**3. Results**

#### *3.1. Analysis of Prepared Nanoscale Hydroxyapatite*

Nano-hydroxyapatite was prepared via the rapid mixing technique previously published [10]. Using TEM, the nanoscale hydroxyapatite particles were visualized and contained crystalline and amorphous regions, typically less than 100 nm in size. Silver was found within the nanoscale hydroxyapatite particles, with the visible formation of nanoparticles of silver. Both materials showed the peaks associated with hydroxyapatite in XRD patterns. XRD showed the presence of silver phosphate peaks but only for the 10 mol.% silver-containing material (Figure 1a). Despite this, in both materials silver nanoparticles were visible under TEM; EDX confirmed that the darker regions found under the TEM contained silver. See Supplementary Figure S1 for EDX mapping of all elements found in sample Figure 1e. The size distribution of particles' diameter between nHA and nHA with 10 mol.% silver substituted in was found to be similar, with an average of 37 and 40 nm, respectively (Figure 1f).

**Figure 1.** Evaluation of silver-containing nanoscale hydroxyapatite (**a**) XRD patterns of 5 and 10 mol.% silver doped nanoscale hydroxyapatite (Ag nHA), identifying peaks for hydroxyapatite Ca10(PO4)6(OH)2 and silver phosphate (Ag3PO4) phases within the sample. Silver phosphate was only identified in the 10 mol.% silver-containing material. Plot line 1 (blue) is 10 mol.% Ag nHA and plot line 2 (green) is 5 mol.% Ag nHA; (**b**) TEM image of nHA sample containing 5 mol.% silver. Silver deposits visible and highlighted by black arrows; (**c**) TEM at higher magnification on one of the silver deposit areas; (**d**) TEM image of nHA sample containing 10 mol.% silver. Larger silver deposit noted by black arrow; (**e**) Spatial EDX map of location of silver, mapped over the same location as that boxed in (**d**); (**f**) Particle diameter comparison between nHA and nHA with 10 mol.% silver shown as a Tukey box plot, ns = not significant; (**g**) TEM micrograph of nHA nanoscale particles.

#### *3.2. Ensuring Encapsulation of nHA by Electrospun Fibers*

Both silver and nondoped nHA were electrospun into polycaprolactone (PCL) scaffolds with a thickness of between 150 and 200 μm. SEM images showed incorporation of large particles in the fibers that were not observed in plain PCL scaffolds (Figure 2a). When digested, ICP analysis showed that plain PCL had very low levels of elements found within Ag-nHA (Figure 2b). The electrospun nHA showed the presence of calcium and phosphorus but not silver. Both electrospun scaffolds with silver-containing nHA showed the presence of silver alongside calcium and phosphorus (Figure 2b). This demonstrated that nHA was successfully incorporated into the electrospun fibers.

**Figure 2.** Scaffold fibers containing nHA (**a**) SEM micrographs of electrospun scaffold. Top: Polycaprolactone (PCL) scaffold. Middle: PCL scaffold containing nHA. Bottom: PCL scaffold containing 10 mol.% Ag nHA. Different magnifications were used to visualize encapsulation of nHA particles into the electrospun scaffold; (**b**) Inductively coupled plasma (ICP) analysis of dissolved electrospun scaffolds showing mg/kg of elements present. All % Ag materials are in mol.%.

#### *3.3. Investigation of Silver Release and Sca*ff*old Loss in Accelerated Degradation Study*

An accelerated degradation assay was performed using sodium hydroxide at 37 ◦C to understand the degradation profile of the electrospun scaffolds and to observe release profiles (Figure 3). All scaffolds degraded at the same rate with the presence of nHA having no apparent effect on degradation profile (Figure 3a). Cumulative release of silver was observed for the full 30 days (Figure 3b). The plot of release rate (Figure 3c) indicated that in the early period of days 1 to 5 the release was from surface-bound origins, whereas from days 7 onwards the release was from silver within the polymer that was then released through degradation. Mass loss from degradation was nonexistent in the 1- to

7-day period and then rapidly degraded from there, which reinforced the conclusions drawn from the silver release rate.

**Figure 3.** Accelerated degradation study of electrospun scaffolds containing nHA, over a 28-day period in 0.1 M sodium hydroxide. (**a**) Remaining weight over degradation time of different mol.% silver scaffolds; (**b**) Release profile of silver from an electrospun scaffold containing 10 mol.% Ag nHA over a 28-day period; (**c**) Plot of the silver release rate per day. All % Ag materials are in mol.%.

