In Vitro Evaluation of Novel Hybrid Cooperative Complexes in a Wound Healing Model: A Step Toward Improved Bioreparation

The effectiveness of hyaluronic acid (HA), also called as hyaluronan, and its formulations on tissue regeneration and epidermal disease is well-documented. High-molecular-weight hyaluronan (HHA) is an efficient space filler that maintains hydration, serves as a substrate for proteoglycan assembly, and is involved in wound healing. Recently, an innovative hybrid cooperative complex (HCC) of high- and low-molecular-weight hyaluronan was developed that is effective in wound healing and bioremodeling. The HCC proposed here consisted of a new formulation and contained 1.6 ± 0.1 kDa HHA and 250 ± 7 kDa LHA (low molecular weight hyaluronic acid). We investigated the performance of this HCC in a novel in vitro HaCaT (immortalized human keratinocytes)/HDF (human dermal fibroblast) co-culture model to assess its ability to repair skin tissue lesions. Compared to linear HA samples, HCC reduced the biomarkers of inflammation (Transforming Growth Factor-β (TGF-β), Tumor Necrosis Factor receptor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8)), and accelerated the healing process. These data were confirmed by the modulation of metalloproteases (MMPs) and elastin, and were compatible with a prospectively reduced risk of scar formation. We also examined the expression of defensin-2, an antimicrobial peptide, in the presence of hyaluronan, showing a higher expression in the HCC-treated samples and suggesting a potential increase in antibacterial and immunomodulatory functions. Based on these in vitro data, the presence of HCC in creams or dressings would be expected to enhance the resolution of inflammation and accelerate the skin wound healing process.

Download Full-text

Gamma-Irradiation-Prepared Low Molecular Weight Hyaluronic Acid Promotes Skin Wound Healing

Polymers ◽

10.3390/polym11071214 ◽

2019 ◽

Vol 11 (7) ◽

pp. 1214 ◽

Cited By ~ 6

Author(s):

Huang ◽

Lew ◽

Fan ◽

Chang ◽

...

Keyword(s):

Molecular Weight ◽

Wound Healing ◽

Hyaluronic Acid ◽

Skin Wound ◽

Cellular Growth ◽

Low Molecular Weight ◽

Γ Irradiation ◽

Skin Wound Healing

In this study, we prepared low-molecular-weight hyaluronic acid (LMWHA) powder by γ-irradiation. The chemical and physical properties of γ-irradiated LMWHA and the in vitro cellular growth experiments with γ-irradiated LMWHA were analyzed. Then, hyaluronic acid exposed to 20 kGy of γ-irradiation was used to fabricate a carboxymethyl cellulose (CMC)/LMWHA fabric for wound dressing. Our results showed that γ-irradiated LMWHA demonstrated a significant alteration in carbon–oxygen double bonding and can be detected using nuclear magnetic resonance and ultraviolet (UV)-visible (Vis) spectra. The γ-irradiated LMWHA exhibited strain rate-dependent Newton/non-Newton fluid biphasic viscosity. The viability of L929 skin fibroblasts improved upon co-culture with γ-irradiated LMWHA. In the in vivo animal experiments, skin wounds covered with dressings prepared by γ-irradiation revealed acceleration of wound healing after two days of healing. The results suggest that γ-irradiated LMWHA could be a potential source for the promotion of skin wound healing.

Download Full-text

A Low Molecular Weight Hyaluronic Acid Derivative Accelerates Excisional Wound Healing by Modulating Pro-Inflammation, Promoting Epithelialization and Neovascularization, and Remodeling Collagen

International Journal of Molecular Sciences ◽

10.3390/ijms20153722 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3722 ◽

Cited By ~ 9

Author(s):

Yin Gao ◽

Yao Sun ◽

Hao Yang ◽

Pengyu Qiu ◽

Zhongcheng Cong ◽

...

