All animal procedures in this study were performed in accordance with the guidelines for care and use of laboratory animals of Fudan University and approved by the animal ethics committee of Fudan University (202206022Z).
In vivo biodegradation and biocompatibility tests were conducted using subcutaneous implantation in Sprague–Dawley (SD) rats. To relieve pain, the rats were anesthetized using intraperitoneal injections of pentobarbital (35 mg kg−1). The dorsal hair of the rats was shaved, and the skin was disinfected with iodophor. After being sterilized, 200 μL of freshly prepared RSF/LAP hydrogel was implanted by injection into individual dorsal subcutaneous pockets. After 14 days, the rats were euthanized, and the constructs and surrounding tissue were explanted and analyzed for biocompatibility using histological and immunohistological evaluations.
After being fixed in 4% (w/v) paraformaldehyde for 24 h, the samples were dehydrated using gradient ethanol solutions (75% (v/v) ethanol 4 h, 85% (v/v) ethanol 2 h, 90% (v/v) ethanol 2 h, 95% (v/v) ethanol 1 h, anhydrous ethanol I 30 min, anhydrous ethanol II 30 min, and anhydrous ethanol III 30 min), cleared in xylene (xylene I 20 min, xylene II 20 min, and xylene III 20 min), embedded in paraffin (48–50 °C melting paraffin I overnight, 56–58 °C melting paraffin II 2 h, and 60–62 °C melting paraffin III 2 h), and sectioned at a thickness of 4 μm by the paraffin slicer (RM2016, Leica, Wetzlar, German). The tissue was flattened when the slice floated on the 40 °C warm water of the spreading machine, and the tissue was picked up using the glass slides and baked in the oven at 60 °C. After the water-baked dried wax was melted, it was taken out and stored at room temperature. After deparaffinization (xylene I 20 min, xylene II 20 min, xylene III 20 min, anhydrous ethanol I 10 min, anhydrous ethanol II 10 min, anhydrous ethanol III 10 min, 95% (v/v) ethanol 10 min, 90% (v/v) ethanol 10 min, 85% (v/v) ethanol 10 min, 75% (v/v) ethanol 10 min, rinsing with tap water), they were stained with hematoxylin–eosin (H&E, Servicebio, Beijing, China), Masson trichrome staining (Servicebio), and immunohistochemical staining. Vessel formation of these samples was examined using immunohistochemical staining with anti-CD31 (Abcam, Cambridge, UK), while the inflammatory response was determined based on the presence of inflammatory markers, including CD3 (Abcam) and CD68 (Abcam).
In vivo biodegradation and biocompatibility tests were conducted using subcutaneous implantation in Sprague–Dawley (SD) rats. To relieve pain, the rats were anesthetized using intraperitoneal injections of pentobarbital (35 mg kg−1). The dorsal hair of the rats was shaved, and the skin was disinfected with iodophor. After being sterilized, 200 μL of freshly prepared RSF/LAP hydrogel was implanted by injection into individual dorsal subcutaneous pockets. After 14 days, the rats were euthanized, and the constructs and surrounding tissue were explanted and analyzed for biocompatibility using histological and immunohistological evaluations.
After being fixed in 4% (w/v) paraformaldehyde for 24 h, the samples were dehydrated using gradient ethanol solutions (75% (v/v) ethanol 4 h, 85% (v/v) ethanol 2 h, 90% (v/v) ethanol 2 h, 95% (v/v) ethanol 1 h, anhydrous ethanol I 30 min, anhydrous ethanol II 30 min, and anhydrous ethanol III 30 min), cleared in xylene (xylene I 20 min, xylene II 20 min, and xylene III 20 min), embedded in paraffin (48–50 °C melting paraffin I overnight, 56–58 °C melting paraffin II 2 h, and 60–62 °C melting paraffin III 2 h), and sectioned at a thickness of 4 μm by the paraffin slicer (RM2016, Leica, Wetzlar, German). The tissue was flattened when the slice floated on the 40 °C warm water of the spreading machine, and the tissue was picked up using the glass slides and baked in the oven at 60 °C. After the water-baked dried wax was melted, it was taken out and stored at room temperature. After deparaffinization (xylene I 20 min, xylene II 20 min, xylene III 20 min, anhydrous ethanol I 10 min, anhydrous ethanol II 10 min, anhydrous ethanol III 10 min, 95% (v/v) ethanol 10 min, 90% (v/v) ethanol 10 min, 85% (v/v) ethanol 10 min, 75% (v/v) ethanol 10 min, rinsing with tap water), they were stained with hematoxylin–eosin (H&E, Servicebio, Beijing, China), Masson trichrome staining (Servicebio), and immunohistochemical staining. Vessel formation of these samples was examined using immunohistochemical staining with anti-CD31 (Abcam, Cambridge, UK), while the inflammatory response was determined based on the presence of inflammatory markers, including CD3 (Abcam) and CD68 (Abcam).
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