Proliferation Of Dermal Fibroblasts Research Articles

Injectable hyaluronate/collagen hydrogel with enhanced safety and efficacy for facial rejuvenation

Collagen, known for its excellent biocompatibility and biological properties, has limited in vivo maintenance duration after implantation, while hyaluronic acid faces challenges such as various complications and insufficient support for cell proliferation. In this study, an injectable hyaluronic acid/collagen (HCol) hydrogel was developed to achieve enhanced cell-material interactions and accelerated skin regeneration. Physical and chemical characterizations demonstrated that the HCol hydrogel was injectable and stable after the implantation. In vitro cell culture results illustrated that the hydrogel promoted the proliferation of human dermal fibroblasts, extracellular matrix expression and angiogenesis. The subcutaneous implantation in rats showed the superior biocompatibility of HCol hydrogel and enhanced secretion and deposition of extracellular matrix, compared with commercial hyaluronic acid dermal filler. MRI analysis showed that the hydrogel stably remained in vivo for at least three months. The histological examination and SHG signals further demonstrated that the hydrogel modulated fibroblast phenotype and stimulated vascular ingrowth and collagen synthesis, without inducing significant inflammation, swelling or erythema in vivo.Graphical

Melt electrowritten poly-lactic acid /nanodiamond scaffolds towards wound-healing patches

Multifunctional wound dressings, enriched with biologically active agents for preventing or treating infections and promoting wound healing, along with cell delivery capability, are highly needed. To address this issue, composite scaffolds with potential in wound dressing applications were fabricated in this study. The poly-lactic acid/nanodiamonds (PLA/ND) scaffolds were first printed using melt electrowriting (MEW) and then coated with quaternized β-chitin (QβC). The NDs were well-dispersed in the printed filaments and worked as fillers and bioactive additions to PLA material. Additionally, they improved coating effectiveness due to the interaction between their negative charges (from NDs) and positive charges (from QβC). NDs not only increased the thermal stability of PLA but also benefitted cellular behavior and inhibited the growth of bacteria. Scaffolds coated with QβC increased the effect of bacteria growth inhibition and facilitated the proliferation of human dermal fibroblasts. Additionally, we have observed rapid extracellular matrix (ECM) remodeling on QβC-coated PLA/NDs scaffolds. The scaffolds provided support for cell adhesion and could serve as a valuable tool for delivering cells to chronic wound sites. The proposed PLA/ND scaffold coated with QβC holds great potential for achieving fast healing in various types of wounds.

CircCDK13-loaded small extracellular vesicles accelerate healing in preclinical diabetic wound models

Chronic wounds are a major complication in patients with diabetes. Here, we identify a therapeutic circRNA and load it into small extracellular vesicles (sEVs) to treat diabetic wounds in preclinical models. We show that circCDK13 can stimulate the proliferation and migration of human dermal fibroblasts and human epidermal keratinocytes by interacting with insulin-like growth factor 2 mRNA binding protein 3 in an N6-Methyladenosine-dependent manner to enhance CD44 and c-MYC expression. We engineered sEVs that overexpress circCDK13 and show that local subcutaneous injection into male db/db diabetic mouse wounds and wounds of streptozotocin-induced type I male diabetic rats could accelerate wound healing and skin appendage regeneration. Our study demonstrates that the delivery of circCDK13 in sEVs may present an option for diabetic wound treatment.

Pulsed Radiofrequency Electromagnetic Fields as Modulators of Inflammation and Wound Healing in Primary Dermal Fibroblasts of Ulcers.

