With advances in tissue engineering, the possibility of regenerating injured tissue or failing organs has become a realistic prospect for the first time in medical history. Tissue engineering - the combination of bioactive materials with cells to generate engineered constructs that functionally replace lost and/or damaged tissue - is a major strategy to achieve this goal. One facet of tissue engineering is biofabrication, where three-dimensional tissue-like structures composed of biomaterials and cells in a single manufacturing procedure are generated. Cell-laden hydrogels are commonly used in biofabrication and are termed "bioinks". Hydrogels are particularly attractive for biofabrication as they recapitulate several features of the natural extracellular matrix and allow cell encapsulation in a highly hydrated mechanically supportive three-dimensional environment. Additionally, they allow for efficient and homogeneous cell seeding, can provide biologically-relevant chemical and physical signals, and can be formed in various shapes and biomechanical characteristics. However, despite the progress made in modifying hydrogels for enhanced bioactivation, cell survival and tissue formation, little attention has so far been paid to optimize hydrogels for the physico-chemical demands of the biofabrication process. The resulting lack of hydrogel bioinks have been identified as one major hurdle for a more rapid progress of the field. In this review we summarize and focus on the deposition process, the parameters and demands of hydrogels in biofabrication, with special attention to robotic dispensing as an approach that generates constructs of clinically relevant dimensions. We aim to highlight this current lack of effectual hydrogels within biofabrication and initiate new ideas and developments in the design and tailoring of hydrogels. The successful development of a "printable" hydrogel that supports cell adhesion, migration, and differentiation will significantly advance this exciting and promising approach for tissue engineering.

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25th Anniversary Article: Engineering Hydrogels for Biofabrication

We demonstrate the additive manufacturing of complex three-dimensional (3D) biological structures using soft protein and polysaccharide hydrogels that are challenging or impossible to create using traditional fabrication approaches. These structures are built by embedding the printed hydrogel within a secondary hydrogel that serves as a temporary, thermoreversible, and biocompatible support. This process, termed freeform reversible embedding of suspended hydrogels, enables 3D printing of hydrated materials with an elastic modulus <500 kPa including alginate, collagen, and fibrin. Computer-aided design models of 3D optical, computed tomography, and magnetic resonance imaging data were 3D printed at a resolution of ~200 μm and at low cost by leveraging open-source hardware and software tools. Proof-of-concept structures based on femurs, branched coronary arteries, trabeculated embryonic hearts, and human brains were mechanically robust and recreated complex 3D internal and external anatomical architectures.

Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.

A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering

Current advances and future perspectives in extrusion-based bioprinting.

Heart valve disease is a serious and growing public health problem for which prosthetic replacement is most commonly indicated. Current prosthetic devices are inadequate for younger adults and growing children. Tissue engineered living aortic valve conduits have potential for remodeling, regeneration, and growth, but fabricating natural anatomical complexity with cellular heterogeneity remain challenging. In the current study, we implement 3D bioprinting to fabricate living alginate/gelatin hydrogel valve conduits with anatomical architecture and direct incorporation of dual cell types in a regionally constrained manner. Encapsulated aortic root sinus smooth muscle cells (SMC) and aortic valve leaflet interstitial cells (VIC) were viable within alginate/gelatin hydrogel discs over 7 days in culture. Acellular 3D printed hydrogels exhibited reduced modulus, ultimate strength, and peak strain reducing slightly over 7-day culture, while the tensile biomechanics of cell-laden hydrogels were maintained. Aortic valve conduits were successfully bioprinted with direct encapsulation of SMC in the valve root and VIC in the leaflets. Both cell types were viable (81.4 ± 3.4% for SMC and 83.2 ± 4.0% for VIC) within 3D printed tissues. Encapsulated SMC expressed elevated alpha-smooth muscle actin, while VIC expressed elevated vimentin. These results demonstrate that anatomically complex, heterogeneously encapsulated aortic valve hydrogel conduits can be fabricated with 3D bioprinting.

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3D bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels.

The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for the long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D printing/photocrosslinking technique for rapidly engineering complex, heterogeneous aortic valve scaffolds. Native anatomic and axisymmetric aortic valve geometries (root wall and tri-leaflets) with 12-22 mm inner diameters (ID) were 3D printed with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels (700 or 8000 MW) supplemented with alginate. 3D printing geometric accuracy was quantified and compared using Micro-CT. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds were cultured for up to 21 days. Results showed that blended PEG-DA scaffolds could achieve over tenfold range in elastic modulus (5.3±0.9 to 74.6±1.5 kPa). 3D printing times for valve conduits with mechanically contrasting hydrogels were optimized to 14 to 45 min, increasing linearly with conduit diameter. Larger printed valves had greater shape fidelity (93.3±2.6, 85.1±2.0 and 73.3±5.2% for 22, 17 and 12 mm ID porcine valves; 89.1±4.0, 84.1±5.6 and 66.6±5.2% for simplified valves). PAVIC seeded scaffolds maintained near 100% viability over 21 days. These results demonstrate that 3D hydrogel printing with controlled photocrosslinking can rapidly fabricate anatomical heterogeneous valve conduits that support cell engraftment.

