Osteoinductive peptide-functionalized nanofibers with highly ordered structure as biomimetic scaffolds for bone tissue engineering

Abstract

The construction of functional biomimetic scaffolds that recapitulate the topographical and biochemical features of bone tissue extracellular matrix is now of topical interest in bone tissue engineering. In this study, a novel surface-functionalized electrospun polycaprolactone (PCL) nanofiber scaffold with highly ordered structure was developed to simulate the critical features of native bone tissue via a single step of catechol chemistry. Specially, under slightly alkaline aqueous solution, polydopamine (pDA) was coated on the surface of aligned PCL nanofibers after electrospinning, followed by covalent immobilization of bone morphogenetic protein-7-derived peptides onto the pDA-coated nanofiber surface. Contact angle measurement, Raman spectroscopy, and X-ray photoelectron spectroscopy confirmed the presence of pDA and peptides on PCL nanofiber surface. Our results demonstrated that surface modification with osteoinductive peptides could improve cytocompatibility of nanofibers in terms of cell adhesion, spreading, and proliferation. Most importantly, Alizarin Red S staining, quantitative real-time polymerase chain reaction, immunostaining, and Western blot revealed that human mesenchymal stem cells cultured on aligned nanofibers with osteoinductive peptides exhibited enhanced osteogenic differentiation potential than cells on randomly oriented nanofibers. Furthermore, the aligned nanofibers with osteoinductive peptides could direct osteogenic differentiation of human mesenchymal stem cells even in the absence of osteoinducting factors, suggesting superior osteogenic efficacy of biomimetic design that combines the advantages of osteoinductive peptide signal and highly ordered nanofibers on cell fate decision. The presented peptide-decorated bone-mimic nanofiber scaffolds hold a promising potential in the context of bone tissue engineering.

Keywords: biomimetic; bone tissue engineering; nanofiber; peptide.

Figure 1

Schematic illustration of (A) fabrication of electrospun nanofibers and (B) surface functionalization of PCL nanofibers with BFP-1 peptides for directing hMSCs osteogenic commitment. Abbreviations: aPCL, aligned PCL nanofibers; PCL, polycaprolactone; BFP-1, bone-forming peptide-1; hMSCs, human mesenchymal stem cells; pDA, polydopamine; pep, peptide; ALP, alkaline phosphatase; OCN, osteocalcin; OPN, osteopontin; h, hour.

Figure 2

Surface characterization of functionalized nanofibers. Notes: (A) SEM images of peptide-decorated nanofibers. The white arrows represent the pDA aggregate particulates. The corresponding SEM images obtained at low magnification are shown as insets. (B) Fiber diameter measurement of nanofibers. (C) Representative surface topographical images of peptide-decorated nanofibers. The white arrows indicate the reference direction for orientation evaluation. The compass images represent the orientation distribution of peptide-decorated nanofibers with random and aligned orientation. (D) Water contact angle and representative images of water droplet on PCL nanofibers with different surface chemistries. (E) Raman spectra analysis of PCL and pDA-coated nanofibers. (F) High-resolution carbon peaks (C1s) from PCL nanofibers with different surface chemistries. The table shows the peak percentage in the carbon peaks (C1s) core-level XPS in different nanofibers. Abbreviations: SEM, scanning electron microscope; pDA, polydopamine; PCL, polycaprolactone; XPS, X-ray photoelectron spectroscopy; R, pure randomly oriented PCL nanofiber; A, pure aligned PCL nanofiber; pep, peptide.

Figure 3

hMSCs’ adhesion, proliferation, and viability on surface-functionalized PCL nanofibers. Notes: (A) SEM observation and (B) CCK-8 assay of adhering hMSCs on samples in serum-free growth media. Scale bars indicate 15 μm. (C) The proliferation of hMSCs on functionalized nanofibers for 1, 3, and 5 days. ^#Compared with R-pDA, P<0.05; *compared with A-pDA, P<0.05. (D) Viability of hMSCs on functionalized nanofibers labeled by FDA/PI staining. Live cells: green; dead cells: red. Scale bars indicate 100 μm. Abbreviations: SEM, scanning electron microscope; CCK-8, Cell Counting Kit-8; hMSCs, human mesenchymal stem cells; pDA, polydopamine; FDA, fluorescein diacetate; PI, propidium iodide; h, hours; TCP, tissue culture plate; PCL, polycaprolactone; R, pure randomly oriented PCL nanofiber; A, pure aligned PCL nanofiber; pep, peptide.

