Sunday, March 31, 2013

Creating a pro-chondrogenic microenvironment

 Ctrl-F for (*NEW*) for the new information.  Several times cellulose is used in biomaterial scaffolds to induce chondrogenesis.  Cellulose is also known as fiber and is not digested.  The question is can we ingest enough of it to build up enough fiber in the bone marrow to build a pro-chondrogenic microenvironment?  Since fiber is something that is studied in the mainstream and not just in the bubble of height increase I open the subject to all of you.

How much fiber can build up in the bone marrow?

How do we create a pro-chondrogenic microenvironment within the epiphyseal bone marrow to allow for the formation of new growth plates?  The goal of LSJL is to induce that environment.

Creation of an in vitro microenvironment to enhance human fetal synovium-derived stem cell chondrogenesis.

"[We] assess the feasibility of the sequential application of extracellular matrix (ECM) and low oxygen to enhance chondrogenesis in human fetal synovium-derived stem cells (hfSDSCs). Human fetal synovial fibroblasts (hfSFs) include hfSDSCs, as evidenced by their multi-differentiation capacity and the surface phenotype markers typical of mesenchymal stem cells. Passage-7 hfSFs were plated on either conventional plastic flasks (P) or ECM deposited by hfSFs (E) for one passage. Passage-8 hfSFs were then reseeded for an additional passage on either P or E. The pellets from expanded hfSFs were incubated in a serum-free chondrogenic medium supplemented with 10 ng/ml transforming growth factor-β3 under either normoxia (21% O(2)) or hypoxia (5% O(2)) for 14 days. Pellets were collected for evaluation of the treatments (EE21, EE5, EP21, EP5, PE21, PE5, PP21, and PP5) on expanded hfSF chondrogenesis. Compared with seeding on conventional plastic flasks, hfSFs expanded on ECM exhibit a lower expression of senescence-associated β-galactosidase and an enhanced level of stage-specific embryonic antigen-4{The Extecellular Matrix has potential to reduce cellular senescence}. ECM-expanded hfSFs show increased cell numbers and an enhanced chondrogenic potential. Low oxygen (5% O(2)) during pellet culture enhances hfSF chondrogenesis. The presence of stem cells in hfSFs, and modulation of the in vitro microenvironment can enhance hfSDSC chondrogenesis."

The two elements of the microenvironment identified as being pro-chondrogenic are hypoxia and ECM.  LSJL heavily upregulates ECM molecules.

"Adult MSCs cultured in vitro lack telomerase activity"<-in contrast to fetal MSCs which have higher telomerase activity and longer telomeres.

Group PE5 had the most positive chondrogenic markers.  Which would be groups first plated on conventional plastic flasks than transferred to ECM by synovial fibroblasts in an hypoxic environment.  EE5 which is ECM for the whole time also had positive measures on all chondrogenic factors.

The reason that Plastic followed by ECM was better than pure ECM could be due to a catch-up growth phenomenon so ECM may be still better at all stages for chondrogenesis overall.

Cellular Response to Hypoxia("Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating lowered oxygen tension. Hypoxia, defined as a decline in O2 levels below normoxic levels of 20.8 - 20.95%, results in metabolic adaptation at both the cellular and organismal level. ") genes for mus musculus also altered in LSJL:
Adam8{down}
Gnb1{down}

ECM stiffness primes the TGFβ pathway to promote chondrocyte differentiation.

"Chondrocytes generate an integrated response to ECM stiffness and transforming growth factor β (TGFβ), a potent agonist of chondrocyte differentiation. Primary murine chondrocytes and ATDC5 cells{ATDC5 cells are progenitor cells to chondrocytes} grown on 0.5-MPa substrates deposit more proteoglycan and express more Sox9, Col2α1, and aggrecan mRNA relative to cells exposed to substrates of any other stiffness{0.5MPa is the optimal stiffness out of those tested to encourage chondrogenesis}. The chondroinductive effect of this discrete stiffness, which falls within the range reported for articular cartilage, requires the stiffness-sensitive induction of TGFβ1. Smad3 phosphorylation, nuclear localization, and transcriptional activity are specifically increased in cells grown on 0.5-MPa substrates{The benefits of this ECM stiffness may be due to an increase in Smad3 phosphorylation}. ECM stiffness primes cells for a synergistic response, such that the combination of ECM stiffness and exogenous TGFβ induces chondrocyte gene expression more robustly than either cue alone through a p38 mitogen-activated protein kinase-dependent mechanism."

"Upon integrin binding to ECM ligands and the generation of internal cell tension, cells develop focal adhesions, a highly ordered array of proteins including focal adhesion kinase (FAK), talin, vinculin, and α-actinin. These proteins interact with small GTPases (i.e., Rho, Rac) and other signaling pathways, facilitating changes in cytoskeletal organization, actinomyosin contractility, and cell shape with even small changes in matrix compliance"

"The activated TβRI phosphorylates Smad2 and Smad3 on the C-terminal domain, causing heteromerization with Smad4 and preferential retention in the nucleus, where Smads act as transcription factors. In chondrocytes, phosphorylated Smad3 recruits CBP to activate Sox9-mediated transcription of Col2α1"

"Cells respond to substrate stiffness by increasing internal cellular tension through stress fiber formation and cell spreading"

"Rho and ROCK participate in stiffness sensing in part through stress fiber formation"

"TGFβ rapidly activates the p38 pathway through the MAPK kinase kinase TAK1 activation of MKK3/6. Phospho-p38 was increased on 0.5-MPa substrates relative to plastic"

"there is an optimal level of ROCK activity on 0.5-MPa substrates that activates chondroinduction, in part, through the induction of TGFβ1 expression on compliant substrates."

