Cell-laden Hydrogel Scaffolds Research Articles

Injectable pH and Thermo-Responsive Hydrogel Scaffold with Enhanced Osteogenic Differentiation of Preosteoblasts for Bone Regeneration.

Bone fractures are common in the geriatric population and pose a great economic burden worldwide. While traditional methods for repairing bone defects have primarily been autografts, there are several drawbacks limiting its use. Bone graft substitutes have been used as alternative strategies to improve bone healing. However, there remain several impediments to achieving the desired healing outcomes. Injectable hydrogels have become attractive scaffold materials for bone regeneration, given their high performance in filling irregularly sized bone defects and their ability to encapsulate cells and bioactive molecules and mimic the native ECM of bone. We investigated the use of an injectable chitosan-based hydrogel scaffold to promote the differentiation of preosteoblasts in vitro. The hydrogels were characterized by evaluating cell homogeneity, cell viability, rheological and mechanical properties, and differentiation ability of preosteoblasts in hydrogel scaffolds. Cell-laden hydrogel scaffolds exhibited shear thinning behavior and the ability to maintain shape fidelity after injection. The CNC-CS hydrogels exhibited higher mechanical strength and significantly upregulated the osteogenic activity and differentiation of preosteoblasts, as shown by ALP activity assays and histological analysis of hydrogel scaffolds. These results suggest that this injectable hydrogel is suitable for cell survival, can promote osteogenic differentiation of preosteoblasts, and structurally support new bone growth.

Fabrication of 3D Bioprinted Bi-Phasic Scaffold for Bone-Cartilage Interface Regeneration.

Treatments for osteochondral defects (OCDs) are mainly palliative and, with the increase in this pathology seen among both young and elderly people, an alternative treatment modality is sought. Many tissue-engineered strategies have been explored for regenerating the cartilage-bone interface; however, they generally fall short of being ideal. Although cell-laden hydrogel scaffolds are a common approach for bone and cartilage tissue regeneration, they usually lack homogenous cell dispersion and patient specificity. In this study, a biphasic 3D bioprinted composite scaffold was fabricated for cartilage-bone interface regeneration. To overcome the shortcoming of both materials, alginate-gelatin (A-G) hydrogel was used to confer a naturally occurring environment for the cells and polycaprolactone (PCL) was used to enhance mechanical stability, thus maximizing the overall performance. Hydroxyapatite fillers were added to the PCL in the bone phase of the scaffold to improve its bioactivity. Physical and biological evaluation of scaffolds in both phases was assessed. The scaffolds demonstrated a desirable biological response both singly and in the combined PCL/A-G scaffolds, in both the short term and longer term, showing promise as an interfacial material between cartilage and bone.

3D-bioprinting of aortic valve interstitial cells: impact of hydrogel and printing parameters on cell viability

Calcific aortic valve disease (CAVD) is a frequent cardiac pathology in the aging society. Although valvular interstitial cells (VICs) seem to play a crucial role, mechanisms of CAVD are not fully understood. Development of tissue-engineered cellular models by 3D-bioprinting may help to further investigate underlying mechanisms of CAVD. VIC were isolated from ovine aortic valves and cultured in Dulbecco’s modified Eagle’s Medium (DMEM). VIC of passages six to ten were dissolved in a hydrogel consisting of 2% alginate and 8% gelatin with a concentration of 2 × 106 VIC ml−1. Cell-free and VIC-laden hydrogels were printed with an extrusion-based 3D-bioprinter (3D-Bioplotter® Developer Series, EnvisionTec, Gladbeck, Germany), cross-linked and incubated for up to 28 d. Accuracy and durability of scaffolds was examined by microscopy and cell viability was tested by cell counting kit-8 assay and live/dead staining. 3D-bioprinting of scaffolds was most accurate with a printing pressure of P < 400 hPa, nozzle speed of v < 20 mm s−1, hydrogel temperature of T H = 37 °C and platform temperature of T P = 5 °C in a 90° parallel line as well as in a honeycomb pattern. Dissolving the hydrogel components in DMEM increased VIC viability on day 21 by 2.5-fold compared to regular 0.5% saline-based hydrogels (p < 0.01). Examination at day 7 revealed dividing and proliferating cells. After 21 d the entire printed scaffolds were filled with proliferating cells. Live/dead cell viability/cytotoxicity staining confirmed beneficial effects of DMEM-based cell-laden VIC hydrogel scaffolds even 28 d after printing. By using low pressure printing methods, we were able to successfully culture cell-laden 3D-bioprinted VIC scaffolds for up to 28 d. Using DMEM-based hydrogels can significantly improve the long-term cell viability and overcome printing-related cell damage. Therefore, future applications 3D-bioprinting of VIC might enable the development of novel tissue engineered cellular 3D-models to examine mechanisms involved in initiation and progression of CAVD.

