Nanoclay-enriched poly(ɛ-caprolactone) electrospun scaffolds for osteogenic differentiation of human mesenchymal stem cells

Abstract

Musculoskeletal tissue engineering aims at repairing and regenerating damaged tissues using biological tissue substitutes. One approach to achieve this aim is to develop osteoconductive scaffolds that facilitate the formation of functional bone tissue. We have fabricated nanoclay-enriched electrospun poly(ɛ-caprolactone) (PCL) scaffolds for osteogenic differentiation of human mesenchymal stem cells (hMSCs). A range of electrospun scaffolds is fabricated by varying the nanoclay concentrations within the PCL scaffolds. The addition of nanoclay decreases fiber diameter and increases surface roughness of electrospun fibers. The enrichment of PCL scaffold with nanoclay promotes in vitro biomineralization when subjected to simulated body fluid (SBF), indicating bioactive characteristics of the hybrid scaffolds. The degradation rate of PCL increases due to the addition of nanoclay. In addition, a significant increase in crystallization temperature of PCL is also observed due to enhanced surface interactions between PCL and nanoclay. The effect of nanoclay on the mechanical properties of electrospun fibers is also evaluated. The feasibility of using nanoclay-enriched PCL scaffolds for tissue engineering applications is investigated in vitro using hMSCs. The nanoclay-enriched electrospun PCL scaffolds support hMSCs adhesion and proliferation. The addition of nanoclay significantly enhances osteogenic differentiation of hMSCs on the electrospun scaffolds as evident by an increase in alkaline phosphates activity of hMSCs and higher deposition of mineralized extracellular matrix compared to PCL scaffolds. Given its unique bioactive characteristics, nanoclay-enriched PCL fibrous scaffold may be used for musculoskeletal tissue engineering.

FIG. 1.

Preparation of electrospun PCL-nanoclay nanocomposite fibers. (a) Schematic representation of the process to generate PCL/nanoclay composite fibers using electrospinning technique. The amount of nanoclay was varied from 0%, 0.1%, 1%, and 10% with regard to PCL. (b) The effect of nanoclay on fiber diameter and surface morphology of fibers. Pure PCL fibers show uniform size distribution and smooth surface morphology. The addition of silicate reduces fiber diameter and induces surface roughness. The effect appears to be prominent in electrospun fibers containing 1% and 10% nanoclay. (c) The effect of nanoclay on fiber diameter was quantified using image analysis. The average fiber diameter of PCL scaffolds is 5.6±0.6 μm. The addition of 1% and 10% nanoclay significantly reduced fiber diameter to 3.6±0.5 and 0.6±0.5 μm. The data represent mean±standard deviation (n=50, ANOVA *p<0.05). The solid bars signify regions indicating 50% of the distribution of fiber diameter. (d) The distribution of nanoclay within the electrospun scaffold was determined by staining the nanoclay with a red dye. The scaffold containing nanoclay-formed beaded structures. ANOVA, analysis of variance; PCL, poly(ɛ-caprolactone). Color images available online at www.liebertpub.com/tea

FIG. 2.

The effect of nanoclay addition on the degradation of electrospun PCL scaffolds. (a) Electrospun PCL scaffolds degrade via surface degradation as depicted from the linear mass loss. The decrease in PCL fiber diameter was observed after 48 h in PCL scaffolds indicating surface degradation characteristic. The addition of nanoclay results in the bulk degradation of electrospun scaffolds. All the nanocomposite scaffolds show breakage of fibers in 24 and 48 h, respectively. In PCL-10% nanoclay fibers, smaller fibers quickly degraded, followed by bulk degradation of the thicker fibers. (b) The accelerated degradation of the electrospun scaffolds was determined by monitoring the weight loss of the fibrous structure over a period of 96 h. All the scaffolds showed a steady weight loss indicating gradual degradation of the scaffold. The scaffold containing 10% nanoclay showed enhanced degradation compared to PCL-only scaffolds indicating that nanoclay might promote the adsorption of water within the structure as well as the bulk degradation of PCL. The data represent mean±standard deviation (n=5).

FIG. 3.

Effect of nanoclay on thermal characterization of electrospun PCL fibers. (a) The differential scanning calorimeter thermograms showing cooling cycle (top panel), the addition of nanoclay-increased crystallization temperature (T_c), and the enthalpy of crystallization (delta H_c) indicate that nanoclay might be restricting polymer chain movements. The electrospinning of PCL results in an increase in polymer crystallinity (χ_c). The addition of nanoclay results in a slight increase in polymer crystallinity. Similarly, the heating cycles (bottom panel) of electrospun PCL and nanoclay-enriched PCL scaffolds indicate a significant increase in the melting temperature (T_m) and enthalpy of melting (delta H_m) when compared to PCL beads. The addition of nanoclay increased the thermal stability of nanocomposite due to an enhanced surface interaction between polymer and nanoclay. (b) TGA thermograph and DTGA profile indicate decreased thermal degradation temperature (T_d) due to the addition of nanoclay to PCL. (c) The summary of thermal properties of PCL and nanoclay-enriched PCL scaffolds. Color images available online at www.liebertpub.com/tea

FIG. 4.

