Split-level folding, step-type tension-relieving suture technique, and the evaluation on scar minimization.

BACKGROUND: Prevailing tension-reducing suture methods have a spectrum of issues. This study presents a straightforward yet highly efficacious suture technique known as the Split-level Folding, Step-type Tension-relieving Suture technique, which could play a pivotal role in preempting incisional scarring. AIMS: To introduce Split-level Folding, Step-type Tension-relieving Suture technique and assess its effect on scar minimization. METHODS: A retrospective analysis of 64 patients who underwent treatment utilizing the proposed suturing methodology. Assessment parameters included the Patient and Observer Scar Assessment Scale (POSAS), the Vancouver Scar Scale (VSS), scar width, complications, and all evaluated at 6- and 12-month postoperatively. RESULTS: At 12-month follow-up, the POSAS and VSS scores in the normal suture group (32.58 ± 5.43, 3.58 ± 1.39) were considerably higher than the step-type suture group (29.75 ± 3.56, p = 0.0007; 2.78 ± 1.17, p = 0.0006). Moreover, the step-type suture group showcased a significantly narrower average incision scar width (1.62 ± 0.36) than the normal suture group (1.87 ± 0.42, p = 0.0004). This novel tension-relieving suture technique that effectively circumvents the occurrence of persistent localized eversion and other complications often associated with traditional tension-relieving sutures. CONCLUSIONS: The Split-level Folding, Step-type Tension-relieving Suture technique emerges as a highly promising option for averting incisional scarring. This suture method works well for incisions on the chest, back, and extremities, resulting in significantly better long-term outcomes.

On-Site Construction of a Full-Thickness Skin Equivalent with Endothelial Tube Networks via Multilayer Electrospinning for Wound Coverage.

Novel full-thickness skin substitutes are of increasing interest due to the inherent limitations of current models lacking capillary networks. Herein, we developed a novel full-thickness skin tissue containing blood capillary networks through a layer-by-layer assembly approach using a handy electrospinning apparatus and evaluated its skin wound coverage potential in vivo. The average diameter and thickness of fabricated poly-ε-caprolactone-cellulose acetate scaffolds were easily tuned in the range of 474 ± 77-758 ± 113 nm and 9.43 ± 2.23-29.96 ± 5.78 µm by varying electrospinning distance and duration, as indicated by FE-SEM. Besides, keratinocytes exhibited homogeneous differentiation throughout the fibrous matrix prepared with electrospinning distance and duration of 9 cm and 1.5 min within five-layer (5L) epidermal tissues with thickness of 135-150 µm. Moreover, coculture of vascular endothelial cells, circulating fibrocytes, and fibroblasts within the 5L dermis displayed network formation in vitro, resulting in reduced inflammatory factor levels and enhanced integration with the host vasculature in vivo. Additionally, the skin equivalent grafts consisting of the epidermal layer, biomimetic basement membrane, and vascularized dermis layer with an elastic modulus of approximately 11.82 MPa exhibited accelerated wound closure effect indicative of re-epithelialization and neovascularization with long-term cell survival into the host, which was confirmed by wound-healing rate, bioluminescence imaging activity, and histological analysis. It is the first report of a full-thickness skin equivalent constructed using a battery-operated electrospinning apparatus, highlighting its tremendous potential in regenerative medicine.

Epidermal stem cells maintain stemness via a biomimetic micro/nanofiber scaffold that promotes wound healing by activating the Notch signaling pathway.

