Wednesday, May 19, 2010

Increase your Stature by stimulating differentiation of stem cells into chondrocytes

The key steps involved in making Lateral Synovial Joint Loading successful at increasing the height of the long bone are: encourage mesenchymal stem cell prliferation and encourage the mesenchymal stem cells to differentiate into chondrocytes at the Hyaline Cartilage growth plate line.  Now we know that loading the articular cartilage alters genetic expression and can stimulate chondrocyte proliferation(plus hopefully differentiation of Mesenchymal Stem Cells into chondrocytes).  But, are there other ways of encouraging Mesenchymal Stem Cells to differentiate into chondrocytes?

Regulation of Chondrogenesis by Transforming Growth Factor-{beta}3 and Insulin-like Growth Factor-1 from Human Mesenchymal Umbilical Cord Blood Cells. 

"Umbilical cord blood is a source of mesenchymal stem cells, which can give rise to cells of different lineages, including cartilage. Cellular condensation is a required step in the initiation of mesenchymal chondrogenesis. We attempted to differentiate cells from umbilical cord blood into chondrocytes with insulin-like growth factor 1 (IGF-1) and transforming growth factor-ss3 (TGF-ss3). 
Cells were grown in high density micromass and monolayer culture systems and then evaluated for expression of type II collagen, aggrecan, and Sox9. Umbilical cord blood from 130 patients was harvested. Expression of type II collagen, aggrecan, and Sox9 was detected after 14 days in TGF-ss3- and IGF-1-stimulated cells in both types of culture (monolayer and micromass). On Day 21 in the micro-mass culture, expression levels were greater than they were at 14 days for all genes. TGF-ss3 was found to be more efficient at promoting chondrogenesis than IGF-1. By western blot, we also found that after 3 weeks, the expression of type II collagen was greater in micromass culture with TGF-ss3. 
TGF-ss3 used in micromass culture is the best growth factor for promoting the proliferation and differentiation of mesenchymal cells from umbilical cord blood during chondrogenesis." 

So, if you want to grow taller don't mess around with IGF-1, get Transforming growth factor-ss3. 

Direct and progressive differentiation of human embryonic stem cells into the chondrogenic lineage. 

"Because of their unlimited capacity for self-renewal and ability to differentiate into multiple lineages, human embryonic stem cells (hESCs) are a potentially powerful tool for repair of cartilage defects. The primary objective of the present study was to develop culture systems and conditions that enable hESCs to directly and uniformly differentiate into the chondrogenic lineage without prior embryoid body (EB) formation, since the inherent cellular heterogeneity of EBs hinders obtaining homogeneous populations of chondrogenic cells that can be used for cartilage repair. To this end, we have subjected undifferentiated pluripotent hESCs to the high density micromass culture conditions we have extensively used to direct the differentiation of embryonic limb bud mesenchymal cells into chondrocytes. Micromass cultures of pluripotent hESCs undergo direct, rapid, progressive, and substantially uniform chondrogenic differentiation in the presence of BMP2 or a combination of BMP2 and TGF-beta1, signaling molecules that act in concert to regulate chondrogenesis in the developing limb. The gene expression profiles of hESC-derived cultures harvested at various times during the progression of their differentiation has enabled us to identify cultures comprising cells in different phases of the chondrogenic lineage ranging from cultures just entering the lineage to well differentiated chondrocytes. Thus, we are poised to compare the abilities of hESC-derived progenitors in different phases of the chondrogenic lineage for cartilage repair." 

"The high density micromass culture system simulates the close juxtaposition of cells and cellular interactions that characterize the onset of the chondrogenic differentiation of mesenchymal progenitor cells in the developing embryonic limb."

The study identifies Brachury as another gene associated with chondrogenesis.  "Brachyury may be involved in the determination process that channels progenitor cells into the chondrogenic lineage."

Sox9 is upregulated until day 7 when it starts to decrease.

LMP1 regulates periodontal ligament progenitor cell proliferation and differentiation. 

"LMP1 is an intracellular scaffold protein that contains a PDZ domain and three LIM domains. LMP1 has multiple functions including regulating mesenchymal stem cell (MSC) osteogenesis. Gene delivery of LMP1 induces bone formation in vivo in heterotopic and orthotopic sites. Periodontal ligament (PDL) cells are a unique progenitor cell population that can differentiate into chondrocytes. LMP1 is upregulated in early stage of PDL cell osteogenic differentiation. Stable gene knockdown of LMP1 by shRNA inhibits DNA synthesis and corresponding cell proliferation in PDL cells, and further leads to decreased mineralization in vitro. Overexpression of LMP1 increases cell proliferation, and PDZ and ww-interacting domains are not sufficient to mediate this effect. Further, we found that in PDL cells, LMP1 is a downstream target gene of TGF-beta1 that is an early signal critical in preosteoblast proliferation and differentiation. TGF-beta1 stimulates PDL cell proliferation, however, this effect is compromised when LMP1 is knocked down. activation of TAK1-JNK/p38 kinase cascade is involved in the LMP1 gene regulation by TGF-beta1." 

