3D bioprinting of an in vitro hepatoma microenvironment model: Establishment, evaluation, and anticancer drug testing.

Tumor behavior, including its response to treatments, is influenced by interactions between mesenchymal and malignant cells, as well as their spatial arrangement. To study tumor biology and evaluate anticancer drugs, accurate 3D tumor models are essential. Here, we developed an in vitro biomimetic hepatoma microenvironment model by combining an extracellular matrix (3DM-7721). Initially, the internal grid structure, composed of 10/6 % GelMA/gelatin loaded with SMMC-7721 cells, was printed using 3D bioprinting. The external component consisted of fibroblasts and human umbilical vein endothelial cells loaded with 10/3 % GelMA/gelatin. A control model (3DP-7721) lacked external cell loading. GelMA/gelatin hydrogels provided robust structural support and biocompatibility. The SMMC-7721 cells in the 3DM-7721 model exhibit superior tumor-associated gene expression and proliferation characteristics when compared to the 3DP-7721 model. Furthermore, the 3DM-7721 type exhibited increased resistance to anticancer agents. SMMC-7721 cells in the 3DM-7721 model exhibit significant tumorigenicity in nude mice. The 3DM-7721 model group showed pathological characteristics of malignant tumors, with a high degree of deterioration, and a significant positive correlation between malignant tumor-related gene pathways. This high-fidelity 3DM-7721 tumor microenvironment model is invaluable for studying tumor progression, devising effective treatment strategies, and discovering drugs. STATEMENT OF SIGNIFICANCE.

A multicrosslinked network composite hydrogel scaffold based on DLP photocuring printing for nasal cartilage repair.

Natural hydrogels are widely employed in tissue engineering and have excellent biodegradability and biocompatibility. Unfortunately, the utilization of such hydrogels in the field of three-dimensional (3D) printing nasal cartilage is constrained by their subpar mechanical characteristics. In this study, we provide a multicrosslinked network hybrid ink made of photocurable gelatin, hyaluronic acid, and acrylamide (AM). The ink may be processed into intricate 3D hydrogel structures with good biocompatibility and high stiffness properties using 3D printing technology based on digital light processing (DLP), including intricate shapes resembling noses. By varying the AM content, the mechanical behavior and biocompatibility of the hydrogels can be adjusted. In comparison to the gelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA) hydrogel, adding AM considerably enhances the hydrogel's mechanical properties while also enhancing printing quality. Meanwhile, the biocompatibility of the multicrosslinked network hydrogels and the development of cartilage were assessed using neonatal Sprague-Dawley (SD) rat chondrocytes (CChons). Cells sown on the hydrogels considerably multiplied after 7 days of culture and kept up the expression of particular proteins. Together, our findings point to GelMA/HAMA/polyacrylamide (PAM) hydrogel as a potential material for nasal cartilage restoration. The photocuring multicrosslinked network ink composed of appropriate proportions of GelMA/HAMA/PAM is very suitable for DLP 3D printing and will play an important role in the construction of nasal cartilage, ear cartilage, articular cartilage, and other tissues and organs in the future. Notably, previous studies have not explored the application of 3D-printed GelMA/HAMA/PAM hydrogels for nasal cartilage regeneration.

3D Bioprinting-Based Dopamine-Coupled Flexible Material for Nasal Cartilage Repair.

INTRODUCTION: Since 3D printing can be used to design implants according to the specific conditions of patients, it has become an emerging technology in tissue engineering and regenerative medicine. How to improve the mechanical, elastic and adhesion properties of 3D-printed photocrosslinked hydrogels is the focus of cartilage tissue repair and reconstruction research. MATERIALS AND METHODS: We established a strategy for toughening hydrogels by mixing GelMA-DOPA (GD), which is prepared by coupling dopamine (DA) with GelMA, with HAMA, bacterial cellulose (BC) to produce composite hydrogels (HB-GD). HB-GD hydrogel scaffolds were characterized in vitro by scanning electron microscopy (SEM), Young's modulus, swelling property and rheological property tests. And biocompatibility and chondrogenic ability were tested by live/dead staining, DNA quantitative analysis and immunofluorescence staining. Combined with 3D bioprinting technology, mouse chondrocytes (ADTC5) were added to form a biological chain to construct an in vitro model, and the feasibility of the model for nasal cartilage regeneration was verified by cytology evaluation. RESULTS: With the increase of GD concentration, the toughness of the composite hydrogel increased (47.0 ± 2.7 kPa (HB-5GD)-158 ± 3.2 kPa (HB-20GD)), and it had excellent swelling properties, rheological properties and printing properties. The HB-GD composite hydrogel promoted the proliferation and differentiation of ATDC5. Cells in 3D printed scaffolds had higher survival rates (> 95%) and better protein expression than the encapsulated cultures. CONCLUSION: The HB-10GD hydrogel can be made into a porous scaffold with precise shape, good internal pore structure, high mechanical strength and good swelling rate through extrusion 3D printing. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

