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    3D bioprinting for skin tissue engineering: Current status and perspectives. Weng Tingting,Zhang Wei,Xia Yilan,Wu Pan,Yang Min,Jin Ronghua,Xia Sizhan,Wang Jialiang,You Chuangang,Han Chunmao,Wang Xingang Journal of tissue engineering Skin and skin appendages are vulnerable to injury, requiring rapidly reliable regeneration methods. In recent years, 3D bioprinting has shown potential for wound repair and regeneration. 3D bioprinting can be customized for skin shape with cells and other materials distributed precisely, achieving rapid and reliable production of bionic skin substitutes, therefore, meeting clinical and industrial requirements. Additionally, it has excellent performance with high resolution, flexibility, reproducibility, and high throughput, showing great potential for the fabrication of tissue-engineered skin. This review introduces the common techniques of 3D bioprinting and their application in skin tissue engineering, focusing on the latest research progress in skin appendages (hair follicles and sweat glands) and vascularization, and summarizes current challenges and future development of 3D skin printing. 10.1177/20417314211028574
    [Influence of the stiffness of three-dimensionally bioprinted extracellular matrix analogue on the differentiation of bone mesenchymal stem cells into skin appendage cells]. ,Zhang Y J,Li J J,Yao B,Song W,Huang S,Fu X B Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns To observe the influence of the stiffness of three-dimensionally bioprinted extracellular matrix analogue on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into skin appendage cells. (1) Sodium alginate of 1 g and 4 g gelatin, 3 g sodium alginate and 8 g gelatin were mixed respectively, and the two mixtures were dissolved in 100 mL ultra-pure water respectively to prepare two sodium alginate-gelatin composite hydrogels, named 1A4G hydrogel and 3A8G hydrogel, which were used in the subsequent experiments. The morphology of the two hydrogels at room temperature, after condensation for 15-30 min at 4 ℃ (the same condensation condition below), after condensation and cross-linking with 25 g/L calcium chloride solution (the same cross-linking condition below), and after condensation and three-dimensional printing with a three-dimensional bioprinter (the same three-dimensional printer below) and cross-linking were observed respectively. Young's modulus (stiffness) of the two kinds of hydrogels was measured by Young's modulus tester after condensation and cross-linking (=3). Two kinds of hydrogels were cross-linked and freeze-dried, and their pore structure was observed by scanning electron microscope. Two hydrogels were cross-linked and freeze-dried, and the porosity was detected by anhydrous ethanol replacement method (=3). (2) BMSCs were isolated from femur and tibia of 20 C57BL/6 mice (no limitation with sex, born 7 days) and cultured, and the second passage of cells was used for further test. The BMSCs single cell suspension (1.0×10(7) /mL) was mixed with 1A4G hydrogel and 3A8G hydrogel respectively at 1∶9 volume ratio to prepare BMSCs-loaded 1A4G hydrogel and BMSCs-loaded 3A8G hydrogel for three-dimensional printing. One construct was printed with 1 mL cell-loaded hydrogel (the same dosage for printing below). Mesenchymal stem cells (MSCs) specific medium was added after cross-linking, and the printed constructs were divided into 1A4G group and 3A8G group according to the hydrogel. One construct of each group cultured for 7 days was tested with live/dead kit to count the live cells and dead cells in 50-fold field of view. Nine printed constructs from each of the two groups were taken, and BMSCs of nine wells (1.0×10(6) per well) cultured with 2 mL MSCs specific medium were set as two-dimensional culture group. After 1, 3, 5 day (s) of culture, three printed constructs from 1A4G group and 3A8G group respectively and three wells of cells from two-dimensional culture group were taken to detect the absorbance value in culture medium by cell counting kit 8, denoting the cell proliferation activity. (3) BMSCs-loaded 1A4G hydrogel and BMSCs-loaded 3A8G hydrogel of 10 mL respectively were prepared as in experiment (2), which were respectively mixed with 0.5 mL