Possible alternative loading device to induce adult height growth?

The key to adult height growth is to induce in vivo chondrogenesis in the bone marrow to get height growth from new endochondral ossification.  LSJL is one method to do such that.  However, there may be another device capable of inducing in vivo chondrogenesis in the bone marrow and therefore other devices capable of adult height growth.

A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells.

"Articular motion is a combination of compressive, tensile and shear deformations; therefore, one can presume that compression alone is unlikely to be a sufficient mechanical signal to generate a cartilage-like tissue in vitro[so compressive, tensile, and shear deformations are likely needed to induce chondrogenesis in bone marrow]. we aimed to determine the role of shear on the fate of stem cell differentiation[LSJL induces fluid based shear strain]. Specifically, we investigated the potential enhancing effect of surface shear, superimposed on cyclic axial compression, on chondrogenic differentiation of human bone marrow-derived stem cells. Using a custom built loading device[could this device be an alternative to LSJL?] we applied compression, shear or a combination of both stimuli onto fibrin/polyurethane composites in which human mesenchymal stem cells were embedded, while no exogenous growth-factors were added to the culture medium. Both compression or shear alone was insufficient for the chondrogenic induction of human mesenchymal stem cells. However, the application of shear superimposed upon dynamic compression led to significant increases in chondrogenic gene expression[LSJL should imply both shear and compressive forces.  Axial loading should only apply compressive forces as there is no pressure gradient hence why axial loading does not induce height growth]. Histological analysis detected sulphated glycosaminoglycan and collagen II only in the compression and shear group."

The device is basically a ball placed directly on the mesenchymal stem cells.  I assume to be like the ball of the knee joint.  So the purpose of the ball was for it to oscillate to generate fluid shear strain.

"Group A was the free-swelling (unloaded) control. Group B was exposed to unconfined dynamic compression at 1 Hz with 0.4 mm sinusoidal strain, superimposed on a 0.4 mm static offset strain, resulting in a strain amplitude of 10-20 % of the scaffold height at the centre of the construct. Group C was exposed to ball oscillation of ±25° at 1 Hz, superimposed on a 0.4 mm static compression offset strain. Group D was a combination of both loading regimes. Mechanical load was applied during 1 h a day for 5 consecutive days per week over 3 weeks"<-LSJL loading applies load for slightly over 1 minute which is much less than 1 hour.

"There was a trend towards higher total GAG/DNA values in the loaded groups which included shear[so shear strain is very important for inducing chondrogenesis, axial loading induces no shear strain thus no chondrogenesis], but no significant difference over the compression only group was detected. All individual donors demonstrated the same trend[so all individual bone marrow sources were capable of chondrogenesis]"

Compression and Sox9 groups expressed much more Sox9 than just shear groups alone.  So compressive forces are still important to chondrogenesis.  If you look at Figure 6D in the study you can almost see a growth plate.

What we can take from this study is that maybe the LSJL clamping routine is not dynamic enough at inducing fluid based shear strain.  Perhaps rapid clamping and unclamping would be a way to make LSJL induce more shear forces.

The ball device is not something that could be used as you'd need to put the ball directly against the bone marrow.  Maybe surgically implanting a ball in the bone marrow may be a possibility for the future.

In Bone Marrow Chondrogenesis of Mesenchymal Stem Cells

The goal of Lateral Synovial Joint Loading is to use lateral loads to induce hydrostatic pressure in the epiphyseal bone marrow.  This hydrostatic pressure results in chondrogenic differentiation.  Unlike with osteoblasts, chondrocytes are both hydrophillic(water loving) and have their hypertrophy and cellular proliferation coupled in columns.  It is likely some combination of these two properties that allow chondrocytes to be much more effective at lengthening bone than osteoblasts.  Thus, studies of in vivo(within the body) studies of chondrogenic differentiation would be very useful in providing insights to the LSJL method.  As they could both tell us how best to induce chondrogenic differentiation and whether the chondrocytes generated by LSJL could increase height.

In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells.