A heparin-functionalized bioink with sustained delivery of vascular endothelial growth factor for 3D bioprinting of prevascularized dermal constructs.

Skin tissue engineering faces challenges due to the absence of vascular architecture, impeding the development of permanent skin replacements. To address this, a heparin-functionalized 3D-printed bioink (GH/HepMA) was formulated to enable sustained delivery of vascular endothelial growth factor (VEGF), comprising 0.3 % (w/v) hyaluronic acid (HA), 10 % (w/v) gelatin methacrylate (GelMA), and 0.5 % (w/v) heparin methacrylate (HepMA). The bioink was then used to print dermal constructs with angiogenic functions, including fibroblast networks and human umbilical vein endothelial cell (HUVEC) networks. GH/HepMA, with its covalently cross-linked structure, exhibits enhanced mechanical properties and heparin stability, allowing for a 21-day sustained delivery of VEGF. Cytocompatibility experiments showed that the GH/HepMA bioink supported fibroblast proliferation and promoted collagen I production. With VEGF present, the GH/HepMA bioink promoted HUVEC proliferation, migration, as well as the formation of a richer capillary-like network. Furthermore, HA within the GH/HepMA bioink enhanced rheological properties and printability. Additionally, 3D-bioprinted dermal constructs showed significant deposition of collagen I and III and mature stable capillary-like structures along the axial direction. In summary, this study offers a promising approach for constructing biomimetic multicellular skin substitutes with angiogenesis-induced functions.

Constructing epidermal rete ridges using a composite hydrogel to enhance multiple signaling pathways for the maintenance of epidermal stem cell niche.

The undulating microstructure rete ridge (RR) located at the junction between the dermis and epidermis plays a crucial role in improving skin mechanical properties and maintaining skin homeostasis. However, the investigation of RR microstructures is usually neglected in current tissue engineering for skin regeneration. Here, to create an epidermal model with RR microstructures, keratinocytes were cultured on a patterned GelMA-PEGDA hydrogel constructed using molding technology. Furthermore, grafting acryloylated Arg-Gly-Asp (RGD) peptides on the hydrogel surface significantly improved cell adhesion, fusion, and development. RT-PCR, Western blot, and immunofluorescence staining confirmed that cells on RR microstructures exhibited higher gene and protein expression associated with epidermal stem cells. RNA sequencing analysis of cells on RR microstructure showed higher gene expression profiles related to stem cell maintenance, basement membrane formation, and epidermal development. Furthermore, RT-PCR analysis of epidermal models of various dimensions demonstrated that smaller microstructures were more conducive to epidermal stem cell marker gene expression, which is analogous to human skin. Overall, we have successfully developed a method for integrating RR microstructures into an epidermal model that mimics natural skin to maintain epidermal stem cell niche, providing a valuable reference for researching skin regeneration within the fields of tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: This study presents a method for precisely fabricating microstructures of skin rete ridges using composite hydrogels, thereby creating a skin model that mimics natural human skin. The findings reveal that this microstructure provides a stem cell niche that regulates the pathways and promotes the expression of proteins related to epidermal stem cells. This work advances the functional properties of tissue engineered skin and holds promise for improving the therapeutic efficacy of artificial skin grafts for the skin wounds.

3D printing to construct in vitro multicellular models of melanoma.

