Smurf2 Regulates Inflammation and Collagen Processing in Cutaneous Wound Healing through Transforming Growth Factor-ß/Smad3 Signaling.

Wound healing is a highly conserved process that restores the integrity and functionality of injured tissues. Transforming growth factor (TGF)-ß is a master regulator of wound healing, whose signaling is attenuated by the E3 ubiquitin ligase Smurf2. Herein, the roles of Smurf2 in cutaneous wound healing were examined using a murine incisional cutaneous model. Loss of Smurf2 increased early inflammation in the wounds and led to narrower wounds with greater breaking strength. Loss of Smurf2 also led to more linearized collagen bundles in normal and wounded skin. Gene expression analyses by real-time quantitative PCR indicated that Smurf2-deficient fibroblasts had increased levels of TGF-ß/Smad3 signaling and changes in expression profile of genes related to matrix turnover. The effect of Smurf2 loss on wound healing and collagen bundling was attenuated by the heterozygous loss of Smad3. Together, these results show that Smurf2 affects inflammation and collagen processing in cutaneous wounds by down-regulating TGF-ß/Smad3 signaling.

Long-Term Sustained Drug Delivery via 3D Printed Masks for the Development of a Heparin-Loaded Interlayer in Vascular Tissue Engineering Applications.

Current approaches in small-diameter vascular grafts for coronary artery bypass surgeries fail to address physiological variations along the graft that contribute to thrombus formation and ultimately graft failure. We present an innovative interlayer drug delivery system that can be utilized for the sustained delivery of heparin through a graft with a high degree of temporal and spatial control. A heparin-loaded gelatin methacrylate (gelMA) interlayer sits within a biohybrid composed of decellularized bovine pericardium (dECM) and poly(propylene fumarate) (PPF), and its UV crosslinking is controlled via three-dimensional (3D) printed shadow masks. The masks can be readily designed to modulate the incident light intensity on the graft, enabling us to control the resultant gelMA crosslinking and properties. A high heparin loading efficiency was obtained in gelMA and was independent of crosslinking. We achieved sustained heparin release over the course of 2 weeks within the biohybrid material using the 3D printed mask patterns. High doses of heparin were observed to have detrimental effects on endothelial cell function. However, when exposed to heparin in a slower, more sustained manner consistent with the masks, endothelial cells behave similarly to untreated cells. Further, slower release profiles cause significantly more release of tissue factor pathway inhibitor, an anticoagulant, than a faster release profile. The heparin-loaded gelMA interlayer we have developed is a useful tool for the temporal and spatial control of heparin release that supports endothelial function and promotes an antithrombotic environment.

Incorporating a structural extracellular matrix gradient into a porcine urinary bladder matrix-based hydrogel dermal scaffold.

The increasing prevalence of chronic, nonhealing wounds necessitates the investigation of full-thickness skin substitutes conducive to host integration and wound closure. Extracellular matrix (ECM)-based hydrogel scaffolds mimic the physiological matrix environment of dermal cells, thereby conferring favorable cellular adhesion, infiltration, and proliferation. However, low-concentration ECM hydrogels rapidly lose mechanical strength as they degrade, leaving them susceptible to shrinkage from fibroblast-mediated contraction. Conversely, high-concentration ECM hydrogels are typically too dense to permit nutrient diffusion and cellular migration. This study investigates the design and fabrication of a graded-concentration hydrogel composed of porcine urinary bladder matrix (UBM) as a dermal scaffold for potential use in chronic wound treatment. Our method of UBM isolation and decellularization effectively removed native DNA while preserving matrix proteins. Hydrogels composed of a range of decellularized UBM (dUBM) concentrations were characterized and used to design a three-tiered gradient hydrogel that promoted cellular activity and maintained structural integrity. The gradient dUBM hydrogel showed stability of cross-sectional area during collagenase degradation, despite considerable loss of mass. The gradient dUBM hydrogel also resisted fibroblast-mediated contraction while supporting high surface cell viability, demonstrating the mechanical support provided by denser layers of dUBM. Overall, incorporation of an ECM concentration gradient into a porcine UBM-based hydrogel scaffold capitalizes on the unique advantages of both high and low-concentration ECM hydrogels, and mitigates the structural weaknesses that have limited the efficacy of hydrogel dermal scaffolds for chronic wounds. Our gradient design shows promise for future development of stable, pro-regenerative wound scaffolds with customized architectures using 3D printing.

