Bioprinted dermis with human adipose tissue-derived microvascular fragments promotes wound healing.

Tissue-engineered skin is an effective material for treating large skin defects in a clinical setting. However, its use is limited owing to vascular complications. Human adipose tissue-derived microvascular fragments (HaMVFs) are vascularized units that form vascular networks by rapid reassembly. In this study, we designed a vascularized bionic skin tissue using a three-dimensional (3D) bioprinter of HaMVFs and human fibroblasts encapsulated in a hybrid hydrogel composed of GelMA, HAMA, and fibrinogen. Tissues incorporating HaMVFs showed good in vitro vascularization and mechanical properties after UV crosslinking and thrombin exposure. Thus, the tissue could be sutured appropriately to the wound. In vivo, the vascularized 3D bioprinted skin promoted epidermal regeneration, collagen maturation in the dermal tissue, and vascularization of the skin tissue to accelerate wound healing. Overall, vascularized 3D bioprinted skin with HaMVFs is an effective material for treating skin defects and may be clinically applicable to reduce the necrosis rate of skin grafts.

Engineered dermis loaded with confining forces promotes full-thickness wound healing by enhancing vascularisation and epithelialisation.

Tissue-engineered skin is ideal for clinical wound repair. Restoration of skin tissue defects using tissue-engineered skin remains a challenge owing to insufficient vascularisation. In our previous study, we developed a 3D bioprinted model with confined force loading and demonstrated that the confined force can affect vascular branching, which is regulated by the YAP signalling pathway. The mechanical properties of the model must be optimised to suture the wound edges. In this study, we explored the ability of a GelMA-HAMA-fibrin scaffold to support the confined forces created by 3D bioprinting and promote vascularisation and wound healing. The shape of the GelMA-HAMA-fibrin scaffold containing 3% GelMA was affected by the confined forces produced by the embedded cells. The GelMA-HAMA-fibrin scaffold was easy to print, had optimal mechanical properties, and was biocompatible. The constructs were successfully sutured together after 14 d of culture. Scaffolds seeded with cells were transplanted into skin tissue defects in nude mice, demonstrating that the cell-seeded GelMA-HAMA-fibrin scaffold, under confined force loading, promoted neovascularisation and wound restoration by enhancing blood vessel connections, creating a patterned surface, growth factors, and collagen deposition. These results provide further insights into the production of hydrogel composite materials as tissue-engineered scaffolds under an internal mechanical load that can enhance vascularisation and offer new treatment methods for wound healing. STATEMENT OF SIGNIFICANCE: Tissue-engineered skin is ideal for use in clinical wound repair. However, treatment of tissue defects using synthetic scaffolds remains challenging, mainly due to slow and insufficient vascularization. Our previous study developed a 3D bioprinted model with confined force loading, and demonstrated that confined force can affect vascular branching regulated by the YAP signal pathway. The mechanical properties of the construct need to be optimized for suturing to the edges of wounds. Here, we investigated the ability of a GelMA-HAMA-fibrin scaffold to support the confined forces created by 3D bioprinting and promote vascularization in vitro and wound healing in vivo. Our findings provide new insight into the development of degradable macroporous composite materials with mechanical stimulation as tissue-engineered scaffolds with enhanced vascularization, and also provide new treatment options for wound healing.

Combined light-cured and sacrificial hydrogels for fabrication of small-diameter bionic vessels by 3D bioprinting.

BACKGROUND: Bionic grafts can replace autologous tissue through tissue engineering in cases of cardiovascular disease. However, small-diameter vessel grafts remain challenging to precellularize. OBJECTIVE: Bionic small-diameter vessels with endothelial and smooth muscle cells (SMCs) manufactured with a novel approach. METHODS: A 1-mm-diameter bionic blood vessel was constructed by combining light-cured hydrogel gelatin-methacryloyl (GelMA) with sacrificial hydrogel Pluronic F127. Mechanical properties of GelMA (Young's modulus and tensile stress) were tested. Cell viability and proliferation were detected using Live/dead staining and CCK-8 assays, respectively. The histology and function of the vessels were observed using hematoxylin and eosin and immunofluorescence staining. RESULTS: GelMA and Pluronic were printed together using extrusion. The temporary Pluronic support was removed by cooling during GelMA crosslinking, yielding a hollow tubular construct. A bionic bilayer vascular structure was fabricated by loading SMCs into the GelMA bioink, followed by perfusion with endothelial cells. In the structure, both cell types maintained good cell viability. The vessel showed good histological morphology and function. CONCLUSION: Using light-cured and sacrificial hydrogels, we formed a small ca bionic vessel with a small caliber containing SMCs and endothelial cells, demonstrating an innovative approach for construction of bionic vascular tissues.

