Identification of mesenchymal stromal cell survival responses to antimicrobial silver ion concentrations released from orthopaedic implants

Antimicrobial silver (Ag+) coatings on orthopaedic implants may reduce infection rates, but should not be to the detriment of regenerative cell populations, primarily mesenchymal stem/stromal cells (MSCs). We determined intramedullary silver release profiles in vivo, which were used to test relevant Ag+ concentrations on MSC function in vitro. We measured a rapid elution of Ag+ from intramedullary pins in a rat femoral implantation model, delivering a maximum potential concentration of 7.8 µM, which was below toxic levels determined for MSCs in vitro (EC50, 33 µM). Additionally, we present in vitro data of the reduced colonisation of implants by Staphylococcus aureus. MSCs exposed to Ag+ prior to/during osteogenic differentiation were not statistically affected. Notably, at clonal density, the colony-forming capacity of MSCs was significantly reduced in the presence of 10 µM Ag+, suggesting that a subpopulation of clonal MSCs was sensitive to Ag+ exposure. At a molecular level, surviving colony-forming MSCs treated with Ag+ demonstrated a significant upregulation of components of the peroxiredoxin/thioredoxin pathway and processes involved in glutathione metabolism compared to untreated controls. Inhibition of glutathione synthesis using l-buthionine sulfoxamine eliminated MSC clonogenicity in the presence of Ag+, which was rescued by exogenous glutathione.


Scientific Reports
| (2020) 10:18950 | https://doi.org/10.1038/s41598-020-76087-1 www.nature.com/scientificreports/ megaprostheses implanted during bone resection as a result of osteosarcoma has proven to reduce the incidence of infection in these compromised patients [21][22][23] . The application of silver to orthopaedic trauma devices such as intramedullary nails, could therefore have a similar clinical impact. Fracture repair requires the action of immune cells, chondrocytes and osteoblasts, the numbers and regulation of which are influenced by mesenchymal stem/stromal cells (MSCs) that act as a source or progenitors, giving rise to reparative osteochondral tissue 24 . MSCs are supplied to the fracture site from the surrounding soft tissue, periosteum and bone marrow, all or which may be disrupted in a severe fracture, therefore preservation of existing MSC populations and their function is of importance 25 . Specifically, the impact of silver ions (Ag + ) on repair of the fracture site and the subsequent generation of ROS that also occurs in eukaryotic cells, needs to be considered, particularly since the level of ROS can affect the lineage divergence of MSCs, with decreased osteogenesis occurring under in vitro conditions of oxidative stress [26][27][28] .
Here, we determined the Ag + release profile from intramedullary implants to identify how exposure to relevant in vivo concentrations of silver ions affected MSC function and survival responses in vitro. Our findings provide insight into the potential clinical use of this metal for biofilm prevention in orthopaedic trauma situations.

Results
Synthesis and characterisation of intramedullary pin implants. Silver ions were integrated on to implant surfaces and inductively coupled plasma mass spectrometry (ICP-MS) quantification of total Ag + confirmed nominal target loading of approximately 50 µg/cm 2 (pins: mean, 50.36 µg/cm 2 SEM ± 0.76, n = 6).
Staphylococcus aureus adhesion to silver coated Ti64 grit blasted pins. Control pin implants, without Ag + coatings, supported bacterial colonisation over the test period, with a mean surface count of 5.00 log 10 CFU/sample after 1 day rising to 7.11 log 10 CFU/sample at day 33 (Fig. 1a). Colonisation was significantly reduced on Ag + coated pins (> 4 log 10 CFU/sample) over control counts at 24 h, maintaining this distinction at day 7 and 14 re-challenges (> 4 log 10 CFU/sample). At these time points all replicates displayed counts at or below the detection limit. At day 21, Ag + coated samples displayed a degree of colonisation, however values remained below control counts by > 4 log 10 CFU/sample. Colonisation was detected in Ag + coated pins at day 33 that was comparable to control samples at earlier time points (day 1, 7 and 14), but still reduced compared to control (Fig. 1a).

