Osseointegration1 is the direct structural and functional bonding between an implant surface and bone, and has had a substantial effect on dental and orthopaedic rehabilitation. In comparison to fitting a limb socket prosthesis over soft-tissue, osseointegration allows for skeletal fixation, resulting in a more comfortable and effective mechanical coupling to transfer load between an artificial limb and the human body2. The artificial limb is connected to the bone via an implant system with implanted and percutaneous components, known as fixture and abutment, respectively. The fixture is the component that osseointegrates within the bone intramedullary canal. The abutment extends from inside the fixture and through the skin to provide mechanical connection for the prosthesis. Recently, one such implant system has been developed that also includes neuromuscular electrodes to record bioelectric signals for control of the artificial limb, and to deliver electrical stimulation to severed nerves for eliciting sensory feedback3.
Commercially-pure titanium and titanium alloys (typically Ti6Al4V) are most frequently used in load-bearing orthopaedic implants due to their biocompatibility, mechanical strength, high corrosion resistance and unique ability to osseointegrate4,5. After surgical implantation of an orthopaedic fixture, there is typically a healing period of 3–12 months prior to loading2,6,7. During which time bone adaptation (osseointegration) to the implant surface occurs; an ideal fixation would mitigate any movement at the bone-implant interface1. Various factors affect peri-implant healing, including implant design and host bone quality8. In conditions where early implant loading is desired, or when the implant is placed in compromised healing conditions, there is a need to stimulate the progression of osseointegration9,10. Reduced healing time, early restoration of function, and prolonged effective lifespan of the prosthesis are the main driving forces behind enhancing osseointegration at the bone-implant interface. To this end, various approaches have been undertaken, including development of different metal alloys, use of macro-porous geometries, manufacturing techniques, surgical techniques, and alteration of implant surface properties such as topography and chemistry11.
Clinically, electrical stimulation has been instrumental in the treatment of a wide spectrum of disorders and disabilities12. Implantable devices that deliver electrical stimulation have shown successful outcomes in applications such as cochlear implants to restore hearing function13, wound-healing therapies intended for the closure of chronic wounds14, and in limb prostheses to restore sensory perception3,15. Electrical stimulation to promote osteogenesis for bone fracture healing has been recognised since the 1950s16, and explored for bone injury treatments including bone healing of union and non-union fractures17. Furthermore, electrical stimulation has been investigated as a potential treatment for bone ingrowth into implants, in vitro and in vivo. Different approaches have been developed by varying the electrode configuration, current type and source, and electrical stimulation parameters (e.g., amplitude and frequency)18,19. Three modalities of electrical stimulation have commonly been used for this purpose: (i) direct stimulation, (ii) indirect stimulation (capacitive or inductive couplings), and (iii) combined stimulation20. Studies reveal significant increases in bone-implant contact4,9,21,22,23, differentiation of preosteoblasts12,24, and increased cell proliferation25 upon application of direct current (DC) stimulation. However, DC stimulation can include pH shifts, accumulation of oppositely charged proteins at the implant surface, and production of reactive oxygen species in the adjacent environment25. Pulsed electrical stimulation overcomes some of these challenges9,25, particularly where pulses of opposite magnitude are used to balance the displacement of charges26. Pulsed electrical stimulation has shown beneficial effects on cell proliferation, in vitro25 and bone-implant contact, in vivo9. However, further investigation of the optimal electrical stimulation parameters is needed18,19.
In this work, we investigated the response of MC3T3-E1 preosteoblasts (precursor cells to osteoblasts) to pulsed electrical stimulation with parameters similar to those used in artificial limbs for sensory feedback through peripheral nerve stimulation3,15,26. We utilised parameters that have been used safely with implanted electrodes for several years3 and are compatible with electronic embedded system for artificial limbs27. A versatile in vitro setup comprising a bespoke, 3D-printed poly(lactic acid) (PLA) chamber was developed to minimise risk of inadvertent motion and enable reproducible positioning of individual components. Flat Ti6Al4V plates represent the implant to be osseointegrated and Ti6Al4V discs represent implanted electrodes that serve as the electrical reference. MC3T3-E1 preosteoblasts cultured on the flat Ti6Al4V plates were exposed to different combinations of pulse amplitude and frequency over a continuous 72-h period. Thereafter we evaluated the pH, cell survival, and collagen production and compared to unstimulated controls (Ctrl). Our results show, for the first time, that pulsed electrical stimulation significantly accelerates collagen production of MC3T3-E1 cells, which is contingent on osteogenic differentiation. In addition, electrical stimulation significantly improves cell survival, without detectable changes in the local pH.
