EFECTO DE LAS INTERVENCIONES SANITARIAS FRENTE AL ESTIGMA

Recent research on electrospun polymer/hydroxyapatite composite scaffolds has been more focussed on the investigation of cell-nanofibre interactions and other characteristics such as mechanical properties and homogeneity of HA particle dispersion in the polymer (Chuenjitkuntaworn et al., 2010; Kim et al., 2006a; Prabhakaran et al., 2009; Thomas et al., 2006) and only few (Sui et al., 2007; Xu et al., 2007; Jose et al., 2010; Ito et al., 2005) have

reported any studies on the degradation properties of these composite scaffolds. HA has been shown to improve cell-scaffold interaction (Peng et al., 2011; Chuenjitkuntaworn et al., 2010) and mechanical properties (Sui et al., 2007) of electrospun polymer/HA scaffolds, however, if these materials are intended for clinical purposes, then it is essential to not only have an understanding of their cell-scaffold interaction behaviour, but an understanding of their degradation behaviour in vitro is also crucial as this may give an indication of their in vivo degradation characteristics.

Sui and co-workers (2007) reported the electrospinning of PLLA and PLLA-HA nanofibre scaffolds and conducted in vitro degradation studies on the scaffolds in PBS (pH 7.4) at 37^oC (Sui et al., 2007). For the PLLA nanofibre scaffold (average fibre diameter of 368 nm), a 10%

loss in mass and a decrease in molecular weight (Mw) from 268 kDa to 201 kDa after 8 weeks were reported. Additionally, a reduction in pH from 7.40 to 7.08 and water uptake of approximately 12% was reported. On the contrary, the PLLA-HA nanofibre scaffold (average fibre diameter of 313 nm) was reported to have lost only approximately 6% of its original mass while the molecular weight had reduced from 268 kDa to 241 kDa. Furthermore, the pH was observed to decrease slightly during the first 4 weeks, and then increase up to the initial pH. According to Sui et al. (2007), the presence of HA particles in the PLLA-HA electrospun scaffold imparted hydrolytic stability to the scaffold, which was attributed to the presence of alkaline HA particles that could possibly help to neutralise the acidic environment caused by the release of the polymer degradation products (Sui et al., 2007).

In another work on the degradation of electrospun PLLA-HA composite, Xu et al. (2007) reported the electrospinning of composite fibres composed of poly(L-lactide)-grafted hydroxyapatite (PLA-g-HAP) nanoparticles within a polylactide matrix (PLA-g-HAP/PLA) (Xu et al., 2007). The authors reported an improvement in wettability as the PLA-g-HAP content

was increased. They further reported that the amount and distribution of PLA-g-HAP nanoparticles affected the degradation rate of the composite fibres, where the degradation rate was determined by calculating the reduction in mass of the electrospun scaffolds. The mass loss observed on the scaffolds was attributed to the hydrolytic degradation of the PLA and the falling-off of the PLA-g-HAP nanoparticles from the fibres. At low PLA-g-HAP content, the degradation rate was reported to be delayed due to the presence of HA particles, which due to their alkaline nature neutralised the acidic products of PLA degradation. However, the degradation rate of the scaffolds increased with an increase in the PLA-g-HAP content. The increased degradation rate with increase in the PLA-g-HAP content was attributed to the enhanced wettability of the composite fibres and the escape of the nanoparticles from the fibre surfaces during incubation. Furthermore, they reported higher pH values for degradation media of PLA-g-HAP/PLA fibres than those of PLA fibres indicating the neutralizing effect of HA particles. Similar to Sui et al. (2007), Xu et al.

attributed the difference in pH values to the presence of HA particles which are alkaline in nature (Xu et al., 2007).

More recently, Jose et al. (2010) reported the electrospinning of scaffolds of PLGA (85:15) and PLGA/collagen/HA with varying PLGA/Collagen:HA ratios, on which degradation studies were conducted in a 1.0% trypsin in PBS solution (pH 7.4) at 37^oC over a 4-week period (Jose et al., 2010). The authors characterised the degradation behaviour of the scaffolds by calculating the reduction in mass of the scaffolds. For the multicomponent scaffolds, a high initial mass loss, followed by gradual decline for the rest of the degradation period, was observed. The PLGA scaffold, on the contrary, exhibited an initial low mass loss during the 1^st day, followed by a tremendous decline in mass over the remainder of the degradation period. The high initial mass loss of the multicomponent scaffolds was ascribed to a

combination of high surface area of nanofibres and the increased hydrophilicity of the scaffolds (due to the presence of nano-HA and collagen). However, it was further noted that the presence of high molecular weight chains of PLGA, cross-linked gelatin and nano-HA (which exhibits an acid-neutralising characteristic) resulted in stable mass retention after the large initial mass loss (Jose et al., 2010).

The method of simply mixing HA particles with polymer solutions prior to electrospinning in order to fabricate electrospun polymer/HA scaffolds has been reported to cause a decrease in fibre diameter. Chuenjitkuntaworn et al. (2010) reported a reduction in fibre diameter from 3.3 ± 1.1 µm to 2.3 ± 0.7 µm when the concentration of HA particles mixed with PLLA prior to electrospinning was increased from 0 wt% to 0.25 wt% (Chuenjitkuntaworn et al., 2010). Chuenjitkuntaworn et al. (2010) attributed the decrease in fibre diameter to an increase in flow restriction of the polymer solution due to the presence of the HA particles.