BACKGROUND: Epidermal stem cells (EpSCs) play a vital role in wound healing and skin renewal. Although biomaterial scaffolds have been used for transplantation of EpSCs in wound healing, the ex vivo differentiation of EpSCs limits their application. METHODS: To inhibit the differentiation of EpSCs and maintain their stemness, we developed an electrospun polycaprolactone (PCL)+cellulose acetate (CA) micro/nanofiber for the culture and transplantation of EpSCs. The modulation effect on EpSCs of the scaffold and the underlying mechanism were explored. Liquid chromatography-tandem mass spectrometry for label-free quantitative proteomics was used to analyze proteomic changes in EpSCs cultured on scaffolds. In addition, the role of transplanted undifferentiated EpSCs in wound healing was also studied. RESULTS: In this study, we found that the PCL+CA micro/nanofiber scaffold can inhibit the differentiation of EpSCs through YAP activation-mediated inhibition of the Notch signaling pathway. Significantly differentially expressed proteomics was observed in EpSCs cultured on scaffolds and IV collagen-coated culture dishes. Importantly, differential expression levels of ribosome-related proteins and metabolic pathway-related proteins were detected. Moreover, undifferentiated EpSCs transplanted with the PCL+CA scaffold can promote wound healing through the activation of the Notch signaling pathway in rat full-thickness skin defect models. CONCLUSIONS: Overall, our study demonstrated the role of the PCL+CA micro-nanofiber scaffold in maintaining the stemness of EpSCs for wound healing, which can be helpful for the development of EpSCs maintaining scaffolds and exploration of interactions between biomaterials and EpSCs.

A Biomimetic Basement Membrane Substitute Based on Tri-Layered Nanofibrous Scaffold for Skin Reconstruction.

Developing basement membranes (BMs) substitute remains major problem for constructing functional tissue engineered skin because of its complex structure and multifunction of regulating cellular behavior. Herein, a stable electrospinning method was employed to generate a biomimetic model of natural BMs based on novel scaffold electrospun from Poly(ɛ-caprolactone) (PCL) and cellulose acetate (CA) incorporated with chitosan (CS). The morphology, structure, surface hydrophilicity, roughness and mechanical tensile strength of prepared monolayer and tri-layered scaffold were comprehensively compared. Besides, co-culture system via seeding keratinocytes (Kcs) and fibroblasts (Fbs) on opposite side of tri-layered scaffold revealed more effective segregation of both cell types within the central nanofibrous barrier together with enhanced cell attachment and proliferation than that on the monolayer scaffold. Moreover, the deposition of type VII collagen and laminin-5 was examined in comparison with normal skin BMs. Furthermore, the histological studies revealed characteristics of reconstructing BM zone at the junction of dermis-epidermis after in vivo implantation for 2 weeks, and wound healing while the seeded cells interacted with the endogenous cells. Additionally, the expression of active integrin ß1 and phosphorylated focal adhesion kinase (FAK) was promoted with treatment of tri-layered scaffold. This study stressed that this tri-layer scaffold might provoke biomimetic responses of Kcs and Fbs and thus be applied for future development of BMs containing tissues.

Spatial-Temporal Changes of Mechanical Microenvironment in Skin Wounds During Negative Pressure Wound Therapy.

Cell migration, proliferation, and differentiation are regulated by mechanical cues during skin wound healing. Negative pressure wound therapy (NPWT) reduces the healing period by optimizing the mechanical microenvironment of the wound bed. Under NPWT, it remains elusive how the mechanical microenvironment (e.g., stiffness, strain gradients) changes both in time and space during wound healing. To illustrate this, the healing time of full-thickness skin wounds under NPWT, with pressure settings ranging from -50 to -150 mm Hg, were evaluated and compared with gauze dressing treatments (control group), and three-dimensional finite element models of full-thickness skin wounds on days 1 and 5 after treatment were developed on the basis of MR 3D imaging data. Shear wave elastography (SWE) was applied to detect the stiffness of wound soft tissue on days 1 and 5, and nonlinear finite element analysis (FEA) was used to represent the spatial-temporal environment of the 3D strain field of the wound under NPWT vs the control group. Compared with the control group, NPWT with -50, -80, and -125 mm Hg promoted wound healing. SWE showed that the elastic modulus of wounded skin increased during healing. Meanwhile, the elastic modulus in wounded skin under NPWT was significantly smaller than in the control group. Strain and its gradient decreased under NPWT during wound healing, while no significant change was observed in the control group. This study, which is based on MR 3D imaging, shear wave elastography, and nonlinear FEA, provides an in-depth understanding of changes of the skin mechanical microenvironment under NPWT in the time-space dimension and the associated wound healing.

Expression of MicroRNA-301a and its Functional Roles in Malignant Melanoma.

