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Mesenchymal stromal cells (MSCs) are multipotent cells obtained from many tissues including bone marrow, adipose tissue, umbilical cord, amniotic fluid, and placenta. MSCs are the leading cell source for stem cell therapy due to their regenerative and immunomodulatory properties, their low risk of tumorigenesis and lack of ethical constraints. However, clinical applications of MSCs remain limited. MSC therapeutic development continues to pose challenges in terms of preparation, purity, consistency, efficiency, reproducibility, processing time and scalability. Additionally, there are issues with their poor engraftment and survival in sites of disease or damage that limit their capacity to directly replace damaged cells. A key recent development in MSC research, however, is the now widely accepted view that MSCs primarily exert therapeutic effects via paracrine factor secretion. One of the major paracrine effectors are extracellular vesicles (EVs). EVs represent a potential cell-free alternative to stem cell therapy but are also rapidly emerging as a novel therapeutic platform in their own right, particularly in the form of engineered EVs (EEVs) tailored to target a broad range of clinical indications. However, the development of EVs and EEVs for therapeutic application still faces a number of hurdles, including the establishment of a consistent, scalable cell source, and the development of robust GMP-compliant upstream and downstream manufacturing processes. In this review we will highlight the clinical challenges of MSC therapeutic development and discuss how EVs and EEVs can overcome the challenges faced in the clinical application of MSCs.

Biomimetic tissue-engineered vascular grafts (TEVGs) have immense potential to replace diseased small-diameter arteries (<4 mm) for the treatment of cardiovascular diseases. However, biomimetic approaches developed thus far only partially recapitulate the physicochemical properties of the native vessel. While it is feasible to fabricate scaffolds that are compositionally similar to native vessels (collagen and insoluble elastic matrix) using freeze-drying, these scaffolds do not mimic the aligned topography of collagen and elastic fibers found in native vessels. Extrusion-based scaffolds exhibit anisotropic collagen orientation but these scaffolds are compositionally dissimilar (cannot incorporate insoluble elastic matrix). In this study, an electrochemical fabrication technique was employed to develop a biomimetic elastin-containing bi-layered collagen scaffold which is compositionally and structurally similar to native vessels and the effect of insoluble elastin incorporation on scaffold mechanics and smooth muscle cell (SMC) response was investigated. Further, the functionality of human umbilical vein endothelial cells (HUVECs) on the scaffold lumen surface was assessed via immunofluorescence. Results showed that incorporation of insoluble elastin maintained the overall collagen alignment within electrochemically aligned collagen (ELAC) fibers and this underlying aligned topography can direct cellular orientation. Ring test results showed that circumferential orientation of ELAC fibers significantly improved scaffold mechanics. Real-time PCR revealed that the expression of α-smooth muscle actin (Acta2) and myosin heavy chain (MyhII) was significantly higher on elastin containing scaffolds suggesting that the presence of insoluble elastin can promote contractility in SMCs. Further, mechanical properties of the scaffolds significantly improved post-culture indicating the presence of a mature cell-synthesized and remodeled matrix. Finally, HUVECs expressed functional markers on collagen lumen scaffolds. In conclusion, electrochemical fabrication is a viable method for the generation of a functional biomimetic TEVG with the potential to be used in bypass surgery.

Tissue engineering approaches for small-diameter arteries require a scaffold that simultaneously maintains patency by preventing thrombosis and intimal hyperplasia, maintains its structural integrity after grafting, and allows integration. While synthetic and extracellular matrix-derived materials can provide some of these properties individually, developing a scaffold that provides the balanced properties needed for vascular graft survival in the clinic has been particularly challenging. After 30 years of research, there are now several scaffolds currently in clinical trials. However, these products are either being investigated for large-diameter applications or they require pre-seeding of endothelial cells. This progress report identifies important challenges unique to engineering vascular grafts for high pressure arteries less than 4 mm in diameter (e.g., coronary artery), and discusses limitations with the current usage of the term "small-diameter." Next, the composition and processing techniques used for generating tissue engineered vascular grafts (TEVGs) are discussed, with a focus on the benefits of blended materials. Other scaffolds for non-tissue engineering approaches and stents are also briefly mentioned for comparison. Overall, this progress report discusses the importance of defining the most critical challenges for small diameter TEVGs, developing new scaffolds to provide these properties, and determining acceptable benchmarks for scaffold responses in the body.

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Modulating the host response, including the accumulation of oxidized lipid species, is important for improving tissue engineered vascular graft (TEVG) viability. Accumulation of oxidized lipids promotes smooth muscle cell (SMC) hyper-proliferation and inhibits endothelial cell migration, which can lead to several of the current challenges for small-diameter TEVGs. Generating biomaterials that reduce lipid oxidation is important for graft survival and this assessment can provide a reliable correlation to clinical situations. In this study, we determined the collagen to poly(ε-caprolactone) (PCL) ratio required to limit the production of pro-inflammatory species, while maintaining the required mechanical strength for the graft. Electrospun conduits were prepared from 0%, 10%, and 25% blends of collagen/PCL (w/w) and implanted in the rat peritoneal cavity for four weeks. The results showed that adding collagen to the PCL conduits reduced the accumulation of oxidized lipid species within the implanted conduits. In addition, the ratio of collagen had a significant impact on the recruited cell phenotype and construct mechanics. All conduits exhibited greater than 44% yield strain and sufficient tensile strength post-implantation. In conclusion, these results demonstrate that incorporating collagen into synthetic electrospun scaffolds, both 10% and 25% blend conditions, appears to limit the pro-inflammatory characteristics after in vivo implantation.

The neoassembly and maturation of elastic matrix is an important challenge for engineering small-diameter grafts for patients with peripheral artery disease. We have previously shown that hyaluronan oligomers and transforming growth factor-β (elastogenic factors or EFs) promote elastogenesis in smooth muscle cell (SMC) culture. However, their combined effects on macrophages and inflammatory cells in vivo are unknown. This information is needed to use the body (e.g., peritoneal cavity) as an "in vivo bioreactor" to recruit autologous cells to implanted EF-functionalized scaffolds. In this study, we determined if peritoneal fluid cells respond to EFs like smooth muscle cells and if these responses differ between cells sourced during different stages of inflammation triggered by scaffold implantation. Electrospun poly(ε-caprolactone)/collagen conduits were implanted in the peritoneal cavity prior to peritoneal fluid collection at 3-42 days postimplantation. Cells from the fluid were cultured in vitro with and without EFs to determine their response. Their phenotype/behaviour was assessed with a DNA assay, quantitative real-time PCR, and immunofluorescence. The EFs reduced peritoneal cell proliferation, maintained cell contractility, and unexpectedly did not exhibit proelastic effects, which we attributed to differences in cell density. We found the greatest elastin deposition in regions containing a high cell density. Further, we found that cells isolated from the peritoneal cavity at longer times after conduit implantation responded better to the EFs and exhibited more CD31 expression than cells at an earlier time point. Overall, this study provides information about the potential use of EFs in vivo and can guide the design of future tissue-engineered vascular grafts.

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