Highly bioactive cell-laden hydrogel constructs bioprinted using an emulsion bioink for tissue engineering applications

The insufficient pore structure of cell-laden hydrogel scaffolds has limited their application in various tissue regeneration applications owing to low cell-to-cell/matrix interactions and low transfer of nutrients and metabolic wastes. Herein, we designed a highly porous cell-laden hydrogel scaffold fabricated using an emulsion bioink consisting of methacrylated collagen (CMA), mineral oil (MO), and human adipose stem cells (hASCs) to induce efficient cell infiltration and cellular activities. By selecting the most appropriate concentration of CMA and MO, the emulsion bioink can be successfully formulated with proper yield stress and printability. The cell-laden scaffold exhibited significantly greater cell growth and cytoskeletal reorganization than the normally printed cell-laden CMA scaffold. Furthermore, two bioactive components (kartogenin and bone morphogenetic protein-2) were physically encapsulated in the oil droplets of the cell construct, and the molecules in the cell constructs enhanced chondrogenic or osteogenic differentiation of hASCs in the printed structure. Based on these results, the cell-printed structure using an emulsion bioink can not only provide a good cellular microenvironment but also be a new potential method to accelerate stem cell differentiation by combining bioactive molecules and cell-laden scaffolds.

Development of agarose–gelatin bioinks for extrusion-based bioprinting and cell encapsulation

Three-dimensional bioprinting continues to advance as an attractive biofabrication technique to employ cell-laden hydrogel scaffolds in the creation of precise, user-defined constructs that can recapitulate the native tissue environment. Development and characterisation of new bioinks to expand the existing library helps to open avenues that can support a diversity of tissue engineering purposes and fulfil requirements in terms of both printability and supporting cell attachment. In this paper, we report the development and characterisation of agarose–gelatin (AG–Gel) hydrogel blends as a bioink for extrusion-based bioprinting. Four different AG–Gel hydrogel blend formulations with varying gelatin concentration were systematically characterised to evaluate suitability as a potential bioink for extrusion-based bioprinting. Additionally, autoclave and filter sterilisation methods were compared to evaluate their effect on bioink properties. Finally, the ability of the AG–Gel bioink to support cell viability and culture after printing was evaluated using SH-SY5Y cells encapsulated in bioprinted droplets of the AG–Gel. All bioink formulations demonstrate rheological, mechanical and swelling properties suitable for bioprinting and cell encapsulation. Autoclave sterilisation significantly affected the rheological properties of the AG–Gel bioinks compared to filter sterilisation. SH-SY5Y cells printed and differentiated into neuronal-like cells using the developed AG–Gel bioinks demonstrated high viability (>90%) after 23 d in culture. This study demonstrates the properties of AG–Gel as a printable and biocompatible material applicable for use as a bioink.

The Application of Cartilage Tissue Engineering with Cell-Laden Hydrogel in Plastic Surgery: A Systematic Review.