"BMP-inducible nuclear translocation of Smad1/5/8 requires sufficient ROCK-dependent cytoskeletal tension. ROCK can also enhance the activity of both Smad3 and Sox9 by phosphorylation of the Smad3 linker region or Sox9 on serine 181"

Mesenchymal stem cells and their microenvironment.

"MSCs are stromal-like cells that are characterized by a CD105+ /CD73+ /CD90+ /CD45- /CD34- /CD11b- /CD19- /HLA-DR- cell surface signature"

"MSCs [may] exist in a perivascular[near or around blood vessels] location and share a number of cell surface markers with pericytes"

"MSC show a greater propensity to differentiate to cartilage and bone, if they are bone marrow derived and to differentiate to fat, if isolated from adipose tissue"

"In order to self renew, stem cells need to be protected from differentiation signals and from apoptosis, and the niche provides the adhesion molecules, soluble factors and conditions that allow this in a concerted fashion. These soluble factors and ECM components activate various kinase cascades including the ERK1/2 MAPK and the PI-3K pathways"<-So not only do we need to induce differentiation signals to induce stem cells to chondrogenesis we need to protect them from differentiation signals that induce differentiation to other cell types.

"EGF, which stimulates the EGFR1 on MSCs, strongly activates the MAP kinases ERK1/2 and Jnk1, the Stat3 and PKC pathways and weakly stimulates the PI-3K pathway, and high concentrations of EGF induce osteogenic differentiation in MSCs"<-Thus we want to avoid high levels of EGF.

"High concentrations of covalently tethered EGF, which restrict signaling to the cell surface, result in increased osteogenic differentiation of MSCs, while low concentrations of soluble EGF, which induce receptor internalization, are anti-osteogenic"<-Reducing EGF levels may be part of the way to grow taller.

"Concomitant PI-3K stimulation prevents ERK1/2-dependent osteogenic differentiation."<-LSJL likely stimulates both PI3K and ERK1/2 which may be why it is more pro-chondrogenic than osteogenic.

"MSCs can produce VEGF, bFGF{up}, PDGF, angiopoietin, CXCL8/IL-8 and other angiogenic factors"

"[MSCs] express Oct4, Nanog, Sox2, SSEA3, SSEA4, Rex1, c-myc, nucleostemin, Nodal, Sca1{only expressed by mice and not humans}, Snail2"

"When chondrogenesis was assessed in cross-linked methacrylated hyaluronic acid hydrogels, the macromer density influenced MSC chondrogenesis: high density macromers resulted in increased chondrogenesis, but of inferior quality than seen with lower density gels"

"early MSC progenitors, defined by their smaller size and expression of podocalyxin-like protein (PODXL), selectively express alpha4 and alpha6 integrins, which are lost during culture. Freshly isolated MSC do not express the vitronectin receptor alphav beta5, but express low levels of the fibronectin receptor alpha5beta1 and the collagen receptors alpha1beta1, alpha2beta1 and alpha3beta1. Upon culture alphavbeta5 is up-regulated. Increased expression of alphavbeta5 and alpha5beta1 is observed during chondrogenic differentiation"

"MSCs do not seem to circulate in the vasculature under physiological conditions, it seems likely that they are released into the vasculature, when there is increased demand for these cells during any kind of tissue injury "

"Although MSCs accumulate in damaged tissue to some degree, recruitment of circulating MSC is very inefficient"

"MSCs seem to be resistant in general to certain apoptotic pathways perhaps due to very low expression of caspase 8 and caspase 9"

"all MSC express cell surface receptors for C3a and C5a"

"genes expressed in MSCs specifically involved in the HSC niche including galectin-1, fibronectin-1, osteopontin, CXCL12, thrombospondin-1 and -2, TGF-beta 2, Angiopoietin-1, ILGFBP-4, FGF-7, SFRP-1 and -2, VCAM-1, and BMPR type 1a"

Redox regulation of stem/progenitor cells and bone marrow niche.

"Bone marrow (BM)-derived stem and progenitor cell functions including self-renewal, differentiation, survival, migration, proliferation, and mobilization are regulated by unique cell-intrinsic and -extrinsic signals provided by their microenvironment. Reactive oxygen species (ROS), especially hydrogen peroxide (H(2)O(2)), play roles in regulating stem and progenitor cell functions in various physiologic and pathologic responses. The low level of H(2)O(2) in quiescent hematopoietic stem cells (HSCs) contributes to maintaining their "stemness," whereas a higher level of H(2)O(2) within HSCs or their niche promotes differentiation, proliferation, migration, and survival of HSCs or stem/progenitor cells. Major sources of ROS are NADPH oxidase and mitochondria. In response to ischemic injury, ROS derived from NADPH oxidase are increased in the BM microenvironment{mechanical loading stimulates ROS release from mitochondria creating a more hypoxic microenvironment}, which is required for hypoxia and hypoxia-inducible factor-1α expression and expansion throughout the BM. This, in turn, promotes progenitor cell expansion and mobilization from BM, leading to reparative neovascularization and tissue repair. Excess amounts of ROS create an inflammatory and oxidative microenvironment, which induces cell damage and apoptosis of stem and progenitor cells."

"ROS such as O2•− and H2O2 are generated from a number of sources including mitochondria, NADPH oxidases (NOXs), xanthine oxidase, cytochrome P450, and nitric oxide synthase (through its uncoupling). Because O2•− is produced from oxygen, the oxygen concentration or hypoxic condition has a significant impact on the total amount of ROS. The O2•− reacts with nitric oxide (NO) to generate peroxynitrite (OONO−), thereby inhibiting endothelial function, whereas it can be quickly converted to H2O2 by superoxide dismutases (SODs) such as MnSOD (SOD2) or Cu/ZnSOD (SOD1) or extracellular SOD (SOD3). H2O2 is catalyzed by catalase, glutathione peroxidases (GPx′s), and the thioredoxin–peroxiredoxin system to nonreactive water"

"Growth factor signaling is mediated through H2O2 production."

