of chondroitin and dermatan sulphate. Homozygous mice lacking the transmembrane and intra-Golgi catalytic domains of this enzyme display severe dwarfism and neonatal death"

"Nkx3/Bapx1 inhibits Runx2 expression and chondrocyte hypertrophy, and appears to be a mediator of the actions of PTHrP "

"Chondrocytes in developing bones of Mef2c-null mice fail to undergo hypertrophy; moreover, they fail to express Runx2, indicating that MEF2C acts upstream of Runx2 in the induction of chondrocyte hypertrophy. Chondrocytes from Mef2c-null mice also fail to express Col10a1, which is a direct transcriptional target of MEF2C. MEF2C and HDAC4 exert antagonistic effects on chondrocyte maturation."

"In most larger mammals (but not in rodents), blood vessels within cartilage canals provide nutrients to growth cartilage"<-This could lead to different effects between LSJL on mice and humans.

"Cathepsin L-null mice have defective metaphyseal ossification, but the cellular defect appears to be a lack of osteoclasts"

"FGF18 induces Vegf expression in chondrocytes, and in Fgf18-null mice, in which vascular invasion is delayed, Vegf expression in hypertrophic chondrocytes is reduced"

Development of the endochondral skeleton.

"Endochondral bone development begins with the condensation of mesenchymal cells of either neural crest in the craniofacial region (e.g., middle ear bones and temporal bones) or mesoderm elsewhere in the body"

"In the limb bud the condensation forms mainly through active congregation of cells without changes in cell proliferation. These condensations can be visualized by the dense packing of cells, the high affinity to the lectin peanut agglutinin, and the transient up-regulation of versican, tenascin, syndecan, N-CAM, and N-cadherin"

"BMP signaling was required for the coalescence of smaller aggregates into a tight cluster with a distinct outer boundary, a prerequisite step for chondrogenic differentiation. This event occurred independently of SOX9, because Sox9-null cells can coalesce, even though they subsequently segregate from the condensations and adopt a distinctive “fibroblastoid” morphology."

"SOX9 is dispensable for the initial formation, but necessary for maintaining the condensation."

"inactivation of both FGFRs 1 and 2 in the limb bud mesenchyme (with Prx1-Cre) results in smaller skeletal elements."

"In chick embryonic limbs and limb bud micromass cultures, ectopic expression of WNT1 or WNT7A inhibited chondrocyte differentiation but did not affect formation of mesenchymal condensations"

"The antichondrogenic role of WNT proteins may be mediated through β-catenin, as overexpression of a stabilized form of β-catenin in mouse embryonic limb mesenchyme resulted in a near complete loss of all limb cartilage elements. Continued exposure to WNT/β-catenin signaling appears to redirect limb mesenchymal cells to the soft connective tissue, but this process can be prevented by FGF signaling. WNT/β-catenin signaling also has further inhibitory action at stages after Col2a1 expression has been activated. Conditional overexpression of a stabilized β-catenin by Col2a1-Cre can lead to severe achondrodysplasia"

"Depicted is a longitudinal section through one of two growth plates of a mouse long bone during late embryogenesis (E15.5–E19). The growth plate at this stage is without a secondary ossification center and is organized into distinct domains as indicated. (1) IHH and PTHrP coordinate chondrocyte proliferation and maturation through a negative-feedback mechanism. IHH produced by pre- and early hypertrophic chondrocytes stimulates chondrocyte proliferation and PTHrP transcription through derepression of GLI3. PTHrP in turn suppresses chondrocyte maturation associated with IHH expression. Direct IHH signaling also regulates the formation of columnar chondrocytes from round chondrocytes (not depicted here). (2) FGF9/18 from the perichondrium suppresses chondrocyte proliferation and maturation. FGFR3 expressed in chondrocytes is a likely receptor for FGF9/18 to suppress proliferation in the growth plate late in embryonic development and during postnatal bone growth. FGF9/18 may use other yet-to-be-established mechanisms to suppress chondrocyte maturation in the early embryo. (3) BMPs expressed by both chondrocytes and perichondrial cells promote proliferation and maturation. (4) NOTCH signaling in chondrocytes promotes proliferation and maturation. (5) WNT5A expressed by prehypertrophic chondrocytes stimulates hypertrophy."

