3D bioprinting of mouse pre-osteoblasts and human MSCs using bioinks consisting of gelatin and decellularized bone particles.

One of the key challenges in biofabrication applications is to obtain bioinks that provide a balance between printability, shape fidelity, cell viability, and tissue maturation. Decellularization methods allow the extraction of natural extracellular matrix, preserving tissue-specific matrix proteins. However, the critical challenge in bone decellularization is to preserve both organic (collagen, proteoglycans) and inorganic components (hydroxyapatite) to maintain the natural composition and functionality of bone. Besides, there is a need to investigate the effects of decellularized bone (DB) particles as a tissue-based additive in bioink formulation to develop functional bioinks. Here we evaluated the effect of incorporating DB particles of different sizes (≤45 and ≤100µm) and concentrations (1%, 5%, 10% (wt %)) into bioink formulations containing gelatin (GEL) and pre-osteoblasts (MC3T3-E1) or human mesenchymal stem cells (hTERT-MSCs). In addition, we propose a minimalistic bioink formulation using GEL, DB particles and cells with an easy preparation process resulting in a high cell viability. The printability properties of the inks were evaluated. Additionally, rheological properties were determined with shear thinning and thixotropy tests. The bioprinted constructs were cultured for 28 days. The viability, proliferation, and osteogenic differentiation capacity of cells were evaluated using biochemical assays and fluorescence microscopy. The incorporation of DB particles enhanced cell proliferation and osteogenic differentiation capacity which might be due to the natural collagen and hydroxyapatite content of DB particles. Alkaline phosphatase activity is increased significantly by using DB particles, notably, without an osteogenic induction of the cells. Moreover, fluorescence images display pronounced cell-material interaction and cell attachment inside the constructs. With these promising results, the present minimalistic bioink formulation is envisioned as a potential candidate for bone tissue engineering as a clinically translatable material with straightforward preparation and high cell activity.

3D printing of piezoelectric and bioactive barium titanate-bioactive glass scaffolds for bone tissue engineering.

Bone healing is a complex process orchestrated by various factors, such as mechanical, chemical and electrical cues. Creating synthetic biomaterials that combine several of these factors leading to tailored and controlled tissue regeneration, is the goal of scientists worldwide. Among those factors is piezoelectricity which creates a physiological electrical microenvironment that plays an important role in stimulating bone cells and fostering bone regeneration. However, only a limited number of studies have addressed the potential of combining piezoelectric biomaterials with state-of-the-art fabrication methods to fabricate tailored scaffolds for bone tissue engineering. Here, we present an approach that takes advantage of modern additive manufacturing techniques to create macroporous biomaterial scaffolds based on a piezoelectric and bioactive ceramic-crystallised glass composite. Using binder jetting, scaffolds made of barium titanate and 45S5 bioactive glass are fabricated and extensively characterised with respect to their physical and functional properties. The 3D-printed ceramic-crystallised glass composite scaffolds show both suitable mechanical strength and bioactive behaviour, as represented by the accumulation of bone-like calcium phosphate on the surface. Piezoelectric scaffolds that mimic or even surpass bone with piezoelectric constants ranging from 1 to 21 pC/N are achieved, depending on the composition of the composite. Using MC3T3-E1 osteoblast precursor cells, the scaffolds show high cytocompatibility coupled with cell attachment and proliferation, rendering the barium titanate/45S5 ceramic-crystallised glass composites promising candidates for bone tissue engineering.

Fish scale containing alginate dialdehyde-gelatin bioink for bone tissue engineering.

