Decellularization of Rat Submandibular Gland for Salivary Gland Tissue-Engineering Applications.

ABSTRACT

OBJECTIVES: This study describes a robust and versatile method for decellularization of rat submandibular glands (SMGs). METHODS: Briefly, rat SMGs were harvested and subjected to perfusion cycles using an anionic detergent. Native and decellularized SMG tissues were subjected to histological analysis using hematoxylin and eosin (H&E) stain and immunohistochemical staining using Hoescht reagent. Further, complementary DNA was synthesized using the native and decellularized SMG tissues and subjected to quantitative reverse transcription polymerase chain reaction (RT-PCR) using rat-specific genes (i.e., α-amylase [Amyl], aquaporin 5 [Aqp5], mucin 19 [Muc19] and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). The total DNA within native and decellularized SMG tissues were also quantified. RESULTS: H&E staining of SMG tissues revealed preserved ECM content. Decellularized SMG scaffolds lacked cellular material but retained collagen bundles similar to native SMGs. Hoechst reagent immunostaining showed cell nuclei and DNA present in native SMG but not in decellularized SMG scaffolds. Quantitative RT-PCR analysis showed specific amplification products of salivary gland-specific genes (Amyl, Muc19 and Aqp5) and GAPDH in the native SMG tissues. However, no amplification product was observed in the cDNAs from the decellularized SMG scaffolds, confirming the absence of DNA. Quantification of the DNA content showed that the decellularized SMG scaffolds had significantly lower DNA content than native SMG tissue. CONCLUSIONS: Results from this study demonstrated that the decellularization protocol was effective in removing cellular material while preserving the extracellular matrix components and structural integrity of the native SMG tissue.