"overexpression of LMP1 in bone marrow stromal stem cells, calvarial osteoblasts, and dermal fibroblasts initiates osteolineage differentiation in vitro"

"TGF-β1 knock-out mice display a 30% decrease in tibial length and a reduction in bone mineral content"

"When TGF-βRI is activated, TAK1 phosphorylates JNK and p38, but not Erk1/2"

"The phosphorylation of JNK and p38 kinase regulates downstream target genes indirectly through activating AP-1 or ATF2 transcription and translation"

"there is an AP-1 binding site in the LMP1 promoter"

Stimulating LMP1 could also increase height...  

So we have five proteins and growth factors to study that could increase height: IGF-1, LMP1, TGF-Beta1, BMP-2, TGF-ss3.  We also know that any exercises or foods that enhance these proteins and growth factors can increase height as well.

ATP oscillations mediate inductive action of FGF and Shh signalling on prechondrogenic condensation.

"Morphogens such as fibroblast growth factor (FGF) and sonic hedgehog (Shh) specify the skeletal patterns in limb development. Synchronized Adenosine triphosphate (ATP) oscillations play a critical role in prechondrogenic condensation. FGF and Shh signalling promoted cellular condensation but not chondrogenic differentiation and also induced ATP oscillations. In addition, blockage of FGF and Shh signalling prevented both ATP oscillations and prechondrogenic condensation. Furthermore, it was found that inhibition of ATP oscillations suppressed FGF/Shh-induced prechondrogenic condensation. ATP oscillations mediate the actions of FGF and Shh signalling on prechondrogenic condensation."

"FGF2 reduced gene expression in the chondrogenic markers"<-thus after condensation do we want FGF2 to be inhibited?

"FGF signalling serves to maintain the expression of Shh, and Shh also acts to maintain the expression of several FGFs by up-regulating Gremlin."

"prechondrogenic condensation is independent of chondrogenic differentiation"<-thus we may need different expression.

"Wnt/β-catenin signalling is required for prechondrogenic condensation but inhibits the progression of chondrocyte differentiation"<-Sox9 inhibits Beta-Catenin so this is easy to get around.

Hematopoietic stem cells give rise to osteo-chondrogenic cells.

"To test the ability of hematopoietic stem cells (HSCs) to give rise to osteo-chondrogenic cells, we used a single HSC transplantation paradigm in uninjured bone and in conjunction with a tibial fracture model. Mice were lethally irradiated and transplanted with a clonal population of cells derived from a single enhanced green fluorescent protein positive (eGFP(+)) HSC. Analysis of paraffin sections from these animals showed the presence of eGFP(+) osteocytes and hypertrophic chondrocytes. To determine the contribution of HSC-derived cells to fracture repair, non-stabilized tibial fracture was created. Paraffin sections were examined at 7days, 2weeks and 2months after fracture and eGFP(+) hypertrophic chondrocytes, osteoblasts and osteocytes were identified at the callus site. These cells stained positive for Runx-2 or osteocalcin and also stained for eGFP demonstrating their origin from the HSC."

"hypertrophic chondrocytes in the callus secrete alkaline phosphatase for mineral deposition and calcification of the matrix. Once this calcification occurs, hypertrophic chondrocytes undergo apoptosis to create cavities in the bone, which are occupied by osteoprogenitor cells"

"cells with the morphology of chondrocytes were also seen in the long bones harvested from clonally transplanted animals"

The mice were ten to fourteen weeks old.  The study makes no mention of chondrocytes that are non hypertrophic.  It could be lack of TGF-Beta to maintain a pre-hypertrophic differentiation state.

In vitro mesenchymal stem cell differentiation after mechanical stimulation.

"Mouse bone marrow-derived MSC were established in vitro by differential adherence to plastic culture plates and grown in low glucose medium with 10% foetal calf serum and growth factors. Cells grew out and were subcultured up to 20 times. Differentiation protocols were followed for several cell lineages. Clones with trilineage potential were seeded in type I collagen gels and incubated in a tensioning force bioreactor and real-time cell-derived forces were recorded. Gels were fixed and sectioned for light and electron microscopy.
Cell monolayers of parent and cloned mouse bone marrow-derived MSC differentiated into adipocytes, osteocytes and chondrocytes, but not into cardiomyocytes, myotubes or neuronal cells. When cast into type I collagen gels and placed in tensioning bioreactors, MSC differentiated into fibroblast-like cells typical of tissue stroma, and upregulated α-smooth muscle actin, but rarely upregulated desmin. Electron microscopy showed collagen and elastin fibre synthesis into the matrix.
MSC cell fate choice depends on minute, cell-derived forces."

