Rizzo Maria Giovanna, Morganti Dario, Smeriglio Antonella, Sciuto Emanuele Luigi, Spata Massimo Orazio, Trombetta Domenico, Fazio Barbara, Guglielmino Salvatore Pietro Paolo, Conoci Sabrina
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d'Alcontres, 31, 98166 Messina, Italy.
CNR-IMM, Institute for Microelectronics and Microsystems, Viale F. Stagno D'Alcontres 31, 98166 Messina, Italy.
Int J Mol Sci. 2025 Sep 1;26(17):8482. doi: 10.3390/ijms26178482.
Cell-cell communication and extracellular matrix (ECM) organization in a bone microenvironment are essential to replicate the bone microenvironment accurately. In this study, the extracellular matrix (ECM) was emulated by incorporating M13 phages, selected through phage display for displaying engineered peptides that mimic bone matrix proteins, into human osteoblast cultures to develop a three-dimensional bone model (3D BMP-Phage). Comprehensive analysis was performed to investigate: (i) the morphological development of spheroids, assessed by optical microscopy and quantified via fractal dimension analysis using box-counting algorithms; (ii) the biochemical composition of the extracellular matrix, evaluated by Raman spectroscopy; (iii) ECM protein deposition, analyzed through immunofluorescence staining; (iv) matrix mineralization, assessed by Alizarin Red staining and alkaline phosphatase (ALP) activity assay; and (v) osteogenic gene expression, measured by quantitative RT-PCR. The findings demonstrate that the 3D BMP-Phage model, facilitated by a cocktail of bone-mimicking peptides, enhances structural integrity, ECM complexity, mineralization, and osteogenic pathways compared to the control. This novel approach replicates key aspects of the bone microenvironment, providing a valuable platform for advanced physiological and regenerative medicine research under controlled conditions.
骨微环境中的细胞间通讯和细胞外基质（ECM）组织对于精确复制骨微环境至关重要。在本研究中，通过将经噬菌体展示筛选出的、用于展示模拟骨基质蛋白的工程肽的M13噬菌体整合到人类成骨细胞培养物中，来模拟细胞外基质（ECM），从而构建三维骨模型（3D BMP - 噬菌体）。进行了全面分析以研究：（i）通过光学显微镜评估并使用盒计数算法通过分形维分析进行量化的球体形态发育；（ii）通过拉曼光谱评估的细胞外基质的生化组成；（iii）通过免疫荧光染色分析的ECM蛋白沉积；（iv）通过茜素红染色和碱性磷酸酶（ALP）活性测定评估的基质矿化；以及（v）通过定量RT - PCR测量的成骨基因表达。研究结果表明，与对照相比，由模拟骨肽混合物促进的3D BMP - 噬菌体模型增强了结构完整性、ECM复杂性、矿化和成骨途径。这种新方法复制了骨微环境的关键方面，为在可控条件下进行先进的生理学和再生医学研究提供了一个有价值的平台。