Keyword(s):

Molecular Weight ◽

Wound Healing ◽

Hyaluronic Acid ◽

Molecular Mechanisms ◽

Healing Process ◽

Low Molecular Weight ◽

Cutaneous Wound Healing ◽

Cutaneous Wound

Recent knowledge of the cellular and molecular mechanisms underlying cutaneous wound healing has advanced the development of medical products. However, patients still suffer from the failure of current treatments, due to the complexity of healing process and thus novel therapeutic approaches are urgently needed. Previously, our laboratories produced a range of low molecular weight hyaluronic acid (LMW-HA) fragments, where a proportion of the glucosamine moieties were chemically N-acyl substituted. Specifically, N-butyrylation results in anti-inflammatory properties in a macrophage system, and we demonstrate the importance of N-acyl substituents in modulating the inflammatory response of LMW-HA. We have set up an inter-institutional collaborative program to examine the biomedical applications of the N-butyrylated LMW-HA (BHA). In this study, the potentials of BHA for dermal healing are assessed in vitro and in vivo. Consequently, BHA significantly promotes dermal healing relative to a commercial wound care product. By contrast, the “parent” partially de-acetylated LMW-HA (DHA) and the re-acetylated DHA (AHA) significantly delays wound closure, demonstrating the specificity of this N-acylation of LMW-HA in wound healing. Mechanistic studies reveal that the BHA-mediated therapeutic effect is achieved by targeting three phases of wound healing (i.e., inflammation, proliferation and maturation), demonstrating the significant potential of BHA for clinical translation in cutaneous wound healing.

Download Full-text

Impairment of the Hif-1α regulatory pathway in Foxn1-deficient (Foxn1−/−) mice affects the skin wound healing process

10.1101/2020.08.04.237388 ◽

2020 ◽

Author(s):

Sylwia Machcinska ◽

Marta Kopcewicz ◽

Joanna Bukowska ◽

Katarzyna Walendzik ◽

Barbara Gawronska-Kozak

Keyword(s):

Wound Healing ◽

Transforming Growth Factor ◽

Healing Process ◽

Transforming Growth Factor Β ◽

Skin Wound ◽

Wound Healing Process ◽

Limiting Factor ◽

Skin Wound Healing ◽

Collagen Iii

ABSTRACTHypoxia and hypoxia-regulated factors [e. g., hypoxia-inducible factor-1α (Hif-1α), factor inhibiting Hif-1α (Fih-1), thioredoxin-1 (Trx-1), aryl hydrocarbon receptor nuclear translocator 2 (Arnt-2)] have essential roles in skin wound healing. Using Foxn1−/− mice that can heal skin injuries in a unique scarless manner, we investigated the interaction between Foxn1 and hypoxia-regulated factors. The Foxn1−/− mice displayed impairments in the regulation of Hif-1α, Trx-1 and Fih-1 but not Arnt-2 during the healing process. An analysis of wounded skin showed that the skin of the Foxn1−/− mice healed in a scarless manner, displaying rapid re-epithelialization and an increase in transforming growth factor β (Tgfβ-3) and collagen III expression. An in vitro analysis revealed that Foxn1 overexpression in keratinocytes isolated from the skin of the Foxn1−/− mice led to reduced Hif-1α expression in normoxic but not hypoxic cultures and inhibited Fih-1 expression exclusively under hypoxic conditions. These data indicate that in the skin, Foxn1 affects hypoxia-regulated factors that control the wound healing process and suggest that under normoxic conditions, Foxn1 is a limiting factor for Hif-1α.

Download Full-text

Wound Healing Promotion by Hyaluronic Acid: Effect of Molecular Weight on Gene Expression and In Vivo Wound Closure

Pharmaceuticals ◽

10.3390/ph14040301 ◽

2021 ◽

Vol 14 (4) ◽

pp. 301

Author(s):

Yayoi Kawano ◽

Viorica Patrulea ◽

Emmanuelle Sublet ◽

Gerrit Borchard ◽

Takuya Iyoda ◽

...

