molecules Article Keratin Scaffolds Containing Casomorphin Stimulate Macrophage Infiltration and Accelerate Full-Thickness Cutaneous Wound Healing in Diabetic Mice Marek Konop 1,2,3, * , Anna K. Laskowska 4 , Mateusz Rybka 1 , Ewa Klodzi ´ nska 5 , Dorota Sulejczak 6 , Robert A. Schwartz 7 and Joanna Czuwara 3   Citation: Konop, M.; Laskowska, A.K.; Rybka, M.; Klodzi ´ nska, E.; Sulejczak, D.; Schwartz, R.A.; Czuwara, J. Keratin Scaffolds Containing Casomorphin Stimulate Macrophage Infiltration and Accelerate Full-Thickness Cutaneous Wound Healing in Diabetic Mice. Molecules 2021, 26, 2554. https:// doi.org/10.3390/molecules26092554 Academic Editor: Giuseppe De Rosa Received: 15 March 2021 Accepted: 21 April 2021 Published: 27 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Laboratory of Centre for Preclinical Research (CePT), Department of Experimental Physiology and Pathophysiology, Medical University of Warsaw, 02-097 Warsaw, Poland;mateuszrybka@mp.pl 2 Mossakowski Medical Research Centre, Department of Neuropeptides, Polish Academy of Sciences, 02-106 Warsaw, Poland 3 Departmentof Dermatology, Medical University of Warsaw, 02-091 Warsaw, Poland;jczuwara@yahoo.com 4 Centre for Preclinical Research and Technology (CePT), Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Warsaw, 02-091 Warsaw, Poland;anna.laskowska@wum.edu.pl 5 Department of Analytical Chemistry and Instrumental Analysis, Institute of Sport-National Research Institute, 03-301 Warsaw, Poland; ewa.klodzinska@insp.waw.pl 6 Department of Experimental Pharmacology, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland; dsulejczak@imdik.pan.pl 7 Dermatology and Pathology, Rutgers New Jersey Medical School, Newark, NJ 07103, USA; raschwartz@gmail.com * Correspondence: marek.konop@wum.edu.pl Abstract: Impaired wound healing is a major medical challenge, especially in diabetics. Over the centuries, the main goal of tissue engineering and regenerative medicine has been to invent bioma- terials that accelerate the wound healing process. In this context, keratin-derived biomaterial is a promising candidate due to its biocompatibility and biodegradability. In this study, we evaluated an insoluble fraction of keratin containing casomorphin as a wound dressing in a full-thickness surgical skin wound model in mice (n = 20) with iatrogenically induced diabetes. Casomorphin, an opioid peptide with analgesic properties, was incorporated into keratin and shown to be slowly released from the dressing. An in vitro study showed that keratin-casomorphin dressing is biocompatible, non-toxic, and supports cell growth. In vivo experiments demonstrated that keratin-casomorphin dressing significantly (p < 0.05) accelerates the whole process of skin wound healing to the its fi- nal stage. Wounds covered with keratin-casomorphin dressing underwent reepithelization faster, ending up with a thicker epidermis than control wounds, as confirmed by histopathological and immunohistochemical examinations. This investigated dressing stimulated macrophages infiltration, which favors tissue remodeling and regeneration, unlike in the control wounds in which neutrophils predominated. Additionally, in dressed wounds, the number of microhemorrhages was significantly decreased (p < 0.05) as compared with control wounds. The dressing was naturally incorporated into regenerating tissue during the wound healing process. Applied keratin dressing favored re- construction of more regular skin structure and assured better cosmetic outcome in terms of scar formation and appearance. Our results have shown that insoluble keratin wound dressing containing casomorphin supports skin wound healing in diabetic mice. Keywords: casomorphin; diabetes; keratin dressing; opioids; tissue regeneration; skin wound healing 1. Introduction Impaired wound healing is a major medical problem, especially in diabetics. Pain associated with chronic non-healing wounds can be particularly difficult to manage. Many patients experience pain despite the use of systemic analgesics. Opioids are the basic Molecules 2021, 26, 2554. https://doi.org/10.3390/molecules26092554 https://www.mdpi.com/journal/molecules