"[What is the effect of] the normal joint cavity environment on chondrocytic differentiation of bone-marrow-derived mesenchymal stem cells (MSCs)[not the epiphyseal bone marrow which is our target area for inducing MSC chondrogenic differentiation]? Autologous bone marrow was aspirated from the iliac crest of male sheep. Labeled MSCs were then grown on a three-dimensional porous scaffold of poly (L-lactic-co-glycolic acid) in vitro and implanted into the joint cavity by a surgical procedure. At 4 or 8 weeks after implantation, the implants were removed for histochemical and immunohistochemical analysis. The [MSCs] in the implants expressed type II collagen and synthesized sulfated proteoglycans. However, the osteoblast-specific marker, osteocalcin, was not detected by immunohistochemistry indicating that the implanted MSCs had not differentiated into osteoblasts by being directly exposed to the normal joint cavity. To investigate the possible factors involved in chondrocytic differentiation of MSCs further, we co-cultured sheep MSCs with the main components of the normal joint cavity, viz., synovial fluid or synovial cells, in vitro. After 1 or 2 weeks of co-culture, the MSCs in both co-culture systems expressed markers of chondrogenesis. These results suggest that synovial fluid and synovium from normal joint cavity are important for the chondrocytic differentiation of adult bone-marrow-derived MSCs[the interstitial fluid flow from LSJL may help synovial fluid get to the epiphysis and synovial tissue might be able to be accessed via the periosteum]."

"in vivo experiments have demonstrated that MSCs implanted into surgically created, osteochondral defects in rabbits are able to differentiate into chondrocytes and improve the quality of cartilaginous repair"<-But not yet whether MSCs in intact epiphyseal bone marrow can differentiate into chondrocytes. Osteochondral defects give more access to various fluids and nutrients. Bone is well vascularized so it may be able to provide many of the nutrients present in osteochondral defects.

Since LSJL involves synovial joint loading it seems likely that loading the synovial joint plays some role in inducing the chondrogenesis than just increasing the hydrostatic pressure in the bone marrow. Loading the synovial joint cavity may enhance the expression of genes that result in the induction of chondrogenesis. Loading the synovial cavity may also create a pressure gradient that increases in hydrostatic pressure in the epiphyseal bone marrow but direct effects on gene expression through the loading of the synovial cavity may play a role as well. To study this, you'd have to perform LSJL on rats, after removing their joint capsule, and then compare the results to the rats in the other study.

The LSJL scientists studied the effects of surgical holes on bone formation but not chondrogenesis.  But it's likely that any chondrogenesis in the bone would've increased the bone measurements like cortical area and cortical thickness.  Thus, the increase in intramedullary pressure may be the main determinant for both chondrogenic and osteogenic differentiation but synovial fluid transport from the joint cavity may enhance chondrogenesis.  Whereas synovial fluid transport from the joint cavity does not encourage osteogenic differentiation.

Here's a study about what happens to extra MSCs that come into the body:

The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion.

"Rat marrow-derived MSCs were ex vivo culture-expanded and infused into syngeneic rats via intra-artery (i.a.), intravenous (i.v.) and intraperitoneal cavity (i.p.) infusions[so put directly into the blood stream basically]. In addition, for i.a. and i.v. infusions, a vasodilator, sodium nitroprusside, was administered prior to the cell infusion and examined for its effect on MSC circulation. The dynamic distribution of infused MSCs was monitored by real-time imaging using a gamma camera immediately after infusion and at 48 h postinfusion. After 48 h, radioactivity in long bones, was measured in a gamma well counter and expressed as a percentage of injected doses. After both i.a. and i.v. infusion, radioactivity associated with MSCs was detected in other organs[including long bones. the homing of labeled MSCs to the marrow of long bones was significantly increased by the pretreatment with vasodilator[Vasodilators widen the blood vessels like Viagra]. These results indicate multiple homing sites for injected MSCs and that the distribution of MSCs can be influenced by administration of vasodilator."

