PhD, Penn State University, Engineering Science and Mechanics (2020)
Ngan Huang, Postdoctoral Faculty Sponsor
Three-dimensional (3D) printing technology is increasingly being employed in biochemical as well as clinical applications and more importantly in fabrication of microfluidic devices. However, the microfluidic community mainly relies on photolithography for fabrication of a defined mask, which is both tedious and expensive requiring clean room settings as well as limited to the generation of two-dimensional (2D) features. In this work, we 3D printed nanoclay-reinforced Pluronic ink as a sacrificial material, which exhibited shear thinning behavior and superior printability allowing the fabrication of unsupported or overhanging templates of channels with uniform diameter and circular cross-sections. To highlight the potential and effectiveness of the presented approach, we fabricated a human blood vessel-on-a-chip model with curved as well as straight channels. These channels were then lined with Human Umbilical Vein Endothelial cells (HUVECs) and subjected to a dynamic culture for 10 days to explore the effect of shear stress on HUVEC morphology based on the location of HUVECs in the devices. Overall, we presented a highly affordable, useful, and practical approach in fabrication of closed microfluidic channels in PDMS based devices, which holds great potential for numerous applications, such as but not limited to tissue/organ-on-chip, microfluidics, point-of-care devices and drug screening platforms.
View details for DOI 10.1088/1748-605X/abe55e
View details for Web of Science ID 000632886700001
View details for PubMedID 33571984
The heterogeneous and anisotropic articular cartilage is generally studied as a layered structure of "zones" with unique composition and architecture, which is difficult to recapitulate using current approaches. A novel hybrid bioprinting strategy is presented here to generate zonally stratified cartilage. Scaffold-free tissue strands (TSs) are made of human adipose-derived stem cells (ADSCs) or predifferentiated ADSCs. Cartilage TSs with predifferentiated ADSCs exhibit improved mechanical properties and upregulated expression of cartilage-specific markers at both transcription and protein levels as compared to TSs with ADSCs being differentiated in the form of strands and TSs of nontransfected ADSCs. Using the novel hybrid approach integrating new aspiration-assisted and extrusion-based bioprinting techniques, the bioprinting of zonally stratified cartilage with vertically aligned TSs at the bottom zone and horizontally aligned TSs at the superficial zone is demonstrated, in which collagen fibers are aligned with designated orientation in each zone imitating the anatomical regions and matrix orientation of native articular cartilage. In addition, mechanical testing study reveals a compression modulus of ?1.1 MPa, which is similar to that of human articular cartilage. The prominent findings highlight the potential of this novel bioprinting approach for building biologically, mechanically, and histologically relevant cartilage for tissue engineering purposes.
View details for DOI 10.1002/adhm.202001657
View details for Web of Science ID 000579169200001
View details for PubMedID 33073548
Osteochondral defects contain damage to both the articular cartilage and underlying subchon- dral bone, which remains a significant challenge in orthopedic surgery. Layered structure of bone, cartilage and the bone-cartilage interface must be taken into account in the case of biofabrication of the osteochondral (OC) interface. In this study, a dual layered OC interface was bioprinted using a newly developed aspiration-assisted bioprinting (AAB) technique, which has been the first time that scaffold-free bioprinting was applied to OC interface engineering. Tissue spheroids, made of human adipose-derived stem cells (ADSCs), were differentiated in three dimensions (3D) into chondrogenic and osteogenic spheroids, which were confirmed by immunostaining and histology qualitatively, and biochemistry assays and gene expression, quantitatively. Remarkably, the OC interface was bioprinted by accurate positioning of a layer of osteogenic spheroids onto a sacrificial alginate support followed by another layer of chondrogenic spheroids overlaid by the same support. Spheroids in individual zones fused and the maintenance of phenotypes in both zones confirmed the successful biofabrication of the histomorphologically-relevant OC interface. The biofabrication of OC tissue model without the use of polymeric scaffolds unveils great potential not only in regenerative medicine but also in drug testing and disease modeling for osteoarthritis.