Venous leg ulcers are one of the most common nonhealing conditions and represent an important clinical problem. The application of pulsed radiofrequency electromagnetic fields (PRF-EMFs), already applied for pain, inflammation, and new tissue formation, can represent a promising approach for venous leg ulcer amelioration. This study aims to evaluate the effect of PRF-EMF exposure on the inflammatory, antioxidant, cell proliferation, and wound healing characteristics of human primary dermal fibroblasts collected from venous leg ulcer patients. The cells' proliferative and migratory abilities were evaluated by means of a BrdU assay and scratch assay, respectively. The inflammatory response was investigated through TNFα, TGFβ, COX2, IL6, and IL1β gene expression analysis and PGE2 and IL1β production, while the antioxidant activity was tested by measuring GSH, GSSG, tGSH, and GR levels. This study emphasizes the ability of PRF-EMFs to modulate the TGFβ, COX2, IL6, IL1β, and TNFα gene expression in exposed ulcers. Moreover, it confirms the improvement of the proliferative index and wound healing ability presented by PRF-EMFs. In conclusion, exposure to PRF-EMFs can represent a strategy to help tissue repair, regulating mediators involved in the wound healing process.

Fe-Porphyrin Cross-Linked Hydrogel for Reactive Oxygen Species Scavenging and Oxygen Generation in Diabetic Wounds.

Healing chronic diabetic wounds is challenging because of excessive reactive oxygen species (ROS) and hypoxia in the wound microenvironment. To address this issue, we propose a hydrogel wound dressing composed of polyethylene glycol (PEG) cross-linked with a biomimetic catalase, Fe-containing porphyrin (FeP) (i.e., FeP hydrogel). The immobilized FeP can serve as a catalyst for both ROS scavenging and O2 generation. The properties of the hydrogels were optimized by varying the composition ratios of the two constituent materials based on their mechanical properties and catalytic activity. Our in vitro cell experiments revealed that the FeP-80 hydrogel enhanced the proliferation and migration of keratinocytes and dermal fibroblasts and promoted the expression of angiogenic growth factors in keratinocytes. When tested with an in vivo diabetic chronic wound model, the FeP-80 hydrogel promoted wound healing by facilitating re-epithelialization, promoting angiogenesis, and suppressing inflammation, compared with other control groups.

Yeast-Derived Nucleotides Enhance Fibroblast Migration and Proliferation and Provide Clinical Benefits in Atopic Dermatitis.

Nucleotides, glycosaminoglycans, and omega-3 essential fatty acids (O3s) could be used for improving skin health, although their modes of action, alone or in combination, are not yet fully understood. To gain some insight into these mechanisms, we performed two in vitro tests and one in vivo pilot trial. The effects on human dermal fibroblast proliferation and migration were evaluated with the following compounds and combinations: 0.156 mg/mL O3s, 0.0017 mg/mL hyaluronic acid (HA), 0.0004 mg/mL dermatan sulfate (DS), 0.0818 mg/mL nucleotides, and [O3s + HA + DS] and [O3s + HA + DS + nucleotides] at the same concentrations. In both in vitro assays, adding nucleotides to [O3s + HA + DS] provided significant improvements. The resulting combination [O3s + HA + DS + nucleotides] was then tested in vivo in dogs with atopic dermatitis by oral administration of a supplement providing a daily amount of 40 mg/kg nucleotides, 0.9 mg/kg HA, 0.18 mg/kg DS, 53.4 mg/kg EPA, and 7.6 mg/kg DHA. After 30 days, the pruritus visual analog scale (pVAS) score was significantly reduced, and no adverse effects were observed. In conclusion, the combination of nucleotides plus glycosaminoglycans and O3s could serve as a useful therapeutic alternative in skin health applications.

