Genevieve E. Romanowicz

Bone homeostasis depends on spatially orchestrated interactions among osteoclasts, osteoblasts, and osteocytes that are embedded within a unique extracellular matrix that is mineralized on the nanoscale. Reconstructing these interactions to enable autonomous cell differentiation and tissue remodeling has remained a significant challenge towards mimicking adequate bone physiology and disease in-vitro. Here, we present an engineered model that spatially defines the paracrine communication of heterogeneous cell populations within bone tissue that supports the rapid maturation of primary osteoblasts into osteocytes, the differentiation of immune cells into osteoclasts, and calcified tissue resorption within a mineralized cell-laden bone-like tissue. We demonstrate that nanoscale mineralization of cell-laden collagen hydrogels on-a-chip enhances osteoblast to osteocyte differentiation, whereas osteocytes in the matrix accelerate osteoclastogenesis and remodeling in a spatially defined manner without the need for exogenous growth factors. Osteocyte-dependent osteoclastogenesis on-a-chip outperformed conventional stimulation with RANKL and M-CSF, reproduced the clinical response of anti-resorptive drugs, and mimicked established tumor-bone interactions observed in invasive oral cancer. By replicating essential aspects of bone composition and function, this system provides a robust, self-regulated microphysiologic model to investigate bone remodeling, cancer-bone crosstalk, and therapeutic interventions.

Up to 40% of elite athletes experience bone stress injuries (BSIs), with 20-30% facing reinjury. Early identification of runners at high risk of subsequent BSI could improve prevention strategies. However, the complex etiology and multifactorial risk factors of BSIs makes identifying predictive risk factors challenging. In a study of 30 female recreational athletes with tibial BSIs, 10 experienced additional BSIs over a 1-year period, prompting investigation of systemic biomarkers of subsequent BSIs using aptamer-based proteomic technology. We hypothesized that early proteomic signatures could discriminate runners who experienced subsequent BSIs. 1,500 proteins related to metabolic, immune, and bone healing pathways were examined. Using supervised machine learning and genetic programming methods, we analyzed serum protein signatures over the 1-year monitoring period. Models were also created with clinical metrics, including standard-of-care blood analysis, bone density measures, and health histories. Protein signatures collected within three weeks of BSI diagnosis achieved the greatest separation by sparse partial least squares discriminant analysis (sPLS-DA), clustering single and recurrent BSI individuals with a mean accuracy of 96 ± 0.02%. Genetic programming models independently verified the presence of candidate biomarkers, including fumarylacetoacetase, osteopontin, and trypsin-2, which significantly outperformed clinical metrics. Time-course differential expression analysis highlighted 112 differentially expressed proteins in individuals with additional BSIs. Gene set enrichment analysis mapped these proteins to pathways indicating increased fibrin clot formation and decreased immune signaling in recurrent BSI individuals. These findings provide new insights into biomarkers and dysregulated protein pathways associated with recurrent BSI and may lead to new preventative or therapeutic intervention strategies. One Sentence Summary Our study identified candidate serum biomarkers to predict subsequent bone stress injuries in female runners, offering new insights for clinical monitoring and interventions.

Bone autografts remain the gold standard for bone grafting surgeries despite having increased donor site morbidity and limited availability. Bone morphogenetic protein‐loaded grafts represent another successful commercial alternative. However, the therapeutic use of recombinant growth factors has been associated with significant adverse clinical outcomes. This highlights the need to develop biomaterials that closely approximate the structure and composition of bone autografts, which are inherently osteoinductive and biologically active with embedded living cells, without the need for added supplements. Here, injectable growth factor‐free bone‐like tissue constructs are developed, that closely approximate the cellular, structural, and chemical composition of bone autografts. It is demonstrated that these micro‐constructs are inherently osteogenic, and demonstrate the ability to stimulate mineralized tissue formation and regenerate bone in critical‐sized defects in‐vivo. Furthermore, the mechanisms that allow human mesenchymal stem cells (hMSCs) to be highly osteogenic in these constructs, despite the lack of osteoinductive supplements, are assessed, whereby Yes activated protein (YAP) nuclear localization and adenosine signaling appear to regulate osteogenic cell differentiation. The findings represent a step toward a new class of minimally invasive, injectable, and inherently osteoinductive scaffolds, which are regenerative by virtue of their ability to mimic the tissue cellular and extracellular microenvironment, thus showing promise for clinical applications in regenerative engineering.

An S-nitroso-N-acetylpenicillamine (SNAP) derivatization approach was used to modify existing free primary amines found in fibrin (a natural protein-based biomaterial) to generate a controlled nitric oxide (NO) releasing scaffold material. The duration of the derivatization reaction affects the NO release kinetics, the induction of controlled NO-release, hydrophobicity, swelling behavior, elastic moduli, rheometric character, and degradation behavior. These properties were quantified to determine changes in fibrin hydrogels following covalent attachment of SNAP. NO-releasing materials exhibited minimal cytotoxicity when cultured with fibroblasts or osteoblasts. Cells maintained viability and proliferative character on derivatized materials as demonstrated by Live/Dead cell staining and counting. In addition, SNAP-derivatized hydrogels exhibited an antimicrobial character indicative of NO-releasing materials. SNAP derivatization of natural polymeric biomaterials containing free primary amines offers a means to generate inducible NO-releasing biomaterials for use as an antimicrobial and regenerative support for tissue engineering.