Currently, there is a lack of suitable models for in-vitro studies of malignant melanoma and traditional single cell culture models no longer reproduce tumor structure and physiological complexity well. The tumor microenvironment is closely related to carcinogenesis and it is particularly important to understand how tumor cells interact and communicate with surrounding nonmalignant cells. Three-dimensional (3D) in vitro multicellular culture models can better simulate the tumor microenvironment due to their excellent physicochemical properties. In this study, 3D composite hydrogel scaffolds were prepared from gelatin methacrylate and polyethylene glycol diacrylate hydrogels by 3D printing and light curing techniques, and 3D multicellular in vitro tumor culture models were established by inoculating human melanoma cells (A375) and human fibroblasts cells on them. The cell proliferation, migration, invasion, and drug resistance of the 3D multicellular in vitro model was evaluated. Compared with the single-cell model, the cells in the multicellular model had higher proliferation activity and migration ability, and were easy to form dense structures. Several tumor cell markers, such as matrix metalloproteinase-9 (MMP-9), MMP-2, and vascular endothelial growth factor, were highly expressed in the multicellular culture model, which were more favorable for tumor development. In addition, higher cell survival rate was observed after exposure to luteolin. The anticancer drug resistance result of the malignant melanoma cells in the 3D bioprinted construct demonstrated physiological properties, suggesting the promising potential of current 3D printed tumor model in the development of personalized therapy, especially for discovery of more conducive targeted drugs.

3D Bioprinting Using Synovium-Derived MSC-Laden Photo-Cross-Linked ECM Bioink for Cartilage Regeneration.

In this study, inspired by the components of cartilage matrix, a photo-cross-linked extracellular matrix (ECM) bioink composed of modified proteins and polysaccharides was presented, including gelatin methacrylate, hyaluronic acid methacrylate, and chondroitin sulfate methacrylate. The systematic experiments were performed, including morphology, swelling, degradation, mechanical and rheological tests, printability analysis, biocompatibility and chondrogenic differentiation characterization, and RNA sequencing (RNA-seq). The results indicated that the photo-cross-linked ECM hydrogels possessed suitable degradation rate and excellent mechanical properties, and the three-dimensional (3D) bioprinted ECM scaffolds obtained favorable shape fidelity and improved the basic properties, biological properties, and chondrogenesis of synovium-derived MSCs (SMSCs). The strong stimulation of transforming growth factor-beta 1 (TGF-ß1) enhanced the aggregation, proliferation, and differentiation of SMSCs, thereby enhancing chondrogenic ECM deposition. In vivo animal experiments and gait analysis further confirmed that the ECM scaffold combined with TGF-ß1 could effectively promote cartilage regeneration and functional recovery of injured joints. To sum up, the photo-cross-linked ECM bioink for 3D printing of functional cartilage tissue may become an attractive strategy for cartilage regeneration.

3D bioprinting of heterogeneous tissue-engineered skin containing human dermal fibroblasts and keratinocytes.

Three-dimensional (3D) printed skin substitutes have great potential for wound healing. However, current 3D printed skin models are limited in simulating heterogeneity and complexity of skin tissue due to the lack of customized bioinks optimized for different skin layers. Herein, different gelatin methacrylate (GelMA)/nano-cellulose (BNC) bioink formulations were used to develop heterogeneous tissue-engineered skin (HTS) containing layers of fibroblast networks with larger pores, basal layers with smaller pores, and multilayered keratinocytes. The results revealed that the 10%GelMA/0.3%BNC bioink was better to model bioprinted dermis due to its high printability and cell-friendly sparse microenvironment. Additionally, the 10%GelMA/1.5%BNC bioink as the basal layer presented a dense network and sufficient material stiffness to support the establishment of keratinocyte confluent monolayers. The HTS not only had the ability to remodel the extracellular matrix but also supported epidermis reconstruction and stratification in vitro, with the epidermal thickness growing to 80 µm after 14 days. Furthermore, the full-thickness wound healing experiments demonstrated that the HTS promoted granulation tissue regeneration and improved wound healing quality. The generated skin of the HTS group had hair follicles and early-stage rete ridge structures, which were similar to normal skin in vivo. The HTS may deliver effective skin grafts for future clinical treatments.

3D printed-electrospun PCL/hydroxyapatite/MWCNTs scaffolds for the repair of subchondral bone.