The development of biomaterial inks suitable for biofabrication and mimicking the physicochemical properties of the extracellular matrix is essential for the application of bioprinting technology in tissue engineering (TE). The use of animal-derived proteinous materials, such as jellyfish collagen, or fish scale (FS) gelatin (GEL), has become an important pillar in biomaterial ink design to increase the bioactivity of hydrogels. However, besides the extraction of proteinous structures, the use of structurally intact FS as an additive could increase biocompatibility and bioactivity of hydrogels due to its organic (collagen) and inorganic (hydroxyapatite) contents, while simultaneously enhancing mechanical strength in three-dimensional (3D) printing applications. To test this hypothesis, we present here a composite biomaterial ink composed of FS and alginate dialdehyde (ADA)-GEL for 3D bioprinting applications. We fabricate 3D cell-laden hydrogels using mouse pre-osteoblast MC3T3-E1 cells. We evaluate the physicochemical and mechanical properties of FS incorporated ADA-GEL biomaterial inks as well as the bioactivity and cytocompatibility of cell-laden hydrogels. Due to the distinctive collagen orientation of the FS, the compressive strength of the hydrogels significantly increased with increasing FS particle content. Addition of FS also provided a tool to tune hydrogel stiffness. FS particles were homogeneously incorporated into the hydrogels. Particle-matrix integration was confirmed via scanning electron microscopy. FS incorporation in the ADA-GEL matrix increased the osteogenic differentiation of MC3T3-E1 cells in comparison to pristine ADA-GEL, as FS incorporation led to increased ALP activity and osteocalcin secretion of MC3T3-E1 cells. Due to the significantly increased stiffness and supported osteoinductivity of the hydrogels, FS structure as a natural collagen and hydroxyapatite source contributed to the biomaterial ink properties for bone engineering applications. Our findings indicate that ADA-GEL/FS represents a new biomaterial ink formulation with great potential for 3D bioprinting, and FS is confirmed as a promising additive for bone TE applications.

3D printed gelatin/decellularized bone composite scaffolds for bone tissue engineering: Fabrication, characterization and cytocompatibility study.

Three-dimensional (3D) printing technology enables the design of personalized scaffolds with tunable pore size and composition. Combining decellularization and 3D printing techniques provides the opportunity to fabricate scaffolds with high potential to mimic native tissue. The aim of this study is to produce novel decellularized bone extracellular matrix (dbECM)-reinforced composite-scaffold that can be used as a biomaterial for bone tissue engineering. Decellularized bone particles (dbPTs, ∼100 â€‹µm diameter) were obtained from rabbit femur and used as a reinforcement agent by mixing with gelatin (GEL) in different concentrations. 3D scaffolds were fabricated by using an extrusion-based bioprinter and crosslinking with microbial transglutaminase (mTG) enzyme, followed by freeze-drying to obtain porous structures. Fabricated 3D scaffolds were characterized morphologically, mechanically, and chemically. Furthermore, MC3T3-E1 mouse pre-osteoblast cells were seeded on the dbPTs reinforced GEL scaffolds (GEL/dbPTs) and cultured for 21 days to assess cytocompatibility and cell attachment. We demonstrate the 3D-printability of dbPTs-reinforced GEL hydrogels and the achievement of homogenous distribution of the dbPTs in the whole scaffold structure, as well as bioactivity and cytocompatibility of GEL/dbPTs scaffolds. It was shown that Young's modulus and degradation rate of scaffolds were enhanced with increasing dbPTs content. Multiphoton microscopy imaging displayed the interaction of cells with dbPTs, indicating attachment and proliferation of cells around the particles as well as into the GEL-particle hydrogels. Our results demonstrate that GEL/dbPTs hydrogel formulations have potential for bone tissue engineering.

3D Bioprinting of Multifunctional Dynamic Nanocomposite Bioinks Incorporating Cu-Doped Mesoporous Bioactive Glass Nanoparticles for Bone Tissue Engineering.