A facile method to fabricate versatile keratin cryogels for tissue engineering applications

Human hair keratin (HHK) has been extensively explored as a biomaterial for soft tissue regeneration due to their excellent bioactivity and biocompatibility. The possibility to fabricate HHK into three-dimensional (3D) hydrogels with physical properties resembling soft tissues has been well demonstrated. However, conventional keratin hydrogels often exhibit a dense architecture that could hinder cell filtration. In the present study, HHK-based cryogels were fabricated using a freeze-thaw (FT) method, where oxidized dopamine (ODA) was employed to covalently crosslink thiol/amine rich-keratin molecules at sub-zero temperatures. The obtained HHK-ODA cryogels have micron-sized pores ranging between 100 and 200 μm and mechanical properties that can be tuned by varying the crosslinking density between ODA and HHK. Through optimization of the weight content of ODA and the number of FT cycles, the compressive strengths and stiffnesses of these cryogels achieved 15-fold increments from ∼1.5 kPa to ∼22 kPa and ∼300 Pa to ∼5000 Pa, respectively. The HHK-ODA cryogels competently supported human dermal fibroblast spreading and proliferation. Overall, this study exhibited a facile method to fabricate mechanically superior keratin-based cryogels with cell compatible microarchitecture, circumventing the need for complicated chemical modifications and the use of cytotoxic crosslinkers.

Impact of topography and added TiN-coating on adult human dermal fibroblasts after seeding on titanium surface in-vitro.

Complications of transcutaneous osseointegrated prosthetic systems (TOPS) focus on the metal-cutaneous interface at the stoma. Besides pain due to scare tissue as well as undefined neuropathic disorders, there is high evidence that the stoma presents the main risk causing hypergranulation and ascending infection. To restore the cutaneous barrier function in this functional area, soft-tissue on- or in-growth providing a vital and mechanically stable bio-artificial conjunction is considered a promising approach. In this study we assessed viability and proliferation of adult human dermal fibroblasts (HDFa) on modifications of a standard prosthetic titanium surface. Un-coated (TiAl6V4) as well as a titanium-nitrite (TiN) coated additive manufactured porous three-dimensional surface structures (EPORE®) were seeded with HDFa and compared to plain TiAl6V4 and polystyrene surfaces as control. Cell viability and proliferation were assessed at 24h and 7days after seeding with a fluorescence-based live-dead assay. Adhesion and cell morphology were analyzed by scanning electron microscopy at the respective measurements. Both EPORE® surface specifications revealed a homogenous cell distribution with flat and spread cell morphology forming filopodia at both measurements. Proliferation and trend to confluence was seen on un-coated EPORE® surfaces with ongoing incubation but appeared substantially lower on the TiN-coated EPORE® specification. While cell viability on both EPORE® specifications was comparable to plain TiAL6V4 and polystyrene controls, cell proliferation and confluence were less pronounced when compared to controls. The EPORE® topography allows for fibroblast adhesion and viability in both standard TiAl6V4 and - to a minor degree - TiN-coated specifications as a proof of principle.

PTEN hinders the formation of scars by regulating the levels of proteins in the extracellular matrix and promoting the apoptosis of dermal fibroblasts through Bcl-xL

Hypertrophic scar (HS) is a dermatological condition characterized by an excessive accumulation of proteins in the extracellular matrix (ECM) and an elevated cell count. The development of HS is thought to be linked to the disruption of dermal fibroblast proliferation and apoptosis. The processes of cell proliferation and apoptosis are notably influenced by PTEN. However, the precise mechanisms by which PTEN regulates hypertrophic scar fibroblasts (HSFs) and its overall role in scar formation are still not fully understood. The objective of this study was to investigate the influence of PTEN on hypertrophic scars(HS) and its function in the regulation of scar formation, with the aim of identifying a pivotal molecular target for scar treatment. Our results demonstrate that the overexpression of PTEN (AdPTEN) significantly suppressed the expression of type I collagen (Col I), type III collagen (Col III), and alpha smooth muscle actin (α-SMA) in HSFs. Furthermore, it was observed that the introduction of AdPTEN resulted in the suppression of Bcl-xL expression, which consequently led to an increase in the apoptosis of HSFs. Similarly, in the inhibition of collagens expression and subsequent increase in HSF apoptosis were also observed upon silencing Bcl-xL (sibcl-xL). Additionally, the in vitro model demonstrated that both AdPTEN and sibcl-xL were effective in reducing the contraction of FPCL. The findings of our study provide validation for the role of PTEN in inhibiting the development of hypertrophic scars (HS) by modulating the expression of extracellular matrix (ECM) proteins and promoting apoptosis in hypertrophic scar fibroblasts (HSFs) via Bcl-xL. These results indicate that PTEN and Bcl-xL may hold promise as potential molecular targets for therapeutic interventions aimed at managing hypertrophic scars.