Rapid elution of Ag + from implants within intramedullary canal environment. To determine
Ag + elution kinetics, Ag + coated titanium pins (Ag + , 50.36 µg/cm 2 SEM ± 0.76) were implanted in to rat femoral intramedullary canals (Fig. 1b,c) and the Ag + determined in the recovered implant (Fig. 1d), the femurs receiving the implants (Fig. 1e) and plasma (Fig. 1f). Results revealed a burst release within the 24 h following implantation, equating to approximately 70% (mean 25.15 μg, SEM ± 1.45 μg) of the pre-implantation content, with little significant release over the remaining study period (Fig. 1d). A corresponding rapid elevation in plasma and femoral Ag + content was detected at Day 1 that remained elevated at Day 3 (Fig. 1e,f). Maximum (a-f) Surface recovery of S. aureus from silver-coated grit blasted Ti64 pins following in vitro challenge (a), line represents the limit of detection of 0.95 CFU/mL, (b) confirmation of pin implantation during in vivo assessment of silver elution, (c) microCT scan of rat femur highlighting intramedullary canal volume. Measurement of total Ag from (d) recovered implants (µg), (e) operated femurs (µg), and silver concentration in (f) plasma (μg/mL), red line represents limit of detection (= 0.00125 μg/mL). All values are means ± SEM (n = 3) determined via ICP-MS for samples taken at termination (Day 1, 2, 3 and 28). Statistical analysis by one-way ANOVA using Tukey correction for multiple comparisons, significance indicated against Day 0 by *p < 0.05, **p < 0.01, ****p < 0.0001, Day 1 by # p < 0.05, Day 2 by $ p < 0.05 and Day 3 by ~p < 0.05.
In vitro susceptibility of MSCs to Ag + is increased at clonal density. The effect of Ag + dose on primary MSC and human osteoblast (hObs) cultures provided calculated EC 50 values of 33.06 μM and 41.4 μM, respectively (Fig. 2a,b). MSC proliferation in the presence of Ag + was unaffected over 3 days at concentrations below the EC 50 as determined by EdU incorporation (Fig. 2c). Colony-forming capacity of MSCs as measured by colony number and percentage area was also unaffected by exposure to sub-EC 50 Ag + concentrations during the initial 3 days of culture (which broadly replicated the exposure period determined during the in vivo elution and CFU-f number (d) and area (e) of MSCs at sub-EC 50 concentrations (≤ 10 µM) during 3 day exposure. CFU-f number (f) and area (g) during continuous Ag + exposure. All values are means ± SEM (n = 3). Images in the right panel are representative of 6-well plates, stained with crystal violet (blue) and analysed (brown) for CFU-f number and area; Ag + concentrations for the plates are (L-R): top row, 0, 0.1, 0.5 μM; bottom row, 1, 5, 10 μM. Statistical analysis was performed for all Ag + groups compared to control (by one-way ANOVA using Dunnett's correction for multiple comparisons), significance indicated by *p < 0.05, **p < 0.01, ****p < 0.0001.
MSC colony formation is unaffected by previous exposure to Ag + . Maintenance of MSC clonal expansion capacity following Ag + exposure is necessary for the continued functioning of bone marrow stroma during and subsequent to fracture repair. This property was determined through the secondary seeding of Ag + exposed CFU-f using a two-stage assay design (Fig. 3a). Essentially, CFU-f were exposed to Ag + at 0, 1 and 10 µM (a-e) Two-stage assay design (a) used to determine the continued MSC colony-forming capacity and Ag + tolerance. Clonal density MSCs were cultured for 14 days ± Ag + after which colonies of treatment groups were pooled and reseeded at clonal density and re-cultured ± Ag + for a further 14 days. www.nature.com/scientificreports/ concentrations for 14 days (Stage 1). After this time, the surviving CFU-fs from each treatment were trypsinised, re-plated at CFU-f density in fresh plates and re-exposed to Ag + at 0, 1 and 10 µM concentrations for a further 14 days (Stage 2). This protocol enabled us to test the hypothesis that colonies surviving an initial Ag + exposure represented an Ag + resistant MSC subpopulation. Stage 2 CFU-f number for untreated controls was unaffected by previous exposure to Ag + . However, the surviving Stage 1 Ag + exposed MSCs remained susceptible to further Ag + exposure in Stage 2 with significant decreases in CFU-f number, irrespective of initial Ag + treatment ( Fig. 3b) indicating that Ag + exposure did not select for a resistant MSC subtype. An inverse correlation was observed between CFU-f diameter and Ag + concentration (Fig. 3c). This relationship was apparent irrespective of pre-exposure to Ag + , however the reduction was only significant for those colonies derived from MSCs previously cultured with silver.
