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1.  Zero Echo Time Magnetic Resonance Imaging of Contrast-Agent-Enhanced Calcium Phosphate Bone Defect Fillers 
Calcium phosphate cements (CPCs) are widely used bone substitutes. However, CPCs have similar radiopacity as natural bone, rendering them difficult to be differentiated in classical X-ray and computed tomography imaging. As conventional magnetic resonance imaging (MRI) of bone is cumbersome, due to low water content and very short T2 relaxation time, ultra-short echo time (UTE) and zero echo time (ZTE) MRI have been explored for bone visualization. This study examined the possibility to differentiate bone and CPC by MRI. T1 and T2* values determined with UTE MRI showed little difference between bone and CPC; hence, these materials were difficult to separate based on T1 or T2 alone. Incorporation of ultra-small particles of iron oxide and gadopentetatedimeglumine (Gd-DTPA; 1 weight percentage [wt%] and 5 wt% respectively) into CPC resulted in visualization of CPC with decreased intensity on ZTE images in in vitro and ex vivo experiments. However, these additions had unfavorable effects on the solidification time and/or mechanical properties of the CPC, with the exception of 1% Gd-DTPA alone. Therefore, we tested this material in an in vivo experiment. The contrast of CPC was enhanced at an early stage postimplantation, and was significantly reduced in the 8 weeks thereafter. This indicates that ZTE imaging with Gd-DTPA as a contrast agent could be a valid radiation-free method to visualize CPC degradation and bone regeneration in preclinical experiments.
doi:10.1089/ten.tec.2011.0745
PMCID: PMC3583250  PMID: 22934755
2.  Tissue-Engineered Constructs of Human Oral Mucosa Examined by Raman Spectroscopy 
A noninvasive quality monitoring of tissue-engineered constructs is a required component of any successful tissue-engineering technique. During a 2-week production period, ex vivo produced oral mucosa-equivalent constructs (EVPOMEs) may encounter adverse culturing conditions that might compromise their quality and render them ineffective. We demonstrate the application of near-infrared Raman spectroscopy to in vitro monitoring of EVPOMEs during their manufacturing process, with the ultimate goal of applying this technology in situ to monitor the grafted EVPOMEs. We identify Raman spectroscopic failure indicators for less-than optimal EVPOMEs that are stressed by higher temperature and exposure to higher than normal concentration of calcium ions. Raman spectra of EVPOMEs exposed to thermal and calcium stress showed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. We compared these results to histology and glucose consumption measurements, demonstrating that Raman spectroscopy is more sensitive and specific to changes in proteins' secondary structure not visible by H&E histology. We also exposed the EVPOMEs to rapamycin, a cell growth inhibitor and cell proliferation capacity preserver, and distinguished between EVPOMEs pretreated with 2 nM rapamycin and controls, using the ratio of the Amide III envelope to the phenylalanine band as an indicator.
doi:10.1089/ten.tec.2012.0287
PMCID: PMC3583251  PMID: 22992065
3.  Attenuated Human Bone Morphogenetic Protein-2–Mediated Bone Regeneration in a Rat Model of Composite Bone and Muscle Injury 
Extremity injuries involving large bone defects with concomitant severe muscle damage are a significant clinical challenge often requiring multiple treatment procedures and possible amputation. Even if limb salvage is achieved, patients are typically left with severe short- and long-term disabilities. Current preclinical animal models do not adequately mimic the severity, complexity, and loss of limb function characteristic of these composite injuries. The objectives of this study were to establish a composite injury model that combines a critically sized segmental bone defect with an adjacent volumetric muscle loss injury, and then use this model to quantitatively assess human bone morphogenetic protein-2 (rhBMP-2)–mediated tissue regeneration and restoration of limb function. Surgeries were performed on rats in three experimental groups: muscle injury (8-mm-diameter full-thickness defect in the quadriceps), bone injury (8-mm nonhealing defect in the femur), or composite injury combining the bone and muscle defects. Bone defects were treated with 2 μg of rhBMP-2 delivered in the pregelled alginate injected into a cylindrical perforated nanofiber mesh. Bone regeneration was quantitatively assessed using microcomputed tomography, and limb function was assessed using gait analysis and muscle strength measurements. At 12 weeks postsurgery, treated bone defects without volumetric muscle loss were consistently bridged. In contrast, the volume and mechanical strength of regenerated bone were attenuated by 45% and 58%, respectively, in the identically treated composite injury group. At the same time point, normalized muscle strength was reduced by 51% in the composite injury group compared to either single injury group. At 2 weeks, the gait function was impaired in all injury groups compared to baseline with the composite injury group displaying the greatest functional deficit. We conclude that sustained delivery of rhBMP-2 at a dose sufficient to induce bridging of large segmental bone defects failed to promote regeneration when challenged with concomitant muscle injury. This model provides a platform with which to assess bone and muscle interactions during repair and to rigorously test the efficacy of tissue engineering approaches to promote healing in multiple tissues. Such interventions may minimize complications and the number of surgical procedures in limb salvage operations, ultimately improving the clinical outcome.
