Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro.

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and craniomaxillofacial implants. To screen biodegradable materials for biomedical applications, it is necessary to evaluate these materials in terms of in vitro cell responses, cytocompatibility, and cytotoxicity. International Organization for Standardization (ISO) standards have been widely utilized in the evaluation of biomaterials. However, most ISO standards were originally established to assess the cytotoxicity of nondegradable materials, thus providing limited value for screening biodegradable materials. This article introduces and discusses three different culture methods, namely, direct culture method, direct exposure culture method, and exposure culture method for evaluating the in vitro cytocompatibility of biodegradable implant materials, including biodegradable polymers, ceramics, metals, and their composites, with different cell types. Research has shown that culture methods influence cell responses to biodegradable materials because their dynamic degradation induces spatiotemporal differences at the interface and in the local environment. Specifically, the direct culture method reveals the responses of cells seeded directly on the implants; the direct exposure culture method elucidates the responses of established host cells coming in contact with the implants; and the exposure culture method evaluates the established host cells that are not in direct contact with the implants but are influenced by the changes in the local environment due to implant degradation. This article provides examples of these three culture methods for studying the in vitro cytocompatibility of biodegradable implant materials and their interactions with bone marrow-derived mesenchymal stem cells (BMSCs). It also describes how to harvest, passage, culture, seed, fix, stain, characterize the cells, and analyze postculture media and materials. The in vitro methods described in this article mimic different scenarios of the in vivo environment, broadening the applicability and relevance of in vitro cytocompatibility testing of different biomaterials for various biomedical applications.

Tunable Crosslinking, Reversible Phase Transition, and 3D Printing of Hyaluronic Acid Hydrogels via Dynamic Coordination of Innate Carboxyl Groups and Metallic Ions.

This article reports tunable crosslinking, reversible phase transition, and three-dimensional printing (3DP) of hyaluronic acid (HyA) hydrogels via dynamic coordination of Fe3+ ions with their innate carboxyl groups for the first time. The concentrations of Fe3+ and H+ ions and the reaction time determine the tunable ratios of mono-, bi-, and tridentate coordination, leading to the low-to-high crosslinking densities and reversible solid-liquid phase transition of HyA hydrogels. At the monodentate-dominant coordination, the liquid hydrogels have low crosslinking densities (HyA_L). At the mixed coordination of mono-, bi-, and tridentate bonding, the solid hydrogels have medium crosslinking densities (HyA_M). At the tridentate-dominant coordination, the solid hydrogels have high crosslinking densities (HyA_H). The reversible solid-liquid phase transitions among HyA_L, HyA_M, and HyA_H were achieved via controlling the concentrations of Fe3+ and H+ ions and reaction time. When the crosslinking densities are between HyA_L and HyA_M, the hydrogels become 3D printable (HyA_P). HyA_P hydrogels were 3D-printed successfully using cold-stage or direct writing methods, and the 3D constructs achieved better structural stability using the latter method. In the direct exposure culture with bone marrow-derived mesenchymal stem cells, the 3D-printed HyA_H (HyA_H_3D) and HyA_H hydrogels showed higher average cell adhesion densities than the HyA_M, HyA_P, and HyA_L hydrogel groups under both direct and indirect contact conditions. For all hydrogel groups, cell adhesion densities under direct contact conditions were statistically lower than the same groups under indirect contact conditions. In this article, we elucidated the mechanisms of dynamic coordination and the relationships among the key parameters in controlling the tunable crosslinking, reversible phase transition, and 3DP of HyA hydrogels without blending with other polymers or adding functional groups. This approach can be potentially adapted to crosslink and 3D print other polymeric hydrogels with carboxyl groups, which is promising for a wide range of applications.