Gaining Stature with Bone Morphogenic Protein-2?

Benzoic Acid a possible BMP-2 stimulator is available for sale: Source Naturals PABA 100mg, 100 Tablets (Pack of 3).

Bone Morphogenic Protein-2 is important for normal height development and could potentially be used for gaining stature.  It is involved in a number of signaling pathways in height development and stimulates stem cells to differentiate into osteoblasts(which can increase height at the top of the head or feet and could conceivably increase height on the top of long bones based on bone deposition patterns).  BMP-2 also helps stem cells differentiate into chondrocytes.  TGF-Beta does not typically encourage osteogenic differentiation which is why it is a better signal for potential chondrogenic differentiation.  How can we utilize Bone Morphogenic Protein-2 to increase our stature?

Effects of low-intensity pulsed ultrasound, dexamethasone/TGF-beta1 and/or BMP-2 on the transcriptional expression of genes in human mesenchymal stem cells: chondrogenic vs. osteogenic differentiation. 

"hMSCs [human Mesenchymal Stem Cells] were subjected to LIPUS [Low Intensity Pulsed Ultrasound] with or without dexamethasone/transforming growth factor-beta1 (TD) or bone morphogenetic protein-2 (BMP-2). TD-treated hMSCs exhibited characteristic chondrogenic morphology and increased messenger RNA (mRNA) expression of chondrogenic markers and LIPUS enhanced the chondrogenic differentiation of hMSCs treated with TD. The expression of Runx2, an osteogenic transcription factor was not altered in either TD treatment group[so TGF-Beta1 did not enhance osteogenesis]; however, a significant increase was detected in the LIPUS only group[LIPUS does enhance osteogenesis]. The osteogenic appearance exhibited 3 days after LIPUS and/or BMP-2 treatment. Increases in the mRNA expression levels of osteogenic markers, Runx2 and ALP were also detected. There was no additive or altered effect with combined LIPUS and BMP-2 treatment. LIPUS alone can increase osteogenic differentiation of hMSCs and LIPUS enhances TD-mediated chondrogenic differentiation of hMSCs[so LIPUS encourages osteogenic differentiation of MSCs but it can enhance chondrogenic differentiation of MSCs so if you want to grow taller you want to upregulate TGF-Beta1 in MSCs by a method like LSJL before applying LIPUS]." 

Ultrasounds are available for purchase: 1MHz Portable Ultrasound Therapy Machine w/ 1 gallon Ultrasound Gel.  From this study, it seems that BMP-2 mainly encourages stem cell differentiation into osteoblasts but BMP-2 has been reported to induce chondrogenic differentiation.

"Chondrogenic differentiation of both animal and human MSCs is enhanced by the application of LIPUS"<-LIPUS can help you grow taller if the cells are encourage to a chondrogenic phenotype such as by TGF-Beta1.

"1 MHz, pulsed 1:4 (2 ms “on” and 8 ms “off”) with a repetition rate of 100 Hz had an optimal effect on osteoblast differentiation. A 200 μs tone burst repeating at 1.0 kHz, 30mW/cm2 may better stimulate aggrecan synthesis. An intensity of 200 mW/cm2 [may result] in more pronounced MSC chondrogenesis in vivo"<-Higher intensities are more likely to result in chondrogenic differentiation.  Dexamethasone and TGF-Beta1 helped encourage more Type II collagen(cartilage) whereas LIPUS increased Type I and Type II Collagen in near equal quantities.  So LIPUS would work better in synergy with another height increasing method specifically targeted to chondrogenesis rather than merely LIPUS alone.

BMP-2 and LIPUS in conjunction seemed to encourage osteogenic markers.  LIPUS seems to act to heighten existing cell stimulants rather than controlling differentiation on it's own.  However, LIPUS was not applied to the whole bone where the effects of LIPUS on the periosteum may increase levels of TGF-Beta1 encouraging more chondrogenic differentiation.  It is unclear whether TGF-Beta or BMP-2 are more important for height increase.  However, TGF-Beta is much easier to induce via mechanical means whereas to increase BMP-2 levels requires chemical means.

Quantitative, structural, and image-based mechanical analysis of nonunion fracture repaired by genetically engineered mesenchymal stem cells. 

"Murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). Nanoindentation studies of bone tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine bone, eight weeks post-treatment.  We investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius bones, following the implantation of MSCs.  [Analysis] was performed 10 and 35 weeks post MSC implantation. regenerated bone tissue remodels over time, as indicated by a significant decrease in bone volume, total volume, and connectivity density combined with an increase in mineral density[This could be bad as bone volume includes bone height]. the axial stiffness of limbs repaired with MSCs was 2-1.5 times higher compared to the contralateral intact limbs, at 10 and 35 weeks post-treatment. These results could be attributed to the fusion that occurred in between the ulna and radius bones. MSCs induce bone formation, which exceeds the fracture site [but] significant remodeling of the repair callus occurs over time.  Limbs treated with an MSC graft demonstrated superior biomechanical properties." 

Long bones ordinarily grow by chondrogenesis rather than osteogenesis but long bones could grow by Mesenchymal Stem Cells over-expressing BMP-2 by injecting these stem cells close to the very ends of the bones.  Preferably the over-expressing BMP-2 cells would be injected at the end of the cortical bone in the long bones or right beneath the periosteum in the short/irregular ones.  There's the problem of remodeling occurring but we don't know why it occurs presumably so that the bone is proportional.  There would have to be a way to get around this remodeling. 

Bone morphogenetic protein-2 delivered by hyaluronan-based hydrogel induces massive bone formation and healing of cranial defects in minipigs. 

"The authors developed a hydrogel that is formed in situ by the cross-linking of multifunctional hyaluronic acid and polyvinyl alcohol derivatives mixed with hydroxyapatite nanoparticles, in the presence of BMP-2. [We] evaluate the suitability of the hydrogel as a carrier for BMP-2 in repairing critical size cranial defects in a minipig model. Cranial defects (2 x 4 cm) were created in 14 minipigs. The experimental groups were as follows: group 1, craniotomy and application of 5 ml of hydrogel with 1.25 mg of BMP-2; group 2, craniotomy and application of 5 ml of hydrogel without BMP-2; and group 3, craniotomy with no further treatment. After 3 months, examinations were performed. There was spontaneous ossification in the untreated group, but the healing was incomplete. The hydrogel alone demonstrated no further effects. The addition of 1.25 mg of BMP-2 to the hydrogel induced a greater than 100 percent increase in bone volume and complete healing of the defects.  Compact lamellar bone [was seen] in the BMP group without intertrabecular fibrous tissue, as was seen in the other groups. The hydrogel was resorbed completely within 3 months and caused no inflammatory reaction." 

"successful bone formation can be obtained using various hydrogels that resemble the natural extracellular matrix in terms of high water content and structural stability."

"The volumes of newly formed bone in animals treated with hydrogel and BMP-2 were 23.4 ± 6.3 cm3 (119 percent ossification of the defects)"<-19% is huge.  Assuming a limb is 12 inches long that would result in a final bone of 14.28.  Over two inches in height from a single bone.

"Hyaluronan [induces] the expression of its own receptor, CD44, specifically in mesenchymal stem cells."<-more benefits of hyaluronic acid supplementation.

"The hyaluronan/CD44 interaction induces adhesion and migration of mesenchymal stem cells to hyaluronan, which suggests a dual capacity of hyaluronan-based biomaterials by functioning as both a matrix for attraction of mesenchymal stem cells and as a carrier and protective container for differentiation factors."

Clearly, BMP-2 use can initiate supernatural height growth.  BMP-2 is involved in chondrogenesis, which increases height in the long bones. 

Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells. 

"Bioactive factors, such as TGF beta and BMP-2, as well as mechanical factors i.e. compressive loading and hydraulic pressure, have been shown to induce and/or modulate chondrogenesis of bone marrow derived mesenchymal stromal cells (BMSCs). Since these factors are intracellularly transduced through different mechanisms, TGF beta, BMP-2 and hydraulic pressure act synergistically on chondrogenic differentiation of BMSCs. Aggregates of bovine BMSC were cultured in the presence of 10 ng/ml TGF beta1, 50 ng/ml BMP-2 or both. Half of the samples were loaded for 4 hours per day with 0.5-3 MPa cyclic hydraulic pressure at 1 Hz. After 14 days of culture/loading, gene expression of chondrogenic genes was assessed. DNA as well as glycosaminoglycan (GAG) content of the pellets were analysed. Neither pressure nor BMP-2 had an influence on GAG/DNA content. cells responded to the presence of TGF beta1 with an up-regulation of chondrogenic genes and GAG/DNA of the aggregates increased compared to controls demonstrating the cells ability to respond to external stimuli. The used concentrations of BMP-2 and parameters for pressure were neither able to induce nor modulate chondrogenesis of bovine BMSCs." 

