Elsevier

Acta Biomaterialia

Volume 6, Issue 7, July 2010, Pages 2448-2456
Acta Biomaterialia

Cyclically stretching developing tissue in vivo enhances mechanical strength and organization of vascular grafts

https://doi.org/10.1016/j.actbio.2010.01.041Get rights and content

Abstract

Tissue-engineered vascular grafts must have qualities that rival native vasculature, specifically the ability to remodel, the expression of functional endothelial components and a dynamic and functional extracellular matrix (ECM) that resists the forces of the arterial circulation. We have developed a device that when inserted into the peritoneal cavity, attracts cells around a tubular scaffold to generate autologous arterial grafts. The device is capable of cyclically stretching (by means of a pulsatile pump) developing tissue to increase the mechanical strength of the graft. Pulsed (n = 8) and unpulsed (n = 8) devices were implanted for 10 days in Lovenaar sheep (n = 8). Pulsation occurred for a period of 5–8 days before harvest. Thick unadhered autologous tissue with cells residing in a collagen ECM was produced in all devices. Collagen organization was greater in the circumferential direction of pulsed tissue. Immunohistochemical labelling revealed the hematopoietic origin of >90% cells and a significantly higher coexpression with vimentin in pulsed tissue. F-actin expression, mechanical failure strength and strain were also significantly increased by pulsation. Moreover, tissue could be grafted as carotid artery patches. This paper shows that unadhered tissue tubes with increased mechanical strength and differentiation in response to pulsation can be produced with every implant after a period of 10 days. However, these tissue tubes require a more fine-tuned exposure to pulsation to be suitable for use as vascular grafts.

Introduction

Vascular diseases are often treated by replacing affected tissue. Synthetic grafts are successful large-diameter blood vessel replacements and for small-diameter grafts, e.g. coronary artery bypass, autologous artery or vein grafts are the major grafts. However, small-diameter synthetic grafts are prone to failure due to a lack of compliance or thrombosis [1], [2]. Synthetic materials do not possess the functional characteristics that make them viable long-term conduits in small-diameter large-flow environments such as the coronary arterial circulation, but especially in vascular shunts used for haemodialysis access in diabetic patients. Consequently, no ideal graftable material has yet been constructed, despite significant advances in polymer chemistry and tissue engineering. In contrast to these synthetic grafts, arteries respond to hemodynamic forces in the circulation by expanding and contracting. Yet they remain compliant in response to these forces because of the presence of elastic fibres and collagen fibrils. Because of these differences between the current grafts and the native tissue, and the lack of suitable small-calibre blood vessel grafts, it is necessary to search for better grafts, a major tissue engineering challenge.

Tissue derived from implantation of a foreign body in the peritoneal cavity is known to contain a population of cells that differentiate into contractile smooth muscle-like cells, producing a strong collagen matrix, and have the ability to synthesize elastin [3], [4], [5]. These cells arise from monocyte/macrophage precursors predominantly, with a mixed population of immune cells [4] and the tissue, produced in a tubular shape, can be used as a vascular graft.

The potential for the tissue derived from the peritoneal cavity to be used as an autologous graft has been explored in various animal models. The tissue capsules produced by the foreign body response in the peritoneal cavity have been grafted as an autologous interposition graft in dog femoral artery [5], rat abdominal aorta [3] and rabbit carotid artery [6]. The myofibroblasts of these grafts were almost indistinguishable from smooth muscle cells (SMCs) of the adjacent native artery into which they were grafted [7] and a 80% patency rate was observed in dogs [5]. Grafting these tubes into the arterial circulation as a vascular substitute has thus far met with limited long-term success as vascular shunts and bypass grafts, partly due to an insufficiently strong and elastic extracellular matrix (ECM) at the time of harvest.

Many groups have reported that stretching cells in vitro can induce a change in cell phenotype and stimulation of production of ECM proteins [7], [8]. A strain as small as 10% at 1 Hz for 8 days on a scaffold with resident SMCs can show a tripling of ultimate tensile strength and ECM deposition [9], [10], [11]. Stretch placed on vascular grafts is so powerful in directing differentiation and secretion of ECM that cells that otherwise do not secrete elastin in vitro will secrete elastin in response to cyclic mechanical force [12], [13]. Myofibroblast differentiation and contractile activity are dependent on mechanical stimulation [14]. It is clear that the production of a tissue-engineered vascular graft that utilizes a large proportion of live cells will need to experience mechanical force to develop a strong ECM and cells with properties of native arterial cells.

