Proliferation Of Human Fibroblasts Research Articles

Silk fibroin microgrooved zirconia surfaces improve connective tissue sealing through mediating glycolysis of fibroblasts

The use of zirconia has significantly enhanced the aesthetic outcomes of implant restorations. However, peri-implantitis remains a challenge to long-term functionality of implants. Unlike the perpendicularly arranged collagen fibers in periodontal tissue, those in peri-implant tissue lie parallel to the abutment surface and contain fewer fibroblasts, making them more prone to inflammation. Studies have shown that microgroove structures on implant abutments could improve surrounding soft tissue structure. However, creating precise microgrooves on zirconia without compromising its mechanical integrity is technically challenging. In this study, we applied inkjet printing, an additive manufacturing technique, to create stable silk fibroin microgroove (SFMG) coatings of various dimensions on zirconia substrates. SFMG significantly improved the hydrophilicity of zirconia and showed good physical and chemical stability. The SFMG with 90 μm interval and 10 μm depth was optimal in promoting the proliferation, alignment, and extracellular matrix production of human gingival fibroblasts (HGFs). Moreover, the in vitro results revealed that SFMG stimulated key glycolytic enzyme gene expression in HGFs via the PI3K-AKT-mTOR pathway. Additionally, the in vivo results of histological staining of peri-abutments soft tissue showed that SFMG promoted the vertical alignment of collagen fibers relative to the abutment surface, improving connective tissue sealing around the zirconia abutment. Our results indicated that SFMG on zirconia can enhance HGF proliferation, migration and collagen synthesis by regulating glycolysis though PI3K-AKT-mTor pathway, thereby improving connective tissue sealing.

152 Three-Dimensional Printing Bespoke Polycaprolactone Tracheal Splint for Treatment of Tracheomalacia

Abstract Aim Tracheomalacia can be a fatal airway disease with limited treatment options. Three-dimensional (3D) printing has led to various patient-specific tracheal stents and splints. However, these are limited by mismatched stiffness and resulting granulation tissue formation and fibrosis. We aimed to develop a non-toxic and biodegradable polycaprolactone expandable tracheal splint by 3D printing. By fine-tuning mechanical properties, we hypothesised that the splint would match the native trachea and accommodate tracheal growth in tracheomalacia patients. Method We designed splints consisting of concave topological patterns with varying angles and aspect ratios. Fabrication occurred using polycaprolactone filaments in a 3D fuse deposition modelling printer. Uniaxial tensile studies were performed on splints to assess Young’s Modulus, Toughness, Ultimate strain, Strength, and Poisson ratio. Splints displaying optimal mechanical properties were tested for human lung fibroblast cell viability, proliferation, and morphology. Results On a mechanical testing rig, uniaxial tensile studies confirmed that splint designs with smaller angles and negative Poisson ratios of up to strains of 25%, better mimicked mechanical properties of native human trachea, accommodating tracheal growth. In vitro studies using these tracheal splints demonstrated successful human lung cell proliferation with low cell toxicity. Conclusions This study characterised 3D printed bespoke tracheal splints with various 2D structures, showing mechanical properties that match the native trachea. However, in vivo studies investigating cellular interactions on our splint must be performed to further evaluate the ability for endogenous tissue formation.

Development of a Cellular Assay as a Personalized Model for Testing Chronic Wound Therapeutics

Exudates of non-healing wounds contain drivers of pathogenicity. We utilized >800 exudates from non-healing and healing wounds of diverse etiologies, collected by three different methods, to develop a wound-specific, cell-based functional biomarker assay. Human dermal fibroblast proliferation served as readout to a) to differentiate between healing and non-healing wounds, b) follow the healing process of individual patients, and c) assess the effects of therapeutics for chronic wounds ex vivo. We observed a strong correlation between wound chronicity and inhibitory effects of individual exudates on fibroblast proliferation, with good diagnostic sensitivity (76-90%, depending on the sample collection method). Transition of a clinically non-healing to a healing phenotype restored fibroblast proliferation and extracellular matrix formation while reducing inflammatory cytokine production. Transcriptional analysis of fibroblasts exposed to ex vivo non-healing wound exudates revealed an induction of inflammatory cytokine- and chemokine pathways and the unfolded protein response, indicating that these changes may contribute to the pathology of non-healing wounds. Testing the wound therapeutics platelet derived growth factor and silver sulfadiazine yielded responses in line with clinical experience and indicate the usefulness of the assay to search for and profile new therapeutics.

