**1. Introduction**

Keratin can be extracted from keratin-rich materials using chemical (reduction, oxidation, hydrolysis, sulphitolysis), physical (steam explosion, microwave irradiation) or biological methods [1]. The properties of keratin such as amino acid content, molecular weight, thermal behavior and bioactivity depend on the extraction and preparation methods [2,3]. Biomaterials prepared from keratin extracted from wool or human hair showed excellent properties regarding biocompatibility and cellular proliferation abilities. These properties make wool keratin an excellent material for tissue engineering and drug delivery systems [4]. In vitro studies have shown that keratin has the ability to scavenge free radicals, similar to that of vitamin C and allows replacing cosmetic preservatives [5].

A study on the anti-inflammatory ability of keratin extracted from human hair showed that it has a more pronounced anti-inflammatory effect on monocytic cell line, than collagen

**Citation:** Olariu, L.; Dumitriu, B.G.; Gaidau, C.; Stanca, M.; Tanase, L.M.; Ene, M.D.; Stanculescu, I.-R.; Tablet, C. Bioactive Low Molecular Weight Keratin Hydrolysates for Improving Skin Wound Healing. *Polymers* **2022**, *14*, 1125. https://doi.org/ 10.3390/polym14061125

Academic Editors: Antonia Ressler, Inga Urlic and Jianxun Ding

Received: 20 January 2022 Accepted: 8 March 2022 Published: 11 March 2022

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**Copyright:** © 2022 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/).

or hyaluronic acid. The study showed that primary macrophage cells are altered when they are exposed to an immobilized keratin biomaterial surface and that these changes appear to target an anti-inflammatory phenotype. This phenomenon appears to be a function of the lower molecular weight of keratin, even if it is recognized that the mechanism of anti-inflammatory process is not fully understood [6]. Tachibana et al. showed also that due to the amino acid sequence with more carboxylic terminal groups, keratin is more effective than collagen in binding osteoblast cells and inducing osteogenesis [7]. Biomaterials based on keratin contain regulatory molecules that make them capable of functioning as synthetic extracellular matrices (ECM) and promoting nerve tissue regeneration due to their capacity to create fibronectin-like cell binding that facilitates cell adhesion [8,9]. Gao et al. conducted in vivo and in vitro studies on the ability of keratin to promote the regeneration of peripheral nerves. The in vitro study showed that it has neuroinducible activity and Schwann cells grown in keratin medium have a number of changes in terms of migration capacity, morphology and proliferation activity, with stimulating effect on neuronal axons extension. In vivo experiments have shown that keratin accelerates the regeneration of the axon in the early stages and has a beneficial effect for subsequent functional recovery [10].