doi:10.1089/ten.tec.2012.0290
PMCID: PMC3583252  PMID: 22992043
4.  Optimized Hyaluronic Acid–Hydrogel Design and Culture Conditions for Preservation of Mesenchymal Stem Cell Properties 
A novel approach that preserved most mesenchymal stem cell (MSC) characteristics was developed using MSC encapsulation in a hydrogel based on hyaluronic acid (HA). An optimized HA–hydrogel composition, whose characteristics were assessed by scanning electron microscopy and viscoelastic property analyses, as well as the more favorable MSC seeding density, was established. These optimal three-dimensional MSC culture conditions allowed morphological cell remodeling, maintained the expression of stem cell markers over 28 days of culture, and preserved MSC differentiation plasticity. In addition, MSCs in HA–hydrogel submitted for 7 days to mechanical constraint that aimed at mimicking in vivo cardiac beat displayed enhanced cell survival by more than 40% compared to static culture conditions. Thus, the optimized HA-based hydrogel provides a niche for MSCs, which preserves their properties and opens ways for cell therapy, in particular in aortic repair medicine.
doi:10.1089/ten.tec.2012.0144
PMCID: PMC3583253  PMID: 22992013
5.  Ultrasonic Bioreactor as a Platform for Studying Cellular Response 
The need for tissue-engineered constructs as replacement tissue continues to grow as the average age of the world's population increases. However, additional research is required before the efficient production of laboratory-created tissue can be realized. The multitude of parameters that affect cell growth and proliferation is particularly daunting considering that optimized conditions are likely to change as a function of growth. Thus, a generalized research platform is needed in order for quantitative studies to be conducted. In this article, an ultrasonic bioreactor is described for use in studying the response of cells to ultrasonic stimulation. The work is focused on chondrocytes with a long-term view of generating tissue-engineered articular cartilage. Aspects of ultrasound (US) that would negatively affect cells, including temperature and cavitation, are shown to be insignificant for the US protocols used and which cover a wide range of frequencies and pressure amplitudes. The bioreactor is shown to have a positive influence on several factors, including cell proliferation, viability, and gene expression of select chondrocytic markers. Most importantly, we show that a total of 138 unique proteins are differentially expressed on exposure to ultrasonic stimulation, using mass-spectroscopy coupled proteomic analyses. We anticipate that this work will serve as the basis for additional research which will elucidate many of the mechanisms associated with cell response to ultrasonic stimulation.
doi:10.1089/ten.tec.2012.0199
PMCID: PMC3557434  PMID: 22873765
6.  Storage Temperatures for Cold-Chain Delivery in Cell Therapy: A Study of Alginate-Encapsulated Liver Cell Spheroids Stored at −80°C or −170°C for Up to 1 Year 
Introduction
A bioartificial liver comprising alginate-encapsulated liver cell spheroids (ELS) could bridge the gap to transplant or spontaneous recovery in acute liver failure, but will be required for emergency use, necessitating cryopreservation. A cryopreservation protocol has been developed, but beyond this, the feasibility of cold-chain storage is considered here. Cryopreservation will be increasingly required for timely delivery of tissue and bioengineered products, and significant, but often, over-looked factors that impact on cost and ease of clinical application are the storage temperature and useful preservation time. Storage in the vapor phase of liquid nitrogen (∼−170°C) is the gold standard, but for safety and economic purposes, storing ELS in electric freezers at −80°C may be preferable.
Methods
ELS were cryopreserved using an optimized protocol and stored at either −80°C or at −170°C for up to 1 year. ELS were removed from storage after 1, 2, 3, 6, 9, or 12 months, and recovery was assessed 24 h postwarming. Cell recovery was assessed using viability (fluorescent staining with image analysis), cell number (nuclei count), and functional (hepatospecific protein enzyme-linked immunosorbent assay) assays.
Results
Viability, the viable cell number, and function of ELS stored at −170°C were maintained at similar values throughout the year. In contrast, ELS stored at −80°C exhibited decreased viability, viable cell numbers, and function by as early as 1 month. Progressive deterioration was subsequently observed. After 12 months of storage at −80°C, viable cell recovery of ELS was ∼15% that of ELS stored at −170°C.
