Can LSJL work on adults?

LSJL was tested on 14 week old male rats.  Many chondrogenic genes were upregulated and studies have shown that Adult Humans BMMSC's are capable of chondroinduction.  However, more evidence would be helpful.

The next step would be to test LSJL on adult female mouse ERalphaAF-1 knockout mice which experience growth plate fusion as described in the study The role of estrogen receptor-α and its activation function-1 for growth plate closure in female mice.  Then see if there's any length increase when LSJL is applied.  You can't apply LSJL to another species because the Yokota lab pizeoelectric loader is custom built for rats and LSJL rat growth plates don't fuse which may alter gene expression.  So by forcing fusion using ERa AF-1 knockout mice, we can test how LSJL would work on results.  Gene expression data would be interesting but length increase would be what is needed.

The transactivating function 1 of estrogen receptor α is dispensable for the vasculoprotective actions of 17β-estradiol explains how to generate ERa AF-1 knockout mice.

"ERα AF-1 was generated through a targeted deletion by using a knockin strategy, through which 441 nt of exon 1 were deleted. The truncated protein lacks the A domain and all three motifs constituting ERα AF-1 (AF-1 boxes 1–3) in the B domain, thus yielding a 451-aa-long, 49-kDa protein"

The cost of these mice is at least $5000 but it should cheaper from there on.  I'll try to do some research and see what can be done.  Then there's the confounding variable that the knockout may affect sensitivity of cells to the stimulus.

Bone mechanotransduction may require augmentation in order to strengthen the senescent skeleton.

"Age-related increases in osteocyte apoptosis is observed and holds potential to alter intercellular signaling and the function of the bone cell syncytium"

"there is a marked age-related increase in marrow adiposity"<-although adiposal stem cells are capable of differentiating into chondrocytes.

"aging is accompanied by a decline in periosteal lining cell numbers"

" Age-related increases in osteocyte apoptosis is also observed and holds potential to alter intercellular signaling and the function of the bone cell syncytium"

"attenuated biophysical stimulation could arise in part from decreases in the surface to volume ratio of bone mineral matrix and increased viscosity of interstitial fluids.  If these physical mechanisms were to occur in bone, the magnitudes of fluid flow induced secondary to skeletal loading could be reduced with age."<-since fluid flow is a vital part of the LSJL stimulus this could reduce LSJL based chondrogenesis.

"PGE2 release has been found to be substantially increased in cells derived from aged vs. young human donors. In contrast, the number of cells displaying spontaneous Ca2+ oscillations and fluid flow induced oscillations were found to be significantly lower in primary cells derived from aged animals, while the amplitudes of Ca2+ oscillations themselves were equivalent"<-Ca2+ oscillations may be critical to induce chondrogenesis.

"Activation of phosphatases and kinases downstream of second messenger signaling including Calcineurin, CAMK, and MAPK are decreased with age. Further downstream (of the phosphatases and kinases), activation and DNA binding by a variety of transcription factors such as NFAT, AP-1, and Wnt/β-catenin also display age-related deficits or suppression"<-LSJL involves both AP-1 and Wnt transcription factors.

"primary cells isolated from young and senescent mice reveals that relative increases in Cox-2 and c-fos gene expression levels in response to fluid flow (vs. no flow controls) were not altered with age "<-Cox-2 and c-fos are both upregulated by LSJL.

"The absolute Cox-2 but not c-fos levels displayed age-related declines in large part due to lower baseline levels of Cox-2 expression in cells derived from aged animals."

"aging markedly diminishes the periosteal response to mechanical stimuli"

"suppression of increased osteoclastic activity [is] associated with aging"

"Cyclosporin A (CsA)[immunosuppresent drug that is available with prescription only], at low-dosages, could be a candidate agent to address age-related deficits in NFAT (or AP-1) activation and transcription"

"senescent mice (22 months) were subjected to mechanical loading in conjunction with low-dose CsA treatment, the resulting periosteal bone formation was significantly increased compared to vehicle-treated aged matched controls subject to loading alone."

CsA inhibits p38, GSK-3beta, and Calcineurin.  Lithium is another GSK-3Beta inhibitor.