Human keratin hair hydrogel efficiency for treatment of thermal or chemical wounds was proved by faster closure and no increase in wound size in the first four days of healing [11]. In the field of skin regeneration, there are several mechanisms working together to restore dramatically affected molecular processes. For example, cellular function is regulated during dermal repair flow by critical interactions between receptors and extracellular matrix proteins. Also, secretory immune cells release cytokines and growth factors (IL-1, IL-4, IL-6, IL-13, TGF-β, TNF) interrelated with structural proteins synthesis [12]. This kind of interactions leads to regenerative processes' activation, including angiogenesis and scar remodeling [13,14]. Integrins are heterodimeric proteins from plasmatic membrane, involved in cellular responses to growth factors, and direct regulation of transcriptional programs, signaling cascades and activation mechanisms [15]. Over-expression of specific integrins directs wound healing, fibrosis and scarring. They are transmembrane structures with α and β subunits assembled to form functional receptors: α3β1 (receptor for laminin 332), α6β5 (hemidesmosome component, receptor for laminin 332), α2β1 (receptor for collagen and laminin, having a pivotal role for wound healing in vivo), αvβ5 (receptor for vitronectin); α9β1 (receptor for tenascin C) [16,17]. A correlation was reported between the level of β1 integrins and proliferation in the interfollicular epidermis [18]. Integrins are also required for an essential step of skin regeneration, namely fibroblast infiltration into the wound clot. Normal fibroblasts and granulation tissue fibroblasts express many types of integrins: α1β1, α2β1, α3β1, α5β1, α11β1, ανβ1, ανβ3, and ανβ5, in order to bind collagens, fibronectins and other blood clot components. For example, fibroblasts interact with fibrillar collagens via α1β1, α2β1, and α11β1 integrins, regulating matrix metalloproteinase (MMP) expression and collagen fibrillogenesis [19]. Another event accompanying skin injuries is inflammation, including cytokines (IL6, IL8, TNFα) signals progression and cellular factors over-expression. Key elements for vascular endothelial inflammation are proteins from CAM family, especially ICAM (intercellular adhesion molecule) and VCAM (vascular cell adhesion molecule) [20]. They play a different role, namely: ICAM-1 specifically participates in trafficking of inflammatory cells, in leukocyte effectors' functions, in adhesion of antigen-presenting cells to T lymphocytes, in microbial pathogenesis, and in signal transduction pathways through outside-in signaling events [21], and VCAM-1 that is one of the major regulators of leukocyte adhesion and transendothelial migration by interacting with α4β1 integration, which in turn activates intracellular signaling that allows transendothelial migration of leukocytes [22].

In our paper we hypothesized that the wool keratin, an easily available byproduct, can be tuned by chemical-enzymatic hydrolyses in different keratin biomolecules so as to stimulate the main skin cell biochemical mechanisms for wound healing. As in many reported research studies, the human hair keratin was extracted and solubilized through complex reduction or oxidizing methods and prepared in hydrogel forms mainly with high

molecular weights [23], the present research proposes alkaline-enzymatic hydrolysates with lower molecular weights, prepared by complete wool solubilization [24] and with preserved bioactive structured molecules.

It is recognized that few commercial products based on keratin reached the market of wound healing biomaterials as compared to other materials due to the complex methods of preparation and the insufficient understanding of cellular interaction with keratin [25,26]. In this regard the present research proposes keratin hydrolysates prepared by a green, reproducible and easy method of solubilization [24] and brings evidences in several molecular factors, decisive for restoring the skin homeostasis as new potential wound dressing active component.

If different keratin dressings with high molecular weights were recognized as activators of keratinocytes wound healing process [27], the present research brings mechanistic insights from damaged skin cells restitution processes induced by two low molecular bioactive keratin hydrolysates with still structured peptides.

Thus, the stimulation effect on β1 glycoprotein expression in pro-inflammatory conditions and α1 and α2 subunits generation in the basal state of fibroblasts were proved by low molecular keratins. The other anti-inflammatory factors involved in wound healing process, like significant down regulation of nonspecific stimulation—TNFα + PMA, and the ICAM expression were also influenced.

According to our knowledge no similar study was performed regarding the influence of low molecular wool keratin hydrolysates on skin cells homeostasis factors.

### **2. Materials and Methods**

### *2.1. Materials*

Raw wool was purchased from a local sheep farmer (Lumina, Constanta, Romania). Chemical reagents of analytical grade like sodium hydroxide (98%), ammonia (25%), sodium carbonate (99.7%), formic acid (85%) and sulfuric acid (92%) were purchased from Chimopar Trading SRL (Bucharest, Romania). Borron SE (ethoxylated alkyl derivatives with 65% concentration) was supplied by SC Triderma SRL (Bucharest, Romania). Esperase® 8.0 L, a serine endo-peptidase from *Bacillus lentus* with activity of 8 KNPU-E/g, working at elevated temperature and pH = 8–12.5, was purchased from Novozymes (Atasehir, Turkey). Valkerase®, a keratinase, serine protease from *B. licheniformis,* with activity of 80,549 U/g at pH = 5.5 and 55 ◦C, was supplied by BioResource International (Durham, NC, USA).
