A brief review of extrusion‐based tissue scaffold bio‐printing

Extrusion‐based bio‐printing has great potential as a technique for manipulating biomaterials and living cells to create three‐dimensional (3D) scaffolds for damaged tissue repair and function restoration. Over the last two decades, advances in both engineering techniques and life sciences have evolved extrusion‐based bio‐printing from a simple technique to one able to create diverse tissue scaffolds from a wide range of biomaterials and cell types. However, the complexities associated with synthesis of materials for bio‐printing and manipulation of multiple materials and cells in bio‐printing pose many challenges for scaffold fabrication. This paper presents an overview of extrusion‐based bio‐printing for scaffold fabrication, focusing on the prior‐printing considerations (such as scaffold design and materials/cell synthesis), working principles, comparison to other techniques, and to‐date achievements. This paper also briefly reviews the recent development of strategies with regard to hydrogel synthesis, multi‐materials/cells manipulation, and process‐induced cell damage in extrusion‐based bio‐printing. The key issue and challenges for extrusion‐based bio‐printing are also identified and discussed along with recommendations for future, aimed at developing novel biomaterials and bio‐printing systems, creating patterned vascular networks within scaffolds, and preserving the cell viability and functions in scaffold bio‐printing. The address of these challenges will significantly enhance the capability of extrusion‐based bio‐printing.     

Supercritical fluid-assisted controllable fabrication of open and highly interconnected porous scaffolds for bone tissue engineering

Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional(3 D)scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we demonstrated the fabrication of porous polycaprolactone(PCL) scaffolds for osteogenic differentiation based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. This eco-friendly process resulted in the highly porous biomimetic scaffolds with open and interconnected architectures. Initially, a 2~3 factorial experiment was designed for investigating the relative significance of various processing parameters and achieving better control over the porosity as well as the compressive mechanical properties of the scaffold. Then, single factor experiment was carried out to understand the effects of various processing parameters on the morphology of scaffolds. On the other hand, we encapsulated a growth factor, i.e., bone morphogenic protein-2(BMP-2), as a model protein in these porous scaffolds for evaluating their osteogenic differentiation. In vitro investigations of growth factor loaded PCL scaffolds using bone marrow stromal cells(BMSCs) have shown that these growth factor-encumbered scaffolds were capable of differentiating the cells over the control experiments. Furthermore, the osteogenic differentiation was confirmed by measuring the cell proliferation, and alkaline phosphatase(ALP) activity, which were significantly higher demonstrating the active bone growth. Together, these results have suggested that the fabrication of growth factor-loaded porous scaffolds prepared by the eco-friendly hybrid processing efficiently promoted the osteogenic differentiation and may have a significant potential in bone tissue engineering.     

Application of polyethyleneimine‐modified scaffolds to the regeneration of cartilaginous tissue

In this study, we analyzed the physicochemical and biophysical properties of three‐dimensional scaffolds modified using polyethyleneimine (PEI) and applied these scaffolds to the cultivation of bovine knee chondrocytes (BKCs). PEI was crosslinked in the bulk or on the surface of the ternary scaffolds comprising polyethylene oxide, chitin and chitosan. The results revealed that when the concentration of PEI was less than 300 μg/mL, the cytotoxicity of a scaffold was on the same order in the two method of modification. An increase in the concentration of PEI favored the adhesion of BKCs. When the amount of PEI in scaffolds is fixed, the surface‐modified scaffolds exhibited a higher adhesion efficiency of BKCs than the bulk‐modified scaffolds. For the regeneration of cartilaginous components, a higher amount of PEI in a scaffold yielded larger amounts of proliferated BKCs, secreted glycosaminoglycans, and produced collagen. In addition, the formation of neocartilage in the surface‐modified scaffolds was more effective than that in the bulk‐modified scaffolds. These tissue‐engineered scaffolds, modified by an appropriate concentration of PEI, can be potentially applied to cartilage repair in clinical trials. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Engineering of vascularized adipose constructs

