The Resting Zone of the Growth Plate

Within the resting zone of the growth plate are stem-like cells which means they are like stem cells but only have limited proliferative capacity.  If we can characterize these resting zone cells we can know more about how to initiate the first stage of the growth plate.

Identification of target genes for wild type and truncated HMGA2 in mesenchymal stem-like cells.

"The HMGA2{up in LSJL} gene [codes] for an architectural transcription factor involved in mesenchymal embryogenesis.
We have over-expressed wild type and truncated HMGA2 protein in an immortalized mesenchymal stem-like cell (MSC) line, and investigated the localisation of these proteins and their effects on differentiation and gene expression patterns.
Over-expression of both transgenes blocked adipogenic differentiation of these cells, and microarray analysis revealed clear changes in gene expression patterns, more pronounced for the truncated protein. Most of the genes that showed altered expression in the HMGA2-overexpressing cells fell into the group of NF-kappaB-target genes, suggesting a central role for HMGA2 in this pathway. Of particular interest was the pronounced up-regulation of SSX1, already implicated in mesenchymal oncogenesis and stem cell functions, only in cells expressing the truncated protein. Furthermore, over-expression of both HMGA2 forms was associated with a strong repression of the epithelial marker CD24, consistent with the reported low level of CD24 in cancer stem cells.:
We conclude that the c-terminal part of HMGA2 has important functions at least in mesenchymal cells, and the changes in gene expression resulting from overexpressing a protein lacking this domain may add to the malignant potential of sarcomas."

"There were several genes up-regulated by HMGA2WT and down- regulated in cells expressing the truncated form, such as FGF13, EHF, HCLS1, MEST, G0S2 and PTPRN2."<-Since the truncated form of HMGA2 can increase height these genes may be important.

Genes downregulated in HMGA2WT-transgenic also downregulated in LSJL:
IL6{up}
Ces1
Thbs2{up}
S100a4{up}
JunB{up}
Has1{up}
Ptgs2{up}
Kynu{up}
Oasl

Upregulated:

Genes downregulated in HMGA2Ttruncated also downregulated in LSJL:
LAMA4{up}
Thbs2{up}
S100a4{up}
JunB{up}
Has1{up}
Ptgs2{up}
Kynu{up}
Oasl

Upregulated:
MMP3
Edn1
Hapln1

"over-expression of truncated HMGA2 induces a more mesenchymal (stem-like) phenotype, characterized by resistance toward differentiation, over-expression of SSX1, lost expression of certain epithelial markers and strengthened expression of mesenchymal markers."

Differential expression of phenotype by resting zone and growth region costochondral chondrocytes in vitro.

"Chondrocytes derived from the resting cell zone and adjacent growth zone of rat costochondral cartilage were compared for retention of phenotype in culture. At third passage confluence, two cell populations differ morphologically and biochemically. Resting zone cells are fibroblast-like, with smooth cell membranes and little rough endoplasmic reticulum. Growth zone cells are more polygonal, smaller in diameter, with numerous cytoplasmic extensions of the plasma membranes and abundant rough endoplasmic reticulum. Both cell populations produce matrix vesicles that are comparable morphologically to matrix vesicles isolated enzymatically from epiphyseal cartilage. While membrane vesicles are released into the media by cells derived from the resting zone as well as from the growth cartilage, alkaline phosphatase activity is enriched in media vesicles produced by growth cartilage cells. Alkaline phosphatase enriched vesicles appear to be preferentially incorporated into the extracellular matrix. Both the plasma membrane marker enzyme activity and the membrane phospholipid composition are differentially expressed in matrix vesicles and plasma membranes and are cell specific. Matrix vesicles produced by resting zone cells are enriched in alkaline phosphatase, 5'-nucleotidase, ouabain sensitive Na+/K+ ATPase and cardiolipin when compared to the cell membrane. In addition, the plasma membranes of these cells contain more phosphatidylcholine plus sphingomyelin than do growth cartilage plasma membranes. Resting zone cell matrix vesicles have less phosphatidylethanolamine than do vesicles from growth cartilage cultures. Matrix vesicles produced by growth cartilage cells contain one proteolipid at 43,000 Mr which comigrates with plasma membrane proteolipid and an additional proteolipid at approximately 3,000 Mr. These data indicate that both cells retain differential expression of phenotype in culture and that one expression of this phenotype is production of specific extracellular matrix vesicles."

"Growth cartilage chondrocyte plasma membranes exhibit higher 5'-nucleotidase activity than do resting cell membranes"

"The resting zone cells membranes contain more phosphatidylcholinc plus sphingomyelin than do the growth zone chondrocyte membranes"

Transforming growth factor-beta1 regulation of resting zone chondrocytes is mediated by two separate but interacting pathways.

" transforming growth factor-beta1 (TGF-beta1) stimulates protein kinase C (PKC) via a mechanism that is independent of phospholipase C or tyrosine kinase, but involves a pertussis toxin-sensitive G-protein. Maximal activation occurs at 12 h and requires new gene expression. To understand the signaling pathways involved, resting zone chondrocytes were incubated with TGF-beta1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7. [(35)S]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-beta1 on matrix production. However, there was little, if any, effect on TGF-beta1-dependent increases in [(3)H]thymidine incorporation, and TGF-beta1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC. TGF-beta1 caused a dose-dependent increase in prostaglandin E(2) (PGE(2)) production which was further increased by PKC inhibition. The increase was regulated by TGF-beta1-dependent effects on phospholipase A(2) (PLA(2)). Activation of PLA(2) inhibited TGF-beta1 effects on PKC, and inhibition of PLA(2) activated TGF-beta1-dependent PKC. Exogenous arachidonic acid also inhibited TGF-beta1-dependent increases in PKC. The effects of TGF-beta1 on PKC involve genomic mechanisms, but not regulation of existing membrane-associated enzyme, since no direct effect of the growth factor on plasma membrane or matrix vesicle PKC was observed.  TGF-beta1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE(2) and protein kinase A (PKA). Inhibition of PKA also decreases TGF-beta1-dependent proliferation. We have previously shown that PGE(2) stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways."

"Resting zone chondrocytes synthesize TGF-β1 in latent form and store it in their extracellular matrix as a 290 kDa complex consisting of latent TGF-β1, latent TGF-β1 binding protein-1 and the latency-associated peptide. Extracellular matrix vesicles produced by these cells can activate latent TGF-β1 when they are exposed to 1,25-(OH)2D3. The interrelationship of TGF-β1 action and vitamin D metabolites is also demonstrated by the fact that TGF-β1 causes resting zone cells to produce increased 1,25-(OH)2D3 within 1 h and increased 24,25-(OH)2D3 at 24 h, which is correlated with TGF-β1-dependent downregulation of the 1α-hydroxylase and upregulation of the 24-hydroxylase in these cells"

"PGE2 has multiple effects on the chondrocytes, promoting differentiation and anabolic responses via cAMP production and PKC activity"

" Resting zone cells have both EP1 and EP2 receptors, as well as an EP1 variant, EP1v. The increase in cAMP leads to increased PKA activity. The importance of this pathway in the response to TGF-β1 is evident in the decrease in proliferation following treatment of the cells with TGF-β1 and the PKA inhibitor, H-8."

Direct effects of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on growth zone and resting zone chondrocyte membrane alkaline phosphatase and phospholipase-A2 specific activities.

"1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 differentially affect the specific activity of alkaline phosphatase (ALPase) and phospholipase-A2 (PLA2) of plasma membranes and extracellular matrix vesicles produced by costochondral reserve zone and growth zone cartilage chondrocytes in culture. In the present study, growth zone and cartilage and reserve zone matrix vesicles and plasma membranes were isolated from confluent chondrocyte cultures and incubated with hormone for 3 and 24 h in vitro. Addition of 1,25-(OH)2D3 to GC matrix vesicles and plasma membranes resulted in dose-dependent increases in ALPase and PLA2 specific activities in both membrane fractions. Addition of 24,25-(OH)2D3 to RC membrane fractions stimulated matrix vesicle ALPase at 10(-7) and 10(-8) M and plasma membrane ALPase at 10(-8) M only. However, 24,25-(OH)2D3 inhibited matrix vesicle and plasma membrane PLA2 activity. The effects of the vitamin D metabolites were noticed after both 3 and 24 h. Neither hormone metabolite had any effect on these enzymes in membrane fractions from cultures of neonatal rat muscle mesenchymal cells, which do not calcify their matrix in vivo. 1,25-(OH)2D3 and 24,25-(OH)2D3 can directly affect chondrocyte membrane enzymes without genomic influence or protein synthesis and that membrane response depends on the stage of chondrocyte differentiation. Changes in PLA2 activity may change membrane fluidity and may be a mechanism by which the hormones affect cell membranes."

