Hybrid extracellular vesicles-liposome incorporated advanced bioink to deliver microRNA.

In additive manufacturing, bioink formulations govern strategies to engineer 3D living tissues that mimic the complex architectures and functions of native tissues for successful tissue regeneration. Conventional 3D-printed tissues are limited in their ability to alter the fate of laden cells. Specifically, the efficient delivery of gene expression regulators (i.e. microRNAs (miRNAs)) to cells in bioprinted tissues has remained largely elusive. In this study, we explored the inclusion of extracellular vesicles (EVs), naturally occurring nanovesicles (NVs), into bioinks to resolve this challenge. EVs show excellent biocompatibility, rapid endocytosis, and low immunogenicity, which lead to the efficient delivery of miRNAs without measurable cytotoxicity. EVs were fused with liposomes to prolong and control their release by altering their physical interaction with the bioink. Hybrid EVs-liposome (hEL) NVs were embedded in gelatin-based hydrogels to create bioinks that could efficiently encapsulate and deliver miRNAs at the target site in a controlled and sustained manner. The regulation of cells' gene expression in a 3D bioprinted matrix was achieved using the hELs-laden bioink as a precursor for excellent shape fidelity and high cell viability constructs. Novel regulatory factors-loaded bioinks will expedite the translation of new bioprinting applications in the tissue engineering field.

Light-controlled growth factors release on tetrapodal ZnO-incorporated 3D-printed hydrogels for developing smart wound scaffold.

Advanced wound scaffolds that integrate active substances to treat chronic wounds have gained significant recent attention. While wound scaffolds and advanced functionalities have previously been incorporated into one medical device, the wirelessly triggered release of active substances has remained the focus of many research endeavors. To combine multiple functions including light-triggered activation, anti-septic, angiogenic, and moisturizing properties, we have developed a 3D printed hydrogel patch encapsulating vascular endothelial growth factor (VEGF) decorated with photoactive and antibacterial tetrapodal zinc oxide (t-ZnO) microparticles. To achieve the smart release of VEGF, t-ZnO was modified by chemical treatment and activated through UV/visible light exposure. This process would also make the surface rough and improve protein adhesion. The elastic modulus and degradation behavior of the composite hydrogels, which must match the wound healing process, were adjusted by changing t-ZnO concentrations. The t-ZnO-laden composite hydrogels can be printed with any desired micropattern to potentially create a modular elution of various growth factors. The VEGF decorated t-ZnO-laden hydrogel patches showed low cytotoxicity and improved angiogenic properties while maintaining antibacterial functions in vitro. In vivo tests showed promising results for the printed wound patches, with less immunogenicity and enhanced wound healing.

Recent advances in 3D bioprinting of musculoskeletal tissues.

The musculoskeletal system is essential for maintaining posture, protecting organs, facilitating locomotion, and regulating various cellular and metabolic functions. Injury to this system due to trauma or wear is common, and severe damage may require surgery to restore function and prevent further harm. Autografts are the current gold standard for the replacement of lost or damaged tissues. However, these grafts are constrained by limited supply and donor site morbidity. Allografts, xenografts, and alloplastic materials represent viable alternatives, but each of these methods also has its own problems and limitations. Technological advances in three-dimensional (3D) printing and its biomedical adaptation, 3D bioprinting, have the potential to provide viable, autologous tissue-like constructs that can be used to repair musculoskeletal defects. Though bioprinting is currently unable to develop mature, implantable tissues, it can pattern cells in 3D constructs with features facilitating maturation and vascularization. Further advances in the field may enable the manufacture of constructs that can mimic native tissues in complexity, spatial heterogeneity, and ultimately, clinical utility. This review studies the use of 3D bioprinting for engineering bone, cartilage, muscle, tendon, ligament, and their interface tissues. Additionally, the current limitations and challenges in the field are discussed and the prospects for future progress are highlighted.

Advancing Frontiers in Bone Bioprinting.

Three-dimensional (3D) bioprinting of cell-laden biomaterials is used to fabricate constructs that can mimic the structure of native tissues. The main techniques used for 3D bioprinting include microextrusion, inkjet, and laser-assisted bioprinting. Bioinks used for bone bioprinting include hydrogels loaded with bioactive ceramics, cells, and growth factors. In this review, a critical overview of the recent literature on various types of bioinks used for bone bioprinting is presented. Major challenges, such as the vascularity, clinically relevant size, and mechanical properties of 3D printed structures, that need to be addressed to successfully use the technology in clinical settings, are discussed. Emerging approaches to solve these problems are reviewed, and future strategies to design customized 3D printed structures are proposed.

Advances in osteobiologic materials for bone substitutes.

