Simultaneous Application of Interstitial Flow and Cyclic Mechanical Strain to a Three-Dimensional Cell-Seeded Hydrogel

The present study describes the design and validation of a simple apparatus to apply simultaneous mechanical and fluidic stress to three-dimensional (3D) cell-seeded collagen hydrogels. Constructs were formed in wells in a silicone substrate that could be stretched cyclically, and were also fitted with inlet ports to apply fluid flow. Acid etching was used to retain adhesion of the gels to the walls of the well, and an acellular layer of collagen hydrogel was used to distribute flow evenly. Finite element modeling showed that 5% uniaxial strain applied to the entire silicone substrate resulted in -6.5% strain in each of the gel constructs. Permeability testing and flow observation showed that acellular hydrogels were fourfold more permeable than cardiac fibroblast-seeded gels, and that the fluid distributed evenly in the acellular layer before entering the cell-seeded gel. Viability testing and imaging demonstrated that cells remained viable with expected fibroblast morphology for the 120-h duration of the experiments. These results demonstrate that this simple bioreactor can be used to study the effects of mechanical strain and interstitial flow in 3D protein hydrogels. Such 3D tissue models have utility in studying cell and tissue responses to their mechanical environment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90463/1/ten-2Etec-2E2010-2E0547.pd

Carbon nanotubes in neural interfacing applications

Carbon nanotubes (CNT) are remarkable materials with a simple and inert molecular structure that gives rise to a range of potentially valuable physical and electronic properties, including high aspect ratio, high mechanical strength and excellent electrical conductivity. This review summarizes recent research on the application of CNT-based materials to study and control cells of the nervous system. It includes the use of CNT as cell culture substrates, to create patterned surfaces and to study cell–matrix interactions. It also summarizes recent investigations of CNT toxicity, particularly as related to neural cells. The application of CNT-based materials to directing the differentiation of progenitor and stem cells toward neural lineages is also discussed. The emphasis is on how CNT surface chemistry and nanotopography can be altered, and how such changes can affect neural cell function. This knowledge can be applied to creating improved neural interfaces and devices, as well as providing new approaches to neural tissue engineering and regeneration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90821/1/1741-2552_8_1_011001.pd

Ultrasound Imaging Techniques for Spatiotemporal Characterization of Composition, Microstructure, and Mechanical Properties in Tissue Engineering

Ultrasound techniques are increasingly being used to quantitatively characterize both native and engineered tissues. This review provides an overview and selected examples of the main techniques used in these applications. Grayscale imaging has been used to characterize extracellular matrix deposition, and quantitative ultrasound imaging based on the integrated backscatter coefficient has been applied to estimating cell concentrations and matrix morphology in tissue engineering. Spectral analysis has been employed to characterize the concentration and spatial distribution of mineral particles in a construct, as well as to monitor mineral deposition by cells over time. Ultrasound techniques have also been used to measure the mechanical properties of native and engineered tissues. Conventional ultrasound elasticity imaging and acoustic radiation force imaging have been applied to detect regions of altered stiffness within tissues. Sonorheometry and monitoring of steady-state excitation and recovery have been used to characterize viscoelastic properties of tissue using a single transducer to both deform and image the sample. Dual-mode ultrasound elastography uses separate ultrasound transducers to produce a more potent deformation force to microscale characterization of viscoelasticity of hydrogel constructs. These ultrasound-based techniques have high potential to impact the field of tissue engineering as they are further developed and their range of applications expands.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140238/1/ten.teb.2015.0453.pd

Collagen I-Matrigel Scaffolds for Enhanced Schwann Cell Survival and Control of Three-Dimensional Cell Morphology

We report on the ability to control three-dimensional Schwann cell (SC) morphology using collagen I Matrigel composite scaffolds for neural engineering applications. SCs are supportive of nerve regeneration after injury, and it has recently been reported that SCs embedded in collagen I, a material frequently used in guidance channel studies, do not readily extend processes, instead adopting a spherical morphology indicative of little interaction with the matrix. We have modified collagen I matrices by adding Matrigel to make them more supportive of SCs and characterized these matrices and SC morphology in vitro. Incorporation of 10%, 20%, 35%, and 50% Matrigel by volume resulted in 2.4, 3.5, 3.7, and 4.2 times longer average SC process length after 14 days in culture than with collagen I only controls. Additionally, only 35% and 50% Matrigel constructs were able to maintain SC number over 14 days, whereas an 88% decrease in cells from initial seeding density was observed in collagen-only constructs over the same time period. Mechanical testing revealed that the addition of 50% Matrigel increased matrix stiffness from 6.4kPa in collagen I only constructs to 9.8kPa. Furthermore, second harmonic generation imaging showed that the addition of Matrigel resulted in non-uniform distribution of collagen I, and scanning electron microscope imaging illustrated distinct differences in the fibrillar structure of the different constructs. Collectively, this work lays a foundation for developing scaffolding materials that are concurrently supportive of neurons and SCs for future neural engineering applications.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78114/1/ten.tea.2008.0406.pd

Comparison of Uncultured Marrow Mononuclear Cells and Culture-Expanded Mesenchymal Stem Cells in 3D Collagen-Chitosan Microbeads for Orthopedic Tissue Engineering

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25?106 cells/mL, containing an estimated 5?104 MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2?105 cells/mL) were added to a 65?35?wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140225/1/ten.tea.2013.0151.pd

