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PURPOSE: The purpose of this study was to evaluate porcine bone marrow-derived progenitor cell (pBMPC) proliferation and penetration into a novel 3-dimensionally printed scaffold. MATERIALS AND METHODS: Four different tissue engineering scaffolds to evaluate pBMPC proliferation and penetration were examined. Scaffolds were fabricated from polycaprolactone (PCL) or the combination of [beta]-tricalcium phosphate ([beta]-TCP) and PCL (50:50), with 2 separate channel sizes (1 mm [small (S)] vs 2 mm [large (L)]). Scaffolds were fabricated into 20 x 20 x 7-mm blocks by use of a TheriForm machine (Integra Life Sciences, Akron, OH). Four groups of scaffolds were examined for pBMPC proliferation and penetration: group 1, [beta]-TCP/PCL S; group 2, [beta]-TCP/PCL L; group 3, PCL S; and group 4, PCL L. Nonparametric mean (Kruskal-Wallis) and multiple comparisons tests were used to compare the 4 groups. RESULTS: No shrinkage or deformation was noted in any of the scaffold groups after 2 weeks of culture. Mean surface cell counts ranged from 13.4 to 87.8 cells/0.57 mm(2), with group 1 ([beta]-TCP/PCL S) having statistically significantly higher counts than the other groups (P < .001). Mean interior cell counts ranged from 10.9 to 75.6 cells/0.57 mm(2), with group 1 having the greatest interior cell count (P < .001). Total collagen formation ranged from 0.2% to 86%, with group 1 having the highest collagen formation (P < .001). CONCLUSIONS: The 3-dimensionally printed scaffold ([beta]-TCP/PCL) with 1-mm channels showed greater cellular proliferation, penetration, and collagen formation after a 2-week in vitro culture than the other scaffolds evaluated. [beta]-TCP/PCL S scaffolds warrant further evaluation for bone tissue engineering in vivo. [ABSTRACT FROM AUTHOR]

PURPOSE: Nonsteroidal anti-inflammatory drugs are commonly prescribed to reduce inflammation and pain. However, little is known about the direct effect of these drugs on the differentiation of bone marrow-derived progenitor cells into osteoblasts. The purpose of this study was to determine the effect of ibuprofen on osteoblast differentiation and proliferation in a minipig model. MATERIALS AND METHODS: Bone marrow was aspirated from the minipig ilium, and porcine bone marrow-derived progenitor cells (pBMPCs) were isolated and expanded in standard culture medium. The pBMPCs were replated and differentiated into osteoblasts by use of osteogenic supplements (OS). Five groups were studied: negative control-pBMPCs in standard medium only; positive control-pBMPCs, standard culture medium, and OS; and 3 experimental groups-pBMPCs, standard culture medium, OS, and ibuprofen added in doses of 0.1, 1.0, and 3.0 mmol/L. Cell cultures were evaluated quantitatively by alkaline phosphatase (ALP) stain, von Kossa stain, and deoxyribonucleic acid (DNA) content. RESULTS: pBMPCs cultured with OS and low-dose ibuprofen (0.1 mmol/L) showed ALP stain, von Kossa stain, and DNA content similar to pBMPCs cultured in OS (positive control). pBMPCs cultured in higher doses of ibuprofen (1.0 and 3.0 mmol/L) produced significantly less positive staining of ALP and von Kossa and decreased DNA content. CONCLUSION: The results indicate that high-dose ibuprofen has a deleterious effect on pBMPC differentiation into osteoblasts whereas low-dose ibuprofen does not. The low dose of 0.1 mmol/L is the typical serum level when prescribed for clinical use. [ABSTRACT FROM AUTHOR]

This study presents the design, fabrication, and evaluation of a general platform for the creation of three-dimensional printed devices (3DPDs) for tissue engineering applications. As a demonstration, we modeled the liver with 3DPDs consisting of a pair of parallel millifluidic channels that function as portal-venous (PV) and hepatobiliary (HB) structures. Perfusion of medium or whole blood through the PV channel supports the hepatocyte-containing HB channel. Device computer-aided design was optimized for structural stability, after which 3DPDs were 3D printed in a polyethylene(glycol) diacrylate photoink by digital light processing and evaluated in vitro. The HB channels were subsequently seeded with hepatic cells suspended in a collagen hydrogel. Perfusion of 3DPDs in bioreactors enhanced the viability and function of rat hepatoma cells and were maintained over time, along with improved liver-specific functions. Similar results were observed with primary rat hepatocytes, including significant upregulation of cytochrome p450 activity. Additionally, coculture experiments involving primary rat hepatocytes, endothelial cells, and mesenchymal stem cells in 3DPDs showed enhanced viability, broad liver-specific gene expression, and histological features indicative of liver tissue architecture. In vivo implantation of 3DPDs in a rat renal shunt model demonstrated successful blood flow through the devices without clot formation and maintenance of cell viability. 3D printed designs can be scaled in 3D space, allowing for larger devices with increased cell mass. Overall, these findings highlight the potential of 3DPDs for clinical translation in hepatic support applications., Competing Interests: Competing interests statement:J.P.V. equity in 3D BioLabs, LLC. T.J.L., T.S., M.J.H., A.P.S., M.S.O., S.C.G., C.M.N., and J.P.V. hold patents and patent applications related to content in this work.