A significant challenge in the current orthopedics is the development of suitable osteobiologic materials that can replace the conventional allografts, autografts, and xenografts and thereby serve as implant materials as bone substitutes for bone repair or remodelling. The complex biology behind the nanostructure and microstructure of bones and their repair mechanisms, which involve various types of chemical and biomechanical signalling amongst different cells, has set strong requirements for biomaterials to be used in bone tissue engineering. This review presents an overview of various types of osteobiologic materials to facilitate the formation of the functional bone tissue and healing of the bone, covering metallic, ceramic, polymeric, and cell-based graft substitutes, as well as some biomolecular strategies including stem cells, extracellular matrices, growth factors, and gene therapies. Advantages and disadvantages of each type, particularly from the perspective of osteoinductive and osteoconductive capabilities, are discussed. Although the numerous challenges of bone regeneration in tissue engineering and regenerative medicine are yet to be entirely addressed, further advancements in osteobiologic materials will pave the way towards engineering fully functional bone replacement grafts.

Visible light crosslinkable human hair keratin hydrogels.

Keratins extracted from human hair have emerged as a promising biomaterial for various biomedical applications, partly due to their wide availability, low cost, minimal immune response, and the potential to engineer autologous tissue constructs. However, the fabrication of keratin-based scaffolds typically relies on limited crosslinking mechanisms, such as via physical interactions or disulfide bond formation, which are time-consuming and result in relatively poor mechanical strength and stability. Here, we report the preparation of photocrosslinkable keratin-polyethylene glycol (PEG) hydrogels via the thiol-norbornene "click" reaction, which can be formed within one minute upon irradiation of visible light. The resulting keratin-PEG hydrogels showed highly tunable mechanical properties of up to 45 kPa in compressive modulus, and long-term stability in buffer solutions and cell culture media. These keratin-based hydrogels were tested as cell culture substrates in both two-dimensional surface seeding and three-dimensional cell encapsulation, demonstrating excellent cytocompatibility to support the attachment, spreading, and proliferation of fibroblast cells. Moreover, the photocrosslinking mechanism makes keratin-based hydrogel suitable for various microfabrication techniques, such as micropatterning and wet spinning, to fabricate cell-laden tissue constructs with different architectures. We believe that the unique features of this photocrosslinkable human hair keratin hydrogel promise new opportunities for their future biomedical applications.

Porous Electrospun Fibers with Self-Sealing Functionality: An Enabling Strategy for Trapping Biomacromolecules.

Stimuli-responsive porous polymer materials have promising biomedical application due to their ability to trap and release biomacromolecules. In this work, a class of highly porous electrospun fibers is designed using polylactide as the polymer matrix and poly(ethylene oxide) as a porogen. Carbon nanotubes (CNTs) with different concentrations are further impregnated onto the fibers to achieve self-sealing functionality induced by photothermal conversion upon light irradiation. The fibers with 0.4 mg mL-1 of CNTs exhibit the optimum encapsulation efficiency of model biomacromolecules such as dextran, bovine serum albumin, and nucleic acids, although their photothermal conversion ability is slightly lower than the fibers with 0.8 mg mL-1 of CNTs. Interestingly, reversible reopening of the surface pores is accomplished with the degradation of PLA, affording a further possibility for sustained release of biomacromolecules after encapsulation. Effects of CNT loading on fiber morphology, structure, thermal/mechanical properties, degradation, and cell viability are also investigated. This novel class of porous electrospun fibers with self-sealing capability has great potential to serve as an enabling strategy for trapping/release of biomacromolecules with promising applications in, for example, preventing inflammatory diseases by scavenging cytokines from interstitial body fluids.

Bioprinted Osteogenic and Vasculogenic Patterns for Engineering 3D Bone Tissue.

Fabricating 3D large-scale bone tissue constructs with functional vasculature has been a particular challenge in engineering tissues suitable for repairing large bone defects. To address this challenge, an extrusion-based direct-writing bioprinting strategy is utilized to fabricate microstructured bone-like tissue constructs containing a perfusable vascular lumen. The bioprinted constructs are used as biomimetic in vitro matrices to co-culture human umbilical vein endothelial cells and bone marrow derived human mesenchymal stem cells in a naturally derived hydrogel. To form the perfusable blood vessel inside the bioprinted construct, a central cylinder with 5% gelatin methacryloyl (GelMA) hydrogel at low methacryloyl substitution (GelMALOW ) was printed. We also develop cell-laden cylinder elements made of GelMA hydrogel loaded with silicate nanoplatelets to induce osteogenesis, and synthesized hydrogel formulations with chemically conjugated vascular endothelial growth factor to promote vascular spreading. It was found that the engineered construct is able to support cell survival and proliferation during maturation in vitro. Additionally, the whole construct demonstrates high structural stability during the in vitro culture for 21 days. This method enables the local control of physical and chemical microniches and the establishment of gradients in the bioprinted constructs.

Engineering Photocrosslinkable Bicomponent Hydrogel Constructs for Creating 3D Vascularized Bone.