Potentiation of Collagen Deposition by the Combination of Substance P with Transforming Growth Factor Beta in Rat Skin Fibroblasts.

A role for substance P has been proposed in musculoskeletal fibrosis, with effects mediated through transforming growth factor beta (TGFβ). We examined the in vitro effects of substance P on proliferation, collagen secretion, and collagen deposition in rat primary dermal fibroblasts cultured in medium containing 10% fetal bovine serum, with or without TGFβ. In six-day cultures, substance P increased cell proliferation at concentrations from 0.0002 to 100 nM. TGFβ increased proliferation at concentrations from 0.0002 to 2 pg/mL, although higher concentrations inhibited proliferation. Substance P treatment alone at concentrations of 100, 0.2, and 0.00002 nM did not increase collagen deposition per cell, yet when combined with TGFβ (5 ng/mL), increased collagen deposition compared to TGFβ treatment alone. Substance P treatment (100 nM) also increased smooth muscle actin (SMA) expression at 72 h of culture at a level similar to 5 ng/mL of TGFβ; only TGFβ increased SMA at 48 h of culture. Thus, substance P may play a role in potentiating matrix deposition in vivo when combined with TGFβ, although this potentiation may be dependent on the concentration of each factor. Treatments targeting substance P may be a viable strategy for treating fibrosis where both substance P and TGFβ play roles.

Omentum Extracellular Matrix-Silk Fibroin Hydroscaffold Promotes Wound Healing through Vascularization and Tissue Remodeling in the Diabetic Rat Model.

Nonhealing diabetic wounds are often associated with significant mortality and cause economic and clinical burdens to the healthcare system. Herein, a biomimetic hydroscaffold is developed using omentum tissue-derived decellularized-extracellular matrix (dECM) and silk fibroin (SF) proteins that associate the behavior of a collagenous fibrous scaffold and a hydrogel to reproduce all aspects of the provisional skin tissue matrix. The chemical cross-linker-free in situ gelation property of the two types of SF proteins from Bombyx mori and Antheraea assamensis ensures the adherence of dECM with surrounding tissue on the wound bed, circumventing further suturing. The physicochemical and mechanical properties of the composite hydroscaffold (SF-dECM) were thoroughly evaluated. The hydroscaffolds were found to support the growth and proliferation of human dermal fibroblasts and influence the angiogenic potential of endothelial cells under in vitro conditions. Furthermore, the healing efficacy of the composites was evaluated by generating full-thickness wounds on a streptozotocin-induced diabetic rat model. The presence of dECM components in the composite facilitated the rate of wound closure, granulation tissue formation, and re-epithelialization by providing intrinsic cues to advance the inflammatory stage and stimulating angiogenesis. Collectively, as an off-the-shelf wound dressing requiring only a single topical administration, the SF-dECM hydroscaffold is a promising, cost-effective dressing for the management of chronic diabetic wounds.

TEM1/endosialin/CD248 promotes pathologic scarring and TGF-β activity through its receptor stability in dermal fibroblasts

BackgroundPathologic scars, including keloids and hypertrophic scars, represent a common form of exaggerated cutaneous scarring that is difficult to prevent or treat effectively. Additionally, the pathobiology of pathologic scars remains poorly understood. We aim at investigating the impact of TEM1 (also known as endosialin or CD248), which is a glycosylated type I transmembrane protein, on development of pathologic scars.MethodsTo investigate the expression of TEM1, we utilized immunofluorescence staining, Western blotting, and single-cell RNA-sequencing (scRNA-seq) techniques. We conducted in vitro cell culture experiments and an in vivo stretch-induced scar mouse model to study the involvement of TEM1 in TGF-β-mediated responses in pathologic scars.ResultsThe levels of the protein TEM1 are elevated in both hypertrophic scars and keloids in comparison to normal skin. A re-analysis of scRNA-seq datasets reveals that a major profibrotic subpopulation of keloid and hypertrophic scar fibroblasts greatly expresses TEM1, with expression increasing during fibroblast activation. TEM1 promotes activation, proliferation, and ECM production in human dermal fibroblasts by enhancing TGF-β1 signaling through binding with and stabilizing TGF-β receptors. Global deletion of Tem1 markedly reduces the amount of ECM synthesis and inflammation in a scar in a mouse model of stretch-induced pathologic scarring. The intralesional administration of ontuxizumab, a humanized IgG monoclonal antibody targeting TEM1, significantly decreased both the size and collagen density of keloids.ConclusionsOur data indicate that TEM1 plays a role in pathologic scarring, with its synergistic effect on the TGF-β signaling contributing to dermal fibroblast activation. Targeting TEM1 may represent a novel therapeutic approach in reducing the morbidity of pathologic scars.

Antioxidative bioactive glass reinforced injectable hydrogel with reactive oxygen species scavenging capacity for diabetic wounds treatment

Diabetic wound healing remains a critical challenge due to its special hyperglycemic environment and disorder in wound healing process. The hyperglycemic microenvironment of diabetic wounds causes inhibitive angiogenesis and overproduction of reactive oxygen species (ROS), which leads to oxidative stress damage to biological macromolecules and cells, and hindrance of wound healing. Herein, an injectable thermo-sensitive methyl cellulose hydrogel with capacities of improving angiogenesis, scavenging ROS and antibiosis is developed to overcome the obstacles of diabetic wound healing. Through loading antioxidative cerium-doped bioactive glass nanoparticles (CeBG), the hydrogel demonstrates efficient ROS scavenging to protect cells from oxidative stress harm, as well as stimulation of dermal fibroblast proliferation and migration. Moreover, addition of quaternary chitosan (QCS) endows the hydrogel with antibacterial ability against E. coli and S. aureus. The hydrogel effectively accelerates diabetic wound healing in a mice model, and more importantly, the wound area presents improved angiogenesis, diminished inflammation and decreased ROS level. These data demonstrate that the hydrogel is a promising biomaterial for the clinical treatment of diabetic wounds, as well as facilitating the therapy of oxidative stress related diseases.

α-Catenin promotes dermal fibroblasts proliferation and migration during wound healing via FAK/YAP activation.

Skin wound healing is a complex and organized biological process, and the dermal fibroblasts play a crucial role. α-Catenin is known to be involved in regulating various cellular signals, and its role in wound healing remains unclear. Here, we have identified the pivotal role of the α-catenin/FAK/YAP signaling axis in the proliferation and migration of dermal fibroblasts, which contributes to the process of skin wound healing. Briefly, when α-catenin was knocked down specifically in dermal fibroblasts, the wound healing rate is significantly delayed. Moreover, interfering with α-catenin can impede the proliferation and migration of dermal fibroblasts both invitro and invivo. Mechanistically, the overexpression of α-catenin upregulates the nuclear accumulation of YAP and transcription of downstream target genes, resulting in enhanced the proliferation and migration of dermal fibroblasts. Furthermore, the FAK Tyr397 phosphorylation inhibitor blocked the promoting effects of α-catenin on YAP activation. Importantly, the continuous phosphorylation mutation of FAK Tyr397 reversed the retardatory effects of α-catenin knockdown on wound healing, by increasing the vitality of fibroblasts. Likewise, α-catenin/FAK was validated as a therapeutic target for wound healing in the db/db chronic trauma model. In summary, our findings have revealed a novel mechanism by which α-catenin facilitates the function of fibroblasts through the activity of the FAK/YAP signaling axis. These findings define a promising therapeutic strategy for accelerating the wound healing process.