#### *3.4. Antimicrobial Impact of Sca*ff*olds*

Both silver-containing scaffolds showed an antibacterial effect on both agar diffusion and on bacterial suspensions. PCL-only scaffolds and those with undoped nHA showed no innate antimicrobial action. Scaffolds containing silver of any concentration showed an antimicrobial response on agar diffusion (Figure 4a,b). Bacterial suspension cultures in PBS exposed to scaffolds for 24 h demonstrated the antimicrobial properties of the scaffolds containing silver. The 10 mol.% silver nHA was studied in a time course assay against bacteria and this showed a significant reduction in bacteria over time (Figure 4e,f). The scaffold reduced the viable bacteria count to undetectable levels by 48 h for *E. coli* and 96 h for *S. aureus*.

**Figure 4.** Antibacterial studies on *E. coli* and *S. aureus* bacteria using the electrospun scaffold. (**a**) Agar diffusion test using disks of the electrospun samples against *E. coli* bacteria. Graph displaying individual data plots with a line demarking the mean value; (**b**) Agar diffusion test using disks of the electrospun samples against *S. aureus*; (**c**) Disks of electrospun samples suspended in PBS containing bacteria, measuring viable colonies after 24 h; (**d**) Optical images of the area of inhibition ring surrounding a 12-mm disk of scaffold containing either 10 mol.% silver or plain PCL and tested against *S. aureus* and *E. coli*; (**e**) Bacteria count after *E. coli* are suspended in PBS and exposed to a disk of electrospun 10 mol.% silver nHA for 3, 18, 24, 48, 72, and 96 h; (**f**) Same as (**e**) but using *S. aureus* bacteria. All graphs show mean ± standard deviation. All % Ag materials are in mol.%.

#### *3.5. Antimicrobial Impact of Sca*ff*olds*

Indirect toxicity assays found that higher silver concentrations were more toxic to both cell types while a longer, 21-day direct growth experiment showed a reduced toxic impact of these high silver samples on cells over time. When tested using fibroblast 3T3 cells, all scaffolds had a significantly higher viability than the negative (cytotoxic) control (Figure 5a). However, the scaffold containing 10 mol.% silver was also significantly different to the cell-only positive control, indicating some toxic impact. When the materials were tested on primary rat MSCs the toxic impact was more pronounced. The scaffold with 10 mol.% silver was significantly different to the cell-only positive control, but no

statistical difference was found with the negative cell death control (Figure 5b). All the remaining materials were statistically different to the negative cell death controls. A longer, 21-day direct growth experiment found that cell metabolic activity for 5 mol.% silver-containing scaffolds were significantly increased compared to the PCL-only scaffolds (Figure 5c). The 10 mol.% silver-containing scaffolds reduced the metabolic activity when compared to 5 mol.% silver but did not show a toxic impact when compared to the PCL-only scaffold. Live cell morphology can be observed from live fluorescent images (Figure 5d) along with the proportion of live to dead cells. Cells appeared to have a more spread out morphology on the nHA silver-containing material than in the PCL-only control.

**Figure 5.** Cell toxicity from the electrospun scaffold with different concentrations of silver nHA assessed by cell metabolic activity via PrestoBlue™ assay. (**a**) A 48-h noncontact toxicity test with the scaffold in the media above a monolayer of 3T3 cells. Graph displaying individual data plots with a line demarking the mean value. Dotted line contains each scaffold under it to be compared to another condition; (**b**) A 48-h noncontact toxicity test with the scaffold in the media above a monolayer of primary rat MSCs. Graph displaying individual data plots with a line demarking the mean value. Dotted line without star contains each scaffold to be compare to another condition; (**c**) Cell metabolism after 21 days of direct culture with the cells seeded on top of the scaffold using metabolic activity at each time point. Statistics (ANOVA) conducted on the day 21 measurements; (**d**) Live/dead staining of MSCs cultured on electrospun scaffolds after 21 days.

#### *3.6. MSC Osteogenic Di*ff*erentiation Study by ALP Quantification*

The impact of silver on the differentiation of primary MSCs down the osteoblast linage was measured after 21 days in culture by quantifying ALP content per unit of DNA (Figure 6). ANOVA showed a significant difference with 5 mol.% and the lower silver concentrations in regard to ALP production. Scaffolds with 10 mol.% silver are not significantly different to 5 mol.% silver scaffolds.

**Figure 6.** Cell differentiation potential of scaffolds containing different silver contents regarding ALP presence per unit of DNA after 21 days in culture with primary MSCs. Cells were cultured on top of the scaffolds. Graph displaying mean value ± standard deviation. N = 2, n = 3.