Bioresponsive microspheres for on‐demand delivery of anti‐inflammatory cytokines for articular cartilage repair

Despite innovations in surgical interventions, treatment of cartilage injury in osteoarthritic joints remains a challenge due to concomitant inflammation. Obstructing a single dominant inflammatory cytokine has shown remarkable clinical benefits in rheumatoid arthritis, and similar strategies are being suggested to target inflammatory pathways in osteoarthritis (OA). Here, we describe the utility of gelatin microspheres that are responsive to proteolytic enzymes typically expressed in arthritic flares, resulting in on‐demand and spatiotemporally controlled release of anti‐inflammatory cytokines for cartilage preservation and repair. These microspheres were designed with a net negative charge to sequester cationic anti‐inflammatory cytokines, and the magnitude of the negative charge potential increased with an increase in crosslinking density. Collagenase‐mediated degradation of the microspheres was dependent on the concentration of the enzyme. Release of anti‐inflammatory cytokines from the loaded microspheres directly correlated with the degradation of the gelatin matrix. Exposure of the IL‐4 and IL‐13 loaded microspheres reduced the inflammation of chondrocytes up to 80%. Hence, the delivery of these microspheres in an OA joint can attenuate the stimulation of chondrocytes and the resulting secretion of catabolic factors such as proteinases and nitric oxide. The microsphere format also allows for minimally invasive delivery and is less susceptible to mechanically induced drug release. Consequently, bioresponsive microspheres can be an effective tool for cartilage preservation and arthritis treatment.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153665/1/jbma36852_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153665/2/jbma36852.pd

Use of Micro-Computed Tomography to Nondestructively Characterize Biomineral Coatings on Solid Freeform Fabricated Poly (L-Lactic Acid) and Poly (ɛ-Caprolactone) Scaffolds In Vitro and In Vivo

Biomineral coatings have been extensively used to enhance the osteoconductivity of polymeric scaffolds. Numerous porous scaffolds have previously been coated with a bone-like apatite mineral through incubation in simulated body fluid (SBF). However, characterization of the mineral layer formed on scaffolds, including the amount of mineral within the scaffolds, often requires destructive methods. We have developed a method using micro-computed tomography (?-CT) scanning to nondestructively quantify the amount of mineral in vitro and in vivo on biodegradable scaffolds made of poly (L-lactic acid) (PLLA) and poly (?-caprolactone) (PCL). PLLA and PCL scaffolds were fabricated using an indirect solid freeform fabrication (SFF) technique to achieve orthogonally interconnected pore architectures. Biomineral coatings were formed on the fabricated PLLA and PCL scaffolds after incubation in modified SBF (mSBF). Scanning electron microscopy and X-ray diffraction confirmed the formation of an apatite-like mineral. The scaffolds were implanted into mouse ectopic sites for 3 and 10 weeks. The presence of a biomineral coating within the porous scaffolds was confirmed through plastic embedding and ?-CT techniques. Tissue mineral content (TMC) and volume of mineral on the scaffold surfaces detected by ?-CT had a strong correlation with the amount of calcium measured by the orthocresolphthalein complex-one (OCPC) method before and after implantation. There was a strong correlation between OCPC pre- and postimplantation and ?-CT measured TMC (R2=0.96 preimplant; R2=0.90 postimplant) and mineral volume (R2=0.96 preimplant; R2=0.89 postimplant). The ?-CT technique showed increases in mineral following implantation, suggesting that ?-CT can be used to nondestructively determine the amount of calcium on coated scaffolds.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140242/1/ten.tec.2012.0495.pd

Noninvasive, Quantitative, Spatiotemporal Characterization of Mineralization in Three-Dimensional Collagen Hydrogels Using High-Resolution Spectral Ultrasound Imaging

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98485/1/ten%2Etec%2E2012%2E0180.pd

The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces

Controlling mesenchymal stromal cell (MSC) shape is a novel method for investigating and directing MSC behaviour in vitro. it was hypothesized that specifigc MSC shapes can be generated by using stiffnessâ defined biomaterial surfaces and by applying cyclic tensile forces. Biomaterials used were thin and thick silicone sheets, fibronectin coating, and compacted collagen type I sheets. The MSC morphology was quantified by shape descriptors describing dimensions and membrane protrusions. Nanoscale stiffness was measured by atomic force microscopy and the expression of smooth muscle cell (SMC) marker genes (ACTA2, TAGLN, CNN1) by quantitative reverseâ transcription polymerase chain reaction. Cyclic stretch was applied with 2.5% or 5% amplitudes. Attachment to biomaterials with a higher stiffness yielded more elongated MSCs with fewer membrane protrusions compared with biomaterials with a lower stiffness. For cyclic stretch, compacted collagen sheets were selected, which were associated with the most elongated MSC shape across all investigated biomaterials. As expected, cyclic stretch elongated MSCs during stretch. One hour after cessation of stretch, however, MSC shape was rounder again, suggesting loss of stretchâ induced shape. Different shape descriptor values obtained by different stretch regimes correlated significantly with the expression levels of SMC marker genes. Values of approximately 0.4 for roundness and 3.4 for aspect ratio were critical for the highest expression levels of ACTA2 and CNN1. Thus, specific shape descriptor values, which can be generated using biomaterialâ associated stiffness and tensile forces, can serve as a template for the induction of specific gene expression levels in MSC. Copyright © 2017 John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/1/term2263.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/2/term2263_am.pd

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches