Kevin A. Rocco

In vivo applications of electrospun tissue-engineered vascular grafts: a review.

There is great clinical demand for synthetic vascular grafts with improved long-term efficacy. The ideal vascular conduit is easily implanted, nonthrombogenic, biocompatible, resists aneurysmal dilatation, and ultimately degrades or is assimilated as the patient remodels the graft into tissue resembling native vessel. The field of vascular tissue engineering offers an opportunity to design the ideal synthetic graft, and researchers have evaluated a variety of methods and materials for use in graft construction. Electrospinning is one method that has received considerable attention within tissue engineering for constructing so-called tissue scaffolds. Tissue scaffolds are temporary, porous structures which are commonly composed of bioresorbable polymers that promote native tissue ingrowth and have degradation kinetics compatible with a patients rate of extracellular matrix production in order to successfully transit from synthetic conduits into neovessels. In this review, we summarize the history of tissue-engineered vascular grafts (TEVG), focusing on scaffolds generated by the electrospinning process, and discuss in vivo applications. We review the materials commonly employed in this approach and the preliminary results after implantation in animal models in order to gauge clinical viability of the electrospinning process for TEVG construction. Scientists have studied electrospinning technology for decades, but only recently has it been orthotopically evaluated in animal models such as TEVG. Advantages of electrospun TEVG include ease of construction, favorable cellular interactions, control of scaffold features such as fiber diameter and pore size, and the ability to choose from a variety of polymers possessing a range of mechanical and chemical properties and degradation kinetics. Given its advantages, electrospinning technology merits investigation for use in TEVG, but an emphasis on long-term in vivo evaluation is required before its role in clinical vascular tissue engineering can be realized.

Evaluation of remodeling process in small-diameter cell-free tissue-engineered arterial graft

OBJECTIVE Autologous grafts are used to repair atherosclerotic cardiovascular diseases; however, many patients lack suitable donor graft tissue. Recently, tissue engineering techniques have emerged to make biologically active blood vessels. We applied this technique to produce arterial grafts using established biodegradable materials without cell seeding. The grafts were evaluated in vivo for vessel remodeling during 12 months. METHODS Poly(L-lactide-co-ε-caprolactone) scaffolds reinforced by poly(lactic acid) (PLA) fiber were prepared as arterial grafts. Twenty-eight cell-free grafts were implanted as infrarenal aortic interposition grafts in 8-week-old female SCID/Bg mice. Serial ultrasound and micro computed tomography angiography were used to monitor grafts after implantation. Five grafts were harvested for histologic assessments and reverse transcription-quantitative polymerase chain reaction analysis at time points ranging from 4 months to 1 year after implantation. RESULTS Micro computed tomography indicated that most implanted mice displayed aneurysmal changes (three of five mice at 4 months, four of five mice at 8 months, and two of five mice at 12 months). Histologic assessments demonstrated extensive tissue remodeling leading to the development of well-circumscribed neovessels with an endothelial inner lining, a neointima containing smooth muscle cells and elastin, and a collagen-rich extracellular matrix. There were a few observed calcified deposits, located around residual PLA fibers at 12 months after implantation. Macrophage infiltration into the scaffold, as evaluated by F4/80 immunohistochemical staining, remained after 12 months and was focused mostly around residual PLA fibers. Reverse transcription-quantitative polymerase chain reaction analysis revealed that gene expression of Itgam, a marker for macrophages, and of matrix metalloproteinase 9 was higher than in native aorta during the course of 12 months, indicating prolonged inflammation (Itgam at 8 months: 11.75 ± 0.99 vs native aorta, P < .01; matrix metalloproteinase 9 at 4 months: 4.35 ± 3.05 vs native aorta, P < .05). CONCLUSIONS In this study, we demonstrated well-organized neotissue of cell-free biodegradable arterial grafts. Although most grafts experienced aneurysmal change, such findings provide insight into the process of tissue-engineered vascular graft remodeling and should allow informed rational design of the next generation of arterial grafts.

Future prospects for tissue engineered lung transplantation: decellularization and recellularization-based whole lung regeneration.

The shortage of donor lungs for transplantation causes a significant number of patient deaths. The availability of laboratory engineered, functional organs would be a major advance in meeting the demand for organs for transplantation. The accumulation of information on biological scaffolds and an increased understanding of stem/progenitor cell behavior has led to the idea of generating transplantable organs by decellularizing an organ and recellularizing using appropriate cells. Recellularized solid organs can perform organ-specific functions for short periods of time, which indicates the potential for the clinical use of engineered solid organs in the future. The present review provides an overview of progress and recent knowledge about decellularization and recellularization-based approaches for generating tissue engineered lungs. Methods to improve decellularization, maturation of recellularized lung, candidate species for transplantation and future prospects of lung bioengineering are also discussed.

