Tissue Printing Research Articles

542 Evaluation of native and non-native bio-inks for 3D printing of human tissues

542 Evaluation of native and non-native bio-inks for 3D printing of human tissues

3D Integrated Tissue and Organ Printing System to Produce Human Body Parts with Structural Integrity

Tissue engineering and regenerative medicine strategies could offer new hope for patients with serious tissue injuries or end‐stage organ failure. Scientists are now applying the principles of cell transplantation, material science, and engineering to create biological substitutes that can restore and maintain normal function in diseased or injured tissues/organs. Among various three‐dimensional (3D) biofabrication technologies developed for the field of tissue engineering, 3D printing technology is one of the most attractive and powerful methods for use in constructing a structure that mimics native tissues, and thus many researchers are focusing on development of new printing technologies and applications. The hypothesis driving development of 3D bioprinting is that by precisely placing cells in relation to each other, an environment that encourages physiologically relevant cues both to and from these cells can be created, resulting in a tissue construct with functionality. Thus, we have developed 3D integrated tissue and organ printing (ITOP) system to allow construction of biological substitutes that mimic the structure and function of native tissues or organs. This system makes it possible to precisely place various cell types and biomaterials in a single 3D architecture. Our proof of concept experiments demonstrates the capabilities of our ITOP by fabricating structures of various tissue constructs of appropriate shape and size needed for clinical use. This novel organ printing system provides a leap forward in our ability to fabricate tissue constructs that could be used in translational applications.Support or Funding InformationThis work was supported, in part, by grants from the Armed Forces Institute of Regenerative Medicine (W81XWH‐08‐2‐0032), the Telemedicine and Advanced Technology Research Center at the US Army Medical Research and Material Command (W81XWH‐07‐1‐0718) and the Defense Threat Reduction Agency (N66001‐13‐C‐2027).

Bioengineering Cardiac Constructs using 3D Printing

To date, 3D bioprinting has found many actual and potential applications in medicine through assembling cells, biomaterials and supporting factors into living tissues. In particular, the combination of bioprinting and tissue engineering has emerged as a new promising strategy to address the growing need for tissues and organs for both transplantation and basic research. This review summarizes the current progress in the design and printing of bioengineered cardiac tissues for various applications. We highlight the specific biological and technical complexities such as the choice of cells and biomaterials, cell viability and function, vasculature design and tissue architecture. Current challenges and future perspectives in the field of cardiovascular tissue printing are also discussed.

Satisfying the fatty acid demand of prostate cancer: De novo synthesis versus uptake as alternative and potentially cooperative prostate cancer phenotypes.

107 Background: Malignant transformation increases cellular demand for fatty acids (FA). Many cancers show increased expression of fatty acid synthase (FASN); FASN catalyzes the de novo synthesis of the FA palmitate. While research has focused on FASN and de novo tumor FA synthesis, observations suggest that alternate mechanisms for FA acquisition are also important, including studies showing that cancer cells can be rescued from FASN inhibition by exogenous palmitate. Using a biopsy-based approach, we identified a PrCa subtype with outlier (>10 fold) overexpression of fatty acid binding protein 5 (FABP5). FABP5 facilitates the utilization of palmitate and other FAs from outside the cell; a FABP5 overexpression phenotype may confer a selective advantage when dietary FAs are abundant or when FASN is targeted as a therapy. Methods: 244 prostate biopsy patients were prospectively enrolled. Tissue prints were collected from each core for RNA and DNA preparation. mRNA was analyzed using Affymetrix arrays and TaqMan qrtPCR assays. Immunohistochemistry (IHC) was performed using archival radical prostatectomy (RP) specimens. Results: mRNA analyses revealed a PrCa subtype with >10 x overexpression of FABP; this phenotype was observed in 7% of 268 cores with Gleason sum (GS) 7-10 PrCa. We also observed a second subtype with >10 x overexpression of FASN in 9% of GS 7-10 cores. With few exceptions these two PrCa subtypes showed an “either-or” pattern with top-quartile overexpression of FABP5 or FASN but not both (Spearman r = - 0.385; P < 0.0001). IHC confirmed variable expression, including “patchwork” PrCas with high Gleason pattern (GP) areas overexpressing FABP5 next to low GP areas overexpressing FASN. The net result may be a selective advantage for the high grade cancer if FABP5 allows it to exploit the FA being synthesized by the adjacent low grade focus Conclusions: Identification of a PrCa subtype with high levels of FABP5 overexpression suggests a previously unrecognized mechanism by which some PrCas can increase FA supply without de novo synthesis; such a PrCa subtype might be particularly sensitive to dietary interventions and relatively insensitive to FASN inhibitors.

