Introduction: A major challenge in the repair of joint injuries is in the reattachment of the soft tissue to the bone[1]. Failure to regenerate these interfaces results in compromised graft stability and issues with long-term clinical outcome[2]. Therefore development of a construct with the ability to promote bone and soft tissue regeneration is a priority.
In this study we propose that the sustained, localised delivery of nanoparticles (NP) comprised of an amphipathic peptide (RALA) and bioactive hydroxyapatite (HA) will promote osteogenesis, thereby enhancing formation of interfacial tissue. NPs will be delivered from a PCL nanofibre reinforced novel Alg-co-PNIPAAm thermo-responsive hydrogel. The addition of PCL nanofibres aids scaffold properties and increases MSC adhesion and directional proliferation.
Materials and Methods: Preparation of hydrogel scaffold: Alginate (Alg) and NIPAAm were synthesised and coupled via free radical polymerisation to form an Alg-co-PNIPAAm hydrogel with various ratios of the constituent parts[3]. Aligned PCL nanofibers were created using a bi-electrospinning method with a rotating needle collector
Formulation of RALA/HA-NPs: NPs were prepared at a range of mass ratios. Particle size and zeta potential (ZP) were measured using a Zetasizer (Malvern, UK) to determine the optimal formulation.
Fluorescent SiHA (f-SiHA) synthesis: f-SiHA for intracellular tracking of NPs was prepared as described by Grover et al[4]. SiHA (6 mg) was suspended in PBS and 7.14 mM stock solution of thiol-reactive Texas Red maleimide (TRM) (Life Technologies Ltd, UK) was added and mixed 2 h at 37°C.
Cell viability and immunocytochemistry for osteogenic markers: Porcine BM-MSC and MG63 cells were cultured with pure HA, RALA/HA-NPs and NP-loaded hydrogel. Cell viability was determined by MTS and live/dead© assays. Expression of osteogenic markers was determined using standard immuno-cytochemistry and real-time PCR methods up to 28 d.
Results: Preliminary studies demonstrate that the Alg-co-PNIPAAm solution exhibits increasing viscosity from 32-40°C as the aqueous solution transformed to an elastic hydrogel, which was indicative of its thermo-responsive properties. NPs prepared with a mass ratio of 5:1 were observed to have a size of 69 ± 11 nm and a zeta potential of 25.7 ± 3.7 mV (Figure 1). The size and shape of the NPs was confirmed by TEM. Cell viability was seen to decrease 24 h post-transfection with the RALA/HA-NP treatment. However by 7 d cytotoxicity decreased to a level similar to that seen in an untreated control group. f-SiHA-TRM NPs were successfully formulated and exhibited fluorescence under excitation at 585 nm (Figure 2).
Discussion: We have successfully produced a nanofibre reinforced thermo-responsive polymer with the potential to behave as both a scaffold and delivery vehicle. Furthermore, RALA peptide was capable of forming biocompatible NPs with HA, which were stable across a range of temperatures and over an extended duration. Transfection of MG63 cells with NPs resulted in an initial increase in cytotoxicity due to cell penetration. However, the cells recover by 7 d and an increase in extracellular collagen-I deposition is observed by 21 d. The degradation pattern of the hydrogel will be studied, as well as fluorescent RALA/SiHA-NP release from the hydrogel in vitro. The dual functionality of the hydrogel as a scaffold for bone regeneration and a delivery vehicle for osteoconductive NPs will finally be assessed in vivo.
US-Ireland Grant (Grant no: 3568); Medical Research Council (Grant no: 1294)
References:
[1] P. Yang et al., Tissue Eng Part B Rev., 2: 127-141, 2009.
[2] H.H. Lu et al., Ann Biomed Eng., 38: 2142-54, 2010.
[3] R. Tan et al., Carbohydr. Polym., 87: 1515-1521, 2012.
[4] Grover L. et al., J. Mater. Chem. B., 1: 4370-4378, 2013.