"Briefly, the loading cycle, which commenced after 12 h of collagen gel contraction by the resident cell population, consisted of an increase in tension of 130 dynes over a 3 min period, followed by a 15 min resting phase. Unloading over a 3 min time period was followed by a further 15 min resting period. This process was repeated for 22 cycles."

"Force-stressed cells generally exhibited gap junctions, organelles and electron-dense granules that were not observed in cells of the undifferentiated population"

"there may be recruitment of MSCs by chemotaxins to a wound site. Cells then attach to connective tissue and are subjected to mechanical stimulation, either from oedema or mediators of inflammation, leading to differentiation into a fibroblastic phenotype"

" the mechanical stimulus-responsive gene Pip4k2b has been identified as being expressed in osteoblastic cell cultures"

Chondrogenesis of mesenchymal stem cells for cartilage tissue engineering.

"Aggrecan molecules are held in place by [a] network of collagen fibres. As their negative charge acts
to draw in water the tissue swells, this swelling pressure produces a tensile load in the cartilage fibres"

"[The] balance between swelling pressure caused by the influx of water and the tensile resistance generated in the collagen fibres allows cartilage to resist compressive loads"

"When cartilage experiences a load greater than the force generated by the swelling pressure, water is
forced out of the tissue. This increases the concentration of proteoglycans and their large negative charge, draws water back into the tissue. This movement of water out of the tissue under load, and its
return during unloading, allows cartilage to act like a shock absorber, dissipating loads applied to it"

Although microfracture tends to lead to fibrocartilage tissue there is evidence that the tissue can mature into more hyaline cartilage like tissue.

"One of the roles of MSCs in vivo is the production of bone marrow stroma. The stroma is made up of the components of bone marrow that facilitate the haematopoietic process and the activity of haematopoietic stem cells and their progeny. The stroma consists of an extracellular matrix which contains type I, III and IV collagen as wells as proteoglycans. The matrix contains adipocytes, osteocytes and other cell types such as endothelial cells, which act together to create an environment suitable for haematopoiesis to occur"

Proteoglycans such as Syndecan and Glypican surround MSCs and may affect their ability to be affected by ligand signaling.

"Within bone marrow there are only one to a hundred MSCs in every five thousand mononuclear cells, so selection is very important"<-Since mesenchymal condensation is an important step to initiate chondrogenesis this is a problem.

"The effects of loading are very specific to individual areas within the musculo-skeletal system; it is, therefore, probable that it is the role of locally acting factors such as cytokines with paracrine signalling roles that mediate the effects of mechanical loading"

"Loading [may stimulate] the endogenous production of TGF-ß in MSCs and chondrocytes, which then much like the exogenous TGF-ß in chondrogenic media, stimulates chondrogenic changes within cells"

"Loaded rabbit bone marrow MSCs show up-regulation of both parts of the dimeric TGF-ß receptor, TßR-I and TßR-II, as well as the up regulation of the c-Jun and c-Fos genes" As well upregulation of Sox9.

"The beneficial effects of loading on the expression of chondrogenic genes were negated it the cells were exposed to TGF-ß within the culture medium."

"IGF is found free in the synovial fluid (up to 50ng/ml) and in the cartilage extracellular matrix (10 ng/ml)."

"When administered to cells in vitro IGF appears to produce a positive feedback loop, causing an increase in the production of IGF mRNA by mature bovine chondrocytes. IGF-1 also produces an
increase in the production of IGF receptor (IGF1R) mRNA and, in doing so, up-regulating matrix

"IGF-1 signals via a tyrosine kinase receptor called IGF-1R. Once IGF binds to this receptor it initiates a signalling cascade by phosphorylating tyrosine residues on molecules in the MEK1/2, Erk1/2, PI3K-Akt and the MAPK pathways, leading to the expression of IGF target genes. Both IGF-1 and IGF-2 which are both found in the growth plate act through the IGF-1R"

Non-specific insulin stimulation of IGF-1R may have negative effects so exogenous levels of general insulin may not be beneficial.

