Osteoporosis is a major healthcare burden, responsible for significant morbidity and mortality. Manipulating bone homeostasis would be invaluable in treating osteoporosis and optimising implant osseointegration. Strontium increases bone density through increased osteoblastogenesis, increased bone mineralisation, and reduced osteoclast activity. However, oral treatment may have significant side effects, precluding widespread use. We have recently shown that controlled disorder nanopatterned surfaces can control osteoblast differentiation and bone formation. We aimed to combine the osteogenic synergy of nanopatterning with local strontium delivery to avoid systemic side effects.

Using a sol-gel technique we developed strontium doped and/or nanopatterned titanium surfaces, with flat titanium controls including osteogenic and strontium doped media controls. These were characterised using atomic force microscopy and ICP-mass spectroscopy. Cellular response assessed using human osteoblast/osteoclast co-cultures including scanning electron microscopy, quantitative immunofluorescence, histochemical staining, ELISA and PCR techniques. We further performed RNAseq gene pathway combined with metabolomic pathway analysis to build gene/metabolite networks.

The surfaces eluted 800ng/cm2 strontium over 35 days with good surface fidelity. Osteoblast differentiation and bone formation increased significantly compared to controls and equivalently to oral treatment, suggesting improved osseointegration. Osteoclast pre-cursor survival and differentiation reduced via increased production of osteoprotegrin. We further delineated the complex cellular signalling and metabolic pathways involved including unique targets involved in osteoporosis.

We have developed unique nanopatterned strontium eluting surfaces that significantly increase bone formation and reduce osteoclastogenesis. This synergistic combination of topography and chemistry has great potential merit in fusion surgery and arthroplasty, as well as providing potential targets to treat osteoporosis.

Background

Following endosteal uncemented orthopaedic device implantation, the initial implant/bone interface retains spaces and deficiencies further exacerbated by pressure necrosis and resultant bone resorption. This implant-bone space requires native bone infill through the process of de novo osteogenesis. New appositional bone formation on the implant surface is known as contact osteogenesis and is generated by osteogenic cells, including skeletal stem cells (SSCs), colonising the implant surface and depositing the extracellular bone matrix. Surface nanotopographies provide physical cues capable of triggering SSC differentiation into osteoblasts, thus inducing contact osteogenesis, translated clinically into enhanced osseointegration and attainment of secondary stability. The current study has investigated the in vitro and in vivo effects of unique nanotopographical pillar substrates on SSC phenotype and function.

We have developed precision-engineered strontium eluting nanopatterned surfaces. Nanotopography has been shown to increase osteoblast differentiation, and strontium is an element similar to calcium, which has been proven to increase new bone formation and mineralization. This combination has great potential merit in fusion surgery and arthroplasty, as well as potential to reduce osteoporosis. However, osteoclast mediated osteolysis is responsible for the aseptic failure of implanted biomaterials, and there is a paucity of literature regarding osteoclast response to nanoscale surfaces. Furthermore, imbalance in osteoclast/osteoblast resorption is responsible for osteoporosis, a major healthcare burden. We aimed to assess the affect of strontium elution nanopatterned surfaces on osteoblast and osteoclast differentiation.

We developed a novel human osteoblast/osteoclast co-culture system without extraneous supplementation to closely represent the in vivo environment. We assessed the surfaces using electron microscopy (SEM), protein expression using immunofluorescence and histochemical staining and gene expression using polymerase chain reaction (PCR).

In complex co-culture significantly increased osteoblast differentiation and bone formation was noted on the strontium eluting, nanopatterned and nanopatterned strontium eluting surfaces, suggesting improved osteointegration. There was a reduction in macrophage attachment on these surfaces as well, suggesting specific anti-osteoclastogenic properties of this surface.

Our results show that osteoblast and osteoclast differentiation can be controlled through use of nanopatterned and strontium eluting surface features, with significant bone formation seen on these uniquely designed surfaces.