View details for DOI 10.1038/s41598-020-69960-6
View details for Web of Science ID 000561102300025
View details for PubMedID 32753630
View details for PubMedCentralID PMC7403300
Extrusion-based bioprinting of hydrogels in a granular secondary gel enables the fabrication of cell-laden three-dimensional (3D) constructs in an anatomically accurate manner, which is challenging using conventional extrusion-based bioprinting processes. In this study, carbohydrazide-modified gelatin (Gel-CDH) was synthesized and deposited into a new multifunctional support bath consisting of gelatin microparticles suspended in an oxidized alginate (OAlg) solution. During extrusion, Gel-CDH and OAlg were rapidly cross-linked because of the Schiff base formation between aldehyde groups of OAlg and amino groups of Gel-CDH, which has not been demonstrated in the domain of 3D bioprinting before. Rheological results indicated that hydrogels with lower OAlg to Gel-CDH ratios possessed superior mechanical rigidity. Different 3D geometrically intricate constructs were successfully created upon the determination of optimal bioprinting parameters. Human mesenchymal stem cells and human umbilical vein endothelial cells were also bioprinted at physiologically relevant cell densities. The presented study has offered a novel strategy for bioprinting of natural polymer-based hydrogels into 3D complex-shaped biomimetic constructs, which eliminated the need for cytotoxic supplements as external cross-linkers or additional cross-linking processes, therefore expanding the availability of bioinks.
View details for DOI 10.1021/acsami.0c05096
View details for Web of Science ID 000535170300017
View details for PubMedID 32274920
Three-dimensional (3D) bioprinting is an appealing approach for building tissues; however, bioprinting of mini-tissue blocks (i.e., spheroids) with precise control on their positioning in 3D space has been a major obstacle. Here, we unveil "aspiration-assisted bioprinting (AAB)," which enables picking and bioprinting biologics in 3D through harnessing the power of aspiration forces, and when coupled with microvalve bioprinting, it facilitated different biofabrication schemes including scaffold-based or scaffold-free bioprinting at an unprecedented placement precision, ~11% with respect to the spheroid size. We studied the underlying physical mechanism of AAB to understand interactions between aspirated viscoelastic spheroids and physical governing forces during aspiration and bioprinting. We bioprinted a wide range of biologics with dimensions in an order-of-magnitude range including tissue spheroids (80 to 600 ?m), tissue strands (~800 ?m), or single cells (electrocytes, ~400 ?m), and as applications, we illustrated the patterning of angiogenic sprouting spheroids and self-assembly of osteogenic spheroids.
View details for DOI 10.1126/sciadv.aaw5111
View details for Web of Science ID 000519001400004
View details for PubMedID 32181332
View details for PubMedCentralID PMC7060055
Bioprinting of cellular aggregates, such as tissue spheroids, to form three-dimensional (3D) complex-shaped arrangements, has posed a major challenge due to lack of robust, reproducible and practical bioprinting techniques. Here, we demonstrate 3D aspiration-assisted freeform bioprinting of tissue spheroids by precisely positioning them in self-healing yield-stress gels, enabling the self-assembly of spheroids for fabrication of tissues. The presented approach enables the traverse of spheroids directly from the cell media to the gel and freeform positioning of the spheroids on demand. We study the underlying physical mechanism of the approach to elucidate the interactions between the aspirated spheroids and the gel's yield-stress during the transfer of spheroids from cell media to the gel. We further demonstrate the application of the proposed approach in the realization of various freeform shapes and self-assembly of human mesenchymal stem cell spheroids for the construction of cartilage and bone tissues.
