The angiogenic potential of three-dimensional open porous synthetic matrix materials

Abstract

Angiogenesis is a complex multistage process involving multiple factors and numerous cells. The use of the Chorioallantoic Membrane (CAM) assay is well documented as a method to investigate angiogenesis. This technique is ideal for screening samples, but requires an objective analysis technique. The angiogenic response of vascular endothelial growth factor (VEGF) was used to confirm that computer-based image analysis was able to quantify angiogenesis. Image analysis was used on samples of increasing porosity of PLLA to determine the effect of pore size on angiogenesis. Another effect also noted was that of an inflammatory response co-incident with angiogenesis. The difference in pore size made a difference to both angiogenesis and inflammation. Real-time polymerase chain reaction (PCR) was used with primers for TNF-α to demonstrate and measure the presence of an inflammatory response.

Introduction

Angiogenesis is a prerequisite for success in many tissue engineering processes, but is usually considered to be only a small component of these processes, which are driven by the need to replace diseased or damaged tissues using a matrix or scaffold within which cells are attached, grown and guided towards the regeneration of new tissue [1], [2]. Porous scaffolds or gels for these purposes are required to be non-toxic and to display the appropriate biocompatibility characteristics. The intrinsic angiogenic properties of a scaffold or gel material, either in its own right or as part of a complex tissue engineered cell-based structure, should be assessed before the deliberate addition of, or invasion by, cells. Modifications to materials should be tested for their angiogenic and inflammatory potential at each stage of material development, as these two components separately, and most significantly, in combination, may control the in vivo fate and function of regenerated tissues placed in a patient. The bulk transport properties of the material and the accessibility of the internal structure to cells are all critical to the success of the material in a tissue engineering construct. Many of the tissue specific objectives in a process will be of secondary concern if the material is implanted and becomes encapsulated by chronic fibrosous tissue or if vascularisation fails to occur in the integrating tissue [3].

Although the availability of commercial in vitro angiogenesis assays has increased recently, there are still limitations to them and none is able to encompass all the facets of the angiogenic response in vivo. The kinetics of processes with different cell types, the flow conditions and the large number of potential growth factors are far too complex for in vitro systems that are ideally suited for examining single parameters [4], [5], [6]. The Chick Chorioallantoic Membrane (CAM) assay is one of the most widely used in vivo assay for determining angiogenesis [7]. In comparison to other animal models the need for a high level of technical skill is not required using the CAM assay as there is for rabbit cornea implantation. The low cost and speed of results using the CAM assay is also an advantage when compared to the skin windows and rat aorta models of angiogenesis [8]. However, there is still a fundamental requirement to apply analytical techniques in order to determine the extent and variation in angiogenesis and to enable the objective evaluation of new therapies and pharmaceutical agents. An objective analysis would allow for unbiased comparisons between samples without external factors affecting the outcome. In addition, the granulomatous tissue associated with an inflammatory response in the CAM assay makes it difficult to characterise the response in detail and, invariably, as with the analysis of the vessel formation on the CAM, this analysis has often been subjective. Histological analysis has been conducted where samples are fixed, sectioned and stained before evaluating the inflammatory infiltrate [9], [10], this has provided useful information in terms of cell types involved in inflammation but it does not provide the facility to identify and quantify key factors involved in inflammation.

Inflammation is an essential function of the body in the defence against pathogens. Angiogenesis is often linked with inflammation, particularly in diseases associated with chronic inflammation such as rheumatoid arthritis, inflammatory bowel disease and diabetic retinopathy [11]. Until recently the links between the two processes of angiogenesis and inflammation had not been established. It has now been demonstrated that angiogenic factors potentiate or act synergistically with proinflammatory mediators [12], [13] to induce inflammation-angiogenesis. The determination of the presence of inflammation is necessary as this may give rise to an angiogenic effect since these two responses are closely associated.

Poly(α-hydroxy esters) such as poly(l-lactic acid) (PLLA) and poly(d,l-lactic acid-co-glycolic acid) (PLGA) are among the few synthetic degradable polymers approved for human clinical use in medical devices and tissue engineering products due to their generally good cellular integration and non-toxic degradation products [14], [15] but they do show a strong inflammatory response in vivo [16]. They also show significant versatility in terms of bulk chemistry with good potential for modification to their physical properties and their ability to be blended with other polymers. In recent years, porous PLLA sponges have been widely used as 3-D scaffolds for the regeneration of many tissues and organs such as bone [17], [18], cartilage [19], liver [20], and skin [21] to guide the activity of cells. The rates of cell expansion and the ingrowth of tissue have been reported to be dependent on the porosity, pore diameter, pore shape, and porous structure of the scaffold [22]. An interconnecting pore network is essential for tissue ingrowth, vascularisation, and diffusion of nutrients.

The hypothesis that angiogenesis is dependant on the pore size of a matrix was tested by investigating the angiogenic potential of PLLA 3D scaffolds as a function of increasing porosity. The CAM assay was combined with a bespoke image analysis routine to quantitatively analyse the induction of angiogenesis. Real time polymerase chain reaction (PCR) determined the presence and extent of inflammation, which itself could induce angiogenesis.