This reason is plausible, considering the relatively large diameter (≈234 nm) (Chuenjitkuntaworn et al., 2010) reported for the HA particles used in their work. Similarly, Jose et al. (2010) reported a reduction in fibre diameter from 269 nm to 179 nm when the concentration of HA particles mixed with PLGA/Collagen was increased from 0 wt% to 1 wt%. The reduction in fibre diameter was attributed to the presence of phosphate and calcium ions of HA in polymer solution which resulted in increased solution conductivity, increased stretching of the electrospinning jet and a consequent decrease in fibre diameter (Jose et al., 2010). As in the work of Chuenjitkuntaworn et al. (2010), it is also possible that the large diameters of the HA particles in this work (100-150 nm) (Jose et al., 2010) caused a restriction in the flow of the polymer solution during electrospinning. Xu et al. (2007) reported a similar reduction in fibre diameter from 1.10 µm to 600 nm when the concentration of PLA-g-HAP particles was increased from 0 wt% to 30 wt%. They also

reported that increasing the PLA-g-HAP concentration resulted in an increase in electrical conductivity and a decrease in viscosity of the polymer solutions. The increase in electrical conductivity was attributed to the polarity of the HAP particles, while the decrease in viscosity was attributed to the molecular aggregation of PLA chains around the PLA-g-HAP particles (Xu et al., 2007). In this case, it was difficult to determine if the PLA-g-HAP particles caused a restriction of polymer solution flow, because although the HAP particle diameter was reported to be in the range of 20-40 nm (Xu et al., 2007), no dimensions were reported for the PLA-g-HAP particles.

The reduction in fibre diameter of electrospun polymers upon inclusion of HA particles may be due to a combination of all of the above reasons given by Jose et al. (2010), Chuenjitkuntaworn et al. (2010) and Xu et al. (2007). The work of Takahashi et al. (1978) established clearly that the OH^- ion is the charge carrier in hydroxyapatite (Bouhaouss et al., 2001; Takahashi et al., 1978), and therefore the increase in solution conductivity may be due to the OH^- ions.

Contrary to the above reports, Sui et al. (2007) reported only a small decrease of the fibre diameter in the composite scaffolds (Mei et al., 2007; Sui et al., 2007). The reason for this may be due to the fact that Sui et al. (2007) did not directly mix HA particles with the polymer solution, but instead they used a two solvent system, where HA particles were dispersed first in 1,4-dioxane prior to the addition of PLLA dissolved in dichloromethane to the suspension. In addition, the diameter of the HA particles reported in their work (15 nm) (Sui et al., 2007) was very small in comparison to the works of Chuenjitkuntaworn et al.

(2010) and Jose et al. (2010), so that the possibility of the HA particles restricting the flow of the polymer solution during electrospinning was much lower.

The reduction in fibre diameter upon the mixing of HA particles with polymer solutions prior to electrospinning could result in a change in their degradation characteristics. Degradation has been shown to vary with fibre diameter. For instance, Bolgen et al. (2005) reported the in vitro degradation of PCL electrospun nanofibres of different diameters for a period of 6

months. The authors reported, that overall, thinner fibres exhibited higher degradation rates in mechanical strength when compared to thicker ones (Bolgen et al., 2005). On the contrary, Dong et al. (2009), in their review, suggested that the degradation rate of electrospun PLGA (both 75:25 and 50:50) nanofibres decreased with reduction in fibre diameter (Dong et al., 2009). Furthermore, in their work on the in vitro degradation of PLLA nanofibre foams produced by liquid-liquid phase separation, Chen and Ma (2006) reported faster degradation rates for PLLA nanofibre foams when compared to solid-walled PLLA foams and attributed the faster degradation rate of the PLLA nanofibre foams to the higher surface area of the nanofibre foams in comparison to the solid-walled foams (Chen and Ma, 2006). Unlike the degradation of millimetre and micron-sized polymers, in which an increase in size has been reported to result in faster degradation rates due to autocatalysis (Grizzi et al., 1995), it has been reported recently, that the probability of the occurrence of autocatalysis during degradation of polymer nanofibres is low because the soluble hydrolytic products can be easily diffused into the degradation medium (Dong et al., 2009;

Dong et al., 2010; Kim et al., 2003; Dahlin et al., 2011; Chen and Ma, 2006). Consequently, since fibres with smaller diameters will possess a higher surface area-to-volume ratio, it may be possible that the thinner fibres formed as a result of the mixing of HA particles with polymer solutions prior to electrospinning may exhibit higher degradation rates in comparison to larger fibres.

However, there have been no studies covering what effect the reduction of fibre diameter upon the inclusion of HA particles in electrospun polymer scaffolds will have on the degradation characteristics of the electrospun scaffold. A study of the in vitro degradation properties of electrospun polymer/HA scaffolds will therefore help to provide some indication into how successful such scaffolds with small average fibre diameters will perform when utilized in vivo.