As a contour-supporting material, the cartilage has a significant application value in plastic surgery. Since the development of hydrogel scaffolds with sufficient biomechanical strength and high biocompatibility, cell-laden hydrogels have been widely studied for application in cartilage bioengineering. This systematic review summarizes the latest research on engineered cartilage constructed using cell-laden hydrogel scaffolds in plastic surgery. A systematic review was performed by searching the PubMed and Web of Science databases using selected keywords and Medical Subject Headings search terms. Forty-two studies were identified based on the search criteria. After full-text screening for inclusion and exclusion criteria, 18 studies were included. Data collected from each study included culturing form, seed cell types and sources, concentration of cells and gels, scaffold materials and bio-printing structures, and biomechanical properties of cartilage constructs. These cell-laden hydrogel scaffolds were reported to show some feasibility of cartilage engineering, including better cell proliferation, enhanced deposition of glycosaminoglycans and collagen type II in the extracellular matrix, and better biomechanical properties close to the natural state. Cell-laden hydrogels have been widely used in cartilage bioengineering research. Through 3-dimensional (3D) printing, the cell-laden hydrogel can form a bionic contour structure. Extracellular matrix expression was observed in vivo and in vitro, and the elastic modulus was reported to be similar to that of natural cartilage. The future direction of cartilage tissue engineering in plastic surgery involves the use of novel hydrogel materials and more advanced 3D printing technology combined with biochemistry and biomechanical stimulation.

Tunable metacrylated hyaluronic acid-based hybrid bioinks for stereolithography 3D bioprinting

Hyaluronic acid is a native extra-cellular matrix derivative that promises unique properties, such as anti-inflammatory response and cell-signaling with tissue-specific applications under its bioactive properties. Here, we investigate the importance of the duration of synthesis to obtain photocrosslinkable methacrylated hyaluronic acid (MeHA) with high degree of substitution. MeHA with high degree of substitution can result in rapid photocrosslinking and can be used as a bioink for stereolithographic (SLA) three dimensional 3D bioprinting. Increased degree of substitution results Our findings show that a ten-day synthesis results in an 88% degree of methacrylation (DM), whereas three-day and five-day syntheses result in 32% and 42% DM, respectively. The rheological characterization revealed an increased rate of photopolymerization with increasing DM. Further, we developed a hybrid bioink to overcome the non-cell-adhesive nature of MeHA by combining it with gelatin methacryloyl (GelMA) to fabricate 3D cell-laden hydrogel scaffolds. The hybrid bioink exhibited a 55% enhancement in stiffness compared to MeHA only and enabled cell-adhesion while maintaining high cell viability. Investigations also revealed that the hybrid bioink was a more suitable candidate for stereolithography (SLA) 3D bioprinting than MeHA because of its mechanical strength, printability, and cell-adhesive nature. This research lays out a firm foundation for the development of a stable hybrid bioink with MeHA and GelMA for first-ever use with SLA 3D bioprinting.

4D biofabrication via instantly generated graded hydrogel scaffolds

Formation of graded biomaterials to render shape-morphing scaffolds for 4D biofabrication holds great promise in fabrication of complex structures and the recapitulation of critical dynamics for tissue/organ regeneration. Here we describe a facile generation of an adjustable and robust gradient using a single- or multi-material one-step fabrication strategy for 4D biofabrication. By simply photocrosslinking a mixed solution of a photocrosslinkable polymer macromer, photoinitiator (PI), UV absorber and live cells, a cell-laden gradient hydrogel with pre-programmable deformation can be generated. Gradient formation was demonstrated in various polymers including poly(ethylene glycol) (PEG), alginate, and gelatin derivatives using various UV absorbers that present overlap in UV spectrum with that of the PI UV absorbance spectrum. Moreover, this simple and effective method was used as a universal platform to integrate with other hydrogel-engineering techniques such as photomask-aided microfabrication, photo-patterning, ion-transfer printing, and 3D bioprinting to fabricate more advanced cell-laden scaffold structures. Lastly, proof-of-concept 4D tissue engineering was demonstrated in a study of 4D bone-like tissue formation. The strategy's simplicity along with its versatility paves a new way in solving the hurdle of achieving temporal shape changes in cell-laden single-component hydrogel scaffolds and may expedite the development of 4D biofabricated constructs for biological applications.