Bioprinting has seen significant progress in recent years for the fabrication of bionic tissues with high complexity. However, it remains challenging to develop cell-laden bioinks exhibiting superior physiochemical properties and bio-functionality. In this study, a multifunctional nanocomposite bioink is developed based on amine-functionalized copper (Cu)-doped mesoporous bioactive glass nanoparticles (ACuMBGNs) and a hydrogel formulation relying on dynamic covalent chemistry composed of alginate dialdehyde (oxidized alginate) and gelatin, with favorable rheological properties, improved shape fidelity, and structural stability for extrusion-based bioprinting. The reversible dynamic microenvironment in combination with the impact of cell-adhesive ligands introduced by aminated particles enables the rapid spreading (within 3 days) and high survival (>90%) of embedded human osteosarcoma cells and immortalized mouse bone marrow-derived stroma cells. Osteogenic differentiation of primary mouse bone marrow stromal stem cells (BMSCs) and angiogenesis are promoted in the bioprinted alginate dialdehyde-gelatin (ADA-GEL or AG)-ACuMBGN scaffolds without additional growth factors in vitro, which is likely due to ion stimulation from the incorporated nanoparticles and possibly due to cell mechanosensing in the dynamic matrix. In conclusion, it is envisioned that these nanocomposite bioinks can serve as promising platforms for bioprinting complex 3D matrix environments providing superior physiochemical and biological performance for bone tissue engineering.

Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Produce Distinct Neural 3D In Vitro Models Depending on Alginate/Gellan Gum/Laminin Hydrogel Blend Properties.

Stable and predictive neural cell culture models are a necessary premise for many research fields. However, conventional 2D models lack 3D cell-material/-cell interactions and hence do not reflect the complexity of the in vivo situation properly. Here two alginate/gellan gum/laminin (ALG/GG/LAM) hydrogel blends are presented for the fabrication of human induced pluripotent stem cell (hiPSC)-based 3D neural models. For hydrogel embedding, hiPSC-derived neural progenitor cells (hiNPCs) are used either directly or after 3D neural pre-differentiation. It is shown that stiffness and stress relaxation of the gel blends, as well as the cell differentiation strategy influence 3D model development. The embedded hiNPCs differentiate into neurons and astrocytes within the gel blends and display spontaneous intracellular calcium signals. Two fit-for-purpose models valuable for i) applications requiring a high degree of complexity, but less throughput, such as disease modeling and long-term exposure studies and ii) higher throughput applications, such as acute exposures or substance screenings are proposed. Due to their wide range of applications, adjustability, and printing capabilities, the ALG/GG/LAM based 3D neural models are of great potential for 3D neural modeling in the future.

Neuronal Differentiation from Induced Pluripotent Stem Cell-Derived Neurospheres by the Application of Oxidized Alginate-Gelatin-Laminin Hydrogels.

Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate-gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.

Electrically Conductive and 3D-Printable Oxidized Alginate-Gelatin Polypyrrole:PSS Hydrogels for Tissue Engineering.

Electroactive hydrogels can be used to influence cell response and maturation by electrical stimulation. However, hydrogel formulations which are 3D printable, electroactive, cytocompatible, and allow cell adhesion, remain a challenge in the design of such stimuli-responsive biomaterials for tissue engineering. Here, a combination of pyrrole with a high gelatin-content oxidized alginate-gelatin (ADA-GEL) hydrogel is reported, offering 3D-printability of hydrogel precursors to prepare cytocompatible and electrically conductive hydrogel scaffolds. By oxidation of pyrrole, electroactive polypyrrole:polystyrenesulfonate (PPy:PSS) is synthesized inside the ADA-GEL matrix. The hydrogels are assessed regarding their electrical/mechanical properties, 3D-printability, and cytocompatibility. It is possible to prepare open-porous scaffolds via bioplotting which are electrically conductive and have a higher cell seeding efficiency in scaffold depth in comparison to flat 2D hydrogels, which is confirmed via multiphoton fluorescence microscopy. The formation of an interpenetrating polypyrrole matrix in the hydrogel matrix increases the conductivity and stiffness of the hydrogels, maintaining the capacity of the gels to promote cell adhesion and proliferation. The results demonstrate that a 3D-printable ADA-GEL can be rendered conductive (ADA-GEL-PPy:PSS), and that such hydrogel formulations have promise for cell therapies, in vitro cell culture, and electrical-stimulation assisted tissue engineering.

