Disposable immunoplatforms for the simultaneous determination of biomarkers for neurodegenerative disorders using poly(amidoamine) dendrimer/gold nanoparticle nanocomposite.

Early diagnosis in primary care settings can increase access to therapies and their efficiency as well as reduce health care costs. In this context, we report in this paper the development of a disposable immunoplatform for the rapid and simultaneous determination of two protein biomarkers recently reported to be involved in the pathological process of neurodegenerative disorders (NDD), tau protein (tau), and TAR DNA-binding protein 43 (TDP-43). The methodology involves implementation of a sandwich-type immunoassay on the surface of dual screen-printed carbon electrodes (dSPCEs) electrochemically grafted with p-aminobenzoic acid (p-ABA), which allows the covalent immobilization of a gold nanoparticle-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM). This scaffold was employed for the immobilization of the capture antibodies (CAbs). Detector antibodies labeled with horseradish peroxidase (HRP) and amperometric detection at - 0.20 V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system were used. The developed methodology exhibits high sensitivity and selectivity for determining the target proteins, with detection limits of 2.3 and 12.8 pg mL-1 for tau and TDP-43, respectively. The simultaneous determination of tau and TDP-43 was accomplished in raw plasma samples and brain tissue extracts from healthy individuals and NDD-diagnosed patients. The analysis can be performed in just 1 h using a simple one-step assay protocol and small sample amounts (5 µL plasma and 2.5 µg brain tissue extracts). Graphical abstract.

The role of nanohydroxyapatite on the morphological, physical, and biological properties of chitosan nanofibers.

OBJECTIVES: This study aimed to evaluate the effects of nanohydroxyapatite (nHAp) particles on the morphological, chemical, physical, and biological properties of chitosan electrospun nanofibers. MATERIALS AND METHODS: nHAp particles with a 1.67 Ca/P ratio were synthesized via the aqueous precipitation method, incorporated into chitosan polymer solution (0.5 wt%), and electrospun into nHAp-loaded fibers (ChHa fibers). Neat chitosan fibers (nHAp-free, Ch fibers) were used as the control. The electrospun fiber mats were characterized using morphological, topographical, chemical, thermal, and a range of biological (antibacterial, antibiofilm, cell viability, and alkaline phosphatase [ALP] activity) analyses. Data were analyzed using ANOVA and Tukey's test (α = 0.05). RESULTS: ChHa fibers demonstrated a bead-like morphology, with thinner (331 ± 110 nm) and smoother (Ra = 2.9 ± 0.3 µm) distribution as compared to the control fibers. Despite showing similar cell viability and ALP activity to Ch fibers, the ChHa fibers demonstrated greater antibacterial potential against most tested bacteria (except for P. intermedia), and higher antibiofilm activity against P. gingivalis biofilm. CONCLUSIONS: The incorporation of nHAp particles did not jeopardize the overall morphology, topography, physical, and biological characteristics of the chitosan nanofibers. CLINICAL RELEVANCE: The combination of nHAp particles with chitosan can be used to engineer bioactive, electrospun composite nanofibers with potential applications in regenerative dentistry.

Advances in dual functional antimicrobial and osteoinductive biomaterials for orthopaedic applications.

A vast growing problem in orthopaedic medicine is the increase of clinical cases with antibiotic resistant pathogenic microbes, which is predicted to cause higher mortality than all cancers combined by 2050. Bone infectious diseases limit the healing ability of tissues and increase the risk of future injuries due to pathologic tissue remodelling. The traditional treatment for bone infections has several drawbacks and limitations, such as lengthy antibiotic treatment, extensive surgical interventions, and removal of orthopaedic implants and/or prosthesis, all of these resulting in long-term rehabilitation. This is a huge burden to the public health system resulting in increased healthcare costs. Current technologies e.g. co-delivery systems, where antibacterial and osteoinductive agents are delivered encounter challenges such as site-specific delivery, sustained and prolonged release, and biocompatibility. In this review, these aspects are highlighted to promote the invention of the next generation biomaterials to prevent and/or treat bone infections and promote tissue regeneration.

Rotary-jet spun polycaprolactone/nano-hydroxyapatite scaffolds modified by simulated body fluid influenced the flexural mode of the neoformed bone.