Current Status and Challenges of Three-Dimensional Modeling and Printing of Tissues and Organs.

Current Status and Challenges of Three-Dimensional Modeling and Printing of Tissues and Organs.

Abstract C40: A biopsy-focused approach to molecular studies of prostate cancer in African Americans: the Birmingham Alabama Prostate Cancer Consortium (BAPrCa)

Abstract Cancer health disparities can arise from research studies in which the study subjects are not representative of the populations affected by the disease. In prostate cancer (PrCa) biorepositories, most of the frozen samples collected for molecular analyses are obtained from radical prostatectomy (RP) specimens. However, tissue collections based on RP specimens do not include patients who are diagnosed with relatively advanced disease and treated with first-line hormonal and/or radiation therapy rather than surgery; African Americans (AA) are more likely than European Americans (EA) to be diagnosed with this type of advanced prostate cancer. With support from the DOD Prostate Cancer Research Program, our research team has established the Birmingham Alabama Prostate Cancer Consortium (BAPrCa) with a major focus on studies that use prostate biopsies for the molecular analysis of PrCa in AAs. To ensure that there is no compromise of biopsy diagnosis, we use innovative tissue print technologies to obtain snap frozen nitrocellulose blots from each of the prostate biopsy cores. These biopsy tissue prints provide high quality RNA and DNA for molecular and genetic studies. Our biopsy-based analysis of high risk cancers has revealed PrCa subtypes that were either unrecognized or significantly underestimated in previous prostatectomy-based studies. As of June 2016 more than 250 prostate biopsy patients have been prospectively enrolled in the BAPrCa prostate biopsy tissue print study; just under half (48%) of these study subjects self-identify as AA. Each biopsy from each study subject was tissue printed to generate more than 3000 unique pairs of high quality RNA and DNA samples. AA men were enrolled from two study sites in Birmingham Alabama, an academic medical center (University of Alabama at Birmingham, UAB) and a large urology private practice (Urology Centers of Alabama, UCA). The prevalence of prostate cancer diagnosed in our AA study subjects undergoing standard biopsies was 51%, with 30% showing cancer of Gleason 7 or more. A higher proportion of AA subjects were diagnosed with PrCa at UAB (61% cancer positive; 45% with Gleason 7 or more) compared to UCA (44% cancer positive; 20% with Gleason 7 or more). The difference between AA cancer diagnoses observed between our two study sites may in part result from differences in the criteria for biopsy; pre-biopsy PSA was less than 4 ng/ml for 25% of UCA subjects but for only 2% of UAB subjects. PSA criteria for prostate biopsy may have a significant and unrecognized impact on the detection of prostate cancer in contemporary AA populations. The BAPrCa study cohort provides a unique opportunity to evaluate the PrCa subtypes diagnosed in AAs using different PSA criteria for biopsy, especially in a deep south AA population. Citation Format: Sandra M. Gaston, Dennis Otali, James Kearns, Kerry Dehimer, George W. Adams, Soroush Rais-Bahrami, Jeffrey Nix, James E. Bryant, Peter Kolettis, William E. Grizzle. A biopsy-focused approach to molecular studies of prostate cancer in African Americans: the Birmingham Alabama Prostate Cancer Consortium (BAPrCa). [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C40.

Abstract IA24: Reduced Availability for Research of Human Tissues from Minority Populations

Abstract IA24: Reduced Availability for Research of Human Tissues from Minority Populations

Three-Dimensional Cell Printing of Large-Volume Tissues: Application to Ear Regeneration.