"Different from phagocytic NADPH oxidase that is normally quiescent but generates a large burst of O2•− (the “oxidative burst”) upon activation, the NOXs constitutively produce low levels of O2•− or H2O2 intracellularly in the basal state and are further stimulated acutely by various agonists and growth factors. NOXs are now recognized to have specific subcellular localizations, which is required for localized H2O2 production and activation of specific redox signaling pathways to mediate various functions"  NOXs are located in MSCs.

"Once O2•− is generated, it is immediately converted into H2O2 by MnSOD or Cu/ZnSOD"

"To avoid the potential damaging effects of H2O2, mitochondria express other antioxidant enzymes such as peroxiredoxin (Prx) 3 and Prx5 and glutathione peroxidase."

"hypoxic conditions increase mitochondrial ROS production, which stabilizes HIF-1α protein expression. "

"optimal levels of ROS are required for normal responses, whereas excess or insufficient levels of ROS are associated with cellular dysfunction and reduced growth factor signaling, respectively"<-So we want to maximize ROS signaling before cellular dysfunction.

"PTEN is a negative regulator of the PI3K–Akt pathway and contains catalytic cysteine residues that are highly susceptible to oxidation by H2O2. Therefore, PTEN inhibition stabilizes the active phosphorylated form of Akt. "

(*NEW*)
Directing chondrogenesis of stem cells with specific blends of cellulose and silk.

"We systematically prepared cellulose, blends with silk at different compositions using a method based on ionic liquids as a common solvent. We tested the effect of blend compositions on the physical properties of the materials as well as on their ability to support mesenchymal stem cell (MSC) growth and chondrogenic differentiation. The stiffness and tensile strength of cellulose was significantly reduced by blending with silk. The characterised materials were tested using MSCs derived from four different patients. Growing MSCs on a specific blend combination of cellulose and silk in a 75:25 ratio significantly upregulated the chondrogenic marker genes SOX9, aggrecan and type II collagen in the absence of specific growth factors{so the stiffness of this environment is likely optimal for chondrogenesis}. This chondrogenic effect was not found with neat cellulose or cellulose/silk 50:50 blend composition. No adipogenic or osteogenic differentiation is detected on the blends suggesting that cellulose/silk 75:25 blend induces specific stem cell differentiation into the chondrogenic lineage without addition of the soluble growth factor TGF-β."

"Cellulose is a linear homopolymer of glucose."  Cellulose is also called fiber.  It can't be digested so if you can get it to your bone marrow it may help create a pro-chondrogenic microenvironment.

"Cellulose, which comprises three hydroxyl groups per repeating unit, is theoretically a good choice as an initiator of chondrogenesis"

"Silane-treated glass surfaces functionalized with carboxyl (–COOH) and hydroxyl (–OH) groups initiated chondrogenic marker mRNA expression in MSCs in the absence of chondrogenic growth factors, whereas amine (–NH2) functional groups encouraged osteogenic differentiation of stem cells in the absence of osteogenic supplements"

Compounds with Hydroxyl groups:
Alcohol
Sugars

Compounds with Carboxyl groups:
Sugars
Vinegar
Goat fat
Breast milk
Coconut/Palm/Peanut oil
Nutmeg

Amino acids contain the amine functional group.

The study Galactooligosaccharides improve mineral absorption and bone properties in growing rats through gut fermentation. found that "Galactooligosaccharides (GOS), prebiotic nondigestible oligosaccharides derived from lactose" had no effect on femur length.

According to Dietary cellulose, zinc and copper: effects on tissue levels of trace minerals in the rat.,  Fiber only increased tibia dry weight with a diet deficient in zinc and copper.  Dry weight should but not necessarily correlate with bone length.  The mice were below 9 weeks old so they were growing.  The study was done in 1979 so there's no hope to contact for length data.

The emergence of mechanoregulated endochondral ossification in evolution.

"stable fractures with small gaps between bone ends can undergo intramembranous healing, involving ossification on a fibrous membrane; in larger fracture gaps and more straining mechanical environments, endochondral ossification is necessary for healing where a cartilage matrix is laid down which later, if the soft tissue succeeds in stabilising the fracture, provides a template for ossification. It is likely that endochondral bone emerged in evolution because it confers a greater fitness in a more demanding mechanical environment."
Full-size image (31 K)So we want shear strain and fluid velocity at an amount that causes cartilage tissue to form.

Thursday, March 28, 2013

Lengthening your Body with a Hyperbaric Chamber?

Request for help:  Does anyone know of any athletes who regularly use hyperbaric chambers so we can study changes in their bone phenotype?
 
The hyperbaric chamber makes you breathe solely oxygen and increases the atmospheric pressure surrounding you.  Now cartilage is an avascular tissue but can hyperbaric chamber treatment increase chondroinduction or increase the growth from growth plates?

Many athletes use hyperbaric chambers so if hyperbaric chambers could chondroinduce on their own there would be some sort of anecdotal evidence.

Hyperbaric chambers may be an interesting possible way to enhance growth but are too cost prohibitive.

The effect of hyperbaric oxygen and air on cartilage tissue engineering.

"Under hyperbaric oxygen and air stimulation, the cell number of chondrocytes in cartilage matrix was not significantly increased, but the glycosaminoglycans syntheses markedly increased compared to the control group.  The chondrogenic-specific gene expression of SOX9, aggrecan, and COL2A1 were compared respectively. Within the limitation of this study, it was concluded that 2.5 atmosphere absolute oxygen and air may provide a stress environment to help cartilage tissue engineering development."