This study presents a novel surgical model developed to provide hematological support for implanted cellularized devices augmenting or replacing liver tissue function. Advances in bioengineering provide tools and materials to create living tissue replacements designed to restore that lost to disease, trauma, or congenital deformity. Such substitutes are often assembled and matured in vitro and need an immediate blood supply upon implantation, necessitating the development of supporting protocols. Animal translational models are required for continued development of engineered structures before clinical implementation, with rodent models often playing an essential early role. Our long-term goal has been generation of living tissue to provide liver function, utilizing advances in additive manufacturing technology to create 3D structures with intrinsic micron to millimeter scale channels modeled on natural vasculature. The surgical protocol developed enables testing various design iterations in vivo by anastomosis to the host rat vasculature. Lobation of rodent liver facilitates partial hepatectomy and repurposing the remaining vasculature to support implanted engineered tissue. Removal of the left lateral lobe exposes the underlying hepatic vasculature and can create space for a device. A shunt is created from the left portal vein to the left hepatic vein by cannulating each with separate silicone tubing. The device is then integrated into the shunt by connecting its inflow and outflow ports to the tubing and reestablishing blood flow. Sustained anticoagulation is maintained with an implanted osmotic pump. In our studies, animals were freely mobile after implantation; devices remained patent while maintaining blood flow through their millifluidic channels. This vascular anastomosis model has been greatly refined during the process of performing over 200 implantation procedures. We anticipate that the model described herein will find utility in developing preclinical translational protocols for evaluation of engineered liver tissue. Impact statement Tissue and organ transplantation are often the best clinically effective treatments for a variety of human ailments. However, the availability of suitable donor organs remains a critical problem. Advances in biotechnology hold potential in alleviating shortages, yet further work is required to surgically integrate large engineered tissues to host vasculature. Improved animal models such as the one described are valuable tools to support continued development and evaluation of novel therapies.

The field of Tissue Engineering and Regenerative Medicine has evolved rapidly over the past thirty years. This review will summarize its history, current status and direction through the lens of clinical need, its progress through science in the laboratory and application back into patients. We can take pride in the fact that much effort and progress began with the surgical problems of children and that many surgeons in the pediatric surgical specialties have become pioneers and investigators in this new field of science, engineering, and medicine. Although the field has yet to fulfill its great promise, there have been several examples where a therapy has progressed from the first idea to human application within a short span of time and, in many cases, it has been applied in the surgical care of children.

Extracellular matrix materials mechanically dissociated into submillimeter particles have a larger surface area than sheet materials and enhanced cellular attachment. Decellularized porcine mesothelial extracellular matrix microparticles were seeded with bone marrow-derived mesenchymal stromal cells and cultured in a rotating bioreactor. The mesenchymal stromal cells attached and grew to confluency on the microparticles. The cell-seeded microparticles were then encapsulated in varying concentrations of fibrin glue, and the cells migrated rapidly off the microparticles. The combination of microparticles and mesenchymal stromal cells was then applied to a splinted full-thickness cutaneous in vivo wound model. There was evidence of increased cell infiltration and collagen deposition in mesenchymal stromal cells-treated wounds. Cell-seeded microparticles have potential as a cell delivery and paracrine therapy in impaired healing environments.

Objectives: Creation of functional, durable vasculature remains an important goal within the field of regenerative medicine. Engineered biological vasculature has the potential to restore or improve human tissue function. We hypothesized that the pleotropic effects of insulin-like growth factor 1 (IGF1) would enhance the engineering of capillary-like vasculature., Materials and Methods: The impact of IGF1 upon vasculogenesis was examined in in vitro cultures for a period of up to 40 days and as subcutaneous implants within immunodeficient mice. Co-cultures of human umbilical vein endothelial cells and human bone marrow-derived mesenchymal stem cells in collagen-fibronectin hydrogels were supplemented with either recombinant IGF1 protein or genetically engineered cells to provide sustained IGF1. Morphometric analysis was performed on the vascular networks that formed in four concentrations of IGF1., Results: IGF1 supplementation significantly enhanced de novo vasculogenesis both in vitro and in vivo. Effects were long-term as they lasted the duration of the study period, and included network density, vessel length, and diameter. Bifurcation density was not affected. However, the highest concentrations of IGF1 tested were either ineffective or even deleterious. Sustained IGF1 delivery was required in vivo as the inclusion of recombinant IGF1 protein had minimal impact., Conclusion: IGF1 supplementation can be used to produce neovasculature with significantly enhanced network density and durability. Its use is a promising methodology for engineering de novo vasculature to support regeneration of functional tissue., (© 2017 John Wiley & Sons Ltd.)