Other genes that may be differentially regulated due to aging in LSJL:

Turner's axial loading study used 20 week old mice versus 14 week old.  So we can also study whether genes were deferentially regulated in the two studies due to age rather than due to the different loading modalaties(axial vs. lateral).

MicroRNA-199a-3p, microRNA-193b, and microRNA-320c are correlated to aging and regulate human cartilage metabolism.

"The expression of two miRNAs (miR-199a-3p and miR-193b) was upregulated with age and that of one miRNA (miR-320c) was downregulated with age."

"Type 2 collagen, aggrecan, and SOX9 expression were downregulated in the miR-199a-3p mimic group[axial loading 20 week old mice upregulated COL2A1 and acan but not Sox9, whereas all three were upregulated in LSJL thus the significant upregulation of Sox9 versus axial loading is likely due to the different modalaties and not aging] but [were] upregulated in the inhibitor group. Similar results were observed for miR-193b. By contrast, ADAMTS5 expression was downregulated in the miR-320c mimic group and upregulated in the inhibitor group. Cell proliferative activity was upregulated significantly in the miR-193b inhibitor group compared with the control group. miR-199a-3p and miR-193b are involved in the senescence of chondrocytes, and miR-320c is involved in the juvenile properties of chondrocytes."

LSJL upregulates the anti aging genes Lin-28b and HMGA2.

Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells.

"chondrogenic potential did not change [with aging]"

The growth of adult(25-50) MSCs was only slightly than child MSCS(0-12) but old had significantly less growth of MSCs.

Thus LSJL inducible chondrogenesis may not change due to alterations in chondrogenic potential with aging but rather due to secondary signaling from osteocytes which do experience alterations in aging.

Effects of treadmill exercise and training frequency on anabolic signaling pathways in the skeletal muscle of aged rats.

"protein levels of IRS-1 and p-mTOR as well as COX activity were reduced in advanced age."  IRS1 was deferentially expressed in the two studies up in axial loading and down in LSJL despite the axial loading rats being older.  The tissues are different, muscle versus bone, but the insulin pathway has a general anabolic effect in all tissues.

Effect of aging on the basal expression of c-Fos, c-Jun, and Egr-1 proteins in the hippocampus. found that aging decreased egr1 expression in the hippocampus.  egr1 was downregulated in axial loading whereas it was upregulated in LSJL.  The differential expressions of egr1 in the two studies could be based on aging rather than loading modality.

Age-related changes in rat bone-marrow mesenchymal stem cell plasticity.

"The expression profile of stemness markers was altered in BM-MSCs derived from old rats. BM-MSCs from young rats (4 months) expressed Oct-4, Sox-2 and NANOG, but we failed to detect Sox-2 and NANOG in BM-MSCs from older animals (15 months). Chondrogenic, osteogenic and adipogenic potential is compromised in old BM-MSCs{chondrogenic potential is not compromised in human BM-MSCs so maybe epiphyseal fusion adult female mice would not be the best model}. Stimulation with a cocktail mixture of bone morphogenetic protein (BMP-2), fibroblast growth factor (FGF-2) and insulin-like growth factor (IGF-1) induced cardiomyogenesis in young BM-MSCs but not old BM-MSCs. Significant differences in the expression of gap junction protein connexin-43 were observed between young and old BM-MSCs. Young and old BM-MSCs fused with neonatal ventricular cardiomyocytes in co-culture and expressed key cardiac transcription factors and structural proteins. Cells from old animals expressed significantly lower levels of VEGF, IGF, EGF, and G-CSF. Significantly higher levels of DNA double strand break marker γ-H2AX and diminished levels of telomerase activity were observed in old BM-MSCs."

"Old BM-MSCs displayed spread out, flat enlarged morphology which is consistent with late passage and extensively cultured BM-MSCs"

"Human fetal bone marrow is known to contain approximately 1 in 10, 000 MSCs in comparison to 1 in 250, 000 in adults "

Bone marrow stromal cells from aged male rats have delayed mineralization and reduced response to mechanical stimulation through nitric oxide and ERK1/2 signaling during osteogenic differentiation.