Conclusions
While convenience and cost might support the use of −80°C for storage of multicellular bioengineered products such as ELS, results indicate rapid deterioration in functional recoveries after only a few weeks. This study demonstrates that storage temperature is an important consideration in regenerative medicine and caution should be applied by limiting storage at −80°C to only a few weeks.
doi:10.1089/ten.tec.2012.0307
PMCID: PMC3557435  PMID: 22834979
7.  Micro-Computed Tomography Evaluation of Human Fat Grafts in Nude Mice 
Background
Although autologous fat grafting has revolutionized the field of soft tissue reconstruction and augmentation, long-term maintenance of fat grafts is unpredictable. Recent studies have reported survival rates of fat grafts to vary anywhere between 10% and 80% over time. The present study evaluated the long-term viability of human fat grafts in a murine model using a novel imaging technique allowing for in vivo volumetric analysis.
Methods
Human fat grafts were prepared from lipoaspirate samples using the Coleman technique. Fat was injected subcutaneously into the scalp of 10 adult Crl:NU-Foxn1nu CD-1 male mice. Micro-computed tomography (CT) was performed immediately following injection and then weekly thereafter. Fat volume was rendered by reconstructing a three-dimensional (3D) surface through cubic-spline interpolation. Specimens were also harvested at various time points and sections were prepared and stained with hematoxylin and eosin (H&E), for macrophages using CD68 and for the cannabinoid receptor 1 (CB1). Finally, samples were explanted at 8- and 12-week time points to validate calculated micro-CT volumes.
Results
Weekly CT scanning demonstrated progressive volume loss over the time course. However, volumetric analysis at the 8- and 12-week time points stabilized, showing an average of 62.2% and 60.9% survival, respectively. Gross analysis showed the fat graft to be healthy and vascularized. H&E analysis and staining for CD68 showed minimal inflammatory reaction with viable adipocytes. Immunohistochemical staining with anti-human CB1 antibodies confirmed human origin of the adipocytes.
Conclusions
Studies assessing the fate of autologous fat grafts in animals have focused on nonimaging modalities, including histological and biochemical analyses, which require euthanasia of the animals. In this study, we have demonstrated the ability to employ micro-CT for 3D reconstruction and volumetric analysis of human fat grafts in a mouse model. Importantly, this model provides a platform for subsequent study of fat manipulation and soft tissue engineering.
doi:10.1089/ten.tec.2012.0371
PMCID: PMC3557441  PMID: 22916732
8.  Effect of Surface Pore Structure of Nerve Guide Conduit on Peripheral Nerve Regeneration 
Polycaprolactone (PCL)/Pluronic F127 nerve guide conduits (NGCs) with different surface pore structures (nano-porous inner surface vs. micro-porous inner surface) but similar physical and chemical properties were fabricated by rolling the opposite side of asymmetrically porous PCL/F127 membranes. The effect of the pore structure on peripheral nerve regeneration through the NGCs was investigated using a sciatic nerve defect model of rats. The nerve fibers and tissues were shown to have regenerated along the longitudinal direction through the NGC with a nano-porous inner surface (Nanopore NGC), while they grew toward the porous wall of the NGC with a micro-porous inner surface (Micropore NGC) and, thus, their growth was restricted when compared with the Nanopore NGC, as investigated by immunohistochemical evaluations (by fluorescence microscopy with anti-neurofilament staining and Hoechst staining for growth pattern of nerve fibers), histological evaluations (by light microscopy with Meyer's modified trichrome staining and Toluidine blue staining and transmission electron microscopy for the regeneration of axon and myelin sheath), and FluoroGold retrograde tracing (for reconnection between proximal and distal stumps). The effect of nerve growth factor (NGF) immobilized on the pore surfaces of the NGCs on nerve regeneration was not so significant when compared with NGCs not containing immobilized NGF. The NGC system with different surface pore structures but the same chemical/physical properties seems to be a good tool that is used for elucidating the surface pore effect of NGCs on nerve regeneration.
doi:10.1089/ten.tec.2012.0221
PMCID: PMC3557444  PMID: 22871377
9.  Investigation of Neovascularization in Three-Dimensional Porous Scaffolds In Vivo by a Combination of Multiscale Photoacoustic Microscopy and Optical Coherence Tomography 
It is a grand challenge to visualize and assess in vivo neovascularization in a three-dimensional (3D) scaffold noninvasively, together with high spatial resolution and deep penetration depth. Here we used multiscale photoacoustic microscopy (PAM), including acoustic-resolution PAM (AR-PAM) and optical-resolution PAM (OR-PAM), to chronically monitor neovascularization in an inverse opal scaffold implanted in a mouse model up to 6 weeks by taking advantage of the optical absorption contrast intrinsic to hemoglobin molecules in red blood cells. By combining with optical coherence tomography (OCT) based on optical scattering contrast, we also demonstrated the capability to simultaneously image and analyze the vasculature and the scaffold in the same mouse. The hybrid system containing OR-PAM and OCT offered a fine lateral resolution of ∼5 μm and a penetration depth of ∼1 mm into the scaffold/tissue construct. AR-PAM further extended the penetration depth up to ∼3 mm at a lateral resolution of ∼45 μm. By quantifying the 3D PAM data, we further examined the effect of pore size (200 vs. 80 μm) of a scaffold on neovascularization. The data collected from PAM were consistent with those obtained from traditional invasive, labor-intensive histologic analyses.