Bone marrow-derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell-based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte-conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor-beta 3 (TGF-β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real-time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real-time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF-β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF-β3. Osteogenesis was only observed in the SM group with TGF-β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF-β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Decellularized extracellular matrix (ECM) biomaterials are increasingly used in regenerative medicine for abdominal tissue repair. Emerging ECM biomaterials with greater compliance target surgical procedures like breast and craniofacial reconstruction to enhance aesthetic outcome. Clinical studies report improved outcomes with newly designed ECM scaffolds, but their comparative biological characteristics have received less attention. In this study, we investigated scaffolds derived from dermis (AlloDerm Regenerative Tissue Matrix), small intestinal submucosa (Surgisis 4-layer Tissue Graft and OASIS Wound Matrix), and mesothelium (Meso BioMatrix Surgical Mesh and Veritas Collagen Matrix) and evaluated biological properties that modulate cellular responses and recruitment. An assay panel was utilized to assess the ECM scaffold effects upon cells. Results of the material-conditioned media study demonstrated Meso BioMatrix and OASIS best supported cell proliferation. Meso BioMatrix promoted the greatest migration and chemotaxis signaling, followed by Veritas and OASIS; OASIS had superior suppression of cell apoptosis. The direct adhesion assay indicated that AlloDerm, Meso BioMatrix, Surgisis, and Veritas had sidedness that affected cell-material interactions. In the chick chorioallantoic membrane assay, Meso BioMatrix and OASIS best supported cell infiltration. Among tested materials, Meso BioMatrix and OASIS demonstrated characteristics that facilitate scaffold incorporation, making them promising choices for many clinical applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 585-593, 2017., (© 2015 Wiley Periodicals, Inc.)

In pre-clinical safety studies, drug-induced vascular injury (DIVI) is defined as an adverse response to a drug characterized by degenerative and hyperplastic changes of endothelial cells and vascular smooth muscle cells. Inflammation may also be seen, along with extravasation of red blood cells into the smooth muscle layer (i.e., hemorrhage). Drugs that cause DIVI are often discontinued from development after considerable cost has occurred. An in vitro vascular model has been developed using endothelial and smooth muscle cells in co-culture across a porous membrane mimicking the internal elastic lamina. Arterial flow rates of perfusion media within the endothelial chamber of the model induce physiologic endothelial cell alignment. Pilot testing with a drug known to cause DIVI induced extravasation of red blood cells into the smooth muscle layer in all devices with no extravasation seen in control devices. This engineered vascular model offers the potential to evaluate candidate drugs for DIVI early in the discovery process. The physiologic flow within the co-culture model also makes it candidate for a wide variety of vascular biology investigations.

Extracellular matrix (ECM) materials from animal and human sources have become important materials for soft tissue repair. Microparticles of ECM materials have increased surface area and exposed binding sites compared to sheet materials. Decellularized porcine peritoneum was mechanically dissociated into 200 µm microparticles, seeded with fibroblasts and cultured in a low gravity rotating bioreactor. The cells avidly attached and maintained excellent viability on the microparticles. When the seeded microparticles were placed in a collagen gel, the cells quickly migrated off the microparticles and through the gel. Cells from seeded microparticles migrated to and across an in vitro anastomosis model, increasing the tensile strength of the model. Cell seeded microparticles of ECM material have potential for paracrine and cellular delivery therapies when delivered in a gel carrier. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1728-1735, 2016., (© 2016 Wiley Periodicals, Inc.)

Background Aims: The use of bone marrow-derived mesenchymal stromal cells (MSCs) in cell-based therapies is currently being developed for a number of diseases. Thus far, the clinical results have been inconclusive and variable, in part because of the variety of cell isolation procedures and culture conditions used in each study. A new isolation technique that streamlines the method of concentration and demands less time and attention could provide clinical and economic advantages compared with current methodologies. In this study, we evaluated the concentrating capability of an integrated centrifuge-based technology compared with standard Ficoll isolation., Methods: MSCs were concentrated from bone marrow aspirate using the new device and the Ficoll method. The isolation capabilities of the device and the growth characteristics, secretome production, and differentiation capacity of the derived cells were determined., Results: The new MSC isolation device concentrated the bone marrow in 90 seconds and resulted in a mononuclear cell yield 10-fold higher and with a twofold increase in cell retention compared with Ficoll. The cells isolated using the device were shown to exhibit similar morphology and functional activity as assessed by growth curves and secretome production compared to the Ficoll-isolated cells. The surface marker and trilineage differentiation profile of the device-isolated cells was consistent with the known profile of MSCs., Discussion: The faster time to isolation and greater cell yield of the integrated centrifuge-based technology may make this an improved approach for MSC isolation from bone marrow aspirates., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Adequate biomaterials for tissue engineering bone and replacement of bone in clinical settings are still being developed. Previously, the combination of mesenchymal stem cells in hydrogels and calcium-based biomaterials in both in vitro and in vivo experiments has shown promising results. However, results may be optimized by careful selection of the material combination., Methods: β-Tricalcium phosphate scaffolds were three-dimensionally printed with five different hydrogels: collagen I, gelatin, fibrin glue, alginate, and Pluronic F-127. The scaffolds had eight channels, running throughout the entire scaffold, and macropores. Mesenchymal stem cells (2 × 10) were mixed with each hydrogel, and cell/hydrogel mixes were dispersed onto the corresponding β-tricalcium phosphate/hydrogel scaffold and cultured under dynamic-oscillating conditions for 6 weeks. Specimens were harvested at 1, 2, 4, and 6 weeks and evaluated histologically, radiologically, biomechanically and, at 6 weeks, for expression of bone-specific proteins by reverse-transcriptase polymerase chain reaction. Statistical correlation analysis was performed between radiologic densities in Hounsfield units and biomechanical stiffness., Results: Collagen I samples had superior bone formation at 6 weeks as demonstrated by volume computed tomographic scanning, with densities of 300 HU, similar to native bone, and the highest compression values. Bone specificity of new tissue was confirmed histologically and by the expression of alkaline phosphatase, osteonectin, osteopontin, and osteocalcin. The bone density correlated closely with histologic and biomechanical testing results., Conclusion: Bone formation is supported best by β-tricalcium phosphate/collagen I hydrogel and mesenchymal stem cells in collagen I hydrogel., Clinical Question/level of Evidence: Therapeutic, V.

