Elsevier

Biomaterials

Volume 31, Issue 34, December 2010, Pages 8854-8863
Biomaterials

An in vitro assessment of titanium functionalized with polysaccharides conjugated with vascular endothelial growth factor for enhanced osseointegration and inhibition of bacterial adhesion

https://doi.org/10.1016/j.biomaterials.2010.08.006Get rights and content

Abstract

The long-term success of orthopedic implants may be compromised by defective osseointegration and bacterial infection. An effective approach to minimize implant failure would be to modify the surface of the implant to make it habitable for bone-forming cells and anti-infective at the same time. In this in vitro study, the surfaces of titanium (Ti) substrates were functionalized by first covalently grafting either dopamine followed by carboxymethyl chitosan (CMCS) or hyaluronic acid-catechol (HAC). Vascular endothelial growth factor (VEGF) was then conjugated to the polysaccharide-grafted surface. Antibacterial assay with Staphylococcus aureus (S. aureus) showed that the polysaccharide-modified substrates significantly decrease bacterial adhesion. The CMCS-functionalized Ti demonstrated better antibacterial property than the HAC-functionalized Ti since CMCS is bactericidal while HA only inhibits the adhesion of bacteria without killing them. Osteoblast attachment, as well as alkaline phosphatase (ALP) activity and calcium deposition were enhanced by the immobilized VEGF on the polysaccharide-grafted Ti. Thus, Ti substrates modified with polysaccharides conjugated with VEGF can promote osteoblast functions and concurrently reduce bacterial adhesion. Since VEGF is also known to enhance angiogenesis, the VEGF-polysaccharide functionalized substrates will have promising applications in the orthopedic field.

Introduction

Numerous regulators of angiogenesis have been identified and characterized over the last decades. Among these, vascular endothelial growth factor (VEGF) appears especially important in normal development and disease processes [1], [2], because it is recognized as a powerful mitogen and chemoattractant for endothelial cells through interactions with VEGF receptors [3]. Recently, immobilized VEGF was used in the development of advanced tissue scaffolds, and it was shown to have a greater angiogenic effect than repeated doses of soluble VEGF [4]. Furthermore, immobilization of soluble protein ligands may provide extended signaling since the ligand will not be internalized as a ligand/receptor complex. In this regard, immobilization of VEGF on biomaterials has attracted much attention as a strategy that can enhance angiogenesis [5], [6]. Our group recently immobilized VEGF on titanium (Ti) surface, and demonstrated that the attachment, viability and proliferation of endothelial cells increase significantly on the functionalized surfaces compared to the pristine substrate [7].

In addition to its reported effects on endothelial cells, VEGF has been recently identified as an essential factor for endochondral ossification, a necessary process for growth of long bones whereby cartilage is replaced by bone [8], [9]. VEGF may couple angiogenesis to osteogenesis both indirectly through its effects on endothelial cells, and directly by modulating chondrocytes, and osteoclasts since all of them express VEGF receptors [10]. VEGF can indirectly induce proliferation and differentiation of osteoblasts by stimulating endothelial cells to produce osteoanabolic growth factors [11]; but whether VEGF has a direct effect on osteoblast is still under debate. VEGF was reported to induce alkaline phosphatase (ALP) activity in primary osteoblasts and to enhance their responsiveness to parathormone. Furthermore, high affinity sites for VEGF have been identified on primary osteoblasts, indicating the presence of functional VEGF receptors on osteoblasts [12]. Deckers et al. found that osteoblasts can express VEGF and VEGF receptors during differentiation, and exogenous VEGF can stimulate nodule formation [13]. This suggests that besides its established effects on endothelial cells, VEGF may play a role in osteoblast differentiation. To test whether VEGF have direct effects on osteoblasts, Street et al. investigated primary human osteoblasts in vitro, and discovered that VEGF plays a direct autocrine role in osteoblast differentiation, and increases nodule formation and ALP activity in a dose-dependent way [14]. However, there are some controversial results regarding the effect of VEGF in directly managing osteoblast behavior. In studying the roles of VEGF in osteoblasts-endothelial cells communication, Clarkin et al. showed that VEGF does not promote osteoblast differentiation directly and this enhancement effect is present only in osteoblast-endothelial cells coculture [15]. Therefore, further studies to establish the effects of VEGF on osteoblasts are necessary.

Ti and its alloys are popular biomaterials used in bone repair and replacement due to their high strength, low weight and excellent corrosion resistance [16]. Despite the existing good biocompatibility of these materials, there is still a need to improve bone covering of the implants (bone–implant contact percentage) [17]. In addition, the pristine Ti implant is susceptible to bacterial infection which is considered to be the second main cause of revision after instability [18]. Since total hip and knee arthroplasties in US alone exceed half a million each year, even a low risk of infection (0.5–5%) will be a serious problem for public health [19]. To overcome these two major problems for Ti implants, a promising approach is to modify the implant surface with a growth factor and antibacterial agent simultaneously [20], [21], [22].

In this study, VEGF was immobilized on Ti surface via a grafted layer of either carboxymethyl chitosan (CMCS) or hyaluronic acid-catechol (HAC). Chitosan, hyaluronic acid (HA) and their derivatives are widely applied in biomaterials [23], and have been proven to suppress the adhesion of bacteria by our group [24]. The effects of the polysaccharide coating with conjugated VEGF on the proliferation, differentiation and mineralization of osteoblasts as well as the antibacterial property were investigated in the present study. VEGF was selected because of its potential in enhancing both osteogenesis and angiogenesis. The VEGF-modified implant is expected to improve not only the surface bone coverage, but also angiogenesis in the injured tissue adjacent to the implant by shortening the healing phase and substituting the fibrous layer often observed around metal implants with functional bone tissue. A reduction in infection probability with concurrent enhancement of osteogenesis and angiogenesis will be a significant achievement and pave the way for the development of a new generation of implants.

Section snippets

Materials

Ti foils were purchased from Goodfellow Inc., UK. Dopamine hydrochloride, hyaluronic acid sodium salt from Streptococcus equi (HA), chitosan (deacetylation degree ≥75%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino)-ethanesulfonic acid (MES), and Alizarin Red S were purchased from Sigma–Aldrich Chemical Co. Recombinant human VEGF was obtained from R&D Systems, US. Staphylococcus aureus (ATCC 25923), and osteoblast cells (MC3T3-E1 subclone 14)

Surface characterization

The elemental composition of the surfaces after the various surface modification steps was determined by XPS. The XPS widescan spectra of the pristine Ti, Ti-Dopa, Ti-CMCS, Ti-CMCS-VEGF, Ti-HAC, and Ti-HAC-VEGF, and their corresponding surface elemental compositions are shown in Fig. 2 and Table 1, respectively. Carbon is typically present in the widescan spectrum of the pristine Ti due to unavoidable hydrocarbon contamination, and it was used as an internal reference at 284.6 eV for peak

Conclusion

Titanium substrates functionalized with VEGF and either carboxymethyl chitosan or hyaluronic acid promote osteoblast functions and inhibit S. aureus adhesion. The use of carboxymethyl chitosan results in a higher antibacterial efficacy than the use of hyaluronic acid. The conjugated VEGF does not affect the antibacterial efficacy, and is equally effective in promoting osteoblast functions (cell attachment, ALP activity and calcium mineralization) on the carboxymethyl chitosan and hyaluronic

Acknowledgment

This work was financially supported by the Singapore Stem Cell Consortium Grant SSCC/09/019.

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