Cadherin-11 binds to PDGFRß and enhances cell proliferation and tissue regeneration via the PDGFR-AKT signaling axis.

Intercellular adhesion through homotypic interaction between cadherins regulates multiple cellular processes including cytoskeletal organization, proliferation, and survival. In this paper, we provide evidence that cadherin-11 (CDH11) binds to and promotes cell proliferation both in vitro and in vivo in synergy with the platelet-derived growth factor receptor beta (PDGFRß). Engagement of CDH11 increased the sensitivity of cells to PDGF-BB by 10- to 100-fold, resulting in rapid and sustained phosphorylation of AKT, ultimately promoting and cell proliferation and tissue regeneration. Indeed, wound healing experiments showed that healing was severely compromised in Cdh11-/- mice, as evidenced by significantly decreased proliferation, AKT phosphorylation, and extracellular matrix (ECM) synthesis of dermal cells. Our results shed light into understanding how intercellular adhesion can promote cell proliferation and may have implications for tissue regeneration and cancer progression.

Cell-free vascular grafts that grow with the host.

Cell-free small diameter vascular grafts, based on small intestinal submucosa (SIS) functionalized with heparin and vascular endothelial growth factor (VEGF) manufactured and implanted successfully into the arterial system of neonatal lambs, where they remained patent and grew in size with the host to a similar extent and with similar rate as native arteries. Acellular tissue engineered vessels (A-TEV) integrated seamlessly into the native vasculature and developed confluent, functional endothelium that afforded patency. The medial layer was infiltrated by smooth muscle cells, showed no signs of calcification and developed contractile function. The vascular wall underwent remarkable extracellular matrix remodeling exhibiting elastin fibers and even inner elastic lamina within six months. Taken together, our results suggest that VEGF-based A-TEVs may be suitable for treatment of congenital heart disorders to alleviate the need for repeated surgeries, which are currently standard practice.

Cadherin-11 is a novel regulator of extracellular matrix synthesis and tissue mechanics.

We discovered that Cadherin-11 (CDH11) regulates collagen and elastin synthesis, both affecting the mechanical properties and contractile function of animal tissues. Using a Cdh11-null mouse model, we observed a significant reduction in the mechanical properties [Youngs' modulus and ultimate tensile strength (UTS)] of Cdh11(-/-) as compared to wild-type (WT) mouse tissues, such as the aorta, bladder and skin. The deterioration of mechanical properties (Youngs' modulus and UTS) was accompanied by reduced collagen and elastin content in Cdh11(-/-) mouse tissues as well as in cells in culture. Similarly, knocking down CDH11 abolished collagen and elastin synthesis in human cells, and consequently reduced their ability to generate force. Conversely, engagement of CDH11 through homophilic interactions, led to swift activation of the TGF-ß and ROCK pathways as evidenced by phosphorylation of downstream effectors. Subsequently, activation of the key transcription factors, MRTF-A (also known as MKL1) and MYOCD led to significant upregulation of collagen and elastin genes. Taken together, our results demonstrate a novel role of adherens junctions in regulating extracellular matrix (ECM) synthesis with implications for many important biological processes, including maintenance of tissue integrity, wound healing and tissue regeneration.

Animal models of cardiovascular disease as test beds of bioengineered vascular grafts.

The last two decades have seen many advances in regenerative medicine, including the development of tissue engineered vessels (TEVs) for replacement of damaged or diseased arteries or veins. Biomaterials from natural sources as well as synthetic polymeric materials have been employed in engineering vascular grafts. Recently, cell-free grafts have become available opening new possibilities for the next generation, off-the-shelf products. These TEVs are first tested in small or large animal models, which are usually young and healthy. However, the majority of patients in need of vascular grafts are elderly and suffer from comorbidities that may complicate their response to the implants. Therefore, it is important to evaluate TEVs in animal models of vascular disease in order to increase their predictive value and learn how the disease microenvironment may affect the patency and remodeling of vascular grafts. Small animals with various disease phenotypes are readily available due to the availability of transgenic or gene knockout technologies and can be used to address mechanistic questions related to vascular grafting. On the other hand, large animal models with similar anatomy, hematology and thrombotic responses to humans have been utilized in a preclinical setting. We propose that large animal models with certain pathologies or age range may provide more clinically relevant platforms for testing TEVs and facilitate the clinical translation of tissue engineering technologies by increasing the likelihood of success in clinical trials.

A novel ovine ex vivo arteriovenous shunt model to test vascular implantability.

The major objective of successful development of tissue-engineered vascular grafts is long-term in vivo patency. Optimization of matrix, cell source, surface modifications, and physical preconditioning are all elements of attaining a compatible, durable, and functional vascular construct. In vitro model systems are inadequate to test elements of thrombogenicity and vascular dynamic functional properties while in vivo implantation is complicated, labor-intensive, and cost-ineffective. We proposed an ex vivo ovine arteriovenous shunt model in which we can test the patency and physical properties of vascular grafts under physiologic conditions. The pressure, flow rate, and vascular diameter were monitored in real-time in order to evaluate the pulse wave velocity, augmentation index, and dynamic elastic modulus, all indicators of graft stiffness. Carotid arteries, jugular veins, and small intestinal submucosa-based grafts were tested. SIS grafts demonstrated physical properties between those of carotid arteries and jugular veins. Anticoagulation properties of grafts were assessed via scanning electron microscopy imaging, en face immunostaining, and histology. Luminal seeding with endothelial cells greatly decreased the attachment of thrombotic components. This model is also suture free, allowing for multiple samples to be stably processed within one animal. This tunable (pressure, flow, shear) ex vivo shunt model can be used to optimize the implantability and long-term patency of tissue-engineered vascular constructs.

