Low-Temperature Extrusion of Waterborne Polyurethane-Polycaprolactone Composites for Multi-Material Bioprinting of Engineered Elastic Cartilage.

3D bioprinting of elastic cartilage tissues that are mechanically and structurally comparable to their native counterparts, while exhibiting favorable cellular behavior, is an unmet challenge. A practical solution for this problem is the multi-material bioprinting of thermoplastic polymers and cell-laden hydrogels using multiple nozzles. However, the processing of thermoplastic polymers requires high temperatures, which can damage hydrogel-encapsulated cells. In this study, the authors developed waterborne polyurethane (WPU)-polycaprolactone (PCL) composites to allow multi-material co-printing with cell-laden gelatin methacryloyl (GelMA) hydrogels. These composites can be extruded at low temperatures (50-60 °C) and high speeds, thereby reducing heat/shear damage to the printed hydrogel-capsulated cells. Furthermore, their hydrophilic nature improved the cell behavior in vitro. More importantly, the bioprinted structures exhibited good stiffness and viscoelasticity compared to native elastic cartilage. In summary, this study demonstrated low-temperature multi-material bioprinting of WPU-PCL-based constructs with good mechanical properties, degradation time-frames, and cell viability, showcasing their potential in elastic cartilage bio-fabrication and regeneration to serve broad biomedical applications in the future.

3D-bioprinted human lipoaspirate-derived cell-laden skin constructs for healing of full-thickness skin defects.

29Three-dimensional (3D)-printed bioactive scaffolds that can be produced rapidly could offer an individualized approach for treating full-thickness skin defects. Decellularized extracellular matrix (dECM) and mesenchymal stem cells have been proven to support wound healing. Adipose tissues obtained by liposuction are rich in adipose-derived dECM (adECM) and adipose-derived stem cells (ADSCs) and thus represent a natural source of bioactive materials for 3D bioprinting. Herein, ADSC-laden 3D-printed bioactive scaffolds consisting of gelatin methacryloyl (GelMA), hyaluronic acid methacryloyl (HAMA), and adECM were fabricated with dual properties of photocrosslinking in vitro and thermosensitive crosslinking in vivo. adECM was prepared by decellularization of human lipoaspirate and mixed as a bioactive material with GelMA and HAMA to form a bioink. Compared with the GelMA-HAMA bioink, the adECM-GelMA-HAMA bioink had better wettability, degradability, and cytocompatibility. Full-thickness skin defect healing in a nude mouse model showed that ADSC-laden adECM-GelMA-HAMA scaffolds accelerated wound healing by promoting faster neovascularization, collagen secretion, and remodeling. ADSCs and adECM collectively conferred bioactivity on the prepared bioink. This study represents a novel approach to enhancing the biological activity of 3D-bioprinted skin substitutes by adding adECM and ADSCs derived from human lipoaspirate and may provide a promising therapeutic option for full-thickness skin defects.

Application of 3D-printed tissue-engineered skin substitute using innovative biomaterial loaded with human adipose-derived stem cells in wound healing.