"Here, we investigated the ability of bone MSCs from mature and aged rats to differentiate into osteoblasts and to respond to short and long periods of mechanical stimulation through signaling by ERK1/2, nitric oxide (NO), and prostaglandin E(2) (PGE(2)) during differentiation[It's osteoblasts and not chondrocytes but it should be indicative of the ability for MSCs to respond to NO, ERK1/2, and PGE2]. Mineralization was delayed and reduced, but extracellular matrix production appeared less affected by increased age. Differentiating MSCs from aged animals had a decreased response to short and long periods of mechanical stimulation through ERK1/2 signaling, and to long periods of mechanical loading through NO signaling early and late during differentiation. Increases in relative PGE(2) signaling were higher in MSCs from aged animals, which could compensate for reduced ERK1/2 and NO signaling. The decreased mineralization may decrease the ability of cells from aged animals to respond to mechanical stimulation through ERK1/2 and NO signaling, with increased impairment over differentiation time. Decreasing the delay in mineralization of MSCs from aging animals might improve their ability to respond to mechanical stimulation."

"Calcium concentration also increased linearly in differentiating MSCs from both aged and mature rats over the time course, but it was consistently lower in cells from aged animals; the difference was statistically significant at day 28"<-This may be applicable to chondrogenic differentiation as calcium secretion may help trigger chondrogenesis.

Aged animals had increased expression of the pro-chondrogenic protein BMP-2.

"Differentiating cells from aged rats expressed higher p21 and p53 levels at day 7 therefore senescence could play a role in the age-related changes in mineralization and response to mechanical stimulation observed."

Time course of epiphyseal growth plate fusion in rat tibiae.

"there has been a question as to when or to what extent the rat growth plate fuses. To investigate this question, we used microcomputed X-ray tomography, at voxel resolutions ranging from (5.7 micro m)(3) to (11 micro m)(3), to image the proximal epiphyseal growth plates of both male (n = 19) and female (n = 15) rat tibiae, ranging in age from 2 to 25 months. The three-dimensional images were used to evaluate fusion of the epiphyseal growth plate by quantitating the amount of cancellous bone that has bridged across the growth plate. The results suggest that the time course of fusion of the epiphyseal growth plate follows a sigmoidal pattern, with 10% of the maximum number of bridges having formed by 3.9 months in the male tibiae and 5.8 months in the female tibiae, 50% of the maximum number of bridges having formed by 5.6 months in the male tibiae and 5.9 months in the female tibiae, and 90% of the total maximum of bridges have formed by 7.4 months for the males and 6.5 months for the females. The total volume of bridges per tibia at the age at which the maximum number of bridges per tibia has first formed is 0.99 mm(3)/tibia for the males and 0.40 mm(3)/tibia for the females. After the maximum number of bridges (-290 for females, -360 for males) have formed the total volume of bridges per tibia continues to increase for an additional 7.0 months in the males and 17.0 months for the females until they reach maximum values (-1.5 mm(3)/tibia for the males and -2.2 mm(3)/tibia for the females)."

No bony bridges were observed for the female rat until 5 months.

"the growth cartilage of female Sprague–Dawley rats to be bridged by bone between 6 and 18 months of age"

"Although the tibiae from the 6-month-old rats exhibited an increase in number and volume of bridges when compared to the younger rats, the growth plates still appeared to show proliferative and hypertrophic zonest hence, there is a local limited potential for further elongation. Conversely, there seemed to be an absence of all four of the distinct zones of an active growth plate (zones of resting cells, cell proliferation, cell maturation, and lacunar hypertrophy) in the 9-, 13-, and 24-month-old male growth plates and the 8-, 12-, and 25-month-old female growth plates, as demonstrated by conventional histological evaluation "<-So you can grow taller despite some briding.

16 weeks the oldest LSJL mice age is 4 months. Note that the C57BL/6J female used have delayed senescence.  Here's the growth rate for C57BL/6J mice according to Growth of C57Bl/6 mice and the material and mechanical  properties of cortical bone from the tibia:

Because of the loss of chondrogenic differentiation potential in aged rats but not adult humans it's necessary to have more adult humans test LSJL(read: you).  It's possible that the existing growth plates in the rats inhibited chondrogenic differentiation and thus if growth plates were removed in old rats this would not be the case.  This theory is supported by research on the tide mark.  However, the stem cells were cultured outside of the rat bone and differentiation was induced there where there would be no growth plate.