Conditioned medium from MSCs maintains clonogenicity during exposure to Ag + . We have found that colony-forming MSCs grown at single cell densities are much more sensitive to Ag + exposure than confluent or near confluent MSC cultures. We hypothesised that this may be due to the decreased availability of secreted, protective signalling factors in CFU-f cultures compared to more densely packed MSC monolayers. To test this hypothesis, conditioned medium (CM) was collected from confluent MSC cultures and applied to the CFU-f assays in the presence and absence of silver ions. We found that the CFU-f number reduction observed at 10 µM Ag + could be rescued by the addition of CM (Fig. 3d). Both human primary MSCs and the Y201 human MSC line (used here as a reproducible model human MSC clonal line 29 ) showed recovery of CFU-f number near to that of control when treated with Ag + in CM. There was also a notable increase in colony size of the CM groups compared to unconditioned medium controls (Fig. 3e).
Glutathione synthesis is upregulated in MSCs of surviving CFU-f in response to Ag + . We have shown that a proportion of MSCs persist as CFU-fs following exposure to Ag + ions, suggesting that Ag + may activate survival mechanisms in MSCs, depending on cell density. To identify these mechanisms, Y201 MSCs were used in a proteomic screen of Ag + exposed CFU-f compared to untreated control CFU-f. Liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomic analysis identified 5050 quantifiable proteins, with peak area-based label-free relative quantification applied using Progenesis QI software (Fig. 4a). Of those identified, 89 were significantly upregulated in Ag + exposed CFU-f compared to controls, while 21 were significantly downregulated (FDR < 0.05) (Fig. 4b). Supplementary figures S1(a-d) demonstrate the differentiation of sample groups and consistency of biological replication in the proteomic data set. Gene Ontology (GO) analysis identified several differentially regulated functional responses including several metabolic pathways, as well as cellular detoxification (GO:1990748), response to stress (GO:0006950) and response to xenobiotic stimulus (GO:0009410) (Fig. 4c). Comparison of proteomic data to known KEGG pathways highlighted glutathione metabolism as the major upregulated cellular response to Ag + exposure. Further KEGG pathway analysis drew attention to the upregulation of proteins involved with DNA replication and nucleotide excision repair (PCNA, LIG1, CUL4A).
Validation of the proteomic evidence was provided by quantitative Polymerase Chain Reaction (qPCR) analysis of the oxidative stress response of three primary MSC donors (Fig. 4d). Data were normalised to B2M, which identified 39 genes showing mean fold-change > 1.5 (Fig. 4e). Genes of glutathione synthesis/metabolism (GPX3, GPX5, GPX6, GR, GCLM), peroxiredoxin (PRDX1) and thioredoxin (TXN, TXNRD1) showed activation of these pathways upon Ag + exposure. Additionally, increases in the stress inducible regulator of NRF2, sequestome 1 (SQSTM1), heme oxygenase-1 (HMOX1), NAD(P)H:quinone oxidoreductase-1 (NQO1) and SOD1 were also noted for all donors. GO functional analysis related to biological processes was performed on the qPCR data, with an FDR (false discovery rate) below 5% the threshold for significance ( Supplementary Fig. S2). Highlights of those terms include: the removal of superoxide radicals (GO:0019430), response to hydrogen peroxide (GO:0042542), regulation of reactive oxygen species metabolic process (GO:2000377) and glutathione metabolic process (GO:0006749). Furthermore, cellular response to cadmium (GO:0071276) and cellular copper homeostasis (GO:0006878) all indicate at the mechanisms employed to ensure management of metal ions that could result in toxicity. Of those identified, glutathione metabolic process (GO:0006749) was replicated from the proteomic analysis ( Supplementary Fig. S3), the only functional process to be so.