"Enzymes present in membranes isolated from the less differentiated mesenchymal cells do not respond to either vitamin D3 metabolite tested, although both metabolites stimulate ALPase gene expression in cultures of these cells"

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3.

"To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10(-10)-10(-8) M 1,25-(OH)2D3 or 10(-9)-10(-7) M 24,25-(OH2)D3 and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)2D3 and 24,25-(OH)2D3 in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [35S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)2D3; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)2D3. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification."

"Resting zone cells exhibit greater sensitivity to BMP-2 than do cells derived from the prehypertrophic and upper hypertrophic zones"

Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes.

"rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3[the inactive form of Vitamin D]. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness."

The 24,25-form tends to correlate with chondrogenesis whereas the 1,25 form tends to correlate with osteogenesis.  The 24,25-form downregulates it's own production in resting zone chondrocytes but upregulates the active form by growth chondrocytes.

Monocarboxylate transporter 10 functions as a thyroid hormone transporter in chondrocytes.

"untreated congenital hypothyroidism is marked by severe short stature. The monocarboxylate transporter 8 (MCT8) is a highly specific transporter for thyroid hormone. The hallmarks of Allan-Herndon-Dudley syndrome, caused by MCT8 mutations, are severe psychomotor retardation and elevated T(3) levels. However, growth is mostly normal. We therefore hypothesized that growth plate chondrocytes use transporters other than MCT8 for thyroid hormone uptake. Extensive analysis of thyroid hormone transporter mRNA expression in mouse chondrogenic ATDC5 cells revealed that monocarboxylate transporter 10 (Mct10) was most abundantly expressed among the transporters known to be highly specific for thyroid hormone, namely Mct8, Mct10, and organic anion transporter 1c1. Expression levels of Mct10 mRNA diminished with chondrocyte differentiation in these cells. Accordingly, Mct10 mRNA was expressed most abundantly in the growth plate resting zone chondrocytes in vivo. Small interfering RNA-mediated knockdown of Mct10 mRNA in ATDC5 cells decreased [(125)I]T(3) uptake up to 44% compared with negative control. Moreover, silencing Mct10 mRNA expression abolished the known effects of T(3), i.e. suppression of proliferation and enhancement of differentiation, in ATDC5 cells. Mct10 functions as a thyroid hormone transporter in chondrocytes and can explain at least in part why Allan-Herndon-Dudley syndrome patients do not exhibit significant growth impairment."

"TH inhibits proliferation and promotes differentiation of chondrocytes and is indispensable for normal growth"

"The SLC16A10 gene, which encodes MCT10, localizes to 6q21-q22 [and is associated with height growth]"

"in RZ chondrocytes, TH exerts its actions via TRα1."

[Histology and proliferative capability of thoracic vertebral body growth plates of rats at different ages].

"The thoracic VBGPs obtained from rats aged 1 day and 1, 4, 8, 16 and 28 weeks were identified using safranin O-fast green staining, and the height of the hypertrophic zone, proliferative zone, and resting zone were measured. The chondrocytes were isolated from these VBGPs with a modified trypsin-collagenase type II digestion method for primary culture in vitro. The expressions of proliferating cell nuclear antigen (PCNA) mRNA and protein was detected by real time-PCR and Western blotting, respectively.

The 1-day- and 1-week-old rats showed significantly greater hypertrophic zone and proliferative zone in the VBGPs than older rats; the proliferative zone was significantly greater in rats aged 4 weeks than in those aged 28 weeks. The resting zone was obviously greater in rats aged 1 day and 1 week than in older rats, and also greater in rats aged 4 weeks than in those aged 16 and 28 weeks. Obvious ossification in the resting zone occurred at 16 weeks, and most of the resting zone became ossified at 28 weeks. The expression of PCNA decreased at both the mRNA and protein levels as the rats grew.

The 3 zones of VBGPs are greater in rats aged 1 day and 1 week than in older ones. Ossification in the resting zone begins at 16 weeks, and till 28 weeks, most of the resting zone is ossified. The proliferation ability of VBGP chondrocytes decreases with the increase of age of the rats."

Study is in a foreign language unfortunately.

Distribution of type I and type II collagen gene expression during the development of human long bones.

"The temporal and spatial gene expression of collagen type I and type II during the development of the human long bones was studied by the technique of in situ hybridization covering the period from the cartilagenous bone anlage to the formation of a regular growth plate in the newborn. Analysis of the early stages around the seventh week of gestation revealed for type II collagen a strong hybridization signal limited to the chondrogenic tissue. The surrounding connective tissue and the perichondrium showed weak type I collagen expression, while the zones of desmal ossification like the clavicle gave a strong signal. Beginning with the eighth week of gestation, type I collagen mRNA was detectable in newly formed osteoblasts at the diaphysis and appeared along with the formation bone marrow, in the areas of enchondral ossification. Parallel to the development of the different zones of cartilage differentiation, a specific pattern of type II expression could be observed: type II was mainly found in the chondrocytes of the hypertrophic zone and to a lesser degree in the zone of proliferation, while the resting zone and the zone of provisional calcification showed little activity. This segregation of type II expression was most pronounced in the early stages of cartilage calcification and in the growth plate of the newborn."

"As prechondrogenic mesenchyme cells develop to chondrocytes, a dramatic increase in the cytoplasmatic volume, the rough endoplasmatic reticulum and the Golgi apparatus takes place. This is

paralleled by the switch from collagen type I, the predominant collagen of fibroblasts, to collagen type II, the major collagen found in cartilage"

"Limbs of human fetuses between the 7th and 15th menstrual weeks"  Mature chondrocytes never displayed Type I Collagen activity.  Type II collagen negative cells occur at the osteochondral junction.

Mesenchymal chondroprogenitor cell origin and therapeutic potential.

"In embryonic limb development, FGF-4 stimulates Sonic hedgehog (Shh) expression in a positive feedback loop that coordinates proximal-distal and anterior-posterior patterning of the cartilaginous anlagen"

According to the paper 20-50% of cells in the bone marrow have the ability to differentiate into chondrocytes.

"progenitor cells with chondrogenic capacity have been isolated from the superficial zone of articular cartilage"

"chondroprogenitors have been identified in arthritic cartilage after their migration from the bone marrow through breaks in the tidemark and into the diseased cartilage"<-meaning chondroprogenitors exist in the bone marrow.

Morphogenetic and regulatory mechanisms during developmental chondrogenesis: new paradigms for cartilage tissue engineering.

Some great diagrams in this paper.

"When the cells aggregate, MCs[mesenchymal cells] begin to produce collagen I, fibronectin, and proteoglycans. The result of the strong interactions that cells establish with their environment is the formation of a dense mass of MCs that immediately begins to differentiate into chondroblasts. Condensed MCs start expressing mainly the transcription factor Sox9 that controls downstream genes involved in chondrogenesis, promoting these progenitor cells to secrete cartilage-specific ECM molecules"

"MMP1 and MMP2 have the capacity to degrade cartilage matrix, and they are characterized as the MMPs that are involved in earlier chondrogenesis. Specifically, blockage of MMP2 function supports precartilage condensation and chondrogenesis, and MMP1 knockout mice show decreased chondrocyte proliferation in the proliferative zone of the growth plates of long bones." MMP2 is increased in LSJL so perhaps we should find a way to decrease it's expression.

"overexpression of human Sox9 in murine ESCs (mESCs) leads to upregulated expression of the cartilage markers collagen IIA, aggrecan, and pax1 even in undifferentiated ESCs"

"fibroblasts can undergo spontaneous chondrogenesis in simple three-dimensional culture conditions"

Inhibiting Estrogen to Increase Height

It's important to evaluate inhibiting Estrogen to increase height after we found that estrogen inhibits TGF-Beta induced Smad3 signaling which induces chondrogenesis.