Keyword(s):

Gene Expression ◽

Molecular Weight ◽

Wound Healing ◽

Hyaluronic Acid ◽

Healing Process ◽

Dermal Fibroblasts ◽

Water Soluble ◽

Wound Healing Process ◽

And Migration ◽

Proliferation And Migration

Hyaluronic acid (HA) has been known to play an important role in wound healing process. However, the effect of molecular weight (MW) of exogenously administered HA on the wound healing process has not been fully understood. In this study, we investigated HA with different MWs on wound healing process using human epidermal keratinocytes and dermal fibroblasts. Cell proliferation and migration ability were assessed by water soluble tetrazolium (WST) assay and wound scratch assay. We examined the effect of HA addition in a full-thickness wound model in mice and the gene expression related to wound healing. Proliferation and migration of HaCaT cells increased with the increase of MW and concentration of HA. Interleukin (IL-1β), IL-8 and vascular endothelial growth factor (VEGF) as well as matrix metalloproteinase (MMP)-9 and MMP-13 were significantly upregulated by high molecular weight (HMW) HA in keratinocytes. Together with VEGF upregulation and the observed promotion of HaCaT migration, HA with the MW of 2290 kDa may hold potential to improve re-epithelialization, a critical obstacle to heal chronic wounds.

Download Full-text

Keratin Scaffolds Containing Casomorphin Stimulate Macrophage Infiltration and Accelerate Full-Thickness Cutaneous Wound Healing in Diabetic Mice

Molecules ◽

10.3390/molecules26092554 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2554

Author(s):

Marek Konop ◽

Anna K. Laskowska ◽

Mateusz Rybka ◽

Ewa Kłodzińska ◽

Dorota Sulejczak ◽

...

Keyword(s):

Wound Healing ◽

Wound Dressing ◽

Healing Process ◽

Skin Wound ◽

Wound Healing Process ◽

Diabetic Mice ◽

Full Thickness ◽

Skin Wound Healing ◽

Impaired Wound Healing

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 biomaterials 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 final 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 reconstruction 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.

Download Full-text

Systemic and topical administration of spermidine accelerates skin wound healing

10.21203/rs.3.rs-111107/v1 ◽

2020 ◽

Author(s):

Daisuke Ito ◽

Hiroyasu Ito ◽

Takayasu Ideta ◽

Ayumu Kanbe ◽

Soranobu Ninomiya ◽

...

Keyword(s):

Cell Proliferation ◽

Wound Healing ◽

Wound Repair ◽

Healing Process ◽

Topical Administration ◽

Skin Wound ◽

Wound Healing Process ◽

Skin Wound Healing

Abstract Background The skin wound healing process is regulated by various cytokines, chemokines, and growth factors. Recent reports have demonstrated that spermine/spermidine (SPD) promote wound healing through urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) signaling in vitro. Here, we investigated whether the systemic and topical administration of SPD would accelerate the skin wound-repair process in vivo.Methods A skin wound repair model was established using C57BL/6 J mice. SPD was mixed with white petrolatum for topical administration. For systemic administration, SPD mixed with drinking water was orally administered. Changes in wound size over time were calculated using digital photography.Results Systemic and topical SPD treatment significantly accelerated skin wound healing. The administration of SPD promoted the uPA/uPAR pathway in wound sites. Moreover, topical treatment with SPD enhanced the expression of IL-6 and TNF-α in wound sites. Scratch and cell proliferation assays revealed that SPD administration accelerated scratch wound closure and cell proliferation in vitro.Conclusion These results indicate that treatment with SPD promotes skin wound healing through activation of the uPA/uPAR pathway and induction of the inflammatory response in wound sites. The administration of SPD might contribute to new effective treatments to accelerate skin wound healing.

Download Full-text

The effect of earthworm extract on promoting skin wound healing

Bioscience Reports ◽

10.1042/bsr20171366 ◽

2018 ◽

Vol 38 (2) ◽

Cited By ~ 4

Author(s):

Zhen-han Deng ◽

Jian-jian Yin ◽

Wei Luo ◽

Ronak Naveenchandra Kotian ◽

Shan-shan Gao ◽

...

