Incorporation of poly(lactide-co-glycolide) microparticles in collagen-based scaffolds for tissue engineering applications

GALLO LOREANA; MARTA MADAGHIELE; LUCA SALVATORE; AMILCARE BARCA; STEFANIA SCIALLA; TIZIANO VERRI; VERÓNICA BUCALÁ; ALESSANDRO SANNINO

Bahía Blanca

Congreso; IX Congreso Argentino de Ingeniería Química; 2017

Asociación Argentina de Ingenieros Químicos- Planta Piloto de Ingeniería Química

Tissue engineering and regenerative medicine are interdisciplinary fields that aim at inducing the regeneration of adult tissues or organs, by using a porous resorbable matrix, termed scaffold (SC), able to host cells and guide them towards the synthesis of physiological tissue. Porous SCs provide mechanical stability, an initial framework for cells to migrate within the lesion and facilitate vascular infiltration [1]. Sustained delivery of exogenous bioactive molecules at the defect site may be also particular important for the regeneration of very large tissue defects and/or tissues that do not show capability for spontaneous regeneration [2,3]. In this context, the goal of the present work was the fabrication of highly porous collagen-based SCs incorporating uniformly dispersed poly(lactide-co-glycolide) (PLGA) microparticles (MPs) as depots for the sustained and localized delivery of bioactive molecules. Collagen SCs loaded with different amounts of PLGA-MPs were prepared by freeze-drying and crosslinking. The SC microstructure wasassessed to evaluate the spatial distribution of MPs and the achieved pore size. The impact of the MPs on the SC stiffness was investigated through compression tests.Preliminarily, the cell-MPs interactions were also evaluated by imaging of cellmorphology in vitro, adopting the Caco-2 human derived epithelial cell model.The PLGA-MPs were produced by a double emulsion technique (w/o/w) [3]. Sizedistribution of MPs was determined by laser diffractometry. For SCs production, type I collagen was dispersed in distilled water at given concentration (1 and 2% w/v) and different amount of MPs was added (1, 2, 4 and 6 mg/ml). The suspensions were kept on stirring for 3 hours. Then, collagen slurries were degassed via centrifugation, cast in Petri dishes, and subjected to freeze-drying [4]. Part of the SCs was crosslinked by watersoluble carbodiimide and re-lyophilized [5]. SCs devoid of MPs were also produced for comparative purposes.The SC microstructure and the MPs distribution were evaluated by scanning electron microscopy (SEM). Total porosity (TP) was measured gravimetrically, as TP= 1-rsc /rcollagen, where rcollagen is the density of collagen and rsc is the apparent density of the SC. The mechanical properties of the SCs were tested by a universal testing machine; the elastic modulus E was calculated as the slope of the stress-strain curve at low deformation (0-5%) [6]. Administration of MPs to Caco-2 cells was performed by adding 3 mg/ml MPs to the standard culture medium (Eagle?s Minimum Essential Medium). MPs were administered simultaneously with seeding and 48 h after seeding. 7 days after MPs administration, standard protocols for phalloidin-FITC staining of cell cytoskeleton and DAPI nuclearstaining were assay.PLGA-MPs which showed a mean size of approximately 20 μm, were added to thecollagen slurry at different amounts. While the 1% w/v collagen suspension was found to induce MPs precipitation upon centrifugation, due to its low viscosity, the 2% w/v suspension provided a satisfactory inclusion of the MPs. This was confirmed by SEM,which showed that the MPs were uniformly dispersed into the porous structure of the SCs, both before and after the crosslinking. Moreover, the SCs presented a pore size between 90 and 150 μm, in accordance with previous studies [4]. The TP of the SCs without MPs was about 97%, as expected [4], and was not significantly affected by crosslinking.Conversely, the SCs with MPs showed a different TP value before and aftercrosslinking, which was about 94% and 96% respectively. However, this difference was not detected for the SCs having the highest MPs loading, which showed anapproximately constant TP of 94%. Overall, the TP of the SCs tended to slightly decrease as the MPs concentration increased.The modulus E of the SCs without MPs increased from about 1.4 to 4 kPa aftercrosslinking demonstrating the efficacy of the treatment. Of particular note was that the presence of MPs in the samples further increased the modulus (up to 7.5-10 kPa),suggesting that the MPs enhanced the mechanical strength of the SCs. However, the different amounts MPs did not produce significant differences among samples.Preliminary analysis of cell-MPs interactions showed that the presence of MPs did not affect cell morphology and cytoskeleton organization, giving hints of proper cytocompatibility. Interestingly, cell adhesion dynamics were shown to be optimal and similar to the untreated control cells, also when MPs were administered simultaneously to cell seeding (i.e. during the period of high sensitivity of the attachment/adhesion phase).Collagen SCs with different amounts of uniformly dispersed PLGA-MPs weresuccessfully produced. The MPs did not negatively affect the porous structure of the SCs while acting as a mechanical reinforcement. Additionally, MPs show high permissiveness to cell adhesion, and the interactions between MPs and epithelial cell membranes do not interfere with the correct cells morphological differentiation. Such promising results suggest the potential of the developed scaffolds for tissue engineering applications.References1. Yannas,2001.2. Giampetruzzi et al.,2017.3. Ungaro et al.,2006.4. O?Brien et al.,2005.5. Damink et al.1996.6. Monaco et al.,2017.