Adipose tissue engineering offers a promising alternative to the current surgical techniques for the treatment of soft tissue defects. It is a challenge to find the appropriate scaffold that not only represents a suitable environment for cells but also allows fabrication of customized tissue constructs, particularly in breast surgery. We investigated two different scaffolds for their potential use in adipose tissue regeneration. Sponge-like polyurethane scaffolds were prepared by mold casting with methylal as foaming agent, whereas polycaprolactone scaffolds with highly regular stacked-fiber architecture were fabricated with fused deposition modeling. Both scaffold types were seeded with human adipose tissue-derived precursor cells, cultured and implanted in nude mice using a femoral arteriovenous flow-through vessel loop for angiogenesis. In vitro, cells attached to both scaffolds and differentiated into adipocytes. In vivo, angiogenesis and adipose tissue formation were observed throughout both constructs after 2 and 4?weeks, with angiogenesis being comparable in seeded and unseeded constructs. Fibrous tissue formation and adipogenesis were more pronounced on polyurethane foam scaffolds than on polycaprolactone prototyped scaffolds. In conclusion, both scaffold designs can be effectively used for adipose tissue engineering.     

A porous medium‐chain‐length poly(3‐hydroxyalkanoates)/hydroxyapatite composite as scaffold for bone tissue engineering

Polyhydroxyalkanoates (PHA) are hydrophobic biopolymers with huge potential for biomedical applications due to their biocompatibility, excellent mechanical properties and biodegradability. A porous composite scaffold made of medium‐chain‐length poly(3‐hydroxyalkanoates) (mcl‐PHA) and hydroxyapatite (HA) was fabricated using particulate leaching technique and NaCl as a porogen. Different percentages of HA loading was investigated that would support the growth of osteoblast cells. Ultrasonic irradiation was applied to facilitate the dispersion of HA particles into the mcl‐PHA matrix. The different P(3HO‐co‐3HHX)/HA composites were investigated using field emission scanning electron microscopy (FESEM), X‐ray diffraction (XRD) and energy dispersive X‐ray analysis (EDXA). The scaffolds were found to be highly porous with interconnecting pore structures and the HA particles were homogeneously dispersed in the polymer matrix. The scaffolds biocompatibility and osteoconductivity were also assessed following the proliferation and differentiation of osteoblast cells on the scaffolds. From the results, it is clear that scaffolds made from P(3HO‐co‐3HHX)/HA composites are viable candidate materials for bone tissue engineering applications.     

On‐demand three‐dimensional freeform fabrication of multi‐layered hydrogel scaffold with fluidic channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186. © 2009 Wiley Periodicals, Inc.     

Controlled release of neurotrophin‐3 from fibrin‐based tissue engineering scaffolds enhances neural fiber sprouting following subacute spinal cord injury

This study investigated whether delayed treatment of spinal cord injury with controlled release of neurotrophin‐3 (NT‐3) from fibrin scaffolds can stimulate enhanced neural fiber sprouting. Long Evans rats received a T9 dorsal hemisection spinal cord injury. Two weeks later, the injury site was re‐exposed, and either a fibrin scaffold alone, a fibrin scaffold containing a heparin‐based delivery system with different concentrations of NT‐3 (500 and 1,000 ng/mL), or a fibrin scaffold containing 1,000 ng/mL of NT‐3 (no delivery system) was implanted into the injury site. The injured spinal cords were evaluated for morphological differences using markers for neurons, astrocytes, and chondroitin sulfate proteoglycans 2 weeks after treatment. The addition of 500 ng/mL of NT‐3 with the delivery system resulted in an increase in neural fiber density compared to fibrin alone. These results demonstrate that the controlled release of NT‐3 from fibrin scaffolds can enhance neural fiber sprouting even when treatment is delayed 2 weeks following injury. Biotechnol. Bioeng. 2009; 104: 1207–1214. © 2009 Wiley Periodicals, Inc.     