Using primary MSCs, we identified increased GCLM immunostaining, a component of glutathione synthesis, and thioredoxin in Ag + exposed CFU-f compared to untreated controls (Fig. 4f). Analysis of whole colony area did not reveal any variation in the expression of either protein.
We used a pharmacological inhibitor of glutathione synthesis, l-buthionine sulfoxamine (BSO), to determine the functional effects of glutathione on CFU-f survival while under Ag + exposure conditions. As observed before, a significant reduction in CFU-f number was noted at 10 µM Ag + in the absence of glutathione inhibition. However with the addition of 1 µM BSO, a significant reduction in CFU-f was observed with Ag + ≥ 5 µM, compared to their equivalent Ag + only control (Fig. 4g). In the case of 10 µM Ag + , CFU-f formation was eliminated in the presence of BSO. Recovery of CFU-f number was achieved through the concomitant treatment of 10 µM Ag + / BSO with exogenous glutathione (GSH-MEE).
Effects of silver on adipogenic differentiation is dependent on exposure time. The level of lipid deposition by MSCs as measured by Oil Red O staining revealed a reduction in adipogenesis when exposed to silver compared to controls. While a short term exposure during the initial 3 days of differentiation had no effect, continuous silver treatment during differentiation resulted in significant declines compared to untreated control and short term sliver exposure (p < 0.001) (Supplementary Fig. S4a). In contrast, pre-treatment of MSCs www.nature.com/scientificreports/ with silver during expansion, with subsequent silver-free differentiation, caused a slight but significant increase in adipogenesis compared to control (p < 0.05, Supplementary Fig. S4b).
Osteogenic differentiation of MSCs is unaffected by Ag + exposure. The effect of Ag + exposure on MSC osteogenic differentiation capacity was assessed by measurement of alkaline phosphatase (ALP) activity. Osteogenic differentiation of MSCs was performed in the presence and absence of 10 µM Ag + , with ALP activity normalised to DNA determined for short-term (3 day) exposure and continual Ag + exposure groups (Fig. 5a).
Cultures treated during the initial 3-day period exhibited a 16% decline from control, while a 26% reduction was observed for cultures treated for the entire differentiation period, however both findings did not reach significance (p = 0.582 and p = 0.246, respectively) ( Fig. 5a and a,i). Furthermore, analysis of DNA data provided additional evidence of the long-term viability of confluent cultures at continual sub-EC 50 Ag + concentrations (Fig. 5a, ii). We had previously found that Ag + exposure could inhibit MSC CFU-f capacity. We therefore determined if the osteogenic differentiation of MSCs was affected following CFU-f culture in the presence of Ag + . We found that osteogenic capacity (as measured by normalised and total ALP activity) was unaffected by previous exposure to Ag + during CFU-f ( Fig. 5b and b,i). However, and in contrast to previous observations, DNA counts (relating to cell number) were significantly elevated in cultures from MSCs previously exposed to Ag + (Fig. 5b,ii).
CFU-Ob capacity is inhibited during Ag + exposure. MSCs were plated at colony-forming seeding density and then exposed to osteogenic conditions to determine the effect of Ag + exposure on the formation of CFU-Ob. In a manner similar to the effects of Ag + exposure on CFU-f growth, the generation of ALP-positive CFU-Ob was decreased in the presence of continuous Ag + . Colony number in the 10 µM Ag + treatment group declined by 62% from control but did not reach statistical significance (p = 0.112, Fig. 5c), however total CFU-Ob area was significantly reduced at this sub-EC 50 concentration (Fig. 5d).

Discussion
The consequences of infection of orthopaedic trauma devices are both severe for the patient and costly to the healthcare system 2,30 . Although the risk of infection for open tibial fractures is as high as 36%, the primary aim of treating all at risk injuries with implants comprising an anti-microbial technology should not jeopardise successful union 31 . Here, we have investigated the use of silver as a broad spectrum antimicrobial in the prevention of biofilm formation, specifically its elution within an intramedullary setting. We then used the in vivo elution profile data to determine in vitro effects on MSC colony-forming capacity and differentiation, the mechanisms of MSC tolerance to Ag + induced oxidative stress, and the outcomes of fracture healing.
The elution profile of any additive from an implant is of importance for not only efficacy but also determining the safety of such products during pre-clinical evaluations. With regards to testing silver-coated implants, valuable data can be gained from laboratory-based testing, for example using saline or simulated body fluid as carrier solutions. However due to the chemical complexities of using this transition metal, the analysis of silver elution from implants placed in clinically relevant in vivo localities is essential 32,33 . Previous reporting of release profiles from silver technologies have shown a rapid increase in silver at day 2 following sub-cutaneous implantation, declining thereafter from day 6 15 . Consistent with those data, we also found a rapid elevation in silver concentration at the implantation site, additionally correlating this to the concomitant rise in plasma concentrations and a large decline of implant-associated Ag + . Importantly, this was mapped during the initial period following implantation and allowed calculation of the maximal Ag + burst release within the free canal space, which was applied in our in vitro investigation. While the elution of some pharmaceuticals has been assessed in this way, to our knowledge, the determination of silver elution from such an implant location has not previously been described 34 . Importantly, the same implants proved capable of reducing in vitro colonisation by S. aureus over a 14 day period, a pathogen commonly the cause of implant related infection 3 . Figure 4. (a-g) Proteomic and molecular identification of the oxidative stress response of MSCs during exposure to Ag + . (a) Protein relative abundance heat map from Y201 CFU-f under control and Ag + exposure conditions (green < 15, red > 20). (b) Volcano plot of 5050 quantified proteins, negative/positive log 2 data equate to Ag + group down/upregulated, respectively. 15.19% of proteins were significantly differentially regulated accepting Progenesis QI calculated q-value of < 0.05 (orange), reducing to 2.17% (green) when applying additional multiple test correction to 5% FDR (Benjamini and Hochberg). (c) GO terminology of biological processes (FDR < 0.05) associated with differentially upregulated proteins of Ag + cultured Y201 CFU-f. (d) Fold change of oxidative stress response genes in 3 primary MSC donors (grey, pink/red and green; dots represent different genes), the red line represents 1.5 fold-change. (e) Oxidative stress genes exhibiting ≥ 1.5 fold-change following Ag + exposure versus controls, values are mean ± SEM (n = 3). (f) Immunostaining of CFU-f for GCLM and TRX (both red), counterstained with DAPI (blue). Scale bar: 50 µm. (g) Ag + induced reduction of CFU-f is potentiated by inhibition of glutathione synthesis with BSO, with recovery aided by exogenous GSH. Results represent the mean CFU-f number ± SEM (n = 3). Statistical analysis by one-way ANOVA using Sidak's correction for multiple comparisons. Comparisons performed against control represented by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Additional comparisons made against treatment groups containing the same level of Ag + (e.g. 1 μM Ag + v 1 μM Ag + /1 μM BSO), significance indicated compared to 5 μM Ag + ($) and 10 μM Ag + (#). Exogenous GSH in the presence/absence of 10 µM Ag + and 1 µM BSO. Comparisons performed against control as before, with additional comparisons made against Ag + (@) and Ag + /BSO (&). www.nature.com/scientificreports/ MSCs account for < 0.01% of the cells isolated from the bone marrow, therefore the capacity to proliferate from a low density population provides the niche with a rapid source of progenitors required for skeletal remodelling and repair 35 . The maintenance of a viable population of colony-forming stromal cells is of paramount importance, and the CFU-f assay can be used as an in vitro assessment of this ability 36,37 . Through this assay we revealed a reduction of MSC colony-forming capacity in the presence of sub-toxic Ag + that prompted the analysis of the mechanisms of tolerance employed by the surviving colonies. ROS production following heavy metal ion exposure is regulated by superoxide dismutase (SOD) and catalase (CAT) in addition to several other antioxidant enzymes and their components, such as the predominant source of cellular thiol groups, glutathione 38 . However, variations in oxidative stress protein synthesis between tissues have been identified during in vivo cadmium exposure 39 . In vitro molecular and protein profiling of these cellular mechanisms has ordinarily been performed after short term exposure using confluent cultures, while investigating specific proteins involved in oxidative www.nature.com/scientificreports/ stress management [40][41][42] . Significantly, for orthopaedic applications, the data presented here provides an insight into the global mechanisms employed by chronically exposed Ag + tolerant clonogenic MSCs. The glutathione synthesis inhibitor, BSO, elevates intracellular ROS and sensitises systems to sub-toxic doses of oxidative agents; recovery of cell viability can be achieved through the administration of exogenous glutathione 43,44 . For the first time with MSCs, we used this technique to confirm the findings of the proteomic, bioinformatics and qPCR analysis and in doing so revealed the importance of glutathione to the clonogenic survival of MSCs following exposure to Ag + ions. Our data also highlighted a reduction in colony number during prolonged BSO treatment, although reduction in in vitro cell viability was not observed by other researchers investigating the effects over shorter time periods, with a greater BSO concentration 45,46 . Additionally, the inhibition of the glutathione system potentiated the effect of sub-toxic Ag + concentrations, which overwhelmed the remaining defence mechanisms of the normally sliver-tolerant sub-population.