"In the presence of IHH, the repressor Gli3 is down-regulated whilst the activator Gli1 is upregulated"

"PTHrP leads to an up-regulation of the chondrogenic markers type II collagen and SOX9, in a dose dependent fashion. There was also a decrease in the marker of hypertrophy type X collagen, and a PKC
related decrease in the expression of Runx2, another hypertrophic marker"

"If applied early in differentiation, PTHrP will inhibit chondrogenesis"

"[If] PTHrP is applied at an intermediate time point, PTHrP will enhance the proliferation of the cells and the expression of chondrogenic genes in chondrocytes and support the differentiation of MSCs into chondroblasts"

"The growth plate is a band of hyaline cartilage that sits in the metaphysis of long bones; it has a clearly
defined structure that allows the elongation of the epiphyseal aspect of the plate, and the calcification of
the diaphyseal aspect of the plate, resulting in elongation of the bone."

"IHH is produced by hypertrophic chondrocytes of including those cells entering hypertrophy in the prehypertrophic and hypertrophic zones of the growth plate, this signalling acts to increases the number of chondrocytes in the proliferating zone entering terminal differentiation. The expression of IHH by these cells also leads to the expression of PTHrP from the periarticular cells. The PTHrP, in turn, prevents the proliferating chondrocytes from entering terminal differentiation by up-regulating the expression of chondrogenic genes, and blocking the action of IHH signalling by binding to PTHR1 and up-regulating the expression of Gli3, forming a negative feedback loop"

"The expression of ALP by hypertrophic chondrocytes is key to the mineralisation of the hypertrophic zone, as the enzymatic activity of ALP clears the way for the process of calcification. ALP encourages
the formation of calcified tissue from the cartilaginous matrix by cleaving inorganic phosphates to form organic phosphates, and in doing so affects the balance of these two compounds in the developing bone"
"The balance of these two forms of phosphate determines whether or not hydroxyapatite crystals can form. By cleaving inorganic phosphate to organic phosphate ALP helps to provide conditions suitable to calcification"

"As chondrocytes progress through hypertrophy cytomorphologic changes occur and two distinct
populations begin to form, they are termed light and dark cells based on their differences when observed under a transmission electron microscope. These two groups of chondrocytes undergo cell death in different ways. Neither of the cells undergoes apoptosis; instead they have their own programs of cell death which vary from the classical form of apoptosis, referred to as ‘chondroptosis’. Both the dark and light cells demonstrate chromatin condensation as part of the cells death"

"unlike the chromatin condensation seen at the periphery of the nucleus during the initial stages of apoptosis the chromatin of chondroptotic cells forms islands all through the nucleus. Dark cells then begin to develop endoplasmic compartments and autophagic vacuoles that digest the cells contents, any remnants are then ejected into the cells lacunae. Light cells under-go a process in which the expansion of the rough endoplasmic leads to the formation of sacks around the organelles and other contents of the cell. Lysosomes then release digestive enzymes that lead to the digestion of the cells contents within these cytoplasmic bodies. The advantage of chondroptosis may be that autophagy removes the need for the inflammatory response required to remove the apoptotic bodies left behind by classical apoptosis"

"MSCs unlike chondrocytes undergo hypertrophy when they are induced into chondrogenesis using TGF-ß in vitro."<-this could be epigenetically caused.

Induction of mesenchymal stem cell chondrogenesis by polyacrylate substrates.

"polyacrylate substrates modelled on the functional group composition and distribution of the Arg-Gly-Asp (RGD) integrin-binding site induce MSCs to undergo chondrogenesis in the absence of exogenous TGF-β. Within a few days of culture on the biomimetic polyacrylates, both mouse and human MSCs, and a mesenchymal-like mouse-kidney-derived stem cell line, began to form multi-layered aggregates and started to express the chondrocyte-specific markers, Sox9, collagen II and aggrecan. Moreover, collagen II tended to be expressed in the centre of the aggregates, similarly to developing limb buds in vivo. Surface analysis of the substrates indicated that those with the highest surface amine content were most effective at promoting MSC chondrogenesis."

"the integrin-binding Arg-Gly-Asp (RGD) motif of fibronectin is critical for mesenchymal cell aggregation"

Here's the content of the substrates but I don't understand how they were made: "The following polyacrylates were fabricated using the Biomer Technology Ltd (BTL) proprietary polymerization technique: BTL15, ESP03, ESP04, ESP07. Each batch was tested for consistency of chain length by gel permeation chromatography. Polyacrylates differed in the proportion and distribution of amine, carboxyl and hydroxyl functional groups and the degree of steric hindrance present in the polymer chain, modulated by a ratio of ethyl to butyl side groups. All polyacrylate materials were supplied by BTL. Each of the polyacrylate-coated discs (Borosilicate Glass Co. UK) were prepared from the same w/w concentration of polymer in solvent using the same dip coating programme, at a thickness of ∼2 μm. The mechanical properties were not measured, but as the polyacrylates are all rigid glassy materials, their mechanical properties are expected to be similar. Substrates were sterilized prior to cell culture with ultraviolet light (265 nm)."