BMP-2 may be involved in chondrogenesis but it doesn't seem to induce it in this case.  Hydraulic pressure is similar to the fluid pressure generated during LSJL.  So, for LSJL to be effective the pressure should be more than 1 Hz(anyway to measure the pressure?).  However LSJL may operate by mechanisms other than increasing fluid pressure(like trabecular microfracture, this study was performed in petri dishes so increase in fluid pressure could have had other effects not noticeable outside a living host).   Hydraulic pressure alone may not upregulate chondrogenic genes but LSJL has also been shown to upregulate TGF-Beta1 which is effective in upregulating chondrogenic genes.

"cyclic compressive loading promoted chondrogenic differentiation of rabbit BMSCs in the absence of TGFβ emphasising the inductive role of the mechanical environment."<-LSJL is like cyclic compressive loading.  So too is LSJL likely effective in promoting chondrogenic differentiation in the absence of TGF-Beta.

"TGF β, BMP-2 and mechanical factors influence chondrogenic differentiation of BMSCs via different mechanisms. TGF β and BMP-2 belong both to the TGF β superfamily of ligands. These ligands bind to a type II receptor which phosphorylates a type I receptor. Type I receptor phoshorylates receptor-regulated SMAD (R-SMAD) which then bind to common-partner SMAD 4 (co-SMAD 4). Finally these complexes act as transcription factors in the nucleus. Although TGF β and BMP-2 belong to the same superfamily of ligands, they bind to different receptors (TGF β receptor and BMP-2 receptor, respectively) which lead to activation of different R-SMAD molecules: SMAD 2 and 3 are TGF β receptor-associated SMAD and SMAD 1, 5 and 8 are BMP-associated SMAD[SMAD 2 and 3 versus SMAD 1/5/8 affects terminal differentiation of chondrocytes, however it is likely that achieving terminal differentiation of chondrocytes may be beneficial given various knockout studies]. All activated SMAD bind to the co-SMAD 4. Binding of either ligand can result in an up-regulation of so-called chondrogenic genes Sox-9, collagen type II and aggrecan. For the mechanical factors, cyclic compressive loading leads to up-regulation of endogenous TGF β gene expression as well as upregulation of type I and type II and TGF β receptors. TGF β signal transduction seems to be involved in chondrogenesis of BMSCs induced by compressive loading[which can be exemplified by the evidence that TGF-Beta1 is upregulated during LSJL]. bioactive factors and mechanical factors act through similar but different pathways to influence chondrogenesis of BMSCs"

BMP-2 had chondrogenic effects in conjunction with TGF-Beta but osteogenic effects without TGF-Beta.

Mesenchymal Stem Cells hyper-expressing BMP-2 may be a new mechanism of increasing height growth in the future.  They could be injected at where the cortical bones end to induce new height growth.

However, in already active growth plates BMP-2 accelerates terminal differentiation.  You may only want BMP-2 when you're trying to induce new chondrogenic differentiation of stem cells like with LSJL.

Interaction of TGFβ and BMP signaling pathways during chondrogenesis.

"TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. conditional deletion of Smad1{LSJL downregulates Smad1 but the shortening of the growth plate was not observed} in chondrocytes resulted in a shortening of the growth plate[remember Smad1 phosphorylation affects height in active growth plates(Phosphorylation in this case refers to activation)]. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components[But does this increase final height?  Just because there were no TGF-Beta receptors in cartilage doesn't mean there were none in stem stems which is where they are needed for chondrogenic differentiation].  in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. During endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo."

 "TGFβ or BMP ligands bind to specific type II receptors to recruit the corresponding type I receptor to initiate a cascade of events leading to phosphorylation of their specific receptor-Smads (R-Smads). Generally, TGFβ signaling depends on Smad2 and Smad3, while BMP signaling depends on Smad1, 5 and 8"<-remember we don't want Smad 1/5/8 phosphorylation(deactivation) but we do want Smad 2 and 3 phosphorylation in active growth plates(when the skeleton is still developing).  So we don't want fully functional TGF-Beta signaling(unless we're trying to form new growth plates like with LSJL).  Remember when TGF-Beta signaling was decreased an elongated growth plate was the result.

