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New strategies for promoting angiogenesis in biodegradable polymeric tissue engineering scaffolds: A materials science approach

Research Area: Biomaterials, polymers, materials science and engineering, physics, chemistry, biochemistry, plasma polymerization, biotechnology

Supervisor: Prof Namita Roy Choudhury

Description: The emergence of the tissue engineering (TE) field in the last few years has resulted in the development of various interdisciplinary strategies primarily aimed at meeting the need to replace organs and tissues lost due to diseases or trauma [1]. In essence, the main TE approach is centred on seeding biodegradable scaffolds (both organic and inorganic such as poly (lactide-co-glycolide) and apatites) with donor cells, possibly appropriate growth factor/s, followed by culturing and implantation of the scaffolds to induce and direct the growth of new, functional tissue. The scaffold material eventually disappears through biodegradation and is replaced by the specific tissue. This scaffold-guided TE approach is aimed at creating tissues such as skin, cartilage, bone, liver, heart, breast, etc.

Despite success with small (thin) tissue-engineered constructs, perhaps the biggest roadblock in scaffold-guided TE is engineering large tissue volumes. This challenge arises due to the lack of rapid vascularization (angiogenesis) of large three-dimensional (3-D) scaffold constructs [2,3]. Accordingly, angiogenesis is a pre-requisite for scaffold-guided TE of large tissue volumes. Hence there is a strong need for developing strategies that can promote angiogenesis in 3-D scaffold constructs of biodegradable polymeric scaffolds; this equally applies to other scaffold materials (such as hydroxyapatite and metals). This particular need forms the basis of the proposed project.

A number of biomolecules, which induce or promote angiogenesis in tissues, have been identified. The most prominent of these are: growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factors (PDGFs) and transforming growth factors (TGFs) [4]; and nitric oxide (NO) [5]. The current worldwide approaches aimed at promoting angiogenesis in the TE field can be summarised into three main categories: (i) delivery of angiogenic growth factors using synthetic and natural polymeric scaffolds; (ii) delivery of plasmids containing DNA that encodes for angiogenic proteins; and (iii) combined delivery of angiogenic molecules and endothelial cell transplantation. Category (i) above is relevant to the proposed project, with the exception that we propose to load the scaffolds with NO molecules instead of growth factors. It is proposed that the loaded NO can be released under physiological conditions, thus promoting localised, rapid angiogenesis.

NO is an important signalling molecule that acts in many tissues to regulate a diverse range of physiological functions [6]. For example, NO is known to play many important roles in wound healing, right from the inflammatory phase through to scar remodelling [7]. NO is also considered to be an important signalling molecule for regulation of bone growth. Stimulation of osteoblasts (bone depositing cells) with NO has shown increased bone matrix turnover and corresponding mineralization of bone whilst having a suppressive effect on bone resorption (carried out by osteoclasts) [8].

The use of NO as an angiogenic biomolecule offers several advantages over angiogenic growth factors. The main advantage is that owing to the simple structure and configuration of NO, the possibility of prion-based infections/diseases can be ruled out if NO can be loaded from a protein-free source. Our interest in the NO molecule is primarily centred not only on its loading in 3-D polymeric scaffolds but also on its role in bone TE and speedy wound repair in bone [9,10].

In view of the potent role of NO in promoting angiogenesis, we raise the following key question:
Is it possible to promote angiogenesis in 3-D scaffold constructs of biodegradable polymeric scaffolds by loading them with NO, followed by NO release under physiological conditions?
Based on our experience with the plasma (ionised gas) processing of polymeric scaffolds and in materials science and organic polymers in general, our answer to this question is YES, and this forms the basis of the proposed project, which has the following general aims:
To develop strategies for loading and subsequent release of NO in biodegradable 3-D polymeric TE scaffolds for promoting angiogenesis; and
to characterise the NO-loaded scaffolds for their microstructural, mechanical and surface chemistry properties as these are crucial to bio-integration of the scaffolds.

For achieving the above listed project aims, the 3-D scaffold material to be used for the proposed project is a copolymer PLGA [poly (lactide-co-glycolide), 75/25] (Figure 1). This choice is governed by the facts that PLGA (i) is a biodegradable material approved by FDA (Federal Drugs Agency, USA) and hence has been in use for several years as surgical screws, rods and pins, (ii) is a linear polyester and hence easy to surface functionalise using low-power, low-temperature radio frequency glow discharge (RFGD) plasmas, and (iii) has a glass transition C temperature of about 55o(PLGA 75/25) which is well suited for the processing conditions that normally exist in radio frequency glow discharge (RFGD) plasmas to be employed by us.



Figure 1 Poly(D,L-lactide-co-glycolide) used for preparing polymer scaffolds.

The amine functionality required for attaching NO within the PLGA scaffolds will be obtained by using the RFGD approach. This plasma-based materials science approach to be employed by us offers numerous advantages such as attendant plasma sterilisation of the scaffold materials, surface functionalisation within the scaffold and a precise control over the density of plasma generated functional groups. Thus the density of NO molecules covalently attached within the scaffolds and their subsequent release can be precisely controlled, unlike the generally physisorbed growth factors. The proposed project is designed such that a foundation is first laid by developing the necessary materials science expertise, with follow-on projects involving in vitro and in vivo studies for assessing the bio-integration of the modified scaffolds in question.

The successful realisation of the project aims will involve preparation, characterisation and optimisation of NO-releasing PLGA scaffold samples. The measurement and optimisation of microstructural, mechanical and surface chemistry properties of the above samples form the core of this project, as these are crucial to bio-integration of the scaffolds. All these testing and analysis facilities are available at The Wark/UniSA. The three phases of the project can be summed as: (i) amine plasma deposition on the PLGA scaffolds; (ii) Formation of the diazeniumdiolate complex on the PLGA scaffolds; and (iii) NO release properties.

References
1. R Langer and J P Vacanti, Science 260 (1993) 920.
2. S Yang, K F Leong, Z Du and C K Chua, Tissue Eng. 7 (2001) 679.
3. J D Sipe, Ann. NY Acad. Sci. 961 (2002) 1.
4. K H Bouhadir and D J Mooney, J. Drug Target. 9 (2001) 397.
5. J P Cooke and D W Losordo, Circulation 105 (2002) 2133.
6. J Garthwaite, S L Charles and R Chess-Williams, Nature 336 (1988) 385.
7. A Schwentker, Y Vodovotz, R Weller and T R Billiar, Nitric Oxide 7 (2002) 1.
8. M L Brandi, M Hukkanen and T Umeda, Pharmacology 92 (1995) 2954.
9. J R Abbott, R St C Smart, M Henneberg, T Yuen, S Kumar, C M Leigh, G M Hermanis and D J David, Proc. 9th International Congress on Cleft Palate and Related Craniofacial Anomalies, Goteberg, Sweden, 24-29 June 2001 (Ed. J. Lilja) pp. 527-29.
10. PCT patent 'Bioactive Coating of Biomedical Implants'. 
No. 2004900202, 19 January 2004. Principal Inventors: Sunil Kumar and Roger St C Smart.

Funding: International students should apply for an International Postgraduate Research Scholarship (IPRS) and a UniSA President's Scholarship (UPS). To be eligible for UPS, applicants must have a supervisor willing to nominate them for consideration.

Australian students should apply for an Australian Postgraduate Award (APA) and a UniSA Australian Postgraduate Research Award (USAPRA).

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