Elsevier

Biomaterials

Volume 27, Issue 33, November 2006, Pages 5708-5714
Biomaterials

The effect of collagen-targeting platelet-derived growth factor on cellularization and vascularization of collagen scaffolds

https://doi.org/10.1016/j.biomaterials.2006.07.023Get rights and content

Abstract

Platelet-derived growth factor BB (PDGF-BB) was an important inductive factor during wound healing, but the lack of efficient delivery system limited its clinical application. Here, a peptide of seven amino acids was firstly utilized to engineer PDGF-BB to construct a collagen-targeting system. This peptide functioned as collagen-binding domain (CBD) to specially target the PDGF-BB to the collagen and restrict its diffusion. In our study, in vitro activity assay showed that the CBD-fused PDGF (CBD-PDGF) and native PDGF (NAT-PDGF) possessed similar activity to stimulate the human fibroblast proliferation. As expected, this peptide promoted the binding of PDGF to collagen scaffolds, and CBD-PDGF caused more cells to proliferate on the collagen gel than NAT-PDGF when the same amounts of PDGF were added. In the in vivo experiment, CBD-PDGF-loaded collagen scaffolds were uniformly cellularized and vascularized compared to that of NAT-PDGF-loaded scaffolds. Our study demonstrated that the CBD targeted PDGF to the collagen scaffold to exert its functions, and it suggested this could be an effective system for targeting tissue regeneration and wound repair.

Introduction

The weak antigenecity, excellent biocompatibility and biodegradability have made collagen a widely used biomaterial [1]. In the tissue engineering of skin, bone, vascular and nerve, many types of collagen-based scaffolds have been fabricated, and they have showed good characters in wound repair [2], [3], [4], [5]. During the process, collagen played an important role in providing cell anchorage site, mechanical stability and structural guidance. They also provided the interface to respond to physiological and biological changes, and to remodel the extracellular matrix to integrate with the surrounding native tissues [6], [7], [8]. Therefore, it was critical to promote cell attachment and migration into collagen scaffold to regenerate new tissues [9], [10].

To accelerate the repair process, it was common to incorporate growth factors to activate scaffolds [11], [12]. Among these growth factors, platelet-derived growth factor-BB (PDGF-BB) was particularly important because it could directly or indirectly promote most cellular processes associated with wound healing, including chemotaxis, stimulation of cell proliferation, and extracellular matrix production [13]. However, simple absorption of PDGF to collagen scaffold would allow the diffusion of PDGF into extracellular fluids, and lose its activity rapidly [14]. Therefore, the therapeutic concentrations of PDGF were difficult to maintain in this manner, and the amount of PDGF had to be tremendous.

In this work, the collagen-targeting PDGF and the collagen scaffold were studied as a targeting wound repair system. The basic conception of such system was fusing a collagen-binding domain (CBD) with PDGF to increase its collagen-binding ability. Epidermal growth factor, transforming growth factor-β1 and basic fibroblast growth factor had been added with a collagen-binding peptide derived from von Willebrand factor (vWF), and results showed the modified growth factors could achieve better repairs compared to the native growth factors at the same concentration [15], [16], [17], [18], [19]. However, these studies emphasized the controlled release of growth factors from collagen scaffolds, and the tissue regenerations outside or inside the scaffold were failed to be discussed.

Our aim here was to construct an effective collagen-based PDGF targeting system to activate the collagen scaffold to accelerate the tissue reconstruction. In this system, a novel CBD, TKKTLRT, which could bind in a saturable fashion to the native collagen was utilized [20]. For the first time, the peptide was fused to the PDGF to construct a PDGF targeting system (Fig. 1A). It was predicated that the CBD-fused PDGF (CBD-PDGF) would be concentrated in the collagen scaffold, avoid being washed away or diluted extensively by extracellular fluids. At the same concentration, fusion proteins would promote and accelerate more cells to grow into the collagen scaffold than the native PDGF (NAT-PDGF). Thus, a more rapid and homologous repair by the activated collagen scaffold was expected.

Section snippets

Engineering and preparation of NAT-PDGF and CBD-PDGF

The gene of CBD together with the linker domain was synthesized and digested with NdeI and HindIII, then inserted into the NdeI-HindIII site of pET-28a (Novage,USA). The vector was named pET-CBD. A human PDGF-BB DNA encoding a mature form (108 amino acids) was amplified by PCR with extra 5′ NdeI and 3′XhoI sequences (NAT-PDGF) and 5′ HindIII and 3′XhoI sequences (CBD-PDGF), respectively. The amplified cDNA was digested and the cDNA fragments were inserted individually into the NdeI-XhoI site of

PDGF-dimmer formation after renaturing

When induced by IPTG, E. coli expressed the recombinant proteins largely but only in inclusion bodies (Fig. 1B). Western-blot assay showed little PDGF were expressed as soluble form (Fig. 1C). Because insoluble proteins possessed no biological activities, renaturing was a critical step. Based on our study, dilution method was the most suitable for PDGF renaturing. After refolding, the dimmer conformations (the active form) in two types of PDGF were formed as about 30–40% in the final resoluble

Discussion

Restricting growth factors in scaffolds to avoid being diffused into the body fluids or washed was very important to maintain their concentrations. In this way, the amount of growth factors could be greatly reduced [24]. Many researches had focused on how to modify the scaffolds to achieve the goal. Here, our strategy was to modify the growth factors to increase its binding to collagen. CBD domains derived from von Willebrand coagulation factor (vWF) or collagenase have been studied for several

Conclusions

We have constructed a unique collagen-targeting PDGF system using a novel collagen binding peptide. The engineered CBD-PDGF could specially bind to collagen scaffold. We also demonstrated that the fusion of CBD to the PDGF did not significantly influence its bioactivity. More importantly, the targeting system promoted the cellularization of the collagen scaffold in vivo. The well-established uniformly cellularization and vascularization of CBD-PDGF-loaded scaffolds suggested CBD-PDGF and

Acknowledgments

This work was supported by Chinese Academy of Sciences’ “100 Talented Scholar Program”; Grants (KSCX2-SW-205; KSCW2-SW-218) from Chinese Academy of Sciences “Knowledge Innovation Program”, and Grants (30428017; 2005CB522603) from NSFC and The Chinese 973 program, and also supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. The authors also gratefully acknowledge the support of K.C. Wong Education Foundation, Hong Kong.

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These authors contributed equally to this work.

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