Production and in vitro evaluation of macroporous, cell-encapsulating alginate fibres for nerve repair

The prospects for successful peripheral nerve repair using fibre guides are considered to be enhanced by the use of a scaffold material, which promotes attachment and proliferation of glial cells and axonal regeneration. Macroporous alginate fibres were produced by extraction of gelatin particle porogens from wet spun fibres produced using a suspension of gelatin particles in 1.5% w/v alginate solution. Gelatin loading of the starting suspension of 40.0, 57.0, and 62.5% w/w resulted in gelatin loading of the dried alginate fibres of 16, 21, and 24% w/w respectively. Between 45 and 60% of the gelatin content of hydrated fibres was released in 1 h in distilled water at 37 °C, leading to rapid formation of a macroporous structure. Confocal laser scanning microscopy (CLSM) and image processing provided qualitative and quantitative analysis of mean equivalent macropore diameter (48–69 μm), pore size distribution, estimates of maximum porosity (14.6%) and pore connectivity. CLSM also revealed that gelatin residues lined the macropore cavities and infiltrated into the body of the alginate scaffolds, thus, providing cell adhesion molecules, which are potentially advantageous for promoting growth of glial cells and axonal extension. Macroporous alginate fibres encapsulating nerve cells [primary rat dorsal root ganglia (DRGs)] were produced by wet spinning alginate solution containing dispersed gelatin particles and DRGs. Marked outgrowth was evident over a distance of 150 μm at day 11 in cell culture, indicating that pores and channels created within the alginate hydrogel were providing a favourable environment for neurite development. These findings indicate that macroporous alginate fibres encapsulating nerve cells may provide the basis of a useful strategy for nerve repair

Gravity spinning of polycaprolactone fibres for applications in tissue engineering

Poly(var epsilon-caprolactone) (PCL) fibres have been produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. The tensile strength and stiffness of as-spun fibres were highly dependent on the concentration of the spinning solution. Use of a 6% w/v solution resulted in fibres having strength and stiffness of 1.8 MPa and 0.01 GPa, respectively, whereas these values increased to 9.9 MPa and 0.1 GPa when fibres were produced from 20% w/v solutions. Cold drawing to an extension of 500% resulted in further increases in fibre strength (up to 50 MPa) and stiffness (0.3 GPa). The surface morphology of as-spun fibres was modified, to yield a directional grooved pattern by drying in contact with a mandrel having a machined topography characterised by a peak–peak separation of 91 μm and a peak height of 30 μm. Limited in vitro studies of cell behaviour in contact with the fibres were performed using cell culture. The number of attached fibroblasts and myoblasts on as-spun PCL fibres after 5 days in cell culture was lower than on tissue culture plastic by a factor 2 and 1.5, respectively, but higher than on Dacron monofilament by a factor of 4 and 11, respectively. The high fibre compliance and the potential for controlling the fibre surface architecture to promote contact guidance effects together with the maintained proliferation of fibroblasts and myoblasts on as-spun PCL fibres in vitro recommends their use for 3-D scaffold production in soft tissue engineering

Micro-CT in drug delivery

Micro-computed tomography (micro-CT) has not to date been fully exploited in the area of controlled drug delivery despite its capability for providing detailed, 3-D images of morphology and the opportunity this presents for exploring the relationships between delivery device formulation, structure and performance. Micro-CT was used to characterize the internal structure of polycaprolactone (PCL) matrix-type devices incorporating soluble particulates (lactose Mw 342.30, gelatin Mw 20–25 kDa) as models of hydrophilic bioactives or pore-forming excipients. Micro-CT images confirmed that the lactose and gelatin particles were uniformly dispersed throughout the PCL phase and that efficient delivery of 95–100% of each species in 9 days involved transport from the matrix core. Quantitative analysis of micro-CT images provided values for matrix macroporosity, which were within 15% of the theoretical value and revealed uniform porosity throughout the samples. Total release of protein occurred in 9 days (PBS, 37 °C) from matrices containing a high protein load (44% w/w) and was independent of particle size. Measurements of equivalent pore diameter and frequency distribution identified a large population of sub-40 μm pores in each material, indicative of a high density of connecting channels between particles which facilitates protein transport through the matrices

Delivery of bioactive macromolecules from microporous polymer matrices: release and activity profiles of lysozyme, collagenase and catalase

Microporous polycaprolactone (PCL) matrices containing lysozyme, collagenase and catalase respectively with molecular weight covering a wide range from 14.3 to 240kDa were produced by a novel method involving rapid cooling of particle suspensions in dry ice. The enzyme loading efficiency (lysozyme (50%), collagenase (75%) and catalase (90%)) depended on the enzyme molecular weight and the non-solvent used to extract acetone from the hardened matrices. Sustained enzyme release occurred from the PCL matrices over 11 days with retained activity dependent on the particular enzyme used (collagenase 100% activity at 11 days, lysozyme 75-80% at 11 days, catalase 10-20% at 5 days). The present findings confirm the potential of microporous PCL matrices for delivering bioactive macromolecules from implantable/insertable depot-type formulations and tissue engineering scaffolds and recommend catalase as a challenging model protein for evaluating such devices