"Vascular cell adhesion molecule-1 (VCAM-1) is expressed by the marrow stromal cells and may play an important role in the interchange of cells between bone marrow and blood."<-So areas with bone marrow already like the long bones express VCAM-1. The more vascularized the blood the more likely it is for MSCs in the blood to bind to the bone marrow.

So to get more MSCs to the epiphysis you'd want more epiphyseal bone marrow to be there and to take a vasodilator.

Induction of chondrogenesis in muscle, skin, bone marrow, and periodontal ligament bydemineralized dentin and bone matrix in vivo and in vitro.

"Induction of chondrogenesis in vivo by rolls of demineralized dentin[dentin is a part of your tooth] implanted in muscle, subcutaneous connective tissue of skin, medullary cavity of femur[not the epiphysis though], and periodontal ligament of rat was investigated. Specimens were examined at various times up to 21 days after implantation. Induction of cartilage occurred most quickly in muscle, followed by subcutaneous connective tissue of skin and medullary cavity of femur, and most slowly in periodontal ligament. Significantly more cartilage was found in muscle than in subcutaneous connective tissue of skin and medullary cavity of femur at the times examined, and least of all in periodontal ligament. Outgrowth of cells from rat muscle, dermis and subcutaneous tissue, bone marrow and periodontal ligament cultured in vitro on demineralized bone matrix for up to 35 days produced similar results."

"Implant in medullary cavity of femur. Wounds were made. A skin incision, approximately 3.0 cm in length,
was made along the lateral aspect of the right thigh, and the femoral muscles were retracted to expose the lateral aspect of the femur. A defect approximately 1.4 mm in diameter was cut in the mid-region of the lateral aspect of the femoral diaphysis, by means of a #3 round bur mounted in a slow-running dental handpiece cooled with phosphate-buffered saline, pH 7.0. The underlying bone marrow was excised with a dental excavator, and debris was removed by being washed with phosphate-buffered saline. One dentin roll was implanted in the medullary cavity proximal to the wound, and parallel to the long axis of the bone. The muscle was sutured with 3.0 silk and the skin with 4.0 silk."

It took 10 days for cartilage to form in the bone.  The cartilage formed directly next to the dentin roll.  No chondrogenesis was evident after day 21.

"demineralized bone matrix is mitogenic for mesenchymal cells; and second, that demineralized bone matrix binds fibronectin, an event that is necessary for induction of cartilage"<-It's possible that hydrostatic pressure demineralizes bone matrix which allows for new cartilage growth to occur.  Acid is also a way to dimeralize bone matrix.

In this study cartilage only formed directly next to the dimineralized dentin roll.  Thus dimineralized matrix is likely necessary for cartilage growth.  Thus for LSJL to be more effective we may need to dimineralize the bone matrix.  Hydrostatic pressure may already contribute to demineralization of the bone as bone tissue is hydrophobic.

Lactic Acid is naturally produced in response to exercise.  Ascorbic acid(Vitamin C), which can induce chondrogenesis, is naturally occurring.  The main minerals that give bone it's stiffness are Vitamin D, phosphorus, and Vitamin C.  However, people with osteomalacia are not growing taller.  In fact people with osteomalacia have stunted height.  So, we want only partial bone demineralization only on the surface area of the bone to allow for chondrogenesis to occur on it.  Hydrostatic pressure may demineralize the surface area of the bone without affecting the mineralization of the interior of the bone providing a safe way to increase height.

The longitudinal ends of the bones may provide a demineralized bone portion for mesenchymal stem cells to differentiate into chondrocytes in the joint capsule.  Osteoarthritis in fact involves changes in subchondral bone mineralization which may affect the stem cells ability to undergo chondrogenesis.

Osteoclasts operate by using an electron transport pump to acidify the surface of the bone.  So osteoclasts would also be a way to dimineralize a bone surface to allow for chondrogenesis.

Surgically, you could insert a roll of demineralized dentin into your growth plate and then take stem cells followed by a vasodilator.

Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells.

