Decellularized matrix-hyaluronic acid-alginate hybrid hydrogels to enable a multi-layered full-thickness oral mucosa-on-a-chip

Abstract

Objective: Oral mucosa-on-a-chip (OMoC) devices mimic oral epithelium but require continuous nutrient support from underlying lamina propria. However, the common Matrigel or Basement membrane extract (BME) may not effectively support the oral mucosa (OM) native architecture. This study investigated whether a hybrid hydrogel composed of OM decellularized extracellular matrix (OM dECM), hyaluronic acid (HA), and alginate (Alg) surpasses Matrigel and BME for OMoC applications. Methods: Porcine OM tissues were decellularized using 0.1-1 % sodium dodecyl sulfate (SDS), then freeze-dried, and pepsin-digested to produce OM dECM hydrogels (5, 10, and 20 mg/mL). DNA quantification, histological, biochemical and rheological assays, electron microscopy, and mass spectrometry were performed for dECM/ hydrogels. Normal oral keratinocytes (NOK), gingival fibroblasts (HGF), and human umbilical vein endothelial cells (HUVEC) cultured in hydrogels were evaluated with proliferation, cytotoxicity, and immunocytochemistry (ICC) assays, and compared to Matrigel/BME. Hybrid hydrogels were used to tri-culture NOK, HGF, and HUVEC in a polydimethylsiloxane (PDMS)-based microfluidic chip for OM modeling. Results: The 1 % SDS decellularization preserved greater ECM components. OM dECM hydrogels enhanced NOK, HGF, and HUVEC viability/proliferation, outperforming Matrigel/BME. Expression of OM-related markers (K14, Ki67, p63, Involucrin, E-cadherin, Von Willebrand factor) increased with hydrogels. Hybrid hydrogel of HA/Alg and 10 mg/mL dECM better supported NOK stratification while maintaining the native phenotype of HGF and HUVEC compared to Matrigel. Within our PDMS-based OMoC, the hybrid hydrogel facilitated the formation of a multi-layered microtissue construct with a stratified epithelial layer, a stromal compartment, and an endothelial network, better replicating the structural features of native OM as compared to other matrices and commercial microfluidic devices. Conclusions: A 1 % SDS decellularization protocol effectively preserved OM ECM, enabling development of an injectable OM dECM hydrogel. The optimized dECM-HA/Alg hybrid hydrogel outperformed Matrigel in supporting oral microtissue stratification, and phenotype maintenance, making it a superior alternative for OMoC applications. Clinical Significance: This study developed a pre-clinical hybrid hydrogel-based OMoC that can more accurately replicate the oral mucosa ECM and tissue architecture when compared to Matrigel/BME. By enhancing the ECM mimicry, the dECM-HA/Alg hybrid hydrogel supported the development of an optimized OMoC for cytotoxicity screening of oral drugs and potential disease modeling of oral mucosal conditions.