Are bone marrow cells capable of chondrogenesis?

The goal of LSJL is to induce chondrogenic differentiation of the mesenchymal stem cells in the epiphyseal bone marrow.  The chondrogenic potential of bone marrow cells is therefore of interest to us.  Are adult bone marrow cells capable of differentiating into chondrocytes?

Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors.

"the cardio-vascular reparative effects attributed to MSCs appear to be mediated predominantly through the secretion of factors targeting cells at the site of repair"<-so what MSCs differentiate into depends on what factors are secreted at certain sites.  So at the sites where we want to grow taller we want our cells to secrete pro-chondrogenic factors.  LSJL encourages secretion of TGF-Beta which is pro-chondrogenic.

"MSC populations are heterogeneous cell populations whose composition depends on isolation methods and expansion conditions that differ largely among investigators. Nearly 50% of CFU-Fs from BM were tripotent MSCs while the remaining population of cells showed varied phenotypes"<-So the ability of MSCs to undergo chondrogenesis may vary by individual.

"SM[synovial membrane] MSCs [contain] two populations: 30% of cells were tripotent while the remainder displayed only osteo-chondral differentiation potential"<-so all synovial membrane cells have the ability to differentiate into chondrocytes.  However, the synovial membrane lines the articular cartilage and does not necessarily have access to epiphyseal bone marrow.

"embryonic stem (ES) cells can clearly be discriminated from MSCs by specific hypermethylation of numerous genes. The comparison of AT[adipose tissue] and BM MSCs revealed few differences. specific hypermethylation of numerous genes [occurs] in HSCs while the methylation patterns of MSCs from different sources were very similar"<-So DNA Methylation affects the differences between embryonic and adult stem cells much more than methylation differences between individuals.  We need to find out which specific methylations affect height.

" promoter hypomethylation is not predictive for the differentiation potential of cells, while hyper-methylation sets restrictions that define frames for differentiation potentials"<-so we may want to un hyper-methylate some genes.

"two cytosines in the COL10A1(Collagen Type X or terminal differentiation) promoter were consistently hypomethylated in MSCs in comparison with articular chondrocytes, correlating to the inducibility of COL10A1 expression and hypertrophy during in vitro chondrogenesis of MSCs "->so the hypomethylation of the COL10A1 gene in articular chondrocytes could be why articular chondrocytes do not ossify and undergo terminal differentiation.  For LSJL to work properly we need to ensure that the COL10A1 gene is hypomethylated.

"Histone modifications and histone-modifying molecules are regulated, while MSCs enter senescence in vitro and could be involved in the ensuing loss of differentiation potential. They are also actively involved in differentiation. Histone deacetylases, in particular HDAC4, may represent important regulators of chondrogenesis "<-So we need to worry about histones as well for inducing chondrogenesis.

"During embryogenesis the development of cartilage is initiated by a phase of condensation of mesenchymal precursor cells, and the cell-cell contact arising from condensation appears to be crucial for the onset of chondrogenesis. N-cadherin seems to be involved in cell-cell contact in pre-cartilage condensations, and functional N-cadherin was necessary for chondrogenesis of chick limb mesenchymal cells in vitro and in vivo. In human MSCs, N-cadherin is strongly up-regulated during the condensation phase during the first few days of chondrogenic induction in vitro . When MSCs are submitted to chondrogenic conditions in monolayer culture, they begin to condensate in response to the stimulus and form high-density three-dimensional cell aggregates. proper chondrogenic differentiation occurs also for MSCs embedded in gel-like biomaterials that keep cells apart from each other and thus limit direct cell-cell contact. This suggests that, although cell-cell contact facilitates chondrogenic induction of MSCs compared with monolayer culture, it does not represent an absolute requirement for in vitro chondrogenic differentiation of human MSCs in a three-dimensional structure."<-So cells may be able to differentiate into chondrocytes without contact with other MSCs.  This is good for LSJL as we only have to encourage chondrogenesis(and subsequent endochondral ossification) of one cell rather than several.

So evidence supporting chondrogenesis in epiphyseal bone marrow.  We need to learn more about methylation and histones that encourage height growth as we can manipulate those to grow taller.

