As catheter-based structural heart interventions become increasingly complex, the ability to effectively model patient-specific valve geometry as well as the potential interaction of an implanted ...device within that geometry will become increasingly important. Our aim with this investigation was to combine the technologies of high-spatial resolution cardiac imaging, image processing software, and fused multi-material 3D printing, to demonstrate that patient-specific models of the mitral valve apparatus could be created to facilitate functional evaluation of novel trans-catheter mitral valve repair strategies. Clinical 3D transesophageal echocardiography and computed tomography images were acquired for three patients being evaluated for a catheter-based mitral valve repair. Target anatomies were identified, segmented and reconstructed into 3D patient-specific digital models. For each patient, the mitral valve apparatus was digitally reconstructed from a single or fused imaging data set. Using multi-material 3D printing methods, patient-specific anatomic replicas of the mitral valve were created. 3D print materials were selected based on the mechanical testing of elastomeric TangoPlus materials (Stratasys, Eden Prairie, Minnesota, USA) and were compared to freshly harvested porcine leaflet tissue. The effective bending modulus of healthy porcine MV tissue was significantly less than the bending modulus of TangoPlus (
p
< 0.01). All TangoPlus varieties were less stiff than the maximum tensile elastic modulus of mitral valve tissue (3697.2 ± 385.8 kPa anterior leaflet; 2582.1 ± 374.2 kPa posterior leaflet) (
p
< 0.01). However, the slopes of the stress-strain toe regions of the mitral valve tissues (532.8 ± 281.9 kPa anterior leaflet; 389.0 ± 156.9 kPa posterior leaflet) were not different than those of the Shore 27, Shore 35, and Shore 27 with Shore 35 blend TangoPlus material (
p
> 0.95). We have demonstrated that patient-specific mitral valve models can be reconstructed from multi-modality imaging datasets and fabricated using the multi-material 3D printing technology and we provide two examples to show how catheter-based repair devices could be evaluated within specific patient 3D printed valve geometry. However, we recognize that the use of 3D printed models for the development of new therapies, or for specific procedural training has yet to be defined.
This study investigated the chondrogenic activity of encapsulated mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) and its impact on the mechanical properties of injectable ...poly(N‐isopropylacrylamide)‐based dual‐network hydrogels loaded with poly(
l
‐lysine) (PLL). To this effect, an ex vivo study model was employed to assess the behavior of the injected hydrogels—specifically, their surface stiffness and integration strength with the surrounding cartilage. The highest chondrogenic activity was observed from AC‐encapsulated hydrogels, while the effect of PLL on MSC chondrogenesis was not apparent from biochemical analyses. Mechanical testing showed that there were no significant differences in either surface stiffness or integration strength among the different study groups. Altogether, the results suggest that the ex vivo model can allow further understanding of the relationship between biochemical changes within the hydrogel and their impact on the hydrogel's mechanical properties.
An ex vivo model was leveraged to investigate chondrogenesis and tissue integration of cell‐laden thermoresponsive poly(N‐isopropylacrylamide)‐based hydrogels of cocultures of mesenchymal stem cells and articular chondrocytes. Hydrogels loaded with a polycationic compound, poly(L‐lysine) were injected into cartilage explants, and the chondrogenic activity of the encapsulated cell populations was examined together with the hydrogel integration with the surrounding cartilage tissue. This work demonstrates the utility of the ex vivo model to characterize tissue‐engineered constructs in a more in vivo‐like environment.