Novel Insights into Phaseolus vulgaris L. Sprouts: Phytochemical Analysis and Anti-Aging Properties.

Skin aging is an inevitable and intricate process instigated, among others, by oxidative stress. The search for natural sources that inhibit this mechanism is a promising approach to preventing skin aging. The purpose of our study was to evaluate the composition of phenolic compounds in the micellar extract of Phaseolus vulgaris sprouts. The results of a liquid chromatography-mass spectrometry (LC-MS) analysis revealed the presence of thirty-two constituents, including phenolic acids, flavanols, flavan-3-ols, flavanones, isoflavones, and other compounds. Subsequently, the extract was assessed for its antioxidant, anti-inflammatory, anti-collagenase, anti-elastase, anti-tyrosinase, and cytotoxic properties, as well as for the evaluation of collagen synthesis. It was demonstrated that micellar extract from common bean sprouts has strong anti-aging properties. The performed WST-8 (a water-soluble tetrazolium salt) assay revealed that selected concentrations of extract significantly increased proliferation of human dermal fibroblasts compared to the control cells in a dose-dependent manner. A similar tendency was observed with respect to collagen synthesis. Our results suggest that micellar extract from Phaseolus vulgaris sprouts can be considered a promising anti-aging compound for applications in cosmetic formulations.

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.

Using network pharmacology to discover potential drugs for hypertrophic scars.

Hypertrophic scar is a disease of abnormal skin fibrosis caused by excessive fibroblast proliferation, and existing drugs cannot achieve satisfactory therapeutic effects. This study aimed to explore the molecular pathogenesis of hypertrophic scars and screen effective drugs for hypertrophic scars. Existing human hypertrophic scar RNA sequencing data were utilized to search for hypertrophic scar-related gene modules and key genes through weighted gene co-expression network analysis (WGCNA). Candidate compounds were screened in a compound library. Potential drugs were screened by molecular docking and verified in human hypertrophic scar fibroblasts and a mouse mechanical force hypertrophic scar model. WGCNA showed that hypertrophic scar-associated gene modules influence focal adhesion, transforming growth factor β (TGF-β) signaling pathway, and other biological pathways. Integrin β1 (ITGB1) is the hub protein. Among the candidate compounds obtained by computer virtual screening and molecular docking, crizotinib, sorafenib, and SU11274 can inhibit the proliferation and migration of human hypertrophic scar fibroblasts and pro-fibrotic gene expression. Crizotinib had the best effect on hypertrophic scar attenuation in mouse models. At the same time, mouse ITGB1 small interfering RNA (siRNA) can also inhibit mouse scar hyperplasia. ITGB1 and TGF-β signaling pathways are important for hypertrophic scar formation. Crizotinib could serve as a potential drug for hypertrophic scars.

Analysis of Gingival Fibroblasts Behaviour in the Presence of 3D-Printed versus Milled Methacrylate-Based Dental Resins-Do We Have a Winner?

Computer-aided design and computer-aided manufacturing (CAD/CAM) techniques are based on either subtractive (milling prefabricated blocks) or additive (3D printing) methods, and both are used for obtaining dentistry materials. Our in vitro study aimed to investigate the behavior of human gingival fibroblasts exposed to methacrylate (MA)-based CAD/CAM milled samples in comparison with that of MA-based 3D-printed samples to better elucidate the mechanisms of cell adaptability and survival. The proliferation of human gingival fibroblasts was measured after 2 and 24 h of incubation in the presence of these samples using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the membrane integrity was assessed through the lactate dehydrogenase release. The level of reactive oxygen species, expression of autophagy-related protein LC3B-I, and detection of GSH and caspase 3/7 were evaluated by fluorescence staining. The MMP-2 levels were measured using a Milliplex MAP kit. The incubation with MA-based 3D-printed samples significantly reduced the viability, by 16% and 28% from control after 2 and 24 h, respectively. There was a 25% and 55% decrease in the GSH level from control after 24 h of incubation with the CAD/CAM milled and 3D-printed samples, respectively. In addition, higher levels of LC3B-I and MMP-2 were obtained after 24 h of incubation with the MA-based 3D samples compared to the CAD/CAM milled ones. Therefore, our results outline that the MA-CAD/CAM milled samples displayed good biocompatibility during 24-h exposure, while MA-3D resins are proper for short-term utilization (less than 24 h).

