A new austenitic stainless steel with negligible nickel content: an in vitro and in vivo comparative investigation

Abstract

New nickel (Ni)-reduced stainless-steel metals have recently been developed to avoid sensitivity to Ni. In the present study, an austenitic Ni-reduced SSt named P558 (P558, Böhler, Milan, Italy) was studied in vitro on primary osteoblasts and in vivo after bone implantation in the sheep tibia, and was compared to ISO 5832-9 SSt (SSt) and Ti6Al4V. Cells were cultured directly on P558 and Ti6Al4V. Cells cultured on polystyrene were used as controls. Osteoblast proliferation, viability and synthetic activity were evaluated at 72 h by assaying WST1, alkaline phosphatase activity (ALP), nitric oxide, pro-collagen I (PICP), osteocalcin (OC), transforming growth factor-β1 (TGFβ-1) and interleukin-6 (IL-6) after 1.25(OH)₂D₃ stimulation. Under general anaesthesia, four sheep were submitted for bilateral tibial implantation of P558, SSt and Ti6Al4V rods. In vitro results demonstrated that the effect of P558 on osteoblast viability, PICP, TGF β-1, tumor necrosis factor-α production did not significantly differ from that exerted by Ti6Al4V and controls. Furthermore, P558 enhanced osteoblast differentiation, as confirmed by ALP and OC levels, and reduced IL-6 production. At 26 weeks, the bone-to-implant contact was higher in P558 than in SSt (28%, p<0.005) and Ti6Al4V (4%, p<0.05), and was higher in Ti6Al4V than in SSt (22%, p<0.005). The tested materials did not affect bone microhardness in pre-existing host bone as evidenced by the measurements taken at 1000 μm from the bone–biomaterial interface (F=1.89, ns). At the bone–biomaterial interface the lowest HV value was found for SSt, whereas no differences in HV were observed between materials (F=1.55, ns). The current findings demonstrate P558 biocompatibility both in vitro and in vivo, and osteointegration processes are shown to be significantly improved by P558 as compared to the other materials tested.

Introduction

Since the mean life expectation in humans is constantly increasing, the working life of orthopaedic implants should be likewise prolonged. The mechanical properties of biomaterials, as well as corrosion resistance and biocompatibility, therefore take on a growing importance in the choice of the materials to be used for implant manufacturing.

Fracture fixation, joint replacement and traumatic and iatrogenic segmental bone losses usually require the implantation of metallic devices [1]. Consequently, local and systemic metallosis, as well as the products of metal corrosion and ion release, has been widely investigated over the last decades [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. In severe cases, notable and permanent tissue changes can occur around metallic implants resulting in the clinical entity of inflammation, pain and, at worst, implant failure [11].

Standard metallic orthopaedic materials include stainless steels (SSt), cobalt (Co)-based alloys, commercially pure (cp) titanium (Ti) and Ti-based alloys, with an increasing number of devices being made of cpTi and Ti alloys. Regarding dental and non-cemented orthopaedic implants, cpTi and Ti alloys are generally preferred to SSt and Co-alloys because of their lower modulus, superior biocompatibility and corrosion resistance [12]. However, SSt is still the most used metal for internal fixation devices thanks to a favourable combination of mechanical properties, acceptable biocompatibility and cost effectiveness when compared to other metallic implant materials [13], [14].

A new class of austenitic SSt with interesting mechanical and electrochemical properties has been ISO-standardized (ISO 5832-9). The high nitrogen content of ISO 5832-9 SSt explains its superior corrosion resistance compared with traditional ISO 5832-1/D and ISO 5832-1/E SSt and the higher mechanical properties (Rmin 770 Mpa, Rs min 465 Mpa, A min 35%) observed even in the annealed state.

However, a disadvantage seen for SSt is its tendency towards corrosion under physiological conditions causing a release of metal ions such as those of nickel (Ni) and chromium (Cr) [14]. This effect takes on a growing importance in the case of ISO 5832-1/D and ISO 5832-1/E SSt. Both types of steel, in fact, are not immune to crevice corrosion in the human body, which can increase ion release in the surrounding tissues by several orders of magnitude.

Ni has been reported to be the most common metal sensitizer in humans [15] and some concern has been expressed regarding toxicity, susceptibility to bacterial infection and cancerogenous effects, even though no evidence of any direct relationship between implants and cancer development exists [1], [11], [16], [17]. In vitro studies have shown that 10–50 μg/ml of Ni ions cause fibroblast, endothelial cell and monocyte total suppression of mitochondrial function [16], [18], [19], [20]. In vivo investigations have demonstrated that more than 25 μg/g of Ni implanted in soft tissue may elicit severe inflammation and necrosis [16] and are therefore consistent with in vitro citotoxicity studies. Furthermore, a concentration of less than 10 μg/g can cause a severe inflammatory response depending on the length of tissue exposure [16]. This specific problem has led researchers to investigate valid alternatives to these materials, in accordance with the EU Directives on the usage of Ni-alloyed materials for manufacturing objects to come into contact with the human body (EU Dir. 94/27/CEE, para.2).

To avoid the above-mentioned severe side effects of SSt, Ti and its alloys have been considered the metal of choice for bone implants because of their high osteointegration properties, suitable modulus of elasticity, lower density, improved biocompatibility and MRI compatibility [21], [22], [23]. However, one might argue that Ti and Ti alloys are known for their relatively poor wear properties; moreover, the tissue reactions around Ti-based implants, as well as the tendency to leave the orthopaedic devices in the body, have resulted in a number of studies on biocompatibility [14], [24], [25]. Some recent in vitro data have shown the negative effect of Ti particles on osteoblast gene expression and on the release of proinflammatory cytokines [26], [27], [28], [29], [30]. Ti and Ti alloys have been associated with inflammation and corrosion [14] and the toxicity of vanadium and aluminium has increased the interest in developing new Ti-based alloys [12]. Furthermore, a shorter time of failure in total hip arthroplasties with Ti alloy versus cobalt chrome alloy has been reported, together with a higher loosening rate and peri-prosthetic osteolysis [31].

Ni-reduced SSt metals with high nitrogen content have recently been developed to address the issue of sensitivity to Ni and appear to have superior mechanical properties and better corrosion resistance [13].

A new austenitic SSt named P558 (Bohler, Milan, Italy), which has been recently patented, may provide an interesting alternative to conventional SSt, Co-based alloys, and Ti and Ti-alloys. P558 has a high Mn and N content and a negligible Ni (<0.20%) content (in accordance with ASTM E 112: 4–5). Such a low Ni content does not induce Ni ion release and, consequently, prevents allergic reactions to Ni, as confirmed by a previous in vivo maximization-sensitization test performed in guinea pigs (in accordance with ISO 10993-10, 1995; Biological evaluation of medical devices, Part.10: Test for irritation and sensitization) (unpublished data).

The aim of the present study was to evaluate in vitro and in vivo biocompatibility in terms of osteoblast proliferation, differentiation and synthetic activity, as well as in vivo osteointegration, through comparison of a Ni-reduced SSt (P558) with the Ti alloy Ti6Al4V and ISO 5832-9 SSt (SSt).