Cell-free vascular grafts: Recent developments and clinical potential.

Recent advances in vascular tissue engineering have led to the development of cell-free grafts that are available off-the-shelf for on demand surgery. Challenges associated with cell-based technologies including cell sourcing, cell expansion and long-term bioreactor culture motivated the development of completely cell-free vascular grafts. These are based on decellularized arteries, decellularized cultured cell-based tissue engineered grafts or biomaterials functionalized with biological signals that promote in situ tissue regeneration. Clinical trials undertaken to demonstrate the applicability of these grafts are also discussed. This comprehensive review summarizes recent developments in vascular graft technologies, with potential applications in coronary artery bypass procedures, lower extremity bypass, vascular injury and trauma, congenital heart diseases and dialysis access shunts, to name a few.

A Simple Method for Fabrication of Microstructures Using a PDMS Stamp.

We report a simple method to fabricate PDMS (polydimethylsiloxane) microwell arrays on glass by using a PDMS stamp to study cell-to-cell adhesion. In the cell-to-cell study, a glass substrate is required since glass has better cell attachment. The microwell arrays are replicated from an SU-8 master mold, and then are transferred to a glass substrate by lifting the PDMS stamp, followed by oxygen plasma bonding of the PDMS stamp on the glass substrate. For the cell-to-cell adhesion, four different types of PDMS arrays (e.g., rectangle, bowtie, wide-rhombus, and rhombus) were designed to vary the cell-to-cell contact length. The transfer success rates of the microwell arrays were measured as a function of both the contact area of the PDMS and the glass substrate and the different ratios between the base polymers and the curing agent. This method of generating the microwell arrays will enable a simple and robust construction of PDMS-based devices for various biological applications.

Successful endothelialization and remodeling of a cell-free small-diameter arterial graft in a large animal model.

The large number of coronary artery bypass procedures necessitates development of off-the-shelf vascular grafts that do not require cell or tissue harvest from patients. However, immediate thrombus formation after implantation due to the absence of a healthy endothelium is very likely. Here we present the successful development of an acellular tissue engineered vessel (A-TEV) based on small intestinal submucosa that was functionalized sequentially with heparin and VEGF. A-TEVs were implanted into the carotid artery of an ovine model demonstrating high patency rates and significant host cell infiltration as early as one week post-implantation. At one month, a confluent and functional endothelium was present and the vascular wall showed significant infiltration of host smooth muscle cells exhibiting vascular contractility in response to vaso-agonists. After three months, the endothelium aligned in the direction of flow and the medial layer comprised of circumferentially aligned smooth muscle cells. A-TEVs demonstrated high elastin and collagen content as well as impressive mechanical properties and vascular contractility comparable to native arteries. This is the first demonstration of successful endothelialization, remodeling, and development of vascular function of a cell-free vascular graft that was implanted in the arterial circulation of a pre-clinical animal model.

Surgical technique for the implantation of tissue engineered vascular grafts and subsequent in vivo monitoring.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Arterial grafts exhibiting unprecedented cellular infiltration and remodeling in vivo: the role of cells in the vascular wall.

OBJECTIVE: To engineer and implant vascular grafts in the arterial circulation of a pre-clinical animal model and assess the role of donor medial cells in graft remodeling and function. APPROACH AND RESULTS: Vascular grafts were engineered using Small Intestinal Submucosa (SIS)-fibrin hybrid scaffold and implanted interpositionally into the arterial circulation of an ovine model. We sought to demonstrate implantability of SIS-Fibrin based grafts; examine the remodeling; and determine whether the presence of vascular cells in the medial wall was necessary for cellular infiltration from the host and successful remodeling of the implants. We observed no occlusions or anastomotic complications in 18 animals that received these grafts. Notably, the grafts exhibited unprecedented levels of host cell infiltration that was not limited to the anastomotic sites but occurred through the lumen as well as the extramural side, leading to uniform cell distribution. Incoming cells remodeled the extracellular matrix and matured into functional smooth muscle cells as evidenced by expression of myogenic markers and development of vascular reactivity. Interestingly, tracking the donor cells revealed that their presence was beneficial but not necessary for successful grafting. Indeed, the proliferation rate and number of donor cells decreased over time as the vascular wall was dominated by host cells leading to significant remodeling and development of contractile function. CONCLUSIONS: These results demonstrate that SIS-Fibrin grafts can be successfully implanted into the arterial circulation of a clinically relevant animal model, improve our understanding of vascular graft remodeling and raise the possibility of engineering mural cell-free arterial grafts.