Use of aromatase inhibitors to increase final height. 

"During puberty in both sexes, the mechanism involved in epiphyseal fusion is mediated by the action of estrogen through a cascade of events including proliferation, differentiation, and apoptosis of chondrocytes. The enzyme P450 aromatase catalyzes the aromatization of C19 androgens (androstenedione and testosterone) to C18 estrogens (estrone and estradiol). Inhibition of estrogen action by aromatase inhibitors (AIs) appears to decelerate the process of growth plate fusion[Note delaying growth plate fusion may not increase final height if growth velocity does not also increase], and thus AIs may be used therapeutically to increase adult height. The clinical experience with AIs in the pediatric setting is limited to testolactone, fadrozole, letrozole, and anastrozole. Testolactone, a nonselective steroidal AI, has been used successfully as an adjunct to antiandrogen and gonadotropin-releasing hormone analogue (GnRHa), therapy for children with familial male-limited precocious puberty (FMPP) and congenital adrenal hyperplasia (CAH), and with some success in girls with McCune-Albright syndrome. The limitations of testolactone include its relatively low potency and the need for frequent dosing. Results of a randomized placebo-controlled trial in boys with delayed puberty treated with letrozole, a selective nonsteroidal AI, found that boys treated with letrozole + testosterone experienced delayed bone maturation and good growth response and achieved an increase in predicted adult height[keyword: PREDICTED adult height]. In this study, only minor differences in bone density were seen between the placebo and letrozole treatment groups[bone density takes into account bone size as well so that means that bone size did not increase], both of which were receiving concomitant testosterone therapy. No adverse effects on testis size or inhibin B concentration were noted." 

"bone in both men and women has the capacity to convert androgen to estrogen in the bone"

"mean predicted adult stature was 143.0 ± 7.8 cm before treatment and 147.3 ± 11.5 cm after 3 years" with AI treatment.

"an increase in predicted adult height was seen for all but one boy in the letrozole + testosterone group (range, 2.5–8.8 cm); predicted adult height decreased by 3.5 cm for the one remaining boy."

Impact of growth plate senescence on catch-up growth and epiphyseal fusion. 

"In mammals, longitudinal bone growth occurs rapidly in prenatal and early postnatal life, but then slows and eventually ceases. This deceleration, which reflects a decline in chondrocyte proliferation, was previously attributed to a hormonal or other systemic mechanism. However, new evidence suggests that it is due to a local mechanism within the growth plate[See not estrogen, GH, IGF-1, or testosterone]. growth plate chondrocytes have a finite proliferative capacity that is gradually exhausted, causing growth to slow and finally stop." 

"With age, there is a decrease in the overall height of the growth plate, associated with a decline in the number of proliferative and hypertrophic chondrocytes per column. In addition, the individual hypertrophic cells do not grow as large in an older animal, and the columns become more widely spaced with more intervening cartilage matrix. The number of apoptotic growth plate chondrocytes detected by the TUNEL assay increases with age and might thus contribute to the age-dependent decline in growth rate"

"in rabbits, estrogen accelerates growth plate senescence despite decelerating longitudinal bone growth"

"estrogen does not stimulate ossification of cartilage directly but instead accelerates the normal process of growth plate senescence, secondarily inducing earlier fusion"

So, this study states that estrogen can not help increase height because final height is due to a finite proliferative capacity of chondrocytes within the growth plates.  What does that mean for Lateral Synovial Joint Loading? 

Fundamental limits on longitudinal bone growth: growth plate senescence and epiphyseal fusion. 

"The decline in [long bone] growth rate is caused primarily by a decrease in the rate of chondrocyte proliferation and is accompanied by structural changes in growth plate cartilage. This programmed senescence does not appear to be caused by hormonal or other systemic mechanisms but is intrinsic to the growth plate itself. In particular, recent evidence indicates that senescence might occur because stem-like[mesenchymal red bone marrow stem] cells in the resting zone have a finite proliferative capacity, which is exhausted gradually. In some mammals, including humans, proliferative exhaustion is followed by epiphyseal fusion, an abrupt event in which the growth plate cartilage is replaced completely by bone." 

Lateral synovial joint loading helps transport new stem-like cells to the resting zone by distracting the growth plates and increasing interstitial fluid flow which sends red bone marrow stem cells into the growth plates thus potentially restoring proliferative capacity!  Here's a study that states that estrogen inhibition does not impact final height and only growth rate: 

Depletion of resting zone chondrocytes during growth plate senescence. 

"With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2'-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate. Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation. Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate[i.e. estrogen only slows down growth rate, it does not eliminate the proliferate capacity of the growth plate]. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle." 

"the resting zone chondrocytes serve as a pool of stem-like cells that generate columnar clones of proliferative zone chondrocytes"

"Estrogen treatment did not significantly affect the number of resting zone chondrocytes"  "Serum estradiol concentration, measured 7 days after the second injection of estradiol cypionate, was 11 ± 2 pg/mL, compared to < 5 pg/mL in animals treated with the vehicle. Estrogen treatment did not significantly affect serum IGF-I concentration (88 ± 6 ng/mL vs 108 ± 6 ng/mL, estrogen vs control, P=NS)."<-This amount seems significant to me though.

"The decline in number of resting zone chondrocytes could also affect growth rate by a paracrine mechanism. For example, a decrease in the number of resting zone chondrocytes might decrease the overall production of parathyroid hormone-related protein (PTHrP), which could lead to earlier hypertrophy of proliferative zone chondrocytes."

"[estrogen] decreased proliferation rate and had no significant effect on cell numbers"  No obvious mechanisms as to how estrogen affects growth plate senescence were observed.

"We speculate that estrogen might increase the loss of proliferative capacity that occurs with each cell cycle, or that estrogen might cause loss of proliferative capacity by a cell-cycle-independent mechanism. For example, if senescence is caused by epigenetic changes such as loss of DNA methylation with each cell cycle, then estrogen might act by decreasing expression of maintenance methylases, causing greater loss of methylation with each cell replication. The effects of estrogen on the resting zone chondrocytes could be mediated by the estrogen receptor-α or -β, both of which are expressed by resting zone chondrocytes in humans, rabbits, and rats. The combination of a decreased rate of growth (in most mammals) and an increased rate of senescence seems to be an effect specific to estrogen"

"Estrogen appears to accelerate growth plate senescence without accelerating resting zone chondrocyte proliferation or accelerating the numerical depletion of these cells"<-Thus estrogen may decrease height.

They do state however that estrogen might accelerate growth plate fusion by a method not dependent on proliferation or that estrogen might reduce final height by another mechanism. 

17β-Estradiol regulates rat growth plate chondrocyte apoptosis through a mitochondrial pathway not involving nitric oxide or MAPKs.

"Estrogens cause growth plate closure in both males and females, by decreasing proliferation and inducing apoptosis of postproliferative growth plate chondrocytes. In vitro studies using 17β-estradiol (E(2)) conjugated to bovine serum albumin (E(2)-BSA) show that rat costochondral growth plate resting zone chondrocytes also respond to E(2). Moreover, they are regulated by E(2)-BSA via a protein kinase C and ERK MAPK signaling pathway that is functional only in female cells.  Rat resting zone chondrocytes cells were treated with E(2) or E(2)-BSA. E(2) caused apoptosis in male and female resting zone and growth zone chondrocytes in a dose-dependent manner, based on elevated DNA fragmentation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation[Now you don't really want apoptosis in the resting zone but still studies have shown positive benefits from lower estrogen levels and these rats could have already been above equilibrium estrogen]. E(2) also up-regulated p53 and Bax protein (Bcl-2-associated X protein) levels and induced release of cytochrome C from the mitochondria, indicating a mitochondrial apoptotic pathway. The apoptotic effect of E(2) did not involve elevated nitric oxide production or MAPKs. It was reduced by ICI 182780, which is an estrogen receptor (ER) antagonist and blocked by antibodies to Erα36, a membrane-associated ER. E(2)-BSA reduced cell viability and increased caspase-3 activity; ICI 182780 had no effect, but anti-ERα36 antibodies blocked the effect. The results indicate that estrogen is able to directly affect the cell population kinetics of growth plate chondrocytes by regulating apoptosis, as well as proliferation and differentiation in both resting zone and growth zone cells."