doi:10.1089/ten.tec.2012.0326
PMCID: PMC3557912  PMID: 22838500
10.  Effect of Oxygen Concentration on Viability and Metabolism in a Fluidized-Bed Bioartificial Liver Using 31P and 13C NMR Spectroscopy 
Many oxygen mass-transfer modeling studies have been performed for various bioartificial liver (BAL) encapsulation types; yet, to our knowledge, there is no experimental study that directly and noninvasively measures viability and metabolism as a function of time and oxygen concentration. We report the effect of oxygen concentration on viability and metabolism in a fluidized-bed NMR-compatible BAL using in vivo 31P and 13C NMR spectroscopy, respectively, by monitoring nucleotide triphosphate (NTP) and 13C-labeled nutrient metabolites, respectively. Fluidized-bed bioreactors eliminate the potential channeling that occurs with packed-bed bioreactors and serve as an ideal experimental model for homogeneous oxygen distribution. Hepatocytes were electrostatically encapsulated in alginate (avg. diameter, 500 μm; 3.5×107 cells/mL) and perfused at 3 mL/min in a 9-cm (inner diameter) cylindrical glass NMR tube. Four oxygen treatments were tested and validated by an in-line oxygen electrode: (1) 95:5 oxygen:carbon dioxide (carbogen), (2) 75:20:5 nitrogen:oxygen:carbon dioxide, (3) 60:35:5 nitrogen:oxygen:carbon dioxide, and (4) 45:50:5 nitrogen:oxygen:carbon dioxide. With 20% oxygen, β-NTP steadily decreased until it was no longer detected at 11 h. The 35%, 50%, and 95% oxygen treatments resulted in steady β-NTP levels throughout the 28-h experimental period. For the 50% and 95% oxygen treatment, a 13C NMR time course (∼5 h) revealed 2-13C-glycine and 2-13C-glucose to be incorporated into [2-13C-glycyl]glutathione (GSH) and 2-13C-lactate, respectively, with 95% having a lower rate of lactate formation. 31P and 13C NMR spectroscopy is a noninvasive method for determining viability and metabolic rates. Modifying tissue-engineered devices to be NMR compatible is a relatively easy and inexpensive process depending on the bioreactor shape.
doi:10.1089/ten.tec.2011.0629
PMCID: PMC3540897  PMID: 22835003
11.  Benefits of Spine Stabilization with Biodegradable Scaffolds in Spinal Cord Injured Rats 
Spine stabilization upon spinal cord injury (SCI) is a standard procedure in clinical practice, but rarely employed in experimental models. Moreover, the application of biodegradable biomaterials for this would come as an advantage as it would eliminate the presence of a nondegradable prosthesis within the vertebral bone. Therefore, in the present work, we propose the use of a new biodegradable device specifically developed for spine stabilization in a rat model of SCI. A 3D scaffold based on a blend of starch with polycaprolactone was implanted, replacing delaminated vertebra, in male Wistar rats with a T8-T9 spinal hemisection. The impact of spinal stabilization on the locomotor behavior was then evaluated for a period of 12 weeks. Locomotor evaluation—assessed by Basso, Beatie, and Bresnahan test; rotarod; and open field analysis—revealed that injured rats subjected to spine stabilization significantly improved their motor performance, including higher coordination and rearing activity when compared with SCI rats without stabilization. Histological analysis further revealed that the presence of the scaffolds not only stabilized the area, but also simultaneously prevented the infiltration of the injury site by connective tissue. Overall, these results reveal that SCI stabilization using a biodegradable scaffold at the vertebral bone level leads to an improvement of the motor deficits and is a relevant element for the successful treatment of SCI.