Polycaprolactone (PCL) is a biocompatible, biodegradable synthetic polymer which in combination with nanohydroxyapatite (nHAp) can give rise to a low cost, nontoxic bioactive product with excellent mechanical properties and slow degradation. Here we produced, characterized and evaluated in vivo the bone formation of PCL/nHAp scaffolds produced by the rotary jet spinning technique. The scaffolds produced were firstly soaked into simulated body fluid for 21 days to also obtain nHAp onto PCL/nHAp scaffolds. Afterwards, the scaffolds were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy and Raman spectroscopy. For in vivo experiments, 20 male Wistar rats were used and randomly divided in 4 experimental groups (n = 5). A critical defect of 3 mm in diameter was made in the tibia of the animals, which were filled with G1 control (clot); G2-PCL scaffold; G3-PCL/nHAp (5%) scaffold; G4-PCL/nHAp (20%) scaffold. All animals were euthanized 60 days after surgery, and the bone repair in the right tibiae were evaluated by radiographic analysis, histological analysis and histomorphometric analysis. While in the left tibias, the areas of bone repair were submitted to the flexural strength test. Radiographic and histomorphometric analyses no showed statistical difference in new bone formation between the groups, but in the three-point flexural tests, the PCL/nHAp (20%) scaffold positively influenced the flexural mode of the neoformed bone. These findings indicate that PCL/nHAp (20%) scaffold improve biomechanical properties of neoformed bone and could be used for bone medicine regenerative.

Rotary jet-spun porous microfibers as scaffolds for stem cells delivery to central nervous system injury.

Stem cell transplantation is a promising strategy to treat brain injuries. However, cell-based therapies are limited because poor local cell engraftment. Here, we present a polylactic acid (PLA) scaffold to support mesenchymal stem cells (MSCs) delivery in stroke. We isolated bone marrow MSCs from adult C57/Bl6 mice, cultured them on PLA polymeric rough microfibrous (PRM) scaffolds obtained by rotary jet spinning, and transplanted over the brains of adult C57/Bl6 mice, carrying thermocoagulation-induced cortical stroke. No inflammatory response to PRM was found. MSCs transplantation significantly reduced the area of the lesion and PRM delivery increased MSCs retention at the injury site. In addition, PRM upregulated α6-integrin and CXCL12 production, which may be the cause for greater cell retention at the lesion site and may provide additional benefit to MSCs transplantation procedures. We conclude that PRM scaffolds offer a promising new system to deliver stem cells to injured areas of the brain.

In vitro and in vivo evaluation of rotary-jet-spun poly(ɛ-caprolactone) with high loading of nano-hydroxyapatite.

Herein, poly(ɛ-caprolactone) (PCL) mats with different amounts of nanohydroxyapatite (nHAp) were produced using rotary-jet spinning (RJS) and evaluated in vitro and in vivo. The mean fiber diameters of the PCL, PCL/nHAp (3%), PCL/nHAp (5%), and PCL/nHAp (20%) scaffolds were 1847 ± 1039, 1817 ± 1044, 1294 ± 4274, and 845 ± 248 nm, respectively. Initially, all the scaffolds showed superhydrophobic behavior (contact angle around of 140oC), but decreased to 80° after 30 min. All the produced scaffolds were bioactive after soaking in simulated body fluid, especially PCL/nHAp (20%). The crystallinity of the PCL scaffolds decreased progressively from 46 to 21% after incorporation of 20% nHAp. In vitro and in vivo cytotoxicity were investigated, as well as the mats' ability to reduce bacteria biofilm formation. In vitro cellular differentiation was evaluated by measuring alkaline phosphatase activity and mineralized nodule formation. Overall, we identified the total ideal amount of nHAp to incorporate in PCL mats, which did not show in vitro or in vivo cytotoxicity and promoted lamellar bone formation independently of the amounts of nHAp. The scaffolds with nHAp showed reduced bacterial proliferation. Alizarin red staining was higher in materials associated with nHAp than in those without nHAp. Overall, this study demonstrates that PCL with nHAp prepared by RJS merits further evaluation for orthopedic applications.

Polypyrrole increases branching and neurite extension by Neuro2A cells on PBAT ultrathin fibers.