"High doses of estrogens also induce activation of caspase-3 in the growth plate, indicating involvement of apoptosis"

"The effects of estrogen on proliferation and differentiation of rat costochondral resting zone (RC) chondrocytes involve a membrane receptor-related pathway, which acts through activation of protein kinase C (PKC) and MAPK"

"Pi[extracellular inorganic phosphate] is able to induce resting zone chondrocyte apoptosis in an NO-dependent pathway"

"Resting zone chondrocytes differ from growth zone chondrocytes with respect to their morphology, the composition of their extracellular matrix, the phospholipid composition and levels of alkaline phosphatase activity in their matrix vesicles, and the production and activity of neutral vs. acid matrix metalloproteinases"

"resting zone chondrocytes respond to the vitamin D metabolite 24R,25(OH)2D3, whereas the growth zone chondrocytes respond to 1α,25(OH)2D3. Both vitamin D metabolites are produced actively in the growth plate in a regulated way by TGFβ1 in a zone-specific manner"<-There have been evidence of Vitamin D effects on growth plates but also studies have shown that Vitamin D polymorphisms have a "negligible effect" on human height.  Vitamin D regulates IL-1, PGE2, and PKC in GP chondrocytes.

"The 1α,25(OH)2D3-dependent activation of PKC involves activation of phospholipase A2 (PLA2) and arachidonic acid production, whereas 24R,25(OH)2D3-dependent activation of PKC involves PLD activation "

"E2 caused a dose-dependent decrease in cell viability based on MTT activity and in cell number in both male and female resting zone chondrocytes [in addition to the dose dependent effects on apoptosis]"<-Note only three dosages were used so this does not disprove that there is an equilibrium level of estrogen that increases height.

E2 does not affect apoptosis by a nitric oxide or inorganic phosphate mechanism.  The apoptosis mechanism is related to caspase-3 and caspase-1.

Now here's a study that mentions Stanozol to increase cellular proliferation.  Stanozol is a controlled substance.

Stanozolol regulates proliferation of growth plate chondrocytes via activation of ERalpha in GnRHa-treated adolescent rats.

"Stanozolol (ST) is a synthetic derivative of androgen. In this study, we investigated the effects and the mechanisms of ST on the proliferation of growth plate chondrocytes isolated from adolescent rats treated with gonadotropin-releasing hormone analogue (GnRHa). Treatment with ST resulted in time- and concentration-dependent effects on proliferation[the effectiveness of Stanozol was dependent on the concentration and the time that stanozol was in the system note that it's unclear whether this is a linear relationship]. ST increased the phosphorylation level of the estrogen receptor alpha (ERalpha)[so if there was a legal substance that increased the phosphorylation of ERalpha that would be beneficial as well], but not the androgen receptor (AR). Pharmacological inhibition of ERalpha and mitogen-activated protein kinase (MAPK) attenuated the effects of ST on the proliferation of growth plate chondrocytes[If ERalpha was inhibited by other means than the effects of Stanozol were diminished(attenuated).  Also, Stanozol seems to operate along the MAPK pathway]. A molecular dynamics simulation showed hydrophobic interactions between ST and ERalpha[cartilage is hydrophillic(water loving) so stanozol is likely effective at bypassing water and getting straight to ERalpha]. These results suggested that ERalpha, but not AR, partially mediates the ST-driven proliferation of growth plate chondrocytes."

So increasing phosphorylation of ERalpha may be one way to increase height growth if the increase in cellular proliferation also increases chondrocyte proliferative capacity.

"in some patients, growth is suppressed to subnormal velocity during the GnRHa therapy."

"mini-dose of estrogen replacement can normalize the slowdown of the growth rate during GnRHa therapy in girls with CPP"

"[ST] may impair adult height"

"In pediatric patients with Turner syndrome who treated with oxandrolone, height velocity increased without significant bone age progression"

"ST promoted chondrocytes proliferation independent of the AR."  Possibly via ER or MAPK pathways

"ER α phosphorylation can mediate the promotion or inhibition of long bone growth and epiphysis fusion"

High circulating Estradiol levels may inhibit chondrogenesis:

Estradiol inhibits chondrogenic differentiation of mesenchymal stem cells via nonclassic signaling.

"The existence of intracellular and membrane-associated E2 receptors was shown at various stages of chondrogenesis. Smaller aggregates and significantly lower type II collagen and sGAG content were detected after treatment with E2 and E2-BSA in a dose-dependent manner. Furthermore, E2 enhanced type X collagen and MMP-13 expression. Compared with estradiol alone, the coincubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis. Treatment with specific GPR-30 agonists alone (G-1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis. In addition, we found an enhancement of hypertrophy by G-1. Furthermore, the specific GPR-30 antagonist G15 reversed the GPR-30-mediated inhibition of chondrogenesis and up-regulation of hypertrophic gene expression.
The experiments revealed a suppression of chondrogenesis by estradiol via membrane receptors (GPR-30)."

So reducing estradiol levels or GPR-30 receptor levels may be a way to increase height.

"MMP-13 expression was significantly up-regulated after treatment with E2 or G-1 as compared with control"<-Maybe the increase in MMP-13 expression is related in somehow to the inhibition of chondrogenesis(MMP-13 gets beat up on a lot).

"During chondrogenesis, no proliferative or apoptotic effects of estradiol were detected"

"estradiol showed a dose-dependent inhibition of 3-D chondrogenesis with macroscopically smaller aggregates as well as reduced GAG and type II collagen deposition as compared with controls. Estradiol treatment did not disturb aggregate condensation and did not completely suppress chondrogenic differentiation."<-Thus high Estradiol levels should not be the cause of lack of LSJL results but they could be the cause of the reduction of the results.

"extracellular matrix can “store” E2"<-If there's no growth plate in the epiphysis then estrogen can't really be stored there.

Here's a study that shows that estrogen may accelerate closure but that doesn't mean that inhibiting estrogen can allow you to go beyond senescence.

The role of estrogen receptor-α and its activation function-1 for growth plate closure in female mice.

"High estradiol levels in late puberty induce growth plate closure and thereby cessation of growth in humans[cessation of growth can occur before closure however]. In mice, the growth plates do not fuse after sexual maturation, but old mice display reduced longitudinal bone growth and high-dose estradiol treatment induces growth plate closure. Estrogen receptor (ER)-α stimulates gene transcription via two activation functions (AFs), AF-1 and AF-2. To evaluate the role of ERα and its AF-1 for age-dependent reduction in longitudinal bone growth and growth plate closure, female mice with inactivation of ERα (ERα(-/-)) or ERαAF-1 (ERαAF-1(0)) were evaluated. Old (16- to 19-mo-old) female ERα(-/-) mice showed continued substantial longitudinal bone growth, resulting in longer bones (tibia: +8.3%, P < 0.01) associated with increased growth plate height (+18%, P < 0.05) compared with wild-type (WT) mice. In contrast, the longitudinal bone growth ceased in old ERαAF-1(0) mice (tibia: -4.9%, P < 0.01). Importantly, the proximal tibial growth plates were closed in all old ERαAF-1(0) mice while they were open in all WT mice. Growth plate closure was associated with a significantly altered balance between chondrocyte proliferation and apoptosis in the growth plate. In conclusion, old female ERα(-/-) mice display a prolonged and enhanced longitudinal bone growth associated with increased growth plate height, resembling the growth phenotype of patients with inactivating mutations in ERα or aromatase.  ERαAF-1 deletion results in a hyperactive ERα, altering the chondrocyte proliferation/apoptosis balance, leading to growth plate closure. This suggests that growth plate closure is induced by functions of ERα that do not require AF-1 and that ERαAF-1 opposes growth plate closure."

So you actually want ERalphaAF-1 to grow taller.

"A patient with a point mutation in exon 2 of the estrogen receptor (ER)α (estrogen-resistant man) experienced a continued growth long into adulthood, but at the age of 33.5 yr his growth plates were nearly fused."<-the man grew to about 80 inches tall.