doi:10.1089/ten.tec.2012.0264
PMCID: PMC3540933  PMID: 22779715
12.  Optimization and Scale-up Culture of Human Endometrial Multipotent Mesenchymal Stromal Cells: Potential for Clinical Application 
We have previously identified and purified multipotent mesenchymal stromal cell (MSC)-like cells in the highly regenerative endometrial lining of the human uterus (eMSC) as CD140b+CD146+ cells. Due to ease of accessibility with minimal morbidity via biopsy, we are proposing to use eMSC in cell-based therapies; however, culture conditions compliant with Good Manufacturing Practice have not been established for eMSC. The aim of this study was to optimize serum-free and xeno-free culture conditions for expansion of eMSC for potential clinical use. Real-time cell assessment (Xcelligence) and MTS viability assays were used to measure attachment and proliferation of freshly isolated, flow cytometry-sorted CD140b+CD146+ eMSC cultured in several commercially available and in-house serum-free and xeno-free media in combination with five attachment matrices (fibronectin, collagen, gelatin, laminin, and Cell Start-XF®). Comparisons were made with a standard serum-containing medium, DMEM/F-12/10% fetal bovine serum. Under all conditions examined, eMSC attachment and proliferation was greatest using a fibronectin matrix, with Lonza TP-SF® and our in-house DMEM/SF/FGF2/EGF serum-free xeno-product-containing medium similar to serum-containing medium. Hypoxia increased eMSC proliferation in the DMEM/SF/FGF2/EGF serum-free medium. Culture of eMSC for 7 days on a fibronectin matrix in DMEM/SF/FGF2/EGF serum-free media in 5% O2 maintained greater numbers of undifferentiated eMSC expressing CD140b, CD146, and W5C5 compared to culture under similar conditions in Lonza TP-SF medium. However, the percentage of cells expressing typical MSC phenotypic markers, CD29, CD44, CD73, and CD105, were similar for both media. EMSC showed greater expansion in 2D compared to 3D culture on fibronectin-coated microbeads using the optimized DMEM/SF/FGF2/EGF medium in 5% O2. In the optimized 2D culture conditions, eMSC retained CFU activity, multipotency, and MSC surface phenotype, representing the first steps in their preparation for potential clinical use.
doi:10.1089/ten.tec.2011.0718
PMCID: PMC3522126  PMID: 22738377
13.  Characterization of Dental Epithelial Stem Cells from the Mouse Incisor with Two-Dimensional and Three-Dimensional Platforms 
Dental epithelial stem cells (DESCs) drive continuous growth in the adult mouse incisors. To date, a robust system for the primary culture of these cells has not been reported, and little is known about the basic molecular architecture of these cells or the minimal extracellular scaffolding that is necessary to maintain the epithelial stem cell population in an undifferentiated state. We report a method of isolating DESCs from the cervical loop of the mouse mandibular incisor. Cells were viable in a two-dimensional culture system and did not demonstrate preferential proliferation when grown on top of various substrates. Characterization of these cells indicated that E-cadherin, integrin alpha-6, and integrin beta-4 mark the DESCs both in vivo and in vitro. We also grew these cells in a three-dimensional microenvironment and obtained spheres with an epithelial morphology and expression patterns. Insights into the mechanisms of stem cell maintenance in vitro will help lay the groundwork for the successful generation of bioengineered teeth from adult DESCs.
doi:10.1089/ten.tec.2012.0232
PMCID: PMC3522131  PMID: 22742471
14.  Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold 
A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver.
doi:10.1089/ten.tec.2011.0723
PMCID: PMC3522132  PMID: 22799502
15.  Two-Dimensional Polymer-Based Cultures Expand Cord Blood-Derived Hematopoietic Stem Cells and Support Engraftment of NSG Mice 
Currently, ex vivo expansion of hematopoietic stem cells (HSC) is still insufficient. Traditional approaches for HSC expansion include the use of stromal cultures, growth factors, and/or bioreactors. Biomaterial-based strategies provide new perspectives. We focus on identifying promising two-dimensional (2D) polymer candidates for HSC expansion. After a 7-day culture period with cytokine supplementation, 2D fibrin, poly(D,L-lactic-co-glycolic acid; Resomer® RG503), and Poly(ɛ-caprolactone; PCL) substrates supported expansion of cord blood (CB)-derived CD34+ cells ex vivo. Fibrin cultures achieved the highest proliferation rates (>8700-fold increase of total nuclear cells, p<0.001), high total colony-forming units (3.6-fold increase, p<0.001), and highest engraftment in NSG mice (7.69-fold more donor cells compared with tissue culture polysterene, p<0.001). In addition, the presence of multiple human hematopoietic lineages such as myeloid (CD13+), erythroid (GypC+), and lymphoid (CD20+/CD56+) in murine transplant recipients confirmed the multilineage engraftment potential of fibrin-based cultures. Filopodia development in fibrin-expanded cells was a further indicator for superior cell adhesion capacities. We propose application of fibrin, Resomer® RG503, and PCL for future strategies of CB-CD34+ cell expansion. Suitable polymers for HSC expansion might also be appropriate for future drug discovery applications or for studies aimed to develop hematological therapies.
doi:10.1089/ten.tec.2011.0706
PMCID: PMC3522133  PMID: 22712684
16.  Adipocyte Induction of Preadipocyte Differentiation in a Gradient Chamber 
Tissue Engineering. Part C, Methods  2012;18(12):958-967.