Oxidative DNA damage resulting from ROS can be induced in cells exposed to Ag +47 . While the risk of transversion mutations is abrogated by several DNA repair mechanisms, the upregulation of proteins involved in global genome nucleotide excision repair were identified in our assessment of MSC clonogenicity following silver treatment. This suggests that neutralisation of ROS alone is not sufficient to enable colony formation in sliver-treated MSCs and that other pathways are employed in order to maintain the MSC population.
Increased oxidative stress impairs fracture healing in rodents 48,49 . In a study by Lippross et al. a decline in the quality of bone remodelling was described and has been supported by evidence of reduced osteogenic differentiation of MSCs in vitro while under conditions of oxidative stress, including those caused by Ag + exposure [49][50][51][52] . Osteoblast activity plays an important role in the formation of the hard callus during secondary fracture healing, an event that occurs several weeks following the initial injury. We determined that the retention time of silver within the bone was minimal in our model, additionally, we found that adipogenesis was differentially altered dependent on the timing of exposure. While a limitation of our study is our measurement of one indicator of osteogenesis (ALP), with further work investigating other markers, for example by qPCR, required to provide more robust information, we report that little effect on the osteogenic capacity of confluent MSCs and cell viability was observed even while under the 10 µM Ag + conditions, a concentration that is in line with the maximum silver elution characteristics measured using the rat in vivo model. Of interest, however, was the significant decline in MSC clonogenicity that was discovered while under these same conditions. The importance of MSC number at a fracture site was highlighted by Hernigou et al. and it is conceivable that our observation of reduced clonogenicity during conditions of oxidative stress may explain the reports of reduced healing in fracture models with localised induced ROS 48,49,53 . Further investigation of MSC function following in vivo exposure to the silver coated implants would have provided valuable additional insight.
Together, our data indicate that clinically relevant silver concentrations remain sub-toxic to MSCs at high density, but can result in a decreased colony-forming capacity. Tolerant CFU-fs show upregulated mechanisms to neutralise ROS and minimise the impact of DNA damage, with the implication that those cells maintain their osteogenic capacity, allowing the progression of fracture healing. These findings indicate that silver may have a role to play in reducing the incidence of fracture fixation related infection, bringing potential benefits to both patients and healthcare system.

Materials and methods
The following methods describe the investigation of Ag + release from intramedullary implants, translating this to in vitro methods that assessed the effect on MSC function.
Total silver analysis was determined pre-and post-implantation (where appropriate). Individual implants were placed in 1:2 (v/v) nitric acid/dH 2 O and incubated overnight at room temperature (RT). Solutions were vortexed and diluted 1:1000 (v/v) in 1% nitric acid. Silver content was determined against an Ag + standard (0.2-20 ppb) using the Agilent 8800 ICP-MS Triple Quad (Stockport, UK) in the presence of a 500 ppb rhodamine internal standard. For each time point, samples allotted for analysis were transferred to a fresh cryovial and 1 mL of fresh inoculum added to each. Following a further 24 h incubation, the pins were placed in to clean tubes, washed 6 × with sterile PBS before transfer to neutraliser (0.85% NaCl, 0.4% sodium thioglycollate, 1% Tween 20) and the colonising bacteria from the surface were recovered as described in ASTM E2871-13. Briefly this comprised vortex mixing for 30 s, followed by sonication for 30 s (45 kHz), this was repeated, with a final vortex before serial dilution in MRD and plating on to Petrifilm (3M, UK). All Petrifilm were incubated for at least 48 h at 32 °C before counting. Twenty-four hour samples underwent the same recovery process, without the additional 24 h re-challenge following initial incubation.

