Hydrogel-Based Engineering of Beige Adipose Tissue.

Brown and beige adipose tissues have a significant capacity for energy expenditure that may be exploited as a treatment for obesity and metabolic disease. However, the limited volumes of these tissues in adults hinders realization of this potential. Engineering beige adipose tissue may provide an alternative source of this tissue. In this paper we describe the preparation of poly(ethylene glycol) (PEGDA) hydrogels with mechanical properties similar to native adipose tissue. Adipose derived stem cells (ASC) were cultured in hydrogels without adhesive sequences or degradable monomers. Cells were able to differentiate, independent of scaffold properties and were maintained as a viable and functioning adipose tissue mass. The cells expressed their own basement membrane proteins consistent with the composition of adipose tissue. The ASCs could be induced to express uncoupling protein-1 (UCP-1) and cIDEA, makers of beige adipocytes with expression level varying with hydrogel stiffness. This hydrogel-based culture system serves as a first step in engineering beige adipose tissue.

Design of a composite biomaterial system for tissue engineering applications.

Biomaterials that regulate vascularized tissue formation have the potential to contribute to new methods of tissue replacement and reconstruction. The goal of this study was to develop a porous, degradable tissue engineering scaffold that could deliver multiple growth factors and regulate vessel assembly within the porous structure of the material. Porous hydrogels of poly(ethylene glycol)-co-(L-lactic acid) (PEG-PLLA) were prepared via salt leaching. The degradation time of the hydrogels could be controlled between 1 and 7 weeks, based on hydrogel composition. Fibrin was incorporated into the interconnected pores of the hydrogels to promote neovascularization and as a reservoir for rapid (<5 days) growth factor delivery. Poly(lactic-co-glycolic acid) (PLGA) microspheres were incorporated into the degradable polymeric hydrogel scaffold to allow sustained (>30 days) growth factor delivery. Fibroblast growth factor-1 (FGF-1) and platelet-derived growth factor-BB (PDGF-BB) were delivered from the system owing to their roles in the promotion of angiogenesis and vascular stabilization, respectively. Hydrogels tested in vivo with a subcutaneous implantation model were selected based on the results from in vitro degradation and growth factor release kinetics. Dual growth factor delivery promoted significantly more tissue ingrowth in the scaffold compared with blank or single growth factor delivery. The sequential delivery of FGF-1 following PDGF-BB promoted more persistent and mature blood vessels. In conclusion, a biomaterials system was developed to provide structural support for tissue regeneration, as well as delivery of growth factors that stimulate neovascularization within the structure prior to complete degradation.

Evaluation of MMP substrate concentration and specificity for neovascularization of hydrogel scaffolds.

Controlled vascular response in scaffolds following implantation remains a significant clinical challenge. A critical biomaterial design criterion is the synchronization of the rates of scaffold degradation and vascularized tissue formation. Matrix metalloproteinases (MMPs) are key enzymes that regulate neovascularization and extracellular matrix remodelling. Synthetic protease-sensitive hydrogels offer controllable environments for investigating the role of matrix degradation on neovascularization. In this study, PEG hydrogels containing MMP-sensitive peptides with increased catalytic activity for MMPs expressed during neovascularization were investigated. Scaffolds were functionalized with MMP-2-, MMP-14- or general collagenase-sensitive peptides and with varying peptide concentration using crosslinkers containing one (SSite) or multiple (TSite) repeats of each protease-sensitive sequence. Increasing peptide concentration enhanced the degradation kinetics of scaffolds functionalized with MMP-specific sequences while 80% of the collagenase-sensitive scaffolds remained upon exposure to MMP-2 and MMP-14. In vitro neovascularization was consistent with in vivo tissue invasion with significantly increased invasion occurring within SSite MMP-specific as compared to collagenase-sensitive hydrogels and with further invasion in TSite as compared to SSite hydrogels regardless of peptide specificity. All scaffolds supported in vivo neovascularization; however, this was not dependent on peptide specificity. These findings demonstrate that peptide concentration and specificity regulate in vivo scaffold degradation, neovascularization and matrix remodelling.

An initial evaluation of analyser-based phase-contrast X-ray imaging of carotid plaque microstructure.