BACKGROUND/AIMS: Although microRNA-301a has been reported to function as an oncogene in many human cancers, the roles of miR-301a in malignant melanoma (MM) is unclear. The present study aims to investigate the functional roles of miR-301a in MM and its possible molecular mechanisms. METHODS: Quantitative real-time PCR (qRT-PCR) assay was performed to detect the expression of miR-301a in MM tissues, and analyze its correlation with metastasis and prognosis of MM patients. In vitro, miR-301a was ectopically expressed using overexpression and knock-down strategies, and the effects of miR-301a expression on growth, apoptosis, migration, invasion and chemosensitivity of MM cells were further investigated. Furthermore, the potential and functional target gene was identified by luciferase reporter, qRT-PCR, Western blot assays. RESULTS: We showed that the expression of miR-301a was significantly upregulated in MM tissues, and upregulation of miR-301a correlated with metastasis and poor prognosis of MM patients. Transfection of miR-301a/inhibitor significantly inhibited growth, colony formation, migration, invasion and enhanced apoptosis and chemosensitivity in MM cells, while transfection of miR-301a/mimic could induce the inverse effects on phenotypes of MM cells. Luciferase reporter, qRT-PCR and Western blot assays showed that phosphatase and tensin homolog (PTEN) was a direct and functional target of miR-301a. It was also observed that the Akt and FAK signaling pathways were involved in miR-301/PTEN-promoting MM progression. CONCLUSION: Taken together, our study suggests that miR-301a may be used as a potential therapeutic target in the treatment of human MM.

A Comparative Study on the Biological Characteristics of Human Adipose-Derived Stem Cells from Lipectomy and Liposuction.

PURPOSES: To compare the biological behaviors of human adipose-derived stem cells (ADSCs) isolated from adipose tissue by lipectomy and liposuction, with the purpose of providing the basis for clinical application. METHODS: The proliferation and apoptosis of ADSCs were analyzed by CCK-8 assay and flow cytometry. Cell migration was measured by a wound healing assay. An ELISA assay was used to evaluate paracrine functions. SOD and MDA were tested by xanthine oxidase and thiobarbituric acid reactions, respectively. In addition, we used a CCK-8, LDH assay and flow cytometry to analyze the proliferation and apoptosis of ADSCs treated with lidocaine or adrenaline. RESULTS: The viable ADSCs yield from liposuction was significantly lower than that from lipectomy, while the apoptosis of cells from liposuction was significantly higher than from lipectomy. The paracrine secretion of the two sources of ADSCs was highest when treated with 10-7 mol/L insulin and 10 ng/mL TGF-α, but there were no significant differences in VEGF, IL-6, IL-8 or HGF levels. The ADSCs from lipectomy migrated faster than those from liposuction, and SOD in the lipectomy group was higher than in the liposuction group, whereas MDA of the lipectomy group was lower than that of the liposuction group. The proliferation ADSCs treated with lidocaine or adrenaline was greatly decreased, while apoptosis was significantly increased, and cytotoxicity of lidocaine or adrenaline to ADSCs was dose-dependent. CONCLUSIONS: Compared with ADSCs from liposuction, the ADSCs from lipectomy have better biological characteristics. Lidocaine and adrenaline decreased the viability of ADSCs, and their cytotoxicity to ADSCs was dose-dependent.

Biomimetic LBL structured nanofibrous matrices assembled by chitosan/collagen for promoting wound healing.

This paper reports the fabrication of biomimetic nanofibrous matrices via co-electrospinning of polycaprolactone (PCL)/cellulose acetate (CA) and layer-by-layer self-assembly (LBL) of positively charged chitosan (CS) and negatively charged Type Ⅰ collagen on the nanofibrous matrix. FE-SEM images indicate that the average fiber diameter increased from 392 to 541 nm when the coating bilayers varied from 5 to 20.5. Besides, the excellent biocompatibility and enhanced attachment and spreading of normal human dermal fibroblasts (NHDFs) of prepared nanofibrous mats are confirmed by MTT and SEM results. Furthermore, the LBL structured (CS/collagen)n nanofibrous mats greatly improve the cell migration in vitro, promote re-epithelialization and vascularization in vivo, and up-regulate the expression of collagen Ⅳ and α-tubulin, as well as the Integrin ß1 and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. The levels of expressed protein are significantly enhanced with increasing coating bilayers via immunohistochemistry and western blotting analyses. Collectively, these results suggest that the LBL structured biomimetic nanofibrous matrices may enhance cell migration and further promote the skin regeneration by up-regulating the secretion of ECM protein and triggering Integrin/FAK signaling pathway, which demonstrate the potential use of the nanofibrous mats to rapidly restore the structural and functional properties of wounded skin.

Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns.

The potential of acellular dermal matrix (ADM) to improve cosmetic and functional outcomes has been demonstrated; however, there have been few clinical comparative studies assessing the long-term morphological, histological and functional changes after ADM placement. This study was designed to retrospectively evaluate the long-term outcomes of the cograft acellular dermal matrix with autologous thin split-thickness skin for the coverage of wounds in extensively burned patients. Thirty burn patients treated with a composite graft of ADM with autologous split-thickness skin from January 2007 to December 2009 were enrolled in this study. Another group of thirty patients who received only an autogenous split-thickness skin implant served as the control. Our study revealed that the collagen in the dermis treated with ADM were ordered, and the proportion of collagen III/I was much higher in the control group than in the ADM group. The basement membrane was prominent and continuous. Meanwhile, the VBSS (Vancouver Burn Skin Score) was used to evaluate skin quality, which shows a significant differences between the two group (P<0.001). Then the functional level was evaluated by the BI (Barthel Index), and the ADM group was much better than the control group (P=0.005). Based on these results, we concluded that the composite graft of ADM with autologous thin split-thickness skin was suitable for repairing the defects in functional areas after a burn. This technique might facilitate wound management with acceptable esthetic outcomes, good functional recovery and less scar hyperplasia at the donor site.

Novel layer-by-layer structured nanofibrous mats coated by protein films for dermal regeneration.

Layer-by-layer coating technique is effective in modifying the surface of nanofibrous mats, but overmuch film-coating makes the mats less porous to hardly suit the condition for tissue engineering. We developed novel nanofibrous mats layer-by-layer coated by silk fibroin and lysozyme on the cellulose electrospun template via electrostatic interaction. The film-coating assembled on the mats was not excessive because the charge of the proteins varied in the coating process due to different pH value. In addition, pure nature materials made the mats nontoxic, biodegradable and low-cost. The morphology and composition variation during layer-by-layer coating process was investigated and the results showed that the structure and thickness of film-coatings could be well-controlled. The antibacterial assay and in vitro cell experiments indicated that the mats could actively inhibit bacteria and exhibit excellent biocompatibility. In vivo implant assay further verified the mats cultured with human epidermal cells could promote wound healing and avoid wound infection. Therefore, these mats showed promising prospects when performed for dermal reconstruction.

KGM and PMAA based pH-sensitive interpenetrating polymer network hydrogel for controlled drug release.

Sequential interpenetrating polymer networks (IPN) hydrogels based on konjac glucomannan (KGM) and poly(methacrylic acid) (PMAA) were prepared by immersion of a solution of methacrylic acid (MAA) monomer with cross-linker N,N'-methylenebisacrylamide (MBAAm) and initiating into a pre-fabricated dried KGM gel. Polymerization and cross-linking of MAA inside the KGM network resulted in a novel biodegradable pH-sensitive IPN hydrogel. The studies on the swelling behavior of IPN hydrogels reveal their sensitive response to environment pH value. It was possible to modulate the degree of swelling of the IPN gels by changing the KGM/PMAA ratio and the cross-linking density of the PMAA component. The KGM component in the IPN can be degraded by ß-glycosidase Mannaway25L. In vitro drug release behavior of IPN hydrogels were investigated under different environments using model drugs 5-fluorouracil. The results suggested that such an IPN hydrogel can be exploited as carrier candidate for colon-specific drug delivery.

The roles of autophagy and apoptosis in burn wound progression in rats.