Scientific Reports
| (2020) 10:18950 | https://doi.org/10.1038/s41598-020-76087-1 www.nature.com/scientificreports/ In vitro cell culture. Mesenchymal stem/stromal cells (MSCs) of human origin were isolated from bone marrow aspirate (Lonza) or excised bone donated by patients undergoing primary arthroplasty surgery (NHS, Clifton Park Hospital, UK). Informed consent was obtained from all subjects. Collections and all methods were carried out in accordance with relevant guidelines and regulations under approval from the University of York Biology Ethical Committee Review Board and NHS Local Research Ethics Committee. Clonal MSCs, termed Y201, were generated from primary bone marrow derived MSCs engineered to overexpress human telomerase reverse transcriptase (hTERT) 29 . The use of Y201 MSCs as a model system has been indicated where appropriate. Both primary MSCs and Y201 MSCs were expanded in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 Units/mL Penicillin and 100 μg/mL Streptomycin (37 °C, 5% CO 2 ). Human osteoblasts (hObs) were purchased from PromoCell (Germany) and cultured in osteoblast growth media (Pro-moCell). Cells were passaged using 0.25% Trypsin-EDTA once 90% confluent. All reagents purchased from Sigma (Poole, UK) unless otherwise stated.
In vitro viability and proliferation. Primary MSCs seeded at 3.125 × 10 4 cells/cm 2 in 96-well plates were incubated for 24 h before addition of an Ag + dose response (range 8.75-100 μM, Ag 2 SO 4 , Alfa Aesar, Heysham, UK). After a further 24 h, WST-1 reagent was added and the optical density read at 440 nm and 600 nm after 1 h.
Results were converted to viability as a percentage of the untreated control. Proliferation over 72 h was assessed using 5-ethynyl-2′-deoxyuridine (EdU) incorporation (ThermoFisher, Loughborough, UK). MSCs were seeded for 24 h in to 24-well plates (2.5 × 10 3 cells/cm 2 ) before addition of Ag + (0-10 µM). After 72 h, cultures were counterstained with Hoechst and image analysis performed using ImageJ, providing the percentage of EdU positive cells.

CFU-f and CFU-Ob formation.
Primary MSC cultures at clonal density (10 cells/cm 2 ) were established in DMEM + 20% HyClone FBS (GE Healthcare, Little Chalfont, UK) to generate CFU-f (colony-forming unit fibroblast). As for the assessment of viability and proliferation, medium was changed 24 h after seeding for that containing Ag + (0-10 µM). Medium was changed twice per week, with one subset of plates receiving Ag + for the first 3 days only, after which medium was without silver. Colony formation (defined as ≥ 50 cells) was assessed after 14 days following crystal violet staining (0.05% w/v crystal violet) for 20 min. CFU-Ob (colony-forming unit osteoblast) were generated as described for CFU-f, but supplementing the medium with osteogenic additives at the medium change 24 h after seeding (50 μg/mL l-ascorbic acid, 10 -8 M dexamethasone, 3 mM β-glycerophosphate). Alkaline phosphatase (ALP) positive colonies were identified after 14 days using 1% Fast Red TR/0.2% Naphthol AS-MX applied for 2 min.

Differentiation of MSCs.
Primary MSCs were culture expanded, seeded in to well-plates for 24 h prior to the addition of osteogenic differentiation medium (described above) ± 10 µM Ag + . As before, one group received Ag + free medium after 3 days while maintaining 10 µM Ag + in the second treatment group. Adipogenic differentiation media (10 -6 M Dexamethasone, 500 µM 3-Isobutyl-1-methylxanthine, 1 µg/mL Insulin from bovine pancreas, 100 µM Indomethacin) and osteogenic media, were changed twice per week with time points of 21 days (adipogenic) and seven and 14 days (osteogenic). Controls underwent the same medium changes (with/without Ag + ) and time points.