Numerical Simulations as Means for Tailoring Electrically Conductive Hydrogels Towards Cartilage Tissue Engineering by Electrical Stimulation.

Cartilage regeneration is a clinical challenge. In recent years, hydrogels have emerged as implantable scaffolds in cartilage tissue engineering. Similarly, electrical stimulation has been employed to improve matrix synthesis of cartilage cells, and thus to foster engineering and regeneration of cartilage tissue. The combination of hydrogels and electrical stimulation may pave the way for new clinical treatment of cartilage lesions. To find the optimal electric properties of hydrogels, theoretical considerations and corresponding numerical simulations are needed to identify well-suited initial parameters for experimental studies. We present the theoretical analysis of a hydrogel in a frequently used electrical stimulation device for cartilage regeneration and tissue engineering. By means of equivalent circuits, finite element analysis, and uncertainty quantification, we elucidate the influence of the geometric and dielectric properties of cell-seeded hydrogels on the capacitive-coupling electrical field stimulation. Moreover, we discuss the possibility of cellular organisation inside the hydrogel due to forces generated by the external electric field. The introduced methodology is easily reusable by other researchers and allows to directly develop novel electrical stimulation study designs. Thus, this study paves the way for the design of future experimental studies using electrically conductive hydrogels and electrical stimulation for tissue engineering.

3D printing and characterization of human nasoseptal chondrocytes laden dual crosslinked oxidized alginate-gelatin hydrogels for cartilage repair approaches.

As cartilage is one of the few tissues in the human body that is not vascularized, the body has very limited capabilities to repair cartilage defects. Hence, novel condro-instructive biomaterials facilitating cartilage formation by implanted chondrocytes are required. In this work, an oxidized alginate-gelatin hydrogel system, alginate-di-aldehyde (ADA) and gelatin (GEL), was used to fabricate 3D printed grid-like structures for cartilage tissue engineering. Enzymatic and ionic crosslinking techniques using microbial transglutaminase (mTG) and divalent ions (CaCl2) were combined to ensure long-term stability of the 3D printed structures. Human nasoseptal chondrocytes were embedded in ADA-GEL prior to 3D printing. Cell viability, proliferation, and metabolic activity were analyzed after 7 and 14 days. The influence of the enzymatic crosslinking and the 3D printing process on the primary human chondrocytes were investigated. It was found that neither the 3D printing process nor the crosslinking by mTG impaired chondrocyte viability. The formation of the main cartilage-specific extracellular matrix components collagen type II and cartilage proteoglycans was shown by immunohistochemical staining. The combination of enzymatic and ionic crosslinking for the 3D printing of ADA-GEL hydrogels is therefore a promising approach for the 3D cultivation of primary human chondrocytes for cartilage tissue engineering.

Polymer-Bioactive Glass Composite Filaments for 3D Scaffold Manufacturing by Fused Deposition Modeling: Fabrication and Characterization.

Critical size bone defects are regularly treated by auto- and allograft transplantation. However, such treatments require to harvest bone from patient donor sites, with often limited tissue availability or risk of donor site morbidity. Not requiring bone donation, three-dimensionally (3D) printed implants and biomaterial-based tissue engineering (TE) strategies promise to be the next generation therapies for bone regeneration. We present here polylactic acid (PLA)-bioactive glass (BG) composite scaffolds manufactured by fused deposition modeling (FDM), involving the fabrication of PLA-BG composite filaments which are used to 3D print controlled open-porous and osteoinductive scaffolds. We demonstrated the printability of PLA-BG filaments as well as the bioactivity and cytocompatibility of PLA-BG scaffolds using pre-osteoblast MC3T3E1 cells. Gene expression analyses indicated the beneficial impact of BG inclusions in FDM scaffolds regarding osteoinduction, as BG inclusions lead to increased osteogenic differentiation of human adipose-derived stem cells in comparison to pristine PLA. Our findings confirm that FDM is a convenient additive manufacturing technology to develop PLA-BG composite scaffolds suitable for bone tissue engineering.