Osteochondral defect caused by trauma or osteoarthritis exhibits a major challenge in clinical treatment with limited symptomatic effects at present. The regeneration and remodeling of subchondral bone play a positive effect on cartilage regeneration and further promotes the repair of osteochondral defects. Making use of the strengths of each preparation method, the combination of 3D printing and electrospinning is a promising method for designing and constructing multi-scale scaffolds that mimic the complexity and hierarchical structure of subchondral bone at the microscale and nanoscale, respectively. In this study, the 3D printed-electrospun poly(ɛ-caprolactone)/nano-hydroxyapatites/multi-walled carbon nanotubes (PCL/nHA/MWCNTs) scaffolds were successfully constructed by the combination of electrospinning and layer-by-layer 3D printing. The resulting dual-scale scaffold consisted of a dense layer of disordered nanospun fibers and a porous microscale 3D scaffold layer to support and promote the ingrowth of subchondral bone. Herein, the biomimetic PCL/nHA/MWCNTs scaffolds enhanced cell seeding efficiency and allowed for higher cell-cell interactions that supported the adhesion, proliferation, activity, morphology and subsequently improved the osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. Together, this study elucidates that the construction of 3D printed-electrospun PCL/nHA/MWCNTs scaffolds provides an alternative strategy for the regeneration of subchondral bone and lays a foundation for subsequent in vivo studies.

Collagen-coated agarose-chitosan scaffold used as a skin substitute.

A single biomaterial is disadvantageous for constructing skin in vitro, so a mixed biomaterial is more conducive to skin research. In this study, agarose-chitosan scaffolds with a final concentration of 4% were constructed by freeze-drying, in which the concentration ratios of agarose to chitosan were 1:3, 2:2, and 3:1. The scaffolds were coated with a 3 mg/ml collagen solution, and the mechanical properties were evaluated by studying density, porosity, swelling rate, and degradation rate. The results demonstrated that the agarose-chitosan scaffolds were porous, with porosity reaching 93%. Their densities ranged from 0.1 to 0.16 g/cm3 . Analysis of Young's modulus showed that the mechanical properties of the agarose-chitosan scaffolds were significantly enhanced when the agarose content in the agarose-chitosan scaffolds was increased. Moreover, the density and Young's modulus of the agarose-chitosan scaffolds of different concentration ratios were significantly different (p < 0.01). These scaffolds can withstand a certain amount of external pressure, such as that of human skin, making them more suitable for further skin replacement research. In addition, the results of thiazolyl blue tetrazolium bromide (MTT) cell assay and immunofluorescence staining showed that the collagen-coated agarose-chitosan scaffolds were conducive to keratinocyte proliferation and differentiation. The MTT results revealed significant differences between the agarose-chitosan scaffolds coated with collagen and the agarose-chitosan scaffolds without collagen (p < 0.05). This study provides the potential for in vitro skin research and applications.

Rete ridges: Morphogenesis, function, regulation, and reconstruction.

Rete ridges (RRs) are distinct undulating microstructures at the junction of the dermis and epidermis in the skin of humans and certain animals. This structure is essential for enhancing the mechanical characteristics of skin and preserving homeostasis. With the development of tissue engineering and regenerative medicine, artificial skin grafts have made great progress in the field of skin healing. However, the restoration of RRs has been often disregarded or absent in artificial skin grafts, which potentially compromise the efficacy of tissue repair and regeneration. Therefore, this review collates recent research advances in understanding the structural features, function, morphogenesis, influencing factors, and reconstruction strategies pertaining to RRs. In addition, the preparation methods and limitations of tissue-engineered skin with RRs are discussed. STATEMENT OF SIGNIFICANCE: The technology for the development of tissue-engineered skin (TES) is widely studied and reported; however, the preparation of TES containing rete ridges (RRs) is often ignored, with no literature reviews on the structural reconstruction of RRs. This review focuses on the progress pertaining to RRs and focuses on the reconstruction methods for RRs. In addition, it discusses the limitations of existing reconstruction methods. Therefore, this review could be a valuable reference for transferring TES with RR structure from the laboratory to clinical applications in skin repair.

Circ_0004712 Silencing Suppresses the Aggressive Changes of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Targeting miR-633/TRAF6 Axis.