Advancement of engineered ear in clinical practice is limited by several challenges. The complex, largely unsupported, three-dimensional auricular neocartilage structure is difficult to maintain. Neocartilage formation is challenging in an immunocompetent host due to active inflammatory and immunological responses. The large number of autologous chondrogenic cells required for engineering an adult human-sized ear presents an additional challenge because primary chondrocytes rapidly dedifferentiate during in vitro culture. The objective of this study was to engineer a stable, human ear-shaped cartilage in an immunocompetent animal model using expanded chondrocytes. The impact of basic fibroblast growth factor (bFGF) supplementation on achieving clinically relevant expansion of primary sheep chondrocytes by in vitro culture was determined. Chondrocytes expanded in standard medium were either combined with cryopreserved, primary passage 0 chondrocytes at the time of scaffold seeding or used alone as control. Disk and human ear-shaped scaffolds were made from porous collagen; ear scaffolds had an embedded, supporting titanium wire framework. Autologous chondrocyte-seeded scaffolds were implanted subcutaneously in sheep after 2 weeks of in vitro incubation. The quality of the resulting neocartilage and its stability and retention of the original ear size and shape were evaluated at 6, 12, and 20 weeks postimplantation. Neocartilage produced from chondrocytes that were expanded in the presence of bFGF was superior, and its quality improved with increased implantation time. In addition to characteristic morphological cartilage features, its glycosaminoglycan content was high and marked elastin fiber formation was present. The overall shape of engineered ears was preserved at 20 weeks postimplantation, and the dimensional changes did not exceed 10%. The wire frame within the engineered ear was able to withstand mechanical forces during wound healing and neocartilage maturation and prevented shrinkage and distortion. This is the first demonstration of a stable, ear-shaped elastic cartilage engineered from auricular chondrocytes that underwent clinical-scale expansion in an immunocompetent animal over an extended period of time.

One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

Chronic lower respiratory disease is highly prevalent in the United States, and there remains a need for alternatives to lung transplant for patients who progress to end-stage lung disease. Portable or implantable gas oxygenators based on microfluidic technologies can address this need, provided they operate both efficiently and biocompatibly. Incorporating biomimetic materials into such devices can help replicate native gas exchange function and additionally support cellular components. In this work, we have developed microfluidic devices that enable blood gas exchange across ultra-thin collagen membranes (as thin as 2 μm). Endothelial, stromal, and parenchymal cells readily adhere to these membranes, and long-term culture with cellular components results in remodeling, reflected by reduced membrane thickness. Functionally, acellular collagen-membrane lung devices can mediate effective gas exchange up to ∼288 mL/min/m(2) of oxygen and ∼685 mL/min/m(2) of carbon dioxide, approaching the gas exchange efficiency noted in the native lung. Testing several configurations of lung devices to explore various physical parameters of the device design, we concluded that thinner membranes and longer gas exchange distances result in improved hemoglobin saturation and increases in pO2. However, in the design space tested, these effects are relatively small compared to the improvement in overall oxygen and carbon dioxide transfer by increasing the blood flow rate. Finally, devices cultured with endothelial and parenchymal cells achieved similar gas exchange rates compared with acellular devices. Biomimetic blood oxygenator design opens the possibility of creating portable or implantable microfluidic devices that achieve efficient gas transfer while also maintaining physiologic conditions.

Background: Carved autologous costal cartilage and porous polyethylene implants (Medpor) are the most common approaches for total ear reconstruction, but these approaches may have inconsistent cosmetic outcomes, a high risk of extrusion, or other surgical complications. Engineering ear cartilage to emulate native auricular tissue is an appealing approach, but often the cell-seeded scaffolds are susceptible to shrinkage and architectural changes when placed in vivo. The aim of this study was to assess the most favorable conditions for in vitro pre-culture of cell-seeded type I collagen scaffolds prior to in vivo implantation., Methods: Sheep auricular chondrocytes were seeded into this type I collagen scaffold. The cell-seeded constructs were cultured in either static or dynamic conditions for two days or two weeks and then implanted into nude mice for another six weeks. The harvested constructs were evaluated histologically, immunohistochemically, and biochemically., Results: Robust neo-cartilage formation was found in these collagen scaffolds seeded with auricular chondrocytes, which was comparable to native cartilage morphologically, histologically, and biochemically. Culture under dynamic conditions prior to implantation improved the neo-cartilage formation histologically and biochemically., Conclusion: Dynamic culture of this cell-seeded fibrous collagen material could permit predictable engineered auricular cartilage and a promising approach for external ear reconstruction., (Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Background: Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs)., Methods: Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs, which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position., Results: Activin-A treatment, followed by transforming growth factor-β inhibition, induced differentiation of human iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2), SRY (Sex Determining Region Y)-Box 17 (SOX17), and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after 5 days. Cells expressing Nkx2.1 were identified at 40% to 60% efficiency. Within whole-lung scaffolds and under perfusion culture, cells further upregulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated by host vasculature and airways., Conclusions: Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further in vivo graft maturation., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objective: Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice., Design: Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed., Results: Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks., Conclusions: Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