Interpreting CARS images of tissue within the C–H‐stretching region

Single band coherent anti‐Stokes Raman scattering (CARS) microscopy is one of the fastest implementation of nonlinear vibrational imaging allowing for video‐rate image acquisition of tissue. This is due to the large Raman signal in the C—H‐stretching region. However, the chemical specificity of such images is conventionally assumed to be low. Nonetheless, CARS imaging within the C—H‐stretching region enables detection of single cells and nuclei, which allows for histopathologic grading of tissue. Relevant information such as nucleus to cytoplasm ratio, cell density, nucleus size and shape is extracted from CARS images by innovative image processing procedures. In this contribution CARS image contrast within the C—H‐stretching region is interpreted by direct comparison with Raman imaging and correlated to the tissue composition justifying the use of CARS imaging in this wavenumber region for biomedical applications. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Fabrication techniques of biomimetic scaffolds in three‐dimensional cell culture: A review

In the last four decades, several researchers worldwide have routinely and meticulously exercised cell culture experiments in two‐dimensional (2D) platforms. Using traditionally existing 2D models, the therapeutic efficacy of drugs has been inappropriately validated due to the failure in generating the precise therapeutic response. Fortunately, a 3D model addresses the foregoing limitations by recapitulating the in vivo environment. In this context, one has to contemplate the design of an appropriate scaffold for favoring the organization of cell microenvironment. Instituting pertinent model on the platter will pave way for a precise mimicking of in vivo conditions. It is because animal cells in scaffolds oblige spontaneous formation of 3D colonies that molecularly, phenotypically, and histologically resemble the native environment. The 3D culture provides insight into the biochemical aspects of cell–cell communication, plasticity, cell division, cytoskeletal reorganization, signaling mechanisms, differentiation, and cell death. Focusing on these criteria, this paper discusses in detail, the diversification of polymeric scaffolds based on their available resources. The paper also reviews the well‐founded and latest techniques of scaffold fabrication, and their applications pertaining to tissue engineering, drug screening, and tumor model development.     

Exploring cellular adhesion and differentiation in a micro‐/nano‐hybrid polymer scaffold

Polymer scaffolds play an important role in three dimensional (3‐D) cell culture and tissue engineering. To best mimic the archiecture of natural extracellular matrix (ECM), a nano‐fibrous and micro‐porous combined (NFMP) scaffold was fabricated by combining phase separation and particulate leaching techniques. The NFMP scaffold possesses architectural features at two levels, including the micro‐scale pores and nano‐scale fibers. To evaluate the advantages of micro/nano combination, control scaffolds with only micro‐pores or nano‐fibers were fabricated. Cell grown in NFMP and control scaffolds were characterized with respect to morphology, proliferation rate, diffentiation and adhesion. The NFMP scaffold combined the advantages of micro‐ and nano‐scale structures. The NFMP scaffold nano‐fibers promoted neural differentiation and induced “3‐D matrix adhesion”, while the NFMP scaffold micro‐pores facilitated cell infiltration. This study represents a systematic comparison of cellular activities on micro‐only, nano‐only and micro/nano combined scaffolds, and demonstrates the unique advantages of the later. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Efficient Plastic Perovskite Solar Cell with a Low‐Temperature Processable Electrodeposited TiO2 Compact Layer and a Brookite TiO2 Scaffold

Recent research on fabricating scaffold‐type perovskite solar cells on plastic substrates has reported noteworthy progress in replacing the high‐temperature processing of TiO₂ scaffolds and compact layers with various low‐temperature processes. Herein, recent progress in the laboratory is reported regarding the development of electrodeposited TiO_x compact layers and brookite TiO₂ scaffolds, both of which can be processed under 150 °C without greatly sacrificing their photovoltaic performance. Through systematic characterization of device properties and careful optimization of the fabrication conditions, a record‐high 15.76% power conversion efficiency of a plastic TiO₂ scaffold‐type perovskite solar cell is demonstrated. In addition, bending durability and preliminary stability tests on this plastic perovskite solar cell show promising results and indicate clear directions for future improvement.     

Processing/structure/property relationship of multi-scaled PCL and PCL-HA composite scaffolds prepared via gas foaming and NaCl reverse templating

In this study, we investigated the processing/structure/property relationship of multi-scaled porous biodegradable scaffolds prepared by combining the gas foaming and NaCl reverse templating techniques. Poly(ε-caprolactone) (PCL), hydroxyapatite (HA) nano-particles and NaCl micro-particles were melt-mixed by selecting different compositions and subsequently gas foamed by a pressure-quench method. The NaCl micro-particles were finally removed from the foamed systems in order to allow for the achievement of the multi-scaled scaffold pore structure. The control of the micro-structural properties of the scaffolds was obtained by the optimal combination of the NaCl templating concentration and the composition of the CO2-N2 mixture as the blowing agent. In particular, these parameters were accurately selected to allow for the fabrication of PCL and PCL-HA composite scaffolds with multi-scaled open pore structures. Finally, the biocompatibility of the scaffolds has been assessed by cultivating pre-osteoblast MG63 cells in vitro, thus demonstrating their potential applications for bone regeneration.     

