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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2005 as doi:10.1096/fj.05-4802fje. |
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,1,
* Clinic of Vascular Surgery;
Department of Histology, Microbiology, and Medical Biotechnologies; and
Clinic of Plastic Surgery, University of Padova, Padova, Italy
1Correspondence: Viale G. Colombo 3, Padova, PD, 35121 Italy. E-mail: g.abatanelo{at}unipd.it
SPECIFIC AIMS
The aim of the present study was to develop a prosthetic graft that could perform as a small diameter vascular conduit. Up to now, the major concern about the use of small diameter prostheses (<6 mm) derives from their relatively high thrombogeneity due to the inability of the material to permit a rapid and complete endothelial layer regeneration. Despite numerous investigations, the development of a prosthetic graft capable of performing adequately as a small diameter conduit has been elusive. To address this lack of a successful vascular prosthesis, we decided to reproduce and positively guide the remodeling process directly in vivo, using a 2 mm diameter and 1 cm in length hyaluronic acid-based tubular scaffold (HYAFF 11) that functioned only as a temporary absorbable guide, similar to an in vivo "artery-bioregeneration assist tube" (ABAT).
PRINCIPAL FINDINGS
Tubular structures of hyaluronan (HYAFF-11TM tubules, 2 mm diameter, 1 cm length) were grafted in the abdominal aorta of 30 rats. Five animals were killed at 5, 15, 30, 60,120, and 180 days. Sections of the explanted graft were stained with hematoxylin-eosin (HE) and Weighert solution, and evaluated for host cell infiltration. Immunofluorescent studies of endothelial cells used antibodies to von Willebrand factor (vWF), CD34, and vascular endothelial growth factor receptor-2 (VEGFR-2). To characterize vascular smooth muscle cells, cells were investigated for expression of myosin light chain kinase (MLCK). Primary antibodies used to determine presence of collagens were anti-collagen I, anti-collagen III, and anti-collagen VI. Samples were also processed for transmission electron microscopy.
Results assessed the feasibility of creating a completely biodegradable vascular regeneration guide in vivo, and three novel findings stood out: 1) endothelialization of the tube luminal surface within 5 days; 2) sequential regeneration of the other vascular components that leads to a complete vascular walls regeneration after 15 days from surgery; 3) temporariness of the tube; biomaterial was entirely degraded after 4 months from implantation, and after that, a new artery remained to connect artery stumps.
1. Endothelialization of the prosthesis luminal surface
Figure 1
illustrates the prosthesis 5 days after surgery. The biomaterial was clearly evident and maintained its structure (blue arrows in Fig. 1A
, B). Longitudinal sectioning revealed the presence of a thin layer of newly formed tissue both on the luminal and external surface of the tubule. Regeneration of neovascular tissue originated from proximal and distal anastomotic sites (black arrows in Fig. 1A
), growing inside the tube without signs of infiltration into the prosthesis wall (Fig. 1A, B
), and converging in the middle. Simultaneously, an adventitial tissue grew from the aorta and externally enveloped the vascular conduit at the anastomotic site (Fig. 1A, B
). Figure 1C, D
illustrates the absence of a muscular and elastic component in the new vessel at this time. Positive staining for elastic fibers and vascular smooth muscle cells was confined to the original artery tract and did not enter anastomotic sites (arrows in Fig. 1C, D
). Regenerated tissue, which lined the luminal surface of the tube, was principally comprised of vWF+ and VEGFR-2+ endothelial cells (Fig. 1E, F
) and by CD34+ progenitor cells (Fig. 1G
). In particular, the presence of CD34+ and VEGFR-2+ cells attested to the participation of circulating endothelial progenitor cells in these early regenerative events (Fig. 1F, G
). Positive staining for CD34 was confined to the neo-artery tract and was negative in the original artery tract (white arrow in Fig. 1G
marks the transition point between original and new artery).