Thus, there is not likely to be an age related difference in LSJL, independent on the loss of the growth plates of course, in humans but there is in rats with respect to chondrocyte differentiation potential but there may be an indirect effect due to secondary osteocyte signaling and changes in fluid viscosity and bone shape.

Impact of aging on rat bone marrow-derived stem cell chondrogenesis.

"We measured the responses of rat bone marrow-derived mesenchymal stem cells (BMSCs) to chondrogenic induction in vitro. BMSCs from immature rats (1 week old), young adult rats (12 weeks old), and old adult rats (1 year old) were analyzed for cartilage extracellular matrix (ECM) production. Histologic analysis showed strong cartilage ECM formation by BMSCs from 1-week-old rats, but not by BMSCs from 12-week-old[LSJL has been proven effective on 8 and 16 week old mice. or 1-year-old rats. Age-related declines [were observed] in messenger RNA encoding type II collagen, aggrecan, and link protein, three major cartilage ECM components. [There were] significant age-related differences in the expression of genes that influence cartilage ECM formation. These findings support the hypothesis that the chondrogenic potential of mesenchymal stem cells declines with age."

"BMSC isolates contain large, slowly replicating cells and small, rapidly growing cells, which retain multipotential differentiation status longer than do larger cells"

"The level of c-Kit, a protein tyrosine kinase receptor for stem cell factor appeared to decrease with increasing age."

Type II collagen gene expression was decreased in 1 year old rats.  Aggrecan expression decreased in twelve week old rats.  Link protein decreased in one year old rats.  Sox9 expression decreased in 1 year old rats but was still greater than that of 1 week old rats.

The decrease in Aggrecan is significant but not the decrease in collagen.  Most genes related to chondrogenesis did not change significantly in the microarray data(with the noted exception of aggrecan) except that many chondrogenic genes increased in expression between 1 week old and 12 week old mice.

Increasing Aggrecan may be a way to enhance the chondrogenic potential of MSCs in adults.

Here's a grant related to the effects of aging on Mechanotransduction:

AGING-RELATED DEGRADATION IN BONE MECHANOTRANSDUCTION

"Mechanical loading and physical exercise hold promise for enhancing bone mass and morphology. [Previously], we examined how real-time Ca2+ signaling/NFAT pathway activation induced acutely by mechanical stimuli influences bone formation and the degraded response of bone at senescence. [Our] hypothesis [is] that age-related decline in bone formation induced by mechanical loading arises primarily via deficits in activation of the Ca2+/NFAT pathway{NFAT5 and NFATC3 are downregulated by LSJL}. We will quantify agerelated alterations in gene expression downstream of the Ca2+/NFAT pathway and their relation to deficits in cell function and bone formation. We will also demonstrate the requirement for this pathway in bone mechanotransduction by examining the impact of inhibiting (using NFATc1 knock-out mice and high dose Cyclosporine A; CsA) or enhancing its activation (using low-dose CsA). These experimental data will in-turn be used to develop multi-scale simulations for how modulating activation of the Ca2+/NFAT pathway influences the dynamics of cell function and bone formation in young adult and aged animals. In the final S. Aim, [we] will optimize activation of the Ca2+/NFAT pathway with the objective of restoring bone response to loading in the aged skeleton to levels observed in the young adult skeleton. Success of this project would clarify the Ca2+/NFAT pathway as a critical mechanism underlying the agerelated degradation in bone's ability to respond to physical stimuli, and will demonstrate the benefits that are anticipated via interventions in this pathway. Cyclosporine A (one of the proposed interventions) is currently approved for clinical use."

Here's a study detailing the properties of 17 week old female mice tibias(LSJL has induced length gain in female mice ages up to 16 weeks):

Intrinsic material properties of cortical bone.