Large-scale skin injuries are usually accompanied by impaired wound healing, resulting in scar formation, or significant morbidity and mortality. The aim of this study is to explore the in vivo application of 3D-printed tissue-engineered skin substitute using innovative biomaterial loaded with human adipose-derived stem cells (hADSCs) in wound healing. Adipose tissue was decellularized, and extracellular matrix components were lyophilized and solubilized to obtain adipose tissue decellularized extracellular matrix (dECM) pre-gel. The newly designed biomaterial is composed of adipose tissue dECM pre-gel, methacrylated gelatin (GelMA), and methacrylated hyaluronic acid (HAMA). Rheological measurement was performed to evaluate the phase-transition temperature and the storage and loss modulus at this temperature. Tissue-engineered skin substitute loaded with hADSCs was fabricated by 3D printing. We used nude mice to establish full-thickness skin wound healing model and divided them into four groups randomly: (A) Full-thickness skin graft treatment group, (B) 3D-bioprinted skin substitute treatment group as the experimental group, (C) microskin graft treatment group, and (D) control group. The amount of DNA in each milligram of dECM was 24.5 ± 7.1 ng, fulfilling the currently accepted decellularization criteria. The solubilized adipose tissue dECM was thermo-sensitive biomaterial and underwent a sol-gel phase transition when temperature rises. The dECM-GelMA-HAMA precursor undergoes a gel-sol phase transition at 17.5°C, where the storage and loss modulus of the precursor is about 8 Pa. The scanning electron microscope showed that the interior of crosslinked dECM-GelMA-HAMA hydrogel is 3D porous network structure with suitable porosity and pore size. The shape of the skin substitute is stable with regular grid-like scaffold structure. Wound healing in the experimented animals was accelerated after being treated with 3D-printed skin substitute, which attenuate inflammatory response, increase blood perfusion around the wound, as well as promote re-epithelialization, collagen deposition and alignment, and angiogenesis. In summary, 3D-printed dECM-GelMA-HAMA tissue-engineered skin substitute loaded with hADSCs, which can be fabricated by 3D printing, can accelerate wound healing and improve healing quality by promoting angiogenesis. The hADSCs and the stable 3D-printed stereoscopic grid-like scaffold structure play a critical role in promoting wound healing.

Chondrogenic Differentiation of Adipose-Derived Stromal Cells Induced by Decellularized Cartilage Matrix/Silk Fibroin Secondary Crosslinking Hydrogel Scaffolds with a Three-Dimensional Microstructure.

Finding an ideal scaffold is always an important issue in the field of cartilage tissue engineering. Both decellularized extracellular matrix and silk fibroin have been used as natural biomaterials for tissue regeneration. In this study, a secondary crosslinking method of γ irradiation and ethanol induction was used to prepare decellularized cartilage extracellular matrix and silk fibroin (dECM-SF) hydrogels with biological activity. Furthermore, the dECM-SF hydrogels were cast in custom-designed molds to produce a three-dimensional multi-channeled structure to improve internal connectivity. The adipose-derived stromal cells (ADSC) were seeded on the scaffolds, cultured in vitro for 2 weeks, and implanted in vivo for another 4 and 12 weeks. The double crosslinked dECM-SF hydrogels exhibited an excellent pore structure after lyophilization. The multi-channeled hydrogel scaffold presents higher water absorption ability, surface wettability, and no cytotoxicity. The addition of dECM and a channeled structure could promote chondrogenic differentiation of ADSC and engineered cartilage formation, confirmed by H&E, safranin O staining, type II collagen immunostaining, and qPCR assay. In conclusion, the hydrogel scaffold fabricated by the secondary crosslinking method has good plasticity and can be used as a scaffold for cartilage tissue engineering. The multi-channeled dECM-SF hydrogel scaffolds possess a chondrogenic induction activity that promotes engineered cartilage regeneration of ADSC in vivo.

Bacterial nanocellulose-reinforced gelatin methacryloyl hydrogel enhances biomechanical property and glycosaminoglycan content of 3D-bioprinted cartilage.

Tissue-engineered ear cartilage scaffold based on three-dimensional (3D) bioprinting technology presents a new strategy for ear reconstruction in individuals with microtia. Natural hydrogel is a promising material due to its excellent biocompatibility and low immunogenicity. However, insufficient mechanical property required for cartilage is one of the major issues pending to be solved. In this study, the gelatin methacryloyl (GelMA) hydrogel reinforced with bacterial nanocellulose (BNC) was developed to enhance the biomechanical properties and printability of the hydrogel. The results revealed that the addition of 0.375% BNC significantly increased the mechanical properties of the hydrogel and promoted cell migration in the BNC-reinforced hydrogel. Constructs bioprinted with chondrocyte-laden BNC/GelMA hydrogel bio-ink formed mature cartilage in nude mice with higher Young's modulus and glycosaminoglycan content. Finally, an auricle equivalent with a precise shape, high mechanics, and abundant cartilage-specific matrix was developed in vivo. In this study, we developed a potentially useful hydrogel for the manufacture of auricular cartilage grafts for microtia patients.

Bioprinting and regeneration of auricular cartilage using a bioactive bioink based on microporous photocrosslinkable acellular cartilage matrix.

Tissue engineering provides a promising strategy for auricular reconstruction. Although the first international clinical breakthrough of tissue-engineered auricular reconstruction has been realized based on polymer scaffolds, this approach has not been recognized as a clinically available treatment because of its unsatisfactory clinical efficacy. This is mainly since reconstruction constructs easily cause inflammation and deformation. In this study, we present a novel strategy for the development of biological auricle equivalents with precise shapes, low immunogenicity, and excellent mechanics using auricular chondrocytes and a bioactive bioink based on biomimetic microporous methacrylate-modified acellular cartilage matrix (ACMMA) with the assistance of gelatin methacrylate (GelMA), poly(ethylene oxide) (PEO), and polycaprolactone (PCL) by integrating multi-nozzle bioprinting technology. Photocrosslinkable ACMMA is used to emulate the intricacy of the cartilage-specific microenvironment for active cellular behavior, while GelMA, PEO, and PCL are used to balance printability and physical properties for precise structural stability, form the microporous structure for unhindered nutrient exchange, and provide mechanical support for higher shape fidelity, respectively. Finally, mature auricular cartilage-like tissues with high morphological fidelity, excellent elasticity, abundant cartilage lacunae, and cartilage-specific ECM deposition are successfully regenerated in vivo, which provides new opportunities and novel strategies for the fabrication and regeneration of patient-specific auricular cartilage.

The Role of Verteporfin in Prevention of Periprosthetic Capsular Fibrosis: An Experimental Study.

BACKGROUND: Capsular contracture (CC) characterized by excessive fibrosis is one of the most common complications after silicone implant surgery. Verteporfin (VP), an inhibitor of Yes-associated protein 1 (YAP1), has recently been found to reduce the fibrotic process. OBJECTIVES: The aim of this study was to use an in vivo rabbit model to evaluate the efficacy of VP for the prevention of CC. METHODS: Twenty-four New Zealand rabbits received 10-cc smooth saline silicone implants inserted in the dorsal skin and were randomly divided into 2 groups to receive 2 mL VP (1.5 mg/mL) or 2 mL phosphate-buffered saline solution instillation in the implant pocket. When the animals were killed on Day 60, capsule formation was observed both macroscopically and microscopically. Histologic evaluation included capsule thickness, fibrosis degree, and myofibroblast (α smooth muscle actin positive) content. In addition, the YAP1 expression level was examined by immunofluorescence staining. Transforming growth factor ß1, collagen I, and connective tissue growth factor expression were measured by real-time quantitative polymerase chain reaction. RESULTS: The VP-treated group exhibited thinner, more transparent capsules and less fibrosis than the control group at 60 days postsurgery (P < 0.05). Moreover, the VP treatment significantly reduced α smooth muscle actin, YAP1, transforming growth factor ß1, collagen I, and connective tissue growth factor expression levels in the capsular tissues (P < 0.05). CONCLUSIONS: VP reduced capsule formation after silicone implantation by inhibiting YAP1-mediated mechanical signaling, thereby attenuating excessive collagen deposition in the rabbit model. This preclinical study may provide a feasible strategy to prevent periprosthetic capsular fibrosis in clinical application.

[Experimental study on tissue engineered cartilage constructed by three-dimensional bioprinted human adipose-derived stem cells combined with gelatin methacryloyl].