Mesenchymal bone marrow stromal cells may be a source of cells to preseed decellularized biologic mesh materials for improved cellularization and promote a more physiologic tissue after remodeling. Spontaneous differentiation of mesenchymal stromal cells on the decellularized material would be undesirable. Conversely, induced differentiation of mesenchymal stem cells (MSC) on the material would suggest that these materials may have promise as scaffold materials for bone, cartilage, or adipocyte formation. Two sources of mesenchymal cells were evaluated for induced differentiation in control wells. These MSCs were also evaluated for spontaneous or induced differentiation on decellularized porcine dermis and mesothelium materials. Primarily harvested bone marrow MSCs and commercially obtained MSCs were induced into osteoblasts and adipocytes on decellularized dermis and mesothelium materials. The MSCs were able to be induced into chondrocytes in pellet form but not when grown as a monolayer on the materials. The MSCs did not undergo spontaneous differentiation when grown on the materials for up to four weeks. MSC grown on decellularized porcine dermis or mesothelium do not spontaneously differentiate and may serve as a source of autologous cells for preseeding these extracellular matrix materials prior to implantation., (© 2013 Wiley Periodicals, Inc.)

Liver transplantation remains the only definitive treatment for liver failure and is available to only a tiny fraction of patients with end-stage liver diseases. Major limitations for the procedure include donor organ shortage, high cost, high level of required expertise, and long-term consequences of immune suppression. Alternative cell-based liver therapies could potentially greatly expand the number of patients provided with effective treatment. Investigative research into augmenting or replacing liver function extends into three general strategies. Bioartificial livers (BALs) are extracorporeal devices that utilize cartridges of primary hepatocytes or cell lines to process patient plasma. Injection of liver cell suspensions aims to foster organ regeneration or provide a missing metabolic function arising from a genetic defect. Tissue engineering recreates the organ in vitro for subsequent implantation to augment or replace patient liver function. Translational models and clinical trials have highlighted both the immense challenges involved and some striking examples of success., (Copyright © 2014. Published by Elsevier Inc.)

Lung tissue engineering is an emerging field focused on the development of lung replacement devices and tissue to treat patients with end stage lung disease. Microfluidic based lung assist devices have been developed that have biomimetically designed vascular networks that achieve physiologic blood flow. Gas exchange in these devices occurs across a thin respiratory membrane. Designed for intrathoracic implantation as a bridge to transplant or destination therapy, these lung assist devices will allow ambulation and hospital discharge for patients with end stage lung disease. Decellularized lungs subsequently recellularized with epithelial and endothelial cells have been implanted in small animal models with demonstration of initial gas exchange. Further development of these tissues and scaling to large animal models will validate this approach and may be an organ source for lung transplantation. Initial clinical success has been achieved with decellularized tracheal implants using autologous stem cells. Development of microfluidic lung models using similar architecture to the lung assist device technology allows study of lung biology and diseases with manipulation of lung cells and respiratory membrane strain.

Background: Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for recellularization with selected cell types. We aimed to up-scale this process, generating biocompatible scaffolds of a clinically relevant scale., Methods: Rat, porcine, and human lungs were decellularized by detergent perfusion at constant pressures. Collagen, elastin, and glycosaminoglycan content of scaffolds were quantified by colorimetric assays. Proteomic analysis was performed by microcapillary liquid chromatography tandem mass spectrometry. Extracellular matrix (ECM) slices were cultured with human umbilical vein endothelial cells (HUVEC), small airway epithelial cells (SAEC), or pulmonary alveolar epithelial cells (PAECs) and evaluated by time-lapse live cell microscopy and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Whole-organ culture was maintained under constant-pressure media perfusion after seeding with PAECs., Results: Rat lungs were decellularized using: (1) sodium dodecyl sulfate (SDS), (2) sodium deoxycholate (SDC), or (3) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Resulting scaffolds showed comparable loss of DNA but greatest preservation of ECM components in SDS-decellularized lungs. Porcine (n = 10) and human (n = 7) lungs required increased SDS concentration, perfusion pressures, and time to achieve decellularization as determined by loss of DNA, with preservation of intact matrix composition and lung architecture. Proteomic analysis of human decellularized lungs further confirmed ECM preservation. Recellularization experiments confirmed scaffold biocompatibility when cultured with mature cell phenotypes and scaffold integrity for the duration of biomimetic culture., Conclusions: SDS-based perfusion decellularization can be applied to whole porcine and human lungs to generate biocompatible organ scaffolds with preserved ECM composition and architecture., (© 2013 International Society for Heart and Lung Transplantation Published by International Society for the Heart and Lung Transplantation All rights reserved.)

Objectives: We developed a large animal model for auricular reconstruction with engineered cartilage frameworks and evaluated the performance of porous polyethylene auricular implants in this model., Methods: Eighteen high-density porous polyethylene auricular frameworks were implanted subcutaneously in the infra-auricular areas of 9 sheep. The implants were harvested 17 weeks later for gross and histologic examination. The perioperative and postoperative courses were carefully documented., Results: Five implants became exposed, and 2 implants needed to be removed at 7 weeks. Additionally, 1 infected implant was removed at 2 weeks. Seromas developed in 2 implants because of drain failures and were drained successfully during the first postoperative week. There were no other surgical site complications. The remaining 10 implants had an acceptable cosmetic appearance at 17 weeks., Conclusions: The perioperative complication rate in the ovine porous polyethylene auricular implant model was higher than that reported for auricular reconstructions in humans. The implant exposures were likely caused by ischemia and excessive stress on the thin overlying skin, because vascularized flap coverage was not used. The histologic findings were comparable to the results reported for other animal models. This large animal model is appropriate for auricular reconstruction experiments, including engineered constructs.

Tissue-engineered cartilage has historically been an attractive alternative treatment option for auricular reconstruction. However, the ability to reliably generate autologous auricular neocartilage in an immunocompetent preclinical model should first be established. The objectives of this study were to demonstrate engineered autologous auricular cartilage in the immunologically aggressive subcutaneous environment of an immunocompetent animal model, and to determine the impact of in vitro culture duration of chondrocyte-seeded constructs on the quality of neocartilage maturation in vivo. Auricular cartilage was harvested from eight adult sheep; chondrocytes were isolated, expanded in vitro, and seeded onto fibrous collagen scaffolds. Constructs were cultured in vitro for 2, 6, and 12 weeks, and then implanted autologously in sheep and in control nude mice for 6 and 12 weeks. Explanted tissue was stained with hematoxylin and eosin, safranin O, toluidine blue, collagen type II, and elastin. DNA and glycosaminoglycans (GAGs) were quantified. The quality of cartilage engineered in sheep decreased with prolonged in vitro culture time. Superior cartilage formation was demonstrated after 2 weeks of in vitro culture; the neocartilage quality improved with increased implantation time. In nude mice, neocartilage resembled native sheep auricular cartilage regardless of the in vitro culture length, with the exception of elastin expression. The DNA quantification was similar in all engineered and native cartilage (p>0.1). All cartilage engineered in sheep had significantly less GAG than native cartilage (p<0.02); significantly more GAG was observed with increased implantation time (p<0.02). In mice, the GAG content was similar to that of native cartilage and became significantly higher with increased in vitro or in vivo durations (p<0.02). Autologous auricular cartilage was successfully engineered in the subcutaneous environment of an ovine model using expanded chondrocytes seeded on a fibrous collagen scaffold after a 2-week in vitro culture period.

New techniques for tissue engineering (TE) are rapidly emerging. The basic concept of autologous TE is to isolate cells from small biopsy specimens, and to expand these cells in culture for subsequent seeding onto biodegradable scaffolds. Nanocrystalline diamond films have attracted the attention of researchers from a variety of different areas in recent years, due to their unique and exceptional properties. In this approach, human dental stem cells (hDSCs) were characterized by flow cytometry and grown on diamond films with hydrogen (H)-terminated and oxygen (O)-terminated surfaces for 28 days, and then removed by lysis and washing with distilled water. Energy dispersive spectroscopy analysis was performed, showing that the regions with O-terminated surfaces contained much higher levels of deposited calcium, oxygen, and phosphorus. These results suggest that the extracellular matrix was considerably more developed in the O-terminated regions, as compared with the H-terminated regions. In addition, optical microscopy of hDSCs cultured on the diamond substrate with H- and O-terminated surfaces, before washing with distilled water, showed preferential directions of the cells arrangement, where orthogonal lines suggest that the cells appeared to be following the O-terminated regions or hydrophilic surface. These findings suggest that O-terminated diamond surfaces prepared on biodegradable scaffolds can be useful for mineralized dental tissue formation.

Engineered cartilage is a promising option for auricular reconstruction. We have previously demonstrated that a titanium wire framework within a composite collagen ear-shaped scaffold helped to maintain the gross dimensions of the engineered ear after implantation, resisting the deformation forces encountered during neocartilage maturation and wound healing. The ear geometry was redesigned to achieve a more accurate aesthetic result when implanted subcutaneously in a nude rat model. A non-invasive method was developed to assess size and shape changes of the engineered ear in three dimensions. Computer models of the titanium framework were obtained from CT scans before and after implantation. Several parameters were measured including the overall length, width and depth, the minimum intrahelical distance and overall curvature values for each beam section within the framework. Local curvature values were measured to gain understanding of the bending forces experienced by the framework structure in situ. Length and width changed by less than 2%, whereas the depth decreased by approximately 8% and the minimum intrahelical distance changed by approximately 12%. Overall curvature changes identified regions most susceptible to deformation. Eighty-nine per cent of local curvature measurements experienced a bending moment less than 50 µN-m owing to deformation forces during implantation. These quantitative shape analysis results have identified opportunities to improve shape fidelity of engineered ear constructs.

Surgical scaffold materials manufactured from donor human or animal tissue are increasingly being used to promote soft tissue repair and regeneration. The clinical product consists of the residual extracellular matrix remaining after a rigorous decellularization process. Optimally, the material provides both structural support during the repair period and cell guidance cues for effective incorporation into the regenerating tissue. Surgical scaffold materials are available from several companies and are unique products manufactured by proprietary methodology. A significant need exists for a more thorough understanding of scaffold properties that impact the early steps of host cell recruitment and infiltration. In this study, a panel of in vitro assays was used to make direct comparisons of several similar, commercially-available materials: Alloderm, Medeor Matrix, Permacol, and Strattice. Differences in the materials were detected for both cell signaling and scaffold architecture-dependent cell invasion. Material-conditioned media studies found Medeor Matrix to have the greatest positive effect upon cell proliferation and induction of migration. Strattice provided the greatest chemotaxis signaling and best suppressed apoptotic induction. Among assays measuring structure-dependent properties, Medeor Matrix was superior for cell attachment, followed by Permacol. Only Alloderm and Medeor Matrix supported chemotaxis-driven cell invasion beyond the most superficial zone. Medeor Matrix was the only material in the chorioallantoic membrane assay to support substantial cell invasion. These results indicate that both biologic and structural properties need to be carefully assessed in the considerable ongoing efforts to develop new uses and products in this important class of biomaterials., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Approximately 100,000 individuals in the United States currently await kidney transplantation, and 400,000 individuals live with end-stage kidney disease requiring hemodialysis. The creation of a transplantable graft to permanently replace kidney function would address donor organ shortage and the morbidity associated with immunosuppression. Such a bioengineered graft must have the kidney's architecture and function and permit perfusion, filtration, secretion, absorption and drainage of urine. We decellularized rat, porcine and human kidneys by detergent perfusion, yielding acellular scaffolds with vascular, cortical and medullary architecture, a collecting system and ureters. To regenerate functional tissue, we seeded rat kidney scaffolds with epithelial and endothelial cells and perfused these cell-seeded constructs in a whole-organ bioreactor. The resulting grafts produced rudimentary urine in vitro when perfused through their intrinsic vascular bed. When transplanted in an orthotopic position in rat, the grafts were perfused by the recipient's circulation and produced urine through the ureteral conduit in vivo.

Background: Despite advances in surgical technique, reconstruction of a mandibular condyle still causes significant donor-site morbidity. The purpose of this study was to compare the effect of 3 different growth factors and define optimal cell culture conditions for bone marrow-derived progenitor cells to differentiate into chondrocytes for mandibular condyle reconstruction., Methods: Porcine bone marrow-derived progenitor cells (pBMPCs) were cultured as a pellet for 2, 3, and 4 weeks under the following conditions: group 1, TGF-β3 + standard medium; group 2, TGF-β3 + BMP-2 + standard medium; group 3, TGF-β3 + IGF-1 + standard medium; and group 4, TGF-β3 + BMP-2 + IGF-1 + standard medium. Chondrogenic differentiation was evaluated using 3 lineage differentiation markers., Results: The mean type II collagen positive area increased over weeks 2, 3, and 4 in group 4 compared to all the other groups (ANOVA; P = 0.005). At week 4, there was significantly greater type II collagen production in group 4 compared to all the other groups (ANOVA; P = 0.003). The medium in group 4 produces the greatest amount of cartilage when compared to groups 1, 2, and 3, and that 4 weeks produces the greatest amount of type II collagen., Conclusions: The results of this study indicate that the most efficacious medium for chondrogenic differentiation of pBMPCs was group 4 medium and the most type II collagen was produced at 4 weeks.

Object: Recurrence after endovascular coiling of intracranial aneurysms is reported in up to 42% of cases and is attributed to the lack of endothelialization across the neck. In this study the authors used a novel tissue engineering approach to promote endothelialization by seeding endothelial progenitor cells (EPCs) within a fibrin polymer injected endovascularly into the aneurysm., Methods: Experimental aneurysms were created in New Zealand White rabbits and were left untreated, surgically clipped, or embolized with platinum coils, fibrin biopolymer alone, or fibrin combined with autologous cultured EPCs., Results: In aneurysms treated with EPCs, a confluent monolayer of endothelial cells with underlying neointima was demonstrated across the neck at 16 weeks posttreatment, which was not observed with aneurysms treated using the other methods., Conclusions: This novel technique may address reasons for the limited durability of standard coil embolization and provides further avenues for the development of improved devices for the care of patients with aneurysms.

Purpose: The purpose of this study was to evaluate porcine bone marrow-derived progenitor cell (pBMPC) proliferation and penetration into a novel 3-dimensionally printed scaffold., Materials and Methods: Four different tissue engineering scaffolds to evaluate pBMPC proliferation and penetration were examined. Scaffolds were fabricated from polycaprolactone (PCL) or the combination of β-tricalcium phosphate (β-TCP) and PCL (50:50), with 2 separate channel sizes (1 mm [small (S)] vs 2 mm [large (L)]). Scaffolds were fabricated into 20 × 20 × 7-mm blocks by use of a TheriForm machine (Integra Life Sciences, Akron, OH). Four groups of scaffolds were examined for pBMPC proliferation and penetration: group 1, β-TCP/PCL S; group 2, β-TCP/PCL L; group 3, PCL S; and group 4, PCL L. Nonparametric mean (Kruskal-Wallis) and multiple comparisons tests were used to compare the 4 groups., Results: No shrinkage or deformation was noted in any of the scaffold groups after 2 weeks of culture. Mean surface cell counts ranged from 13.4 to 87.8 cells/0.57 mm(2), with group 1 (β-TCP/PCL S) having statistically significantly higher counts than the other groups (P < .001). Mean interior cell counts ranged from 10.9 to 75.6 cells/0.57 mm(2), with group 1 having the greatest interior cell count (P < .001). Total collagen formation ranged from 0.2% to 86%, with group 1 having the highest collagen formation (P < .001)., Conclusions: The 3-dimensionally printed scaffold (β-TCP/PCL) with 1-mm channels showed greater cellular proliferation, penetration, and collagen formation after a 2-week in vitro culture than the other scaffolds evaluated. β-TCP/PCL S scaffolds warrant further evaluation for bone tissue engineering in vivo., (Copyright © 2012 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.