"Biomechanical forces may be one of the major epigenetic factors that determine the form and differentiation of skeletal tissues. In order to test the hypothesis that static compressive forces are transduced into molecular signals during early chondrogenesis, we have developed a unique three-dimensional collagen gel cell culture system which is permissive for the proliferation and differentiation of chondrocytes. Mouse embryonic day 10 (E10) limb buds were microdissected and dissociated into cells which were then cultured within a collagen gel matrix and maintained for up to 10 days. Static compressive forces were exerted onto these cultures. The time course for expression pattern and level for cartilage specific markers, type II collagen and aggrecan, and regulators of chondrogenesis, Sox9 and IL-1beta, were analyzed and compared with non-compressed control cultures. Under compressive conditions, histological evaluation showed an apparent acceleration in the rate and extent of chondrogenesis. Quantitatively, there was a significant 2- to 3-fold increase in type II collagen and aggrecan expression beginning at day 5 of culture and the difference was maintained through 10 days of cultures. Compressive force also causes an elevated level of Sox9, a transcriptional activator of type II collagen. In contrast, the expression and accumulation of IL-1beta, a transcriptional repressor of type II collagen was down-regulated. We conclude that static compressive forces promote chondrogenesis in embryonic limb bud mesenchyme, and propose that the signal transduction from a biomechanical stimuli can be mediated by a combination of positive and negative effectors of cartilage specific extracellular matrix macromolecules."

"During chondrogenesis, it was proposed that the osmotic changes during hyaluronic acid synthesis and degradation could produce sufficient swelling and deswelling to regulate the balance between cell-cell versus cell-ECM contacts"

"[mesenchymal] condensation is promoted by a regulated balance between cell-ECM and cell-cell adhesion involving type I and type III collagen, tenascin, fibronectin, cellular receptors for these matrix proteins such as integrins, and other non-integrin adhesion molecules, such as cell-cell adhesion molecules N-CAM and N-cadherin"

"As matrix molecules are deposited between cells, cell-cell contacts are lost"

"Static compressive forces were applied by the use of a weight placed on the lid of the culture plate exerted through a well insert which fitted on top of the collagen gel"

"1, 1.5, and 2 kPa was empirically determined to yield a 20-30% deformation of the gel"<-Can LSJL induce a 20-30% deformation of the epiphyseal bone marrow?

"When 1.5 and 2 kPa of force was loaded, type II collagen mRNA increased 1.5- and 3.8-fold, respectively, over transcript levels detected in samples subjected to 1 kPa of force magnitude"

"A collagen gel matrix under compressive force is likely to become more compact when compared with non-compressed matrix. Compaction may lead to changes in gel density and cell number per unit volume."<-The study also mentions that high cell density promotes chondrogenesis.

"Possible mechanisms of how compressive stimuli are translated into biochemical signals might involve integrin or non-integrin ECM receptors, cell-cell adhesion molecules or alteration of cytoskeletal architecture mediated by MAP kinase, rho kinase or focal adhesion associated tyrosine kinase signal transduction pathways "

Here's a diagram of the bone marrow of the intramedullary cavity, the epiphyseal bone marrow cavity should be similar from the study(Hierarchical organization and regulation of the hematopoietic stem cell osteoblastic niche):

Schematic of osteoblastic niche in bone marrow. Osteoblastic niche locates in trabecular region of bone marrow. Active osteoblats (OB), bone-lining cells (BLC), and osteoclasts (OC) constitutively line its bony boundary. Hematopoietic stem cell (HSC)–mesenchymal stem cell (MSC) pairings share common niches. Hierarchical regulators from immune, humoral, and neural systems are engaged in the osteoblastic niche and are represented by FoxP3 regulatory T (Treg), parathyroid hormone (PTH), and β-adrenergic nerve cell (β-ANC) with ensheathing glial cells (GC) respectively. Abbreviations: MK, megakaryocytes; OM, endosteal macrophages; EDL, endothelium; PPR, PTH/PTHRP receptor; brown dot ()—Ca²⁺; blue dot ()—PTH; —PPR.