Roles of transactivating functions 1 and 2 of estrogen receptor-alpha in bone.

"estrogen receptor-α (ERα) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal and AF-2 in the ligand binding domain. To evaluate the role of ERα AF-1 and ERα AF-2 for the effects of estrogen in bone in vivo, we analyzed mouse models lacking the entire ERα protein (ERα(-/-)), ERα AF-1 (ERαAF-1(0)), or ERα AF-2 (ERαAF-2(0)). ERα AF-2 is required for the estrogenic effects on all parameters evaluated, whereas the role of ERα AF-1 is tissue-specific Selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial actions."

"a normal negative feedback regulation of serum sex steroids requires an intact AF-1 and an intact AF-2 in ERα"

Tamoxifen is a selective ERalpha inhibitor but tamoxifen may decrease height.  One study found tamoxifen increasing longitudinal growth in female rats whereas the other study found tamoxifen decreasing longitudinal growth in male rats.  This could be due to differing responses to hormones or that females were more likely to be above equilibrium due to higher endogenous estrogen production.

Gender- and region-specific variations of estrogen receptor α and β expression in the growth plate of spine and limb during development and adulthood.

"The effects of ER inactivation on bone growth are sex and age dependent, and may differ between the axial and appendicular regions. In this study, the spatial and temporal expression of ERα and β in the tibial and spinal growth plates of the female and male rats during postnatal development was examined to explore the possible mechanisms. The level of mRNA was examined and compared with quantitative real-time PCR. The spatial location was determined by immunohistochemical analysis. The 1-, 4-, 7-, 12- and 16-week age stages correspond to early life, puberty and early adulthood after puberty, respectively. Gender- and region-specific differences in ERα and β expression were shown in the growth plates. Mainly nuclear staining of ERα and β immunoreactivity was demonstrated in the spinal and tibial growth plate chondrocytes for both genders. [There's a] significant effect of gender on temporal ERα and β expression and of region on temporal ERα/ERβ expression ratio. However, spatial differences of region-related ERα and β expression were not observed. Gender-related spatial changes were detected only at 16 weeks of both spine and limb growth plates. ERα and β immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in the early life stage and during puberty. After puberty, ERα expression was mainly located in the late proliferative and hypertrophic chondrocytes in female, whereas the expression still extended from the resting to hypertrophic chondrocytes in males."

"a significant decrease in the length of femoral bone was observed in ERα-knockout (ERKO) females; in contrast, a slight decrease was shown in ERKO males"

"In the female rat, gonadectomy [removal of the ovaries] resulted in a bigger femur"

"In growing female rats, estrogen deficiency leads to an increase in the length of appendicular bones, while growth was normal at spine"

"Compared with WT controls, the axial skeletal growth increase was bigger than that of the appendicular in BERKO female mice"

"a significant decrease in length of lumbar vertebrae was demonstrated in ERKO male and female mice"

This study finds that ERalpha knockout may increase height:

The role of estrogen receptor α in growth plate cartilage for longitudinal bone growth.

"we developed a mouse model with cartilage-specific inactivation of ERα. Although mice with total ERα inactivation displayed affected longitudinal bone growth associated with alterations in the GH/IGF-1 axis, the skeletal growth was normal during sexual maturation in mice with cartilage-specific ERα inactivation. High-dose estradiol treatment of adult mice reduced the growth plate height as a consequence of attenuated proliferation of growth plate chondrocytes in control mice but not in cartilage-specific ERα(-/-) mice. Adult cartilage-specific ERα(-/-) mice continued to grow after 4 months of age, whereas growth was limited in control mice, resulting in increased femur length in 1-year-old cartilage-specific ERα(-/-) mice compared with control mice. We conclude that during early sexual maturation, ERα in growth plate cartilage is not important for skeletal growth. In contrast, it is essential for high-dose estradiol to reduce the growth plate height in adult mice and for reduction of longitudinal bone growth in elderly mice."

"female GPR30−/− mice displayed reduced longitudinal bone growth and reduced growth plate height"

"reduced femur and crown-rump lengths in the 17-week-old male total ERα−/− mice were associated with a significant reduction of serum IGF-1 levels (–20% ± 6% versus control littermates, p < .01), whereas serum IGF-1 levels were unchanged in Col2α1-ERα−/− mice (14% ± 7% versus control littermates, nonsignificant)."

Maybe you can increase IGF-1 levels exogenously to make up for loss of systemic ERalpha to get a total increase in height(since it will be hard to generate an ERalpha cartilage specific knockout).

The genetic basis of human height : the role of estrogen

" It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively{about 4 inches}. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. Another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER , BMP6, SOX9 and TGF 1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate{so estrogen likely affects peak chondrocyte hypertrophy and optimizing growth per hypertrophic chondrocyte}. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells{Thus MMP13 may be a pro-height protein}. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA{up in LSJL}, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. "

In GH transgenic mice, SOCS2 overexpression further increases height.  BCL2 is protective against apoptosis in hypertrophic chondrocytes while Caspase 3 is pro-apoptotic.  CBFA1 induces Ihh expression.

FGFR3 knockout increases Ihh levels.  Retinoic Acid increases MMP2 and can induce chondrocyte hypertrophy.  BCL2 may increase Sox9 activity.  BCL2 knockout decreases height.  PTHrP increases BCL2 in chondrocytes.

An ERa mutation increases height in males and females.  An ERb mutation increases height in females.  BSM I(associated with VDR) knockout decreases height.  VDR mutations associated with increased height.  Vitamin D deficiency decreases CBFA1.  PPARG3 mutation that decreases activity resulted in increased height.  DRD2(associated with dopamine) knockout decreases height.  Mutation in PTHR1 increases height.

Stage specific effect of leptin on the expressions of estrogen receptor and extracellular matrix in a model of chondrocyte differentiation.

"The aim of our study was to investigate the effect of leptin on the expression of estrogen receptors and extracellular matrix in ATDC5 cells, an in vitro model of endochondral ossification. First, we quantified the physiological expressions of estrogen receptors α, β (ERα, ERβ), leptin receptor (Ob-Rb), type II and type X collagens in definite stages of endochondral ossification in ATDC5 cells using real-time PCR. Dynamic and stage specific expression characteristics of these target genes were observed. Simultaneous expressions of Ob-Rb with ERα or ERβ in ATDC5 cells were also found with dual-label confocal immunofluorescency. Then using Western blotting analysis and/or real-time PCR, we detected that, leptin treatment up-regulated the expressions of ERα, ERβ and type II collagen, but down-regulated type X collagen expression and the ERα/ERβ ratio in the chondrogenic differentiation stage. Meanwhile, leptin down-regulated the expressions of ERα, type II and type X collagens, and the ERα/ERβ ratio, but up-regulated the expression of ERβ in the hypertrophic differentiation stage. Significant positive correlation existed between ERα and type II collagen expression, and between the ratio of ERα/ERβ and type X collagen production. In summary, the crosstalk between leptin and estrogen receptor might be differentiation stage specific in ATDC5 cells."

"In a mandibular condyle organ culture model, leptin treatment increased overall condylar height"

The role of estrogen receptor α in the regulation of bone and growth plate cartilage.

"rapid responses to E2 are mediated by second messenger systems, e.g., cAMP and protein kinase A (PKA), while other responses are mediated by membrane-based ion fluxes, involving, e.g., Ca2+ and Ca2+-dependent K+ channels, which are capable of responding to estrogens"

"A mouse model, believed to lack the entire ERα (K-ERα−/−) had an opposite growth plate phenotype compared to the estrogen-resistant man, in that these mice completely fused their growth plates"

"Young adult (4 months old) female ERα−/− mice had normal longitudinal bone growth. Interestingly, old (16–19 months) female ERα−/− mice showed continued longitudinal bone growth, resulting in longer bones, associated with increased growth plate height compared with WT mice "

"male ERα−/− mice had a reduced longitudinal bone growth during sexual maturation, resulting in shorter bone length, while the male Col2α1-ERα−/− mice had a normal growth"

Membrane Effects of Sex Hormones on Growth Plates Chondrocytes 

"E2 caused rapid increases in Ca++ ion transport and activation of protein kinase C (PKC) and phospholipase A2 in female chondrocytes but not in male cells."