The three-dimensional (3D) printing of large-volume cells, printed in a clinically relevant size, is one of the most important challenges in the field of tissue engineering. However, few studies have reported the fabrication of large-volume cell-printed constructs (LCCs). To create LCCs, appropriate fabrication conditions should be established: Factors involved include fabrication time, residence time, and temperature control of the cell-laden hydrogel in the syringe to ensure high cell viability and functionality. The prolonged time required for 3D printing of LCCs can reduce cell viability and result in insufficient functionality of the construct, because the cells are exposed to a harsh environment during the printing process. In this regard, we present an advanced 3D cell-printing system composed of a clean air workstation, a humidifier, and a Peltier system, which provides a suitable printing environment for the production of LCCs with high cell viability. We confirmed that the advanced 3D cell-printing system was capable of providing enhanced printability of hydrogels and fabricating an ear-shaped LCC with high cell viability. In vivo results for the ear-shaped LCC also showed that printed chondrocytes proliferated sufficiently and differentiated into cartilage tissue. Thus, we conclude that the advanced 3D cell-printing system is a versatile tool to create cell-printed constructs for the generation of large-volume tissues.

3D Bioprinting of Spatially Heterogeneous Collagen Constructs for Cartilage Tissue Engineering.

3D printing of biological tissues has been of increasing interest to the biomaterials community in part because of its potential to produce spatially heterogeneous constructs. Such technology is particularly promising for orthopedic applications, which require the generation of complex geometries to match patient anatomy and complex microstructures to produce spatial heterogeneity and anisotropy. Prior research has demonstrated the capacity to create precisely shaped 3D printed constructs using biocompatible alginate hydrogels. However, alginate is extremely compliant and brittle, and high-density collagen hydrogels could be a preferable option for load-bearing applications. This research focused on developing and evaluating a method of printing soft tissue implants with high-density collagen hydrogels using a commercially available 3D printer, modified for tissue-engineering purposes. The tissue constructs, seeded with primary meniscal fibrochondrocytes, were evaluated using measures of geometric fidelity, cell viability, mechanical properties, and fiber microstructure. The constructs were found to be mechanically stable and were able to support and maintain cell growth. Furthermore, heterogeneous 3D-printed constructs were generated, consisting of discrete domains with distinct mechanical properties.

NR2F2 regulates chondrogenesis of human mesenchymal stem cells in bioprinted cartilage.

Bioprinting as an advanced enabling technology has the capacity to construct tissues with respective anatomical structures. In order to maintain the precise printing resolution for anatomical tissue printing, cell seeding density in bioink is limited. Bone marrow derived mesenchymal stem cells (MSCs) are widely used for cartilage tissue engineering. However, the approach of ideal chondrogenic differentiation of MSCs without hypertrophy still remains elusive. Here, we reported NR2F2 plays a crucial role in MSC chondrogenesis in bioprinted cartilage. NR2F2 over-expressed MSCs showed significantly enhanced chondrogenesis and NR2F2 knockdown cells demonstrated the exactly opposite behavior. We evaluated the cells cultured in monolayer, 3D pellet, and bioprinted 3D scaffold. All observations were consistent among gene expression, biochemical analysis, histological assay, and biomechanical evaluation. The data also revealed possible involvement of NR2F2 in mechanism of MSC chondrogenic differentiation under hypoxic culture condition. Biotechnol. Bioeng. 2017;114: 208-216. © 2016 Wiley Periodicals, Inc.

Serological detection of 'Candidatus Liberibacter asiaticus' in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens.

We describe experiments with antibodies against ‘Candidatus Liberibacter asiaticus used to detect the pathogen in infected plants. We used scFv selected to bind epitopes exposed on the surface of the bacterium in tissue prints, with secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary antibody produced positive results with CaLas diseased samples when the primary scFv were not used. The anti-FLAG monoclonal antibody (Mab) also identified plants infected with other vascular pathogens. We then identified a paralogous group of secreted chaperone proteins in the HSP-90 family that contained the amino acid sequence DDDDK identical to the carboxy-terminal sequence of the FLAG epitope. A rabbit polyclonal antibody against one of the same epitopes combined with a goat anti-rabbit secondary antibody produced very strong purple color in individual phloem cells, as expected for this pathogen. These results were entirely specific for CaLas-infected citrus. The simplicity, cost and ability to scale the tissue print assay makes this an attractive assay to complement PCR-based assays currently in use. The partial FLAG epitope may itself be useful as a molecular marker for the rapid screening of citrus plants for the presence of vascular pathogens.

3D Bioprinting Using a Templated Porous Bioink.

