Lanthanides-Substituted Hydroxyapatite/ Aloe vera Composite Coated Titanium Plate for Bone Tissue Regeneration

Abstract

Purpose: To develop the surface-treated metal implant with highly encouraged positive properties, including high anti-corrosiveness, bio-activeness and bio-compatibleness for orthopedic applications.

Methods: In this work, the surface of commercially pure titanium (Ti) metal was treated with bio-compatible polydopamine (PD) by merely immersing the Ti plate in PD solution. The composite of trivalent lanthanide minerals (La³⁺, Ce³⁺ and Gd³⁺)-substituted hydroxyapatite (MHAP) with Aloe vera (AV) gel was prepared and coated on the PD-Ti plate by electrophoretic deposition (EPD) method. The choice of trivalent lanthanide ions is based on their bio-compatible nature and bone-seeking properties. The formation of the PD layer, composites, and composite coatings on Ti plate and PD-Ti surface was confirmed by FT-IR, XRD, SEM and HR-TEM observations. In-vitro assessments such as osteoblasts like MG-63 cell viability, alkaline phosphatase activity and mineralization ability of the MHAP/AV composite were tested, and the composite-coated plate was implanted into a rat bone defect model for in-vivo bone regeneration studies.

Results: The coating ability of the MHAP/AV composite was highly preferred to PD-treated Ti plate than an untreated Ti plate due to the metal absorption ability of PD. This was confirmed by SEM analysis. The in-vitro and in-vivo studies show the better osteogenic ability of MHAP/AV composite at 14^th day and 4^th week of an experimental period, respectively.

Conclusion: The osteoblast ability of the fabricated device without producing any adverse effect in the rat model recommends that the fabricated device would serve as a better platform on the hard tissue regeneration for load-bearing applications of orthopedics.

Keywords: bio-compatible; electrophoretic deposition; hydroxyapatite; mussel adhesive protein; polydopamine; surface treatment.

Figure 1

Schematic representation of the present study.

Figure 2

(A) FT-IR spectra of (a) HAP, (b) MHAP, (c) HAP/AV composite and (d) MHAP/AV composite. (B) Indication of pectin absorption peaks for the composites (a) HAP/AV and (b) MHAP/AV composite at the range of 2000–1000 cm⁻¹.

Figure 3

XRD patterns of (A) HAP, (B) MHAP, (C) HAP/AV composite and (D) MHAP/AV composite.

Figure 4

SEM morphology of (A) HAP, (B) MHAP, (C) Acid-etched surface of Ti plate after sintering at 400 ºC for 5 h, (D) PD-treated Ti, (E) MHAP/AV, (F) MHAP/AV composite coated on Ti plate (Absence of PD treatment) (G) Surface of PD-Ti plate after coating with MHAP/AV composite and (H) Cross-section image of MHAP/AV composite coated on PD-treated Ti plate.

Figure 5

EDX spectra for the confirmation of elements present in the MHAP/AV composite coated on PD-Ti plate.

Figure 6

HR-TEM images of (A) HAP, (B) HAP/AV, (C) MHAP and (D) MHAP/AV nano rods and the SAED patterns of the all prepared compounds were given in insets of their corresponding TEM images.

Figure 7

(A–D) Diameter distributions of HAP, HAP/AV, MHAP and MHAP/AV nano rods, respectively, (E–H) Length distributions of HAP, HAP/AV, MHAP and MHAP/AV nano rods, respectively.

Figure 8

Zeta potential mobility distribution curve of (A) MHAP and (B) MHAP/AV composite.

Figure 9

Formation of apatite crystals on the surface of MHAP/AV composite-coated PD-Ti plate after immersion in SBF for (A) 1 day, (B) 3 days and (C) 7 days. The formation of apatite crystals over the rod morphology is indicated in a black circle. (D) XRD spectra of MHAP/AV composite-coated PD-Ti plate after immersion in SBF for (a) 1 day and (b) 7 days.

Figure 10

MG-63 osteoblast cells proliferation evaluated by MTT assay (A) and ALP activity of the MG-63 osteoblast cells (B) on different testing samples. *Comparison of the denoted group with the same set at 1 day of treatment time *p < 0.05. ^#Comparison of the denoted group with the HAP control at the same time period ^#p < 0.05.

Figure 11

Optical microscopic images of MG-63 osteoblast cells after treated with HAP (A–D), MHAP (E–H), HAP/AV (I–L) and MHAP/AV (M–P) for different time days such as 1, 3, 7 and 14 days.

Figure 12

Mineralization ARS staining of MG-63 osteoblast cells cultured on HAP (A–D), MHAP (E–H), HAP/AV (I–L), and MHAP/AV (M–P) for 1, 3, 7 and 14 days. (Q) Quantification of deposition of minerals.*Comparison of the denoted group with the same set at 1^st day of treatment time *p < 0.05. ^#Comparison of the denoted group with the HAP control at the same time period ^#p < 0.05.

Figure 13

Representative MT (A–E) and HE staining (F–J) of retrieved implant section at 1^st, 2^nd, 3^rd, and 4^th week of surgical experiment with MHAP/AV coated on PD-Ti implant. Red arrow indicates Osteoblasts; Blue arrow indicates Osteoids.

Figure 14

X-ray images of (A) defected rat and (B) 28th day of treatment with MHAP/AV-coated PD-Ti plate implantation.