"Testosterone had no effect on PKC in male chondrocytes at any of the times tested, whereas DHT caused a rapid increase in PKC activity at 9 minutes that was still present at 90 minutes"

"DHT induced the PKC activity in dose dependent manner, which was significant at 10-9 to 10-7 M DHT"

 

"DHT induced PKC activity via two mechanisms: influx of extracellular Ca2+ via voltage gated calcium ion channels, and Ca2+ mobilization from the endoplasmic reticulum. Thapsigargin abolished the effect of DHT on PKC activity on a dose dependent manner"

 

"Once PKC is activated, it can initiate a protein phosphorylation cascade that ultimately affects cell differentiation."

"DHT signaling via the AR plays a role in chondrocyte maturation by increasing [35S]-sulfate incorporation, suggesting an increase in the synthesis of a sulfated proteoglycan extracellular matrix."

How to Grow Taller if your Growth Plates are still open

Here's a list of supplements by risk versus reward stated as benefit.  The most beneficial supplements have the most validated effects, tend to always be beneficial without needing an equilibrium value, and the most pro-chondrogenic:

Most Beneficial: Milk(Cycle), Choindroitin, Hyaluronic Acid
Medium: LSJL, PEMF, LIPUS, Lovastatin, Creatine
Least Beneficial: Other(See LSJL supplement page)

First, the growth plate closing does not inhibit height increase from occuring.  Their are still mesenchymal stem cells in the epiphyseal bone marrow.  It's just that there are no chondrocytes proliferating and differentiating.  If new stem cells proceeded to differentiate into chondrocytes then growth plate activity would be restored. This is what Lateral Synovial Joint Loading does, it encourages stem cells to differentiate into chondrocytes by altering intracellular calcium secretions via interstitial fluid flow and hydrostatic pressure.

The best supplements to increase height are hyaluronic acid and choindroitin.  Their are countless studies that show the pro-chondrogenic effects of hyaluronic acid and choindroitin and their ability to induce chondrogenic differentiation.  Their are even studies that show that Hyaluronic Acid supplementation does increase serum levels of HA.

LIPUS is also a potential way to grow taller for open plates as well as epiphysis with no active proliferating and differentiating chondrocytes.  Their are countless studies that show the prochondrogenic effects of LIPUS.  Unfortunately, the optimal setting has not been determined.  An experimental routine is provided here.

Yet another way to increase height is anabolic(anabolic = increase cellular proliferation and differentiation) factors like IGF-1 and Insulin.  Milk increases IGF-1 but needs to be cycled after age 5.  It would be better for somehow the milk to be lactose free as lactose enhances FGF21 signaling. I'm not sure how it should be cycled.  Insulin is controversial as there is evidence that insulin resistance increases height.  IGF-1 is pro-chondrogenic

Whether you still have active proliferating chondrocytes or not, what you want to do to achieve growth is pretty much the same.  First, the most important thing to do is to inhibit myostatin.  Myostatin is a regulator of stem cell proliferation and differentiation.  Now that doesn't mean that you can't achieve extra stem cell proliferation and differentiation in the presence of myostatin.  Compare it to muscle growth, it's easier if myostatin is inhibited but it's still possible if it's not.  Creatine is a myostatin inhibitor that has other pro-chondrogenic effects as well.

Lovastatin which is in Red Yeast Rice may help increase height as well but it hasn't been well documented.

There's also electrical methods.

You would also want to find a way to enhance the amount of C-type nautriuretic peptides that you express.  There's a product coming out that releases guanyl Cyclase B which enhances your expression of C-type nautriuretic peptides.  CNP increases height by increasing chondrocyte hypertrophy and inhibits FGFR3 and keeps Sox9 in check when it wants to inhibit the cell cycle by inhibiting CyclinD

But even if you are doing LSJL to try to re-activate your growth plates you still ideally want both of these substances.  You want your stem cells to proliferate and differentiate.  You want your chondrocytes to proliferate or differentiate when the stem cells differentiate into them.  The difference between growth plates open or not is that if your growth plates are "closed" you need to first initiate reform growth plates via mesenchymal condensation and chondrogenesis.

Another way to grow is to inhibit estrogen.  There's an equilibrium quantity of estrogen however so you have to be careful not to go below this quantity.  However, being at too high level of estrogen is more damaging to height than too low way.

The problem with this is negative feedback.  Scientists have tried injecting extraneous HGH into children and only found an increase in growth rate but not final adult height.  The reason that extraneous HGH doesn't work is negative feedback.  Most of the causes of Gigantism involve an alteration in the bodies homeostatic mechanisms(like the pituitary gland).  They have even found causes of Gigantism with low HGH levels due to an increase in tissue sensitivity to HGH.  There is evidence that the genes related to HGH is involved in height growth but there's also a lot of evidence that HGH isn't the be-all/end-all to height growth.

Here are other supplements that may help.

So, pretty much everything you want to do to grow taller is pretty much the same whether you have existing chondrocytes proliferating and differentiating in the growth plate or not.  The difference is getting new MSCs to differentiate into chondrocytes and form ectopic growth plates within bone in fused bone.

Chondrocyte injection in distraction epiphysiolysis (rabbit model).

"The purpose of this study was to determine if cultured chondrocytes could prevent premature closure of the physis after physeal distraction in a rabbit model. Epiphyseal distraction at the proximal tibial epiphysis was performed in 24 immature rabbits. Chondrocytes were harvested from the iliac apophysis. The animals were divided into three groups: A, epiphyseal distraction alone; B, injection of phosphate buffered saline (PBS) after epiphyseal distraction; and C, injection of cultured chondrocytes after epiphyseal distraction. After epiphyseal separation was noted radiographically, each animal was evaluated at routine intervals. At 2 to 4 weeks, significant tibial lengthening as compared to the contralateral tibia was noted in all three groups. At 10 weeks, lengthening was apparent only in group C; in groups A and B, the operated tibia was shorter than the unoperated one. Histologic examination at 4 weeks revealed partial bony bridge formation with cell clustering in the fibrocartilaginous matrix in groups A and B. In group C, the matrix showed a typical hyaline aspect with cells organized in columns at the injection site. The zone of hypertrophic chondrocytes was enlarged. It appears that the injected cells differentiated into hypertrophic chondrocytes and delayed premature physeal closure. At 10 weeks after distraction of the physis, the width of the physis was restored to 75% of normal, but disordered chondrocyte growth with cell clustering was present in the cell-injected group."

"Physeal distraction is a relatively simple method of limb lengthening in which the epiphysis is pulled away from the metaphysis at a slow, controlled rate. The physis separates without osteotomy and distraction can be applied through an external fixator."

"the lengthening obtained often is lost by growth retardation caused by premature physeal closure after removal of the fixator."

"Skeletally immature, 6-week-old New Zealand white rabbits were ideal for study because these animals approach physeal closure (maturity) at 4 to 6 months."

"In group C, physeal defects were primarily filled with abundant hypertrophic cartilage. A few islands of calcified cartilage were seen in the separated defect in the lower portion of the separated physis at 4 weeks
The matrix showed a typical hyaline aspect with cells organizein columns at the injection site. Most of the cells were viable and exhibited a delimited nucleus. The cellularity of the repair tissue was greater than that of the adjacent normal cartilage.  The injected cells seemed to attach to the bottom of the separated physis and incorporate as hypertrophic chondrocytes, thus delaying premature closure of the physis.At 10 weeks after initiation of distraction, the defect was filled primarily with mixed fibro-hyaline cartilage and the thickness of the physis was restored to 75% of normal, but chondrocyte growth remained disordered with cell clustering"

"Injected chondrocytes (group C) prevented bony bridge formation in all specimens, even in those
few where cell survival appeared to be minimal.  Increased thickness of physeal cartilage produced by distraction was not due to increased cell proliferation in the physis. On the contrary, cell division appeared to be reduced with zonal separation. In 7 of 8 animals with injected chondrocytes, the cells continued to maintain a proteoglycan-rich cartilage matrix for the duration of the experimental period and in some cases showed ordered maturation. Evaluation at longer postoperative intervals showed an increase in vascularity at the distraction site, as fibrous stroma extending into the defects was replaced
with calcified tissue."