Silica's silent threat: Contributing to skin fibrosis in systemic sclerosis by targeting the HDAC4/Smad2/3 pathway

Nowadays, silica products are widely used in daily life, especially in skin applications, which inevitably increases the risk of silica exposure in general population. However, inadequate awareness of silica's potential hazards and lack of self-protection are of concern. Systemic sclerosis (SSc) is characterized by progressive tissue fibrosis under environmental and genetic interactions. Silica exposure is considered an important causative factor for SSc, but its pathogenesis remains unclear. Within this study, we showed that lower doses of silica significantly promoted the proliferation, migration, and activation of human skin fibroblasts (HSFs) within 24 h. Silica injected subcutaneously into mice induced and exacerbated skin fibrosis. Notably, silica increased histone deacetylase-4 (HDAC4) expression by inducing its DNA hypomethylation in normal HSFs. The elevated HDAC4 expression was also confirmed in SSc HSFs. Furthermore, HDAC4 was positively correlated with Smad2/3 phosphorylation and COL1, α-SMA, and CTGF expression. The HDAC4 inhibitor LMK235 mitigated silica-induced upregulation of these factors and alleviated skin fibrosis in SSc mice. Taken together, silica induces and exacerbates skin fibrosis in SSc patients by targeting the HDAC4/Smad2/3 pathway. Our findings provide new insights for evaluating the health hazards of silica exposure and identify HDAC4 as a potential interventional target for silica-induced SSc skin fibrosis.

CircNSD1 promotes cardiac fibrosis through targeting the miR-429-3p/SULF1/Wnt/β-catenin signaling pathway.

Cardiac fibrosis is a detrimental pathological process, which constitutes the key factor for adverse cardiac structural remodeling leading to heart failure and other critical conditions. Circular RNAs (circRNAs) have emerged as important regulators of various cardiovascular diseases. It is known that several circRNAs regulate gene expression and pathological processes by binding miRNAs. In this study we investigated whether a novel circRNA, named circNSD1, and miR-429-3p formed an axis that controls cardiac fibrosis. We established a mouse model of myocardial infarction (MI) for in vivo studies and a cellular model of cardiac fibrogenesis in primary cultured mouse cardiac fibroblasts treated with TGF-β1. We showed that miR-429-3p was markedly downregulated in the cardiac fibrosis models. Through gain- and loss-of-function studies we confirmed miR-429-3p as a negative regulator of cardiac fibrosis. In searching for the upstream regulator of miR-429-3p, we identified circNSD1 that we subsequently demonstrated as an endogenous sponge of miR-429-3p. In MI mice, knockdown of circNSD1 alleviated cardiac fibrosis. Moreover, silence of human circNSD1 suppressed the proliferation and collagen production in human cardiac fibroblasts in vitro. We revealed that circNSD1 directly bound miR-429-3p, thereby upregulating SULF1 expression and activating the Wnt/β-catenin pathway. Collectively, circNSD1 may be a novel target for the treatment of cardiac fibrosis and associated cardiac disease.

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.

Inhibition of activin receptor 2 signalling ameliorates metabolic dysfunction–associated steatotic liver disease in western diet/L-NAME induced cardiometabolic disease

ObjectiveBlockade of activin 2 receptor (ACVR2) signaling has been shown to improve insulin sensitivity and aid in weight loss. Inhibition of ACVR2 signaling restores cardiac function in multiple heart failure models. However, its potential in the treatment of obesity-related cardiometabolic disease remains unknown. Here, we investigated targeting ACVR2 signaling in cardiometabolic disease manifested with metabolic dysfunction–associated steatotic liver disease (MASLD). MethodsMice were fed a high-fat, high-sugar diet combined with the administration of nitric oxide synthase inhibitor L-NAME in drinking water, which causes hypertensive stress. For the last eight weeks, the mice were treated with the soluble ACVR2B decoy receptor (sACVR2B-Fc). ResultssACVR2B-Fc protected against the development of comorbidities associated with cardiometabolic disease. This was most pronounced in the liver where ACVR2 blockade attenuated the development of MASLD including cessation of pro-fibrotic activation. It also significantly reduced total plasma cholesterol levels, impeded brown adipose tissue whitening, and improved cardiac diastolic function. In vitro, ACVR2 ligands activin A, activin B and GDF11 induced profibrotic signaling and the proliferation of human cardiac fibroblasts. ConclusionsBlockade of ACVR2B exerts broad beneficial effects for therapy of cardiometabolic disease. By reducing obesity, ameliorating cardiovascular deterioration and restraining MASLD, blockade of ACVR2B signaling proves a potential target in MASLD and its comorbidities.