"All substrates tested were significantly more hydrophobic than glass controls, with the higher amine content polyacrylates (ESP03, ESP04 and ESP07) having higher contact angles than BTL15. The trend from most hydrophilic to most hydrophobic was BTL15 > ESP04 > ESP03 > ESP07. Little difference in elemental composition was demonstrated."

" amine content was lowest in BTL15 (0.6%) followed by ESP03 (1.4%) and highest in ESP04 (1.6%), and oxygen content was highest in BTL15 (21.1%) followed by ESP03 and ESP04 (20%). Interestingly, in ESP03 and ESP04, the proportion of oxygen was ∼ 3% higher at the surface (15°) than in the bulk (45°) and nitrogen was 0.1% lower at the surface than in the bulk, whereas no difference was observed in BTL15."

"Following an 18 day culture period, large aggregates were visible across ESP04 substrates, which typically ranged from 300 μm to 600 μm in diameter. Both collagen II and osteocalcin were observed within the aggregates on ESP04, but were barely detectable within aggregates on ESP03."

"collagen II was mainly present at the centre of the aggregates, whereas osteocalcin was predominantly localized at the periphery"<-consistent with endochondral ossification.  So it seems ESP04 was the most chondrogenic.

"Col2a1 (collagen II-encoding gene) and Acan (aggrecan-encoding gene) increased 2.3- and 2-fold, respectively, in mMSCs cultured on ESP04 in comparison to negative controls"  After 2 days Sox9 and Cdh2 increased in ESP04.

"chondrogenic induction of mMSCs on ESP04 occurs prior to aggregation."

ESP07 was also highlighted as being chondroinductive.

"there did appear to be a positive correlation between chondroinductive ability and surface amine content"

"aggregation on amine surfaces [may be due] to reduced cell adhesion or altered protein adhesion"

"MSC interaction with the RGD peptides mimics the interaction that occurs between mesenchymal cells and the RGD motif of fibronectin in the developing limb bud"

Cyclic strain dominates over microtopography in regulating cytoskeletal and focal adhesion remodeling of human mesenchymal stem cells.

"Human bone marrow-derived mesenchymal stem cell (hMSCs) function depends on chemical factors and also on the physical cues of the microenvironmental niche. Here, this physical microenvironment is recapitulated with controlled modes of mechanical strain applied to substrata containing three-dimensional features in order to analyze the effects on cell morphology, focal adhesion distribution, and gene expression. Ten percentage of strain at 1Hz is delivered for 48h to hMSCs cultured on flat surfaces, or on substratate with 15μm-high microtopographic posts spaced 75μm apart. Adding strain to microtopography produced stable semicircular focal adhesions, and actin spanning from post to post. Strain dominated over microtopography for expression of genes for the cytoskeleton (caldesmon-1 and calponin 3), cell adhesion (integrin-α2, vinculin, and paxillin), and extracellular matrix remodeling (MMP13){MMP13 is endochondral ossification related gene}."

"Actin distribution was changed with respect to both strain and microtopography. Cells on flat surfaces contained thin layers of actin; however, by straining the cells (FS), intensely-stained bundles of actin stress fibers were observed, which elongated the cell shape. The cells in unstrained 3D microtopography had a high intensity of the actin wrapped closely around the post. Straining hMSCs on the microtopographic substrata resulted in actin stress fibers that span the 75 μm from post to post. The actin distribution was similar with or without strain nearer to the post (15 and 22.5 μm), but significant differences between the post and post-strain groups were found further away (30 and 37.5 μm). Adding strain to microtopography shifted the nuclei midway between the adjacent posts in the cell center "

"On flat, unstrained surfaces, paxillin is sparse; while with strain the paxillin is aligned with the long axis of the cell"

"when hMSCs are strained, the paxillin adopts a semicircular appearance around the post"

Microarray data to be analyzed as there is an error in the study right now.

1 comment:

  1. finding the food that contains these protein effectively is hard? on the other hand, the body absorbing utilising is another larger hurdle. This is not easy, at least for me.

    aniway, tyler. jus wondering about the lsjl. If i conduct loading on both the ankles and knees simultaneously while pushing them apart with both forces directions slightly slanted away, perhaps it can achieve the same bone stretching effect as sky shinbone routine, while stimulating the MSCs as well. What do you tink? tyler.