"the injected cells attached to the bottom of the separated physis and behaved as hypertrophic chondrocytes to delay premature physeal closure. The enlargement of the hypertrophic chondrocyte region was similar to that seen after slow distraction"

"this proliferation of hypertrophic chondrocytes is derived partly from the injected cells and partly from the tension effect on the physis."

Growth Plate transplants not so far fetched

Note that I believe that hydrostatic pressure induced chondrogenic differentiation of stem cells is currently the best way to grow taller.  That doesn't mean the science of stem cells in growth plate repair can't help us in our quest for height. 

Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy. 

"The potentials of mesenchymal stem cells (MSCs) in cartilage repair include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling[if they identify some carriers that are already in the body we could take advantage of that to gain height]. Gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells [can be used to regenerate cartilage]. [What is] the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes? As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone." 

What we're doing with LSJL is mobilizing endogenous MSCs from the bone marrow. 

Let's look at the three criteria for successful growth plate cartilage repair(and by extension successful formation of new growth plate cartilage): 

1) MSCs capable of chondrogenic capacity.  This should be true of bone marrow MSCs as they are capable of mesenchymal chondrosarcoma(which involves chondrogenic differentiation). Telomere Length and Methylation Status play a role also. 

2) The second factor is a way of encouraging chondrogenic differentiation.  This is hydrostatic pressure.  Which we induce by laterally compressing the bone to increase the fluid pressure within the bone marrow.  There are alternatives to this such as LIPUS plus TGF-Beta.  

3)The scaffolds for transplantation or direct filling.  Well, the MSCs are already in the body so there's no need for transplantation or filling.  They are already in a position to increase height. 

In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes.

"A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP])[MSCs were co-cultured with chondrocytes]. Committed MSCs were seeded into a collagen scaffold[bone is technically already a collagen scaffold Type II collagen] and analyzed for their neocartilage-forming ability.
Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules[studying these chondrogenic adhesion molecules could be a way to help us gain height] and stimulated cartilage developmental growth factors, including members of the transforming growth factor beta superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold.
Human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes."

Now, LSJL can't co-culture MSCs with growth plate chondrocytes as there are no growth plate chondrocytes left after fusion.  However, LSJL can induce chondrogenesis by other means.  This does mean that maybe pre-fusion that something like LIPUS can increase height on it's own as LIPUS causes shear strain in the bone disrupting the actin cytoskeleton and could allow for adhesion of MSCs to chondrocytes. 

Application of autologous bone marrow derived mesenchymal stem cells to an ovine model of growth plate cartilage injury. 

"Injury to growth plate cartilage in children can lead to bone bridge formation and result in bone growth deformities. Mesenchymal stem/stromal cells (MSC) offer a promising therapeutic option for regeneration of damaged cartilage, due to their self renewing and multi-lineage differentiation attributes. Our laboratory has recently characterised MSCs derived from ovine bone marrow, and demonstrated these cells form cartilage-like tissue when transplanted within the gelatin sponge, Gelfoam[Will have to explore height increase applications of Gelfoam], in vivo. In the current study, autologous bone marrow MSC were seeded into Gelfoam scaffold containing TGF-beta1, and transplanted into a surgically created defect of the proximal ovine tibial growth plate. Examination of implants at 5 week post-operatively revealed transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Importantly, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated due to transplantation of MSCs or the gelatin scaffold." 

So the transplant failed to induce chondrogenesis and instead underwent fibrogenesis.  Note that they didn't use shear strain at all and from the previous research shear strain plus TGF-Beta is needed for chondrogenesis.  Also the gelatin scaffold may have been insufficient. 

"Analysis of growth plate defects treated with Gelfoam scaffold and autologous MSC revealed a tissue composition consisting of dense fibrous (38.2 ± 6.1%), fibrous (29.6 ± 10.0%), fat (24.2 ± 6.6%) and bone (7.9 ± 1.8%)."

Also they inserted the growth plate scaffold directly into the growth plate defect that may be a part of the problem and they would've had better luck with a scaffold in a bone fracture.

But at least the technology is there.  They just need better ways to induce chondrogenesis.  Of course, you could just do LSJL. 

Here's a growth plate transplant experiment in action:


Assessment of epiphyseal plate allograft viability and function after ex vivo storage in university of wisconsin solution.

"Compromised epiphyseal plate function can result in limb deformities. Microvascular transplantation of an epiphyseal plate allograft is a potentially effective approach to reestablish longitudinal limb growth. The goal of this study was to determine a time frame for which proximal tibial epiphyseal plate allografts could be stored in University of Wisconsin Preservation Solution (UWPS) and remain functional in vivo after microvascular transplantation.

Proximal tibial epiphyseal plate allografts from skeletally immature female New Zealand White rabbits (10 to 12 wk of age) were used. Allografts (isolated on the popliteal arteriovenous pedicle) were stored ex vivo in cold UWPS for periods of up to 21 days. Chondrocyte viability, phenotype, and extracellular matrix composition of growth plate cartilage was assessed. Microvascular transplantations of nonstored or prestored (3 d) allografts were performed and analysis of bromodeoxyuridine and calcein incorporation was done to determine chondrocyte proliferation and new bone growth, respectively.

In vitro analysis showed that, compared with control tissue, epiphyseal plate chondrocyte viability, organization, and collagen extracellular matrix was preserved up to 4 days in cold UWPS. Microvascular transplantation of nonstored epiphyseal plate allografts was successful[scientists can transfer growth plates that have not been stored]. Despite care being taken to ensure vascular patency during the microvascular procedure, transplantation of prestored allografts failed due to absent flow in the larger vessels and in the allograft based upon the visualization of organized thrombus within the vascular pedicle, and absent flow within the composite graft itself. However, growth plate viability and function was detected in a peripheral region of a single allograft where partial blood flow had been maintained during the transplantation period.

Ex vivo storage in cold UWPS for 3 days maintains growth plate chondrocyte viability and function in vivo. However, future studies must be directed toward investigating the direct effect of ex vivo storage on the integrity and function of the vascular pedicles."

So growth plate transplantation is possible along as the growth plate is not in storage for more than 3 days.  Although I don't see why you wouldn't just differentiate a new growth plate in the bone marrow.

Here's a study related to formation of new growth plates so they can be available for transplant:

Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage.

"We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). [Are] chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP? hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6.  A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. We compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development."

"Pellet cultures were used to induce chondrogenic differentiation of hfMSCs"

"The mean diameter of the pellets increased with time, as well as the amount of glycosaminoglycans, a major constituent of the cartilaginous extracellular matrix. Immunofluorescent staining for collagen type II demonstrated the presence of chondrocytes after 1 week of pellet culture. The expression of collagen type II increased over time. Hypertrophic chondrocytes were first detected after 3 weeks, as evidenced by immunohistochemical staining for collagen type X. These collagen type X positive cells were located in a discrete ring-like zone surrounded by collagen type II positive chondrocytes. In all stages of differentiation, the chondrogenic core of the pellets was surrounded by a thin layer of two to three undifferentiated cells"

"Global gene expression microarray analysis showed that the Wnt antagonist DKK1 and FRZB and the BMP antagonist GREM1 are highly expressed in articular cartilage as compared to growth plate cartilage."

"PANX3, EPYC{up 6 fold in LSJL}, WNT11 and LEF1 are highly expressed in growth plate cartilage as compared to articular cartilage"

KEGG Pathways expressed in growth plates:

Focal Adhesion

Cytokine and Cytokine Receptor Interaction

Wnt and IHH signaling

Complement and coagulation

TGFBeta Signaling

Cell Communication and Extracellular Matrix Interaction

B-cell receptor signaling

Click on the image to see it enlarged.