"In approximately 2.5 ATA[absolute atmosphere] HBO–treated group, the SOX9 and aggrecan expressed significantly at days 9 and 12, but there was no increase with the type I collagen–related gene COL1A2. Alternately, in 2.5 ATA hyperbaric air–treated group, the SOX9 increased with the time and type II collagen–related gene COL2A1 showed significant over expression at days 6, 9, and 15, with no manifest increase with the COL1A2 "

Since hyperbaric chambers go to 2-3 times normal pressure maybe this would would be sufficient.

"Fifteen hASCs contained biocomposites and were equally separated into 3 groups and subsequently treated with 1 ATA air (as control), 2.5 ATA HBO, and 2.5 ATA hyperbaric air operations which were performed in a hyperbaric chamber (MEDITT, Republic of China). The duration of treatment was 1 hour a day for 5 days, and samples were observed at days 6, 9, 12, and 15, respectively, after induction."

Equine peripheral blood-derived mesenchymal stem cells: isolation, identification, trilineage differentiation and effect of hyperbaric oxygen treatment. states that hyperbaric oxygen increased the concentration of mesenchymal stem cells.  Mesenchymal condensation is an important part of new growth plate formation.  Increased MSC concentration would facilitate mesenchymal condensation.

Osteodistraction of a previously irradiated mandible with or without adjunctive hyperbaric oxygenation: an experimental study in rabbits. states that mandibular osteodistraction resulted in cartilagenous tissue in all the experimented groups.  Only irradiation increased the size of chondroid islands and not hyperbaric oxygen.

According to Hyperbaric oxygen-stimulated proliferation and growth of osteoblasts may be mediated through the FGF-2/MEK/ERK 1/2/NF-κB and PKC/JNK pathways., stimulates the growth and proliferation of osteoblasts but can that apply to chondrocytes or stem cells?

Age-dependent response of murine female bone marrow cells to hyperbaric oxygen.

"We treated 2- and 18-month old C57BL/6 female mice by HBO[Hyperbaric Oxygen]. Hematopoietic stem cells and progenitors, enumerated as colony-forming units in culture, were doubled only in peripheral leukocytes and BM cells of young mice receiving HBO. In old mice colony-forming unit fibroblast numbers, a measure of mesenchymal stromal cells (MSCs) from BM, were high but unaffected by HBO. To further explore this finding, in BM-MSCs we quantified the transcripts of adipocyte early-differentiation genes peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein-β and fatty-acid binding protein 4; these transcripts were not affected by age or HBO. However, osteoblast gene transcripts runt-related transcription factor 2, osterix (OSX) and alkaline phosphatase (AP) were twofold to 20-fold more abundant in MSCs from old control mice relative to those of young control mice. HBO affected expression of osteoblast markers only in old MSCs (OSX gene expression was reduced by twofold and AP expression was increased threefold)."

Unfortunately no chondrogenic markers were measured.  The study did say the HBO increased the mobilization of MSCs.

Monday, March 25, 2013

Grow Taller by your periosteum?

 In the *NEW*(ctrl-f for *NEW*) section of this article I provide articles supporting Natural Height Growth's theory that periosteal stripping could help increase height.  When cartilage first condenses in the embryo there is no periosteum therefore the periosteum could serve as a mechanism to inhibit growth related to maturity.  Can rapid repetive loading(like via a chisel and hammer method) be akin to periosteal stripping?  One of the papers mentioned in the aforementioned page is on adult rats and seems to possibly have longitudinal bone growth.

We already know it's possible to grow taller by increasing the periosteal width of your certain bones that have periosteum oriented in the longitudinal direction such as the flat bone of the skull .  We also know that the periosteum is key in distraction osteogenesis surgery.  Tissue damage is highly anabolic.  How does muscular hypertrophy occur?  By damage to the myosin-actin bridge.  Does this damage just repair what was done before?  No it increases the size of your muscle according to the cellular signals as regulated by myostatin(testesterone inhibits myostatin) plus other factors.  Some of the cells that are released from damage to the muscle tissue go to repair but others go to build new muscle.

There are studies that show that bone can increase in size.  Tissue damage is highly anabolic and bones can increase in size, the periosteum is a tissue and has been shown to be highly important in limb lengthening surgery, therefore the periosteum is likely to have anabolic effects on your bone.  The periosteum contains progenitor cells which are like stem cells.

Even though the progenitor cells from the periosteum are not as potent as the stem cells from trabecular, it still has anabolic effects.  The periosteum also contains fibroblasts which are anabolic for connective tissues and what can possibly account for the increase in periosteal width in runners.

One problem is the location of the most easily accessible periosteum(in the tibia) which damage to the tissues should only increase bone width unless the periosteal progenitor cells somehow differentiate into chondrocytes.  Lateral Synovial Joint loading would definitely cause shear strain on the periosteum thus causing anabolic effects on the periosteum that way.  However, it's unclear whether LSJL would cause hydrostatic pressure in the periosteum as the periosteum is far more malleable than the hard tissue of the bone surrounding the bone marrow.

Here's an article about the direct effect of the periosteum on growth plate development:

Tissue engineering models of human digits: effect of periosteum on growth plate cartilage development.

"Tissue-engineered middle phalanx constructs of human digits were investigated to determine whether periosteum wrapped partly about model midshafts mediated cartilage growth plate formation. Models were fabricated by suturing ends of polymer midshafts in a human middle phalanx shape with polymer sheets seeded with heterogeneous chondrocyte populations from bovine articular cartilage. Half of each midshaft length was wrapped with bovine periosteum{if periosteum was wrapped on the midshaft ends would the bone than grow longer?}. Constructs were cultured, implanted in nude mice for up to 20 weeks, harvested and treated histologically to assess morphology and cartilage proteoglycans. After 20 weeks of implantation, chondrocyte-seeded sheets adjacent to periosteum-wrapped midshaft halves established cartilage growth plates resembling normal tissue in vivo. Sheets adjacent to midshafts without periosteum had disorganized cells and no plate formation. Proteoglycans were present at both midshaft ends. Periosteum appears to guide chondrocytes toward growth plate cartilage organization and tissue engineering provides means for carefully examining construct development of this tissue."