Bioprinting a skin patch with dual-crosslinked gelatin (GelMA) and silk fibroin (SilMA): An approach to accelerating cutaneous wound healing.

Clinical settings often face significant obstacles in treating large acute wounds. The alternative of therapeutic approach is needed urgently. Hydrogels derived from natural or synthetic materials may be designed to perform a variety of functions for promoting wound healing. Herein, a 3D bioprinted hydrogel patch is designed for accelerating acute wound healing, which is fabricated with methacryloyl-substituted gelatin (GelMA) and silk fibroin (SilMA) dual-cross-linked by ultraviolet (UV) light. The GelMA with added silk fibroin (GelSilMA) shows improved biodegradation and mechanical properties. Furthermore, SilMA hydrogel can maintain a moisturized healing environment in wound area persistently with adequate degradation capacity. In vivo, GelSilMA (G-S) hydrogel can help to speed wound closure by the improved microenvironment for epidermal tissue regeneration and endogenous collagen generation accordingly. In summary, the G-S hydrogel patch can accelerate acute wound healing efficiently in a relatively simple and inexpensive manner.

miR-100-5p Promotes Epidermal Stem Cell Proliferation through Targeting MTMR3 to Activate PIP3/AKT and ERK Signaling Pathways.

Skin epidermal stem cells (EpSCs) play a critical role in wound healing and are ideal seed cells for skin tissue engineering. Exosomes from human adipose-derived stem cells (ADSC-Exos) promote human EpSC proliferation, but the underlying mechanism remains unclear. Here, we investigated the effect of miR-100-5p, one of the most abundant miRNAs in ADSC-Exos, on the proliferation of human EpSCs and explored the mechanisms involved. MTT and BrdU incorporation assays showed that miR-100-5p mimic transfection promoted EpSC proliferation in a time-dependent manner. Cell cycle analysis showed that miR-100-5p mimic transfection significantly decreased the percentage of cells in the G1 phase and increased the percentage of cells in the G2/M phase. Myotubularin-related protein 3 (MTMR3), a lipid phosphatase, was identified as a direct target of miR-100-5p. Knockdown of MTMR3 in EpSCs by RNA interference significantly enhanced cell proliferation, decreased the percentage of cells in the G1 phase and increased the percentage of cells in the S phase. Overexpression of MTMR3 reversed the proproliferative effect of miR-100-5p on EpSCs, indicating that miR-100-5p promoted EpSC proliferation by downregulating MTMR3. Mechanistic studies showed that transfection of EpSCs with miR-100-5p mimics elevated the intracellular PIP3 level, induced AKT and ERK phosphorylation, and upregulated cyclin D1, E1, and A2 expression, which could be attenuated by MTMR3 overexpression. Consistently, intradermal injection of ADSC-Exos or miR-100-5p-enriched ADSC-Exos into cultured human skin tissues significantly reduced MTMR3 expression and increased the thickness of the epidermis and the number of EpSCs in the basal layer of the epidermis. The aforementioned effect of miR-100-5p-enriched ADSC-Exos was stronger than that of ADSC-Exos and was reversed by MTMR3 overexpression. Collectively, our findings indicate that miR-100-5p promotes EpSC proliferation through MTMR3-mediated elevation of PIP3 and activation of AKT and ERK. miR-100-5p-enriched ADSC-Exos can be used to treat skin wound and expand EpSCs for generating epidermal autografts and engineered skin equivalents.

Bioprinting small-diameter vascular vessel with endothelium and smooth muscle by the approach of two-step crosslinking process.

Three-dimensional bioprinting shows great potential for autologous vascular grafts due to its simplicity, accuracy, and flexibility. The 6-mm-diameter vascular grafts are used in clinic. However, producing small-diameter vascular grafts are still an enormous challenge. Normally, sacrificial hydrogels are used as temporary lumen support to mold tubular structure which will affect the stability of the fabricated structure. In this study, we have developed a new bioprinting approach to fabricating small-diameter vessel using two-step crosslinking process. The » lumen wall of bioprinted gelatin mechacrylate (GelMA) flat structure was exposed to ultraviolet (UV) light briefly for gaining certain strength, while ¾ lumen wall showed as concave structure which remained uncrosslinked. Precrosslinked flat structure was merged towards the uncrosslinked concave structure. Two individual structures were combined tightly into an intact tubular structure after receiving more UV exposure time. Complicated tubular structures were constructed by these method. Notably, the GelMA-based bioink loaded with smooth muscle cells are bioprinted to form the outer layer of the tubular structure and human umbilical vein endothelial cells were seeded onto the inner surface of the tubular structure. A bionic vascular vessel with dual layers was fabricated successfully, and kept good viability and functionality. This study may provide a novel idea for fabricating biomimetic vascular network or other more complicated organs.

Bioprinting small diameter blood vessel constructs with an endothelial and smooth muscle cell bilayer in a single step.