3D printed oxidized alginate-gelatin bioink provides guidance for C2C12 muscle precursor cell orientation and differentiation via shear stress during bioprinting.

Biofabrication can be a tool to three-dimensionally (3D) print muscle cells embedded inside hydrogel biomaterials, ultimately aiming to mimic the complexity of the native muscle tissue and to create in-vitro muscle analogues for advanced repair therapies and drug testing. However, to 3D print muscle analogues of high cell alignment and synchronous contraction, the effect of biofabrication process parameters on myoblast growth has to be understood. A suitable biomaterial matrix is required to provide 3D printability as well as matrix degradation to create space for cell proliferation, matrix remodelling capacity, and cell differentiation. We demonstrate that by the proper selection of nozzle size and extrusion pressure, the shear stress during extrusion-bioprinting of mouse myoblast cells (C2C12) can achieve cell orientation when using oxidized alginate-gelatin (ADA-GEL) hydrogel bionk. The cells grow in the direction of printing, migrate to the hydrogel surface over time, and differentiate into ordered myotube segments in areas of high cell density. Together, our results show that ADA-GEL hydrogel can be a simple and cost-efficient biodegradable bioink that allows the successful 3D bioprinting and cultivation of C2C12 cells in-vitro to study muscle engineering.

3D Printing of Piezoelectric Barium Titanate-Hydroxyapatite Scaffolds with Interconnected Porosity for Bone Tissue Engineering.

The prevalence of large bone defects is still a major problem in surgical clinics. It is, thus, not a surprise that bone-related research, especially in the field of bone tissue engineering, is a major issue in medical research. Researchers worldwide are searching for the missing link in engineering bone graft materials that mimic bones, and foster osteogenesis and bone remodeling. One approach is the combination of additive manufacturing technology with smart and additionally electrically active biomaterials. In this study, we performed a three-dimensional (3D) printing process to fabricate piezoelectric, porous barium titanate (BaTiO3) and hydroxyapatite (HA) composite scaffolds. The printed scaffolds indicate good cytocompatibility and cell attachment as well as bone mimicking piezoelectric properties with a piezoelectric constant of 3 pC/N. This work represents a promising first approach to creating an implant material with improved bone regenerating potential, in combination with an interconnected porous network and a microporosity, known to enhance bone growth and vascularization.

Ionically and Enzymatically Dual Cross-Linked Oxidized Alginate Gelatin Hydrogels with Tunable Stiffness and Degradation Behavior for Tissue Engineering.

Hydrogels that allow for the successful long-term in vitro culture of cell-biomaterial systems to enable the maturation of tissue engineering constructs are highly relevant in regenerative medicine. Naturally derived polysaccharide-based hydrogels promise to be one material group with enough versatility and chemical functionalization capability to tackle the challenges associated with long-term cell culture. We report a marine derived oxidized alginate, alginate dialdehyde (ADA), and gelatin (GEL) system (ADA-GEL), which is cross-linked via ionic (Ca2+) and enzymatic (microbial transglutaminase, mTG) interaction to form dually cross-linked hydrogels. The cross-linking approach allowed us to tailor the stiffness of the hydrogels in a wide range (from <5 to 120 kPa), without altering the initial ADA and GEL hydrogel chemistry. It was possible to control the degradation behavior of the hydrogels to be stable for up to 30 days of incubation. Increasing concentrations of mTG cross-linker solutions allowed us to tune the degradation behavior of the ADA-GEL hydrogels from fast (<7 days) to moderate (14 days) and slow (>30 days) degradation kinetics. The cytocompatibility of mTG cross-linked ADA-GEL was assessed using NIH-3T3 fibroblasts and ATDC-5 mouse teratocarcinoma cells. Both cell types showed highly increased cellular attachment on mTG cross-linked ADA-GEL in comparison to Ca2+ cross-linked hydrogels. In addition, ATDC-5 cells showed a higher proliferation on mTG cross-linked ADA-GEL hydrogels in comparison to tissue culture polystyrene control substrates. Further, the attachment of human umbilical vein endothelial cells (HUVEC) on ADA-GEL (+) mTG was confirmed, proving the suitability of mTG+Ca2+ cross-linked ADA-GEL for several cell types. Summarizing, a promising platform to control the properties of ADA-GEL hydrogels is presented, with the potential to be applied in long-term cell culture investigations such as cartilage, bone, and blood-vessel engineering, as well as for biofabrication.

