In vivo evaluation of biocompatibility and immune modulation potential of poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone)-gelatin hydrogels enriched with nano-hydroxyapatite in the model of mouse.

Biocompatible, biodegradable, and injectable hydrogels are a novel and promising approach for bone regeneration. In this study, poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone) (PCL-PEG-PCL), PCL-PEG-PCL-gelatin (Gel), PCL-PEG-PCL-Gel/nano-hydroxyapatite (nHA) injectable hydrogels were synthesized and evaluated in a mouse model of subcutaneous transplantation after 14 days. PCL-PEG-PCL-Gel and PCL-PEG-PCL-Gel/nHA hydrogels were fabricated with in situ precipitation method. Structure, intermolecular interaction, and the reaction between the PCL-PEG-PCL, Gel, and nHA were evaluated using a scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (H-NMR), and carbon nuclear magnetic resonance (C-NMR). Fourteen days after subcutaneous injection, the existence of an immune system reaction was investigated using Hematoxylin and Eosin (H&E) staining. Using immunofluorescence imaging, the number of CD68+ cells was determined in the periphery of the hydrogel. The CD8/CD4 lymphocyte ratio was also calculated in blood samples. We monitored the expression of CCL-2, BCL-2, IL-10, and CD31 using real-time PCR assay. The chemical evaluation revealed the successful integration of Gel and nHA to the PCL-PEG-PCL backbone. Histological examination showed the lack of inflammation at the site of injection. No toxicological effects were determined in hepatic and renal tissues. The addition of nHA to the PCL-PEG-PCL-Gel decreased biodegradation time. None of the hydrogels caused statistically significant differences in the number of CD68 cells (p > 0.05). The CD8/CD4 lymphocyte ratio remained unchanged in all groups (p > 0.05). Compared to the PCL-PEG-PCL group, the addition of nHA and Gel increased the expression of CCL-2, BCL-2, IL-10, and CD31 (p < 0.05). In conclusion, the current study showed that PCL-PEG-PCL-Gel/nHA hydrogels could be used in in vivo conditions without prominent toxic effects and inflammatory responses.