Electrospun radially oriented berberine-PHBV nanofiber dressing patches for accelerating diabetic wound healing.

A dressing patch made of radially oriented poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers was successfully manufactured with a modified electrospinning strategy. The as-electrospun PHBV radially oriented nanofiber dressing patch exhibited uniform and bead-free nanofibrous morphology and innovative radially oriented arrangement, which was demonstrated to possess obviously improved mechanical property, increased surface hydrophilicity and enhanced biological properties compared to the PHBV nanofiber dressing patch control with traditionally randomly oriented pattern. Interestingly, it was found that the radially oriented pattern could induce the cell migration from the periphery to the center along the radially oriented nanofibers in a rapid manner. To further improve the biofunction of PHBV radially oriented nanofiber dressing patch, berberine (Beri, an isoquinoline alkaloid) with two different concentrations were encapsulated into PHBV nanofibers during electrospinning, which were found to present a sustained drug release behavior for nearly one month. Importantly, the addition of Beri could impart the dressing patch with excellent anti-inflammatory property by significantly inhibiting the secretion of pro-inflammatory factors of M1 macrophages, and also showed an additive influence on promoting the proliferation of human dermal fibroblasts (HDFs), as well as inhibiting the growth of E. coli, S. aureus and C. albicans, compared with the Beri-free dressing patch. In the animal studies, the electrospun PHBV radially oriented nanofiber dressing patch loading with high Beri content was found to obviously accelerate the healing process of diabetic mouse full-thickness skin wound with shortened healing time (100% wound closure rate after 18 days' treatment) and improved healing quality (improved collagen deposition, enhanced re-epithelialization and neovascularization and increased hair follicles). In all, this study reported an innovative therapeutic strategy integrating the excellent physical cues of electrospun PHBV radially oriented nanofiber dressing patch with the multiple biological cues of Beri for the effective treatment of hard-to-heal diabetic wounds.

Bio-response of copper-magnesium co-substituted mesoporous bioactive glass for bone tissue regeneration.

Mesoporous bioactive glass (MBG) is widely acknowledged in bone tissue engineering due to its mesoporous structure, large surface area, and bioactivity. Recent research indicates that introduction of metallic ions has beneficial impacts on bone metabolism and angiogenesis. Thus, the features of MBG can be modified by incorporating combinations of ions, such as magnesium (Mg) and copper (Cu), which can play a considerable role in bone formation, influencing angiogenesis, osteogenesis, as well as antibacterial properties. In this study, Mg and Cu were co-doped for the first time (in a ratio of 1 : 1) in 80SiO2-5P2O5-(15 - 2x)CaO-xMgO-xCuO glass composition with x = 0, 0.5, 1, and 2 mol%, synthesized using the sol-gel and evaporation-induced self-assembly method. X-ray diffraction analysis confirmed the amorphous nature of the powders, while inductively coupled plasma-optical emission spectrometry verified the existence of dopant ions in the respective amounts. The nitrogen sorption method indicated the formation of uniform cylindrical mesopores which are open at both ends and a high surface area of the powders. TEM images show fringes, indicating an ordered mesoporous structure in all MgCu co-doped systems. In vitro bioactivity was observed in all MBG powders, confirmed by the formation of an apatite phase when placed in simulated body fluid (SBF). Flake-like microstructure characteristics of HAp crystals found on the surface of MBG powders were visualized using FESEM. Cytotoxicity tests at lower concentrations (0.1 and 1 wt/vol%) of co-doped 2MC MBG (co-doping up to 2 mol%) showed cell proliferation and viability of osteoblast-like MG-63 cells and normal human dermal fibroblast (NHDF) cells similar to the basic glass 80S. Antibacterial study of MBG pellets showed an increment in the zone of inhibition with the sequential addition of doping ions. The turbidity measurement of bacterial cultures revealed that the optimal concentration for effectively inhibiting bacterial growth was 1 wt/vol% (i.e., 10 mg mL-1) concentration of MBG extracts. The result suggested that the incorporation of Mg and Cu ions in MBG in lower concentrations of up to 2 mol% can be useful in bone regeneration owing to bioactivity, cell proliferation, and antibacterial characteristics.