plantar dermis homogenate extracted from 10 C57BL/6 mice of 1 day postnatal with unknown sex, then three-dimensionally printed, cross-linked, cultured with MSCs specific medium for 3 days and then changed to sweat gland specific medium. The printed constructs were divided into 1A4G group and 3A8G group according to their hydrogel. After 7 days of culture with sweat gland specific medium, the expressions of epithelial cell surface markers cytokeratin-5 (CK5) and CK14, sweat gland cell surface markers CK18 and Na(+) /K(+) -ATPase (NKA), and hair follicle cell surface markers CK17 and alkaline phosphatase (ALP) at protein level in cells of printed constructs in the two groups were detected by immunofluorescence method. The expressions of CK5, CK14, CK18, NKA (detecting ATP1a1), CK17, and ALP at mRNA level in cells of printed constructs in the two groups were detected with real-time fluorescent quantitative reverse transcription polymerase chain reaction (=3). Data were statistically analyzed with independent sample test, Fisher's exact probability test, analysis of variance for factorial design, and Bonferroni method. (1) Compared with that of 3A8G hydrogel, 1A4G hydrogel had lower viscosity and better fluidity at room temperature. Both kinds of hydrogels were gel-like after condensation, based on which, the shape of cross-linked hydrogels was uniform and regular, with three-dimensional printing and cross-linking made hrdrogels forming solid crisscross cylindrical constructs. The Young's modulus of 1A4G hydrogel was (52±6) kPa, which was obviously lower than (218±5) kPa of 3A8G hydrogel (=40.470, <0.01). The pore structure of the two hydrogels was similar, with all the cross-sections showing porous network structure. The porosity of the two hydrogels was similar (=0.930, >0.05). (2) The distribution of live/dead cells between 1A4G group and 3A8G group was similar after 7 days of culture (>0.05), most of which were live cells. The absorbance value in culture medium of printed constructs among 1A4G group, 3A8G group, and two-dimensional culture group didn't show statistically significant differences after 1, 3, 5 day (s) of culture (>0.05). Compared with that after 1 day of culture within each group, the absorbance value in culture medium of printed constructs in 1A4G group and 3A8G group was significantly increased after 3 and 5 days of culture (<0.05 or <0.01), and the absorbance value in culture medium of cells in two-dimensional culture group was significantly increased after 5 days of culture (<0.01). Compared with that after 3 days of culture within each group, the absorbance value in culture medium of printed constructs in 1A4G group and 3A8G group and that of cells in two-dimensional culture group was significantly increased after 5 days of culture (<0.01). (3) After 7 days of culture with sweat gland specific medium, the CK5, CK14, CK18, NKA, CK17, and ALP were positively expressed at protein level in cells of printed constructs in the two groups. After 7 days of culture with sweat gland specific medium, the expressions of CK5, CK14, CK18, and NKA at mRNA level in cells of printed constructs were close between the two groups (=0.362, 0.807, 0.223, 1.356, >0.05); the expressions of CK17 and ALP at mRNA level in cells of printed constructs in 3A8G group were 1.96±0.21 and 55.57±11.49, respectively, which were significantly higher than 1.05±0.42 and 2.01±0.27 in 1A4G group (=3.333, 8.074, <0.05 or <0.01). BMSCs cultured three-dimensionally in 1A4G and 3A8G hydrogels tend to differentiate into sweat gland cells, but the BMSCs cultured three-dimensionally in 3A8G hydrogel show a stronger tendency to differentiate into hair follicle cells than the cells cultured in 1A4G hydrogel. It suggests that relatively high stiffness of three-dimensionally bioprinted extracellular matrix analogue facilitates not only differentiation of BMSCs into sweat gland cells, but also their differentiation into hair follicle cells. 10.3760/cma.j.cn501120-20200811-00375
