The primary graft-related complication during the first clinical trial evaluating the use of tissue-engineered vascular grafts (TEVGs) was stenosis. We investigated the role of macrophages in the formation of TEVG stenosis in a murine model. We analyzed the natural history of TEVG macrophage infiltration at critical time points and evaluated the role of cell seeding on neovessel formation. To assess the function of infiltrating macrophages, we implanted TEVGs into mice that had been macrophage depleted using clodronate liposomes. To confirm this, we used a CD11b-diphtheria toxin-receptor (DTR) transgenic mouse model. Monocytes infiltrated the scaffold within the first few days and initially transformed into M1 macrophages. As the scaffold degraded, the macrophage infiltrate disappeared. Cell seeding decreased the incidence of stenosis (32% seeded, 64% unseeded, P=0.024) and the degree of macrophage infiltration at 2 wk. Unseeded TEVGs demonstrated conversion from M1 to M2 phenotype, whereas seeded grafts did not. Clodronate and DTR inhibited macrophage infiltration and decreased stenosis but blocked formation of vascular neotissue, evidenced by the absence of endothelial and smooth muscle cells and collagen. These findings suggest that macrophage infiltration is critical for neovessel formation and provides a strategy for predicting, detecting, and inhibiting stenosis in TEVGs.

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We developed a tissue-engineered vascular graft composed of biodegradable scaffold seeded with autologous bone marrow-derived mononuclear cells (BMMCs) that is currently in clinical trial and developed analogous mouse models to study mechanisms of neovessel formation. We previously reported that seeded human BMMCs were rapidly lost after implantation into immunodeficient mice as host macrophages invaded the graft. As a consequence, the resulting neovessel was entirely of host cell origin. Here, we investigate the source of neotissue cells in syngeneic BMMC-seeded grafts, implanted into immunocompetent mouse recipients. We again find that seeded BMMCs are lost, declining to 0.02% at 14 d, concomitant with host macrophage invasion. In addition, we demonstrate using sex-mismatched chimeric hosts that bone marrow is not a significant source of endothelial or smooth muscle cells that comprise the neovessel. Furthermore, using composite grafts formed from seeded scaffold anastomosed to sex-mismatched natural vessel segments, we demonstrate that the adjacent vessel wall is the principal source of these endothelial and smooth muscle cells, forming 93% of proximal neotissue. These findings have important implications regarding fundamental mechanisms underlying neotissue formation; in this setting, the tissue-engineered construct functions by mobilizing the body's innate healing capabilities to "regenerate" neotissue from preexisting committed tissue cells.

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Summary Objective The development of a living, tissue engineered vascular graft (TEVG) holds great promise for advancing the field of cardiovascular surgery. However, the ultimate source and time needed to procure these cells remain problematic. Induced puripotent stem (iPS) cells have recently been developed and have the potential for creating a pluripotent cell line from a patient’s own somatic cells. In this study we evaluated the use of a sheet created from iPS cell-derived vascular cells as a potential source for the construction of TEVGs. Methods Male mouse iPS cells were differentiated into embryoid bodies with the hanging-drop method. Cell differentiation was confirmed by a decrease in the proportion of SSEA-1 positive cells over time using FACS. Expression of endothelial cell and smooth muscle cell markers was detected by Real-Time PCR. The differentiated iPS cell sheet was made using temperature-responsive dishes and then seeded onto a biodegradable scaffold composed of PGA-P(CL/LA) with a diameter of 0.8mm. These scaffolds were implanted as interposition grafts in the inferior vena cava of female SCID/bg mice (N=15). Graft function was serially monitored using ultrasound. The grafts were analyzed at 1, 4, and 10 weeks with histology and immunohistochemistry. The behavior of seeded differentiated iPS cells was tracked using Y-chromosome FISH and SRY Real- Time PCR. Results All mice survived without thrombosis, aneurysm formation, graft rupture or calcification. PCR evaluation of iPS cell sheets in vitro demonstrated increased expression of endothelial cell markers. Histological evaluation of the grafts demonstrated endothelialization with VWF and an inner layer with SMA and calponin positive cells at 10 weeks. The number of seeded differentiated iPS cells was found to decrease over time by Real-Time PCR (42.2% at 1wk, 10.4% at 4wks, 9.8% at 10wks). A fraction of the iPS cells were found to be TUNEL positive at 1 week. No iPS cells were found to co-localize with VWF or SMA positive cells at 10 weeks. Conclusions Differentiated iPS cells offer an alternative cell source for constructing TEVGs. Seeded iPS cells exerted a paracrine effect to induce neotissue formation in the acute phase and were reduced in number by apoptosis at later time points. Sheet seeding of our TEVG represents a viable mode of iPS cell delivery over time.