Adipogenic cultures were rinsed (1xPBS) and fixed for ten minutes in 10% formaldehyde (Polysciences, USA) followed by a rinse with dH 2 O and the addition of Oil Red O working solution for five minutes. The stain was removed and the wells washed with 60% Isopropanol before a final rinse with tap water. Lipid bound Oil Red O was quantified through removal with 99% Isopropanol and the optical density measured at 520 nm (Multiskan GO, Thermo Fisher, UK,). For osteogenic analysis, at the specified time points, cultures were lysed and ALP activity analysed against a p-nitrophenol standard (pNP, 0-250 nmol/mL) using a pNPP substrate (5 mM). Optical density (405 nm) was measured after 1 h (37 °C). Data were normalised to DNA, quantified using PicoGreen (ThermoFisher), measured at Ex: 485 nm/Em: 538 nm. Differentiation following MSC expansion as CFU-f was determined, with MSCs seeded at clonal density ± 10 μM Ag + for 14 days, before differentiation in Ag + free medium. Time points and analysis were as previously described.
CFU-f formation following Ag + exposure. The continued clonogenic capacity of MSCs following Ag + treatment and the existence of an Ag + tolerant subpopulation that maintained colony formation under further Ag + exposure were assessed using a two-stage CFU-f assay (see Fig. 3a for schematic description). In brief, primary MSCs seeded at clonal density were cultured in medium (0, 1 or 10 µM Ag + ) and assigned as 'Stage 1' . Medium was changed twice per week for 14 days, after which, cells from the same treatment group (e.g. control) were pooled. MSCs were re-seeded in to further 6-well plates at clonal density ('Stage 2') and medium (± Ag + ) changed as before. After 14 days, the colonies of the 'Stage 2' plates were counted and diameters measured. Assessment of CFU-f diameter was performed using the Zeiss Zen 2.3 Lite software.
CFU-f formation in conditioned medium. The effect of environmental signals on MSC (primary and Y201 MSCs) clonogenicity was determined by culturing clonally seeded MSCs in conditioned medium (MSC-CM). Tissue culture flasks of MSCs at 70% confluence were rinsed with phosphate buffered saline (PBS) and further culture in serum free DMEM for 24 h. Medium was removed, centrifuged and filtered (0.45 µm) and HyClone FBS added to give 20% (stored 2-8 °C). 'Non-conditioned' (i.e. basal) or 'Conditioned' medium were used during media changes (± 10 µM Ag + ) with colonies assessed as before after 14 days.  In vivo Ag + release from intramedullary implants. Study design was approved by the Smith and Nephew Animal Welfare and Ethical Review Board in compliance with the Animals (Scientific Procedures) Act 1986, taking into consideration the requirements for reduction, replacement and refinement. Twelve male Sprague Dawley rats (250-300 g) were randomised in to four groups consisting of three animals per group. Each group was allocated to a time point of 24, 48, 72 h or 28 days. Animals were group housed in temperature controlled rooms at 21 ± 2 °C with a relative humidity of 55 ± 10% and artificial lighting cycle of 12 h light/dark. Sub-cutaneous antibiotic prophylaxis (Septrin, 24 mg/kg), was provided prior to surgery and twice per day for the initial 2 days of the study. In addition, intra-peritoneal injections of Buprenorphine analgesia (0.05 mg/kg) and medetomidine sedative (0.1 mg/kg) were provided. Following surgery, buprenorphine (0.05 mg/kg) was administered every eight hours for a minimum of 48 h. Animals were anaesthetised using an Isoflurane/Oxygen/ Nitrous oxide mixture. Access to the femoral canal was via an entry hole in the inter-condylar notch of the left hind leg. The test article was inserted and the wound closed using absorbable sub-articular sutures. Following surgery, the animals underwent X-rays and were returned to individual housing to recover, after which they were returned to the group. Animals were sacrificed at allocated time points and plasma samples prepared from whole blood collected in heparin sodium salt anticoagulant. The femurs were removed and the implants retrieved.
Silver quantification of plasma and femurs. Plasma  www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.