View details for DOI 10.1038/s42005-020-00449-4
View details for PubMedID 33251340
View details for PubMedCentralID PMC7695349
Current technologies for manufacturing of microfluidic devices include soft-lithography, wet and dry etching, thermoforming, micro-machining and three-dimensional (3D) printing. Among them, soft-lithography has been the mostly preferred one in medical and pharmaceutical fields due to its ability to generate polydimethylsiloxane (PDMS) devices with resin biocompatibility, throughput and transparency for imaging. It is a multi-step process requiring the preparation of a silicon wafer pattern, which is fabricated using photolithography according to a defined mask. Photolithography is a costly, complicated and time-consuming process requiring a clean-room environment, and the technology is not readily accessible in most of the developing countries. In addition, generated patterns on photolithography-made silicon wafers do not allow building 3D intricate shapes and silicon direct bonding is thus utilized for closed fluid channels and complex 3D structures. 3D Printing of PDMS has recently gained significant interest due to its ability to define complex 3D shapes directly from user-defined designs. In this work, we investigated Carbopol as a sacrificial gel in order to create microfluidic channels in PDMS devices. Our study demonstrated that Carbopol ink possessed a shear-thinning behavior and enabled the extrusion-based printing of channel templates, which were overlaid with PDMS to create microfluidic devices upon curing of PDMS and removal of the sacrificial Carbopol ink. To demonstrate the effectiveness of the fabricated devices, channels were lined up with human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (BMECs) in separate devices, where both HUVECs and BMECs demonstrated the formation of endothelium with highly aligned cells in the direction of fluid flow. Overall, we here present a highly affordable and practical approach in fabrication of PDMS devices with closed fluid channels, which have great potential in a myriad of applications from cancer treatments to infectious disease diagnostics to artificial organs.
View details for DOI 10.1088/1758-5090/ab10ae
View details for Web of Science ID 000465013000001
View details for PubMedID 30884470
Hernia repair is a common surgical procedure with polypropylene (PP) mesh being the standard material for correction because of its durability. However, complications such as seroma and pain are common, and repair failures still approach 15% secondary to poor tissue integration. In an effort to enhance mesh integration, we evaluated the applicability of a squid ring teeth (SRT) protein coating for soft-tissue repair in an abdominal wall defect model. SRT is a biologically derived high-strength protein with strong mechanical properties. We assessed tissue integration, strength, and biocompatibility of a SRT-coated PP mesh in a first-time pilot animal study.PP mesh was coated with SRT (SRT-PP) and tested for mechanical strength against uncoated PP mesh. Cell proliferation and adhesion studies were performed in vitro using a 3T3 cell line. Rats underwent either PP (n = 3) or SRT-PP (n = 6) bridge mesh implantation in an anterior abdominal wall defect model. Repair was assessed clinically and radiographically, with integration evaluated by histology and mechanical testing at 60 days.Cell proliferation was enhanced on SRT-PP mesh. This was corroborated in vivo by abdominal wall histology, dramatically diminished craniocaudal mesh contraction, improved strength testing, and higher tissue failure strain. There was no increase in seroma or visceral adhesion formation. No foreign body reactions were noted on liver histology.SRT applied as a coating appears to augment mesh-tissue integration and improve abdominal wall stability following bridged repair. Further studies in larger animals will determine its applicability for hernia repair in patients.
View details for DOI 10.1097/GOX.0000000000001881
View details for Web of Science ID 000444969900019
View details for PubMedID 30254828
View details for PubMedCentralID PMC6143318
Despite the recent achievements in cell-based therapies for curing type-1 diabetes (T1D), capillarization in beta (?)-cell clusters is still a major roadblock as it is essential for long-term viability and function of ?-cells in vivo. In this research, we report sprouting angiogenesis in engineered pseudo islets (EPIs) made of mouse insulinoma ?TC3 cells and rat heart microvascular endothelial cells (RHMVECs). Upon culturing in three-dimensional (3D) constructs under angiogenic conditions, EPIs sprouted extensive capillaries into the surrounding matrix. Ultra-morphological analysis through histological sections also revealed presence of capillarization within EPIs. EPIs cultured in 3D constructs maintained their viability and functionality over time while non-vascularized EPIs, without the presence of RHMVECs, could not retain their viability nor functionality. Here we demonstrate angiogenesis in engineered islets, where patient specific stem cell-derived human beta cells can be combined with microvascular endothelial cells in the near future for long-term graft survival in T1D patients.
View details for DOI 10.1088/1758-5090/aab002
View details for Web of Science ID 000427721000002
View details for PubMedID 29451122
Three-dimensional (3D) organoids have shown advantages in cell culture over traditional two-dimensional (2D) culture, and have great potential in various applications of tissue engineering. However, there are limitations in current organoid fabrication technologies, such as uncontrolled size, poor reproductively, and inadequate complexity of organoids. In this chapter, we present the existing techniques and discuss the major challenges for 3D organoid biofabrication. Future perspectives on organoid bioprinting are also discussed, where bioprinting technologies are expected to make a major contribution in organoid fabrication, such as realizing mass production and constructing complex heterotypic tissues, and thus further advance the translational application of organoids in tissue engineering and regenerative medicine as well drug testing and pharmaceutics.
