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. 2019 Jul 4:14:4881-4893.
doi: 10.2147/IJN.S210687. eCollection 2019.

Encapsulation of a nanoporous simvastatin-chitosan composite to enhance osteointegration of hydroxyapatite-coated polyethylene terephthalate ligaments

Xiaoquan Ding  1 , Siheng Wang  1 , Wenhe Jin  1 , Xingwang Liu  1 , Jun Chen  1 , Shiyi Chen  1
Affiliations

Encapsulation of a nanoporous simvastatin-chitosan composite to enhance osteointegration of hydroxyapatite-coated polyethylene terephthalate ligaments

Xiaoquan Ding et al. Int J Nanomedicine. .

Abstract

Purpose: This study was designed to evaluate the in vitro and in vivo biocompatibility and osteointegration of plasma-sprayed hydroxyapatite (HA)-coated polyethylene terephthalate (PET) ligaments encapsulated with a simvastatin (SV)-chitosan (CS) composite.

Methods: This study compared the in vitro and in vivo bone responses to three different PET ligaments: SV/CS/PET-HA, CS/PET-HA and PET-HA. A field emission scanning electron microscope was used to characterize the morphology, and the in vitro SV release profile was analyzed. MC3T3 cells were cocultured with SV/CS/PET-HA, CS/PET-HA and PET-HA to test their biocompatibility using CCK-8 tests. Osteogenic differentiation was investigated by the expression of marker genes using qPCR. Osteointegration was performed by implanting the PET ligaments into the proximal tibia bone tunnels of male Sprague-Dawley rats for 3 weeks and 6 weeks. The bone-implant interface was evaluated by micro-computed tomography (micro-CT) and histological analysis.

Results: The characteristic nanoporous structures mainly formed on the surface of the plasma-sprayed HA particles in the SV/CS/PET-HA and CS/PET-HA groups. The SV release test showed that the sustained release of simvastatin lasted for 25 days in the SV/CS/PET-HA group. The in vitro studies demonstrated that the SV/CS/PET-HA ligaments induced osteogenic differentiation in the MC3T3 cells, with higher mRNA expression levels of collagen-1, bone morphogenetic protein-2, osteocalcin and alkaline phosphatase than those in the CS/PET-HA and PET-HA ligament groups. The in vivo tests showed that both micro-CT analysis (bone mineral density and bone volume per total volume) and histological analysis (bone implant contact and interface area) revealed significantly higher peri-implant bone formation and less interface area in the SV/CS/PET-HA group than in the other groups.

Conclusion: The SV-CS composite nanoporous structure was associated with the improved biocompatibility and osteogenic differentiation in vitro and enhanced osteointegration process in vivo of plasma-sprayed HA-coated PET ligaments.

Keywords: chitosan; hydroxyapatite; osteointegration; polyethylene terephthalate; simvastatin.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Schematic of the study: the process of preparing the SV/CS/PET-HA ligaments, in vitro osteogenic differentiation and in vivo rat model. Abbreviations: PET, polyethylene terephthalate; HA, hydroxyapatite; CS, chitosan; SV, simvastatin.
Figure 2
Figure 2
Characterizations of control PET (A1-2), nontreated PET-HA (B1-2), CS/PET-HA (C1-2) and SV/CS/PET-HA (D1-2). Diameter distribution of HA nanoparticles in nontreated PET-HA (E) and distribution of nanopore size of the SV/CS/PET-HA sheet (F). Abbreviations: CS, chitosan; SV, simvastatin; HA, hydroxyapatite; PET, polyethylene terephthalate; FESEM, Field Emission Scanning Electron Microscope.
Figure 3
Figure 3
Drug release and CCK-8 assay. (A) In vitro cumulative release profile of SV. (B) Results of the CCK-8 cell proliferation assay of MC3T3-E1 preosteoblast cells cocultured with PET-HA, CS/PET-HA and SV/CS/PET-HA sheets after 1, 2, 4 and 7 days. Notes: *p<0.05; **p<0.01, compared with that of PET-HA. Abbreviations: CS, chitosan; SV, simvastatin; HA, hydroxyapatite; PET, polyethylene terephthalate.
Figure 4
Figure 4
mRNA expression levels of osteogenic-related genes. qPCR analysis was performed for COL-1 (A, E), BMP-2 (B, F), OCN (C, G) and ALP (D, H) on the 4th and 7th days. Notes: *p<0.05; **p<0.01, compared with that of PET-HA. Abbreviations: CS, chitosan; SV, simvastatin; HA, hydroxyapatite; PET, polyethylene terephthalate; COL-1, collegen-1; BMP-2, bone morphogenetic protein-2; OCN, osteocalcin, ALP, alkaline phosphatase.
Figure 5
Figure 5
Extra-articular traverse bone tunnel model. (A) A PET ligament passing through the tibia bone tunnel. Representative reconstructed 2D micro-CT images of the bone tunnel at the 6th week in the sagittal (B), coronal (C), and transverse (D) planes. Notes: The red region contoured the PET ligament in bone tunnel. Abbreviations: PET, polyethylene terephthalate; PT, patellar tendon; Sag, sagittal; Cor, coronal; Tra, transverse.
Figure 6
Figure 6
Representative reconstructed 3D micro-CT images of the bone-implant integration at the 3rd (A1, B1, C1) and 6th (A2, B2, C2) weeks after surgery. Quantitative analysis of the parameters (BMD, BV/TV, and BS/BV) of peri-implant bone at the 3rd (D1, E1, F1) and 6th (D2, E2, F2) weeks. Notes: *p<0.05; **p<0.01, compared with that of PET-HA. Abbreviations: CS, chitosan; SV, simvastatin; HA, hydroxyapatite; PET, polyethylene terephthalate; BMD, bone mineral density; BV/TV, bone volume per total volume; BS/BV, bone surface per bone volume.
Figure 7
Figure 7
Images of the histological sections and quantitative analysis of the BIC and interface area of bone formation around the implants at the 3rd and 6th weeks after surgery. (A1, C1, E1) HE staining and (A2, C2, E2) Masson’s trichrome staining of corresponding sections showed the bone-implant interface at the 3rd week after surgery. (B1, D1, F1) HE staining and (B2, D2, F2) Masson’s trichrome staining of the corresponding sections showed the bone-implant interface at the 6th week after surgery. Quantitative analysis of the BIC (G, I) and interface area (H, J). Notes: The yellow dotted lines contoured the interface area. The original magnification is ×5 for A1-F2, and the bar is 500 μm. *p<0.05; **p<0.01, compared with that of PET-HA. Abbreviations: CS, chitosan; SV, simvastatin; HA, hydroxyapatite; PET, polyethylene terephthalate; BIC, bone interface contact.
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