OBJECTIVE: To explore the feasibility of three-dimensional (3D) bioprinted adipose-derived stem cells (ADSCs) combined with gelatin methacryloyl (GelMA) to construct tissue engineered cartilage. METHODS: Adipose tissue voluntarily donated by liposuction patients was collected to isolate and culture human ADSCs (hADSCs). The third generation cells were mixed with GelMA hydrogel and photoinitiator to make biological ink. The hADSCs-GelMA composite scaffold was prepared by 3D bioprinting technology, and it was observed in general, and observed by scanning electron microscope after cultured for 1 day and chondrogenic induction culture for 14 days. After cultured for 1, 4, and 7 days, the composite scaffolds were taken for live/dead cell staining to observe cell survival rate; and cell counting kit 8 (CCK-8) method was used to detect cell proliferation. The composite scaffold samples cultured in cartilage induction for 14 days were taken as the experimental group, and the composite scaffolds cultured in complete medium for 14 days were used as the control group. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect cartilage formation. The relative expression levels of the mRNA of cartilage matrix gene [(aggrecan, ACAN)], chondrogenic regulatory factor (SOX9), cartilage-specific gene [collagen type Ⅱ A1 (COLⅡA1)], and cartilage hypertrophy marker gene [collagen type ⅩA1 (COLⅩA1)] were detected. The 3D bioprinted hADSCs-GelMA composite scaffold (experimental group) and the blank GelMA hydrogel scaffold without cells (control group) cultured for 14 days of chondrogenesis were implanted into the subcutaneous pockets of the back of nude mice respectively, and the materials were taken after 4 weeks, and gross observation, Safranin O staining, Alcian blue staining, and collagen type Ⅱ immunohistochemical staining were performed to observe the cartilage formation in the composite scaffold. RESULTS: Macroscope and scanning electron microscope observations showed that the hADSCs-GelMA composite scaffolds had a stable and regular structure. The cell viability could be maintained at 80%-90% at 1, 4, and 7 days after printing, and the differences between different time points were significant ( P<0.05). The results of CCK-8 experiment showed that the cells in the scaffold showed continuous proliferation after printing. After 14 days of chondrogenic induction and culture on the composite scaffold, the expressions of ACAN, SOX9, and COLⅡA1 were significantly up-regulated ( P<0.05), the expression of COLⅩA1 was significantly down-regulated ( P<0.05). The scaffold was taken out at 4 weeks after implantation. The structure of the scaffold was complete and clear. Histological and immunohistochemical results showed that cartilage matrix and collagen type Ⅱ were deposited, and there was cartilage lacuna formation, which confirmed the formation of cartilage tissue. CONCLUSION: The 3D bioprinted hADSCs-GelMA composite scaffold has a stable 3D structure and high cell viability, and can be induced differentiation into cartilage tissue, which can be used to construct tissue engineered cartilage in vivo and in vitro.

A novel waterborne polyurethane with biodegradability and high flexibility for 3D printing.

Three-dimensional (3D) printing provides a new approach of fabricating implantable products because it permits a flexible manner to extrude complex and customized shapes of the tissue scaffolds. Compared with other printable biomaterials, the polyurethane elastomer has several merits, including excellent mechanical properties and good biocompatibility. However, some intrinsic behavior, especially its high melting point and slow rate of degradation, hampered its application in 3D printed tissue engineering. Herein, we developed a 3D printable amino acid modified biodegradable waterborne polyurethane (WBPU) using a water-based green chemistry process. The flexibility of this material endows better compliance with tissue during implantation and prevents high modulus transplants from scratching surrounding tissues. The histocompatibility experiments show that the WBPU induces no apparent acute rejection or inflammation in vivo. We successfully fabricated a highly flexible WBPU scaffold by deposition 3D printing technology at a low temperature (50°C ~ 70 °C), and the printed products could support the adhesion and proliferation of chondrocytes and fibroblasts. The printed blocks possessed controllable degradability due to the different amounts of hydrophilic chain extender and did not cause accumulation of acidic products. In addition, we demonstrated that our WBPU is highly applicable for implantable tissue engineering because there is no cytotoxicity during its degradation. Taken together, we envision that this printable WBPU can be used as an alternative biomaterial for tissue engineering with low temperature printing, biodegradability, and compatibility.