3D tissue printing with adult stem cells is reported. A novel cell-containing multicomponent bioink is used in a two-step 3D printing process to engineer bone and cartilage architectures.

3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening.

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D vs. 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs.

Investigating the Role of Extensin Proteins in Poplar Biomass Recalcitrance

The biological conversion of cellulosic biomass to biofuel is hindered by cell wall recalcitrance, which can limit the ability of cellulases to access and break down cellulose. The purpose of this study was to investigate whether hydroxyproline-rich cell wall proteins (extensins) are present in poplar stem biomass, and whether these proteins may contribute to recalcitrance. Three classical extensin genes were identified in Populus trichocarpa through bioinformatic analysis of poplar genome sequences, with the following proposed names: PtEXTENSIN1 (Potri.001G019700); PtEXTENSIN2 (Potri.001G020100); PtEXTENSIN3 (Potri.018G050100). Tissue print immunoblots localized the extensin proteins in poplar stems to regions near the vascular cambium. Different thermochemical pretreatments reduced but did not eliminate hydroxyproline (Hyp, a proxy for extensins) from the biomass. Protease treatment of liquid hot water-pretreated poplar biomass reduced Hyp content by a further 16% and increased subsequent glucose yield by 20%. These data suggest that extensins may contribute to recalcitrance in pretreated poplar biomass, and that incorporating protease treatment into pretreatment protocols could result in a small but significant increase in the yield of fermentable glucose.

Regenerative medicine: The future of 3D printing of human tissues is taking shape.

Regenerative medicine: The future of 3D printing of human tissues is taking shape.

Abstract C72: Improving the accuracy and diagnostic power of prostate biopsy for African American patients: The Birmingham Alabama Prostate Cancer (BAPrCa) Consortium

Abstract Study Purpose: Both incidence and mortality data show that the burden of prostate cancer (PrCa) is greater in African Americans (AA) than in European Americans (EA). Socioeconomic factors contribute to this health disparity, but do not fully account for observations that AA are more likely than others to be diagnosed with more aggressive and life threatening forms of PrCa. Prostate biopsies usually establish the diagnosis of PrCa and are used to estimate the extent of the disease (based on the number and location of cores with cancer and involvement of individual cores) and its potential aggressiveness (based on Gleason scores). Health policy groups recommend that men with limited low grade prostate cancer be managed by active surveillance (AS) rather than immediate surgical or radiation treatment. However, the standard-of-care prostate biopsy is limited by sampling error and the possibility that a high grade PrCa might have been missed is a significant concern for many patients who are considering AS; this concern is heightened for AA because of their higher risk of aggressive disease. Moreover, AA are more likely to be diagnosed with high grade/high stage prostate cancer that is not treated surgically and thus not well represented in molecular studies that utilize radical prostatectomy specimens. Our research team established the Birmingham Alabama Prostate Cancer (BAPrCa) Consortium with a major focus on the molecular analysis of prostate biopsies in order to increase the clinically actionable information that can be obtained from these specimens. We use an ancestry-informed approach that is specifically designed to improve the accuracy and diagnostic power of prostate biopsy for AA patients. Experimental Procedures: The BAPrCa Consortium implemented an innovative prostate biopsy “tissue print” technology that permits collection of snap-frozen nitrocellulose blots of biopsy cores without diagnostically compromising these specimens. Tissue prints provide high quality RNA and DNA from biopsies from the full range of patients, including AAs whose cancer is too advanced at diagnosis for radical prostatectomy; this permits the molecular characterization of PrCa subtypes in men diagnosed with high volume/high grade disease who have not been adequately represented in previous molecular profiling studies. Our BAPrCa research protocols include informed consent for genetic ancestry admixture studies. Gene expression analysis of prostate biopsy tissue prints is correlated with histopathology and multiparametric prostate MRI. Results: Our data suggest that in the Birmingham area, higher prostate cancer risk in AA is associated with increasing proportion of West African (WA) ancestry, which may reflect the prevalence of population-specific genetic mutations or variations that contribute to the development of more aggressive disease. As a group, the men diagnosed with high grade PrCa showed a significantly higher level of WA ancestry than the men diagnosed with no cancer (P = 0.001). A similar pattern is observed in comparisons of AA men diagnosed with high grade cancer vs low grade PrCa. Inasmuch as our AIMs genotyping panel uses a small number of well-established AIMS markers, our observation of significantly increased risk of PrCa in AA men with high %WA AIMS ancestry may, if confirmed, have immediate potential clinical applications for improving prostate cancer screening and active surveillance. Moreover, gene expression profiles of biopsies from BAPrCa patients diagnosed with high volume/high grade PrCa revealed two subtypes of high grade PrCa with striking differences in the pathways that drive a shift in tumor fatty acid metabolism; one is a fatty acid synthase (FASN) dominant phenotype and the other a previously unrecognized fatty acid binding protein (FABP5) dominant phenotype. Our data suggest that the FABP5 dominant PrCa subtype is more common in AA and the FASN dominant subtype more common in EA. These findings may provide the basis for more effective dietary interventions and targeted therapies for AA and EA patients with high grade PrCa. Conclusions: By utilizing innovative tissue print techniques for the molecular analyses of prostate biopsies and using an ancestry informed approach in our study designs, the Birmingham Alabama Prostate Cancer (BAPrCa) Consortium has identified new and potentially actionable PrCa signatures that may improve the accuracy and diagnostic power of prostate biopsy for AA patients. Citation Format: Sandra M. Gaston, Soroush Rais-Bahrami, Rick Kittles, Kerry Dehimer, Dennis Otali, Jeffrey W. Nix, Peter N. Kolettis, George Adams, William E. Grizzle. Improving the accuracy and diagnostic power of prostate biopsy for African American patients: The Birmingham Alabama Prostate Cancer (BAPrCa) Consortium. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C72.

