Magoosh GRE

The Differentiation between cartilage and Bone

| March 14, 2015

Introduction
Treatment of cartilage damage presents one of the significant problems in orthopedics. Cartilage damage, though not life threatening, affects the quality of life significantly. In most cases patients suffering from degenerative disorders such as osteoarthritis are forced to undergo expensive joint replacement surgeries. With advancements in biomedical science there is an increasing focus on the possibilities of nonsurgical and regenerative therapies for cartilage damage. It is well known that both bone as well as cartilage tissues are derived from the same Mesenchymal stem cells (MSC). Therefore understanding the biological mechanisms and the various signaling pathways that underlie chondrogenic differentiation of MSCs is vital to developing effective cartilage regenerative therapy.
AIM
This research aims to identify gene markers that are involved in both cartilage as well as bone differentiation. The research hopes to highlight the signaling pathways that are involved with genes responsible for osteogenesis or chondrogenesis. Inferences from the various studies that are reviewed during the course of this research would also help to identify potential new markers for cartilage formation and bone formation. Also by including a brief explanation of the various stages of chondrocytic differentiation and the associated markers this research aims to understand the signaling pathways and the end products of the various markers involved in each stage.

Method
To identify the various important genes and their markers the array expression database of ‘European Bioinformatics’ was used. The gene expression atlas was searched for bone and cartilage genes. Of the hundreds of markers that were listed in the array expression database only a selected few, based on their high expression values were included for further research. From this initial search in the database and the screening process, multiple markers were identified for each stage of the chondrocyte differentiation.

The following table gives a list of genes that were up regulated in chondrocyte formation. These genes were chosen from the initial search in the array express database using the keyword ‘Cartilage formation’ and the ‘Up In’ option. From the hundreds of gene markers only the first 50 with high P values were chosen. The following tables show 20 of these genes that were selected.!@_001!!!!!!!!!!!!#@

Signaling Pathways
ACAN -The over expression of aggrecan promotes Chondrogenesis. The expression of this gene is mediated by the transforming growth factor-β (TGF-β). Aggrecan also interacts with Hyaluronan (HA) to form supramolecular ternary complex.
ANXA6- Cholesterol is essential for the autocatalytic process and it affects chondrogenesis.

COL5A2 – This gene expression results in the secretion of pro-alpha2(V) chain which constitutes the collagen fibers of the cartilage.
CD44- This gene impacts the cell matrix interactions by affecting the cellular reaction to inductive signals. High CD44 expression is critical for the proteoglycan binding of the cellular matrix.
ITM2A – The expression of this gene has been linked with suppressing collagen type X and consequently the formation of hypertrophic cells. (endochondral ossification)
Markers across the Stages of Chondrogenesis
Several markers are known to be specific for the various stages of chondrogenesis. For instance, the Sox9, COL2A1 (IIa) , Ncad, tnc , Ncam1 are some of the markers that are highly expressed in proliferative mesenchymal chondrocyte cells. A low expression of Ptc1, Fgfr3 and the NKX3-2 was indentified during this stage. (Zuscik et.al ) Sox9 belongs to the family of transcription factors. This protein is usually used as a marker for cell differentiation along the stages of development. The Col2a1 belongs to the fibrillar Collagen family and the gene controls the production of apha 1 chain of type II collagen. The Ncam1, a cell adhesion molecule, belongs to the immunoglobulin (Ig) superfamily. In the highly proliferative flat columnar chondrocyte cells there is an observed over expression of NKX3-2 , Ptc1, Fgfr3 while there is low expression of Vegf, Runx2 and Osx. Sox9 and Col2a1(IIb) and Agc1 were also found to be common markers at this stage of mesenchymal cell condensation. (Zuscik , 2008) The aggrecan gene (Agc1) belongs to the proteoglycan family and the encoded protein constitutes an important part of the extra cellular matrix. The Fgfr3 gene belongs to the fibroblast growth receptor family and is identified to play a vital; role in chondrogenesis. Mutations of this gene is an identified cause for skeletal dysplasia. The prehypertrophic chondrocyte stage is marked by increased expression of Ihh and Fgfr3. Studies have also revealed expression of Col2a1(IIb), Agc1 and Col10a1. (Lefebvre & Smits, 2005) A relatively high expression of Runx2, Vegf and Col10a1 is observed in the hypertrophic chondrocytes. The vascular endothelial growth factor (Vgf) belongs to the family of platelet growth factors. Runx2 belongs to the family of transcription factors known as RUNX. These markers were chosen based on their high P values in the array expression database suggesting their important role in the chondrogenesis process. (Array Expression Database).@ani