About the Authors: Pamela K. Kreeger * E-mail: kreeger@wisc.edu Affiliation: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States of America ORCID ...logo https://orcid.org/0000-0001-8193-1007 Amy Brock Affiliation: Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, United States of America ORCID logo https://orcid.org/0000-0001-8255-9024 Holly C. Gibbs Affiliation: Microscopy and Imaging Center, Texas A&M University, College Station, Texas, United States of America K. Jane Grande-Allen Affiliation: Department of Bioengineering, Rice University, Houston, Texas, United States of America Alice H. Huang Affiliation: Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America ORCID logo https://orcid.org/0000-0002-5037-6829 Kristyn S. Masters Affiliation: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States of America ORCID logo https://orcid.org/0000-0001-6911-3116 Padmini Rangamani Affiliation: Department of Mechanical and Aerospace Engineering, University of California San Diego, San Diego, California, United States of America ORCID logo https://orcid.org/0000-0001-5953-4347 Michaela R. Reagan Affiliation: Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, United States of America Shannon L. Servoss Affiliation: Ralph E. Martin Department of Chemical Engineering, University of Arkansas, Fayetteville, Arkansas, United States of America Introduction In the spring of 2020, nearly all academic institutions went to some level of shutdown/quarantine in order to slow the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes Coronavirus Disease 2019 (COVID-19). Consistent with this, some of the authors experienced or observed messaging from department chairs, center leaders, or mentors telling principal investigators (PIs) that the pandemic situation has likely created “extra time” for them to focus on writing grants and developing new ideas. Discussions of these data have focused primarily on the fact that women do a disproportionate amount of house and childcare 5–7, and options used to provide support for this unpaid work have essentially evaporated (e.g., limiting outside workers into the home for cleaning, day cares not accessible to children of nonessential workers, and school and summer camp closures). ...the term “staff” refers to administrative staff, whether in support of the research or teaching missions of the university.
Thermogelling hydrogels based on poly(N‐isopropyl acrylamide) (pNiPAAm) and crosslinked with a peptide‐bearing macromer poly(glycolic acid)‐poly(ethylene glycol)‐poly(glycolic ...acid)‐di(but‐2‐yne‐1,4‐dithiol) (PdBT) were fabricated to assess the role of hydrogel charge and lower critical solution temperature (LCST) over time in influencing cellular infiltration and tissue integration in an ex vivo cartilage explant model over 21 days. The p(NiPAAm)‐based thermogelling polymer was synthesized to possess 0, 5, and 10 mol% dimethyl‐γ‐butyrolactone acrylate (DBA) to raise the LCST over time as the lactone rings hydrolyzed. Further, three peptides were designed to impart charge into the hydrogels via conjugation to the PdBT crosslinker. The positively, neutrally, and negatively charged peptides K4 (+), zwitterionic K2E2 (0), and E4 (−), respectively, were conjugated to the modular PdBT crosslinker and the hydrogels were evaluated for their thermogelation behavior in vitro before injection into the cartilage explant models. Samples were collected at days 0 and 21, and tissue integration and cellular infiltration were assessed via mechanical pushout testing and histology. Negatively charged hydrogels whose LCST changed over time (10 mol% DBA) were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation due to increased transport and diffusion across the hydrogel‐tissue interface. Indeed, the negatively charged thermogelling polymer groups containing 5 and 10 mol% DBA demonstrated cellular infiltration and cartilage‐like matrix deposition via histology. This study demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future cartilage tissue engineering strategies.
Transcatheter mitral valve repair (TMVR) therapies offer a minimally invasive alternative to surgical mitral valve (MV) repair for patients with prohibitive surgical risks. Pre-procedural planning ...and associated medical device modeling is primarily performed in silico, which does not account for the physical interactions between the implanted TMVR device and surrounding tissue and may result in poor outcomes. We developed 3D printed tissue mimics for modeling TMVR therapies. Structural properties of the mitral annuli, leaflets, and chordae were replicated from multi-material blends. Uniaxial tensile testing was performed on the resulting composites and their mechanical properties were compared to those of their target native components. Mimics of the MV annulus printed in homogeneous strips approximated the tangent moduli of the native mitral annulus at 2% and 6% strain. Mimics of the valve leaflets printed in layers of different stiffnesses approximated the force–strain and stress–strain behavior of native MV leaflets. Finally, mimics of the chordae printed as reinforced cylinders approximated the force–strain and stress–strain behavior of native chordae. We demonstrated that multi-material 3D printing is a viable approach to the development of tissue phantoms, and that printed patient-specific geometries can approximate the local deformation force which may act upon devices used for TMVR therapies.
Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical ...nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.