Engineering bone tissue requires the generation of a highly organized vasculature. Cellular behavior is affected by the respective niche. Directing cellular behavior and differentiation for creating mineralized regions surrounded by vasculature can be achieved by controlling the pattern of osteogenic and angiogenic niches. This manuscript reports on engineering vascularized bone tissues by incorporating osteogenic and angiogenic cell-laden niches in a photocrosslinkable hydrogel construct. Two-step photolithography process is used to control the stiffness of the hydrogel and distribution of cells in the patterned hydrogel. In addittion, osteoinductive nanoparticles are utilized to induce osteogenesis. The size of microfabricated constructs has a pronounced effect on cellular organization and function. It is shown that the simultaneous presence of both osteogenic and angiogenic niches in one construct results in formation of mineralized regions surrounded by organized vasculature. In addition, the presence of angiogenic niche improves bone formation. This approach can be used for engineered constructs that can be used for treatment of bone defects.

Direct 3D bioprinting of perfusable vascular constructs using a blend bioink.

Despite the significant technological advancement in tissue engineering, challenges still exist towards the development of complex and fully functional tissue constructs that mimic their natural counterparts. To address these challenges, bioprinting has emerged as an enabling technology to create highly organized three-dimensional (3D) vascular networks within engineered tissue constructs to promote the transport of oxygen, nutrients, and waste products, which can hardly be realized using conventional microfabrication techniques. Here, we report the development of a versatile 3D bioprinting strategy that employs biomimetic biomaterials and an advanced extrusion system to deposit perfusable vascular structures with highly ordered arrangements in a single-step process. In particular, a specially designed cell-responsive bioink consisting of gelatin methacryloyl (GelMA), sodium alginate, and 4-arm poly(ethylene glycol)-tetra-acrylate (PEGTA) was used in combination with a multilayered coaxial extrusion system to achieve direct 3D bioprinting. This blend bioink could be first ionically crosslinked by calcium ions followed by covalent photocrosslinking of GelMA and PEGTA to form stable constructs. The rheological properties of the bioink and the mechanical strengths of the resulting constructs were tuned by the introduction of PEGTA, which facilitated the precise deposition of complex multilayered 3D perfusable hollow tubes. This blend bioink also displayed favorable biological characteristics that supported the spreading and proliferation of encapsulated endothelial and stem cells in the bioprinted constructs, leading to the formation of biologically relevant, highly organized, perfusable vessels. These characteristics make this novel 3D bioprinting technique superior to conventional microfabrication or sacrificial templating approaches for fabrication of the perfusable vasculature. We envision that our advanced bioprinting technology and bioink formulation may also have significant potentials in engineering large-scale vascularized tissue constructs towards applications in organ transplantation and repair.

Detachment of cells adhered on the photoreactive phospholipid polymer surface by photoirradiation and their functionality.

Regulating the detachment of adhered living cells from a surface is a key technological requirement to obtain specific cells in the field of cellular engineering. Here, we describe dynamic control of cell adhesion and detachment at a photoreactive and cytocompatible phospholipid polymer surface. The surface was prepared using the amphiphilic and water-insoluble substance poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) bearing 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (PL) groups in its side chain (PMB-PL). On this prepared surface, the photoinduced control of human epidermoid carcinoma cancer cells (A431 cells) and primary murine embryonic fibroblast (PMEF) cells was examined. The PMB-PL surface allowed successful control of living cells adhesion with photoreactivity. The efficiency of cell detachment obtained was approximately 50% of the initial number of adhered cells. The PL groups at the surface provide adhesion points for cells, as evidenced by the fact that after photoreaction of PL groups by photoirradiation, the number of adhered cells on the surface considerably decreased. Additionally, when the polymer surface was re-used after the first photoirradiation, cells did not adhere to it, and low detachment was observed. The functionalities of the cells detached by photoreaction were evaluated. The proliferation rate and morphological changes of cells were as the same as those of cells detached by conventional enzymatic digestion. Moreover, the extracellular expression levels of transmembrane proteins on cells detached by photoirradiation showed no significant difference with those seen on normal cells. From these results, we conclude that the PMB-PL surface is a suitable platform to regulate cell adhesion and detachment via photochemical reaction.

Cell adhesion control on photoreactive phospholipid polymer surfaces.

Non-invasive and effective cell recovery from culture substrates is important for the passage and characterization of cells. In this study, a photoreactive polymer surface, which uses UV-irradiation to control substrate cell adhesion, was prepared. The photoreactive phospholipid polymer (PMB-PL) reported herein, was composed of a both 2-methacryloyloxyethyl phosphorylcholine (MPC) unit as a cytocompatible unit and methacrylate bearing a photolabile nitrobenzyl group. The PMB-PL polymer was used to coat a cell culture substrate thus affording a photoreactive surface. Surface analysis of the PMB-PL coating indicated a strong photoresponse owing to the sensitivity of the PL unit. Before light exposure, the PMB-PL surface provided cell adhesion. Following UV-irradiation, the PMB-PL coating was converted to a neutral ζ-potential and hydrophilic surface. The photoreactive surface conversion process allowed for the detachment of adhered cells from the PMB-PL surface while maintaining cell viability. This study demonstrates the promise and significance of the PMB-PL photoreactive surface as a method to control cell attachment and detachment for cell function investigation.