Generating adipose stem cell-laden hyaluronic acid-based scaffolds using 3D bioprinting via the double crosslinked strategy for chondrogenesis

Bioprinting of most cell-laden hydrogel scaffolds with the required structural integrity, mechanical modulus, cell adhesion, cell compatibility, and chondrogenic differentiation are still significant issues that affect the application of bioinks in cartilage tissue engineering. This study focuses on constructing printable bioinks by combining adipose-derived stem cells (ADSCs), hyaluronic acid (HA)-based hydrogels and analyzing their ability to induce chondrogenesis using 3D bioprinting technology. First, biotinylated hyaluronic acid was synthesized via an adipic acid dihydrazide (ADH) linker with amide bond formation to form HA-biotin (HAB). Both HAB and the as-received streptavidin were mixed to form a partially cross-linked HA-biotin-streptavidin (HBS) hydrogel through noncovalent bonding. After that, the partially cross-linked HBS hydrogel was mixed with sodium alginate and subsequently printed to form the HBSA hydrogel 3D scaffolds using a bioprinter. Finally, the 3D scaffolds of the HBSA (HBS + alginate) hydrogel were submerged into CaCl2 solution to achieve a stable 3D HBSAC (HBSA + Ca2+) hydrogel scaffold through ion transfer crosslinking. The physical-chemical characteristics of the hybrid bioink compositions have been evaluated to determine the desired 3D bioprinting structure. Cytotoxicity and chondrogenic differentiation were also assessed to confirm that the double cross-linked HBSAC hydrogel scaffold was useful for chondrogenic formation. The results showed that partially crosslinking the biotinylated HA-based hydrogel with streptavidin has a significant effect on printability and structural integrity. Morphological analysis of a suitable 3D printed HBSAC hydrogel scaffold showed visible pores with the desired shape and geometry. We have concluded that the HBSAC hydrogel possesses a favorable biocompatibility profile. The HBSAC hydrogel can also secrete significantly higher amounts of chondrogenic marker genes at day 5 and sulfated glycosaminoglycans (sGAGs) from days 7 to 14 compared to the HA hydrogel, as determined via quantitative real-time PCR assay and Alcian blue staining and the DMMB assay.

Cyclic tensile stimulation enrichment of Schwann cell-laden auxetic hydrogel scaffolds towards peripheral nerve tissue engineering

The nervous system in the body is a complex network of nerves and cells that regulates several functions. Neural regeneration is a complex process that involves matrix secretion and remodeling, in which growth factors play a huge role in regulating such processes. Recent studies demonstrated the structures and topography of scaffolds are expected to provide a spectrum of unique biomimetic 3D microenvironments to regulate cell behavior. In this study, we fabricated auxetic scaffolds using fish gelatin methacrylamide and evaluated the effects of cyclic tensile stimulation effects on the neural differentiation capabilities of human Schwann cells. The auxetic hydrogels were found to withstand up to 20% tensile strain without tears, and the hydrogels had lost only about 10% weight after immersed for 14 days. The tensile forces were able to enhance cell viability and proliferation as compared to a static culture. In addition, the secretion of neural regeneration-related proteins was enhanced in the tensile stimulation group. The cell-laden auxetic scaffold with tensile stimulation caused improvement in the nerve growth factor and TRKA receptor expression. This is the first study to combine tensile stimulation with NGF. The initial results showed the positive potential of such conditions being applied in clinical applications.

Determining How Human Mesenchymal Stem Cells Change Their Degradation Strategy in Response to Microenvironmental Stiffness.