Background: The extracellular matrix (ECM) is a critical determinant of neovessel integrity. Materials and Methods: Thirty-six (polyglycolic acid + polycaprolactone and poly lactic acid) tissue-engineered vascular grafts seeded with syngeneic bone marrow mononuclear cells were implanted as inferior vena cava interposition grafts in C57BL/6 mice. Specimens were characterized using immunohistochemical staining and qPCR for representative ECM components in addition to matrix metalloproteinases (MMPs). Total collagen, elastin, and glycosaminoglycan (GAG) contents were determined. MMP activity was measured using zymography. Results: Collagen production on histology demonstrated an initial increase in type III at 1 week followed by type I production at 2 weeks and type IV at 4 weeks. Gene expression of both type I and type III peaked at 2 weeks, whereas type IV continued to increase over the 4-week period. Histology demonstrated fibrillin-1 deposition at 1 week followed by elastin production at 4 weeks. Elastin gene expression significantly increased at 4 weeks, whereas fibrillin-1 decreased at 4 weeks. GAG demonstrated abundant production at each time point on histology. Gene expression of decorin significantly increased at 4 weeks, whereas versican decreased over time. Biochemical analysis showed that total collagen production was greatest at 2 weeks, and there was a significant increase in elastin and GAG production at 4 weeks. Histological characterization of MMPs showed abundant production of MMP-2 at each time point, while MMP-9 decreased over the 4-week period. Gene expression of MMP-2 significantly increased at 4 weeks, whereas MMP-9 significantly decreased at 4 weeks. Conclusions: ECM production during neovessel formation is characterized by early ECM deposition followed by extensive remodeling.

A major limitation of tissue engineering research is the lack of noninvasive monitoring techniques for observations of dynamic changes in single tissue-engineered constructs. We use cellular magnetic resonance imaging (MRI) to track the fate of cells seeded onto functional tissue-engineered vascular grafts (TEVGs) through serial imaging. After in vitro optimization, murine macrophages were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles and seeded onto scaffolds that were surgically implanted as inferior vena cava interposition grafts in SCID/bg mice. Serial MRI showed the transverse relaxation times (T(2)) were significantly lower immediately following implantation of USPIO-labeled scaffolds (T(2) = 44 ± 6.8 vs. 71 ± 10.2 ms) but increased rapidly at 2 h to values identical to control implants seeded with unlabeled macrophages (T(2) = 63 ± 12 vs. 63 ± 14 ms). This strongly indicates the rapid loss of seeded cells from the scaffolds, a finding verified using Prussian blue staining for iron containing macrophages on explanted TEVGs. Our results support a novel paradigm where seeded cells are rapidly lost from implanted scaffolds instead of developing into cells of the neovessel, as traditionally thought. Our findings confirm and validate this paradigm shift while demonstrating the first successful application of noninvasive MRI for serial study of cellular-level processes in tissue engineering.

Objectives: To determine if children with laryngeal penetration on videofluoroscopic swallow study (VFSS) who received feeding interventions (thickened liquids, change in liquid flow rate and/or method of liquid delivery) had improved symptoms and decreased hospitalizations compared to those without intervention. Methods: We performed a retrospective cohort study of children under 2 years with laryngeal penetration on VFSS at our institution in 2015 to determine initial and follow-up VFSS findings, symptom improvement at follow-up, and hospitalization risk before and after VFSS. Proportions were compared with Fisher's exact test and hospitalizations with paired t-tests. Results: We evaluated 137 subjects with age 8.93±0.59 months who had laryngeal penetration without aspiration on VFSS. 55% had change in management, with 40% receiving thickening and 15% a change in flow rate. There was significant improvement in symptoms for children that had feeding intervention and this improvement was greatest with thickening (OR 41.8, 95% CI 12.34-141.69, p<0.001). On repeat VFSS, 26% had evidence of aspiration that was not captured on initial VFSS. Subjects had decreased total and pulmonary hospitalizations with feeding intervention and decreased pulmonary nights with thickening (p<0.05). Conclusions: Laryngeal penetration appears to be clinically significant in children with oropharyngeal dysphagia and interventions to decrease its occurrence are associated with improved outcomes including decreased symptoms of concern and hospitalization nights. Thickening or other feeding intervention should be considered for all symptomatic children with laryngeal penetration on swallow study.