Photothermal modulation of gingival fibroblasts via polydopamine-coated zirconia: A novel approach for promoting peri-implant soft tissue integration

Achieving robust soft tissue integration around dental implants is crucial for long-term clinical success, as it forms a protective biological seal against bacterial invasion. However, the soft tissue attachment to implants is relatively deficient compared to natural teeth, particularly in the connective tissue region lacking sufficient gingival fibroblasts and collagen fiber alignment. This study proposed an innovative strategy to enhance peri‑implant soft tissue integration by modulating gingival fibroblast behavior via photothermal conversion. Zirconia surfaces were coated with polydopamine (PDA), a melanin-like polymer exhibiting near-infrared (NIR) absorption for photothermal conversion. Under NIR irradiation, the PDA coating enabled mild hyperthermia (42–43 °C) on the zirconia surface. Remarkably, this mild photothermal stimulation significantly promoted human gingival fibroblast proliferation, adhesion, and collagen production compared to unmodified zirconia in vitro. By utilizing the photothermal properties of PDA coatings to modulate cellular behaviors beneficial for connective tissue formation, this approach provides a promising avenue to achieve improved soft tissue integration and long-term stability of dental implants. The findings highlight the innovative potential of combining biomaterial surface engineering with photothermal therapy for applications in implant dentistry.

Achieving Complete Human Gingival Fibroblast Collagen Coverage on Implant Abutments through Vacuum Ultraviolet (VUV) Photofunctionalization.

The formation of a biological seal between implant abutments and the surrounding soft tissue is a preventive strategy against peri-implantitis. The aim of this study is to test the hypothesis that surfaces of prosthetic implant abutments treated with vacuum ultraviolet (VUV) light enhance the growth and function of human gingival fibroblasts. Implant abutments were treated with 172 nm VUV light for one minute. Untreated abutments were subjected as controls. Their surface properties were characterized using SEM, contact angle measurements, and chemical composition analysis. Human gingival fibroblasts were cultured on both untreated and VUV-treated abutments to evaluate cell attachment, proliferation, distribution, and collagen production. Cell detachment assays were also performed under various mechanical and chemical stimuli. After VUV treatment, implant abutments demonstrated a notable transition from hydrophobic to hydrophilic wettability. Surface element analysis revealed a considerable reduction in surface carbon and increases in oxygen and titanium elements on the VUV-treated surfaces. On day 1 of culture, 3.9 times more fibroblasts attached on VUV-treated abutments than on untreated control abutments. Fibroblastic proliferation increased 1.9-3.1 times on VUV-treated abutments, along with a significant improvement in the distribution of populating cells. Collagen production on VUV-treated abutments increased by 1.5-1.7 times. While untreated abutment surfaces showed voids and limited spread of collagen deposition, dense and full coverage of collagen was observed on VUV-treated abutments, with a great contrast in the challenging axial surface zone. Cell retention against mechanical and chemical detaching stimuli was increased 11.3 and 4.3 times, respectively, by VUV treatment. Treatment of implant abutments with VUV light for one minute resulted in a reduction of surface carbon and a transformation of the surface from hydrophobic to hydrophilic. This led to enhanced attachment, proliferation, and retention of human gingival fibroblasts, along with nearly complete collagen coverage on implant abutments. These in vitro results indicate the promising potential of utilizing VUV photofunctionalized implant abutments to enhance soft tissue reaction and sealing mechanisms.

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.

ZNF862 induces cytostasis and apoptosis via the p21-RB1 and Bcl-xL-Caspase 3 signaling pathways in human gingival fibroblasts.