Three-dimensional printing of biological matters

Three-dimensional (3D) printing of human tissues and organ has been an exciting research topic in the past three decades. However, existing technological and biological challenges still require a significant amount of research. The present review highlights these challenges and discusses their potential solutions such as mapping and converting a human organ onto a 3D virtual design, synchronizing the virtual design with the printing hardware. Moreover, the paper discusses in details recent advances in formulating bio-inks and challenges in tissue construction with or without scaffold. Next, the paper reviews fusion processes effecting vascular cells and tissues. Finally, the paper deliberates the feasibility of organ printing with state-of-the-art technologies.

Olten Meeting 2015 – Antibiotics and Bioprinting – for a better life

The ever lurking danger of antibiotic resistance and the potential of bioprinting are on everyone's lips. But where do we stand in the battle against antibiotic-resistant pathogens? And what are the opportunities for biotech in the 3D printing of biological tissues and organs through the layering of living cells? At the Olten Meeting 2015 scientists and entrepreneurs met to throw light on the current situation.

3D printing of bone tissue engineering scaffolds with osteoconductivity and osteorinductivity

3D printing of bone tissue engineering scaffolds with osteoconductivity and osteorinductivity

A Review of Inkjet, Microextrusion and Laser 3D Bioprinting Techniques for Tissue Engineering

Bioprinting is considered to be an emerging field that is having a revolutionary impact on the medical science. Three-dimensional (3D) bioprinting is the process of creating spatially controlled cell patterns, in which the behaviour of biological tissues can be reproduced. Over the last few decades, cell-printing techniques are emerging as one of the most promising technologies for organising cells in 2D and 3D to form tissues and organ. This emerging biotechnology appears to be promising for advancing tissue engineering and organ fabrication for organ transplantation, drug testing, research investigations and cancer or disease modelling, and therefore, it has attracted growing interest worldwide among researchers and the general public. Recent advances in science and technology has enabled 3D printers to use biocompatible materials, cells and supporting components and construct a complex 3D functional living tissues. 3D bioprinting has already been used to generate several tissues, tracheal splints, bone, vascular grafts, multilayered skin, heart tissue and cartilaginous structures for transplantation, but the idea extends to the printing of complete, viable organs and tissues for human transplant. Bioprinted organs could significantly decrease transplant organ rejections and also increase the availability of organs for critically ill patients. There are three main techniques of 3D bioprinting: inkjet, microextrusion and laser assisted. In this review, we focus on these techniques of bioprinting and their current applications. We also highlight the cell source required for bioprinting, and briefly review its market size and economic value. We conclude with future prospect of 3D bioprinting.