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2. Sequential regeneration of the other vascular components that leads to a complete regeneration of vascular walls after 15 days from surgery
Fifteen days after implantation, the biomaterial was still present, its structure was intact, and it enveloped the neo-vessel with no signs of vascular tissue infiltration. Connective tissue surrounded the biomaterial externally, resembled an adventitial wall, and isolated the biomaterial from the external environment. Flattened cells were observed on the luminal surface surrounded by other cell components, which collectively resembled a natural artery wall with no signs of intimal hyperplasia. Vascular lumen was lined with cells that stained positively for vWF. Endothelial cells were also clearly illustrated by transmission electron microscopy. Immunofluorescence confirmed the presence of a continuous thin layer of vascular smooth muscle cells, as indicated by positive staining with an antibody specific for MLCK. Elastic components of the neo-artery wall progressively started to regenerate from the anastomotic site.
3. Biomaterial was entirely degraded after 4 months from implantation, and after that, a new artery remained to connect artery stumps
By the 120th day, the biomaterial was completely degraded. The neo-artery macroscopically maintained its anatomical structure and the artery lumen was patent with no signs of dilatation or collapse (Fig. 2
A). At the 180th day (Fig. 2A, B
), walls of neo-tissue were well integrated and of the same diameter as the original artery (Fig. 2A, B
). The biomaterial was absent and substituted by neo-adventitial tissue that was completely fused with media (black arrows in Fig. 2B
). Macrophages were still present; however, monocytes and neutrophils were absent or scarcely visible. Immunofluorescence confirmed the presence of a continuous thin layer of mature endothelial cells (Fig. 2C
) and vascular smooth muscle cells (Fig. 2D
). Weighert's stain demonstrated a thin but linear and continuous elastic component and muscle layer (Fig. 2E
).
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CONCLUSIONS AND SIGNIFICANCE
This study assessed the feasibility of creating a completely biodegradable vascular regeneration guide in vivo. The difficulty of obtaining significant long-term patency and good wall mechanical strength in vivo has been a significant obstacle in achieving this goal. Results of this study demonstrate that the vascular prosthesis tested was able to sequentially orchestrate the vascular regeneration events needed for very small artery reconstruction (2 mm diameter). A tubular scaffold was used as a temporary absorbable guide that served as an in vivo ABAT to promote the complete regeneration of the vascular structures. Gross observations showed that ABAT was strong enough to withstand internal pressure for the entire regenerative process, and sutures supported the prosthesis anchored to artery stumps until the complete regeneration of the neo-artery segment. The prosthesis also isolated the regeneration process from the external environment and its potentially damaging intervention.
Macroscopic and histological results confirmed the ability of the prosthesis to create a biomimetic environment during tissue formation. The regenerated vessel grew inside the tubule without signs of infiltration into the biomaterial wall. The ABAT demonstrated good in vivo durability until the complete regeneration of the neo-vascular tract, preventing common complications such as abnormal dilatation and acute thrombosis.
After 5 days, the endothelial layer was evident along the luminal surface. Participation of endothelial progenitor cells (EPCs) in regenerative events was confirmed by the presence of CD34+ and VEGFR-2+ cells. When regenerated vascular tissue growing from the proximal and distal anastomotic sites joined at the midline, cells lost CD34 expression and were positive only for vWF, which is the typical marker of mature endothelial cells. This indicated that the biomaterial surface promoted the anchoring of EPCs to its surface, favoring the regeneration process.
After 15 days, all vascular structures were regenerated. A layer of vascular smooth muscle cells and extracellular matrix components, such as collagen and elastin, were visible.
After 180 days, the biomaterial was completely substituted by adventitial tissue that fused with the inner layers of the regenerated artery. The survival rate was 100% and there were no signs of insufficient vascular perfusion in peripheral regions.
The hyaluronic acid-derived tubular prostheses used in the preliminary experimental model described above seemed to fulfill the major requirements of a microvascular scaffold: it allowed rapid vascular tissue ingrowths, facilitated rapid endothelialization of the luminal surface, and withstood pulsatile stress. These findings are an encouraging step toward the possible use of this vascular conduit in clinical situations, and they provide a tool to study the mechanisms and phases of artery regeneration and its pharmacological modulation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4802fje;
This article has been cited by other articles:
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  B. Zavan, V. Vindigni, S. Lepidi, I. Iacopetti, G. Avruscio, G. Abatangelo, and R. Cortivo Neoarteries grown in vivo using a tissue-engineered hyaluronan-based scaffold FASEB J, August 1, 2008; 22(8): 2853 - 2861. [Abstract] [Full Text] [PDF]
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