Extracellular Matrix for Small-Diameter Vascular Grafts.

To treat coronary heart disease, coronary artery bypass grafts are used to divert blood flow around blockages in the coronary arteries. Autologous grafts are the gold standard of care, but they are characterized by their lack of availability, low quality, and high failure rates. Alternatively, tissue-engineered small-diameter vascular grafts made from synthetic or natural polymers have not demonstrated adequate results to replace autologous grafts; synthetic grafts result in a loss of patency due to thrombosis and intimal hyperplasia, whereas scaffolds from natural polymers are generally unable to support the physiological conditions. Extracellular matrix (ECM) from a variety of sources, including cell-derived, 2D, and cannular tissues, has become an increasingly useful tool for this application. The current review examines the ECM-based methods that have recently been investigated in the field and comments on their viability for clinical applications.

Concurrent multi-lineage differentiation of mesenchymal stem cells through spatial presentation of growth factors.

Severe tendon and ligament injuries are estimated to affect between 300 000 and 400 000 people annually. Surgical repairs of these injuries often have poor long-term clinical outcomes because of resection of the interfacial tissue-the enthesis-and subsequent stress concentration at the attachment site. A healthy enthesis consists of distinct regions of bone, fibrocartilage, and tendon, each with distinct cell types, extracellular matrix components, and architecture, which are important for tissue function. Tissue engineering, which has been proposed as a potential strategy for replacing this tissue, is currently limited by its inability to differentiate multiple lineages of cells from a single stem cell population within a single engineered construct. In this study, we develop a multi-phasic gelatin methacrylate hydrogel construct system for spatial presentation of proteins, which is then validated for multi-lineage differentiation towards the cell types of the bone-tendon enthesis. This study determines growth factor concentrations for differentiation of mesenchymal stem cells towards osteoblasts, chondrocytes/fibrochondrocytes, and tenocytes, which maintain similar differentiation profiles in 3D hydrogel culture as assessed by qPCR and immunofluorescence staining. Finally, it is shown that this method is able to guide heterogeneous and spatially confined changes in mesenchymal stem cell genes and protein expressions with the tendency to result in osteoblast-, fibrochondrocyte-, and tenocyte-like expression profiles. Overall, we demonstrate the utility of the culture technique for engineering other musculoskeletal tissue interfaces and provide a biochemical approach for recapitulating the bone-tendon enthesis in vitro.

Assessment of decellularized pericardial extracellular matrix and poly(propylene fumarate) biohybrid for small-diameter vascular graft applications.

Autologous grafts are the current gold standard of care for coronary artery bypass graft surgeries, but are limited by availability and plagued by high failure rates. Similarly, tissue engineering approaches to small diameter vascular grafts using naturally derived and synthetic materials fall short, largely due to inappropriate mechanical properties. Alternatively, decellularized extracellular matrix from tissue is biocompatible and has comparable strength to vessels, while poly(propylene fumarate) (PPF) has shown promising results for vascular grafts. This study investigates the integration of decellularized pericardial extracellular matrix (dECM) and PPF to create a biohybrid scaffold (dECM+PPF) suitable for use as a small diameter vascular graft. Our method to decellularize the ECM was efficient at removing DNA content and donor variability, while preserving protein composition. PPF was characterized and added to dECM, where it acted to preserve dECM against degradative effects of collagenase without disturbing the material's overall mechanics. A transport study showed that diffusion occurs across dECM+PPF without any effect from collagenase. The modulus of dECM+PPF matched that of human coronary arteries and saphenous veins. dECM+PPF demonstrated ample circumferential stress, burst pressure, and suture retention strength to survive in vivo. An in vivo study showed re-endothelialization and tissue growth. Overall, the dECM+PPF biohybrid presents a robust solution to overcome the limitations of the current methods of treatment for small diameter vascular grafts. STATEMENT OF SIGNIFICANCE: In creating a dECM+PPF biohybrid graft, we have observed phenomena that will have a lasting impact within the scientific community. First, we found that we can reduce donor variability through decellularization, a unique use of the decellularization process. Additionally, we coupled a natural material with a synthetic polymer to capitalize on the benefits of each: the cues provided to cells and the ability to easily tune material properties, respectively. This principle can be applied to other materials in a variety of applications. Finally, we created an off-the-shelf alternative to autologous grafts with a newly developed material that has yet to be utilized in any scaffolds. Furthermore, bovine pericardium has not been investigated as a small diameter vascular graft.