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Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds

In this paper we present bioengineering and biomedical research performed with Fab@Home and modifications to the system which improve its performance for these applications. At Cornell University, the Cardiovascular Developmental Bioengineering Laboratory uses the Fab@Home system for tissue engineering research to fabricate replicas of native cardiac valves using hydrogel mixes that include poly(ethylene glycol) diacrylate (PEG-DA), poly-(ester amide) (PEA), alginate and a commercial photoinitiator. The printed porcine aortic valve demonstrates good shape fidelity and dimensional accuracy compared to medical scans of the valve. CTI has a team working to improve Fab@Home systems for biomedical applications by installing a linear guide with limit switches for deposition tools, attaching a device to align the tip for better deposition with two syringes and making improvements to the software for importing and manipulation of 3D models using high quality open source libraries The second deposition tool is loaded with a solution of 2% LF10/60 alginate and 3% Agarose Type IX-A (Sigma-Aldrich, St. Louis, MO). Agarose Type IX-A solutions have a gelling temperature of ≤17°C and melting temperature of ≤60°C. These temperature characteristics allow the agarose to serve as a washable support for unstable or nonfreestanding elements of the valve structure during fabrication. 1.2 Replica Porcine Aortic Valves Aortic valves were harvested from freshly slaughtered pigs at Shirk Meats (Himrod, NY), rinsed with cold sterile PBS, and preserved in formalin. The GE eXplore CT120 μCT (micro computed tomography) system (General Electric Medical Systems, London, ON) was used to scan the tissue at 100 μm resolution using 80 keV, 32 mA, 220 angles, and 16ms exposure time. The dataset was reconstructed using a modified Feldkamp method and exported to DICOM images using Microview (General Electric Healthcare) v2.2 software. The DICOM image set was analyzed via thresholding and region growing algorithms, and the set was then exported into stereolithography (STL) format. A mold structure was designed to support valve leaflets during the layered manufacturing process and also exported in STL. For fabrication, a secondary platform was mounted onto the standard z-stage of the Model 1 system. The porcine aortic valve replica and the mold structure were fabricated directly onto this secondary platform using PEG-DA mixture and liquid agar solution at room temperature. The secondary platform was cooled using dry ice to maintain a surface temperature capable of immediately gelling deposited agarose during fab. After printing each layer, a handheld 365 nm UV light was exposed onto the hydrogel to allow crosslinking. The fabricated valve and support mold were then submerged in a water bath and gently heated to 60°C to melt away the agarose mold.

Construction and adaptation of an open source rapid prototyping machine for biomedical research purposes—a multinational collaborative development

Objectives To investigate the efficacy and safety of lung stereotactic body radiation therapy (SBRT) for non-small cell lung cancer (NSCLC) including oligorecurrent and oligoprogressive disease. Methods Single-institution retrospective analysis of 60 NSCLC patients with 62 discrete lesions treated with SBRT between 2008 and 2017. Patients were stratified into three groups, including early stage, locally recurrent, and oligoprogressive disease. Group 1 included early stage local disease with no prior local therapy. Group 2 included locally recurrent disease after local treatment of a primary lesion, and group 3 included regional or well-controlled distant metastatic disease receiving SBRT for a treatment naive lung lesion (oligoprogressive disease). Patient/tumor characteristics and adverse effects were recorded. Local failure free survival (LFFS), progression free survival (PFS), and overall survival (OS) were estimated using the Kaplan Meier method. Results At median follow-up of 34 months, 67% of the study population remained alive. The estimated 3-year LFFS for group 1, group 2, and group 3 patients was 95% (95% CI: 86%-100%), 82%(62% - 100%), and 83% (58-100%), respectively. The estimated 3-year PFS was 59% (42-83%), 40% (21%-78%), and 33% (12%-95%), and the estimated 3-year OS was 58% (41-82%), 60% (37-96%), and 58% (31-100%)), respectively for each group. When adjusted for age and size of lesion, no significant difference in OS, LFFS, and PFS emerged between groups (p > 0.05). No patients experienced grade 3 to 5 toxicity. Eighteen patients (29%) experienced grade 1 to 2 toxicity. The most common toxicities reported were cough and fatigue. Conclusions Our data demonstrates control rates in group 1 patients comparable to historical controls. Our study also reveals comparable clinical results for SBRT in the treatment of NSCLC by demonstrating similar rates of LFFS and OS in group 2 and group 3 patients with locally recurrent and treatment naïve lung lesion with well-controlled distant metastatic disease.

Stereotactic Body Radiation Therapy for the Treatment of Locally Recurrent and Oligoprogressive Non-Small Cell Lung Cancer: A Single Institution Experience

N. W. Colangelo

Papers

Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds

Construction and adaptation of an open source rapid prototyping machine for biomedical research purposes—a multinational collaborative development

Stereotactic Body Radiation Therapy for the Treatment of Locally Recurrent and Oligoprogressive Non-Small Cell Lung Cancer: A Single Institution Experience