Fabrication and characterization of aligned nanofibrous PLGA/Collagen blends as bone tissue scaffolds

Abstract

Aligned nanofibrous blends of poly (d, l-lactide-co-glycolide) (PLGA) and collagen with various PLGA/collagen compositions (80/20, 65/35 and 50/50) were fabricated by electrospinning and characterized for bone tissue engineering. Morphological characterization showed that the addition of collagen to PLGA resulted in narrowing of the diameter distribution and a reduction in average diameter. Differential scanning calorimetric (DSC) studies showed that the triple helix structure of the native collagen was not destroyed during the fabrication process. However, the blending had a marked effect on the overall enthalpy of the blends, whereby the total enthalpy decreased as the collagen content decreased. Thermogravimetric analysis showed the addition of collagen increased the hydrophilicity of the scaffolds. The crosslinking of collagen to increase the biostability was done using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and an overall ∼25% degree of crosslinking was achieved. The EDC crosslinking had little effect on the nanofibrous morphology of the 80/20 blend system; however, the nanofibrous features were compromised to some extent at higher collagen concentrations. The mechanical characterization under dry and wet conditions showed that increasing collagen content resulted in a tremendous decrease in the mechanical properties. However, crosslinking resulted in the increase in elastic modulus from 47 MPa to 83 MPa for the wet PLGA/Collagen 80/20 blend system, with little effect on the tensile strength. In conclusion, the aligned nanofibrous scaffold used in this study constitutes a promising material for bone tissue engineering.

Introduction

Bone is a complex tissue that serves multiple functions, including supporting the body, protecting organs, and storing nutrients. It has a highly anistropic morphology, which results in a range of mechanical properties. When considering engineering bone tissue, particularly for musculoskeletal tissues, matching the anisotropy and properties of the tissue scaffold is a key [1], [2]. In addition, aligning the scaffolds can assist in guided growth for the cells as well as increased cell proliferation [3] and higher natural extracellular matrix (ECM) production [4], compared to random fibers. Recently, electrospinning has emerged as a solution for fabricating scaffolds with nanofibrous features which can mimic the ECM. The inherent nonwoven nature of the electrospun nanofibers results in interconnected pores sufficient for cell attachment and nutrient transfer [5]. Orientation in electrospun nanofibers can be achieved by adapting various collector designs [6].

In addition to the morphology, another important criterion for any scaffold is the choice of materials, and for bone tissue engineering, an obvious choice is type I collagen. Collagen is a major ECM component of bone (30%) and it possesses natural binding sites for the adhesion of osteoblasts and fibroblasts [2]. However, the use of collagen alone as a scaffold material is limited due to its poor mechanical properties and rapid degradation behavior [7], [8]. Blending a bioabsorbable polymer with collagen is expected to modulate its degradation rate, while the collagen should improve the bioactivity of the synthetic polymers. Significant increases in cell adhesion and proliferation has been reported in blends of collagen with polymers [9], [10]. Meng et al. [9] studied electrospun blends of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and collagen and found that the blending (30% collagen) resulted in significant increase in cell adhesion and growth compared to PHBV nanofibers. Zhang et al. [11] found that the addition of gelatin (denatured collagen) to poly(ɛ-caprolactone) (PCL) resulted in not only better hydrophilicity but a threefold increase in cell infiltration into the depth of the scaffolds. Similarly higher hydrophilicity and an 82% increase in cell proliferation were observed by Venugopal et al. also upon addition of collagen to PCL [12]. Chiu et al. [13] showed the incorporation of type I collagen (<1.0 wt %) in electrospun poly(l-lactide) (PLLA) scaffolds increased both cell attachment and migration tremendously. After 1 week, cells migrated through 85% of the blend scaffold whereas only 32% was observed in the PLLA scaffold. In addition to blending with slowly degradable synthetic polymers, the rapid hydrolysis of a collagenous matrix can be prevented via crosslinking. Several different physical as well as chemical crosslinking methods have been reported for collagen-crosslinking [14], [15], [16], [17], [18]. Recently, use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol has been studied by various researchers [17], [19], [20], [21], [22]. Powell et al. [21] have used EDC crosslinking to stabilize collagen-chondroitin-6-sulfate (GAG) scaffolds with different concentrations of EDC. The characterization of scaffolds revealed no difference in scaffold morphology between control and crosslinked scaffolds, almost no degradation for 30 days at high concentration (50 mM), increase in mechanical properties and cell density at low EDC concentration (5 mM) and a decrease in mechanical properties at higher concentration. Also, Barnes et al. [17] successfully used EDC in ethanol medium for crosslinking type II collagen and showed enhanced structural stability, greater degree of crosslinking and mechanical properties compared to glutaraldehyde crosslinking.

In this study, we prepared aligned nanofibrous scaffolds based on blends of poly (d,l-lactide-co-glycolide) (PLGA) and type I collagen with different blend compositions (PLGA/Collagen: 80/20, 65/35, 50/50). These collagen blended scaffolds were crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and the mechano-morphological properties were compared with those of non-crosslinked scaffolds. We report, for the first time, aligned collagen–polymer nanofiber blends in which the morphology was preserved by crosslinking with EDC.