Well-organized neointima of large-pore poly(l-lactic acid) vascular graft coated with poly(l-lactic-co-ε-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(l-lactic acid) graft in a mouse aortic implantation model

OBJECTIVE Tissue engineering techniques have emerged that allow bioresorbable grafts to be implanted that restore function and transform into biologically active arteries. However, these implants are susceptible to calcification during the remodeling process. The objective of this study was to evaluate the role of pore size of bioabsorbable grafts in the development of calcification. METHODS Two types of grafts were prepared: a large-pore graft constructed of poly(L-lactic acid) (PLA) fibers coated with poly(L-lactide-co-ε-caprolactone) (PLCL) (PLA-PLCL), and a small-pore graft made of electrospun PLA nanofibers (PLA-nano). Twenty-eight PLA-PLCL grafts and twenty-five PLA-nano grafts were implanted as infra-renal aortic interposition conduits in 8-week-old female SCID/Bg mice, and followed for 12 months after implantation. RESULTS Large-pore PLA-PLCL grafts induced a well-organized neointima after 12 months, and Alizarin Red S staining showed neointimal calcification only in the thin neointima of small-pore PLA-nano grafts. At 12 months, macrophage infiltration, evaluated by F4/80 staining, was observed in the thin neointima of the PLA-nano graft, and there were few vascular smooth muscle cells (VSMCs) in this layer. On the other hand, the neointima of the PLA-PLCL graft was composed of abundant VSMCs, and a lower density of macrophages (F4/80 positive cells, PLA-PLCL; 68.1 ± 41.4/mm(2) vs PLA-nano; 188.3 ± 41.9/mm(2), p = 0.007). The VSMCs of PLA-PLCL graft expressed transcription factors of both osteoblasts and osteoclasts. CONCLUSION These findings demonstrate that in mouse arterial circulation, large-pore PLA-PLCL grafts created a well-organized neointima and prevented calcific deposition compared to small-pore, electrospun PLA-nano grafts.

Development of tissue engineered vascular grafts and application of nanomedicine

Vascular Tissue Engineering belongs to a rapidly expanding discipline. Tissue engineered vascular grafts (TEVG) have a broad range of clinical application extending from use as small diameter vascular grafts in adult peripheral vasculature to serving as large vessel conduits in pediatric cardiovascular surgery. Several approaches have been utilized by different groups to design these grafts. Preliminary outcomes are exceedingly promising. These grafts have demonstrated the ability to transform into living blood vessels with growth potential and while the underlying mechanisms remain to be elucidated, it has been shown that inflammatory pathways may play an important role. Small animal experiments, development of cell seeding techniques and the application of nanotechnology have all contributed vastly to our understanding of the mechanisms involved in TEVG remodeling. The application of nanomedicine in TEVG design continues to expand at a rapid rate and has provided some clues as to how vascular graft design can be pursued in the future. In this review we discuss the current state of the field of tissue engineered vascular grafts and how the principles of nanomedicine are being applied to aid in the design of second-generation grafts.

Development of small diameter nanofiber tissue engineered arterial grafts.

The surgical repair of heart and vascular disease often requires implanting synthetic grafts. While synthetic grafts have been successfully used for medium-to-large sized arteries, applications for small diameter arteries (<6 mm) is limited due to high rates of occlusion by thrombosis. Our objective was to develop a tissue engineered vascular graft (TEVG) for small diameter arteries. TEVGs composed of polylactic acid nanofibers with inner luminal diameter between 0.5 and 0.6 mm were surgically implanted as infra-renal aortic interposition conduits in 25 female C17SCID/bg mice. Twelve mice were given sham operations. Survival of mice with TEVG grafts was 91.6% at 12 months post-implantation (sham group: 83.3%). No instances of graft stenosis or aneurysmal dilatation were observed over 12 months post-implantation, assessed by Doppler ultrasound and microCT. Histologic analysis of explanted TEVG grafts showed presence of CD31-positive endothelial monolayer and F4/80-positive macrophages after 4, 8, and 12 months in vivo. Cells positive for α-smooth muscle actin were observed within TEVG, demonstrating presence of smooth muscle cells (SMCs). Neo-extracellular matrix consisting mostly of collagen types I and III were observed at 12 months post-implantation. PCR analysis supports histological observations. TEVG group showed significant increases in expressions of SMC marker, collagen-I and III, matrix metalloproteinases-2 and 9, and itgam (a macrophage marker), when compared to sham group. Overall, patency rates were excellent at 12 months after implantation, as structural integrity of these TEVG. Tissue analysis also demonstrated vessel remodeling by autologous cell.