View details for DOI 10.1007/5584_2018_216
View details for Web of Science ID 000458010000005
View details for PubMedID 29855825
Introduction: Although there have been significant contributions from the pharmaceutical industry to clinical practice, several diseases remain unconquered, with the discovery of new drugs remaining a paramount objective. The actual process of drug discovery involves many steps including pre-clinical and clinical testing, which are highly time- and resource-consuming, driving researchers to improve the process efficiency. The shift of modelling technology from two-dimensions (2D) to three-dimensions (3D) is one of such advancements. 3D Models allow for close mimicry of cellular interactions and tissue microenvironments thereby improving the accuracy of results. The advent of bioprinting for fabrication of tissues has shown potential to improve 3D culture models. Areas covered: The present review provides a comprehensive update on a wide range of bioprinted tissue models and appraise them for their potential use in drug discovery research. Expert opinion: Efficiency, reproducibility, and standardization are some impediments of the bioprinted models. Vascularization of the constructs has to be addressed in the near future. While much progress has already been made with several seminal works, the next milestone will be the commercialization of these models after due regulatory approval.
View details for DOI 10.1080/17460441.2018.1542427
View details for Web of Science ID 000455587600006
View details for PubMedID 30384781
View details for PubMedCentralID PMC6494715
Craniofacial (CF) tissue is an architecturally complex tissue consisting of both bone and soft tissues with significant patient specific variations. Conditions of congenital abnormalities, tumor resection surgeries, and traumatic injuries of the CF skeleton can result in major deficits of bone tissue. Despite advances in surgical reconstruction techniques, management of CF osseous deficits remains a challenge. Due its inherent versatility, bioprinting offers a promising solution to address these issues. In this review, we present and analyze the current state of bioprinting of bone tissue and highlight how these techniques may be adapted to serve regenerative therapies for CF applications. Biotechnol. Bioeng. 2017;114: 2424-2431. © 2017 Wiley Periodicals, Inc.
View details for DOI 10.1002/bit.26349
View details for Web of Science ID 000411699200001
View details for PubMedID 28600873
Successful launch of a commercial drug requires significant investment of time and financial resources wherein late-stage failures become a reason for catastrophic failures in drug discovery. This calls for infusing constant innovations in technologies, which can give reliable prediction of efficacy, and more importantly, toxicology of the compound early in the drug discovery process before clinical trials. Though computational advances have resulted in more rationale in silico designing, in vitro experimental studies still require gaining industry confidence and improving in vitro-in vivo correlations. In this quest, due to their ability to mimic the spatial and chemical attributes of native tissues, three-dimensional (3D) tissue models have now proven to provide better results for drug screening compared to traditional two-dimensional (2D) models. However, in vitro fabrication of living tissues has remained a bottleneck in realizing the full potential of 3D models. Recent advances in bioprinting provide a valuable tool to fabricate biomimetic constructs, which can be applied in different stages of drug discovery research. This paper presents the first comprehensive review of bioprinting techniques applied for fabrication of 3D tissue models for pharmaceutical studies. A comparative evaluation of different bioprinting modalities is performed to assess the performance and ability of fabricating 3D tissue models for pharmaceutical use as the critical selection of bioprinting modalities indeed plays a crucial role in efficacy and toxicology testing of drugs and accelerates the drug development cycle. In addition, limitations with current tissue models are discussed thoroughly and future prospects of the role of bioprinting in pharmaceutics are provided to the reader.Present advances in tissue biofabrication have crucial role to play in aiding the pharmaceutical development process achieve its objectives. Advent of three-dimensional (3D) models, in particular, is viewed with immense interest by the community due to their ability to mimic in vivo hierarchical tissue architecture and heterogeneous composition. Successful realization of 3D models will not only provide greater in vitro-in vivo correlation compared to the two-dimensional (2D) models, but also eventually replace pre-clinical animal testing, which has their own shortcomings. Amongst all fabrication techniques, bioprinting- comprising all the different modalities (extrusion-, droplet- and laser-based bioprinting), is emerging as the most viable fabrication technique to create the biomimetic tissue constructs. Notwithstanding the interest in bioprinting by the pharmaceutical development researchers, it can be seen that there is a limited availability of comparative literature which can guide the proper selection of bioprinting processes and associated considerations, such as the bioink selection for a particular pharmaceutical study. Thus, this work emphasizes these aspects of bioprinting and presents them in perspective of differential requirements of different pharmaceutical studies like in vitro predictive toxicology, high-throughput screening, drug delivery and tissue-specific efficacies. Moreover, since bioprinting techniques are mostly applied in regenerative medicine and tissue engineering, a comparative analysis of similarities and differences are also expounded to help researchers make informed decisions based on contemporary literature.