Circular RNA_0004712 (circ_0004712) is reported to be up-regulated in rheumatoid arthritis (RA) patients. Nevertheless, its role and mechanism in RA pathology remain to be clarified. RNA and protein expression was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Cell viability, proliferation, apoptosis, migration, and inflammation were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 5-ethynyl-20-deoxyuridine assay, flow cytometry, scratch test, and enzyme-linked immunosorbent assay. The target correlation between microRNA-633 (miR-633) and circ_0004712 or TNF receptor associated factor 6 (TRAF6) was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Circ_0004712 was up-regulated in RA synovial tissues and RA fibroblast-like synoviocytes (RA-FLSs). Circ_0004712 silencing suppressed the viability, proliferation, migration and inflammatory response and facilitated the apoptosis of RA-FLSs. miR-633 was confirmed to be a direct target of circ_0004712, and miR-633 knockdown reversed circ_0004712 silencing-mediated protective effects on the dysfunction and inflammation of RA-FLSs. TRAF6 was a direct target of miR-633, and miR-633 overexpression suppressed the aggressive changes of RA-FLSs by down-regulating TRAF6. Circ_0004712 could up-regulate TRAF6 expression by sponging miR-633 in RA-FLSs. Circ_0004712 interference inactivated nuclear factor (NF)-κB signaling by targeting miR-633/TRAF6 axis. Circ_0004712 silencing inhibited the aggressive changes of RA-FLSs by targeting miR-633/TRAF6 axis and NF-κB signaling, which provided new targets for RA therapy.

Photo-crosslinked hydrogels for tissue engineering of corneal epithelium.

The vast majority of patients with corneal blindness cannot recover their vision due to the serious shortage of donor cornea. However, the technology to construct a feasible corneal substitute is a promising treatment method for corneal blindness. In this paper, methacrylated gelatin (GelMA)-methacrylated hyaluronic acid (HAMA) double network (GHDN) hydrogels were prepared by modifying gelatin and hyaluronic acid with methacrylate anhydride (MA). GHDN hydrogel was compared with GelMA single network and HAMA single network hydrogels through characterization experiments of mechanical properties, optical properties, hydrophilicity and in-situ degradation in vitro. At the same time, the biocompatibility of hydrogel was tested by inoculating rabbit corneal epithelial cells (CEpCs) epidermal cells on hydrogels using CCK-8 test, live/dead staining, immunofluorescence staining and qRT-PCR. It was found that the GHDN hydrogel has optical transparency in the visible region, and its mechanical properties are better than those of GelMA and HAMA hydrogels, and its hydrophilicity is similar to that of normal human corneas. The results of in vitro hydrogel culture of CEpCs showed that the proliferation of CEpCs on GHDN hydrogel was two times higher than that of HAMA hydrogel, and the expression of specific marker Cytokeratin 3 (CK3) and Cytokeratin 12 (CK12) could be better maintained on GHDN hydrogel. All the experimental results proved that GHDN hydrogel has good physical properties and biocompatibility and is a potential candidate for corneal tissue engineering scaffolds.

Biocompatible chitosan/polyethylene glycol/multi-walled carbon nanotube composite scaffolds for neural tissue engineering.

Carbon nanotube (CNT) composite materials are very attractive for use in neural tissue engineering and biosensor coatings. CNT scaffolds are excellent mimics of extracellular matrix due to their hydrophilicity, viscosity, and biocompatibility. CNTs can also impart conductivity to other insulating materials, improve mechanical stability, guide neuronal cell behavior, and trigger axon regeneration. The performance of chitosan (CS)/polyethylene glycol (PEG) composite scaffolds could be optimized by introducing multi-walled CNTs (MWCNTs). CS/PEG/CNT composite scaffolds with CNT content of 1%, 3%, and 5% (1%=0.01 g/mL) were prepared by freeze-drying. Their physical and chemical properties and biocompatibility were evaluated. Scanning electron microscopy (SEM) showed that the composite scaffolds had a highly connected porous structure. Transmission electron microscope (TEM) and Raman spectroscopy proved that the CNTs were well dispersed in the CS/PEG matrix and combined with the CS/PEG nanofiber bundles. MWCNTs enhanced the elastic modulus of the scaffold. The porosity of the scaffolds ranged from 83% to 96%. They reached a stable water swelling state within 24 h, and swelling decreased with increasing MWCNT concentration. The electrical conductivity and cell adhesion rate of the scaffolds increased with increasing MWCNT content. Immunofluorescence showed that rat pheochromocytoma (PC12) cells grown in the scaffolds had characteristics similar to nerve cells. We measured changes in the expression of nerve cell markers by quantitative real-time polymerase chain reaction (qRT-PCR), and found that PC12 cells cultured in the scaffolds expressed growth-associated protein 43 (GAP43), nerve growth factor receptor (NGFR), and class III ß|-tubulin (TUBB3) proteins. Preliminary research showed that the prepared CS/PEG/CNT scaffold has good biocompatibility and can be further applied to neural tissue engineering research.

