To evaluate the biological responses of the subcutaneous tissues to the implanted material, we followed the International Organization for Standardization (ISO)–10993 and notification no. 2014-115 of Korea Ministry of Food and Drug Safety. Following ISO 10993-2, healthy male 6-week-old BALB/c mice with a mean ± SD body weight of 21.3 ± 0.8 g were used. The mice were obtained at 5 weeks of age from Orient Bio Co. (Republic of Korea) and maintained under an artificial 12-hour light/dark cycle at a constant temperature of 22 ± 1°C and constant humidity of 55 ± 10%. The mice were kept in their cages for 1 week to acclimate to the laboratory conditions.

Each mouse was subcutaneously implanted with a piece of CM and poly(dimethylsiloxane) (PDMS; Sylgard s18, Dow Corning, USA; 10:1 weight ratio of prepolymer to cross-linker) as a control following ISO 10993-6. Briefly, the mice were anesthetized with 2% isoflurane and the dorsal hair was removed by shaving. A sagittal skin incision was made on the back, and subcutaneous pouches were made. The CM and PDMS were prepared following ISO 10993-12 and finely cut into 10-mm-diameter circles. The samples were subcutaneously implanted at least 1 cm apart. The skin was sutured with nonabsorbable 6-0 silk. The mice were allowed unrestricted cage activity. Two weeks after surgery, the mice were euthanized in a CO2 chamber and whole skins were freshly sampled for analysis.

To evaluate the local histopathological effects, freshly excised skin samples were fixed with 4% paraformaldehyde for 24 hours. The tissues were processed using routine tissue techniques and embedded in paraffin. The paraffin-embedded specimens were sliced into 4-μm-thick sections. The sections were then transferred to microscope slides. Subsequently, deparaffinized skin sections were stained with H&E. All stained sections were examined under a light microscope to assess histological changes generally indicative of irritation, including polymorphonuclear cell infiltration, mononuclear cell infiltration, necrosis, angiogenesis, fibrosis, and fatty infiltration. All operating procedures for handling of experimental animals were performed in accordance with guidelines and regulations of the Institutional Animal Care and Use Committee (IACUC) of Konkuk University, which was accredited for laboratory animal care by the Korea Ministry of Food and Drug Safety.

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