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VALIDATION OF TISSUE HYDROLYSIS METHODS FOR QUANTIFICATION OF COLLAGEN IN BOVINE PERICARDIUM

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Karthik S, Suneel Rallapalli, Satish Galla, Soma Guhathakurta

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ABSTRACT:Collagen is the most abundant protein in the extracellular matrix of animals and is mainly composed of amino acids glycine, proline and hydroxyproline.Collagen has been used in haemostatic sponges, dermal equivalents, injectables, and as a vehicle for drug delivery. The quantity and properties of the collagen present in biomaterials must be thoroughly evaluated as the collagen may influence the properties of biomaterials.  Hydroxyproline (Hyp) plays key role in collagen stability by permitting the sharp twisting of collagen helix and quantification of Hyp is an indicator of collagen content. The main objective of this study is to validate different tissue hydrolysis methods in order achieve simple, fast and reproducible method for quantification of collagen in biomaterials. Bovine pericardium used in cardiovascular surgery was used as a source material for collagen in the present study. We estimated amounts of collagen present in the bovine pericardium by hydrolysing with collagenase type 1, trypsin, sodium hydroxide (NaOH), hydrochloric acid (HCl) and sulphuric acid (H2SO4) singularly. We have observed 13.5 mg of collagen/100 mg of in pericardial tissue hydrolysed by collagenase type 1 treatment and similarly 4.45 mg by trypsin, 16.9 mg by NaOH, 7.55 mg by HCl and 29.45 mg of collagen / 100 mg tissue by H2SO4 was quantified. Simultaneously we have tested the robustness and reproducibility of H2SO4 hydrolysis method. Our study shows tissue hydrolysis by H2SO4 is more efficient, simple and fast for estimation of collagen in biomaterials.

 

KEY WORDS:Collagen, Hydroxyproline, Bovine pericardium, Sulphuric acid, Hydrolysis and Biomaterials

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