We present a methodology for production and application of electrospun hybrid materials containing commercial polyester (poly (butylene adipate-co-terephthalate; PBAT), and a conductive polymer (polypyrrole; PPy) as scaffold for neuronal growth and differentiation. The physical-chemical properties of the scaffolds and optimization of the electrospinning parameters are presented. The electrospun scaffolds are biocompatible and allow proper adhesion and spread of mesenchymal stem cells (MSCs). Fibers produced with PBAT with or without PPy were used as scaffold for Neuro2a mouse neuroblastoma cells adhesion and differentiation. Neuro2a adhered to PBAT and PBAT/PPy2% scaffolds without laminin coating. However, Neuro2a failed to differentiate in PBAT when stimulated by treatment with retinoic acid (RA), but differentiated in PBAT/PPy2% fibers. We hypothesize that PBAT hydrophobicity inhibited proper spreading and further differentiation, and inhibition was overcome by coating the PBAT fibers with laminin. We conclude that fibers produced with the combination of PBAT and PPy can support neuronal differentiation.

Bioactivity behaviour of nano-hydroxyapatite/freestanding aligned carbon nanotube oxide composite.

Bioactive and low cytotoxic three dimensional nano-hydroxyapatite (nHAp) and aligned carbon nanotube oxide (a-CNTO) composite has been investigated. First, freestanding aligned carbon nanotubes porous scaffold was prepared by large-scale thermal chemical vapour deposition and functionalized by oxygen plasma treatment, forming a-CNTO. The a-CNTO was covered with plate-like nHAp crystals prepared by in situ electrodeposition techniques, forming nHAp/a-CNTO composite. After that nHAp/a-CNTO composite was immersed in simulated body fluid for composite consolidation. This novel nanobiomaterial promotes mesenchymal stem cell adhesion with the active formation of membrane projections, cell monolayer formation and high cell viability.

Confined Water Dynamics in the Scaffolds of Polylactic Acid.

Resorbable polylactic acid (PLA) ultrathin fibers have been applied as scaffolds for tissue engineering applications due to their micro- and nanoporous structure that favor cell adhesion, besides inducing cell proliferation and upregulating gene expression related to tissue regeneration. Incorporation of multiwalled carbon nanotubes into PLA fibers has been reported to increase the mechanical properties of the scaffold, making them even more suitable for tissue engineering applications. Ideally, scaffolds should be degraded simultaneously with tissue growth. Hydration and swelling are factors related to scaffold degradation. Hydration would negatively impact the mechanical properties since PLA shows hydrolytic degradation. Water absorption critically affects the catalysis and allowance of the hydrolysis reactions. Moreover, either mass transport and chemical reactions are influenced by confined water, which is an unexplored subject for PLA micro- and nanoporous fibers. Here, we probe and investigate confined water onto highly porous PLA microfibers containing few amounts of incorporated carbon nanotubes by Fourier transform infrared (FTIR) spectroscopy. A hydrostatic pressure was applied to the fibers to enhance the intermolecular interactions between water molecules and C=O groups from polyester bonds, which were evaluated over the wavenumber between 1600 and 2000 cm-1. The analysis of temperature dependence of FTIR spectra indicated the presence of confined water which is characterized by a non-Arrhenius to Arrhenius crossover at T0 = 190 K for 1716 and 1817 cm-1 carbonyl bands of PLA. These bands are sensitive to a hydrogen bond network of confined water. The relevance of our finding relies on the challenge detecting confined water in hydrophobic cavities as in the PLA one. To the best of our knowledge, we present the first report referring the presence of confined water in a hydrophobic scaffold as PLA for tissue engineering. Our findings can provide new opportunities to understand the role of confined water in tissue engineering applications. For instance, we argue that PLA degradation may be affected the most by confined water. PLA degradation involves hydrolytic and enzymatic degradation reactions, which can both be sensitive to changes in water properties.

Hydrogel Based on Nanoclay and Gelatin Methacrylate Polymeric Matrix as a Potential Osteogenic Application.