"Analysis of protein interactions of all genes that were ≥3.29-fold changed after 5 weeks of chondrogenic differentiation as compared to undifferentiated hfMSC"

Genes changed that were not in highlighted clusters in diagram also altered in LSJL:

CAPN6{up}

LRRC1{down}

CADM1{down}

ITGBL1{up}

ARL6ip1{down}

Acta2{up}

Angptl1{up}

Tmem100{up}

Scn3a{up}

Slc38a4{up}

Dpt{up}

Cluster A(all genes listed genes that were altered by LSJL are noted as these genes may not have been altered by LSJL immediately but may have been altered by LSJL at a later time point or at under 2 fold.  Bolded means that the genes are centrally located in the cluster thus if they are altered by LSJL it's more likely that the other genes are too):

ADAMTS1{up}

OMD

GDF5

Sp7

Acan{up}

Epyc{up}

Col10a1{up}

Adamts5

Col9a2

Ptprz1

Cntnap1

Ctsb

Fap

Comp

Col9a3{up}

KIAA1199

Slc24a2

CHAD

PTH1R

Spp1

Col2a1{up}

Chi3l1

Fmod

Col11a1{up}

29.2%

Cluster B:

S100P

TGFBR3

HSPA8

UQCRFS1

CAV1

CRLF1{up}

LRP4

Basp1{down}

Grem1

Has2

FST

Bambi

Wif1

15.4%

Cluster C:

ID3

Hey1

Slc14a1

F13a1

Wnt11

Vcam1{down}

Nqo1

Akr1c3

Serpina3

MMP1

Plau

Fos{up}

Jun{up}

Sox8

21.4%

Cluster D:

Ccnb1

Prc1

Ndc80

Pbk

Ube2c

Plk2

Ccnc

Orc6l

Dlgap5

Melk{down}

10%

Cluster E:

Pcolce2

Ogn{down}

Smoc2{up}

66%

LSJL likely alters expression of clusters A, C, and E.

Now here's human fetal MSCs gene expression versus growth plate gene expression:

"Analysis of protein interactions of genes that are differentially expressed in undifferentiated hfMSC (week 0) compared to average expression profiles of growth plate cartilage of 3 prepubertal donors "

No Cluster Genes that were altered in growth plate cartilage and LSJL:

Gldn{up}

Fxyd6{up}

BSP{up}

Tagln{up}

Postn{up}

Acta2{up}

Col16a1{up}

Vgll3{up}

Scn3a{up}

Fzd2{up}

Capn6{up}

Arl4c{down}

Tardbp{down}

Ankrd29{up}

Slc5a1{up}

Spp1{up}

Cluster A:

Acan{up}

Omd

Matn2{up}

Adamts1{up}

Frzb

MMP9

Ptprz1

Col9a2

Col10a1{up}

Matn3{up}

Epyc{up}

Csgalnact1

Cntnap1

Phlda2

Col2a1{up}

Sox9{up}

Comp

Matn1

Col11a1{up}

Col11a2

Fmod

MMP13

Ctsk

Chi3l1

Lect1

Itga10

Pth1r

FGFR3

CHAD

Prelp

Gprasp1{down}

Wisp3

Col9a1{up}

33.3%

Cluster B:

Grem1

Rgmb

Sost

Gasp1

Has2

Col15a1{up}

glipr1

bambi

fat3

daam1

Wif1

Tmemff2

C4orf49

7.7%

Cluster C:

MMP1

IGFBP3

GBP1

Fap

Tnfaip8{down}

Fos{up}

Fosb{up}

Serpina3

37.5%

Cluster D:

Clu

Serpine1{up}

Gas6

F13a1

Lpl

Lif

Loxl1

TNIK

Vegfc

Serpina1

PF4

Neto2

CH25H{up}

15.4%

Cluster E:

S100B

Myo5c

Prss23

Man1a1

Boc

Ptx3

Serpina5

CTCFL

Clgn

Rbp4

Ifit1

Cdh13{up}

Adipoq

Gpnmb

Sox8

Cxcl14

Smoc2{up}

Ogn{down}

Pcolce2

Hbb

Hbd

Hba2

F3

TF

IGFBP4

MYO5C

11.5%

Cluster F:

BDNF

Eno4

Uchl1

Scg2

Scrg1

Cnih3

Spry2

Stmn2

0%

LSJL likely alters clusters A and C.

"Growth and differentiation factor 5 (GDF5), previously reported as stimulator of chondrocyte proliferation, was highly expressed at the earliest time point observed and down regulated thereafter."

"Cartilage matrix analysis is often limited to examining collagen type 2 and aggrecan expression. These markers are characteristic for hyaline cartilage and cannot distinguish growth plate cartilage from articular cartilage. Indeed, Huang et al. performed global microarray analysis of adult bovine MSCs at time 0 and after 28 days of differentiation in agarose constructs and compared the gene expression profile to that of chondrocytes isolated from articular cartilage. They showed that chondrogenically differentiating MSC do not form articular cartilage at 28 days in the presence of TGFβ3"

Engineering osteochondral constructs through spatial regulation of endochondral ossification.

"Chondrogenically primed bone marrow derived mesenchymal stem cells (MSCs) have been shown to become hypertrophic and undergo endochondral ossification when implanted in vivo. Modulating this endochondral phenotype may be an attractive approach to engineering the osseous phase of an osteochondral implant. [We engineered] an osteochondral tissue by promoting endochondral ossification in one layer of a bi-layered construct and stable cartilage in the other. The top-half of bi-layered agarose hydrogels were seeded with culture expanded chondrocytes (termed chondral layer) and the bottom half of the bi-layered agarose hydrogels with MSCs (termed osseous layer). Constructs were cultured in a chondrogenic medium for 21 days and thereafter were either maintained in a chondrogenic medium, transferred to a hypertrophic medium, or implanted subcutaneously into nude mice. This structured chondrogenic bi-layered co-culture was found to enhance chondrogenesis in the chondral layer, appearing to help re-establish the chondrogenic phenotype that is lost in chondrocytes during monolayer expansion. The bi-layered co-culture appeared to suppress hypertrophy and mineralisation in the osseous layer. The addition of hypertrophic factors to the media was found to induce mineralisation of the osseous layer in vitro. A similar result was observed in vivo where endochondral ossification was restricted to the osseous layer of the construct leading to the development of an osteochondral tissue."

Fully differentiated chondrocytes do not undergo endochondral ossification upon implantation whereas MSCs derived into chondrocytes do.

"A structured co-culture of chondrocytes and MSCs significantly enhanced collagen synthesis in the top chondral layer of bi-layered engineered constructs compared to single layer constructs that only contained chondrocytes (133.32 ± 21.8 vs. 72.45 ± 18.63 ng/ng).  MSCs in single layer constructs accumulated significantly more collagen compared to MSCs in the bottom osseous layer of bi-layered constructs (154.65 ± 14.53 vs. 83.57 ± 21.38 ng/ng)."

"No evidence of mineralisation was observed in bi-layered constructs maintained in a hypertrophic medium without additional β-glycerophosphate supplementation (HM-). When β-glycerophosphate was added to the hypertophic medium (HM+), mineralisation of the osseous layer was observed"

"Both hypertrophic media formulations resulted in apparent elongation of the interface between the osseous and chondral layer of bi-layered constructs. sGAG accumulation in the chondral layer of the engineered tissue was significantly reduced for constructs maintained in HM+ compared to CM"

"Mineralisation of the osseous layer correlated with significant cell death as evidenced by a reduction in the DNA content in this layer of bi-layered constructs when cultured in a hypertrophic medium with additional β-glycerophosphate supplementation (HM+)"

"Mineral volume, was significantly greater for single layer MSC constructs compared to bi-layered constructs (6.09± 0.59 vs. 1.36 ± 0.42 mm3; n=3)"

"[There was] reduced type X collagen accumulation in the osseous layer of bi-layered constructs while type II collagen accumulation increased in the chondral layer."

"bi-layered coculture suppresses hypertrophy of MSCs and enhances chondrogenesis of chondrocytes"

"Single layer chondrocyte seeded constructs stained weakly for collagen type II, indicating that a certain degree of de-differentiation had occurred prior to hydrogel encapsulation."

"chondrogenically primed MSCs release growth factors and cytokines such as TGF-β3, BMP-2, IGF-1 and FGF-2"

"In hypertrophic media formulations, both with and without β-glycerophosphate supplementation, elongation of the interface between the two cell types was observed, suggesting perhaps that aspects of long bone growth are being mimicked in this culture system."