Tissue engineering using autologous microcirculatory beds as vascularized bioscaffolds

Abstract

Classic tissue engineering paradigms are limited by the incorporation of a functional vasculature and a reliable means for reimplantation into the host circulation. We have developed a novel approach to overcome these obstacles using autologous explanted microcirculatory beds (EMBs) as bioscaffolds for engineering complex three-dimensional constructs. In this study, EMBs consisting of an afferent artery, capillary beds, efferent vein, and surrounding parenchymal tissue are explanted and maintained for 24 h ex vivo in a bioreactor that preserves EMB viability and function. Given the rapidly advancing field of stem cell biology, EMBs were subsequently seeded with three distinct stem cell populations, multipotent adult progenitor cells (MAPCs), and bone marrow and adipose tissue-derived mesenchymal stem cells (MSCs). We demonstrate MAPCs, as well as MSCs, are able to egress from the microcirculation into the parenchymal space, forming proliferative clusters. Likewise, human adipose tissue-derived MSCs were also found to egress from the vasculature and seed into the EMBs, suggesting feasibility of this technology for clinical applications. We further demonstrate that MSCs can be transfected to express a luciferase protein and continue to remain viable and maintain luciferase expression in vivo. By using the vascular network of EMBs, EMBs can be perfused ex vivo and seeded with stem cells, which can potentially be directed to differentiate into neo-organs or transfected to replace failing organs and deficient proteins.

Figure 1.

Bioengineering paradigms. a) The classic tissue engineering paradigm. b) Microvascular beds are a microcosm of the circulatory system, already patterned for nutrient exchange. c) The EMB approach to tissue engineering. d) Schematic illustration of perfusion bioreactor. e) Laboratory setup.

Figure 2.

Perfusion pressures maintained at physiological levels with use of agarose gel. a) Ex vivo perfusion pressure curve demonstrating initial perfusion period with high pressures. After addition of warm PBS-agarose matrix, perfusion pressures decrease (single arrow). Solidifying agarose caused a transient increase in pressures (double arrows), followed by a gradual decrease with the extended perfusion period. b) EMBs are cast into a PBS-agarose matrix at the start of the perfusion period. c) Following extended perfusion, there is little leakage or edema, and flow is preserved through the vascular bed.

Figure 3.

Indicators of EMB viability. a) EMBs cultivated at 6, 12, 18, and 24 h (black plots) were stained with NBT and compared to control EMBs (gray plots). Statistically significant differences in percentage of viable tissue was determined for all perfused EMBs compared to controls; *P < 0.001. EMBs perfused for 12 and 18 h did not show a statistically significant difference in tissue viability; #P > 0.05. b) H&E demonstrates that perfused EMBs (top) maintained cell viability and architecture, permitting extended flow compared to nonperfused EMBs (bottom), which demonstrate loss of architecture, intravascular debris, and apoptotic cells. c) Colocalization of lectin (green, left) and DiI-acLDL uptake (red, right) indicates perfused and functional endothelium, respectively, in the perfused EMBs (top row), compared to nonperfused control EMBs (bottom row). d) Gross appearance of replanted EMB after 18 h of ex vivo perfusion at PRD 0 (immediately after replantation, left) and at PRD 28 (right). e) Nonperfused control EMBs (PRD 0, left) exhibited full thickness necrosis by PRD 2.

Figure 4.

Artificial oxygen carrier augments EMB viability. a) Ex vivo tissue oxygenation with DMEM perfusate only and supplementation with oxyglobin (blue shading indicates physiological oxygenation in vivo). b) Oxygenation (bottom) was directly related to flow and perfusion pressures (top). c) NBT viability staining of EMBs perfused for 24 h with DMEM perfusate alone compared to HBOC supplementation. There was greater EMB viability in the HBOC-supplemented perfused EMBs compared to those perfused with DMEM alone; *P < 0.001. d) Percentage EMB survival at PRD7 after 24-h perfusion with DMEM perfusate alone compared to HBOC supplementation. Seventy-five percent of EMBs subjected to 24-h perfusion with HBOC survived as compared to 50% of those perfused with DMEM alone.

Figure 5.

Immunohistochemistry of MAPCs mobilizing onto vascularized bioscaffold. a, b) MAPCs (red, arrowheads) adhere to (a) and egress from (b) the EMB microcirculation. c) Following reimplantation, MAPC clusters can be identified by serial sections of hematoxylin (top) and DiI fluorescence (bottom, red). d) Bulk egress of MAPCs in EMB parenchymal tissue (arrowheads). Asterisks indicate vessels.

Figure 6.

Engraftment and proliferation of MAPCs and MSCs onto EMBs. a) Following infusion, gender-mismatched DiI-labeled MAPCs (red) can be readily identified by FISH (green, Y chromosome, white arrowheads; inset ×400). b) BrdU staining of MAPC clusters demonstrates proliferation of stem cells following engraftment (black arrowheads). c) MAPC clusters marked with DiI (red) and dapI (blue) staining stimulate angiogenesis or differentiate into functional neovessels (vasculogenesis) seen with lectin (green) perfusion within the EMB. d) Human MSCs engrafted onto EMBs and formed viable clusters, as seen on dapI staining. e) PKH26-fluorescent labeling.

Figure 7.

MSC transfection and luciferase expression in vivo. a) Rat bone marrow-derived MSCs were transfected with a luciferase plasmid and demonstrate luciferase expression in vitro. b) Luciferase expression can be detected in EMBs receiving direct injection of transfected MSCs; however, EMBs seeded with transfected MSCs through the bioreactor demonstrated markedly greater expression of luciferase activity. c) Graphical representation of relative luminescence (photons/s) between MSC injection and perfusion.