"NOTCH signaling may inhibit both mesenchymal condensation and subsequent chondrocyte differentiation."

"PTHrP appears to delay chondrocyte hypertrophy, mainly by activating cAMP-dependent signaling that both increases the activity of SOX9, and regulates the HDAC4–MEF2C complex"

"Like RUNX2, HDAC4 is expressed in the prehypertrophic and early hypertrophic chondrocytes. Genetic deletion of HDAC4, like overexpression of RUNX2, greatly accelerated chondrocyte hypertrophy. Conversely, overexpression of HDAC4 in all chondrocytes inhibited hypertrophy, mimicking the effect of RUNX2 deletion. Biochemically, HDAC4 physically interacts with RUNX2 and reduces RUNX2 binding to DNA. Thus, HDAC4 prevents premature hypertrophy of chondrocytes in part by directly suppressing RUNX2 activity."

"β-catenin signaling appears to function downstream from IHH, as β-catenin deletion does not impair IHH signaling in the perichondrium (containing osteoblast progenitors), whereas IHH removal abolishes β-catenin signaling in that compartment"

"Whereas RUNX2 deletion leads to a hypoplastic perichondrium, loss of OSX causes ectopic cartilage formation beneath a thickened perichondrium at the midshaft of long bones (where a bone collar normally forms)"

"(TWIST1, HAND2, ZFP521, STAT1, Schnurri 3, GLI3, HOXA2, and the HES/HEY proteins) have been found to suppress RUNX2 levels or activit"

A biochemical strategy for simulation of endochondral and intramembranous ossification.

"PTHrP is expressed in the articular perichondrium and Ihh is expressed in the centre of the hyaline cartilage. In this process, these molecular factors diffuse through the bone, where Ihh reaches its highest value in the distal area and PTHrP in the proximal area. Both Ihh and PTHrP are present throughout the epiphysis, reaching a stable equilibrium to changes in the size and shape of the bone."

"Chondrocytes become HCs when the concentration level of PTHrP is lower than a threshold value. During this time, the cells hypertrophy in the centre of the hyaline cartilage where PTHrP is low."

"During the growth stage, the elongation of the cells from proliferation to hypertrophy generates bone growth, as seen in the linear concentration change of cells. The proliferation in the radial direction in the epiphysis causes growth in the diameter of the femoral head."

"we assume the presence of a reaction–diffusion system of two primary molecules, such as BMP2 and Noggin whose distribution in space may lead to a stable pattern in time and an unstable pattern in space, similar to the differentiation patterns of osteoblasts from mesenchymal cells."

Human Long Bone Development in Vivo: Analysis of the Distal Femoral Epimetaphysis on MR Images of Fetuses.

"272 MR imaging examinations (April 2004-July 2011) in 253 fetuses with a mean gestational age (GA) of 26 weeks 6 days (range, 19 weeks 2 days to 35 weeks 6 days) without known musculoskeletal abnormalities. Two independent readers qualitatively analyzed epiphyseal and metaphyseal shape, secondary ossification, and the perichondrium on 1.5-T echo-planar MR images and correlated the results with the GA that was derived from previous fetal ultrasonography (US). Diaphyseal and epiphyseal morphometric measurements were correlated with GA by means of the Pearson correlation and linear regression. MR imaging measurements of diaphyseal length and US normative values were compared graphically. Interreader agreement analysis was performed with weighted κ statistics and the intraclass correlation coefficient.  With advancing GA, the [cartilagenous] epiphyseal shape changed from spherical to hemispherical with a notch, and the metaphyseal shape changed from flat to clearly undulated. Secondary ossification was not observed until 25 weeks 3 days. The perichondrium decreased from 20 weeks onward. Correlation coefficients were 0.897 for diaphyseal length, 0.738 for epiphyseal length, and 0.801 for epiphyseal width with respect to GA. The range of measurements of diaphyseal length was larger than that of the reported US normative values. Interreader agreement was good for bone morphometrics (intraclass correlation coefficient, 0.906-0.976), and moderate for bone characteristics (weighted κ, 0.448-0.848)."

"there was a significant correlation between the epiphyseal length and width"