Background

In 2012, the National Joint Registry recorded 86,488 primary total hip replacements (THR) and 9,678 revisions (1). To date aseptic loosening remains the most common cause of revision in hip and knee arthroplasty, accounting for 40% and 32% of all cases respectively and emphasising the need to optimise osseointegration in order to reduce revisions. Clinically, osseointegration results in asymptomatic stable durable fixation of orthopaedic implants. Osseointegration is a complex process involving a number of distinct mechanisms affected by the implant surface topography, which is defined by surface orientation and surface roughness. Micro- and nano-topography levels have discrete effects on implant osseointegration and yet the role on cell function and subsequent bone implant function is unknown. Nanotopography such as collagen banding is a critical component influencing the SSC niche in vivo and has been shown to influence a range of cell behaviours in vitro (2,3). We have used unique fabricated nanotopographical pillar substrates to examine the function of human bone stem cells on titanium surfaces.

Poly-ether-ether-ketone (PEEK) is a biomaterial commonly used for spinal implants and screws. It is often desirable for orthopaedic implants to osseointegrate, but as PEEK is biologically inert this will not occur. The aim of this project was to determine if injection mould nanopatterning can be used to create a make PEEK bioactive and stimulate osteogenesis in vitro.

PEEK substrates were fabricated by injection mould nanopatterning to produce near-square (NSQ) nanopatterned PEEK and planar (FLAT) PEEK samples. Atomic force microscopy (AFM) and scanning electron microscopy were used to characterize the surface topography. Human bone marrow stromal cells (hBMSCs) were isolated from patients undergoing primary hip replacement operations and seeded onto the PEEK substrates. After 6 weeks the cells were stained using alizarin red S (ARS) stain (to detect calcium) and the von Kossa technique (to detect phosphate) and analyzed using CellProfiler image analysis software to determine: surface coverage; cell number; and expression of either calcium (ARS stain) or phosphate (von Kossa technique).

ARS stain showed calcium expression (quantified relative to the number of cells) was increased on NSQ PEEK compared to FLAT PEEK (not statistically significant) and the surface coverage was similar. Von Kossa staining revealed more surface coverage on FLAT PEEK (69.1% cf. 31.9%), cell number was increased on FLAT PEEK (9803 ± 4066 cf. 4068 ± 1884) and phosphate expression relative to cell number was also increased (seven-fold) on NSQ PEEK (P < 0.05) compared to FLAT PEEK.

Although hBMSCs may adhere to NSQ PEEK in smaller numbers, the cells expressed a relatively larger amount of calcium and phosphate. This indicates that the cells adopted a more osteoblastic phenotype and that nanopatterning PEEK induces hBMSC differentiation and stimulates osteogenesis. Injection mould nanopatterning therefore has the potential to improve osseointegration of PEEK implants in vivo.

Titanium is a popular orthopaedic implant material, but it requires surface modification techniques to improve osseointegration and long term functionality. This project compares a new method of modifying surface topography (nano-patterning) with an existing clinical technology (grit-blasting and acid-etching (GAE)).

Titanium discs were blasted with aluminium oxide and etched in sulphuric and acetic acid. Injection moulded discs (with two different nano-patterns) were coated in titanium by evaporation. The topography and chemistry of the discs was assessed using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle measurements, and X-ray photo-electron spectroscopy (XPS). Two discs were plated bilaterally onto a flattened area of the tibiae of 12 rabbits. Tibiae were removed after 4 and 8 weeks for histological assessment of the bone-implant contact (BIC) ratio.

AFM and SEM demonstrated a difference in pattern between the square array of nano-pits (SQ) and the randomly positioned nano-pits (RAND). The GAE implants exhibited increased surface roughness (Ra = 570nm) compared to the titanium coated SQ and RAND implants (Ra = 12nm). Water contact angle measurements showed the surface had comparable wettability and XPS demonstrated similar chemical compositions, except GAE surfaces contained 6.8% aluminium.

Histological samples analysed at 4 weeks showed a BIC ratio of 36% for GAE, 56% for SQ, and 48% for RAND. At 8 weeks, the BIC ratio was 52% for GAE, 80% for SQ, and 72% for RAND implants. This increase in BIC at 8 weeks for both SQ and RAND implants compared to GAE was statistically significant (P < 0.05).

This project demonstrated there was an increase in interfacial bone to implant contact when using a nano-scale topography incorporating nano-pits compared to conventional grit-blasted acid-etched micro-scale topographies. This enhancement of BIC may reduce long term loosening of orthopaedic implants due to mechanical and biological attrition at the interface.