During the wound healing process, human mesenchymal stem cells (hMSCs) are recruited to the injury where they regulate inflammation and initiate healing and tissue regeneration. To aid in healing, synthetic cell-laden hydrogel scaffolds are being designed to deliver additional hMSCs to wounds to enhance or restart the healing process. These scaffolds are being designed to mimic native tissue environments, which include physical cues, such as scaffold stiffness. In this work, we focus on how the initial scaffold stiffness hMSCs are encapsulated in changes cell-mediated remodeling and degradation and motility. To do this, we encapsulate hMSCs in a well-defined synthetic hydrogel scaffold that recapitulates aspects of the native extracellular matrix (ECM). We then characterize cell-mediated degradation in the pericellular region as a function of initial microenvironmental stiffness. Our hydrogel consists of a 4-arm poly(ethylene glycol) (PEG) end-functionalized with norbornene which is chemically cross-linked with a matrix metalloproteinase (MMP) degradable peptide sequence. This peptide sequence is cleaved by hMSC-secreted MMPs. The hydrogel elastic modulus is varied from 80 to 2400 Pa by changing the concentration of the peptide cross-linker. We use multiple particle tracking microrheology (MPT) to characterize the spatiotemporal cell-mediated degradation in the pericellular region. In MPT, fluorescently labeled particles are embedded in the material, and their Brownian motion is measured. We measure an increase in cell-mediated degradation and remodeling as the post-encapsulation time increases. MPT also measures changes in the degradation profile in the pericellular region as hydrogel stiffness is increased. We hypothesize that the change in the degradation profile is due to a change in the amount and type of molecules secreted by hMSCs. We also measure a significant decrease in cell speed as hydrogel stiffness increases due to the increased physical barrier that needs to be degraded to enable motility. These measurements increase our understanding of the rheological changes in the pericellular region in different physical microenvironments which could lead to better design of implantable biomaterials for cell delivery to wounded areas.

Hyaluronic acid-based shape memory cryogel scaffolds for focal cartilage defect repair

Purpose: Traumatic joint injuries can result in focal cartilage defects that regenerate poorly due to a lack of blood supply and low chondrocyte density. Cell-laden hydrogel scaffolds are under study but provide little flexibility in modulating their pore size, interconnectivity, swelling and stiffness, properties required for a conducive biomimetic environment. In this study, we engineered an injectable and biomimetic shape memory hyaluronic acid (HA)-based cryogel scaffolds with a macroporous and highly interconnected network in three-dimensions seeded with chondrocytes. Due to their unique ability to withstand up to 90% strains and rapidly recover their shape back, they can be arthroscopically delivered to fill up focal chondral defects for cartilage repair. Here we studied whether chondrocytes encapsulated within cryogels remain viable and metabolically active following injection and provide a superior micro-environment than conventional (nanoporous) hydrogels for cartilage regeneration. We also investigated how RGD-functionalized cryogel affects chondrocyte adhesion, migration and proliferation. Methods: Synthesis of HA-based Cryogels and Hydrogels: HA cryogels were prepared using HA modified with glycidyl methacrylate (HAGM) and either Acryl-PEG-G4RGDS to synthesis gels functionalized with RGD (Cryo RGD+) or Acryl-PEG-methoxy to synthesize gels without RGD (Cryo RGD-) in deionized (DI) water. The mixture was cast in 6 mm diam x 1.5 mm cylindrical discs using APS/TEMED as our initiator system. The samples were frozen at -20°C and allowed to cryopolymerize overnight. Next, cryogels were brought to RT and washed/sanitized prior to cell seeding. HA hydrogels (Hydro RGD+) were fabricated via photopolymerization by mixing HAGM, RGD, and lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) photo-initiator in DI water. Chondrocyte seeding and in-vitro culture:Chondrocytes harvested from calf knee femoral condyle cartilage were seeded onto each scaffold and cultured for 15 days. Injectability test: Cell-laden cryogels were incubated overnight to enable complete cell adhesion and subsequently syringe injected through a 16-gauge needle before incubation for another 24h following which cell viability and metabolism were quantified. Biochemical analysis: At the end of 15-day culture, gel samples were digested to quantify the cell metabolism (Alamar Blue), DNA (PicoGreen) and GAG (DMMB) content. Cell viability and cytoskeleton staining: Gel samples were fixed and stained for nucleus, actin, dead cells and confocal imaged at 40x. Results: Shape memory HA-based cryogels fully recovered their shape back following syringe injection through a 16-gauge needle without impacting cell metabolism or viability (Figs 1A-C). Scanning electron microscopy (SEM) images showed an average pore size of 49.2 ± 15 μm (Fig 1D) rendering a macroporous structure with high interconnectivity, which was 7x higher than hydrogels (Fig 1E). This unique macrostructural architecture enables efficient transport of nutrients and metabolites thereby offering a superior microenvironment for chondrocytes; both cell metabolism and GAG content was 2x higher in cryogels than hydrogels after 15-day culture (Figs 1F-G). Additionally, a significant amount of GAG (∼70 μg) was released from both cryogels into media owing to their macroporous structure; no released GAG was measured in media from Hydro RGD+ (Fig 1G). The presence of RGD (cell-adhesion peptide) did not improve cell adhesion or biosynthesis rates. Chondrocytes formed larger organoid-like structures in Cryo RGD- while remaining homogenously dispersed in the presence of RGD in Cryo RGD+ (Fig 1H). Conclusions: Syringe injectable HA-based shape memory cryogels provide a conducive microenvironment for chondrocyte adhesion, proliferation and matrix biosynthesis for use in repair of cartilage defects.