So the periosteum is needed to from growth plates.  Chondrocytes not near periosteum will not form growth plates and will not make you taller.  Adults have periosteum so this is a good sign for the potential for adult growth plates.  There is usually no periosteum surrounding the epiphysis of the bone which could make it difficult to form growth plates there as an adult.  But there is periosteum at the end of the epiphysis when it becomes the diaphysis, so it may be close enough to direct the formation of new growth plates.

"After 20 weeks of implantation, engineered human middle phalanx models were found to have glistening, firm and well defined cartilage on both ends of their individual midshaft regions. The portion of midshaft covered with periosteum consisted of essentially clear tissue having a few red-colored areas over its surface indicative of vascular formation. The midshaft region left unwrapped was notably reddened and vascularized{so the periosteum does not seem to have an effect on growth plate vascularization}. X-ray radiography revealed marked mineral deposition within the midshafts of the models only where periosteum had been placed and sutured. No mineral formation was detectable in the cartilage regions at the ends of the models."

So it could be the periosteum that affects the distinction between articular and growth plate cartilage.  The reason that articular cartilage usually does not ossify could be that it's too far away from periosteum.

"Over identical implantation times, chondrocyte-seeded PGA sheets adjacent to the half of the same model midshafts left uncovered by periosteum had disorganized cells and no growth plate formation or mineralization"<-So you need both chondrocytes and periosteum to grow taller.  And the chondrocytes need to be pretty close to the periosteum as the chondrocytes adjacent to the periosteum did not form growth plates.

"These results support the possibility that periosteal tissue mediates growth plate cartilage formation, perhaps by synthesis and secretion of growth factors and other proteins that provide diffusion-limited regulation and control of neighboring cartilage."<-So we could mimic the benefits of periosteal tissue by increasing serum levels of growth factors and proteins.  It would be hard to mimic the diffusion regulation and control of neighboring cartilage.

Shear Strain from lateral synovial joint loading may help spread periosteal growth factors to the epiphysis.  Periosteum also has the ability to lengthen.

The fact that LSJL targets height growth by stimulating cell differentiation in the epiphysis into chondrocytes and that chondrocytes will not form growth plates unless adjacent to periosteum(and the epiphysis usually has no periosteum) means that it is likely that the periosteum has to be addressed to maximize gains from LSJL.

Here's the diagram of the study of the portions of the bone attached to periosteum:


Since the scientists attached the periosteum themselves it is likely that this does not completely represent a periosteal distribution pattern but you can see some periosteum at the very end of the epiphysis.  Any stem cells that differentiate into chondrocytes at this end zone could have sufficient access to periosteum.  Any other stem cells that differentiate in the remainder of the epiphysis will not form growth plates.  Since only a small portion of the epiphysis is covered by periosteum, this makes only a small portion of stem cells successfully differentiated by LSJL increase height.

This could explain LSJL stagnation as well.  In the beginning, individuals epiphysis may be well oriented to the periosteum making it easy for the chondrocytes to find surrounding periosteum.  However, growing taller changes the epiphysis and periosteum thus perhaps making it harder for chondrocytes to be located next to periosteum.

Also, it was noted that people who performed LSJL got a larger epiphysis.  It was then theorized that this could be due to chondrogenesis in the epiphysis.  This is now not possible as growth plates cannot form unless adjacent to periosteum.  The enlarged epiphysis is likely due to a direct increase in the width of individual osteons and direct bone deposition by osteoblasts.

There are two ingredients to growing taller:  Stem Cells differentiating into chondrocytes and those chondrocytes being adjacent to periosteum.  LSJL and some supplements that increase TGF-Beta1 and BMP-2 can help with the former, now we need to deal with the latter.

Here's a study that shows how the periosteum can cause bone regeneration:

A novel osteogenesis technique: The expansible guided bone regeneration{Go over this study in *Detail*}

"Guided bone regeneration is a unique osteogenesis technique that requires a barrier membrane under periosteum to create space for bone regeneration{if we extend the periosteum over the longitudinal ends of the bones and create a barrier membrane then we can grow taller, also we can grow taller when periosteum is already at the longitudinal location of the bone such as the flat bone of the skull}. However, creating sizeable spaces is clinically not commonly feasible. A titanium plate and a thin silicone membrane were surgically layered on each calvaria of eight rabbits. Then, the periphery of the silicone membrane was fixed by a plastic ring to the underlying bone using titanium micro screws. After 1 week, a 5-mm-length titanium screw was used to elevate the titanium plate, which in turn elevated the silicone membrane together with overlying soft tissue in a rate of 1 mm/day for 5 days to create a secluded space. Animals were killed at 2 months (n = 4, group 1) and 4 months (n = 4, group 2) after the elevation. Histological and microradiographical analyses demonstrated creation of an amount of de novo bone formation (68.2 ± 22 mm3 in group 1 and 70.3 ± 14 mm3 in group 2) in the sizeable created spaces (207.1 ± 31 mm3 in group 1 and 202 ± 21 mm3 in group 2) without exposure of the device. This novel osteogenesis technique, “expansible guided bone regeneration,” created a substantial in vivo incubator without applying growth factors or osteoprogenitor cells. Creating a growing space over the secluded surface allowed the development of normal biological healing process occurring on the bone surface into a regenerative process, generating bone outside the genetically determined skeletal bone{so what we can do is create a growing space between the articular cartilage and the subchondral ends of bone}. This technique is a new tissue engineering approach stimulating endogenous tissue repair without applying cells or factors exogenously."

"large volumes of bone can be produced in a predictive manner without exogenously applying the three key players, if the space is provided by injecting biocompatible gel under periosteum."<-space plus periosteum equals bone growth.  The problem is there's no periosteum at the longitudinal ends of bones.