"TGFβ/BMP signaling can also be mediated by noncanonical MAPK pathways, such as P38, JNK and Erk1/2 signaling pathways, during chondrogenesis"

"In vitro BMPs can promote mesenchymal cells to differentiate into chondrocytes in high-density cultures in part by inducing Sox9 gene expression[remember though you want Beta-Catenin levels to be higher than Sox9 levels]"

"Loss of Noggin, a potent BMP antagonist, leads to overgrowth of skeletal elements in mice"<-so transgenic expression of BMP-2 may increase height as Noggin a BMP antagonist decreases height.

"BMPs promote the differentiation of proliferating chondrocytes to hypertrophic chondrocytes, the chondrocyte specific expression of constitutively active Bmpr-1a in transgenic mice accelerated the maturation and hypertrophy of proliferating chondrocytes"<- you would think accelerating maturation would decrease height

"reduced BMP signaling in proliferating chondrocytes leads to a shortening of the growth plate in part due to decreased cell proliferation, reduced TGFβ signaling results in an increased proliferation rate and an elongated growth plate"<-so maybe we want to enhance BMP signaling while inhibiting TGF-Beta signaling.

"BMP2 can inhibit TGFβ signaling, while TGFβ1 enhances BMP signaling in-vitro"<-Both BMP-2 and TGF-Beta can induce chondrogenic differentiation but BMP-2 is better than TGF-Beta since BMP-2 inhibits TGF-Beta anyways you'd want to increase BMP-2 levels to grow taller.  All the chondrogenic benefits for TGF-Beta may be due to BMP-2 signaling enhancement and not TGF-Beta itself.

However, in the previous study only TGF-Beta1 upregulated chondrogenic genes whereas BMP-2 had no effect.

Bezafibrate may be a way to upregulate BMP-2 expression.

Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation.

"MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined. NO production was evaluated. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured. [We] detect the expression of AMPK and eNOS proteins.
Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression[this increase in BMP-2 could help you grow taller by helping LSJL start that initial differentiation of chondrocytes]."

"Bezafibrate, a dual ligand for peroxisome proliferator-activated receptors α (PPARα) and PPARβ, is a lipid-lowering drug widely used to treat hypertriglyceridemia"

"After addition of bezafibrate, BMP-2 mRNA expression was increased to 1.80- and 1.74-fold of the control group by bezafibrate at d 7 and 14, respectively"

Identification of Novel 2-((1-(Benzyl(2-hydroxy-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid Analogues as BMP-2 Stimulators.

"10 new chemical entities (NCEs) have shown BMP-2 stimulation and osteoblast differentiation. Among these, 2-((1-(benzyl(2-hydroxy-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid (11) was the most effective while its analogue 13 also showed good activity in inducing osteoblast BMP-2 production. Compound 11 induced osteoblast differentiation in vitro, and this effect was abrogated by a physiological BMP-2 inhibitor, noggin."

"Piceatannol stimulates osteoblast differentiation and is associated with the increase in BMP-2 production. Recently, some proteasome inhibitors, viz. epoximycin (a natural product) and bortezomib, showed increased bone formation rates in vitro, with the participation of BMP-2 signaling in osteoblasts.  Wwo series of substituted benzothiophene and benzofuran derivatives [are] BMP-2 up-regulators."

BMP2 induces segment-specific skeletal regeneration from digit and limb amputations by establishing a new endochondral ossification center

"BMP treatment can induce a regeneration response in mouse digits amputated at a proximal level of the terminal phalangeal element (P3) "

"the regeneration-inductive ability of BMP2 extends to amputations at the level of the second phalangeal element (P2) of neonatal digits, and the hindlimb of adult limbs[really 8-10 week old mice]."

"BMP2-induced regeneration is associated with a localized proliferative response and the transient expression of established digit blastema marker genes. This is followed by the formation of a new endochondral ossification center at the distal end of the bone stump. The endochondral ossification center contains proliferating chondrocytes that establish a distal proliferative zone and differentiate proximally into hypertrophic chondrocytes. Skeletal regeneration occurs from proximal to distal with the appearance of osteoblasts that differentiate in continuity with the amputated stump. Using the polarity of the endochondral ossification centers induced by BMP2 at two different amputation levels, we show that BMP2 activates a level-dependent regenerative response indicative of a positional information network."