Bioprinting of glioma stem cells improves their endotheliogenic potential.

Glioblastoma (GBM), with rich blood vessels and high invasiveness, is the most common malignant primary brain tumor. The current treatment strategies are less effective, resulting in tumor recurrence. Tumor angiogenesis plays an important role in the occurrence, development and metastasis of GBM. Currently, GBM has been treated by inhibiting tumor angiogenesis. In-depth study of tumor angiogenesis is of great significance for the treatment of GBM. Recent studies have shown that glioma stem cells (GSCs) are involved in tumor vascularization by secreting vascular endothelial growth factor (VEGF). It is necessary to construct an ideal in vitro model to study the mechanism of GSCs in tumor vascularization. Here we used extrusion-based three-dimensional (3D) bioprinting technology to fabricate GSCs tumor model. In this study, the viability of cells after bioprinting was 86.27 ± 2.41%. Furthermore, compared with traditional suspension culture, the proliferation of 3D printed GSCs was more stable. Through the transmission electron microscopy (TEM), numerous long microvilli of cells cultured in 3D bioprinted scaffolds were observed. 3D bioprinted GSCs also have more abundant mitochondria and rough endoplasmic reticulum. Additionally, the stemness properties, the expression of tumor angiogenesis-related genes and vascularization potential of 3D bioprinted GSCs in vitro were higher than that of suspension cultured cells. In summary, 3D bioprinted cell-laden hydrogel scaffolds provide a proper model for investigating the biological characteristics of GSCs and tumor angiogenesis.

Three dimensional cell printing with sulfated alginate for improved bone morphogenetic protein-2 delivery and osteogenesis in bone tissue engineering