To generate physiologically-relevant experimental models, the study of enteric diarrheal diseases is turning increasingly to advanced
in vitro
models that combine
ex vivo
, stem cell-derived ...“organoid” cell lines with bioengineered culture environments that expose them to mechanical stimuli, such as fluid flow. However, such approaches require considerable technical expertise with both microfabrication and organoid culture, and are, therefore, inaccessible to many researchers. For this reason, we have developed a perfusion system that is simple to fabricate, operate, and maintain. Its dimensions approximate the volume and cell culture area of traditional 96-well plates and allow the incorporation of fastidious primary, stem cell-derived cell lines with only minimal adaptation of their established culture techniques. We show that infections with enteroaggregative
E. coli
and norovirus, common causes of infectious diarrhea, in the system display important differences from static models, and in some ways better recreate the pathophysiology of
in vivo
infections. Furthermore, commensal strains of bacteria can be added alongside the pathogens to simulate the effects of a host microbiome on the infectious process. For these reasons, we believe that this perfusion system is a powerful, yet easily accessible tool for studying host-pathogen interactions in the human intestine.
Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of ...facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.
Display omitted
Biomaterials for tissue engineering that recapitulate the mechanical response and biological function of native tissue are highly sought after to lessen the burden of damaged or ...diseased tissue. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels are a popular candidate because of their favorable bioactive properties. However, their mechanical behavior is very dissimilar to that of biological tissue, which behaves in a mechanically anisotropic, nonlinear, and viscoelastic fashion. It has been previously shown that PEGDA hydrogels can be patterned in alternating linear strips of different stiffnesses to generate anisotropic behavior, but these constructs still have a linear stress-strain response. In this study, we imparted nonlinear mechanical properties to PEGDA hydrogels by fabricating composite hydrogel constructs consisting of a stiff sinusoidal reinforcement embedded into a softer base matrix. This was achieved by polymerizing low molecular weight (MW) PEGDA hydrogel precursor into a stiff sinusoidal shape and then polymerizing this construct into a high MW precursor. Samples were generated with different relative stiffness between the two components and a range of sinusoid periodicities to assess the tunability of the resulting stress-strain curve. Tensile testing indicates that the sinusoidal patterning gives rise to nonlinear stress-strain behavior. Varying the relative stiffness was shown to tune the slope of the linear region of the stress-strain curve, and varying periodicity was shown to affect the length of the toe region of this curve. We conclude that composite hydrogels with stiff sinusoidally-patterned reinforcements display mechanical properties more similar to those of biological tissue than uniform or linearly-patterned hydrogels.
Hydrogel biomaterials are a popular candidate for engineering constructs that can mimic the properties of native tissue for disease modeling and tissue-engineering applications. Studies have shown that poly(ethylene) glycol diacrylate (PEGDA) hydrogels can be fabricated to display many biological aspects of native tissue. However, they are unable to recapitulate fundamental mechanical properties of such tissue, such as anisotropy and nonlinearity. Photolithographic techniques have been employed to generate anisotropic linear PEGDA hydrogels via patterned reinforcement. The present study indicates that such techniques can be modified to generate PEGDA constructs with a sinusoidal reinforcement that display a strongly nonlinear response to tensile loading. This work sets the stage for more intricate patterning for providing increased control over hydrogel mechanical response.
The aortic valve consists of valvular interstitial cells (VICs) and endothelial cells (VECs). While these cells are understood to work synergistically to maintain leaflet structure and valvular ...function, few co-culture models of these cell types exist. In this study, aortic valve co-cultures (AVCCs) were assembled using magnetic levitation and cultured for 3days. Immunohistochemistry and quantitative reverse-transcriptase polymerase chain reaction were used to assess the maintenance of cellular phenotype and function, and the formation of extracellular matrix. AVCCs stained positive for CD31 and α-smooth muscle actin (αSMA), demonstrating that the phenotype was maintained. Functional markers endothelial nitric oxide synthase (eNOS), von Willebrand factor (VWF) and prolyl-4-hydroxylase were present. Extracellular matrix components collagen type I, laminin and fibronectin also stained positive, with reduced gene expression of these proteins in three dimensions compared to two dimensions. Genes for collagen type I, lysyl oxidase and αSMA were expressed less in AVCCs than in 2-D cultures, indicating that VICs are quiescent. Co-localization of CD31 and αSMA in the AVCCs suggests that endothelial–mesenchymal transdifferentiation might be occurring. Differences in VWF and eNOS in VECs cultured in two and three dimensions also suggests that the AVCCs possibly have anti-thrombotic potential. Overall, a co-culture model of the aortic valve was designed, and serves as a basis for future experiments to understand heart valve biology.
PDF