View details for DOI 10.1016/j.actbio.2017.05.025
View details for Web of Science ID 000405041900002
View details for PubMedID 28501712
Bioprinting is a promising technology to fabricate design-specific tissue constructs due to its ability to create complex, heterocellular structures with anatomical precision. Bioprinting enables the deposition of various biologics including growth factors, cells, genes, neo-tissues and extra-cellular matrix-like hydrogels. Benefits of bioprinting have started to make a mark in the fields of tissue engineering, regenerative medicine and pharmaceutics. Specifically, in the field of tissue engineering, the creation of vascularized tissue constructs has remained a principal challenge till date. However, given the myriad advantages over other biofabrication methods, it becomes organic to expect that bioprinting can provide a viable solution for the vascularization problem, and facilitate the clinical translation of tissue engineered constructs. This article provides a comprehensive account of bioprinting of vascular and vascularized tissue constructs. The review is structured as introducing the scope of bioprinting in tissue engineering applications, key vascular anatomical features and then a thorough coverage of 3D bioprinting using extrusion-, droplet- and laser-based bioprinting for fabrication of vascular tissue constructs. The review then provides the reader with the use of bioprinting for obtaining thick vascularized tissues using sacrificial bioink materials. Current challenges are discussed, a comparative evaluation of different bioprinting modalities is presented and future prospects are provided to the reader.Biofabrication of living tissues and organs at the clinically-relevant volumes vitally depends on the integration of vascular network. Despite the great progress in traditional biofabrication approaches, building perfusable hierarchical vascular network is a major challenge. Bioprinting is an emerging technology to fabricate design-specific tissue constructs due to its ability to create complex, heterocellular structures with anatomical precision, which holds a great promise in fabrication of vascular or vascularized tissues for transplantation use. Although a great progress has recently been made on building perfusable tissues and branched vascular network, a comprehensive review on the state-of-the-art in vascular and vascularized tissue bioprinting has not reported so far. This contribution is thus significant because it discusses the use of three major bioprinting modalities in vascular tissue biofabrication for the first time in the literature and compares their strengths and limitations in details. Moreover, the use of scaffold-based and scaffold-free bioprinting is expounded within the domain of vascular tissue fabrication.
View details for DOI 10.1016/j.actbio.2017.01.035
View details for Web of Science ID 000398014000001
View details for PubMedID 28087487
Micro- to nanosized droplets of liquid metals, such as eutectic gallium indium (EGaIn) and Galinstan, have been used for developing a variety of applications in flexible electronics, sensors, catalysts, and drug delivery systems. Currently used methods for producing micro- to nanosized droplets of such liquid metals possess one or several drawbacks, including the lack in ability to control the size of the produced droplets, mass produce droplets, produce smaller droplet sizes, and miniaturize the system. Here, a novel method is introduced using acoustic wave-induced forces for on-chip production of EGaIn liquid-metal microdroplets with controllable size. The size distribution of liquid metal microdroplets is tuned by controlling the interfacial tension of the metal using either electrochemistry or electrocapillarity in the acoustic field. The developed platform is then used for heavy metal ion detection utilizing the produced liquid metal microdroplets as the working electrode. It is also demonstrated that a significant enhancement of the sensing performance is achieved by introducing acoustic streaming during the electrochemical experiments. The demonstrated technique can be used for developing liquid-metal-based systems for a wide range of applications.
View details for DOI 10.1002/smll.201600737
View details for Web of Science ID 000383375100015
View details for PubMedID 27309129
View details for PubMedCentralID PMC6311111