"More recently, we and others have been reported that gradual periosteum elevation creating a space over bone surface results in new bone formation in this space"<-How do we elevate and stretch the periosteum?

"the invasion of the created space with highly competitive nonosteogenic soft tissue and poor quality of the newly formed bone are the main drawbacks of this technique"

In the study they use an elevation screw to lift the periosteum.  Maybe we can mimic this with mechanical stimuli somehow.

"Upon activation of bone surface, biological healing process taking place on the activated surface is kept confined to the surface during the first week. After that the elevation plate is set to move upward, the membrane is gradually elevated and the space attains its maximum size in 5 days."

Now we we need find mechanical stimuli that can stretch periosteum over the longitudinal ends of bones and that can elevate the periosteum.

The nature and role of periosteum in bone and cartilage regeneration.

"[Can] periosteum from different bone sources in a donor [result] in the same formation of bone and cartilage? In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium >> radial ≥ cranial ≥ mandibular; type II collagen, radial > ilium > cranial ≥ mandibular; runx2, cranial >>> radial > mandibular ≥ ilium; and BSP, ilium ≥ radial > cranial > mandibular. The osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively."

Only the madible had no signs of cartilage proteoglycans.

"These results indicate that osteoblasts and chondrocytes derived from sutured periosteum remain viable during implantation and migrate into the constructs. The cells proliferate and secrete matrix that leads to new bone and mineral formation (osteoblasts) and new cartilage (chondrocytes) in interior spaces of the scaffolds as well as in the tissue over the scaffolds"<-With LSJL we have no scaffold.  We're trying to use endogenous tissues as a scaffold.

Multiple exostosis: a short study of abnormalities near the growth plate.

"The pathogenesis of multiple exostosis has been controversial with many theories put forward including the structural/mechanical theory, which emphasizes that the osteochondroma arises in the displaced growth plate cartilage penetrating a defective periosteum. Recently, molecular genetics has offered the neoplastic model with tumor suppressor genes implicated in the development and pathogenesis of exostosis. In this study, we demonstrated the spectrum of histological abnormalities in the developing exostosis present on the surface of the bone at the physis. Seven skeletally immature patients with multiple exostoses were used in this study. The patients' families were advised of and consented to the proposed study. Coincident with removal of symptomatic exostoses that was adjacent to the physis, a thin strip of bone with overlying periosteum was removed to include the edge of the physis. This was followed by formalin fixation and routine paraffin embedding. We demonstrated the earliest lesion as a microchondroma within the periosteum adjacent to the normal physis (also called the 'groove of Ranvier'). More mature progressively larger lesions showing enchondral ossification were seen distally. The periosteum and the perichondrium were intact with normal physis. Our observations give support to the fact that precursor cells in the periosteum adjacent to the physis (also called the 'groove of Ranvier') gives rise to the chondrocytes that clonally expands and develops into exostosis."

"the cause of exostoses was a ‘fault of the epiphyseal plate, nests of cartilage being misplaced’. He indicated that ‘fragments of cartilage around the epiphyseal line become isolated on the surface of the metaphysis, proliferate, and form exostosis’. ‘The periosteum, which is incomplete at the sites of these cartilaginous nests, fails to model the metaphysis in a normal manner’."

"Multiple noncontiguous clusters of cartilage cells of increasing size were found on the surface of the bone. The chondromas increased in size as the distance from the physis increases."

In vivo generation of cartilage from periosteum.

"Damaging the periosteum may be a way to generate ectopic cartilage or bone, which may be useful for the repair of articular cartilage and bone defects. Periosteum was bilaterally dissected from the proximal medial tibia of New Zealand White rabbits. Reactive periosteal tissue was harvested 10, 20, and 40 days postsurgery and analyzed for expression of collagen types I, II, and X, aggrecan, osteopontin, and osteonectin and collagen types I and II. Reactive tissue was present in 93% of cases. Histologically, this tissue consisted of hyaline cartilage at follow-up days 10 and 20. Expression of collagen type II and aggrecan was present at 10 and 20 days postsurgery. Highest expression was at 10 days. Expression of collagen type X increased up to 20 days. No significant changes in the mRNA expression of osteopontin or osteonectin were observed. Cartilage [was present], which was positive for collagen types I and II at 10 days and only for collagen type II at 20 days. At 20 days postsurgery the onset of bone formation was also observed. At 40 days postsurgery, the reactive tissue had almost completely turned into bone."

"cells in the cambial layer of the periosteum have chondrogenic potential in vitro and in vivo"

The ectopic cartilage is at the longitudinal ends of the bones so maybe it can increase height.

*NEW*

Regulation of endochondral cartilage growth in the developing avian limb: cooperative involvement of perichondrium and periosteum.

"To determine if the perichondrium and periosteum regulate growth through the production of diffusible factors, we have tested various conditioned media from these tissues for the ability to modify cartilage growth in tibiotarsal organ cultures from which these tissues have been removed. Both negative and positive regulatory activities were detected. Negative regulation was observed with conditioned medium from (1) cell cultures of the region bordering both the perichondrium and the periosteum, (2) co-cultures of perichondrial and periosteal cells, and (3) a mixture of conditioned media from perichondrial cell cultures and periosteal cell cultures. Positive regulation was observed with conditioned media from several cell types, with the most potent activity being from articular perichondrial cells and hypertrophic chondrocytes."

"At the point where the boney shaft borders the cartilage, the perichondrium (PC) differentiates into the periosteum (PO), whose cells have osteoblastic potential"

"PC/PO-free long bones [had an] increase in overall length of the cartilage [resulting from] increases in the sizes of both the proliferative and hypertrophic zones"

Multiple mechanisms of perichondrial regulation of cartilage growth.