"BMP7 induces bone growth when applied to neonatal limb amputations in mice"

"BMP2-induced skeletal elongation occurs by the regeneration of an endochondral ossification center at the amputated stump that forms an apical growth zone that directs skeletal elongation. "

"BMP2 [induces] regeneration of the amputated distal region of the P2 element."

"Bone formation during digit development involves endochondral ossification and the differentiation of Col2a1 expressing chondroctyes into Col10a1 expressing hypertrophic chondrocytes establishes the polarity of skeletal elongation."

"mammalian tissues that lack regenerative ability still possess and/or are able to re-acquire positional information that is necessary to participate in a functional regenerative response that is appropriate for the amputation level."

"at 1 DPI we found [TWO] blastemal markers were expressed in the distal mesenchyme. Msx1 transcripts were expressed primarily by cells closely associated with the BMP2 bead, whereas Pedf expressing cells were scattered throughout the BMP2 treated amputation wound and not restricted to the region surrounding the BMP2 bead"

"On the other hand amputated limbs treated with BMP2 displayed directed skeletal elongation and a regenerative response of both the tibia and fibula"<-Note the wounds were allowed to heal first and that these were adult amputees.

"differentiation occurs distal to proximal during P3 regeneration and proximal to distal in P2 regeneration. This finding demonstrates that BMP2 is not acting to pattern the regenerative response, but rather appears to activate a level-specific regenerative potential."

"The stimulation of an apical zone of proliferating chondrocytes represents a foundation for exploring segment-specific de novo skeletal regeneration by effectively regenerating a growth plate."

BMP-2 was able to induce endochondral ossification after wound healing had occurred in 8 to 10 week old mice amputations.

Gene expression profiling following BMP-2 induction of mesenchymal chondrogenesis in vitro.

"The experimental system consists of micromass cultures of C3H10T1/2 cells, a murine multipotential embryonic cell line, treated with the chondroinductive growth factor, bone morphogenetic factor-2 (BMP-2). In this system, chondrogenic differentiation characterized by both morphological changes and cartilage matrix gene expression has been shown to be completely dependent upon BMP-2 treatment and the high cell plating density of micromass cultures."

"chondrogenesis can be induced at ectopic extra-skeletal sites by the implantation of demineralized bone matrix led to the isolation of chondroinductive factors"

BMP-2 induced col2a & Col2b however Col2a was already present in controls.  a-actin disrupts some B-catenin activities.

BMP-2 upregulated genes also upregulated by LSJL:
Osteopontin

BMP-2 downregulated genes also downregulated by LSJL:

"BNIP3 is a pro-apoptotic protein that heterodimerizes with Bcl-2 and Bcl-XL. Induction of BNIP3 may be required to control cell number during chondrogenesis.  BNIP3 is induced in this C3H10T1/2 system. hypertrophy [occurs] in late stages of the chondrifying micromass cultures"

This next study involves osteoblasts but I believe information can be inferred to chondrocytes and stem cells:

BMP2 and mechanical loading cooperatively regulate immediate early signalling events in the BMP pathway.

"We use a three-dimensional bioreactor system. Time-dependent phosphorylation of Smad, mitogen-activated protein kinases and Akt in human fetal osteoblasts was investigated under loading and/or BMP2 stimulation conditions. The phosphorylation of R-Smads is increased both in intensity and duration under BMP2 stimulation with concurrent mechanical loading.  The synergistic effect of both stimuli on immediate early Smad phosphorylation is reflected in the transcription of only a subset of BMP target genes, while others are differently affected. Regulation of osteogenesis is guided by both signalling pathways.

Mechanical signals are integrated into the BMP signalling pathway by enhancing immediate early steps within the Smad pathway, independent of autocrine ligand secretion. This suggests a direct crosstalk of both mechanotransduction and BMP signalling, most likely at the level of the cell surface receptors."