Adipose tissue expansion involves enlargement of mature adipocytes and the formation of new adipocytes through the differentiation of locally resident preadipocytes. Factors released by the enlarged adipocytes are potential cues that induce the differentiation of the preadipocytes. Currently, there are limited options to investigate these cues in isolation from confounding systemic influences. A gradient generating microfluidic channel-based cell culture system was designed to enable solution patterning, while supporting long-term culture and differentiation of preadipocytes. Solution patterning was confirmed by selectively staining a fraction of uniformly seeded preadipocytes. An adipogenic cocktail gradient was used to induce the differentiation of a fraction of uniformly seeded preadipocytes and establish a spatially defined coculture of adipocytes and preadipocytes. Varying the adipogenic cocktail gradient generated cocultures of preadipocytes and adipocytes with different compositions. Transient application of the cocktail gradient, followed by basal medium treatment showed a biphasic induction of differentiation. The two phases of differentiation correlated with a spatial gradient in adipocyte size. Our results provide in vitro data supporting the size-dependent release of preadipocyte differentiation factors by enlarged adipocytes. Prospectively, the coculture system developed in this study could facilitate controlled, yet physiologically meaningful studies on paracrine interactions between adipocytes and preadipocytes during adipose tissue development.
doi:10.1089/ten.tec.2012.0168
PMCID: PMC3491637  PMID: 22651694
17.  Charged Nanomatrices as Efficient Platforms for Modulating Cell Adhesion and Shape 
Tissue Engineering. Part C, Methods  2012;18(12):913-923.
In this article, we describe the design and manipulation of charged nanomatrices and their application as efficient platforms for modulating cell behaviors. Using electrospraying technology and well designed biomaterials, poly(ɛ-caprolactone; PCL) and polyethylenimine, the negatively charged PCL nanomatrix (nPCL nanomatrix) and the positively charged PCL nanomatrix (pPCL nanomatrix) were fabricated. It was demonstrated that cell adhesion, affinity, and shape were sensitively modulated in negatively and positively charged nanomatrices. Our results showed that the pPCL nanomatrix promoted adhesion of NIH 3T3 fibroblast cells as compared to the nPCL nanomatrix. When fluid shear stress was applied, cell affinity on the pPCL nanomatrix increased even more. NIH 3T3 fibroblast cells adopted a relatively spherical shape on the pPCL nanomatrix while adopting an aligned, narrow shape on the nPCL nanomatrix. It was also found that charged nanomatrices influenced the cross-sectional cell shape. The cross-sectional cell shape on the pPCL nanomatrix was extremely flattened, whereas the cross-sectional cell shape was relatively round on the nPCL nanomatrix and some of the adhered cells floated. We also showed that the surfaces of the nPCL and pPCL nanomatrices adsorbed the different serum proteins. These results collectively demonstrated a combination of environmental factors including nanoscale structure, electrostatic forces, and absorption of biomolecules on charged substrates affected cell response in terms of cellular adhesion and shape.
doi:10.1089/ten.tec.2011.0731
PMCID: PMC3491638  PMID: 22621374
18.  Noninvasive, Quantitative, Spatiotemporal Characterization of Mineralization in Three-Dimensional Collagen Hydrogels Using High-Resolution Spectral Ultrasound Imaging 
Tissue Engineering. Part C, Methods  2012;18(12):935-946.
As tissue engineering products move toward the clinic, nondestructive methods to monitor their development and ensure quality are needed. In this study, high-resolution spectral ultrasound imaging (SUSI) was used to noninvasively characterize mineral content in collagen hydrogels. SUSI was used to generate three-dimensional (3D) grayscale (GS) images of construct morphology with submillimeter resolution. Spectral analysis of the backscattered radio frequency (RF) ultrasound signals was used to determine the midband fit (MBF) and slope of the linearized RF spectrum. These parameters are operator and instrument independent, and were used to characterize the spatial distribution of mineral in constructs supplemented with hydroxyapatite particles. GS and MBF correlated closely with mineral content, while slope was not dependent on concentration. SUSI also was used to monitor mineralization of collagen constructs by immersion in simulated body fluid (SBF) over 21 days. The construct surface was mineralized before the interior, and there was a dose-dependent effect of SBF concentration on degree of mineralization and deposited particle size. MBF density was closely correlated with the amount of calcium deposited. These data demonstrate that SUSI has utility as a noninvasive imaging method for quantitative analysis of mineralization in 3D protein constructs. Such techniques may assist the development of engineered orthopedic tissues.
doi:10.1089/ten.tec.2012.0180
PMCID: PMC3968959  PMID: 22624791
19.  Cryopreservation of Cell/Scaffold Tissue-Engineered Constructs 
Tissue Engineering. Part C, Methods  2012;18(11):852-858.