Three-dimensional (3D) cell printing is a unique technique that enables free-form fabrication of cell-laden hydrogel scaffolds with controllable features and interconnected pores for tissue engineering applications. To this end, bioink materials able to offer good printability and favorable cellular interaction are highly required. Herein, we synthesized alginate sulfate, which is a structural mimic of heparin that can strongly bind with growth factors to prolong their activities, and studied its feasibility for cell printing applications. Several bio-inks composed of alginate and alginate-sulfate were studied to characterize their material properties and their utilities in 3D printing. The inclusion of alginate-sulfate in bio-inks (alginate/alginate-sulfate) did not significantly influence their rheological properties and allowed for a good 3D printing processibility with distinct pores and features. Moreover, alginate/alginate-sulfate bio-inks exhibited an improved retention of bone morphogenetic protein 2 in 3D-printed scaffolds. Osteoblastic proliferation and differentiation in vitro were promoted by alginate/alginate-sulfate 3D-printed constructs with an optimal composition of 3% alginate and 2% alginate-sulfate. We envision that bio-inks displaying prolonged interactions with growth factors will be useful for tissue engineering applications including bone regeneration.

Iterative feedback bio-printing-derived cell-laden hydrogel scaffolds with optimal geometrical fidelity and cellular controllability

For three-dimensional bio-printed cell-laden hydrogel tissue constructs, the well-designed internal porous geometry is tailored to obtain the desired structural and cellular properties. However, significant differences often exist between the designed and as-printed scaffolds because of the inherent characteristics of hydrogels and cells. In this study, an iterative feedback bio-printing (IFBP) approach based on optical coherence tomography (OCT) for the fabrication of cell-laden hydrogel scaffolds with optimal geometrical fidelity and cellular controllability was proposed. A custom-made swept-source OCT (SS-OCT) system was applied to characterize the printed scaffolds quantitatively. Based on the obtained empirical linear formula from the first experimental feedback loop, we defined the most appropriate design constraints and optimized the printing process to improve the geometrical fidelity. The effectiveness of IFBP was verified from the second run using gelatin/alginate hydrogel scaffolds laden with C3A cells. The mismatch of the morphological parameters greatly decreased from 40% to within 7%, which significantly optimized the cell viability, proliferation, and morphology, as well as the representative expression of hepatocyte markers, including CYP3A4 and albumin, of the printed cell-laden hydrogel scaffolds. The demonstrated protocol paves the way for the mass fabrication of cell-laden hydrogel scaffolds, engineered tissues, and scaled-up applications of the 3D bio-printing technique.

Recent Progress in Developing Injectable Matrices for Enhancing Cell Delivery and Tissue Regeneration.

Biomaterials are key factors in regenerative medicine. Matrices used for cell delivery are especially important, as they provide support to transplanted cells that is essential for promoting cell survival, retention, and desirable phenotypes. Injectable matrices have become promising and attractive due to their minimum invasiveness and ease of use. Conventional injectable matrices mostly use hydrogel precursor solutions that form solid, cell-laden hydrogel scaffolds in situ. However, these materials are associated with challenges in biocompatibility, shear-induced cell death, lack of control over cellular phenotype, lack of macroporosity and remodeling, and relatively weak mechanical strength. This Progress Report provides a brief overview of recent progress in developing injectable matrices to overcome the limitations of conventional in situ hydrogels. Biocompatible chemistry and shear-thinning hydrogels have been introduced to promote cell survival and retention. Emerging investigations of the effects of matrix properties on cellular function in 3D provide important guidelines for promoting desirable cellular phenotypes. Moreover, several novel approaches are combining injectability with macroporosity to achieve macroporous, injectable matrices for cell delivery.

Green bioprinting: extrusion-based fabrication of plant cell-laden biopolymer hydrogel scaffolds

Plant cell cultures produce active agents for pharmaceuticals, food and cosmetics. However, up to now process control for plant cell suspension cultures is challenging. A positive impact of cell immobilization, such as encapsulation in hydrogel beads, on secondary metabolites production has been reported for several plant species. The aim of this work was to develop a method for bioprinting of plant cells in order to allow fabrication of free-formed three-dimensional matrices with defined internal pore architecture for in depth characterization of immobilization conditions, cell agglomeration and interactions. By using extrusion-based 3D plotting of a basil cell-laden hydrogel blend consisting of alginate, agarose and methylcellulose (alg/aga/mc), we could demonstrate that bioprinting is applicable to plant cells. The majority of the cells survived plotting and crosslinking and the embedded cells showed high viability and metabolic activity during the investigated cultivation period of 20 d. Beside its compatibility with the plant cells, the novel alg/aga/mc blend allowed fabrication of defined 3D constructs with open macropores both in vertical and horizontal direction which were stable under culture conditions for several weeks. Thus, Green Bioprinting, an additive manufacturing technology processing live cells from the plant kingdom, is a promising new immobilization tool for plant cells that enables the development of new bioprocesses for secondary metabolites production as well as monitoring methods.