This study investigates the effects of ZNF862 on the proliferation and apoptosis of human gingival fibroblasts and their related mechanisms. As a major transcription factor family, zinc finger proteins (ZFPs) regulate cell differentiation, growth, and apoptosis through their conserved zinc finger motifs, which allow high flexibility and specificity in gene regulation. In our previous study, ZNF862 mutation was associated with hereditary gingival fibromatosis. Nevertheless, little is known about the biological function of ZNF862. Therefore, this study was aimed to reveal intracellular localization of ZNF862, the influence of ZNF862 on the growth and apoptosis of human gingival fibroblasts (HGFs) and its potential related mechanisms. Immunohistochemistry, immunofluorescence staining, and western blotting were performed to determine the intracellular localization of ZNF862 in HGFs. HGFs were divided into three groups: ZNF862 overexpression group, ZNF862 interference group, and the empty vector control group. Then, the effects of ZNF862 on cell proliferation, migration, cell cycle, and apoptosis were evaluated. qRT-PCR and western blotting were performed to further explore the mechanism related to the proliferation and apoptosis of HGFs. ZNF862 was found to be localized in the cytoplasm of HGFs. Invitro experiments revealed that ZNF862 overexpression inhibited HGFs proliferation and migration, induced cell cycle arrest at the G0/G1-phase and apoptosis. Whereas, ZNF862 knockdown promoted HGFs proliferation and migration, accelerated the transition from the G0/G1 phase into the S and G2/M phase and inhibited cell apoptosis. Mechanistically, the effects of ZNF862 on HGFs proliferation and apoptosis were noted to be dependent on inhibiting the cyclin-dependent kinase inhibitor 1A (p21)-retinoblastoma 1 (RB1) signaling pathway and enhancing the B-cell lymphoma-extra-large (Bcl-xL)-Caspase 3 signaling pathway. Our results for the first time reveal that ZNF862 is localized in the cytoplasm of HGFs. ZNF862 can inhibit the proliferation of HGFs by inhibiting the p21-RB1 signaling pathway, and it also promotes the apoptosis of HGFs by enhancing the Bcl-xL-Caspase 3 signaling pathway.

Nicotine reduces cell viability and induces oxidative stress in human gingival fibroblasts

BackgroundNicotine, as the main component of cigarettes, is known to interfere with the proliferation of human gingival fibroblasts (HGFs) and can trigger oxidative stress. This study aimed to analyze the impact of nicotine on viability, expression of the antioxidant Nrf2, levels of the product of oxidative stress malondialdehyde (MDA), and the migration capacity of HGFs. MethodsAn experimental laboratory study used fibroblasts isolated from healthy human gingiva. The cells were grouped into the non-treatment control group (NTC), the solvent control (SC), and the treatment groups, exposed to nicotine at various concentrations for twenty-four hours. Cell viability was assesed using the cell counting kit-8 (CCK-8), Nrf2 expression was examined using ELISA, MDA level was measured using an MDA kit, and migration capacity was assessed using a scratch assay. Statistical analysis used one-way Anova or Kruskal-Wallis test. A p-value of <0.05 was expressed statistically significant. ResultsThe Cell viability was substantially reduced in the nicotine group compared to the untreated group, accompanied by changes in cell morphology. In contrast, Nrf2 expression increased significantly (p=0.010) in the 5 mM nicotine group compared with the control group. The MDA levels were not significantly distinct across groups (p=0.056). Cell migration was delayed significantly in the 5 mM nicotine group at 72 hours after scratching compared to the control group. ConclusionNicotine decreased HGFs viability and increased Nrf2 expression significantly in a dose-dependent manner. Nicotine at 5 mM concentration did not alter MDA levels but delayed cell migration.

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.

Food Safety Assessment and Nutraceutical Outcomes of Dairy By-Products: Ovine Milk Whey as Wound Repair Enhancer on Injured Human Primary Gingival Fibroblasts.

The valorization of milk whey appears to be a promising strategy for managing by-products from dairy food industries, which incur demanding economic costs for treatment and/or disposal. Thanks to its numerous bioactive components, whey is expected to be increasingly incorporated into foods in the future. We investigated the safety of ovine milk whey through in vitro experiments on human primary gingival fibroblast (HGF-1) proliferation and wound healing. Fibroblasts play a crucial role in the repair processes from the late inflammatory phase until the final stages. Cells treated with varying concentrations of ovine whey (0.01%, 0.1%, 1%, and 10%) were able to close wounds more rapidly than vehicle-treated cells. Time- and dose-dependent responses were observed in cell populations exposed to ovine whey. Specifically, wounds treated with 0.1% and 10% milk whey showed better migratory capabilities compared to those treated with 0.01% and 1% milk whey after 24 and 48 h. In addition, ovine milk whey stimulates extracellular matrix deposition, as evidenced by the increasing levels of CD44 antigen density evaluated through FACS analysis, as well as COL1A1 expression measured both via RT-qPCR and immunofluorescence. This phenomenon was particularly evident at concentrations of 0.01% and 10%. Ensuring quality and safety has become a major concern for health authorities in the food industry. Our findings suggest that ovine milk whey is safe and possesses regenerative properties. It facilitates tissue re-establishment following exposure to environmental stress, particularly accelerating gingival wound closure.