"The G171V mutation (high bone mass, HBM) is autosomal dominant and is responsible for high bone mass in humans {The humans are transgenic for LRP5, being transgenic for LRP5 inhibits GSK-3Beta so it may help with height growth}. Transgenic HBM mice in which the human LRP5 G171V gene is inserted also show a similar phenotype with greater bone mass and biomechanical performance than wild-type mice, as determined by whole bone testing. Whole bone mechanics, however, depend jointly on bone mass, architecture, and intrinsic bone tissue mechanical properties. To determine whether the HBM mutation affects tissue-level biomechanical performance, we performed nano-indentation testing of unembedded cortical bone from HBM mice and their nontransgenic (NTG) littermates. Femora from 17-week-old mice (female, 8 mice/genotype) were subjected to nano-indentation using a Triboscope. For each femoral specimen, approximately 10 indentations were made on the midshaft anterior surface with a target force of either 3 or 9 mN{LSJL uses 0.5N of Force} at a constant loading rate of 400 mN/s. The load-displacement data from each test were used to calculate indentation modulus and hardness for bone tissue. The intrinsic material property that reflected the bone modulus was greater (48%) in the HBM as compared to the NTG mice. The greater intrinsic modulus in HBM reflects greater bone mineral content as compared to NTG (wild-type, WT) mice. The greater intrinsic property of cortical bone is derived from the greater bone mineral content and BMD, resulting in greater bone strength in HBM as compared to NTG (WT) mice."

"Elastic depth (nm, deformation) is elastic, that is, the deformation recovers when the load is removed, whereas plastic depth (nm, deformation) is plastic, or the deformation is permanent."<-plastic depth of the long bones in a longitudinal direction may way to growth taller.  And the plasticity and elasticity of the bone may effect the ability of bone to respond to deformation by newly induced growth plate.  And this plasticity and elasticity may be affected by age.

This is what a 17 week old bone looks like.  There seems to be lots of porous areas to induce new growth plates.

"the composition (mineral, collagen, etc.) of bone may be responsible for the differences in the elastic and plastic components of total deformation"<-We can alter this composition to get more permanent changes in bone growth.

Skeletal growth and the changing genetic landscape during childhood and adulthood.

"All processes of skeletal growth (longitudinal growth as well as gains and losses of bone mass) are subjected to environmental and genetic influences. These influences, and their relative contributions to the phenotype, can be asserted at any stage of life."

" The shape and content of bone will change throughout the life span with fluctuating contributions of [environmental and genetic] influences."

"in humans, long bones lengthen with age from birth to ∼18 years (or until skeletal maturity), with fluctuations in growth velocity occurring at various periods therein. Increases in total bone diameter and cortical thickness occurs from birth to approximately 30 years of age"

"Genetic influence on bone changes over the life course. The mechanisms for changes in genetic influence can include developmental timing of gene action, epigenetic modification of the bone-related gene product, or even variation in environmental factors influencing gene expression."

"Quantitative measures of bone size and shape were obtained from the second metacarpal from anteroposterior radiographs of the left hand."

"the genetic contribution to bone length remains high and is relatively consistent throughout the remainder of childhood and into young adulthood (average heritability of 0.92 from age of 7 to 29 years). In later adulthood, the genetic influence on metacarpal length again fluctuates, with heritabilities ranging between 0.64 and 0.88. "

Functional comparison of chronological and in vitro aging: differential role of the cytoskeleton and mitochondria in mesenchymal stromal cells.

"We established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages[a round of cell growth and proliferation in cell culture]. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics{so by reducing some of of these division related changes may enable stem cells to differentiate into chondrocytes once more}, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function{or loss of progenitor characteristics could be due to culture and it's possible similar changes may occur in aging by degeneration of the microenvironment}. Altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels [occurred] in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging."

"Cell roundness increases during long term culture of aMSCs and yMSCs"

Comparison of genes upregulated and downregulated by long term passaging to LSJL to be done.  A number of genes downregulated by passaging are focal adhesion proteins, regulation of actin cytoskeleton, mitochondrion, wnt signaling pathway, and TGF/BMP signaling.

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2.

"We examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25-81 years old), including patients with or without systemic vascular diseases.
All stem cell lines were expanded with fibroblast growth factor-2{upregulated by lsjl} and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels{all upregulated by LSJL}. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining.
Systemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes.
The aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells."

GAG content of the chondrogenic medium decreased fairly dramatically by age and by age 90 was almost non-existent.    But is still quite significant in the ages of 20-40 in life.  Sox9 and Aggrecan were non-existant by age 70.   Whereas, Col2a1 was non-existent by age 65.  Osteogenic potential on the other hand increased with age.

