Cyclooxygenase-2 (COX-2) is a biomolecule known to be overexpressed in inflammation. Therefore, it has been considered a diagnostically useful marker in numerous studies. In this study, we attempted to assess the correlation between COX-2 expression and the severity of intervertebral disc (IVD) degeneration using a COX-2-targeting fluorescent molecular compound that had not been extensively studied. This compound, indomethacin-adopted benzothiazole-pyranocarbazole (IBPC1), was synthesized by introducing indomethacin—a compound with known selectivity for COX-2—into a phosphor with a benzothiazole-pyranocarbazole structure. IBPC1 exhibited relatively high fluorescence intensity in cells pretreated with lipopolysaccharide, which induces inflammation. Furthermore, we observed significantly higher fluorescence in tissues with artificially damaged discs (modeling IVD degeneration) compared to normal disc tissues. These findings indicate that IBPC1 can meaningfully contribute to the study of the mechanism of IVD degeneration in living cells and tissues and to the development of therapeutic agents.

Optimizing cell-matrix interactions for effective bone regeneration remains a significant hurdle in tissue engineering. This study presents a novel approach by developing a hMSC-embedded 3D aligned collagen for enhanced bone regeneration. A one-step mechanical strain was applied to a mixture of hMSCs and collagen, producing an hMSC-embedded, aligned 3D collagen hydrogel patch that mimics the natural bone matrix. Notably, the hMSCs embedded in the aligned collagen spontaneously differentiated into osteoblasts without external inducing reagents. Immunofluorescence analysis revealed that the BMP2-smad1/5 signaling pathway, critical for osteogenic differentiation, were activated by aligned collagen. In vivo experiments using a calvarial defect model confirmed that this approach effectively promotes new bone formation, starting centrally within the defect rather than from the edges adjacent to the existing bone. Our findings suggest that this simple method of pre-straining to create aligned 3D collagen embedded with hMSCs holds promise as a novel cell therapy platform for bone regeneration. STATEMENT OF SIGNIFICANCE: : This study introduces a novel method for enhancing bone regeneration by developing a 3D aligned collagen patch embedded with human mesenchymal stem cells (hMSCs). A single mechanical strain applied to the hMSC-collagen mixture produces an aligned collagen matrix that mimics natural bone tissue. Remarkably, the hMSCs spontaneously differentiate into osteoblasts in the absence of exogenous inducing reagents triggered by activation of the bone morphogenetic protein signaling pathway. In vivo studies using a calvarial defect model confirm effective bone regeneration, initiating the new bone generation from the center of the defect. This approach offers a promising and simple cell therapy platform for bone repair, with broad implications for tissue engineering and regenerative medicine.

Integrin-mediated focal adhesion (FA) and subsequent cytoskeletal reorganization influence cell morphology, migration, and ultimately cell fate. Previous studies have used various patterned surfaces with defined macroscopic cell shapes or nanoscopic FA distributions to explore how different substrates affect the fate of human bone marrow mesenchymal stem cells (BMSCs). However, there is currently no straightforward relationship between BMSC cell fates induced by patterned surfaces and FA distribution substrates. In this study, we conducted single-cell image analysis of integrin α_v-mediated FA and cell morphological features of BMSCs during biochemically induced differentiation. This enabled the identification of distinct FA features that can discriminate between osteogenic and adipogenic differentiation, demonstrating that integrin α_v-mediated focal adhesion (FA) can be used as a non-invasive biomarker for real time observation. Based on these results, we developed an organized microscale fibronectin (FN) patterned surface where the fate of BMSC could be precisely manipulated by these FA features. Notably, even in the absence of any biochemical inducers, such as those contained in the differentiation medium, BMSCs cultured on these FN patterned surfaces exhibited upregulation of differentiation markers comparable to BMSCs cultured using conventional differentiation methods. Therefore, the present study reveals the application of these FA features as universal markers not only for predicting differentiation status, but also for regulating cell fate by precisely controlling the FA features with a new cell culture platform. STATEMENT OF SIGNIFICANCE: Although the effects of material physiochemical properties on cell morphology and subsequent cell fate decisions have been extensively studied, a simple yet intuitive correlation between cellular features and differentiation remains unavailable. We present a single cell image-based strategy for predicting and directing stem cell fate. By using a specific integrin isoform, integrin α_v, we identified distinct geometric features that can be used as a marker for discriminating between osteogenic and adipogenic differentiation in real-time. From these data, new cell culture platforms capable of regulating cell fate by precisely controlling FA features and cell area can be developed.