The aim of this work was to study the effect of cryopreservation over the functionality of tissue-engineered constructs, analyzing the survival and viability of cells seeded, cultured, and cryopreserved onto 3D scaffolds. Further, it also evaluated the effect of cryopreservation over the properties of the scaffold material itself since these are critical for the engineering of most tissues and in particular, tissues such as bone. For this purpose, porous scaffolds, namely fiber meshes based on a starch and poly(caprolactone) blend were seeded with goat bone marrow stem cells (GBMSCs) and cryopreserved for 7 days. Discs of the same material seeded with GBMSCs were also used as controls. After this period, these samples were analyzed and compared to samples collected before the cryopreservation process. The obtained results demonstrate that it is possible to maintain cell viability and scaffolds properties upon cryopreservation of tissue-engineered constructs based on starch scaffolds and goat bone marrow mesenchymal cells using standard cryopreservation methods. In addition, the outcomes of this study suggest that the greater porosity and interconnectivity of scaffolds favor the retention of cellular content and cellular viability during cryopreservation processes, when compared with nonporous discs. These findings indicate that it might be possible to prepare off-the-shelf engineered tissue substitutes and preserve them to be immediately available upon request for patients' needs.
doi:10.1089/ten.tec.2011.0649
PMCID: PMC3483048  PMID: 22676448
20.  Imaging of Poly(α-hydroxy-ester) Scaffolds with X-ray Phase-Contrast Microcomputed Tomography 
Tissue Engineering. Part C, Methods  2012;18(11):859-865.
Porous scaffolds based on poly(α-hydroxy-esters) are under investigation in many tissue engineering applications. A biological response to these materials is driven, in part, by their three-dimensional (3D) structure. The ability to evaluate quantitatively the material structure in tissue-engineering applications is important for the continued development of these polymer-based approaches. X-ray imaging techniques based on phase contrast (PC) have shown a tremendous promise for a number of biomedical applications owing to their ability to provide a contrast based on alternative X-ray properties (refraction and scatter) in addition to X-ray absorption. In this research, poly(α-hydroxy-ester) scaffolds were synthesized and imaged by X-ray PC microcomputed tomography. The 3D images depicting the X-ray attenuation and phase-shifting properties were reconstructed from the measurement data. The scaffold structure could be imaged by X-ray PC in both cell culture conditions and within the tissue. The 3D images allowed for quantification of scaffold properties and automatic segmentation of scaffolds from the surrounding hard and soft tissues. These results provide evidence of the significant potential of techniques based on X-ray PC for imaging polymer scaffolds.
doi:10.1089/ten.tec.2012.0123
PMCID: PMC3483049  PMID: 22607529
21.  In Vitro Differentiation and Mineralization of Dental Pulp Stem Cells on Enamel-Like Fluorapatite Surfaces 
Tissue Engineering. Part C, Methods  2012;18(11):821-830.
Our previous studies have shown good biocompatibility of fluorapatite (FA) crystal surfaces in providing a favorable environment for functional cell–matrix interactions of human dental pulp stem cells (DPSCs) and also in supporting their long-term growth. The aim of the current study was to further investigate whether this enamel-like surface can support the differentiation and mineralization of DPSCs, and, therefore, act as a potential model for studying the enamel/dentin interface and, perhaps, dentine/pulp regeneration in tooth tissue engineering. The human pathway-focused osteogenesis polymerase chain reaction (PCR) array demonstrated that the expression of osteogenesis-related genes of human DPSCs was increased on FA surfaces compared with that on etched stainless steel (SSE). Consistent with the PCR array, FA promoted mineralization compared with the SSE surface with or without the addition of a mineralization promoting supplement (MS). This was confirmed by alkaline phosphatase (ALP) staining, Alizarin red staining, and tetracycline staining for mineral formation. In conclusion, FA crystal surfaces, especially ordered (OR) FA surfaces, which mimicked the physical architecture of enamel, provided a favorable extracellular matrix microenvironment for the cells. This resulted in the differentiation of human DPSCs and mineralized tissue formation, and, thus, demonstrated that it may be a promising biomimetic model for dentin-pulp tissue engineering.
doi:10.1089/ten.tec.2011.0624
PMCID: PMC3483051  PMID: 22563788
22.  Immortalized Functional Endothelial Progenitor Cell Lines from Umbilical Cord Blood for Vascular Tissue Engineering 
Tissue Engineering. Part C, Methods  2012;18(11):890-902.
