The response of annulus fibrosus cell to fibronectin-coated nanofibrous polyurethane-anionic dihydroxyoligomer scaffolds
Introduction
The intervertebral disc (IVDs) imparts flexibility to the spine, allowing for movement and dissipation of mechanical loads [1], [2]. It is a heterogeneous structure composed of three distinct tissues; the inner proteoglycan (PG)-rich nucleus pulposus (NP), the outer collagen-rich annulus fibrosus (AF) and the cartilage endplates (EP). The AF is a highly organized tissue comprised of layers of collagenous lamellae having a specific angle of orientation of 60° to the vertical that alternates with successive lamellae. This fibrous tissue has tensile strength and functions to withstand the forces exerted by the swelling of the NP [3]. The confinement of the NP by the AF allows the IVD to sustain the compressive loads experienced during the activities of daily living [4]. Structural breakdown of one or more of these tissues compromises IVD function and may contribute to the onset of degenerative disc disease (DDD) [2], [5], [6].
The prevalence of DDD is very high as demonstrated in an autopsy study which demonstrated that 97% of individuals 50 years of age or older have some form of the disorder [7]. Although the causes of DDD remain unknown, genetics [1], [6], [8], a decline in cellularity [1], loss of notochordal cells [2], [5], mechanical stress [9], [10], [11], [12] and calcification of the cartilage endplate [2], [13], [14] have all been implicated in the pathogenesis of this disorder. There are various treatment options for DDD, ranging from conservative management (medication and physiotherapy), to surgical intervention such as interbody fusion or total disc replacement. However, these have limited success with the potential for side effects [15], [16]. For these reasons, there is a need to develop novel therapies for the treatment of chronic symptomatic DDD. Tissue engineering an IVD suitable to use as a disc replacement is one approach that shows much promise in that it functions to promote tissue repair and/or regeneration [3], [5], [17].
Although it is possible to generate NP tissue [18], tissue engineering of the AF has proven to be more challenging due to its highly oriented lamellar structure. Various polymeric scaffolds have been used to try to accomplish this, including polyglycolic acid/polylactic acid based materials [19], collagen-hyaluronan [20], PDLLA/45S5 Bioglass® composite films [21], and alginate materials [22], but all show limited success. For example, polylactides and polyglycolides form acidic degradation products resulting in reduced local pH and decreased extracellular matrix synthesis [23]. More recently, others have developed aligned structures using collagen [24] and polycaprolactone [25]. Poly-carbonate-urethane (PU) is also a good scaffold candidate as it is elastic, biodegradable, biocompatible, non-toxic to AF cells and can be electrospun to resemble the structure and orientation of native AF lamellae [26], [27], [28]. Modifying the PU by combining it with an anionic/dihydroxyl oligomeric additive (PU–ADO) results in a higher surface energy and enhanced cell attachment compared to standard PU scaffolds [28]. The anionic dihydroxy oligomers (ADO) promoted the adsorption of protein from serum and newly synthesized protein by the AF cells in order to enhance cell adhesion to the scaffolds and showed that this may be important in enhancing AF tissue formation [28].
Although the proteins involved in modulating AF cell attachment to PU–ADO is unknown, it has been estimated that approximately 150 proteins can participate in cell adhesion [29]. Integrins are commonly involved in mediating cell attachment and also interact with actin to direct cell shape [30], [31], [32]. Studies have shown that specific integrin subunits, such as α5, αv and β1 and β3, are expressed by porcine and human AF cells [33], [34]. Interestingly, integrin activation and consequently changes to the actin cytoskeleton appear to be important for the deposition of oriented collagen as shown in studies of the developing rat IVD [35]. This raises questions in regards to the role of integrins in AF tissue formation. In this study, we sought to determine whether pre-coating PU–ADO with extracellular matrix proteins known to be involved in mediating cell attachment, such as fibronectin, type I collagen, or vitronectin, will influence AF cell attachment, shape and collagen production and orientation. An understanding of these early molecular events with the PU–ADO scaffolds will facilitate developing organized AF tissue in vitro that mimics the native tissue.
Section snippets
Poly-carbonate urethane synthesis and fabrication
PU–ADO was prepared as described previously [28]. Briefly, the base polymer was synthesized from the reaction of poly(1,6-hexyl 1,2-ethyl carbonate)diol, 1,6-hexane diisocyanate and 1,4-butanediol in N, N-dimenthylacetamide solvent at a temperature between 60° and 70 °C. The ADO additive was synthesized by reacting polytetramethylene oxide, hydroxyethylmethacrylate (HEMA) and lysine diisocyanate in N, N-dimethyl acetamide (DMAC) solvent overnight in a temperature range of 50 °C–60 °C, followed
Annulus fibrosus cell attachment on pre-coated PU–ADO
As shown in Fig. 1, pre-coating PU–ADO with ECM proteins (ColI, Fn, a combination of ColI and Fn (CF) and Vn) resulted in a significant increase in AF cell attachment to the scaffold after 6 h as compared to the non-coated (NC) control (31 ± 2.7%) (p < 0.05) or the BSA-coated control (7 ± 2.0%) (p < 0.005). Amongst the various protein coatings, Fn resulted in significantly greater (p < 0.05) cell attachment as compared to all other conditions (Fn: 73 ± 5.6%; ColI: 51 ± 3.4%; CF: 54 ± 2.5%; Vn:
Discussion
The current study used extracellular protein(s) such as collagen type I, alone or in combination with fibronectin, fibronectin alone, or vitronectin (all of which are known to influence the adhesion of different cell types) to examine the regulation of AF cell and collagen orientation on PU scaffolds. The data suggests that Fn plays a pivotal role in influencing the AF cell under the culture conditions studied. The greatest cell attachment occurred when the scaffolds were pre-coated with Fn. Fn
Conclusion
In summary pre-coating aligned PU–ADO scaffolds with Fn provides molecular and topographical cues that allow AF cells to orient themselves parallel to scaffold fibres. This is mediated in part by α5β1 integrins and results in the formation of Fn fibrils and the subsequent deposition of aligned collagen type I. These findings support that Fn-coated PU–ADO aligned nanofibers provide an optimal scaffold construct to engineer AF tissue, in vitro. Additional long term studies are being planned to
Acknowledgements
This research was supported by CIHR (M0P8672). MA was supported by an NSERC CGS scholarship. The authors would also like to thank Harry Bojarski and Ryding-Regency Meat Packers for providing bovine tissues and Dr. Meilin Yang for the synthesis of the scaffolds.
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