Effect of Hydroxyapatite Coating by Er: YAG Pulsed Laser Deposition on the Bone Formation Efficacy by Polycaprolactone Porous Scaffold

Abstract

Composite scaffolds obtained by the combination of biodegradable porous scaffolds and hydroxyapatite with bone regeneration potential are feasible materials for bone tissue engineering. However, most composite scaffolds have been fabricated by complicated procedures or under thermally harsh conditions. We have previously demonstrated that hydroxyapatite coating onto various substrates under a thermally mild condition was achieved by erbium-doped yttrium aluminum garnet (Er: YAG) pulsed laser deposition (PLD). The purpose of this study was to prepare a polycaprolactone (PCL) porous scaffold coated with the hydroxyapatite by the Er: YAG-PLD method. Hydroxyapatite coating by the Er: YAG-PLD method was confirmed by morphology, crystallographic analysis, and surface chemical characterization studies. When cultured on PCL porous scaffold coated with hydroxyapatite, rat bone marrow-derived mesenchymal stem cells adhered, spread, and proliferated well. The micro-CT and staining analyses after the implantation of scaffold into the critical-sized calvaria bone defect in rats indicate that PCL porous scaffold coated with hydroxyapatite demonstrates accelerated and widespread bone formation. In conclusion, PCL porous scaffold coated with hydroxyapatite obtained by the Er: YAG-PLD method is a promising material in bone tissue engineering.

Keywords: Er: YAG laser; bone formation; hydroxyapatite coating; polycaprolactone; porous scaffold; pulsed laser deposition.

Figure 1

Characterization of hydroxyapatite coating onto PCL porous scaffold by Er: YAG PLD method. (A) SEM images of PCL porous scaffold before (a–c) and after (d–f) treatments of irradiation and hydrolysis (g–i). The images were taken at magnifications of ×100 (a,d,g), ×400 (b,e,h), and ×1000 (c,f,i). (B) XRD patterns of PCL porous scaffold before and after treatments of irradiation and hydrolysis. The marks ◆, ▼, and ● indicate the representative patterns of PCL, α-TCP, and hydroxyapatite, respectively. (C) XPS chart of PCL porous scaffold after hydrolysis treatment. (D) ATR-FTIR spectrum of PCL porous scaffold after hydrolysis treatment.

Figure 2

Mechanical and physicochemical surface properties of PCL-based discs/scaffolds. (A) Compressive strain curves of PCL and HAp-PCL scaffolds. (B) Images of water droplet on the surface of PCL and HAp-PCL discs.

Figure 3

Assessment of initial cell attachment and cell viability after seeding on PCL-based discs. (A) Initial attachment of rBMSCs after seeding on discs of PCL or HAp-PCL. *** p < 0.001, **** p < 0.0001: significant difference between two groups at the corresponding time. (B) Viability of rBMSCs cultured on discs of PCL or HAp-PCL for 24 h, evaluated by the live/dead assay. Scale bar is 50 μm.

Figure 4

The behavior of rBMSCs on PCL-based scaffolds. (A) Fluorescent microscopic images of rBMSCs 24 h after culturing on PCL (a,b) or HAp-PCL (c,d). Images were acquired in the outermost (a,c) and interior (b,d) planes. The region between the dashed lines indicates a fiber of the scaffold. Actin filaments and nucleus were stained and are indicated in green and blue, respectively. Scale bar is 50 μm. (B) Time profiles of relative DNA content of BMSCs cultured on PCL or HAp-PCL. *** p < 0.001, **** p < 0.0001: significant difference between two groups at the corresponding time. (C) SEM images of rBMSCs cultured on PCL or HAp-PCL for 3 days.

Figure 5

μ-CT analysis of bone formation at 2, 4, and 8 weeks after implantation of PCL or HAp-PCL into critical-sized calvaria bone defects in rats. The μ-CT images were acquired from the axial (A) and coronal (B) planes of the defective site. (C) Time profiles of bone volume/total volume (BV/TV). The bone volume for the HAp-PCL group were calculated by subtracting the value of scaffold-derived hydroxyapatite from that of the original signal. (D) Bone mineral density (BMD) images obtained from the axial plane of μ-CT. *** p < 0.001, **** p < 0.0001: significant difference between two groups at the corresponding time.

Figure 6

H&E staining for the sectioned tissue inside the scaffold at 2, 4, and 8 weeks after implantation of PCL or HAp-PCL into the critical-sized calvaria bone defect in rats. (A) Representative staining images. The frames in the upper image indicate the region of lower image with high maginification. Scale bars are 100 (lower images) or 900 μm (upper images). (B) Percentage of new bone area formed analyzed by the staining results. **** p < 0.0001: significant difference between two groups at the corresponding time. (C) Staining images of tissue surrounding the implant-host bone interface. The implant-host bone interface is present between two arrows. Scale bar is 100 μm.

Figure 7

Alkaline phosphatase (ALP) staining for the sectioned tissue inside the scaffold at 2, 4, and 8 weeks after implantation of PCL or HAp-PCL into the critical-sized calvaria bone defect in rats. (A) Representative staining images. Scale bar is 100 μm. (B) Time profiles in percentages of ALP-positive area. n.s.; no significance.

Figure 8

Von Kossa staining for the sectioned tissue inside the scaffold at 2, 4, and 8 weeks after implantation of PCL or HAp-PCL into the critical-sized bone defect in rats. The tissues were counterstained with Nuclear Fast Red solution. Scale bar was 100 μm.

Figure 9

Immunohistochemical staining of cytosolic osteocalcin (OCN, red) for the sectioned tissue inside the scaffold at 2, 4, and 8 weeks after implantation of PCL or HAp-PCL into the critical-sized bone defect in rats. (A) Representative staining images. The cell nucleus (blue) was stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bar is 50 μm. (B) Time profiles in the ratio of osteocalcin-positive area. ** p < 0.01, **** p < 0.0001: significant difference between two groups at the corresponding time.

Figure 10

Immunohistochemical staining of von Willebrand factor (brown) for the sectioned tissue inside the scaffold at 2, 4, and 8 weeks after implantation of PCL or HAp-PCL into the critical-sized bone defect in rats. The tissues were counterstained with hematoxylin (purple). Scale bar was 100 μm.