Because of the promising results obtained with foreign body derived arterial grafts and previous work done with in vitro bioreactor stimulus upon peritoneal-derived tissue (unpublished data), we hypothesize that applying cyclic stretching forces within physiological range to the developing foreign body reaction will ameliorate the strength and development of the tissue. This work aims to assess the possibility to strengthen the tissue and shorten the development time, necessary to produce adequate tissue for engraftment as a vascular graft. For this purpose an implantable device was created consisting of an inner stretchable tube, a perforated external tube and a transabdominal tube. Cyclic force was then applied by means of a ventricle pump for a period of 5–8 days. The resultant tissue was then implanted into the circulation of two sheep as a carotid artery patch.

Section snippets

Animals and devices

Lovenaar sheep (n = 8, ewes 30–40 kg) were selected and cared for in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH Publication 85-23, revised 1985). The study was approved by the local Ethics Committee.

The peritoneal implant devices (Fig. 1A) were designed to recruit cells around a pulsating inner tubular scaffold of 100 mm length and 6 mm external diameter and providing protection against adhering to the omentum or visceral organs. The outer tubes had 2 mm diameter

Pulsation and macroscopy

In all implantations, both pulsed devices and unpulsed controls, avascularized unadhered free-floating tissue was produced. The macroscopic integrity of tissue was not different between devices implanted on the left and the right side of the animal. Four animals developed device leaks approximately 4–5 days into the pulsing procedure. These device incubations had to be terminated at this time. However, the sheep’s health and the tissue quality that was produced in unpulsed or pulsed samples

Discussion

These results have shown how pulsatile mechanical stretch can initiate changes in the development of tissue in the peritoneal cavity. At the macroscopic level, the differences between pulsed and unpulsed tissue were remarkable. Pulsed tissue was much paler in colour, more homogenous, dense, less translucent and of a consistent colour that resembled native tissue. In contrast, unpulsed tissue expressed a yellow tinge that was slightly opaque and jelly-like. The pulsed tissue also displayed tone

Conclusions

This study clearly shows that applying cyclic and physiological loading (20% diameter increase at 1 Hz for 8 days) to a tissue that develops in vivo alters the mechanical properties towards a more than twofold increase in tissue strength, which clearly proves our initial hypothesis. This stretch is even sufficient to produce changes in ECM organization and some contractile protein expression. However, mechanical properties were still inferior to native tissue properties and higher-order

Author disclosure statement

No competing financial interests exist.

Acknowledgements

The authors thank Ruth Plusquin, Kristof Reyniers, Veerle Leunens and Grant Edwards for their excellent technical assistance. This project was made possible with the assistance of the University of Queensland Postgraduate School Research Travel Grant scheme and partially funded by FWO (G.0549.06).

References (33)

  • J.L. Efendy et al.

    The effect of environmental cues on the differentiation of myofibroblasts in peritoneal granulation tissue

    J Pathol

    (2000)
  • M.M. Thi et al.

    Fluid shear stress upregulates vascular endothelial growth factor gene expression in osteoblasts

    Ann NY Acad Sci

    (2007)
  • D. Seliktar et al.

    Dynamic mechanical conditioning of collagen-gel blood vessel constructs induces remodeling in vitro

    Ann Biomed Eng

    (2000)
  • D. Seliktar et al.

    The role of matrix metalloproteinase-2 in the remodeling of cell-seeded vascular constructs subjected to cyclic strain

    Ann Biomed Eng

    (2001)
  • B.C. Isenberg et al.

    Long-term cyclic distention enhances the mechanical properties of collagen-based media-equivalents

    Ann Biomed Eng

    (2003)
  • D.J. Johnson et al.

    Decreased elastin synthesis in normal development and in long-term aortic organ and cell cultures is related to rapid and selective destabilization of mRNA for elastin

    Circ Res

    (1995)
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    These authors contributed equally to this work.

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