A nanocomposite hydrogel has potentially applicability in the induction of osteogenesis. The hydrogel was synthesized using 1% gelatin methacrylate (GelMA), a biodegradable and bioactive polymer containing the structure of gelatin, denatured collagen derived from the extracellular bone matrix, and 6% laponite (Lap), a synthetic phyllosilicate of nanosized particles. Initially, 0.6 g of Lap was added to deionized water, and then a solution of GelMA/Igarcure was added under stirring and UV light for crosslinking. The spectra in the Fourier-transform infrared region showed bands that indicate the interaction between gelatin and methacrylate anhydride. X-ray diffraction patterns confirmed the presence of Lap and GelMA in the hydrogel. The thermogravimetric analysis suggested an increase in the thermal stability of the hydrogel with the presence of clay mineral. Rheological analysis showed that the hydrogel had a viscosity that allowed its injectability. The hydrogel did not show acute toxicity at any of the concentrations tested according to the Artemia salina lethality test. It showed cell viability more significant than 80% in the MTT test, which makes it suitable for in vivo osteogenic induction tests. The cell differentiation test showed the differentiation of stem cells into osteogenic cells. It indicates a material with the potential for osteogenic induction and possible application in bone tissue engineering.

Biomineralization of superhydrophilic vertically aligned carbon nanotubes.

Vertically aligned carbon nanotubes (VACNT) promise a great role for the study of tissue regeneration. In this paper, we introduce a new biomimetic mineralization routine employing superhydrophilic VACNT films as highly stable template materials. The biomineralization was obtained after VACNT soaking in simulated body fluid solution. Detailed structural analysis reveals that the polycrystalline biological apatites formed due to the -COOH terminations attached to VACNT tips after oxygen plasma etching. Our approach not only provides a novel route for nanostructured materials, but also suggests that COOH termination sites can play a significant role in biomimetic mineralization. These new nanocomposites are very promising as nanobiomaterials due to the excellent human osteoblast adhesion.

Nanocomposite Hydrogel Produced from PEGDA and Laponite for Bone Regeneration.

Herein, a nanocomposite hydrogel was produced using laponite and polyethylene-glycol diacrylate (PEGDA), with or without Irgacure (IG), for application in bone tissue regeneration. The nanocomposites were characterized by X-ray diffraction (XRD), Fourier-Transform infrared spectroscopy (FTIR), and thermal analysis (TG/DTG). The XRD results showed that the crystallographic structure of laponite was preserved in the nanocomposite hydrogels after the incorporation of PEGDA and IG. The FTIR results indicated that PEGDA polymer chains were entangled on laponite in hydrogels. The TG/DTG found that the presence of laponite (Lap) improved the thermal stability of nanocomposite hydrogel. The toxicity tests by Artemia salina indicated that the nanocomposite hydrogels were not toxic, because the amount of live nauplii was 80.0%. In addition, in vivo tests demonstrated that the hydrogels had the ability to regenerate bone in a bone defect model of the tibiae of osteopenic rats. For the nanocomposite hydrogel (PEGDA + Lap nanocomposites + UV light), the formation of intramembranous bone in the soft callus was more intense in 66.7% of the animals. Thus, the results presented in this study evidence that nanocomposite hydrogels obtained from laponite and PEGDA have the potential for use in bone regeneration.

Rotary Jet-Spun Polycaprolactone/Hydroxyapatite and Carbon Nanotube Scaffolds Seeded with Bone Marrow Mesenchymal Stem Cells Increase Bone Neoformation.

Clinically, bone tissue replacements and/or bone repair are challenging. Strategies based on well-defined combinations of osteoconductive materials and osteogenic cells are promising to improve bone regeneration but still require improvement. Herein, we combined polycaprolactone (PCL) fibers, carbon nanotubes (CNT), and hydroxyapatite (nHap) nanoparticles to develop the next generation of bone regeneration material. Fibers formed by rotary jet spinning (RJS) instead of traditional electrospinning (ES) with embedded bone marrow mesenchymal stem cells (BMMSCs) showed the best outcomes to repair rat calvarial defects after 6 weeks. To understand this, it was observed that different morphologies were formed depending on the manufacturing method used. RJS fibers presented a particular topography with rough fibers, which allowed for better cellular growth and cell spreading in vitro around and into a three-dimensional (3D) mesh, while fibers made by ES were more smooth and cellular growth was only measured on the 3D mesh surface. The fibers with incorporated nHap/CNT nanoparticles enhanced in vitro cell performance as indicated by more cellular proliferation, alkaline phosphatase activity, proliferation, and deposition of calcium. Greater bone neoformation occurred by combining three characteristics: the presence of nHap and CNT nanoparticles, the topography of the RJS fibers, and the addition of BMMSCs. RJS fibers with nanoparticles and seeded with BMMSCs showed 10 136 mm3 of bone neoformation, meaning a 10-fold increase compared to using RJS only and BMMSCs (0.853 mm3) and a 5-fold increase from using ES only (2054 mm3) after 6 weeks of implantation. Conversely, none of these approaches used individually showed any significant difference for in vivo bone neoformation, suggesting that their combination is essential for optimizing bone formation. In summary, our work generated a potential material composed of well-defined combinations of suitable scaffolds seeded with BMMSCs for enhancing numerous orthopedic tissue engineering applications.