Effect of nanoclay on the mechanical properties of electrospun PCL fibers. (a) Stress-strain curve of electrospun scaffolds subjected to uniaxial tensile stress was shown. (b) The addition of nanoclay significantly reduced elastic modulus, ultimate stress, and fracture stress. This is attributed to the decrease in fiber diameter due to the addition of nanoclay. The decrease in elongation was mainly attributed to the deformation of individual fibers. (c) The SEM images indicate that PCL scaffolds undergo uniform deformation when subjected to tensile stress. The addition of nanoclay resulted in a heterogeneous deformation and lower mechanical strength. SEM, scanning electron microscopy. Color images available online at www.liebertpub.com/tea

FIG. 5.

Nanoclay promotes in vitro biomineralization on electrospun scaffold in simulated body fluid (SBF). (a) Since the prepared PCL scaffolds had uniform and smooth surface morphology, however after subjecting these electrospun scaffolds to 10× SBF for 2 h, the formation of mineralized layer on the fibers was observed. SBF is a super saturated solution of calcium and phosphate, and a bioactive surface when submersed in SBF promotes the formation of mineralized structure. (b) The SEM images indicated fiber morphology of the electrospun scaffolds before and after subjecting the scaffold to SBF. The addition of nanoclay to PCL significantly improved the biomineralization ability due to enhanced surface roughness. (c) The chemical nature of the deposited mineralized matrix was evaluated using FTIR. Hydroxyapatite has a strong peak at 1045 cm⁻¹ that corresponds to the P-O stretching band. Both PCL and PCL-Nanoclay composites showed the formation of hydroxyapatite when submersed in SBF. FTIR, Fourier Transform Infrared Spectroscopy. Color images available online at www.liebertpub.com/tea

FIG. 6.

Adhesion and proliferation of hMSCs on PCL and PCL-nanoclay electrospun scaffolds. (a) hMSCs readily attached and spread on the fibrous structure, and the cell bodies were stretched along the fiber axis (Day 3). However, no significant difference in cell attachment was observed due to the addition of nanoclay. (b) The proliferation of hMSCs was monitored using Alamar Blue on day 1 and 7 in normal media. hMSCs readily proliferate on PCL and nanoclay-enriched PCL scaffolds. However, no significant effect of nanoclay on cell proliferation was observed. The data represent mean±standard deviation (n=3, ANOVA *p<0.05). hMSCs, human mesenchymal stem cells. Color images available online at www.liebertpub.com/tea

FIG. 7.

Effect of nanoclay on osteogenic differentiation of hMSCs. (a) hMSCs were stained for surface alkaline phosphatase (ALP)-positive cells after 7 and 14 days. A uniform distribution of the ALP-positive cells on the scaffold can be observed, suggesting that the differentiation occurs in a homogeneous manner throughout the scaffolds. This is in correlation with the ALP activity result, where peak ALP activity in PCL scaffolds was observed on day 14 and in nanoclay-enriched PCL scaffolds, it was observed on day 7. (b) ALP activity of hMSCs seeded on electrospun scaffold was monitored over the period of 21 days. The ALP activity first increases and then decreases, presents a bell shape pattern, compatible with osteogenic differentiation of hMSCs. Briefly, no significant effect of nanoclay was observed on days 3, 14, and 21. However, on day 7, the nanoclay-enriched scaffolds showed significantly higher ALP activity compared to the PCL scaffolds. This indicates that the nanoclay from polymeric scaffold triggers and sustains the osteogenic differentiation of stem cells. Color images available online at www.liebertpub.com/tea

FIG. 8.

Effect of nanoclay on the formation of mineralized matrix. (a) The production of mineralized matrix was evaluated using Alizarin Red S staining on day 21. The results indicate a significantly higher production of mineralized extracellular matrix in PCL-nanoclay composites compared to PCL scaffolds. This indicates that the nanoclay from polymeric scaffold triggers and sustains the osteogenic differentiation of stem cells. (b) The image quantification indicates the production of enhanced mineralized matrix coverage due to the addition of nanoclay. The bars represent mean±standard deviation (n=3; horizontal bars represent significant differences between groups, p<0.05, ANOVA). Color images available online at www.liebertpub.com/tea