Fibroblast Matrix Enhanced Three-Dimensional-Bioprinted Hydrogel for Osteochondral Regeneration

Decellularized extracellular matrix (dECM) plays an important role in tissue engineering by preserving native biochemical and structural cues while removing immunogenic cellular components. Addressing donor shortages, this study develops a standardized, reproducible protocol for producing cell-derived dECM for bone and cartilage applications, focusing on effective deoxyribonucleic acid (DNA) removal to prevent immune responses in 3D-bioprinted hydrogels. We also evaluate dECM's impact on cell viability and differentiation potential. Human dermal fibroblasts were decellularized using nonidet P-40, a nonionic detergent (nonyl phenoxypolyethoxylethanol) (NP-40) lysis buffers (1% or 10%) for 1 or 3 h. Decellularization efficacy was assessed via double-stranded DNA (dsDNA) Qubit assay, gel electrophoresis, immunofluorescence, and bicinchoninic acid protein assay. Hydrogels (5 wt% alginate, 3 wt% gelatin) with/without 1% dECM were extrusion bioprinted. Structural and mechanical properties were analyzed using Raman spectroscopy and rheology. Fibroblast viability within bioprinted constructs was monitored over 21 days. Hoechst staining and Qubit assay confirmed residual DNA after 1-h incubations, but complete removal (<50 ng dsDNA) occurred after 3 h with both NP-40 concentrations. The 10% NP-40/3-h protocol yielded the highest protein content. dECM incorporation did not compromise scaffold properties. Significantly enhanced cell viability and glycosaminoglycan (GAG) content (up to day 6) were observed in dECM hydrogels versus controls. Mechanical testing showed a 33% increase in Young's modulus in dECM-containing hydrogels. Raman spectroscopy confirmed successful dECM integration via a characteristic GAG peak (895 cm^-1). We established an optimized decellularization protocol (10% NP-40, 3 h) that effectively eliminates cellular/nuclear material (DNA <50 ng, RNA undetectable) below immunogenic thresholds while preserving essential extracellular matrix components. Fibroblast-derived dECM significantly enhanced alginate-gelatin hydrogel performance, improving cell viability, GAG synthesis, and early osteogenic markers without compromising structural integrity. This protocol provides a robust and standardized source of bioactive dECM, offering a viable alternative to tissue-derived matrices for advanced bone and cartilage tissue engineering bioinks. While the method demonstrates potential for scale-up, further validation following internationally recognized International Organization for Standardization (ISO) standards would be necessary before production-level implementation.