Catalyst-Free Click Chemistry for Engineering Chondroitin Sulfate-Multiarmed PEG Hydrogels for Skin Tissue Engineering.

The quest for an ideal biomaterial perfectly matching the microenvironment of the surrounding tissues and cells is an endless challenge within biomedical research, in addition to integrating this with a facile and sustainable technology for its preparation. Engineering hydrogels through click chemistry would promote the sustainable invention of tailor-made hydrogels. Herein, we disclose a versatile and facile catalyst-free click chemistry for the generation of an innovative hydrogel by combining chondroitin sulfate (CS) and polyethylene glycol (PEG). Various multi-armed PEG-Norbornene (A-PEG-N) with different molecular sizes were investigated to generate crosslinked copolymers with tunable rheological and mechanical properties. The crosslinked and mechanically stable porous hydrogels could be generated by simply mixing the two clickable Tetrazine-CS (TCS) and A-PEG-N components, generating a self-standing hydrogel within minutes. The leading candidate (TCS-8A-PEG-N (40 kD)), based on the mechanical and biocompatibility results, was further employed as a scaffold to improve wound closure and blood flow in vivo. The hydrogel demonstrated not only enhanced blood perfusion and an increased number of blood vessels, but also desirable fibrous matrix orientation and normal collagen deposition. Taken together, these results demonstrate the potential of the hydrogel to improve wound repair and hold promise for in situ skin tissue engineering applications.

Ionizable Lipid Nanoparticle-Mediated Delivery of Plasmid DNA in Cardiomyocytes.

Introduction: Gene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. However, cardiomyocytes are crucial cell types that are considered hard-to-transfect. The entrapment of nucleic acids in non-viral vectors, such as lipid nanoparticles (LNPs), is an attractive approach for safe and effective delivery. Methods: Here, a mini-library of engineered LNPs was developed for pDNA delivery in cardiomyocytes. LNPs were characterized and screened for pDNA delivery in cardiomyocytes and identified a lead LNP formulation with enhanced transfection efficiency. Results: By varying lipid molar ratios, the LNP formulation was optimized to deliver pDNA in cardiomyocytes with enhanced gene expression in vitro and in vivo, with negligible toxicity. In vitro, our lead LNP was able to reach a gene expression greater than 80%. The in vivo treatment with lead LNPs induced a twofold increase in GFP expression in heart tissue compared to control. In addition, levels of circulating myeloid cells and inflammatory cytokines remained without significant changes in the heart after LNP treatment. It was also demonstrated that cardiac cell function was not affected after LNP treatment. Conclusion: Collectively, our results highlight the potential of LNPs as an efficient delivery vector for pDNA to cardiomyocytes. This study suggests that LNPs hold promise to improve gene therapy for treatment of cardiovascular disease.

Nanohydroxyapatite/Titanate Nanotube Composites for Bone Tissue Regeneration.