The dynamics of surface acoustic wave‐driven scaffold cell seeding

Flow visualization using fluorescent microparticles and cell viability investigations are carried out to examine the mechanisms by which cells are seeded into scaffolds driven by surface acoustic waves. The former consists of observing both the external flow prior to the entry of the suspension into the scaffold and the internal flow within the scaffold pores. The latter involves micro‐CT (computed tomography) scans of the particle distributions within the seeded scaffolds and visual and quantitative methods to examine the morphology and proliferation ability of the irradiated cells. The results of these investigations elucidate the mechanisms by which particles are seeded, and hence provide valuable information that form the basis for optimizing this recently discovered method for rapid, efficient, and uniform scaffold cell seeding. Yeast cells are observed to maintain their size and morphology as well as their proliferation ability over 14 days after they are irradiated. The mammalian primary osteoblast cells tested also show little difference in their viability when exposed to the surface acoustic wave irradiation compared to a control set. Together, these provide initial feasibility results that demonstrate the surface acoustic wave technology as a viable seeding method without risk of denaturing the cells. Biotechnol. Bioeng. 2009;103: 387–401. © 2009 Wiley Periodicals, Inc.     

Biofabrication of antibodies and antigens via IgG‐binding domain engineered with activatable pentatyrosine pro‐tag

We report the assembly of seven different antibodies (and two antigens) into functional supramolecular structures that are specifically designed to facilitate integration into devices using entirely biologically based bottom‐up fabrication. This is enabled by the creation of an engineered IgG‐binding domain (HG3T) with an N‐terminal hexahistidine tag that facilitates purification and a C‐terminal enzyme‐activatable pentatyrosine “pro‐tag” that facilitates covalent coupling to the pH stimuli‐responsive polysaccharide, chitosan. Because we confer pH‐stimuli responsiveness to the IgG‐binding domain, it can be electrodeposited or otherwise assembled into many configurations. Importantly, we demonstrate the loading of both HG3T and antibodies can be achieved in a linear fashion so that quantitative assessment of antibodies and antigens is feasible. Our demonstration formats include: conventional multiwell plates, micropatterned electrodes, and fiber networks. We believe biologically based fabrication (i.e., biofabrication) provides bottom‐up hierarchical assembly of a variety of nanoscale components for applications that range from point‐of‐care diagnostics to smart fabrics. Biotechnol. Bioeng. 2009;103: 231–240. © 2008 Wiley Periodicals, Inc.     

High‐speed X‐ray‐induced luminescence computed tomography

X‐ray‐induced luminescence computed tomography (XLCT) is an emerging molecular imaging. Challenges in improving spatial resolution and reducing the scan time in a whole‐body field of view (FOV) still remain for practical in vivo applications. In this study, we present a novel XLCT technique capable of obtaining three‐dimensional (3D) images from a single snapshot. Specifically, a customed two‐planar‐mirror component is integrated into a cone beam XLCT imaging system to obtain multiple optical views of an object simultaneously. Furthermore, a compressive sensing based algorithm is adopted to improve the efficiency of 3D XLCT image reconstruction. Numerical simulations and experiments were conducted to validate the single snapshot X‐ray‐induced luminescence computed tomography (SS‐XLCT). The results show that the 3D distribution of the nanophosphor targets can be visualized much faster than conventional cone beam XLCT imaging method that was used in our comparisons while maintaining comparable spatial resolution as in conventional XLCT imaging. SS‐XLCT has the potential to harness the power of XLCT for rapid whole‐body in vivo molecular imaging of small animals.     