Endothelial progenitor cells (EPCs) play a significant role in multiple biological processes such as vascular homeostasis, regeneration, and tumor angiogenesis. This makes them a promising cell of choice for studying a variety of biological processes, toxicity assays, biomaterial–cell interaction studies, as well as in tissue-engineering applications. In this study, we report the generation of two clones of SV40-immortalized EPCs from umbilical cord blood. These cells retained most of the functional features of mature endothelial cells and showed no indication of senescence after repeated culture for more than 240 days. Extensive functional characterization of the immortalized cells by western blot, flow cytometry, and immunofluorescence studies substantiated that these cells retained their ability to synthesize nitric oxide, von Willebrand factor, P-Selectin etc. These cells achieved unlimited proliferation potential subsequent to inactivation of the cyclin-dependent kinase inhibitor p21, but failed to form colonies on soft agar. We also show their enhanced growth and survival on vascular biomaterials compared to parental cultures in late population doubling. These immortalized EPCs can be used as a cellular model system for studying the biology of these cells, gene manipulation experiments, cell–biomaterial interactions, as well as a variety of tissue-engineering applications.
doi:10.1089/ten.tec.2011.0482
PMCID: PMC3483052  PMID: 22889128
23.  Rolling the Human Amnion to Engineer Laminated Vascular Tissues 
Tissue Engineering. Part C, Methods  2012;18(11):903-912.
The prevalence of cardiovascular disease and the limited availability of suitable autologous transplant vessels for coronary and peripheral bypass surgeries is a significant clinical problem. A great deal of progress has been made over recent years to develop biodegradable materials with the potential to remodel and regenerate vascular tissues. However, the creation of functional biological scaffolds capable of withstanding vascular stress within a clinically relevant time frame has proved to be a challenging proposition. As an alternative approach, we report the use of a multilaminate rolling approach using the human amnion to generate a tubular construct for blood vessel regeneration. The human amniotic membrane was decellularized by agitation in 0.03% (w/v) sodium dodecyl sulfate to generate an immune compliant material. The adhesion of human umbilical vein endothelial cells (EC) and human vascular smooth muscle cells (SMC) was assessed to determine initial binding and biocompatibility (monocultures). Extended cultures were either assessed as flat membranes, or rolled to form concentric multilayered conduits. Results showed positive EC adhesion and a progressive repopulation by SMC. Functional changes in SMC gene expression and the constructs' bulk mechanical properties were concomitant with vessel remodeling as assessed over a 40-day culture period. A significant advantage with this approach is the ability to rapidly produce a cell-dense construct with an extracellular matrix similar in architecture and composition to natural vessels. The capacity to control physical parameters such as vessel diameter, wall thickness, shape, and length are critical to match vessel compliance and tailor vessel specifications to distinct anatomical locations. As such, this approach opens new avenues in a range of tissue regenerative applications that may have a much wider clinical impact.
doi:10.1089/ten.tec.2012.0119
PMCID: PMC3483053  PMID: 22616610
24.  Decellularized Extracellular Matrix Derived from Porcine Adipose Tissue as a Xenogeneic Biomaterial for Tissue Engineering 
Tissue Engineering. Part C, Methods  2012;18(11):866-876.
Cells in tissues are surrounded by the extracellular matrix (ECM), a gel-like material of proteins and polysaccharides that are synthesized and secreted by cells. Here we propose that the ECM can be isolated from porcine adipose tissue and holds great promise as a xenogeneic biomaterial for tissue engineering and regenerative medicine. Porcine adipose tissue is easily obtained in large quantities from commonly discarded food waste. Decellularization protocols have been developed for extracting an intact ECM while effectively eliminating xenogeneic epitopes and minimally disrupting the ECM composition. Porcine adipose tissue was defatted by homogenization and centrifugation. It was then decellularized via chemical (1.5 M sodium chloride and 0.5% sodium dodecyl sulfate) and enzymatic treatments (DNase and RNase) with temperature control. After decellularization, immunogenic components such as nucleic acids and α-Gal were significantly reduced. However, abundant ECM components, such as collagen (332.9±12.1 μg/mg ECM dry weight), sulfated glycosaminoglycan (GAG, 85±0.7 μg/mg ECM dry weight), and elastin (152.6±4.5 μg/mg ECM dry weight), were well preserved in the decellularized material. The biochemical and mechanical features of a decellularized ECM supported the adhesion and growth of human cells in vitro. Moreover, the decellularized ECM exhibited biocompatibility, long-term stability, and bioinductivity in vivo. The overall results suggest that the decellularized ECM derived from porcine adipose tissue could be useful as an alternative biomaterial for xenograft tissue engineering.
doi:10.1089/ten.tec.2012.0009
PMCID: PMC3483066  PMID: 22559904
25.  Design of a High-Throughput Flow Perfusion Bioreactor System for Tissue Engineering 
Tissue Engineering. Part C, Methods  2012;18(10):817-820.
Flow perfusion culture is used in many areas of tissue engineering and offers several key advantages. However, one challenge to these cultures is the relatively low-throughput nature of perfusion bioreactors. Here, a flow perfusion bioreactor with increased throughput was designed and built for tissue engineering. This design uses an integrated medium reservoir and flow chamber in order to increase the throughput, limit the volume of medium required to operate the system, and simplify the assembly and operation.
doi:10.1089/ten.tec.2012.0037
PMCID: PMC3460612  PMID: 22468910

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