Vascular disease decreased chondrogenic potential whereas it increased the osteogenic potential.  So the aging trends in osteogenic vs. chondrogenic differentiation may be related to vascular issues.

"The age-dependent decrease in SOX9 expression must limit the cartilage regeneration capability of MSCs."

Human fetal and adult bone marrow derived mesenchymal stem cells use different signalling pathways for the initiation of chondrogenesis.

"Human fetal mesenchymal stem cells (MSCs) have been isolated from a range of perinatal tissues including first trimester bone marrow and have demonstrated enhanced expansion and differentiation potential. However their ability to form mature chondrocytes for use in cartilage tissue engineering has not been clearly established. Here we compare the chondrogenic potential of human MSCs isolated from fetal and adult bone marrow and show distinct differences in their responsiveness to specific growth factors. Transforming growth factor beta 3 (TGFβ3) induced chondrogenesis in adult but not fetal MSCs. In contrast, bone morphogenetic protein 2 (BMP2) induced chondrogenesis in fetal but not adult MSCs. When fetal MSCs co-stimulated with BMP2 and TGFβ3 were used for cartilage tissue engineering they generated tissue with type II collagen and proteoglycan content comparable to adult MSCs treated with TGFβ3 alone. Investigation of the TGFβ/BMP signalling pathway showed that TGFβ3 induced phosphorylation of SMAD3 in adult but not fetal MSCs. These findings demonstrate that the initiation of chondrogenesis is modulated by distinct signalling mechanisms in fetal and adult MSCs. This study establishes the feasibility of using fetal MSCs in cartilage repair applications and proposes their potential as an in vitro system for modelling chondrogenic differentiation and skeletal development studies."

"Fetal MSCs have enhanced plasticity, proliferation propensity and expansion potential compared to adult MSCs. They appear to form an intermediate cell type between adult MSCs and embryonic stem cells (ESCs) as they have active telomerase and express pluripotency markers, albeit at a considerably lower level than ESCs"

"Fetal MSCs also lack intracellular HLA class II and have lower HLA class I expression compared to adult MSCs which suggests that these cells may be immunologically inert"

"the bone morphogenetic proteins (BMPs) have been shown to have the ability to induce de novo ectopic cartilage formation in a system that recapitulates endochondral ossification during skeletogenesis. Specifically, BMP2 has been shown to promote condensation of the mesenchymal cells in the developing limb"

"Fetal and adult MSCs both induced strong phosphorylation of BMP-specific SMAD1 and SMAD5 with BMP2 treatment alone or in combination with TGFβ3. However, strikingly we detected TGFβ3-mediated phosphorylation of SMAD1/5 in adult MSCs as well as a marginal level in fetal MSCs. TGFβ-induced signalling is predominantly activated through TGFBR1 and TGFBR2 to phosphorylate SMAD2/3. However, TGFβ has also been shown to activate SMAD1/5 phosphorylation, traditionally activated by BMP signals in numerous cell types. By signalling through TGFBR1, activin A receptor type II-like 1 (ALK1) is recruited and in combination with TGFBR2, activates SMAD1/5 phosphorylation. This dual signalling results in the formation of mixed-receptor SMAD complexes, which may bind to BMP promoters and influence BMP-mediated transcriptional responses. The expression of TGFBR2 and upregulation of BMPR2 in adult MSCs stimulated with TGFβ3 indicates that TGFβ-mediated SMAD1/5 phosphorylation may signal through a receptor complex incorporating TGFBR2-BMPR2 receptors to mediate the initiation of chondrogenesis."

"phosphorylation of both SMAD2/3 and SMAD1/5/8 is essential at the onset of chondrogenic differentiation and these SMADs remain in an active state in differentiated MSCs, while only SMAD2/3 is present in native articular cartilage"

"the initiation of chondrogenic differentiation of MSCs may be mediated by either TGFβ or BMP, but can only be maintained through signalling associated with SMAD3. This may explain the need for TGFβ3 in addition to BMP2 for optimal cartilage tissue engineering by fetal MSCs even though BMP2 alone is sufficient for the initiation of chondrogenic differentiation"