All animal care and experiment procedures were conducted in accordance with the National Institutes of Health guidelines. The animal protocol was approved by the Institutional Animal Care and Use Committee of the Ohio State University. To determine microsphere fluorescent intensity change after implantation, 100 μL of 6 wt% PNAH hydrogel solution with or without 50 mg/mL microspheres was injected into thigh muscles of 8-week-old NCr nude mice (Taconic Biosciences. n =4 for each group). At days 0, 3, 7, 10, 14, 17 and 21, the mice were anesthetized with isoflurane, and imaged using an In Vivo Imaging System (IVIS) Lumina II (PerkinElmer) with DsRed emission filter.
To determine biocompatibility of the microspheres, 200 μL of 6 wt% PNAH hydrogel solution with or without 50 mg/mL microspheres was injected into thigh muscles of 8-week-old C57BL/6 mice (Jackson laboratory. n =4 for each group). The muscles were harvested 3 weeks after the injection. The tissues were fixed with 4% paraformaldehyde overnight, processed and embedded in paraffin. Tissue sections of 5-μm thickness were cut, and stained with mouse monoclonal anti-F4/80 (Santa Cruz Biotechnology). Nuclei were stained by DRAQ5. The cells were imaged using a confocal microscope. F4/80 positive cell density was quantified by ImageJ.
To determine biocompatibility of the microspheres, 200 μL of 6 wt% PNAH hydrogel solution with or without 50 mg/mL microspheres was injected into thigh muscles of 8-week-old C57BL/6 mice (Jackson laboratory. n =4 for each group). The muscles were harvested 3 weeks after the injection. The tissues were fixed with 4% paraformaldehyde overnight, processed and embedded in paraffin. Tissue sections of 5-μm thickness were cut, and stained with mouse monoclonal anti-F4/80 (Santa Cruz Biotechnology). Nuclei were stained by DRAQ5. The cells were imaged using a confocal microscope. F4/80 positive cell density was quantified by ImageJ.