Inorganic Biomaterials Shape the Transcriptome Profile to Induce Endochondral Differentiation.

Minerals play a vital role, working synergistically with enzymes and other cofactors to regulate physiological functions including tissue healing and regeneration. The bioactive characteristics of mineral-based nanomaterials can be harnessed to facilitate in situ tissue regeneration by attracting endogenous progenitor and stem cells and subsequently directing tissue-specific differentiation. Here, cellular responses of human mesenchymal stem/stromal cells to traditional bioactive mineral-based nanomaterials, such as hydroxyapatite, whitlockite, silicon-dioxide, and the emerging synthetic 2D nanosilicates are investigated. Transcriptome sequencing is utilized to probe the cellular response and determine the significantly affected signaling pathways due to exposure to these inorganic nanomaterials. Transcriptome profiles of stem cells treated with nanosilicates reveals a stabilized skeletal progenitor state suggestive of endochondral differentiation. This observation is bolstered by enhanced deposition of matrix mineralization in nanosilicate treated stem cells compared to control or other treatments. Specifically, use of 2D nanosilicates directs osteogenic differentiation of stem cells via activation of bone morphogenetic proteins and hypoxia-inducible factor 1-alpha signaling pathway. This study provides  insight into impact of nanomaterials on cellular gene expression profile and predicts downstream effects of nanomaterial induction of endochondral differentiation.

Inorganic ions activate lineage-specific gene regulatory networks.

Inorganic biomaterials have been shown to direct cellular responses, including cell-cell and cell-matrix interactions. Notably, ions released from these inorganic biomaterials play a vital role in defining cell identity, and promoting tissue-specific functions. However, the effect of inorganic ions on cellular functions have yet to be investigated at the transcriptomic level, representing a critical knowledge gap in the development of next-generation bioactive materials. To address this gap, we investigated the impact of various inorganic ions including silver, copper, titanium, and platinum on human mesenchymal stem cells (hMSCs). Our finding showed that silver and copper induce osteogenic and chondrogenic differentiation respectively, through enrichment of lineage-specific gene expression program. In particular, silver effectively induced Wingless/Integrated (Wnt) and mitogen-activated protein kinase (MAPK) signaling, which are vital for osteogenesis. On the other hand, copper specifically stimulated Transforming growth factor beta (TGFß) signaling, while suppressing Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling, thereby promoting chondrogenesis. In contrast, platinum, and tantalum, ions didn't stimulate regenerative responses. Together, our findings highlight the potential of inorganic biomaterials in tissue regeneration strategies, which currently rely largely on growth factors and small molecule therapeutics. STATEMENT OF SIGNIFICANCE: This research emphasizes the critical role of bioactive inorganic ions in controlling lineage-specific gene expression patterns in mesenchymal stem cells, effectively modulating the transcriptome landscape and directing cell fate. The study lays the foundation for a systematic database of biomaterial candidates and their effects on cellular functions, which will ultimately streamline the translation of new biomaterials into clinical applications.

Stiffness assisted cell-matrix remodeling trigger 3D mechanotransduction regulatory programs.

Engineered matrices provide a valuable platform to understand the impact of biophysical factors on cellular behavior such as migration, proliferation, differentiation, and tissue remodeling, through mechanotransduction. While recent studies have identified some mechanisms of 3D mechanotransduction, there is still a critical knowledge gap in comprehending the interplay between 3D confinement, ECM properties, and cellular behavior. Specifically, the role of matrix stiffness in directing cellular fate in 3D microenvironment, independent of viscoelasticity, microstructure, and ligand density remains poorly understood. To address this gap, we designed a nanoparticle crosslinker to reinforce collagen-based hydrogels without altering their chemical composition, microstructure, viscoelasticity, and density of cell-adhesion ligand and utilized it to understand cellular dynamics. This crosslinking mechanism utilizes nanoparticles as crosslink epicenter, resulting in 10-fold increase in mechanical stiffness, without other changes. Human mesenchymal stem cells (hMSCs) encapsulated in 3D responded to mechanical stiffness by displaying circular morphology on soft hydrogels (5 kPa) and elongated morphology on stiff hydrogels (30 kPa). Stiff hydrogels facilitated the production and remodeling of nascent extracellular matrix (ECM) and activated mechanotransduction cascade. These changes were driven through intracellular PI3AKT signaling, regulation of epigenetic modifiers and activation of YAP/TAZ signaling. Overall, our study introduces a unique biomaterials platform to understand cell-ECM mechanotransduction in 3D for regenerative medicine as well as disease modelling.

Dissociation of nanosilicates induces downstream endochondral differentiation gene expression program.

Bioactive materials harness the body's innate regenerative potential by directing endogenous progenitor cells to facilitate tissue repair. Dissolution products of inorganic biomaterials provide unique biomolecular signaling for tissue-specific differentiation. Inorganic ions (minerals) are vital to biological processes and play crucial roles in regulating gene expression patterns and directing cellular fate. However, mechanisms by which ionic dissolution products affect cellular differentiation are not well characterized. We demonstrate the role of the inorganic biomaterial synthetic two-dimensional nanosilicates and its ionic dissolution products on human mesenchymal stem cell differentiation. We use whole-transcriptome sequencing (RNA-sequencing) to characterize the contribution of nanosilicates and its ionic dissolution products on endochondral differentiation. Our study highlights the modulatory role of ions in stem cell transcriptome dynamics by regulating lineage-specific gene expression patterns. This work paves the way for leveraging biochemical characteristics of inorganic biomaterials to direct cellular processes and promote in situ tissue regeneration.