High-resolution 3D Bioprinting System for Fabricating Cell-laden Hydrogel Scaffolds with High Cellular Activities

Hydrogel scaffolds play vital roles in tissue engineering via providing appropriate microenvironments for cell adhesion, proliferation, and functioning. In this paper, a high-resolution 3D bioprinting system was developed for precise deposition of biomaterials mixed with cells, and thus, freeform fabrication of cell-laden hydrogel scaffolds. The feasibility of the system was validated by experiments. High cell viability was presented in live/dead assay. Cell aggregation and migration were observed after cultured for 2, 7, 14, and 21 days. Results indicated that the system had potential for fabricating cell-laden hydrogel scaffolds which could maintain high cellular activities.

Computer-aided multiple-head 3D printing system for printing of heterogeneous organ/tissue constructs.

Recently, much attention has focused on replacement or/and enhancement of biological tissues via the use of cell-laden hydrogel scaffolds with an architecture that mimics the tissue matrix, and with the desired three-dimensional (3D) external geometry. However, mimicking the heterogeneous tissues that most organs and tissues are formed of is challenging. Although multiple-head 3D printing systems have been proposed for fabricating heterogeneous cell-laden hydrogel scaffolds, to date only the simple exterior form has been realized. Here we describe a computer-aided design and manufacturing (CAD/CAM) system for this application. We aim to develop an algorithm to enable easy, intuitive design and fabrication of a heterogeneous cell-laden hydrogel scaffolds with a free-form 3D geometry. The printing paths of the scaffold are automatically generated from the 3D CAD model, and the scaffold is then printed by dispensing four materials; i.e., a frame, two kinds of cell-laden hydrogel and a support. We demonstrated printing of heterogeneous tissue models formed of hydrogel scaffolds using this approach, including the outer ear, kidney and tooth tissue. These results indicate that this approach is particularly promising for tissue engineering and 3D printing applications to regenerate heterogeneous organs and tissues with tailored geometries to treat specific defects or injuries.

Microfluidic direct writer with integrated declogging mechanism for fabricating cell-laden hydrogel constructs

Cell distribution and nutrient supply in 3D cell-laden hydrogel scaffolds are critical and should mimic the in vivo cellular environment, but been difficult to control with conventional fabrication methods. Here, we present a microfluidic direct writer (MFDW) to construct 3D cell-laden hydrogel structures with openings permitting media exchange. The MFDW comprises a monolithic microfluidic head, which delivers coaxial streams of cell-laden sodium alginate and calcium chloride solutions to form hydrogel fibers. Fiber diameter is controlled by adjusting the ratio of the volumetric flow rates. The MFDW head is mounted on a motorized stage, which is automatically controlled and moves at a speed synchronized with the speed of fiber fabrication. Head geometry, flow rates, and viscosity of the writing solutions were optimized to prevent the occurrence of curling and bulging. For continuous use, a highly reliable process is needed, which was accomplished with the integration of a declogging conduit supplying a solvent to dissolve the clogging gel. The MFDW was used for layer-by-layer fabrication of simple 3D structures with encapsulated cells. Assembly of 3D structures with distinct fibers is demonstrated by alternatively delivering two different alginate gel solutions. The MFDW head can be built rapidly and easily, and will allow 3D constructs for tissue engineering to be fabricated with multiple hydrogels and cell types.