Three dimensional electrospun PCL/PLA blend nanofibrous scaffolds with significantly improved stem cells osteogenic differentiation and cranial bone formation

Abstract

Nanofibrous scaffolds that are morphologically/structurally similar to natural ECM are highly interested for tissue engineering; however, the electrospinning technique has the difficulty in directly producing clinically relevant 3D nanofibrous scaffolds with desired structural properties. To address this challenge, we have developed an innovative technique of thermally induced nanofiber self-agglomeration (TISA) recently. The aim of this work was to prepare (via the TISA technique) and evaluate 3D electrospun PCL/PLA blend (mass ratio: 4/1) nanofibrous scaffolds having high porosity of ∼95.8% as well as interconnected and hierarchically structured pores with sizes from sub-micrometers to ∼300 μm for bone tissue engineering. The hypothesis was that the incorporation of PLA (with higher mechanical stiffness/modulus and bioactivity) into PCL nanofibers would significantly improve human mesenchymal stem cells (hMSCs) osteogenic differentiation in vitro and bone formation in vivo. Compared to neat PCL-3D scaffolds, PCL/PLA-3D blend scaffolds had higher mechanical properties and in vitro bioactivity; as a result, they not only enhanced the cell viability of hMSCs but also promoted the osteogenic differentiation. Furthermore, our in vivo studies revealed that PCL/PLA-3D scaffolds considerably facilitated new bone formation in a critical-sized cranial bone defect mouse model. In summary, both in vitro and in vivo results indicated that novel 3D electrospun PCL/PLA blend nanofibrous scaffolds would be strongly favorable/desired for hMSCs osteogenic differentiation and cranial bone formation.

Keywords: 3D electrospun nanofibrous scaffold; Bone regeneration; Osteogenic differentiation; Polycaprolactone; Polylactic acid.

Fig. 1

SEM images showing the representative morphologies of PCL/PLA blend nanofibers electrospun from the solutions with PCL/PLA concentrations of (A) 7 wt.%, (B) 8 wt.%, (C) 9 wt.%, and (D) 10 wt.%, respectively.

Fig. 2

SEM images showing the typical morphologies of (A1) as-electrospun PCL nanofibrous mat, (A2) fragmented/tiny PCL nanofibrous mat/piece, (A3) short individual PCL nanofibers, (B1) as-electrospun PCL/PLA nanofibrous mat, (B2 and B3) fragmented/tiny PCL/PLA nanofibrous mat/piece; (C1, C2, and C3) outer surface and (D1, D2, and D3) inner surface of 3D electrospun PCL nanofibrous scaffold; (E1, E2, and E3) outer surface and (F1, F2, and F3) inner surface of 3D electrospun PCL/PLA nanofibrous scaffold.

Fig. 3

Compressive stress-strain curves acquired from PCL-3D and PCL/PLA-3D scaffolds under both dry (left) and wet (right) conditions.

Fig. 4

SEM images of PCL-3D and PCL/PLA-3D scaffolds immersed in SBF after 1 day: (A1) PCL, (A2) PCL/PLA; after 7 days: (A3) PCL, (A4) PCL/PLA; and after 14 days: (A5) PCL, (A6) PCL/PLA.

Fig. 5

ATR spectra acquired from PCL-3D (left) and PCL/PLA-3D (right) scaffolds after immersion in SBF for different days (i.e., 0, 4, and 14 days).

Fig. 6

(A) hMSCs viabilities on PCL-3D and PCL/PLA-3D scaffolds after culturing for 1 and 3 days. hMSCs morphologies on (B1) PCL-3D and (B2) PCL/PLA-3D scaffolds after culturing for 16 h. Note that PCL-3D scaffolds are included as control samples. Data are expressed as mean ± SD (n = 3).

Fig. 7

ALP activity of hMSCs cultured on (A) PCL-3D and PCL/PLA-3D scaffolds for 10 days in growth medium (GM) and osteoinductive medium (OI). ALP activity was normalized by total protein content. (B) Calcium contents in PCL-3D and PCL/PLA-3D scaffolds were measured after culturing in GM and OI for 3 weeks. (C1 and C2) Osteogenic marker gene expressions (ALP and BSP) were studied by real-time PCR assay after 7 days of cell culture. Data are expressed as mean ± SD (n = 3). P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 8

(A) ALP activity of hMSCs cultured on PCL-3D and PCL/PLA-3D scaffold extracts, (B) expression of VEGF in hMSCs cultured in PCL-3D and PCL/PLA-3D scaffold extracts.

Fig. 9

Radiographic examination and macro-view of the histological scaffolds of PCL, PCL/PLA, PCL-rhBMP2, and PCL/PAL-rhBMP2 groups after 6 weeks of implantation. Representative data are shown (n = 5–6).

Fig. 10

H&E staining of the repaired calvarias after 6 weeks of implantation in vivo.