Understanding the role of cell death in burn wound progression is crucial for giving appropriate diagnoses and designing therapy regimens for burn patients. A well-described and reliable "comb burns model" was employed to evaluate the roles of autophagy and apoptosis in burn wound progression at 2 h, 6 h, 12 h, 24 h, and 48 h post-burn in a rat model. Immunohistochemistry (IHC) results showed that autophagy was detectable in hair follicle epithelium at 2 h post-burn, peaked at 12 h post-burn, then declined. Conversely, apoptosis was mainly located in the stratum epidermis and took place at low levels until 6 h post-burn, at which point it slowly increased. Bcl-2 and Bax, which are regulators of both processes, showed protein expression level patterns that were consistent with the IHC results. This study of autophagy in burn wound tissue progression represents a conceptual expansion of cell death in burn wounds. Based on these results, we suggest that different treatments should be performed on a specific post-burn time course depending on the most prevalent type of cell death occurring at that time.

Cytotoxicity and antibacterial ability of scaffolds immobilized by polysaccharide/layered silicate composites.

Chitosan and pectin/organic rectorite (OREC) were initially deposited on the surface of cellulose acetate electrospun nanofibers by a layer-by-layer (LBL) technique to fabricate scaffolds for bacterial inhibition, and the cytotoxicity of the LBL structured scaffolds was also investigated. A couple of opposite charged material, pectin and OREC, were firstly used to fabricate the intercalated composites. The intercalated structure was determined by selected area electron diffraction. Field-emission scanning electron microscope, X-ray diffraction and X-ray photoelectron spectroscopy were applied for the characterization of LBL structured nanofibrous scaffolds. Antibacterial assay results showed that the diameters of the inhibition zone increased from 7.6 to 15.8 mm for Escherichia coli, as well as from 7.4 to 14.2 mm for Staphylococcus aureus. Finally, human epidermal (EP) cells grew well on the LBL films coating. These novel scaffolds could be an ideal candidate for wound dressings and food packaging.

Nanofibrous mats coated by homocharged biopolymer-layered silicate nanoparticles and their antitumor activity.

Quaternized chitosan (QC)-organic rectorite (OREC) intercalated composites based nanoparticles were immobilized on cellulose acetate nanofibrous mats by layer-by-layer deposition technique via hydrogen bonds. The key design of those mats was the utilization of all negatively charged materials to reduce the quantity of nanoparticles immobilized on the fibers and then the cytotoxicity of the mats. The intercalated structure in nanoparticles was confirmed by selected area electron diffraction. The morphology and composition of NPs-assembled nanofibrous mats were studied by field emission scanning electron microscopy and X-ray photoelectron spectroscopy. The results trended to give clues as how the assembly process varied by adding OREC. MTT assay and cell culture experiments showed that NPs-assembled nanofibrous mats were commendably compatible with normal cells and could selectively kill human lung carcinoma epithelial cells. Hemolysis test indicated these mats had excellent blood compatibility. As a result, QC-OREC NPs-assembled nanofibrous mats by homocharged deposition process can be considered as a novel alternative method for cancer therapy.

[The effect of microencapsulated NGF-expressing NIH-3T3 cells on bioengineered dermis function in vitro].

Nerve growth factor (NGF) can promote the regeneration of peripheral nerve as well as contraction and reepithelization of wound. We constructed a bioengineered dermis containing microencapsulated NGF-expressing NIH-3T3 cells and study the effect of the microencapsule to the bioengineered dermis and seed cells. NGF gene was transfected into NIH-3T3 cells and enclosed into alginate-poly-L-lysine-alginate (APA) microencapsulation and cultivated in vitro. Content of NGF in microencapsules culturing supernatant was measured by enzyme linked immunosorbent assay (ELISA) method. These microencapsules were co-cultured with epidermic cells and fibroblast cells. Bioengineered dermis was constructed with NGF-expressing micorencapsules as seed cells using tissue engineering method. NIH-3T3 microencapsules, empty microencapsules, normal culture media were control groups. After one week culture, the characteristics of the dermis were described by MTT test, the content of hydroxyproline (HP), HE staining and ultrastructure photograph. We found the NGF-expressing microencapsulates can secret NGF steadly after cultured 8w in vitro, promot the proliferation of epidermic cells and secret collagen of fibroblast cells. These functions can maintaine in bioengineered dermis. So NGF-expressing NIH-3T3 microencapsulates can promote the quality of bioengineered dermis.