Figure 4

The effect of functionalized nanofibers on osteogenic differentiation of hMSCs under osteoinductive condition. Notes: (A) Representative staining of ALP on day 14 and determination of ALP activity at 7 and 14 days. (B) ARS staining and determination of calcium deposition on day 21. (C) RT-qPCR analysis for osteo-specific genes on day 14. (D) Representative immunofluorescent images of OCN (green) and OPN (red) in different groups on day 14. Scale bars indicate 100 μm. (E) Western blot analysis of Col1a1 and Runx2 expression in hMSCs on different samples. ^#Compared with R-pDA, P<0.05; *compared with A-pDA, P<0.05; ^$compared with R-pDA-pep, P<0.05. Abbreviations: hMSCs, human mesenchymal stem cells; ALP, alkaline phosphatase; ARS, Alizarin Red S; RT-qPCR, real-time quantitative polymerase chain reaction; OCN, osteocalcin; OPN, osteopontin; Col1a1, type I collagen alpha 1; Runx2, Runt-related transcription factor 2; pDA, polydopamine; TCP, tissue culture plate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCL, polycaprolactone; R, pure randomly oriented PCL nanofiber; A, pure aligned PCL nanofiber; pep, peptide.

Figure 4

The effect of functionalized nanofibers on osteogenic differentiation of hMSCs under osteoinductive condition. Notes: (A) Representative staining of ALP on day 14 and determination of ALP activity at 7 and 14 days. (B) ARS staining and determination of calcium deposition on day 21. (C) RT-qPCR analysis for osteo-specific genes on day 14. (D) Representative immunofluorescent images of OCN (green) and OPN (red) in different groups on day 14. Scale bars indicate 100 μm. (E) Western blot analysis of Col1a1 and Runx2 expression in hMSCs on different samples. ^#Compared with R-pDA, P<0.05; *compared with A-pDA, P<0.05; ^$compared with R-pDA-pep, P<0.05. Abbreviations: hMSCs, human mesenchymal stem cells; ALP, alkaline phosphatase; ARS, Alizarin Red S; RT-qPCR, real-time quantitative polymerase chain reaction; OCN, osteocalcin; OPN, osteopontin; Col1a1, type I collagen alpha 1; Runx2, Runt-related transcription factor 2; pDA, polydopamine; TCP, tissue culture plate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCL, polycaprolactone; R, pure randomly oriented PCL nanofiber; A, pure aligned PCL nanofiber; pep, peptide.

Figure 5

The effect of functionalized nanofibers on osteogenic differentiation of hMSCs under non-osteoinductive condition. Notes: (A) Representative staining of ALP on day 14 and determination of ALP activity at 7 and 14 days. (B) ARS staining and determination of calcium deposition on day 21. (C) RT-qPCR analysis for osteo-specific genes on day 14. (D) Representative immunofluorescent images of OCN (green) and OPN (red) in different groups on day 14. Scale bars indicate 100 μm. (E) Western blot analysis of Col1a1 and Runx2 expression in hMSCs on different samples. ^#Compared with R-pDA, P<0.05; *compared with A-pDA, P<0.05; ^$compared with R-pDA-pep, P<0.05. Abbreviations: hMSCs, human mesenchymal stem cells; ALP, alkaline phosphatase; ARS, Alizarin Red S; RT-qPCR, real-time quantitative polymerase chain reaction; OCN, osteocalcin; OPN, osteopontin; Col1a1, type I collagen alpha 1; Runx2, Runt-related transcription factor 2; pDA, polydopamine; TCP, tissue culture plate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCL, polycaprolactone; R, pure randomly oriented PCL nanofiber; A, pure aligned PCL nanofiber; pep, peptide.

Figure 6

Effect of fiber topography on the focal adhesion formation of hMSCs. Notes: (A) The location of paxillin in hMSCs cultured on glass. Scale bar indicates 20 μm. (B) Representative immunofluorescent images of paxillin expressed on different samples. Scale bars indicate 100 μm. DAPI (blue), phalloidin (green), and paxillin (red). (C) Western blot analysis of paxillin expression in hMSCs on different samples. GAPDH was used as control. *Compared with R-pDA, P<0.05; ^#compared with R-pDA-pep, P<0.05. Abbreviations: hMSCs, human mesenchymal stem cells; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pDA, polydopamine; PCL, polycaprolactone; R, pure randomly oriented PCL nanofiber; A, pure aligned PCL nanofiber; pep, peptide.