Bioengineered artificial blood vessels have been a major area of interest over the last decade. Of particular interest are small diameter vessels, as surgical options are currently limited. This study aimed to fabricate a small diameter, heterogeneous bilayer blood vessel-like construct in a single step with gelatin methacryloyl (GelMA) bioink using a 3D micro-extrusion bioprinter on a solid platform. GelMA was supplemented with Hyaluronic acid (HA), glycerol and gelatin to form a GelMA bioink with good printability, mechanical strength, and biocompatibility. Two separate concentrations of GelMA bioink with unique pore sizes were selected to fabricate a heterogeneous bilayer. A higher concentration of GelMA bioink (6% w/v GelMA, 2% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load human umbilical vein endothelial cells (HUVECs) and form an inner, endothelial tissue layer. A lower concentration of GelMA bioink (4% w/v GelMA, 4% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load smooth muscle cells (SMCs) and form an outer, muscular tissue layer. Bioprinted blood vessel-like grafts were then assessed for mechanical properties with Instron mechanical testing, and suture-ability, and for biological properties including viability, proliferation, and histological analysis. The resulting 20 mm long, 4.0 mm diameter lumen heterogeneous bilayer blood vessel-like construct closely mimics a native blood vessel and maintains high cell viability and proliferation. Our results represent a novel strategy for small diameter blood vessel biofabrication.

Estradiol inhibits fMLP-induced neutrophil migration and superoxide production by upregulating MKP-2 and dephosphorylating ERK.

Estrogen has been reported to inhibit neutrophil infiltration related inflammation and suppress neutrophils migration in vitro, but the underlying mechanism is not fully understood. By using HL-60 differentiated neutrophil-like cells (dHL-60) and human neutrophils, we examined the effect of 17-ß estradiol (E2) on cell migration and superoxide production in response to chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) and explored the mechanisms involved. We found that fMLP significantly induced dHL-60 cell and neutrophil migration and superoxide production, which was inhibited by ERK inhibitor PD98059. E2 significantly inhibited fMLP-induced dHL-60 cell and neutrophil migration and superoxide production at both physiological and pharmacological concentrations. Mechanistic studies showed that pretreatment of these cells with E2 rapidly elevated the protein level of mitogen-activated protein kinase phosphatase 2 (MKP-2) and inhibited fMLP-induced ERK phosphorylation. Pretreatment of these cells with estrogen receptor (ER) antagonist ICI 182780 reversed the inhibition of fMP-induced cell migration and superoxide production, and the induction of MKP-2 expression and the suppression of fMP-induced ERK phosphorylation by E2. However, pretreatment of cells with G-protein coupled ER antagonist G15 had no such effect. Collectively, these results demonstrate that fMLP stimulates neutrophil chemotaxis and superoxide production through activating ERK, and indicate that ER-mediated upregulation of MKP-2 may dephosphorylate ERK and contribute to the inhibitory effect of E2 on neutrophil activation by fMLP. Our study reveals new mechanisms involved in the anti-inflammatory activity of estrogen.

Estradiol postconditioning relieves ischemia/reperfusion injury in axial skin flaps of rats, inhibits apoptosis and alters the MKP-1/ERK pathway.

Previous studies have suggested that estradiol can reduce the ischemia/reperfusion (I/R) injury in skin flaps. However, the mechanism, particularly the signal pathways involved in this protective effect are not well established. In the current study, an I/R injury model was established in rats to explore the connection between estradiol protection during I/R injury and extracellular signal­regulated kinase (ERK) signaling. Healthy male Wistar rats were divided into five groups (n=10): Control group (group I), I/R group (group II), saline group (group III), estradiol group (group IV) and inhibitor (PD­98059) group (group V). The survival rate of the flap was compared between groups, morphological changes were observed by hematoxylin and eosin staining of sections, and terminal deoxynucleotidyl transferase dUTP nick end labeling was performed to identify apoptotic cells and determine the apoptotic index. To further investigate the mechanism, western blot analysis was performed to assess the protein level of ERK1/2, phospho­ERK1/2, and mitogen­activated protein kinase phosphatase 1 (MKP­1). The results of the present study demonstrated that estradiol therapy can reduce I/R injury and decrease the apoptosis index in an axial skin flap model. The inhibitor of the ERK pathway (PD­98059) partially abolished the effects of estradiol, which involve the phosphatase enzyme MKP­1. Taken together, the findings of the present study indicate that estradiol may act by reducing the expression of MKP­1, mediating the expression/activation changes of the ERK pathway and subsequently reduce the level of apoptosis and the I/R injury the axial flap. Estrogen may be used to mitigate the adverse reaction caused by ischemia­reperfusion injury and effectively improve the survival rate and survival quality of free skin flap and improve patient prognosis.