INTRODUCTION Nearly 900,000 fractures are reported every year in UK with 5-10% being classified as a non-union fracture, which cost on average £43,000, presenting a substantial expense to the healthcare system (1). Injectable hydrogels are an attractive proposition for minimally invasive surgery due to ease of delivery and the potential for homogenous, controlled cargo delivery. Chitosan (Cs) is biocompatible, biodegradable, and has structure resembling that of a native extracellular matrix (2). A thermo-responsive N-isopropylacrylamide (NIPAAm) was grafted onto the Cs backbone and crosslinked with genipin (GP) to form an injectable hydrogel that undergoes in situ sol-gel transition at body temperature and degrades over an 8-week period. Hydroxyapatite (HA) is routinely used in fracture repair. Our group has demonstrated that osteogenesis is significantly increased when the RALA peptide is used to optimise the physiochemical characteristics of nanoscale HA to ensure cellular entry (3). The aim of this study was to synthesise Cs-g-PNIPAAm hydrogel loaded with RALA-HA nanoparticles (NP) as an easy-to-implant device with controlled release of NPs for bone repair and regeneration. MATERIALS AND METHODS NIPAAm, 75-85% deacetylated Cs, ammonium persulfate (APS), N,N,N’,N’-tetramethylenediamine (TEMED) and GP were used (Sigma-Aldrich, UK). Cs-g-PNIPAAm with 10 and 30% of Cs (in relation to NIPAAm) were synthesised by free radical polymerisation: NIPAAm was added to 1% w/v Cs in acetic acid and purged with nitrogen, followed by addition of initiator APS and accelerator TEMED. The copolymer was dialysed in double distilled water (DDW) and recovered by freeze-drying. The GP crosslinker was added to reconstituted hydrogel (0.1% w/v). RALA-HA NPs were formulated via electrostatic attraction by incubating negatively charged HA with the positively charged RALA peptide for 30 min (Fig. 1a). The lyophilised Cs-g-PNIPAAm was solubilised to 5% w/v with NPs suspension (420 μg/mL concentration) at 4°C. 1H-NMR was performed using a Bruker 400 Plus operating at 400 MHz. FTIR analysis was performed using a FTIR 4100. Thermogravimetric analysis was performed using a TGA Q500 under nitrogen at a scan rate of 10°C/min from 20 to 600°C. Rheological properties of 5% w/v hydrogels in DDW were tested using a stress-controlled AR-2000ex rheometer. Temperature sweep from 20 to 50°C was conducted at a rate of 3°C/min in the linear viscoelastic region. Hydrogels (5% w/v) were injected into air through 25G needle at displacement of 1 mm/s for distance of 20 mm. The force was recorded with TA.XT plus Tensile Analyser. In vivo degradation profile was characterised by injecting hydrogel subcutaneously into C57 mice (n=3) and extracted at day 1, 3 and 7. In vivo degradation profile up to day 56 was predicted based on nonlinear regression equations. RESULTS AND DISCUSSION Both 1H-NMR and FTIR confirmed co-polymerisation as Cs and NIPAAm were observed in the Cs-g-PNIPAAM samples. PO43- group of the HA in lyophilised RALA-HA exhibited bands at 1000–1100 cm-1 (Fig. 1b&c). At 600°C the PNIPAAm was fully decomposed and the remaining mass calculated as 9.12, and 25.30 wt.% for 10 and 30% Cs-g-PNIPAAm (Fig. 1d). The successful GP crosslinking of hydrogel was evident by the colour change from white to blue (Fig 2a). 10% and 30%Cs hydrogels ± GP required force below 3.5 N for injection, confirming the formulations were highly injectable (Fig. 2b). All hydrogels exhibited a sol-gel transition at approximately 35°C due to the thermo-responsive nature of PNIPAAm. The sol-gel temperature of 10%Cs was unaffected by crosslinking with 1% GP (35.5 ± 0.5°C, p=0.691) or incorporation of NPs (35.1 ± 0.46°C, p=0.383). Storage modulus of 10%Cs hydrogel with NPs was higher (0.17 ± 0.03 kPa) compared to that of a pristine hydrogel (0.13 ± 0.04 kPa) at 37oC due to an increase in physical cross-links and formation of a particle-polymer network (Fig. 2c) (4). Crosslinking 10%Cs with 1% GP slowed the degradation rate (Fig. 2d), presumably due to the short chains of condensed GP acting as crosslinking bridges between Cs (5). The extrapolated in vivo profiles suggest at D56 the 10%Cs hydrogel will be fully degraded, and the crosslinking will allow extending the degradation up to 12 weeks. CONCLUSION FTIR, 1H-NMR and thermal analysis confirmed incorporation of Cs and NIPAAm into the hydrogel. The extent of Cs grafting was lower compared to theoretical values of 10 & 30% suggesting the residual Cs had been cleared during dialysis. The mechanical stability of the pristine hydrogel was improved via incorporation of NPs – resulting in an increased storage modulus and reinforcement of the microstructure. All hydrogel formulations were highly injectable at 20°C. The extrapolated in vivo degradation rates indicated that 10%Cs will be fully degraded by D56 and slower degradation rate can be achieved though crosslinking the hydrogel with GP, creating a highly tailorable solution for NP delivery. Future work will include tailoring the release profile of RALA-HA NP.
|Publication status||Published - 09 Sep 2019|
|Event||30th Annual Meeting of the European Society for Biomaterials - Dresden, Germany|
Duration: 09 Sep 2019 → 13 Sep 2019
|Conference||30th Annual Meeting of the European Society for Biomaterials|
|Period||09/09/2019 → 13/09/2019|