Tissue engineering in the vasculature.

Tissue engineering holds great promise to address complications and limitations encountered with the use of traditional prosthetic materials, such as thrombogenicity, infection, and future degeneration which represent the major morbidity and mortality after device implant surgery. The general concept of tissue engineering consists of three main components: a scaffold material, a cell type for seeding the scaffold, and biochemical, physio‐chemical signaling and remodeling process. This remodeling process is guided by cell signals derived from both seeded cells and host inflammatory cells that infiltrate the scaffold and deposit extracellular matrix, forming the neotissue. Vascular tissue engineering is at the forefront in the translation of this technology to clinical practice, as tissue engineered vascular grafts (TEVGs) have now been successfully implanted in children with congenital heart disease. In this report, we review the history, advances, and state of the art in TEVGs. Anat Rec, 297:83–97. 2014.

Characterization of evolving biomechanical properties of tissue engineered vascular grafts in the arterial circulation.

We used a murine model to assess the evolving biomechanical properties of tissue engineered vascular grafts (TEVGs) implanted in the arterial circulation. The initial polymeric tubular scaffold was fabricated from poly(lactic acid)(PLA) and coated with a 50:50 copolymer of poly(caprolactone) and poly(lactic acid)(P[PC/LA]). Following seeding with syngeneic bone marrow derived mononuclear cells, TEVGs (n=50) were implanted as aortic interposition grafts in wild-type mice and monitored serially using ultrasound. A custom biaxial mechanical testing device was used to quantify the in vitro circumferential and axial mechanical properties of grafts explanted at 3 or 7 months. At both times, TEVGs were much stiffer than native tissue in both directions. Repeated mechanical testing of some TEVGs treated with elastase or collagenase suggested that elastin did not contribute significantly to the overall stiffness whereas collagen did contribute. Traditional histology and immunostaining revealed smooth muscle cell layers, significant collagen deposition, and increasing elastin production in addition to considerable scaffold at both 3 and 7 months, which likely dominated the high stiffness seen in mechanical testing. These results suggest that PLA has inadequate in vivo degradation, which impairs cell-mediated development of vascular neotissue having properties closer to native arteries. Assessing contributions of individual components, such as elastin and collagen, to the developing neovessel is needed to guide computational modeling that may help to optimize the design of the TEVG.

Biomimetic Culture Reactor for Whole-Lung Engineering.

Abstract Decellularized organs are now established as promising scaffolds for whole-organ regeneration. For this work to reach therapeutic practice, techniques and apparatus are necessary for doing human-scale clinically applicable organ cultures. We have designed and constructed a bioreactor system capable of accommodating whole human or porcine lungs, and we describe in this study relevant technical details, means of assembly and operation, and validation. The reactor has an artificial diaphragm that mimics the conditions found in the chest cavity in vivo, driving hydraulically regulated negative pressure ventilation and custom-built pulsatile perfusion apparatus capable of driving pressure-regulated or volume-regulated vascular flow. Both forms of mechanical actuation can be tuned to match specific physiologic profiles. The organ is sealed in an elastic artificial pleura that mounts to a support architecture. This pleura reduces the fluid volume required for organ culture, maintains the organs position during mechanical conditioning, and creates a sterile barrier allowing disassembly and maintenance outside of a biosafety cabinet. The combination of fluid suspension, negative-pressure ventilation, and physiologic perfusion allows the described system to provide a biomimetic mechanical environment not found in existing technologies and especially suited to whole-organ regeneration. In this study, we explain the design and operation of this apparatus and present data validating intended functions.

Bioengineered lungs generated from human iPSCs-derived epithelial cells on native extracellular matrix

The development of an alternative source for donor lungs would change the paradigm of lung transplantation. Recent studies have demonstrated the potential feasibility of using decellularized lungs as scaffolds for lung tissue regeneration and subsequent implantation. However, finding a reliable cell source and the ability to scale up for recellularization of the lung scaffold still remain significant challenges. To explore the possibility of regeneration of human lung tissue from stem cells in vitro, populations of lung progenitor cells were generated from human iPSCs. To explore the feasibility of producing engineered lungs from stem cells, we repopulated decellularized human lung and rat lungs with iPSC‐derived epithelial progenitor cells. The iPSCs‐derived epithelial progenitor cells lined the decellularized human lung and expressed most of the epithelial markers when were cultured in a lung bioreactor system. In decellularized rat lungs, these human‐derived cells attach and proliferate in a manner similar to what was observed in the decellularized human lung. Our results suggest that repopulation of lung matrix with iPSC‐derived lung epithelial cells may be a viable strategy for human lung regeneration and represents an important early step toward translation of this technology.