Keyword(s):

Wound Healing ◽

Healthcare System ◽

Transforming Growth Factor ◽

Healing Process ◽

White Blood Cells ◽

Skin Wound ◽

Wound Healing Process ◽

Blood Capillary ◽

Skin Wound Healing

Chronic nonhealing wounds pose a significant challenge to healthcare system because of its tremendous utilization of resources and time to heal. It has a well-deserved reputation for reducing the quality of life for those affected and represent a substantial economic burden to the healthcare system overall. Earthworms are used as a traditional Chinese medicine, and have been applied pharmacologically and clinically since a long time in China. However, there is paucity in data regarding its wound healing effects. Therefore, we investigated the effect of earthworm extract (EE) on skin wound healing process. The obtained data showed that EE has healing effects on local wound of mice. It decreased the wound healing time and reduced the ill-effects of inflammation as determined by macroscopic, histopathologic, hematologic, and immunohistochemistry parameters. The potential mechanism could be accelerated hydroxyproline and transforming growth factor-β secretion—thus increasing the synthesis of collagen, promoting blood capillary, and fibroblast proliferation. It could accelerate the removal of necrotic tissue and foreign bodies by speeding up the generation of interleukin-6, white blood cells, and platelets. It thus enhances immunity, reduces the risk of infection, and promotes wound healing. All in all, the obtained data demonstrated that EE improves quality of healing and could be used as a propitious wound healing agent.

Download Full-text

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

AJP Cell Physiology ◽

10.1152/ajpcell.00179.2011 ◽

2012 ◽

Vol 302 (8) ◽

pp. C1213-C1225 ◽

Cited By ~ 17

Author(s):

Chen Zhang ◽

Chek Kun Tan ◽

Craig McFarlane ◽

Mridula Sharma ◽

Nguan Soon Tan ◽

...

Keyword(s):

Wound Healing ◽

Growth Factor ◽

Wound Repair ◽

Transforming Growth Factor ◽

Healing Process ◽

Critical Role ◽

Transforming Growth Factor Β ◽

Skin Wound ◽

Wound Healing Process ◽

Skin Wound Healing

Myostatin (Mstn) is a secreted growth and differentiation factor that belongs to the transforming growth factor-β (TGF-β) superfamily. Mstn has been well characterized as a regulator of myogenesis and has been shown to play a critical role in postnatal muscle regeneration. Herein, we report for the first time that Mstn is expressed in both epidermis and dermis of murine and human skin and that Mstn-null mice exhibited delayed skin wound healing attributable to a combination of effects resulting from delayed epidermal reepithelialization and dermal contraction. In epidermis, reduced keratinocyte migration and protracted keratinocyte proliferation were observed, which subsequently led to delayed recovery of epidermal thickness and slower reepithelialization. Furthermore, primary keratinocytes derived from Mstn-null mice displayed reduced migration capacity and increased proliferation rate as assessed through in vitro migration and adhesion assays, as well as bromodeoxyuridine incorporation and Western blot analysis. Moreover, in dermis, both fibroblast-to-myofibroblast transformation and collagen deposition were concomitantly reduced, resulting in a delayed dermal wound contraction. These decreases are due to the inhibition of TGF-β signaling. In agreement, the expression of decorin, a naturally occurring TGF-β suppressor, was elevated in Mstn-null mice; moreover, topical treatment with TGF-β1 protein rescued the impaired skin wound healing observed in Mstn-null mice. These observations highlight the interplay between TGF-β and Mstn signaling pathways, specifically through Mstn regulation of decorin levels during the skin wound healing process. Thus we propose that Mstn agonists might be beneficial for skin wound repair.