3D Bioprinted GelMA/PEGDA Hybrid Scaffold for Establishing an In Vitro Model of Melanoma.

Due to the high incidence of malignant melanoma, the establishment of in vitro models that recapitulate the tumor microenvironment is of great biological and clinical importance for tumor treatment and drug research. In this study, 3D printing technology was used to prepare GelMA/PEGDA composite scaffolds that mimic the microenvironment of human malignant melanoma cell (A375) growth and construct in vitro melanoma micro-models. The GelMA/PEGDA hybrid scaffold was tested by the mechanical property, cell live/dead assay, cell proliferation assay, cytoskeleton staining and drug loading assay. The growth of tumor cells in two- and three-dimensional culture systems and the anti-cancer effect of luteolin were evaluated using the live/dead staining method and the Cell Counting Kit-8 (CCK-8) method. The results showed a high aggregation of tumor cells on the 3D scaffold, which was suitable for long-term culture. Cytoskeleton staining and immunofluorescent protein staining were used to evaluate the degree of differentiation of tumor cells under 2D and 3D culture systems. The results indicated that 3D bioprinted scaffolds were more suitable for tumor cell expansion and differentiation, and the tumor cells were more aggressive. In addition, luteolin was time- and dose-dependent on tumor cells, and tumor cells in the 3D culture system were more resistant to the drug.

Preparation and In Vitro Characterization of Gelatin Methacrylate for Corneal Tissue Engineering.

BACKGROUND: Corneal disease is second only to cataract considered as the leading cause of blindness in the world, with high morbidity. Construction of corneal substitutes in vitro by tissue engineering technology to achieve corneal regeneration has become a research hotspot in recent years. We conducted in-depth research on the biocompatibility, physicochemical and mechanical properties of rat bone marrow mesenchymal stem cells (rBM-MSCs)-seeded gelatin methacrylate (GelMA) as a bioengineered cornea. METHODS: Four kinds of GelMA with different concentrations (7, 10, 15 and 30%) were prepared, and their physic-chemical, optical properties, and biocompatibility with rBM-MSCs were characterized. MTT, live/dead staining, cell morphology, immunofluorescence staining and gene expression of keratocyte markers were performed. RESULTS: 7%GelMA hydrogel had higher equilibrium water content and porosity, better optical properties and hydrophilicity. In addition, it is more beneficial to the growth and proliferation of rBM-MSCs. However, the 30%GelMA hydrogel had the best mechanical properties, and could be more conducive to promote the differentiation of rBM-MSCs into keratocyte-like cells. CONCLUSION: As a natural biological scaffold, GelMA hydrogel has good biocompatibility. And it has the ability to promote the differentiation of rBM-MSCs into keratocyte-like cells, which laid a theoretical and experimental foundation for further tissue-engineered corneal stromal transplantation, and provided a new idea for the source of seeded cells in corneal tissue engineering.

Construction of tissue-engineered skin with rete ridges using co-network hydrogels of gelatin methacrylated and poly(ethylene glycol) diacrylate.