The Therapeutic Effect of Bone Marrow Derived Stem Cell Implantation Following Epiphyseal Plate Injury is Abrogated by Chondrogenic Pre-Differentiation.

"Chondrogenesis was induced with TGF-Beta1 treatment in high-density monolayer cultures of BMSCs in vitro. The pre-differentiated or undifferentiated BMSCs were either seeded into agarose gels for continued in vitro culture, or injected into growth plate defects via an in situ gelling agarose. Pre-differentiated BMSCs had higher Sox-9, type II collagen, and aggrecan mRNA levels compared to undifferentiated cells after high-density monolayer culture. After transfer to agarose gels, pre-differentiated cells did not produce a cartilaginous matrix{so injecting chondrocytes into the bone may not help you grow taller}, even with continued TGF-Beta1 stimulation, whereas undifferentiated cells produced a cartilaginous matrix in this system. Three-dimensional images of the growth plate showed that delivery of either pre-differentiated or undifferentiated cells to defects resulted in a decrease in mineralized tether formation (fusion) in the growth plate tissue surrounding the defect to normal levels. Limb length discrepancy between injured and control limbs was corrected following treatment with undifferentiated but not pre-differentiated cells."

"delivery of an in situ gelling agarose to centralized growth plate defects in a rat animal model decreased limb length discrepancies, but did not fully restore function to the injured area"

"Growth factor stimulation is required to induce chondrogenesis of stem cells, although the specific treatment protocol depends on the tissue source from which the cells are derived"

"Bone marrow was flushed from diaphyses with alpha-MEM supplemented with 1% antibiotic/antimycotic" Our target is epiphyseal bone marrow but they should be similar to diaphyseal bone marrow stem cells.

"control samples continued to receive 1ng/ml FGF-2 and chondrogenic samples were switched to receiving 10ng/ml TGF-Beta1"

"BMSCs in high density monolayer showed very little proliferation after switching to chondrogenic media in both FGF-2 and TGF-Beta1 treated samples over the culture period"<-Maybe it's better to try to upregulate stem cell proliferation proteins when starting LSJL rather than inducing chondrogenesis?

"The limb length discrepancy was corrected in only the group treated with undifferentiated cells and the defect legs of this group were significantly longer then the empty defect limbs"<-overgrowth

Cultured mesenchymal stem cell transfers in the treatment of partial growth arrest.

"Mesenchymal stem cells were cultured from periosteum harvested from the tibias of New Zealand White (NZW) rabbits. An experimental model for growth arrest was created by excising the medial half of the proximal growth plate of the tibia of 6-week-old NZW rabbits. The cultured mesenchymal stem cells were embedded in agarose and transferred into the growth-plate defect after excision of the bony bridge in established growth arrest. Transfer of agarose alone and a periosteum flap without cells served as the control groups. In cases of transfer of mesenchymal stem cells, growth arrest with angular deformation and loss of length of the tibia was corrected. Transfer of agarose alone and a periosteum flap yielded poor results."

"periosteal derived MSCs delivered in agarose generated a new growth plate and completely corrected angular deformity"

Normal structure, function, and histology of the bone marrow.

Bone marrow accounts for 5% of body weight in humans.

Thus the natural bone marrow MSCs may actually be better than exogenous chondrocytes.

Biological properties of mesenchymal Stem Cells from different sources.

"Bone marrow MSCs (BM-MSCs) are a subpopulation of the stromal cells that line the endosteal surface of the marrow space. Cells in many respects identical to BMMSCs can be isolated from trabecular and compact bone and from non-hematopoietic bone marrow sites, such as the femoral head"

"MSCs have been first isolated from the bone marrow. They have been defined as nonhematopoietic, multipotential cells that support hematopoietic stem cells expansion in vitro and can differentiate into cells of various connective tissues. They are easy to harvest and they are hold in bone marrow in relatively high concentration"

Bone Marrow MSCs are rated as having very high chondrogenic differentiation capability second though to Umbillical Cord, Tendon, and Synovial Stem Cells.