Effects of Osmolarity on Osteoarthritic Human Articular Cartilage

Effects of Osmolarity on Osteoarthritic Human Articular Cartilage

Karthik Raman, H.

Holger, J. (mentor)
Zadpoor, A.A. (mentor)
Weinans, H.H. (mentor)

Mechanical, Maritime and Materials Engineering

BioMechanical Engineering

Master Biomedical Engineering/Specialization - Tissue Biomechanics and Implants

2016-01-14

Chondrocyte is the only cell type which constitutes the articular cartilage, is solely responsible for the turnover of extracellular matrix composed of proteoglycans, type II collagen and water. Chondrocytes experience a unique ionic/osmotic environment, which is attributable to the polyanionic proteoglycan, which carry a high fixed negative charge and attracts cations, (mainly sodium) into the interstitial fluid. Extracellular tonicity in healthy cartilage ranges between 350 – 450 mOsm. The osmotic environment of chondrocytes is dynamic and osmotic perturbations occur in response to loading regimen and in pathophysiology. Articular cartilage defects heal poorly and lead to catastrophic degenerative arthritis. Clinical experience has indicated that no existing medication substantially promotes the healing process and the cartilage defect requires surgical replacement, preferably with an autograft. Some models of articular cartilage regeneration have yielded good repair of cartilage defects, in animal models and clinical settings, but the overall results suggest that there is room for improvement of this technique before its routine clinical application. In this body of work, we hypothesize that the hyper-osmotic stimulus of 380 mOsm is chondro-protective, phenotype preserving and up regulates the gene expression of the critical genes necessary for the maintenance of homeostasis. To test this hypothesis we study the effect of hypo (280 mOsm) and hyper (380 mOsm) osmotic stimulus on chondrogenic markers such as COL2 and AGC1, TGF-? receptors such as ALK1 and ALK5 and members of BMP namely; BMP2 and BMP4. Additionally, we would study the effects of osmolarity on genes synthesizing the GAG molecules. Osteo-arthritic human articular cartilage is obtained from three patients undergoing TKR. The tissue is digested with collagenase to liberate to the cells. The de-differentiation experiment is performed, in which the cells are allowed to proliferate and parallelly, they are cultured in 6 well plates for seven days. The cells used would be from P0 up to P2. RNA isolation and purification, c-DNA synthesis and PCR is performed at each passage with both the osmotic conditions. The final results are tested for significance with SPSS. Chondrocytes cultured in the hyper osmotic environment, proved beneficial for the chondrocytes. The phenotype was preserved to a certain extent and also the up regulation of the aforementioned genes was observed. For the first time, sensitivity of genes responsible for synthesis of GAG molecules was established and was observed, an upregulation in those specific genes. In a nutshell, chondrocytes showed good adaptation to the hyper-osmotic stimulus. It proved beneficial because the 380 mOsm is chondron-protective as it enhances the gene expression of the critical genes and also the preservation of the phenotype is noted. This study opens up a new plethora for research with respect to osmolarity in combination with genes responsible for GAG synthesis. Targeting various critical genes, by varying osmolarity within physiological limits, would aid in engineering phenotypically stable in vitro cartilage constructs.

osmolarity
osteoarthritis
cartilage
glycosaminoglycans
transforming growth factor - beta

http://resolver.tudelft.nl/uuid:03fb6490-33ac-4254-a80f-b5d99295ce0b

2016-06-01

Student theses

master thesis

(c) 2016 Karthik Raman, H.