Strategies for the production of new nanocomposites that promote bone tissue regeneration are important, particularly those that enhance the osteoinduction of hydroxyapatite in situ. Here, we studied and report the synthesis of nanohydroxyapatite and titanate nanotube (nHAp/TiNT) composites formulated at different concentrations (1, 2, 3, and 10 wt % TiNT) by means of a wet aqueous chemical reaction. The addition of TiNT affects the morphology of the nanocomposites, decreasing the average crystallite size from 54 nm (nHAp) to 34 nm (nHAp/TiNT10%), while confirming its interaction with the nanocomposite. The crystallinity index (CI) calculated by Raman spectroscopy and XRD showed that the values decreased according to the increase in TiNT concentration, which confirmed their addition to the structure of the nanocomposite. SEM images showed the presence of TiNTs in the nanocomposite. We further verified the potential cytotoxicity of murine fibroblast cell line L929, revealing that there was no remarkable cell death at any of the concentrations tested. In vivo regenerative activity was performed using oophorectomized animal (rat) models organized into seven groups containing five animals each over two experimental periods (15 and 30 days), with bone regeneration occurring in all groups tested within 30 days; however, the nHAp/TiNT10% group showed statistically greater tissue repair, compared to the untreated control group. Thus, the results of this study demonstrate that the presently formulated nHAp/TiNT nanocomposites are promising for numerous improved bone tissue regeneration applications.

Nanostructured Non-Newtonian Drug Delivery Barrier Prevents Postoperative Intrapericardial Adhesions.

With the increasing volume of cardiovascular surgeries and the rising adoption rate of new methodologies that serve as a bridge to cardiac transplantation and that require multiple surgical interventions, the formation of postoperative intrapericardial adhesions has become a challenging problem that limits future surgical procedures, causes serious complications, and increases medical costs. To prevent this pathology, we developed a nanotechnology-based self-healing drug delivery hydrogel barrier composed of silicate nanodisks and polyethylene glycol with the ability to coat the epicardial surface of the heart without friction and locally deliver dexamethasone, an anti-inflammatory drug. After the fabrication of the hydrogel, mechanical characterization and responses to shear, strain, and recovery were analyzed, confirming its shear-thinning and self-healing properties. This behavior allowed its facile injection (5.75 ± 0.15 to 22.01 ± 0.95 N) and subsequent mechanical recovery. The encapsulation of dexamethasone within the hydrogel system was confirmed by 1H NMR, and controlled release for 5 days was observed. In vitro, limited cellular adhesion to the hydrogel surface was achieved, and its anti-inflammatory properties were confirmed, as downregulation of ICAM-1 and VCAM-1 was observed in TNF-α activated endothelial cells. In vivo, 1 week after administration of the hydrogel to a rabbit model of intrapericardial injury, superior efficacy was observed when compared to a commercial adhesion barrier, as histological and immunohistochemical examination revealed reduced adhesion formation and minimal immune infiltration of CD3+ lymphocytes and CD68+ macrophages, as well as NF-κß downregulation. We presented a novel nanostructured drug delivery hydrogel system with unique mechanical and biological properties that act synergistically to prevent cellular infiltration while providing local immunomodulation to protect the intrapericardial space after a surgical intervention.

Fast preparation of hydroxyapatite/superhydrophilic vertically aligned multiwalled carbon nanotube composites for bioactive application.

A method for the electrodeposition of hydroxyapatite films on superhydrophilic vertically aligned multiwalled carbon nanotubes is presented. The formation of a thin homogeneous film with high crystallinity was observed without any thermal treatment and with bioactivity properties that accelerate the in vitro biomineralization process and osteoblast adhesion.

Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds.

Monolayer formation of SaOS-2 (human osteoblast-like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non-toxicity and the flat spreading with monolayer formation of the SaOs-2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.

3D Bioprinting in Tissue Engineering for Medical Applications: The Classic and the Hybrid.

Three-dimensional (3D) printing, as one of the most popular recent additive manufacturing processes, has shown strong potential for the fabrication of biostructures in the field of tissue engineering, most notably for bones, orthopedic tissues, and associated organs. Desirable biological, structural, and mechanical properties can be achieved for 3D-printed constructs with a proper selection of biomaterials and compatible bioprinting methods, possibly even while combining additive and conventional manufacturing (AM and CM) procedures. However, challenges remain in the need for improved printing resolution (especially at the nanometer level), speed, and biomaterial compatibilities, and a broader range of suitable 3D-printed materials. This review provides an overview of recent advances in the development of 3D bioprinting techniques, particularly new hybrid 3D bioprinting technologies for combining the strengths of both AM and CM, along with a comprehensive set of material selection principles, promising medical applications, and limitations and future prospects.