Regulation of autocrine fibroblast growth factor‐2 signaling by perfusion flow in 3D human mesenchymal stem cell constructs

Perfusion bioreactor systems play a crucial role in mitigating nutrient limitation as well as providing biomechanical stimuli and redistributing regulatory macromolecules that influence human mesenchymal stem cells (hMSC) fate in three‐dimensional (3D) scaffolds. As fibroblast growth factor‐2 (FGF‐2) is known to regulate hMSC phenotype, understanding the role of autocrine FGF‐2 signaling in the 3D construct under the different perfusion flow provides important insight into an optimal bioreactor design. To investigate FGF‐2 signaling inhibition in hMSC cultured in the porous poly(ethylene terephthalate) (PET) scaffolds perfused under two flow configurations, PD173074, an FGFR1 inhibitor, was added in growth media after 7 day of pre‐culture and its impact on hMSC proliferation and clonogenicity during the subsequent 7 days of cultivation was analyzed. Compared with control constructs in growth media, the addition of PD173074 resulted in significant reduction in hMSC proliferation and colony formation in both constructs with a more dramatic reduction in the parallel flow constructs. The results demonstrate that autocrine FGF‐2 plays a significant role in 3D scaffold and suggest modulation of the perfusion flow in the bioreactor as a strategy to influence autocrine actions and cell fate in the 3D scaffold. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Unsupervised organization of cervical cells using bright‐field and single‐shot digital holographic microscopy

We report results on unsupervised organization of cervical cells using microscopy of Pap‐smear samples in brightfield (3‐channel color) as well as high‐resolution quantitative phase imaging modalities. A number of morphological parameters are measured for each of the 1450 cell nuclei (from 10 woman subjects) imaged in this study. The principal component analysis (PCA) methodology applied to this data shows that the cell image clustering performance improves significantly when brightfield as well as phase information is utilized for PCA as compared to when brightfield‐only information is used. The results point to the feasibility of an image‐based tool that will be able to mark suspicious cells for further examination by the pathologist. More importantly, our results suggest that the information in quantitative phase images of cells that is typically not used in clinical practice is valuable for automated cell classification applications in general.      

Real‐time imaging of single cancer‐cell dynamics of lung metastasis

We have developed a new in vivo mouse model to image single cancer‐cell dynamics of metastasis to the lung in real‐time. Regulating airflow volume with a novel endotracheal intubation method enabled controlling lung expansion adequate for imaging of the exposed lung surface. Cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected in the tail vein of the mouse. The right chest wall was then opened in order to image metastases on the lung surface directly. After each observation, the chest wall was sutured and the air was suctioned in order to re‐inflate the lung, in order to keep the mice alive. Observations have been carried out for up to 8 h per session and repeated up to six times per mouse thus far. The seeding and arresting of single cancer cells on the lung, accumulation of cancer‐cell emboli, cancer‐cell viability, and metastatic colony formation were imaged in real‐time. This new technology makes it possible to observe real‐time monitoring of cancer‐cell dynamics of metastasis in the lung and to identify potential metastatic stem cells. J. Cell. Biochem. 109: 58–64, 2010. © 2009 Wiley‐Liss, Inc.     

Quantitative phase microscopy with enhanced contrast and improved resolution through ultra‐oblique illumination (UO‐QPM)

Recent developments in phase contrast microscopy have enabled the label‐free visualization of certain organelles due to their distinct morphological features, making this method an attractive alternative in the study of cellular dynamics. However tubular structures such as endoplasmic reticulum (ER) networks and complex dynamics such as the fusion and fission of mitochondria, due to their low phase contrast, still need fluorescent labeling to be adequately imaged. In this article, we report a quantitative phase microscope with ultra‐oblique illumination that enables us to see those structures and their dynamics with high contrast for the first time without labeling. The imaging capability was validated through comparison to the fluorescence images with the same field‐of‐view. The high image resolution (~270 nm) was validated using both beads and cellular structures. Furthermore, we were able to record the vibration of ER networks at a frame rate of 250 Hz. We additionally show complex cellular processes such as remodeling of the mitochondria networks through fusion and fission and vesicle transportation along the ER without labels. Our high spatial and temporal resolution allowed us to observe mitochondria “spinning”, which has not been reported before, further demonstrating the advantages of the proposed method.