Characterisation of the macroporosity of polycaprolactone-based biocomposites and release kinetics for drug delivery

Microporous, biocomposite matrices comprising a continuous phase of poly(epsilon-caprolactone) (PCL) and a dispersed phase of lactose or gelatin particles with defined size range (45-90, 90-125 and 125-250 microm) were produced by precipitation casting from solutions of PCL in acetone. Scanning electron microscopy (SEM) analysis revealed a characteristic surface morphology of particulates interspersed amongst crystalline lamellae of the polymer phase. Rapid release of around 80% of the lactose content occurred in PBS at 37 degrees C in 3 days, whereas biocomposites containing gelatin particles of size range 90-125 and 125-250 microm, respectively, displayed gradual and highly efficient release of around 90% of the protein phase over 21 days. A highly porous structure was obtained on extraction of the water-soluble phase. Micro-computed tomography (Micro-CT) and image analysis enabled 3-D visualisation and quantification of the internal pore size distribution. A maximum fractional pore area of 10.5% was estimated for gelatin-loaded matrices. Micro-CT analysis confirmed the presence of an extensive system of macropores, sufficiently connected to permit protein diffusion, but an absence of high volume, inter-pore channels. Thus tissue integration would be confined to the matrix surface initially if the designs investigated were used as tissue-engineering scaffolds, with the core potentially providing a depot system for controlled delivery of growth factors

Characterising the macroporosity Of polymeric matrices designed for protein delivery and tissue engineering

Microporous, biocomposite matrices comprising a continuous phase of poly(ε-caprolactone) (PCL) and a dispersed phase of lactose or gelatin particles with defined size range (45–90, 90–125 and 125–250 μm) were produced by precipitation casting from solutions of PCL in acetone. Scanning electron microscopy (SEM) analysis revealed a characteristic surface morphology of particulates interspersed amongst crystalline lamellae of the polymer phase. Rapid release of around 80% of the lactose content occurred in PBS at 37 °C in 3 days, whereas biocomposites containing gelatin particles of size range 90–125 and 125–250 μm, respectively, displayed gradual and highly efficient release of around 90% of the protein phase over 21 days. A highly porous structure was obtained on extraction of the water-soluble phase. Micro-computed tomography (Micro-CT) and image analysis enabled 3-D visualisation and quantification of the internal pore size distribution. A maximum fractional pore area of 10.5% was estimated for gelatin-loaded matrices. Micro-CT analysis confirmed the presence of an extensive system of macropores, sufficiently connected to permit protein diffusion, but an absence of high volume, inter-pore channels. Thus tissue integration would be confined to the matrix surface initially if the designs investigated were used as tissue-engineering scaffolds, with the core potentially providing a depot system for controlled delivery of growth factors

An evaluation of polycaprolactone matrices for vaginal delivery of the antiviral, tenofovir, in preventing heterosexual transmission of HIV

Nevirapine (NVP) was loaded in polycaprolactone (PCL) matrices to produce vaginal inserts with the aim of preventing HIV transmission. NVP dispersions in PCL were prepared, at 10% (w/w) theoretical loading, measured with respect to the PCL content of the matrices, in the form of (1) NVP only, (2) a physical mixture of NVP with polyethylene glycol (PEG) 6000 or (c) a solid dispersion (SD) with PEG produced by co-dissolution in ethanol. Characterisation of SD by differential scanning calorimetry and attenuated total reflectance-Fourier transform infrared spectroscopy suggested transformation of the crystalline structure of NVP to an amorphous form which consequently increased the dissolution rate of drug. A low-loading efficiency of 13% was obtained for NVP-loaded matrices and less than 20% for matrices prepared using physical mixtures of drug and PEG. The loading efficiency was improved significantly to around 40% when a 1:4 NVP-PEG SD was used for matrix production. After 30 days, 40% of the drug content was released from NVP-loaded matrices, 55% from matrices containing 1:4 NVP-PEG physical mixtures and 60% from matrices loaded with 1:4 NVP-PEG SDs. The in vitro anti-viral activity of released NVP was assessed using a luciferase reporter gene assay following the infection of HeLa cells with pseudo-typed HIV-1. NVP released from PCL matrices in simulated vaginal fluid retained over 75% anti-HIV activity compared with the non-formulated NVP control. In conclusion, 1:4 NVP-PEG SDs when loaded in PCL matrices increase drug loading efficiency and improve release behaviour