What's important to note is that the ratio of MSCs to other cells is very small so work will be needed to achieve mesenchymal condensation to induce chondrogenesis.

Other facts of note from the paper: "BMMSCs preferentially locate in developing sinusoids[a blood vessel], their own microvascular niche, which is a primary producer of angiopoietin-1, a vital molecule for HSCs vascular niche remodeling"

"increased osteoclastogenesis decreases bone trabecular acreage, as well as hematopoietic stem and progenitor cell (HSPC) pools"<-So osteoclastgenesis may be helpful for inducing chondrogenesis by increasing the number of potentially chondrogenic cells.

Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells--potential and pitfalls.

"the triple positive subset (CD56+, CD271+, TNAP+) displayed a pronounced chondrogenic potential"

Inferred Functions of "Novel" Genes Identified in Fibroblasts Chondroinduced by Demineralized Bone

" several functional classes of genes (cytoskeletal and matrix elements, cell adhesion proteins, peptide growth factors, and signal transduction proteins) were found to be altered in human dermal fibroblasts (hDFs) cultured in porous collagen sponges with chondroinductive demineralized bone powder (DBP) for 3 days. In addition, a number of "novel" sequences were identified. In this study, molecular techniques were combined with computational methods to characterize those sequences. Gene expression of all 10 novel sequences tested was found in hDFs by RT-PCR. The sequences were compared to the human genome, and their cellular functions were inferred from genes that mapped to the same chromosomal coordinates. Only one of the novel sequences contained a protein-coding region (kinesin superfamily protein 26B){up in LSJL}. The others contained 3′ untranslated (osteonectin, alpha-V integrin, RAP2B) or other untranslated regions (PTPN21, GAS6) of mRNAs."

"DBP induces osteogenesis by an endochondral mechanism.  Mesenchymal cells are attracted to DBP, differentiate into chondroblasts, and secrete a cartilage matrix. The cartilage matrix is then calcified, resorbed, and replaced by bone and hematopoietic bone marrow"

Demineralized bone alters expression of Wnt network components during chondroinduction of post-natal fibroblasts.

"Human dermal fibroblasts (hDFs) were cultured in porous, three-dimensional (3D) collagen sponges with or without chondroinductive DBP. In some experiments, lithium chloride (LiCl), an agonist of the Wnt/beta-catenin signaling pathway, was added to the culture media. Sponges were cultured for intervals (0.5-21 days) before processing for molecular, histologic, and biochemical analyses. Expression of wnt, fzd, and sfrp genes was characterized by semi-quantitative RT-PCR. Fibroblasts' contacts with DBP were documented by histology. Accumulation of proteoglycan in extracellular matrix was evaluated by histology (metachromasia in toluidine blue-stained sections) and quantitative immunoassay (chondroitin 4-sulfate ELISA).

Expression of 15 wnt, fzd, and sfrp family members was detected in hDFs by RT-PCR. A subset of those genes (wnt2b, wnt5b, wnt10b, fzd6, fzd7) showed altered expression in hDFs exposed to DBP for 3 days. wnt and fzd gene expression was not altered before hDFs contacted the DBP within the collagen sponge. Human DFs cultured in plain collagen sponges and treated with LiCl accumulated significantly more metachromatic matrix than NaCl-treated controls on day 10, and showed a trend towards increased matrix chondroitin-4 sulfate content.

These data suggest that changes in Wnt signaling contribute to chondroinduction of post-natal fibroblasts by DBP. This is the first evidence that Wnt components, which are essential regulators of pre-natal chondrocyte differentiation, may also influence post-natal chondrocyte differentiation induced by DBP."

Lithium chloride is an activator of the Wnt pathway.

