Decellularised Sheep Tendon Derived Extracellular Matrix: An In-vitro Biochemical and Histological Analysis

Abstract

Introduction: Demineralised ovine cartilage is derived from sheep cartilage with its mineral content removed, preserving the collagen-rich organic matrix. It provides an osteoconductive scaffold that supports cell attachment, proliferation, and differentiation. Its biocompatibility and structural similarity to human bone Extracellular Matrix (ECM) make it suitable for periodontal, promotes new bone formation and periodontal ligament attachment. Aim: To fabricate decellularised sheep tendon derived ECM and to analyse Deoxyribonucleic Acid (DNA) and Glycosaminoglycan (GAG) content using histological Haematoxylin & Eosin (H&E), Alizarin Red and Alcian Blue staining and DNA quantification. Materials and Methods: This in-vitro study was done in July 2024 at Saveetha Dental College and Hospitals in Chennai,Tamil Nadu, India. Fresh ovine/sheep tendon samples were obtained, cleaned of extraneous tissues, and stored at -20°C. They were then cut into small pieces (1.5×1.5 mm) using a 15-number size BP blade scalpel. The tendon fragments were submerged in a decellularisation fluid consisting of 10% Phosphate-Buffered Saline (PBS), Sodium Dodecyl Sulfate (SDS), and Triton-X. The mixture was shaken at 37°C until foam formed, with the froth being removed and the solution replenished every six hours for three days. After decellularisation, the tendon pieces were thoroughly washed with distilled water, freeze-dried, and the resulting ECM was refrigerated. Morphology of one sample was evaluated using a scanning electron microscope. Histological analysis was performed by haematoxylin & eosin, Alizarin Red and Alcian Blue staining to detect GAG content. DNA quantification was done to confirm the remaining DNA percentage. Results: Scanning Electron Microscopy (SEM) image revealed that the ECM exhibited a fibrous shape with a high density of linked fibers. Haematoxylin and eosin staining revealed 90% decellularisation of the sheep tendon. Alizarin red staining showed that almost no mineral content remained after decellularisation. Alcian blue staining is used to detect the presence of GAG and shows that almost 90% of the GAG has been removed from the ovine sheep/tendon by decellularisation. This result is further substantiated by the DNA quantification which shows only around 10-15% DNA remaining in the decellularised tissue. The GAG quantification also gives similar observations which prove the almost complete removal of cells from the sheep tendon (100% naive tendon and 15% decellularised tendon). Conclusion: Decellularised ovine/sheep tendon may prove as a useful material for tissue regeneration as it helps in collagen synthesis, combining effective decellularisation with the preservation of key ECM components. Further studies are needed to evaluate its potential use as a graft material and for various clinical applications.

Keywords

• bone regeneration
• periodontal attachment
• tissue engineering
• tissue regeneration