Threshold Optimization of In Situ <scp>HAPnanoclay</scp> in Polymeric Scaffolds to Enhance Biomechanical Response

Developing bone-mimetic tissue engineering scaffolds with tunable mechanical and biological properties is vital to overcoming obstacles in bone repair and creating realistic 3D bone models. This study utilizes montmorillonite clay (MMT) modified with amino valeric acid, featuring in situ HAP mineralization within the clay galleries, henceforth referred to as in situ HAPnanoclay. Three-dimensional scaffolds were fabricated from polymer clay nanocomposites (PCNs), where varying the amount of in situ HAPnanoclay influenced both their mechanical and biological performance. SEM and EDS analyses confirmed that the in situ HAPnanoclay was uniformly dispersed within the PCL matrix. Despite being present in small amounts, the in situ HAPnanoclay significantly enhanced the scaffolds' mechanical behavior. Incorporating as little as 1% in situ HAPnanoclay established the threshold for noticeable improvements in mechanical properties compared to pure PCL scaffolds. Cell viability studies demonstrated the scaffolds' biocompatibility, showing significantly increased cell viability when the HAPnanoclay content exceeded 3%. Additionally, the scaffolds supported osteogenic differentiation of human mesenchymal stem cells (hMSCs), with ECM mineralization improving across all HAPnanoclay loadings. Moreover, scaffolds with 5% or more in situ HAPnanoclay exhibited a substantial increase in mineralization after 23 days, identifying 5% loading as a critical threshold for enhanced biomineralization. 3D PCL/in situ HAPnanoclay scaffolds demonstrated tunable mechanical and biological properties through varying clay contents. This study is the first to report the threshold percentages of in situ HAPnanoclay modified with amino valeric acid necessary to significantly improve mechanical strength and biological performance in PCN-based scaffolds for bone regeneration.