Download Full-text

Collagen-Derived Di-Peptide, Prolylhydroxyproline (Pro-Hyp): A New Low Molecular Weight Growth-Initiating Factor for Specific Fibroblasts Associated With Wound Healing

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.548975 ◽

2020 ◽

Vol 8 ◽

Author(s):

Kenji Sato ◽

Tomoko T. Asai ◽

Shiro Jimi

Keyword(s):

Molecular Weight ◽

Wound Healing ◽

Chronic Wounds ◽

Therapeutic Potential ◽

Healing Process ◽

Collagen Fibrils ◽

Wound Healing Process ◽

Low Molecular Weight ◽

Collagen Hydrolysate ◽

Weight Growth

Many cells and soluble factors are involved in the wound healing process, which can be divided into inflammatory, proliferative, and remodeling phases. Fibroblasts play a crucial role in wound healing, especially during the proliferative phase, and show heterogeneity depending on lineage, tissue distribution, and extent of differentiation. Fibroblasts from tissue stem cells rather than from healthy tissues infiltrate wounds and proliferate. Some fibroblasts in the wound healing site express the mesenchymal stem cell marker, p75NTR. In the cell culture system, fibroblasts attached to collagen fibrils stop growing, even in the presence of protein growth factors, thus mimicking the quiescent nature of fibroblasts in healthy tissues. Fibroblasts in wound healing sites proliferate and are surrounded by collagen fibrils. These facts indicate presence of new growth-initiating factor for fibroblasts attached to collagen fibrils at the wound healing site, where the collagen-derived peptide, prolyl-hydroxyproline (Pro-Hyp), is generated. Pro-Hyp triggers the growth of p75NTR-positive fibroblasts cultured on collagen gel but not p75NTR-negative fibroblasts. Thus, Pro-Hyp is a low molecular weight growth-initiating factor for specific fibroblasts that is involved in the wound healing process. Pro-Hyp is also supplied to tissues by oral administration of gelatin or collagen hydrolysate. Thus, supplementation of gelatin or collagen hydrolysate has therapeutic potential for chronic wounds. Animal studies and human clinical trials have demonstrated that the ingestion of gelatin or collagen hydrolysate enhances the healing of pressure ulcers in animals and humans and improves delayed wound healing in diabetic animals. Therefore, the low molecular weight fibroblast growth-initiating factor, Pro-Hyp, plays a significant role in wound healing and has therapeutic potential for chronic wounds.

Download Full-text

Cooperative Regulation of Substrate Stiffness and Extracellular Matrix Proteins in Skin Wound Healing of Axolotls

BioMed Research International ◽

10.1155/2015/712546 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Ting-Yu Huang ◽

Cheng-Han Wu ◽

Mu-Hui Wang ◽

Bo-Sung Chen ◽

Ling-Ling Chiou ◽

...

Keyword(s):

Wound Healing ◽

Healing Process ◽

Ambystoma Mexicanum ◽

Wound Healing Process ◽

Body Parts ◽

Skin Wound Healing ◽

Skin Explants ◽

Wound Epithelium

Urodele amphibians (Ambystoma mexicanum), unique among vertebrates, can regenerate appendages and other body parts entirely and functionally through a scar-free healing process. The wound epithelium covering the amputated or damaged site forms early and is essential for initiating the subsequent regenerative steps. However, the molecular mechanism through which the wound reepithelializes during regeneration remains unclear. In this study, we developed anin vitroculture system that mimics anin vivowound healing process; the biomechanical properties in the system were precisely defined and manipulated. Skin explants that were cultured on 2 to 50 kPa collagen-coated substrates rapidly reepithelialized within 10 to 15 h; however, in harder (1 GPa) and other extracellular matrices (tenascin-, fibronectin-, and laminin-coated environments), the wound epithelium moved slowly. Furthermore, the reepithelialization rate of skin explants from metamorphic axolotls cultured on a polystyrene plate (1 GPa) increased substantially. These findings afford new insights and can facilitate investigating wound epithelium formation during early regeneration using biochemical and mechanical techniques.

Download Full-text