Effect of cleaning and sterilization on titanium implant surface properties and cellular response

Abstract

Titanium (Ti) has been widely used as an implant material due to the excellent biocompatibility and corrosion resistance of its oxide surface. Biomaterials must be sterile before implantation, but the effects of sterilization on their surface properties have been less well studied. The effects of cleaning and sterilization on surface characteristics were bio-determined using contaminated and pure Ti substrata first manufactured to present two different surface structures: pretreated titanium (PT, Ra=0.4 μm) (i.e. surfaces that were not modified by sandblasting and/or acid etching); (SLA, Ra=3.4 μm). Previously cultured cells and associated extracellular matrix were removed from all bio-contaminated specimens by cleaning in a sonicator bath with a sequential acetone-isopropanol-ethanol-distilled water protocol. Cleaned specimens were sterilized with autoclave, gamma irradiation, oxygen plasma, or ultraviolet light. X-ray photoelectron spectroscopy (XPS), contact angle measurements, profilometry, and scanning electron microscopy were used to examine surface chemical components, hydrophilicity, roughness, and morphology, respectively. Small organic molecules present on contaminated Ti surfaces were removed with cleaning. XPS analysis confirmed that surface chemistry was altered by both cleaning and sterilization. Cleaning and sterilization affected hydrophobicity and roughness. These modified surface properties affected osteogenic differentiation of human MG63 osteoblast-like cells. Specifically, autoclaved SLA surfaces lost the characteristic increase in osteoblast differentiation seen on starting SLA surfaces, which was correlated with altered surface wettability and roughness. These data indicated that recleaned and resterilized Ti implant surfaces cannot be considered the same as the first surfaces in terms of surface properties and cell responses. Therefore, the reuse of Ti implants after resterilization may not result in the same tissue responses as found with never-before-implanted specimens.

Fig. 1

(A) Illustration of events at the Ti surface during cleaning, sterilization, and implantation. (B) Cleaning procedures (CP) and sterilization methods used in this study.

Fig. 2

Effect of cleaning procedures on surface properties of used PT and SLA disks. Surface properties of PT and SLA surfaces before and after cleaning with CP2 were examined using SEM (A), XPS low resolution spectra (B, C), water contact angle (n = 2) (D) and surface roughness (n = 6) (E). *, p < 0.05, PT vs. SLA; #, p < 0.05, vs. before cleaning.

Fig. 3

Effect of sterilization method on surface chemical composition of cleaned PT and SLA surfaces (n = 2). Atomic percentages (%) of components on the surface after sterilization were obtained by XPS for the PT-CP2 surfaces (A), the PT-CP3 surfaces (B), the SLA-CP2 surfaces (C), the SLA-CP3 surfaces (D). PT and SLA are new, unused and sterilized with gamma irradiation surfaces. Before cleaning (BC); autoclave (AC); gamma irradiation (GI); oxygen plasma (OP); and ultraviolet (UV).

Fig. 4

Effect of sterilization method on water contact angle of cleaned PT and SLA surfaces. Contact angle was measured on PT and SLA disks after cleaning with CP2 (A) and CP3 (B). Control surfaces are new, unused PT and SLA surfaces. (C) PT-CP3 surfaces were sterilized with oxygen plasma and contact angle was measured as a function of hours after oxygen plasma. *, p < 0.05, PT vs. SLA control surface; #, p < 0.05, vs. control, ■, p < 0.05, vs. CP, $, p < 0.05, vs. GI, ^, p < 0.05, vs. AC, &, p < 0.05 vs. OP. Cleaning protocol (CP); autoclave (AC); gamma irradiation (GI); oxygen plasma (OP); and ultraviolet (UV).

Fig. 5

Effect of sterilization method on roughness of cleaned PT and SLA surfaces. Surface roughness of PT and SLA surfaces was measured after cleaning with CP2 (A) or CP3 (B). Control surfaces are new, unused PT and SLA surfaces. *, p < 0.05, PT vs. SLA control surface; #, p < 0.05, vs. control, ■, p < 0.05, vs. CP, $, p < 0.05, vs. GI, ^, p < 0.05, vs. AC. Cleaning protocol (CP); autoclave (AC); gamma irradiation (GI); oxygen plasma (OP); and ultraviolet (UV).

Fig. 6

Surface morphology of PT and SLA surfaces. (A) SEM images of new PT (left) and SLA (right) surfaces. (B) SEM images of PT and SLA surfaces after cleaning (CP2 or CP3) and sterilization. Autoclave (AC); gamma irradiation (GI); oxygen plasma (OP); ultraviolet (UV).

Fig. 7

Effect of sterilization method of PT and SLA on MG63 cell number. MG63 cells were cultured on TCPS, PT and SLA surfaces and grown to confluence. At confluence, cell number on (A) new, unused, and sterilized with gamma irradiation PT and SLA surfaces,*, p < 0.05 Ti vs. TCPS; #, p < 0.05 SLA vs. PT, (B) cleaned with CP2 and then sterilized, and (C) cleaned with CP3 and sterilized. *, p < 0.05, PT vs. SLA; #, p < 0.05, vs. GI, ■, p < 0.05, vs. AC; $, p < 0.05, vs. OP.

Fig. 8

Effect of PT and SLA surface treated with different sterilization methods on MG63 cells. MG63 cells were plated on TCPS, PT and SLA surfaces and grown to confluence. At confluence, alkaline phosphatase (ALP) specific activity, osteocalcin (OCN), and osteoprotegerin (OPG) for new surface (A), (D), and (G), respectively. *, p < 0.05 Ti vs. TCPS; #, p < 0.05 SLA vs. PT. Alkaline phosphatase specific activity with CP2 and CP3 is (B) and (C), respectively. Osteocalcin with CP2 and CP3 is (E) and (F), respectively. Osteoprotegerin with CP2 and CP3 is (H) and (I), respectively. *, p < 0.05, PT vs. SLA; #, p < 0.05, vs. GI, ■, p < 0.05, vs. AC; $, p < 0.05, vs. OP. Inserted red and blue dotted lines indicate PT and SLA control surfaces, respectively.