The reconstruction, repair, and regeneration of the external auricular framework continue to be one of the greatest challenges in the field of tissue engineering. To replace like with like, we should emulate the native structure and composition of auricular cartilage by combining a suitable chondrogenic cell source with an appropriate scaffold under optimal in vitro and in vivo conditions. Due to the fact that a suitable and reliable substitute for auricular cartilage has yet to be engineered, hand-carved autologous costal cartilage grafts and ear-shaped porous polyethylene implants are the current treatment modalities for auricular reconstruction. However, over the last decade, significant advances have been made in the field of regenerative medicine and tissue engineering. A variety of scaffolds and innovative approaches have been investigated as alternatives to using autologous carved costal cartilage or porous polyethylene implants. A review of recent developments and the current state of the art and science is presented, focusing on scaffolds, cell sources, seeding densities, and mechanical characteristics of tissue-engineered auricular cartilage.

Blood oxygenators are vital for the critically ill, but their use is limited to the hospital setting. A portable blood oxygenator or a lung assist device for ambulatory or long-term use would greatly benefit patients with chronic lung disease. In this work, a biomimetic blood oxygenator system was developed which consisted of a microfluidic vascular network covered by a gas permeable silicone membrane. This system was used to determine the influence of key microfluidic parameters-channel size, oxygen exposure length, and blood shear rate-on blood oxygenation and carbon dioxide removal. Total gas transfer increased linearly with flow rate, independent of channel size and oxygen exposure length. On average, CO(2) transfer was 4.3 times higher than oxygen transfer. Blood oxygen saturation was also found to depend on the flow rate per channel but in an inverse manner; oxygenation decreased and approached an asymptote as the flow rate per channel increased. These relationships can be used to optimize future biomimetic vascular networks for specific lung applications: gas transfer for carbon dioxide removal in patients with chronic obstructive pulmonary disease or oxygenation for premature infants requiring complete lung replacement therapy.

Background: More than 11 million Americans live with chronic lung disease; in search for an alternative to donor organs, we attempted to regenerate lungs based on perfusion decellularized lung scaffolds that can be transplanted similar to a donor organ., Methods: Cadaveric rat lungs were decellularized by detergent perfusion. Resulting scaffolds were mounted in bioreactors and seeded with endothelial and fetal lung cells. Biomimetic organ culture was maintained for 7 days. Resulting bioartificial left lungs were transplanted in orthotopic position after left pneumonectomy in rats. Cadaveric left lung transplants and pneumonectomies served as controls. Blood gas analyses, compliance testing, and fluoroscopies were performed on postoperative days 1, 7, and 14. Lungs were removed for final analysis on day 14., Results: Perfusion decellularization of cadaveric lungs yielded acellular scaffolds with intact architecture and matrix composition. Alveolar volumes, number, and size were comparable in bioartificial and native lungs, as were gas exchange, vital capacity and compliance in vitro. After using improved graft preservation and postoperative weaning protocols, animals could be fully recovered, and bioartificial lung constructs provided oxygenation as long as 7 days at levels comparable to cadaveric lung transplants. Compliance, gas exchange, and radiographic appearance gradually declined over the subsequent 7 days owing to progressive graft consolidation and inflammation., Conclusions: Perfusion decellularization of cadaveric lungs yields intact scaffolds that can be seeded with cells to generate bioartificial lung grafts. After orthotopic transplantation, grafts are perfused by the recipient's circulation, ventilated through the recipient's airway and provide gas exchange in vivo for 7 days., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications., (© 2011, Copyright the Authors. Artificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Membranes for a lung assist device must permit the exchange of gaseous O₂ and CO₂ while simultaneously acting as a liquid barrier, so as to prevent leakage of blood and its components from passing from one side to the other. Additionally, these membranes must be capable of being integrated into microfluidic devices possessing a vascular network. In this work, uniform, large-area, ultra-thin, polymeric free-standing membranes (FSMs) and composite membranes (CMs) are reproducibly fabricated by initiated Chemical Vapor Deposition (iCVD). The 5 μm thick FSMs remained intact during handling and exhibited a CO₂ permeance that was 1.3 times that of the control membrane (8 μm thick spun-cast membrane of silicone). The CMs consisted of a dense iCVD skin layer (0.5-3 μm thick) deposited on top of a polytetrafluoroethylene (PTFE) support membrane (20 μm thick, 100 nm pores). The CMs exhibited CO₂ and O₂ permeance values 50-300 times that of the control membrane. The FSMs were subjected to mechanical testing to assess the impact of the absence of an underlying support structure. The CMs were subjected to liquid barrier tests to ensure that while they were permeable to gases, they acted as barriers to liquids. Both FSMs and CMs were integrated into silicone microfluidic devices and tested for bond integrity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)