The expression of different markers during different stages of chondrogenesis.
Zuscik et.al (2008)

Pubmed database was searched for the above markers and results were screened based on their relevance to chondrogenesis. This search pointed to some new areas of interest. Particularly, the role of BMP signaling pathways in the regulation of Sox9, among other markers, and its effect on chondrogenesis. Several transforming growth factors including Bone Morphogenetic Proteins (BMP) such as BMP 12, 13 and 14 are being studied and results suggest a positive correlation between the expression of these genes and chondrogenesis. Several micro RNA’s have been shown to impact protein synthesis and Chondrogenic differentiation. For instance, miR-145 is shown to have a negative impact on chondrogenesis. Mouse based experiments have also confirmed that the presence of growth factor beta 3 (TGF-β3) promoted chondrogenic differentiation. The role of miR-145 and many chondrogenic marker genes such as (Col2a1), (Agc1), cartilage oligomeric matrix protein (COMP) and (Sox9) are being studied intensely and results clearly suggest miR-145 as a controlling factor of chondrogenesis. (Yang B, 2011)
Recent research has also shown that polyhydroxyalkanoates (PHA) induce chondrogenesis and formation of chondrocytic extra cellular matrix (ECM). Also, miR-29a and miR-29b are found to directly affect chondrocyte differentiation. (Yan et.al, 2011) Research is also focusing on genetic manipulation of the MSCs to promote chondrogenetic differentiation. In particular, adenovirus mediated expression of the human fibroblast growth factor 2 (hFGF-2) and its effect on chondrogenesis are being studied. (Cucchiarini, et.al 2011) Also, transcription factors such as Arid5a and Sox9 and their influence on chondrocyte stimulation are recent areas of interest. (Amano et.al, 2011) This paper would review several research studies pertaining to the chondrogenesis and osteogenesis with an attempt to clearly understand the respective protein biomarkers. This would help in the development of regenerative therapy for the treatment of articular cartilage defects

References (to be expanded)
1) Amano K, Hata K, & Muramatsu S et.al (Apr 2011), Arid5a cooperates with Sox9 to stimulate chondrocyte specific transcription, Mol Biol Cell 15;22(8):1300-11
2) Yang B, Guo H, Zhang Y et.al (2011), MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9. PLoS One. ;6(7):e21679
3) Yan C, Wang Y, Shen XY (2011), MicroRNA regulation associated Chondrogenesis of Mouse MSCs grown on Ployhydroxyalkanoates. Biomaterials. 2011 Sep;32(27):6435-44
4) Cucchiarini M, Ekici M, Schetting S, Kohn D, Madry H.( Aug 2011) , Metabolic activities and chondrogenic differentiation of human Mesenchymal stem calls following recombinant adeno associated virus- mediated gene transfer and over expression of fibroblast growth factor 2. Tissue Eng Part A. 17(15-16):1921-33.
5) Michael J. Zuscik, Matthew J Hilton, Xinping Zhang et.al (2008), Regulation of Chondrogenesis and Chondrocyte differentiation by stress, J Clin Invest. Vol 118, Issue 2.
6) Ve´ ronique Lefebvre and Patrick Smits, (2005), Transcriptional control of Chondrocyte fate and Differentiation, Birth Defects Research (Part C) 75:200–212

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