Bioactive electrospun nanoyarn-constructed textile dressing patches delivering Chinese herbal compound for accelerated diabetic wound healing

The treatment of tenacious diabetic wounds still remains an enormous challenge in clinics, originated from the complicated pathological microenvironment of wound sites. Therefore, it's urgently required to develop one type of innovative dressing patch with appropriate microstructure and multifunctions to regulate the pathological microenvironment and promote the regeneration of diabetic wounds. In this study, novel gelatin (Gel)/poly (L-lactic acid) (PLLA) nanofibrous yarns loading with or without Salvia miltiorrhiza Bunge-Radix Puerariae herbal compound (SRHC) are fabricated by using our modified electrospinning strategy, which are further interlaced into nanofibrous woven fabrics respectively, serving as biofunctional dressing patches for potential diabetic wound treatment application. The actual photographs and SEM images confirm that all the different nanofibrous textiles with or without SRHC exhibit a uniform interwoven structure of warp and weft, and the internal nanofibers present bead-free morphology and uniaxially oriented structure along the longitudinal axis of nanofibrous yarns. Moreover, all the different nanofibrous woven fabrics are demonstrated to possess strong mechanical properties and great surface wettability. The in vitro cell characterization shows that the addition of SRHC can significantly promote the attachment and proliferation of human dermal fibroblasts (HDFs), and also dramatically inhibit the secretion levels of proinflammatory factors of M1 macrophages. The in vivo diabetic mouse full-thickness skin model experiments reveal that the as-developed SRHC-loaded Gel/PLLA nanofibrous textile shows the best performances referring to shorten wound healing time (100 % wound closure after 18 days of treatment) and high-quality regeneration (i.e., enhance collagen deposition, improve re-epithelialization and neovascularization, and increase hair follicles), which assuredly finds great interests serving as an innovative dressing patch for the treatment of hard-to-heal diabetic wounds.

MiR-21 Expressed by Dermal Fibroblasts Enhances Skin Wound Healing Through the Regulation of Inflammatory Cytokine Expression.

The management of skin wound healing is still a challenge. MicroRNA-21 (miR-21) has been reported to play important roles in wound repair; however, the underlying mechanism needs to be further clarified. The present study aimed to study the direct role of miR-21 in skin wound healing in miR-21 KO mice and to investigate the role of miR-21 in controlling the migration and proliferation of primary human skin cells and its underlying mechanism(s). miR-21 KO and wild-type (WT) mice were used for in vivo wound healing assays, while mouse and human primary skin cells were used for in vitro assays. miR-21 inhibitors or mimics or negative control small RNAs were transfected to either inhibit or enhance miR-21 expression in the human primary dermal fibroblasts or epidermal cells. RNA sequencing analysis was performed to identify the potential molecular pathways involved. We found that the loss of miR-21 resulted in slower wound healing in miR-21 KO mouse skin and especially delayed the healing of dermal tissue. In vitro assays demonstrated that the reduced expression of miR-21 caused by its inhibitor inhibited the migration of human primary dermal fibroblasts, which could be enhanced by increased miR-21 expression caused by miR-21 mimics. RNA-sequence analysis revealed that the inhibition of miR-21 expression downregulated the inflammatory response pathways associated with the decreased expression of inflammatory cytokines, and the addition of IL-1β into the culture medium enhanced the migration and proliferation of dermal fibroblasts in vitro. In conclusion, miR-21 in dermal fibroblasts can promote the migration and growth of epidermal and dermal cells to enhance skin wound healing through controlling the expression of inflammatory cytokines.