"hDFs cultured in LiCl-treated sponges were surrounded by significantly more metachromatix matrix"

"In the chick, chondrogenesis in embryonic mesenchymal cells is enhanced by ectopic expression of wnt5b and inhibited by fzd7"

"human bone marrow stromal cells induced with TGF-β [to chondrogenesis] show decreased fzd2{up} and increased wnt11 mRNAs"

"In human bone marrow stromal cells, LiCl-treatment increases the chondrogenic response to TGF-β. In the embryonic mouse limb, chondrogenic cells do not express Dkk-1, an inhibitor of the Wnt/β-catenin pathway. In the murine mesenchymal cell line, C3H10T1/2, overexpression of wnt3a increases nuclear β-catenin levels and these cells’ chondrogenic response to bone morphogenetic protein 2 (BMP-2). Curiously, exposure of C3H10T1/2 cells to LiCl inhibits BMP-2-induced chondrogenesis. Chick embryonic limb mesenchymal cells exposed to LiCl also show reduced chondrogenesis. Finally, β-catenin expression inversely correlates with expression of the cartilage matrix protein, collagen type II, during in vitro chondrogenesis of chick embryonic mesenchymal cells"

"During mouse limb development, loss of calcium-pathway-signaling Wnt5a increases β-catenin and inhibits chondrogenesis"

In vitro model of mesenchymal condensation during chondrogenic development.

"The extracellular matrix (ECM) secreted by cells are macromolecules with nanofibrillar features. The ECM plays a crucial role in tissue development, by providing a natural ‘scaffold’ to guide cell responses, in terms of regulation of cell anchorage, migration, organization and triggering signals that govern cell survival, cell cycle progression and expression of differentiated phenotypes during development. Basement membranes (BM) are 50-100 nm thick nano-fibrous sheet-like ECM architectures that underpin epithelial and endothelial cells. BM is also found supporting several mesenchymal cell types, such as smooth muscle cells, adipocytes- the cells which are derived from the same progenitor cells as chondrocytes."

"Chondrocytes are terminally differentiated cells originating from pluripotent mesenchymal stem cells. Mature human Chondrocytes are not in direct contact with BM in cartilage."

"individual BM components [are present] in adult cartilage matrix, such as laminin, perlecan, nidogen-2  and collagen type IV"

"With progression of age this extensive ECM layer gradually becomes less and less distinct. In the cartilage of newborn mice, BMs are widespread in surrounding matrices, whereas in mature cartilage of adult mice BMs are localized mainly to a narrow pericellular zone"

"on the 30 cm matrix (malleable nanofibrous matrix) cells initially assumed a spread morphology but after 3-4 days started to assume a rounded morphology. After 8-10 days of culture in the presence of chondrogenic media, cells migrated and formed aggregates on the 30 cm mats."

"As H9 ES cells (passage 35) were seeded on the 30 cm mat in aggregated form, thus no significant modulation in cell migration or aggregate size was noticed over 10 days of culture. TEM images of the ES cell clusters showed that initially all the ES cells were in close contact with each other, maintaining a rounded cell shape typical of ES cells. After 10 days within the aggregates individual cells were separated by putative ECM, which may be simulating “chondron”-like morphology. The prominent presence of lipid vacuoles in the cytoplasm was also noticed in the ES aggregates, as reported earlier for chondrogenic differentiation of mouse ES cells"

"The level of chondrogenesis regulating transcripts, such as collagen-II, Sox-9, aggrecan and fibronectin were significantly lower in nanofibrillar matrices with higher stiffness, where cells maintained their spread morphology and hence, poor tissue forming ability, compared to the cells grown on nanofibrillar matrices with relatively reduced stiffness"

Gene-Induced Chondrogenesis of Mesenchymal Stem Cells: A Comparative Analysis of Candidate cDNAs

[This is not a full study but just a paper]