Carotid artery plaque instability can result in rupture and lead to ischaemic stroke. Stability of plaques appears to be a function of composition. Current non-invasive imaging techniques are limited in their ability to classify distinct histological regions within plaques. Phase-contrast (PC) X-ray imaging methods are an emerging class of techniques that have shown promise for identifying soft-tissue features without use of exogenous contrast agents. This is the first study to apply analyser-based X-ray PC imaging in CT mode to provide three-dimensional (3D) images of excised atherosclerotic plaques. The results provide proof of principle for this technique as a promising method for analysis of carotid plaque microstructure. Multiple image radiography CT (MIR-CT), a tomographic implementation of X-ray PC imaging that employs crystal optics, was employed to image excised carotid plaques. MIR-CT imaging yields three complementary images of the plaque's 3D X-ray absorption, refraction and scatter properties. These images were compared with histological sections of the tissue. X-ray PC images were able to identify the interface between the plaque and the medial wall. In addition, lipid-rich and highly vascularized regions were visible in the images as well as features depicting inflammation. This preliminary research shows MIR-CT imaging can reveal details about plaque structure not provided by traditional absorption-based X-ray imaging and appears to identify specific histological regions within plaques. This is the first study to apply analyser-based X-ray PC imaging to human carotid artery plaques to identify distinct soft-tissue regions.

New alginate microcapsule system for angiogenic protein delivery and immunoisolation of islets for transplantation in the rat omentum pouch.

Severe hypoxia caused by a lack of vascular supply and an inability to retrieve encapsulated islets transplanted in the peritoneal cavity for biopsy and subsequent evaluation are obstacles to clinical application of encapsulation strategies for islet transplantation. We recently proposed an omentum pouch model as an alternative site of encapsulated islet transplantation and have also described a multi-layer microcapsule system suitable for coencapsulation of islets with angiogenic protein in which the latter could be encapsulated in an external layer to induce vascularization of the encapsulated islet graft. The purpose of the present study was to determine the angiogenic efficacy of fibroblast growth factor (FGF-1) released from the external layer of the new capsule system in the omentum pouch graft. We prepared 2 groups of alginate microspheres, each measuring ∼600 µm in diameter with a semipermeable poly-L-ornithine (PLO) membrane separating 2 alginate layers. While one group of microcapsules contained no protein (control), FGF-1 (1.794 µg/100 microcapsules) was encapsulated in the external layer of the other (test) group. From each of the 2 groups, 100 microcapsules were transplanted separately in an omentum pouch created in each normal Lewis rat and were retrieved after 14 days for analysis of vessel density using the technique of serial sample sections stained for CD31 with quantitative three-dimensional imaging. We found that FGF-1 released from the external layer of the test microcapsules induced a mean ± SD vessel density (mm(2)) of 198.8 ± 59.2 compared with a density of 128.9 ± 10.9 in pouches measured in control capsule implants (P = .03; n = 5 animals/group). We concluded that the external layer of our new alginate microcapsule system is an effective drug delivery device for enhancement of graft neovascularization in a retrievable omentum pouch.

FRNK overexpression limits the depth and frequency of vascular smooth muscle cell invasion in a three-dimensional fibrin matrix.

Pathological vascular smooth muscle cell (VSMC) behavior after vascular interventions such as angioplasty or bypass is initiated within the 3D environment of the vessel media. Here VSMCs proliferate, invade the surrounding matrix, migrate adluminally, and deposit substantial amounts of matrix, leading to myointimal hyperplasia and decreased blood flow to critical organs and tissue. Since focal adhesion kinase (FAK) mediates many of the VSMC responses to these pathologic events, it provides a reasonable pharmacologic target to limit this invasive VSMC behavior and to better understand the cellular pathophysiology of this disease. Here we quantified the effectiveness of disabling FAK in VSMCs with its dominant-negative inhibitor, FAK-related nonkinase (FRNK), in a clinically relevant 3D assay. We found that FRNK overexpression decreased VSMC invasion (both the length and frequency) in this matrix. These effects were demonstrated in the presence and absence of chemical mitotic inhibition, suggesting that FAK's effect on cellular matrix invasion, migration, and proliferation utilize separate and/or redundant signaling cascades. Mechanistically, FAK inhibition decreased its localization to focal adhesions which led to a significant decrease in FAK autophosphorylation and the phosphorylation of the serine/threonine kinase, AKT. Together these findings suggest that disruption of FAK signaling may provide a pharmaceutical tool that limits pathological VSMC cell behavior.

Cardiovascular gene delivery: The good road is awaiting.

Atherosclerotic cardiovascular disease is a leading cause of death worldwide. Despite recent improvements in medical, operative, and endovascular treatments, the number of interventions performed annually continues to increase. Unfortunately, the durability of these interventions is limited acutely by thrombotic complications and later by myointimal hyperplasia followed by progression of atherosclerotic disease over time. Despite improving medical management of patients with atherosclerotic disease, these complications appear to be persisting. Cardiovascular gene therapy has the potential to make significant clinical inroads to limit these complications. This article will review the technical aspects of cardiovascular gene therapy; its application for promoting a functional endothelium, smooth muscle cell growth inhibition, therapeutic angiogenesis, tissue engineered vascular conduits, and discuss the current status of various applicable clinical trials.