Osteoinductive potential of CS/Gel/PVA hydrogel with the CEMP-1-P1 peptide evaluated using human mesenchymal stem cells

Abstract

Introduction: Bone injuries are a significant clinical problem; bone tissue is the second most frequently transplanted tissue worldwide. Although bone grafts remain a common therapeutic option, they have limitations such as low availability, donor site morbidity, immune response, and incomplete integration. In this context, tissue engineering has driven the development of three-dimensional biomaterials capable of functioning as cellular scaffolds and promoting tissue repair. Among these, hydrogels stand out for their biocompatibility, high water content, porosity, and ability to mimic the extracellular matrix. Furthermore, the incorporation of bioactive molecules, such as peptides, can enhance osteogenic differentiation. Objective: To evaluate in vitro the biocompatibility and osteoinductive potential of the CS/Gel/PVA hydrogel containing the CEMP-1-p1 peptide using independently cultured human adipose-derived mesenchymal stem cells (AD-MSCs) and periosteal mesenchymal stem cells (Pr-MSCs). Methodology: After obtaining informed consent, AD-MSCs were isolated from abdominoplasty remnants and Pr-MSCs from cruciate ligament repair remnants. The cells were expanded to 90% confluence in DMEM-F12 with 10% fetal bovine serum and 1% antibiotic. Proliferation curves were generated with both cell types to identify the optimal concentration of the CEMP-1-p1 peptide. The CS/Gel/PVA hydrogel was prepared at a 1:1:1 ratio according to the method described by. The hydrogel was cut into 8 mm diameter cylinders; 7 x 10⁵ cells were injected into each hydrogel and cultured for 5, 10, and 15 days. Three experimental groups were established: (1) negative control, (2) positive control with BMP-7 (10 ng/mL), and (3) stimulation with CEMP-1-P1. Cell viability was assessed using calcein; cell adhesion was evaluated by scanning electron microscopy (SEM); protein expression was assessed by immunofluorescence; and tissue formation was assessed by hematoxylin and eosin (H&E) staining. Results: SEM revealed an irregular porous structure in the hydrogel, with pores up to 500 μm in size, a characteristic favorable for cell migration and allowing for nutrient exchange and the elimination of metabolic waste. MTT assays showed increased cell proliferation in the presence of CEMP-1-P1, with a statistically significant difference at a concentration of 10 μg/mL. Cell viability was greater than 90% in all three experimental groups. SEM analysis also revealed the presence of cell extensions (filopodia and lamellipodia), indicating adequate cell adhesion to the hydrogel. Immunofluorescence showed expression of proteins characteristic of the osteogenic lineage, such as osteopontin and type I collagen, in both cell types under negative control, BMP-7 stimulation, and CEMP-1-P1 stimulation conditions, evaluated at 5, 10, and 15 days. Finally, H&E staining allowed observation of de novo tissue formation and organization after 1 and 2 months of culture on the hydrogel. Conclusion: The CEMP-1-P1-enhanced Qs/Gel/PVA hydrogel showed adequate biocompatibility, promoted cell viability and adhesion, and enhanced the expression of osteogenic markers in AD-MSC and Pr-MSC, forming a promising strategy for the development of bone substitutes and their future in vivo evaluation.
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