"he perichondrium (PC) and the periosteum (PO) negatively regulate endochondral cartilage growth through secreted factors. Conditioned medium from cultures of PC and PO cells when mixed (PC/PO-conditioned medium) and tested on organ cultures of embryonic chicken tibiotarsi from which the PC and PO have been removed (PC/PO-free cultures) effect negative regulation of growth. Of potential importance, this regulation compensates precisely for removal of the PC and PO, thus mimicking the regulation effected by these tissues in vivo. We have now examined whether two known negative regulators of cartilage growth (retinoic acid [RA] and transforming growth factor-beta1 [TGF-beta1]) act in a manner consistent with this PC/PO-mediated regulation. The results suggest that RA and TGF-beta1, per se, are not the regulators in the PC/PO-conditioned medium. Instead, they show that these two factors each act in regulating cartilage growth through an additional, previously undescribed, negative regulatory mechanism(s) involving the perichondrium. When cultures of perichondrial cells (but not periosteal cells) are treated with either agent, they secrete secondary regulatory factors into their conditioned medium, the action of which is to effect precise negative regulation of cartilage growth when tested on the PC/PO-free organ cultures. This negative regulation through the perichondrium is the only activity detected with TGF-beta1. Whereas, RA shows additional regulation on the cartilage itself. However, this regulation by RA is not "precise" in that it produces abnormally shortened cartilages. Overall, the precise regulation of cartilage growth effected by the action of the perichondrial-derived factor(s) elicited from the perichondrial cells by treatment with either RA or TGF-beta1, when combined with our previous results showing similar--yet clearly different--"precise" regulation by the PC/PO-conditioned medium suggests the existence of multiple mechanisms involving the perichondrium, possibly interrelated or redundant, to ensure the proper growth of endochondral skeletal elements."

"RA has been reported to be both an inhibitor and promoter of cartilage development. In developing embryos of various species, both hypervitaminosis A and hypovitaminosis A greatly disturb the organization of the growth plate. In cell cultures, low doses (50 nM) of RA promote cartilage differentiation. However, in organ cultures, the addition of RA produces the opposite effect: a dose-dependent inhibition of longitudinal bone growth. This finding is due to decreases in both chondrocyte proliferation and hypertrophy"<-one difference between an organ culture and cell culture is the presence of the periosteum.

"RA treatment of the intact cultures produced a reduction in cartilage length from 3.77 mm for the controls to 2.96 mm for the RA-treated. This reduction of 0.81 mm is an even greater overcompensation than for the PC/PO-free cultures, suggesting that RA must have another mechanism of action in addition to that which acts directly on cartilage"

"TGF-β1 showed negative regulation only with the intact organ cultures—not with the PC/PO-free ones. When 10 ng/ml TGF-β1 was added to the intact cultures, the lengths of the cartilage was reduced to 3.48 mm for the treated vs. 3.81 mm for the controls. However, the PC/PO-free organ cultures showed no response to TGF-β1 treatment, with both treated and untreated cultures growing to 4.0 mm"

"FGF-2 acts solely on the cartilage, resulting in identical cartilage lengths between intact and PC/PO-free cultures when treated with FGF-2."

"one of the three nuclear RARs (RARβ) has been shown to be expressed at high levels in the perichondrium, as is RA itself; the remaining two RARs (RARα and RARγ) are expressed in cartilage."

Intracellular tension in periosteum/perichondrium cells regulates long bone growth.

"erichondrium/periosteum cells were cultured on substrates with different stiffness. The medium produced by these cultures was added to embryonic chick tibiotarsi from which perichondrium/periosteum was either stripped or left intact. After 3 culture days, long bone growth was proportionally related to the stiffness of the substrate on which perichondrium/periosteum cells were grown while they produced conditioned medium. A second set of experiments demonstrated that the effect occurred through expression of a growth-inhibiting factor, rather than through the reduction of a stimulatory factor. Finally, evidence for the importance of intracellular tension was obtained by showing that the inhibitory effect was abolished when perichondrium/periosteum cells were treated with cytochalasin D, which disrupts the actin microfilaments. Modulation of long bone growth occurs through release of soluble inhibitors by perichondrium/periosteum cells, and that the ability of cells to develop intracellular tension through their actin microfilaments is at the base of this mechano-regulated control pathway."

"periosteum [may regulate] growth via a direct mechanical feedback mechanism where pressure in growing cartilage, balanced by tension in the periosteum, [modulating] growth processes of chondrocytes. "

" after 3 days of culture, distal cartilage length was significantly longer in stripped versus intact tibiotarsi in non-conditioned medium and in conditioned medium obtained from periosteum/perichondrium cell cultures on 3, 14, 21, and 48 kPa stiff substrates. The difference in distal length between stripped and intact tibiotarsus decreased with increasing stiffness and was no longer significant on 80 kPa gels and on glass"<-Thus the stiffness of the periosteum may affect the height reduction.

"Both the variations in substrate stiffness and applying cytochalasin D in culture modulated the ability of periosteum cells to actively develop intracellular tension via their actin microfilament network."

Stripped periosteum cartilage was about 33% higher than intact periosteum.

You can see here that axial loading increases periosteal thickness(From Cortical and trabecular bone adaptation to incremental load magnitudes using the mouse tibial axial compression loading model). This serves to contrast LSJL images here where there is less visible periosteal thickness in the without drilling mice.  One reason for this difference could be that LSJL involves less force as in the axial loading study periosteal thickness increased with increasing force.  So less increase of periosteal thickness could be one possibility in why LSJL can increase height but why axial loading does not.  Which leads to the question of whether axial loading can increase height with periosteal stripping.

Thursday, March 7, 2013

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."


"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.


"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.