    Development of a Multi-Layer Skin Substitute Using Human Hair Keratinic Extract-Based Hybrid 3D Printing. Choi Won Seok,Kim Joo Hyun,Ahn Chi Bum,Lee Ji Hyun,Kim Yu Jin,Son Kuk Hui,Lee Jin Woo Polymers Large-sized or deep skin wounds require skin substitutes for proper healing without scar formation. Therefore, multi-layered skin substitutes that mimic the genuine skin anatomy of multiple layers have attracted attention as suitable skin substitutes. In this study, a novel skin substitute was developed by combining the multi-layer skin tissue reconstruction method with the combination of a human-derived keratinic extract-loaded nano- and micro-fiber using electrospinning and a support structure using 3D printing. A polycaprolactone PCL/keratin electrospun scaffold showed better cell adhesion and proliferation than the keratin-free PCL scaffold, and keratinocytes and fibroblasts showed better survival, adhesion, and proliferation in the PCL/keratin electrospun nanofiber scaffold and microfiber scaffold, respectively. In a co-culture of keratinocytes and fibroblasts using a multi-layered scaffold, the two cells formed the epidermis and dermal layer on the PCL/keratin scaffold without territorial invasion. In the animal study, the PCL/keratin scaffold caused a faster regeneration of new skin without scar formation compared to the PCL scaffold. Our study showed that PCL/keratin scaffolds co-cultured with keratinocytes and fibroblasts promoted the regeneration of the epidermal and dermal layers in deep skin defects. Such finding suggests a new possibility for artificial skin production using multiple cells. 10.3390/polym13162584
    Three-Dimensional Tissue-Engineered Grafts for Hair Follicle Regeneration. Ishack Stephanie,Lipner Shari R Dermatologic surgery : official publication for American Society for Dermatologic Surgery [et al.] 10.1097/DSS.0000000000003142
    3D Bioprinting of a Gelatin-Alginate Hydrogel for Tissue-Engineered Hair Follicle Regeneration. Kang Deni,Liu Zhen,Qian Chuanmu,Huang Junfei,Zhou Yi,Mao Xiaoyan,Qu Qian,Liu Bingcheng,Wang Jin,Hu Zhiqi,Miao Yong Acta biomaterialia Hair follicle (HF) regeneration remains challenging, principally due to the absence of a platform that can successfully generate the microenvironmental cues of hair neogenesis. Here, we demonstrate a 3D bioprinting technique based on a gelatin/alginate hydrogel (GAH) to construct a multilayer composite scaffold simulating the HF microenvironment in vivo. Fibroblasts (FBs), human umbilical vein endothelial cells (HUVECs), dermal papilla cells (DPCs), and epidermal cells (EPCs) were encapsulated in GAH (prepared from a mixture of gelatin and alginate) and respectively 3D-bioprinted into the different layers of a composite scaffold. The bioprinted scaffold with epidermis- and dermis-like structure was subsequently transplanted into full-thickness wounds in nude mice. The multilayer scaffold demonstrated suitable cytocompatibility and increased the proliferation ability of DPCs (1.2-fold; P < 0.05). It also facilitated the formation of self-aggregating DPC spheroids and restored DPC genes associated with hair induction (ALP, β-catenin, and α-SMA). The dermal and epidermal cells self-assembled successfully into immature HFs in vitro. HFs were regenerated in the appropriate orientation in vivo, which can mainly be attributed to the hierarchical grid structure of the scaffold and the dot bioprinting of DPCs. Our 3D printed scaffolds provide a suitable microenvironment for DPCs to regenerate entire HFs and could make a significant contribution in the medical management of hair loss. This method may also have broader applications in skin tissue (and appendage) engineering. STATEMENT OF SIGNIFICANCE: Hair loss remains a challenging clinical problem that influences quality of life. Three-dimensional (3D) bioprinting has become a useful tool for the fabrication of tissue constructs for transplantation and other biomedical applications. In this study, we used a 3D bioprinting technique based on a gelatin/alginate hydrogel to construct a multi-layer composite scaffold with cuticular and corium layers to simulate the microenvironment of dermal papilla cells (DPCs) in the human body. This new approach permits the controllable formation of self-aggregating spheroids of DPCs in a physiologically relevant extracellular matrix and the initiation of epidermal-mesenchymal interactions, which results in HF formation in vivo. The ability to regenerate entire HFs should have a significant impact on the medical management of hair loss. 10.1016/j.actbio.2022.03.011