"roughly 1,000 times the normal physiological concentration has to be administered [for various BMP2 and BMP7 applications]"

"Id1 expression was slightly induced by BMP2 stimulation after 90 minutes"

"Dlx5, [under] both under loading and non-loading conditions, BMP2 led to enhanced gene expression"

"Id2{down} expression was also induced by BMP2 but the enhancing effect of mechanical loading was not present. c-fos{up} and osteopontin{up} expression was strongly up-regulated by mechanical loading, while BMP treatment exhibited no effect. By contrast, Runx2 expression, that was induced after 90 minutes, was down-regulated by mechanical loading after 24 hours. Gene expression of members of the Distal-less homeobox family, Dlx2 and Dlx3, was induced by BMP2 but expression was not significantly enhanced by concurrent mechanical loading. Dlx5 expression after 24 hours of stimulation was not regulated by the different treatments."

"Both BMP type I and type II receptors co-localize with αvβ integrins"

"BMP4 and BMP7 tend to be down-regulated under loading conditions, BMP6 expression was positively affected by mechanical loading, even more so when BMP2 was present"

Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology.

"we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID-1, ID-2{down}, ID-3, and ID-4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1{UP}, STAT1, and JunB{up}."

"core binding factor beta (CBFbeta), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment."

Vcam1 which was downregulated by LSJL had a rendency to be downregulated by BMP-2 treatment.  IL6 tended to be downregulated by BMP-2 but was upregulated by LSJL.


Chondrogenesis of human bone-marrow derived mesenchymal stem cells is modulated by complex mechanical stimulation and adenoviral-mediated overexpression of bone-morphogenetic protein 2.

"This study strived to investigate the combined effect of complex mechanical stimulation and adenoviral-mediated over-expression of bone morphogenetic protein 2 (BMP-2) on hMSC chondrogenesis. hMSCs were encapsulated in a fibrin hydrogel and seeded into biodegradable polyurethane (PU) scaffolds. A novel three-dimensional transduction protocol was used to transduce cells with an adenovirus encoding for BMP-2 (Ad.BMP-2). Control cells were left un-transduced. Cells were cultured for 7 or 28 days in chondropermessive medium, which lacks any exogenous growth factors. Thereby, the in vivo situation is mimicked more precisely. hMSCs in fibrin-PU composite scaffolds were either left as free-swelling controls or mechanically stimulated using a custom built bioreactor system which is able to generate joint-like forces. Outcome parameters measured were BMP-2 concentration within the culture medium, biochemical and gene expression analysis. Mechanical stimulation resulted in an up-regulation of chondrogenic genes. Further, GAG/DNA ratios were elevated in mechanically stimulated groups. Transduction with Ad.BMP-2 led to a pronounced up-regulation of the gene aggrecan and an up-regulation of Sox9 message after 7 days. Furthermore, a synergistic effect in combination with mechanical stimulation on collagen 2 message was detected after 7 days. This synergistic increase was more than 8-fold if compared to the additive effect of the application of each stimulus on its own. However, BMP-2 over-expression consistently resulted in a trend towards decreased GAG/DNA ratios in both mechanical stimulated and unloaded groups."

"[Gene transfer] is based on the delivery of cDNA (encoding a specific transgene) to a target cell. This procedure enables the cell to produce the desired transgene."

"Chondropermessive medium: Serum-free DMEM, 1% insulin-transferrin selenium premix, 50 μg/ml ascorbate-2-phosphate, 10-7 M dexamethasone and 1% MEM non-essential amino acids. P/S was replaced with 100 μg/ml of Primocin. This was necessary in order to prevent possible contaminations as the bioreactor system is not a closed system. A further 5 μM of 6-aminocaproic acid was added, in order to prevent fibrin degradation"

"Mechanical stimulation was executed for either 7 or 28 days using a custom built bioreactor system 37 and a ceramic hip ball with a diameter of 32 mm. Unconfined, dynamic compression strain was generated by pressing the ceramic hip ball onto the cell-seeded fibrin-PU scaffolds. Shear stresses were generated by rotating the ball around an axis perpendicular to the scaffold axis. Both stimuli were superimposed on a static offset strain of 0.4 mm. The following loading protocol was used: Compression 1Hz 0.4-0.8 mm; Rotation 1 Hz ± 25°"  The lowest MSCs used were from a 41 year old.

"In the unloaded groups, a trend towards a decrease in BMP-2 release into the culture medium was monitored if compared to the loaded groups. However, this trend did not reach statistical significance. Furthermore, BMP-2 concentrations steadily increased until week 4 in the loaded groups."

"The peak BMP-2 concentrations were 130±37.3.4 ng/ml in the unloaded groups and 339.8±186 ng/ml in the loaded groups."