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"

LSJL progress update 2-23-13

Unfortunately on one has been able to explain how I can retrieve x-rays from Sharp-Reese Steeley Online and the internet has been no luck either. The threshold required for growth to show up via x-ray is pretty high.  No micro-growth plates are going to show up.  So until someone can explain how I can access my x-ray records via the Sharp Reese Steeley medical system there can be no X-rays for now. I've been using an allen wrench and a hammer to test out Natural Height Grow's Pick Axe method in addition to LSJL: Bondhus 12116 1/2-Inch Long Hex L-Wrench. Since Michael is now performing the LSJL method maybe that is the solution to proving the LSJL method. My tests so far show that the allen wrench + hammer has been effective in creating sensations in the bone. In fact, I was worried that doing this may actually fracture the bone but I think it would take tons of hard taps to create enough residual strain to fracture the bone. And it may be necessary to fracture the bone as there is a different microenvironment involved in a microfracture versus a macrofracture. A microenvironment that is more chondrogenic. It's unclear whether this method has synergy with LSJL as drilling the bone in LSJL has been shown to reduce LSJL effectiveness. However this method may be a way to induce (-micro)fractures without penetrating the skin. There might be a region between micro- and macro-fracture that can generate a pro-chondrogenic microenvironment without the disability caused by a full blown macro-fracture.
The current progress pic is above.
Here's the last set of pictures.

I know it's grainy but if you look at these pics from 2010:

So here's me trying to match that measurement today at 13 1/4 inches:
The ruler was too far to the side so I tried to move it in:
You can see that 13 1/4 no longer quite covers end of the ankle to where the calf stops sloping in and where the next muscle slopes out.  And I think this is true even if you account for the account that the tibia is rotated slightly outward in the present pics.

You can also see an increase in ankle width even if the foot in the before pic is rotated slightly inward.  So I definitely gained some height with LSJL.

Wednesday, March 6, 2013

How much does frequency matter for LSJL?

In the LSJL lengthening studies a frequency of 5 Hz is used.  Frequency is the hardest thing to mimic in our home made clamping method.  Even though this is an axial loading study it is still by the LSJL scientist Hiroki Yokota and it'll provide insights on how important frequency is for LSJL results.

Resonance in the mouse tibia as a predictor of frequencies and locations of loading-induced bone formation

"we conducted axial tibia loading using low, medium, or high frequency to the mouse tibia. The experimental data demonstrated dependence of the maximum bone formation on location and frequency of loading{But does frequency influence chondrogenic differentiation?}. Samples loaded with the low-frequency waveform exhibited peak enhancement of bone formation in the proximal tibia, while the high-frequency waveform offered the greatest enhancement in the midshaft and distal sections. Furthermore, the observed dependence on loading frequencies was correlated to the principal strains in the first five resonance modes at 8.0–42.9 Hz. Collectively, the results suggest that resonance is a contributor to the frequencies and locations of maximum bone formation."

"When loading is applied to such a material at or near its resonant frequencies, additional energy is absorbed and the material tends to vibrate at greater amplitude than when loading is applied at other frequencies. These vibrations propagate through the material in specific ways, or modes, based on the geometry and characteristics of the material."

"he tibia is composed of a shell of dense, stiff cortical bone that is thinnest on the outside of each epiphysis and thickest throughout the diaphysis. Inside the epiphysis a matrix of less dense, weaker trabecular bone is present. An epiphyseal plate is found at the border between the each epiphysis and diaphysis, which consists of hyaline cartilage. Each type of tissue likely contributes to the frequency response of the tibia."<-So when the epiphyseal plate is absent that affects the optimal frequency for chondroinduction.

"(C57BL/6 male, ∼13 weeks old) were used in this study."<-So these mice were definitely growing.

7N were used versus 0.5N in LSJL.

Low Frequency: 1-17Hz<-So LSJL is in this range
Medium Frequency: 18-34Hz
High Frequency: 35-51Hz

The animals experienced all 200 repetitions at each of the frequencies within the range.

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So 8% region would be the region closest to the growth plate range and that was the range the responded most to low frequency.
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It's hard to tell how chondrogenesis was affected in the 8% region.

"When a periodic load is applied at or near one of an object’s resonant frequencies, it tends to absorb more energy and oscillate at greater amplitudes than at other loading frequencies. In the case of the tibia, loading at frequencies near the resonant frequencies of the bone may be causing more energy to be dissipated and larger displacements in certain areas of the bone than loading at other frequencies with equal amounts of force. This may lead to increased strain rates, amplified intramedullary fluid flow, increased fluid shear stresses on bone cells, and enhanced cellular response in areas that absorb the most energy"<-So the correct frequency is a bonus but is not required.

Monday, March 4, 2013

LSJL Prototype Device designed by Yokota/Zhang

This study was actually published in 2005 but it's in an obscure space journal so I didn't find it until now.

Development of a Knee-Loading Joint Supporter for Potential Use in Preventing Bone Loss during Spaceflight/Aging

14 week old female C57/BL6 mice were used.  Knee loading was applied for 3 minutes for 3 consecutive days.  Peak force of 0.5N was used.  Groups were 5, 10, 15Hz.  5Hz was the one used to generate the most bone formation which doesn't mean it will translate into the most length but 5 Hz was used in the lengthening study.

Fig1 provides a diagram with a loaded mouse.

In Figure 3 they give the knee prototype.  In this study they blocked copy and pasting so you'll have to read the full study.

In the device there is a pad to avoid a local stress concentration on the knee.  Perhaps for LSJL foam could be placed in between the clamp and the knee.  Although the study above did not study any lengthening effects of LSJL.

Yokota/Zhang mention 10N being what is required for humans but again this was before they noted any LSJL lengthening effects.

Usage mentioned being approx. 30 min daily...per knee.  In this study the the outer part of the knee was the one loaded. Here's how a gear/cam mechanism works:



This ratcheting motion is very similar to a clamp.