3D printing of electrically conductive hydrogels for tissue engineering and biosensors - A review.

Electrically conductive biomaterials are gaining increasing interest owing to their potential to be used in smart, biosensoric and functional tissue-engineered scaffolds and implants. In combination with 3D printing technology, this class of materials might be one of the most advanced approaches towards future medical implants regarding potential functionalities and design possibilities. Conductive hydrogels themselves have been researched for potential sensoric and tissue engineering applications for more than a decade, while the 3D printing of such functional materials is still under early exploration. This review aims to provide a short insight into the most recent developments of 3D printable and electrically conductive hydrogels. It also provides a summary of the last few years of research in this field, with key scope on 3D printing for biomedical applications. The final literature search was conducted in May 2019, with the specific keywords '3D', 'printing', 'conductive', 'hydrogel', 'biocompatible' and combinations of the latter, using advanced search in the databases Scopus®, Web of Science® (Web of Knowledge®) and Google Scholar®. A total of 491 results were gained, while 19 recent publications were identified with the above-mentioned criteria and keywords, which are the studies finally discussed in the paper. The key results have been summarised, and the remaining challenges in the field and the scope for future research activities have been discussed. STATEMENT OF SIGNIFICANCE: Hydrogels are among the most frequently used biomaterials in tissue engineering (TE). A new class of hydrogels, namely, electrically conductive hydrogels (ECHs), has been introduced in recent years. Although ECHs have been comprehensively reviewed in the literature, the combination of ECHs with 3D printing technology has emerged only recently, representing a promising key development toward the fabrication of functional 3D TE constructs. In this review, we cover for the first time the state of the art in the field of 3D printing of ECHs. Previous advances are presented, reviewing the 3D printing technologies utilised, spatial resolution and electrical conductivity values achieved, in addition to discussing the obtained mechanical properties and emerging applications of these materials.

Development of 3D Biofabricated Cell Laden Hydrogel Vessels and a Low-Cost Desktop Printed Perfusion Chamber for In Vitro Vessel Maturation.

The vascular system represents the key supply chain for nutrients and oxygen inside the human body. Engineered solutions to produce sophisticated alternatives for autologous or artificial vascular implants to sustainably replace diseased vascular tissue still remain a key challenge in tissue engineering. In this paper, cell-laden 3D bioplotted hydrogel vessel-like constructs made from alginate di-aldehyde (ADA) and gelatin (GEL) are presented. The aim is to increase the mechanical stability of fibroblast-laden ADA-GEL vessels, tailoring them for maturation under dynamic cell culture conditions. BaCl2 is investigated as a crosslinker for the oxidized alginate-gelatin system. Normal human dermal fibroblast (NHDF)-laden vessel constructs are optimized successfully in terms of higher stiffness by increasing ADA concentration and using BaCl2 , with no toxic effects observed on NHDF. Contrarily, BaCl2 crosslinking of ADA-GEL accelerates cell attachment, viability, and growth from 7d to 24h compared to CaCl2 . Moreover, alignment of cells in the longitudinal direction of the hydrogel vessels when extruding the cell-laden hydrogel crosslinked with Ba2+ is observed. It is possible to tune the stiffness of ADA-GEL by utilizing Ba2+ as crosslinker. In addition, a customized, low-cost 3D printed polycarbonate (PC) perfusion chamber for perfusion of vessel-like constructs is introduced.