Osteogenic genes Collagen I and Runx2 were unresponsive to mechanical loading but were upregulated by BMP-2.  Collagen X is mechanically upregulated but BMP-2 counteracts this mechanical upregulation.

"[A] synergistic effect of [mechanical and BMP-2] stimuli was detected on day 7. The n-fold up-regulation in Col 2 message was 2655±7417 (control loaded), 936±1785 (BMP unloaded) respectively 29878±86410 (BMP-2 loaded)."

BMP-2 but not loading seem to upregulate Sox9.  Aggren is upregulated by BMP-2 and loading independently but not synergestically.

"A trend towards an increased GAG release in loaded vs. unloaded groups was detected."

"Mechanical stimulation is known to lead to the paracrine production of TGF-β1, inducing chondrogenesis of hMSCs in [a] fibrin-PU composite system"

"in the unloaded group, the synthesized BMP-2 can leave the scaffold only by means of passive diffusion. In the loaded group however, application of mechanical forces will likely squeeze out most of the BMP-2 which is retained within the scaffold."

"An up-regulation in gene expression does not always correlate to elevated protein levels."

Latexin is involved in bone morphogenetic protein-2-induced chondrocyte differentiation.

"Latexin is the only known carboxypeptidase A inhibitor in mammals. BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin."

"The distribution of latexin-positive cells was similar to that of Sox9-positive cells"

Comparative Analysis of Osteogenic/Chondrogenic Differentiation Potential in Primary Limb Bud-Derived and C3H10T1/2 Cell Line-Based Mouse Micromass Cultures.

"we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules{learning why they failed to form chondrogenic nodules could be key}, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1{all upregulated by LSJL except for Snorc} at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPARγ2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2) were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models."

"the cell line-based colony exhibited a substantially different morphology than the primary model. Cells in the [embryonic] limb bud-derived HDC[High Density Culture] formed numerous nodules with multiple cell layers, while cellular density remained low in the internodular areas. The fact that differentiation of chondroprogenitor cells into chondroblasts primarily occurs within these foci was confirmed by staining with DMMB: on day 3, metachromatic cartilage matrix could only be detected within the aggregates and no metachromasia was visible in the internodular areas. By contrast, the central region of the C3H10T1/2-based culture was densely populated with complete lack of foci and internodular areas. Furthermore, cellular behaviour in terms of migratory characteristics was also different in the two models: although some cells have also migrated to the periphery of the primary HD culture, this area remained relatively sparse compared to the cell line-based HDC in which a very high number of cells have spread from the centre and they even formed dense, multiple cellular layers on the periphery."<-Formation of condrogenic foci and internodular areas could be key to neo-growth plate formation.  You can look at figure 1 for more analysis.

"there is an increasing tendency in the amount of metachromatic[color stained] ECM areas as differentiation proceeds."

"While we observed extensive metachromatic areas within cartilaginous nodules (but not in the internodular areas) in 3-day-old primary HDC, mainly orthochromatic[no color change] staining was visible in the cell line based models on the same culturing day. By days 6 and 10 of culturing the disparity became even more pronounced between primary HDC and the C3H10T1/2 models. Nonetheless, the BMP-2 overexpressing cell line-based cultures presented higher amounts of metachromatic ECM areas in comparison with the control ones. After 15 days of culturing, the ECM was exclusively metachromatic and enlarged, presumably hypertrophic chondrocytes were also visible in the primary HDC. The size of metachromatic matrix areas also increased in the BMP-2 overexpressing C3H10T1/2 colonies{So in the cell line culture it just takes longer}, but it failed to reach the amount detected for primary cultures of the same age. In contrast, no metachromatic territories could be identified in micromass cultures of control C3H10T1/2 cells even on day 15 of culturing. It is of note that the appearance of metachromatic territories in the embryonic limb bud-derived HDC and that in cultures established from the BMP-2 overexpressing variant of C3H10T1/2 was different after staining with DMMB; while the former model exhibited distinct, heavily metachromatic regions that corresponded to cartilaginous nodules, a weaker but relatively homogenous metachromasia was observed in the latter one. Furthermore, considerable orthochromatic territories were also visible throughout the culturing period in colonies of the BMP-2 overexpressing C3H10T1/2."

The embryonic cell culture had higher expression of Prg4, Col10a1, and Acan than the cell line overexpressing BMP2.