Tissue-engineered skin, as a promising skin substitute, can be used for in vitro skin research and skin repair. However, most of research on tissue-engineered skin tend to ignore the rete ridges (RRs) microstructure, which enhances the adhesion between dermis and epidermis and provides a growth environment for epidermal stem cells. Here, we prepared and characterized photocurable gelatin methacrylated (GelMA) and poly(ethylene glycol) diacrylate (PEGDA) co-network hydrogels with different concentrations. Using a UV curing 3D printer, resin molds were designed and fabricated to create three-dimensional micropatterns and replicated onto GelMA-PEGDA scaffolds. Human keratinocytes (HaCaTs) and human skin fibroblasts (HSFs) were co-cultured on the hydrogel scaffold to prepare tissue-engineered skin. The results showed that 10%GelMA-2%PEGDA hydrogel provides the sufficient mechanical properties and biocompatibility to prepare a human skin model with RRs microstructure, that is, it presents excellent structural support, suitable degradation rate, good bioactivity and is suitable for long-term culturing. Digital microscope image analyses showed the micropattern was well-transferred onto the scaffold surface. Both in vitro and in vivo experiments confirmed the formation of the epidermal layer with undulating microstructure. In wound healing experiments, hydrogel can significantly accelerate wound healing. This study provides a simple and powerful way to mimic the structures of human skin and can make a contribution to skin tissue engineering and wound healing.

Mesenchymal stem cells loaded on 3D-printed gradient poly(ε-caprolactone)/methacrylated alginate composite scaffolds for cartilage tissue engineering.

Cartilage has limited self-repair ability due to its avascular, alymphatic and aneural features. The combination of three-dimensional (3D) printing and tissue engineering provides an up-and-coming approach to address this issue. Here, we designed and fabricated a tri-layered (superficial layer (SL), middle layer (ML) and deep layer (DL)) stratified scaffold, inspired by the architecture of collagen fibers in native cartilage tissue. The scaffold was composed of 3D printed depth-dependent gradient poly(ε-caprolactone) (PCL) impregnated with methacrylated alginate (ALMA), and its morphological analysis and mechanical properties were tested. To prove the feasibility of the composite scaffolds for cartilage regeneration, the viability, proliferation, collagen deposition and chondrogenic differentiation of embedded rat bone marrow mesenchymal stem cells (BMSCs) in the scaffolds were assessed by Live/dead assay, CCK-8, DNA content, cell morphology, immunofluorescence and real-time reverse transcription polymerase chain reaction. BMSCs-loaded gradient PCL/ALMA scaffolds showed excellent cell survival, cell proliferation, cell morphology, collagen II deposition and hopeful chondrogenic differentiation compared with three individual-layer scaffolds. Hence, our study demonstrates the potential use of the gradient PCL/ALMA construct for enhanced cartilage tissue engineering.

Actin cytoskeleton reorganization correlates with polarization of secretory vesicle and cell morphology in the degranulation of mast cell subtypes in human colon tissues.

Mast cells play a central role in the intestinal immune response. To investigate the relationship between degranulation, cell polarization and the reorganization of actin cytoskeleton of mast cells, we used fluorescence or gold labeling methods to identify different mast cell subtypes in human colon. The reorganization of filamentous actin was visualized and then the polarization of secretory vesicles, as well as cell surfaces, was analyzed by fluorescence microscopy and electron microscopy. Our results first showed a diversity of filamentous actin assembly or disassembly within the contacting cell membrane of different mast cell subtypes. The polarization and degranulation of secretory vesicles was not only accompanied with the assembly and disassembly of filamentous actin at the cell periphery, but also with changes of cell surface polarization. Our study provides an insight into the local membranous structures and suggested correlations of cytoskeleton arrangement with the polarization of secretory vesicles and cell surface configuration during mast cell degranulation.

[Selective neurotomy of anterior ethmoid nerve and squeezing operation of inferior turbinate for the treatment of perennial allergic rhinitis].

OBJECTIVE: To explore the treatment effect of selective neurotomy of anterior ethmoid nerve and squeezing operation of inferior turbinate in the treatment of perennial allergic rhinitis (PAR). METHOD: Seventy cases of perennial allergic rhinitis were selected and subjected to selective neurotomy of anterior ethmoid nerve and squeezing operation of inferior turbinate,and the treatment effect was observed by analysis of the the symptoms and signs score of all cases preoperatively and postoperatively. RESULT: The total effective rate were 90.0% at 1 year follow-up. CONCLUSION: Selective neurotomy of anterior ethmoid nerve and squeezing operation of inferior turbinate were effective for the patients with PAR. It is worthy to be popularized for its convent and rare complications.