"Gene transfer to MSCs can be used to achieve sustained expression of specific protein factors capable of inducing chondrogenic differentiation. In this study, using adenoviral mediated gene transfer and bone marrow derived MSCs in an aggregate culture system, we compared the relative condroinductive capacity of several cDNAs whose protein products have been associated with the induction or maintenance of the articular cartilage phenotype. First generation, E1, E3 deleted, serotype 5 adenoviral vectors carrying the complete cDNAs for TGF-b1, bone morphogenetic protein (BMP) -2, BMP -4 , BMP- 7, Sox 9, indian hedgehog (IHH), and insulin-like growth factor-1 (IGF-1) were constructed using cre-lox recombination. As a source of MSCs, adherent, colony forming cells from the bone marrow of 3-week-old calves were isolated, cultured and used at low passage. Individual flasks of MSCs were infected with Ad.TGF-b1, Ad.BMP-2, Ad.BMP-4, Ad.BMP-7 and Ad.IGF-1 at doses sufficient to provide low (<10 ng/ml) , medium (10-100 ng/ml) and high (>100 ng/ml) amounts of transgene expression per 24 hrs. Delivery of Sox-9 and Ad.IHH was performed over a wide range of viral doses. At 24 hours post infection, 2.0 x 10^5 cells of each culture were pelleted by centrifugation. The aggregates were cultured for 3 weeks in conical tubes in a defined, serum-free medium containing ascorbate 2-phosphate and dexamethasone. Chondrogenesis was determined using histologic staining for glycosaminoglycans and by immunohistochemistry for collagen types I and II. By this method, gene transfer and expression of BMP-2 and BMP-4 showed the greatest chondrogenic potential. This was followed to a lesser extent by TGF-b1 and then Sox-9 and IGF-1. Delivery of IHH or BMP-7 was met with only minimal chondrogenic activity which was only slightly greater than uninfected control cells. The potent activity of the secreted factors BMPs-2, -4 and TGF-b1 were expected and are consistent with the literature. The capacity to induce chondrogenesis through delivery of the Sox-9 transcription factor offers a promising intracellular alternative to the overexpression of secretable pleiotropic growth factors. The relative lack of chondroinduction following overexpression of IGF-1 and IHH is perhaps not surprising because these products thought to be more important for maintenance than induction of the chondrocytic phenotype."

Differentiation of mesenchymal stem cells to osteoblasts and chondrocytes: a focus on adenosine receptors.

Adenosine receptors signal via cAMP.

"if β-catenin is activated, it upregulates Runx2 expression and MSCs differentiate to osteoblasts, rather than chondrocytes "

"In the prechondrogenic mesenchyme, Runx2 is expressed along with Sox9 [but with Sox9 expression dominant]" "Nkx3.2 inhibits Runx2 transcription, enforcing the differentiation to chondrocytes"

"The expression of Sox9, along with the activation of cAMP response element binding protein (CREB) and c-Fos, maintains the chondrocytes in a proliferative state. In order for the chondrocytes to exit the cell cycle and become hypertrophic, Runx2 must be upregulated and Sox9 suppressed. The exit from the cell cycle and apoptosis is necessary for the eventual invasion by osteoblasts and ossification of the bone matrix "

Hoxa2 inhibits Runx2.

"high expression of the A2AAR and A2BAR receptors [adenosine receptors] can be found in cartilage"

"In human MSCs, A2BAR activation increased osteoblast differentiation, as determined by an increase in alkaline phosphatase activity"

"caffeine impairs new endochondral bone formation by inhibiting the proliferation and differentiation of chondroprogenitor cells."

JunB promotes osteoblast differentiation.

"TNF-α [promotes] Runx2 protein degradation in C2C12 and 2T3 osteoblast precursor cells by upregulating the E3 ligases Smurf1 and Smurf2 "

"High extracellular concentrations of adenosine can be achieved during cell injury or stress"

PKA stimulates Sox9 activation of Col2a1.

Mesenchymal stromal cell-derived extracellular matrix influences gene expression of chondrocytes.