Hypoxic microenvironment promotes dermal fibroblast migration and proliferation via a BNIP3-autophagy pathway.

Upon injury, nearby cells, including fibroblasts at the wound edge, are often found in a hypoxic microenvironment. Nevertheless, the influence of hypoxia on skin fibroblasts is poorly understood. Using previously established mouse full-thickness wounds, we show that Bcl-2 and adenovirus E1B 19-kDa interacting protein 3 (BNIP3) expression was significantly elevated at the wound edge, and hypoxia treatment enhanced BNIP3 expression in fibroblasts. Interestingly, BNIP3 promoted the migration and proliferation, as well as the activation of autophagy, in fibroblasts under hypoxia. The hypoxia-induced autophagy was found to induce the migration and proliferation of fibroblasts, a process that could be reversed by knocking down the autophagy-related gene for autophagy protein 5, ATG5. Furthermore, hypoxia-inducible factor 1 subunit alpha (HIF-1α) was significantly upregulated in fibroblasts under hypoxia treatment, and HIF-1α knockdown attenuated the hypoxia-induced expression of BNIP3 and the migration and proliferation of fibroblasts. Altogether, our results establish the hypoxia-BNIP3-autophagy signaling axis as a newly identified regulatory mechanism of skin fibroblast migration and proliferation upon wounding. Autophagy intervening might thus represent a promising therapeutic strategy for patients with chronic refractory wounds.

In situ photo-crosslinkable hyaluronic acid-based hydrogel embedded with GHK peptide nanofibers for bioactive wound healing

A versatile hydrogel was developed for enhancing bioactive wound healing by introducing the amphiphilic GHK peptide (GHK-C16) into a photo-crosslinkable tyramine-modified hyaluronic acid (HA-Ty). GHK-C16 self-assembled into GHK nanofibers (GHK NF) in HA-Ty solution, which underwent in situ gelation after the wound area was filled with precursor solution. Blue light irradiation (460–490 nm), with riboflavin phosphate as a photoinitiator, was used to trigger crosslinking, which enhanced the stability of the highly degradable hyaluronic acid and enabled sustained release of the nanostructured GHK derivatives. The hydrogels provided a microenvironment that promoted the proliferation of dermal fibroblasts and the activation of cytokines, leading to reduced inflammation and increased collagen expression during wound healing. The complexation of Cu2+ into GHK nanofibers resulted in superior wound healing capabilities compared with non-lipidated GHK peptide with a comparable level of growth factor (EGF). Additionally, nanostructured Cu-GHK improved angiogenesis through vascular endothelial growth factor (VEGF) activation, which exerted a synergistic therapeutic effect. Furthermore, in vivo wound healing experiments revealed that the Cu-GHK NF/HA-Ty hydrogel accelerated wound healing through densely packed remodeled collagen in the dermis and promoting the growth of denser fibroblasts. HA-Ty hydrogels incorporating GHK NF also possessed improved mechanical properties and a faster wound healing rate, making them suitable for advanced bioactive wound healing applications. Statement of significanceBy combining photo-crosslinkable tyramine-modified hyaluronic acid with self-assembled Cu-GHK-C16 peptide nanofibers (Cu-GHK NF), the Cu-GHK NF/HA-Ty hydrogel offers remarkable advantages over conventional non-structured Cu-GHK for wound healing. It enhances cell proliferation, migration, and collagen remodeling—critical factors in tissue regeneration. The incorporation of GHK nanofibers complexed with copper ions imparts potent anti-inflammatory effects, promoting cytokine activation and angiogenesis during wound healing. The Cu-GHK NF/hydrogel's unique properties, including in situ photo-crosslinking, ensure high customization and potency in tissue regeneration, providing a cost-effective alternative to growth factors. In vivo experiments further validate its efficacy, demonstrating significant wound closure, collagen remodeling, and increased fibroblast density. Overall, the Cu-GHK NF/HA-Ty hydrogel represents an advanced therapeutic option for wound healing applications.