Polydopamine-modified poly(l-lactic acid) nanofiber scaffolds immobilized with an osteogenic growth peptide for bone tissue regeneration

Abstract

It is highly desirable for bone tissue engineering scaffolds to have significant osteogenic properties and capability to improve cell growth and thus enhance bone regeneration. In this study, a poly(l-lactic acid) (PLLA) nanofiber scaffold-immobilized osteogenic growth peptide (OGP) was prepared via polydopamine (PDA) coating. X-ray photoelectron spectroscopy (XPS), contact angle measurement, and scanning electron microscopy (SEM) were used to determine the OGP immobilization, hydrophilicity and surface roughness of the samples. The SEM and fluorescence images demonstrate that the PLLA nanofiber scaffolds immobilized with the OGP have excellent cytocompatibility in terms of cell adhesion and proliferation. The ALP activity and the Runx2 and OPN expression results indicated that the PLLA nanofiber scaffolds immobilized with OGP significantly enhanced the osteogenic differentiation and calcium mineralization of hMSCs in vitro. A rat model of critical skull bone defect was selected to evaluate the bone formation capacity of the scaffolds. Micro CT analysis and histological results demonstrated that the PLLA scaffolds immobilized with OGP significantly promoted bone regeneration in critical-sized bone defects. This study verifies that the PLLA scaffold-immobilized OGP has significant potential in bone tissue engineering.

Fig. 1. Microstructure and hydrophilicity of the scaffolds. (a) SEM images and images of water contact angles of the scaffolds. The scale bar indicates 2 μm. (b) Fiber diameters of the scaffolds. (c) Water contact angles of the scaffolds. “*” indicates significant difference as compared to the PLLA-PDA-OGP group, “#” indicates significant difference as compared to the PLLA-PDA group (P < 0.05).

Fig. 2. X-ray photoelectron spectroscopy (XPS) of the sample. (a) XPS peaks of the PLLA, PLLA-PDA and PLLA-PDA-OGP scaffolds. (b) Atomic chemical composition of each sample surface. (c) FTIR analysis of different samples.

Fig. 3. Cumulative release of OGP from the PLLA-OGP scaffolds and PLLA-PDA-OGP scaffolds.

Fig. 4. Proliferation of hMSCs cultured on various scaffolds after 1, 3, and 7 d. “*” indicates significant difference as compared to the PLLA group, “#” indicates significant difference as compared to the PLLA-PDA group (P < 0.05).

Fig. 5. Morphology of hMSCs cultured on the different scaffolds. Fluorescence images of F-actin (green) and nuclei (blue) in hMSCs cultured in scaffolds for 1, 3 and 7 days. The scale bar indicates 100 μm.

Fig. 6. Morphology of the hMSCs cultured on the different scaffolds. SEM images of hMSCs cultured in scaffolds for 1, 3 and 7 days. The scale bar indicates 100 μm.

Fig. 7. Osteogenic differentiation of hMSCs cultured on different scaffolds. (a) Alizarin red S staining of hMSCs cultured on scaffolds for 14 days. The scale bar indicates 100 μm. (b) The ALP activity of hMSCs cultured on the scaffold for 14 days. “*” indicates significant difference as compared to the PLLA scaffold and the PLLA-PDA scaffold.

Fig. 8. In vitro differentiation of hMSCs cultured on different scaffolds. (a) Double immunofluorescence staining of Runx2 and OPN in hMSCs cultured on the scaffolds for 14 days. (b) qRT-PCR analysis to examine the expression of Runx2 and OPN in hMSCs cultured on the scaffolds for 14 days. “*” indicates significant difference as compared to the PLLA scaffold and the PLLA-PDA scaffold (P < 0.05).

Fig. 9. Micro-CT analysis of the bone specimens retrieved eight weeks after the surgery. (a) Reconstructed Micro-CT images of the bone specimens implanted with scaffolds. (b) BV/TV (bone volume/tissue volume) of different groups after eight weeks. “*” indicates significant difference as compared to the defect-only group, “#” indicates significant difference as compared to the PLLA-PDA group (P < 0.05).

Fig. 10. H&E (a) and Masson (b) staining of the samples after eight weeks of scaffold implantation. The scale bar indicates 100 μm (CT = connective tissue, M = material, NB = new bone). Black arrowhead indicates newly formed blood vessels in the regenerated bone.

Scheme 1. Fabrication protocol of PLLA-based scaffolds: (a) schematic of the electrospinning device in this study. (b) Experimental steps of coating PDA and OGP molecules on the PLLA scaffolds. (c) Characterization aspects of the samples.