"the ability of adult human mesenchymal stem cell (hMSC)-derived ECM to rescue the phenotype of osteoarthritic (OA) chondrocytes and to further stimulate the differentiation of healthy (HL) chondrocytes was evaluated. ECMs were prepared by decellularizing hMSCs cultured in basic medium (BM) and chondrogenic medium (CM). The obtained ECM was then combined with a polymeric solution of Poly (ε-caprolactone) (PCL) dissolved in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP) and electrospun meshes were fabricated. Electrospun ECM scaffolds were characterized using scanning electron microscopy (SEM) and picrosirius red staining was used to confirm the presence of collagen. OA and HL chondrocytes were cultured on scaffolds containing hMSC ECM in BM or CM and compared to PCL electrospun scaffolds without ECM. Metabolic activity and chondrogenic gene expression were assessed by Alamar blue assay and quantitative PCR (qPCR) analysis, respectively. The ECM presence resulted in a significant difference in chondrocyte metabolic activity compared to PCL scaffolds alone. HL chondrocytes cultured for 21 days in chondrogenic medium on electrospun scaffolds containing hMSC ECM from BM showed a significant increase in collagen II and aggrecan expression compared to hMSC ECM from CM and PCL scaffolds without ECM incorporation. No significant influence of hMSC ECM presence on the chondrogenic signature of OA chondrocytes was found."

" hMSCs (female, 75 years old) were isolated from the bone marrow"

Aggrecan, Col2a1, Col1a1 were upregulated by healthy chondrocytes in MSC ECM.  Type X Collagen was only upregulated in the presence of chondrogenic medium.  The other three genes were upregulated in basal media but more significantly in chondrogenic medium.

Mechanical Signals As a Non-Invasive Means to Influence Mesenchymal Stem Cell Fate, Promoting Bone and Suppressing the Fat Phenotype.

"MSCs are ideally positioned as mechanosensitive elements central to musculoskeletal adaptation, but that the signals needn't be large to be influential."

The study states that mechanical loading suppresses the adipose phenotype but does not state much of enhancing the chondrogenic phenotype.  Mechanical stimulation also increases the number of progenitor cells which makes mesenchymal condensation easier to occur.

Biophysical Regulation of Stem Cell Differentiation.

"Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro"<-perhaps these mechano-sensing osteocytes can induce stem cell chondrogenesis as well.

"Membrane-bound stretch-activated or voltage-activated cation channels also open as a consequence of membrane deformation, electromagnetic field stimulation, or fluid streaming potentials, initiating an influx of Ca2+. MSCs may also transduce membrane strains directly by the Piezo family of cation-selective mechano-sensitive channels" <-the primary cilium can also detect mechanical stimuli.

"In osteoporotic bone, ImP greatly increases, approaching arterial blood pressure in children with paralytic osteoporosis. Adipose tissue in the marrow was found to increase with age and disease, leading to decreased marrow viscosity and proportional decreases in fluid shear stresses"<-Osteoperosis can be a way to study increases in Intramedullary Pressure and the converse can be studied with increases in adipose tissue.

"Oscillatory strains compressing MSC-seeded fibrin scaffolds by 15 % produced chondrogenic differentiation, but inhibition of ERK1/2 abolished this chondrogenic response in favor of osteogenesis"

New insights into osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells and their potential clinical applications for bone regeneration in pediatric orthopaedics.

" trabecular bone[has] been identified as [a] potential [source] of MSCs"<-very promising for LSJL.  What would be even better if cortical bone was identified as a source for MSCs.

According to the book Stem Cells and Bone Tissue, there is a MSC population within cortical bone.  Trabecular bone MSCs are similar to bone marrow MSCs except they do not have the markers for Hematopoetic stem cells which is actually good as HSCs are not a component of the growth plate.

The study identifies the following transcription factors of regulating chondrogenic differentiation: Sox 5/6/9, Slug,  TSRP1, and GDF5.

"SOX9 specifically interacts with two binding sites for HMG-domain proteins and activates elements in the promoters of Col2a1, Col9a1, Col11a2, and aggrecan. Removal of SOX9 before mesenchymal condensation results in mice without cartilage development, while loss of SOX9 after condensation causes the arrest of chondrocytes differentiation"

"Procedures devoted to recruit stem cells from BM by penetration of the subchondral bone have been widely used to treat localized cartilage defects"