Nanoscale topography increases the bioactivity of a material and stimulates specific responses (third generation biomaterial properties) at the molecular level upon first generation (bioinert) or second generation (bioresorbable or bioactive) biomaterials.

We developed a technique (based upon the effects of nanoscale topography) that facilitated the in vitro expansion of bone graft for subsequent implantation and investigated the optimal conditions for growing new mineralised bone in vitro.

Two topographies (nanopits and nanoislands) were embossed into the bioresorbable polymer Polycaprolactone (PCL). Three dimensional cell culture was performed using double-sided embossing of substrates, seeding of both sides, and vertical positioning of substrates. The effect of Hydroxyapatite, and chemical stimulation were also examined.

Human bone marrow was harvested from hip arthroplasty patients, the mesenchymal stem cells culture expanded and used for cellular analysis of substrate bioactivity.

The cell line specificity and osteogenic behaviour was demonstrated through immunohistochemistry, confirmed by real-time PCR and quantitative PCR. Mineralisation was demonstrated using alizarin red staining.

Results showed that the osteoinduction was optimally conferred by the presence of nanotopography, and also by the incorporation of hydroxyapatite (HA) into the PCL. The nanopit topography and HA were both superior to the use of BMP2 in the production of mineralised bone tissue.

The protocol from shim production to bone marrow harvesting and vertical cell culture on nanoembossed HaPCL has been shown to be reproducible and potentially applicable to economical larger scale production.

Recent studies have shown that random disorder nanotopography increases osteoblast differentiation and bone formation. This has great potential merit in producing surfaces where osteointegration is required such as spinal fusion surgery and arthroplasty. However, the long-term failure of orthopaedic implants is often related to osteoclast mediated osteolysis and loosening. It is vitally important that we understand the effect of nanotopography on osteoclast formation and bone remodeling.

We developed an unique osteoblast/osteoclast co-culture system derived from human mesenchymal and haematopoetic stem cells. This was co-cultured on both nanopatterned and unpatterned polycarbonate substrates. We assessed the co-culture using electron microscopy (SEM), protein expression using immunofluorescence and histochemical staining and gene expression using polymerase chain reaction (PCR).

Co-culture of both osteoclasts and osteoblasts was confirmed with mature bone nodules and resorption pits identified on both surfaces. Significantly increased osteoblast differentiation and bone formation was noted on disordered nanotopography. Antagonistic genes controlling osteoclast activity were both upregulated with no significant difference in osteoclast marker gene expression.

Our results confirm successful co-culture of osteoblasts and osteoclasts using an unique method closely resembling the in vivo environment encountered by orthopaedic implants. Nanotopography increases osteoblast differentiation and bone formation as previously identified, with possible subsequent increase in osteoclast mediated bone turnover.

Polyetheretherketone (PEEK) is a thermoplastic polymer that is predominant in spinal surgery as the material of choice for spinal fusion cages, and is also used for bone anchors, cruciate ligament interference screws, and femoral stems. It has the distinct advantage of having similar mechanical properties to bone, but its clinical application as implant material is limited by a lack of bioactivity. This project aims to create an PEEK surface capable of osseointegration using a surface modification technique known as oxygen plasma treatment.

PEEK surfaces were injection molded, washed and then treated in a plasma chamber for up to 10 min. Surfaces were characterised using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle measurements and X-ray photo-electron spectroscopy (XPS). Human bone marrow cells were cultured on the surfaces and assessed for calcium production (using alizarin red stain).

Water contact angle measurements show that after plasma treatment, the surfaces become very hydrophilic, before developing a meta-stable state at approx. 6 weeks. AFM and SEM showed destruction of the nano-pits at treatment durations longer than 2 mins. XPS detected a progressive increase in the atomic proportion of oxygen at the surface with increasing plasma treatment duration. There was significantly less alizarin uptake (and hence calcium production) on the untreated PEEK compared to the plasma treated PEEK surfaces (p < 0.05).

These results show that oxygen-plasma treatment can increase calcium production on PEEK surfaces and may improve long term osseointegration of PEEK implants.