3.1. Scaffolds Based on Cu-Containing Melt-Derived Glasses
HSM analysis guided the selection of the sintering treatment for the fabrication of SCNA-2Cu and SCNA-5Cu scaffolds. As shown in Figure 1
a, SCNA-2Cu and SCNA-5Cu samples maintained their unaltered cylindrical shape up to 722 and 725 °C, respectively, when first shrinkage occurred. Maximum shrinkage was reached within 820–830 °C and the samples underwent melting above 1190 °C. Figure 1
b compares the thermal shrinkage of melt-derived Cu-doped glasses with that of the parent SCNA material. The thermal behavior of the three glasses is similar as they all exhibit a one-stage shrinkage upon heating, and the maximum shrinkage is followed by a plateau before reaching the melting temperature. The first shrinkage temperature does not significantly change with CuO content. Interestingly, decrease of the melting temperature was observed with the increasing amount of CuO in substitution to CaO in the glass composition. This result is in accordance with those obtained by Wers et al. [25
]. CuO is known to be an intermediate oxide but, if there is a relatively low amount of alkaline ions in the glass, Cu2+
participates only as a network modifier assuming the same role as Ca2+
]. Therefore, the reduction of melting temperature with increasing amount of CuO can be related to the difference of high-temperature stability of CaO and CuO. CaO is a widely accepted refractory compound, conversely to CuO (the refractoriness of a compound reflects its melting point, and in the case of CaO and CuO melting points are 2590 and 1326 °C, respectively). Based on the HSM results, the selected sintering temperatures for making the scaffolds were 1000 and 950 °C for SCNA-2Cu and SCNA-5Cu, respectively (well above the maximum shrinkage and in the middle of the densification region).
As-poured SCNA-2Cu and SCNA-5Cu are completely amorphous materials (glasses), as demonstrated by the broad halo (2θ within 20–40°) in the wide-angle XRD patterns shown in Figure 2
a and Figure 3
a. Analogous results were found elsewhere for the parent SCNA material [27
]. Therefore, it can be stated that the introduction of CuO in the glass matrix does not affect the amorphous character of the glasses. The wide-angle XRD analyses performed on SCNA-2Cu and SCNA-5Cu scaffolds ground in powders (Figure 2
b and Figure 3
b) indicate that wollastonite (CaSiO3
; PDF code: 00-027-0088) is the major crystalline phase developed in both materials upon heat treatment. This is consistent with previous observations on Cu-free crystallized SCNA [27
] as well as with the results reported by Pérez et al. (devitrification of a SiO2
glass to wollastonite within 850–950 °C) [28
]. The biocompatibility of wollastonite is well known and recognized since the 1980s [29
]. Labradorite (Ca0.65
); PDF code: 01-083-1370) was also detected as a secondary crystalline phase in heat-treated SCNA-5Cu. In summary, both scaffolds are actually made of glass–ceramic materials.
Micro-CT investigations allowed visualizing the pore/strut structure of the scaffolds (Figure 4
), characterized by a 3D network of open and interconnected macropores with a size in the range of 450–500 µm (total porosity about 50 vol. %). The connectivity density of the scaffolds is around 3.0 mm−3
which, interestingly, is very close to that of femur trabecular bone from human patients [31
]. In general, pore interconnectivity is a key feature for implantable scaffolds to allow biological fluid perfusion, cell colonization and blood vessel access. A comparison with the pore characteristics of commercial implants supports the architectural suitability of Cu-doped SCNA-based scaffolds for orbital applications (for example, the total porosity and pore size of Medpor®
polyethylene implant are within 30–70 vol. % and 100–1000 µm, while the bioceramic alumina implant has a nominal pore size centered at 500 µm [33
Compressive strength of SCNA-2Cu and SCNA-5Cu scaffolds (23 ± 2 and 21 ± 3 MPa, respectively) is enough to allow safe manipulation of the implants during surgical procedures.
Copper ion release from SCNA-2Cu and SCNA-5Cu scaffolds as a function of immersion time in SBF is shown in Figure 5
. In general, a continuous release over time was observed and the amount of Cu ions in SBF increased with increasing amount of Cu in the as-prepared glass (SCNA-5Cu > SCNA-2Cu). A quantitative evaluation of in vitro release of copper is essential in order to rationalize the effective therapeutic properties of the materials. The therapeutic behavior, in fact, will depend on the amount of copper ions released. Most of the studies existing in the literature reveal that an antimicrobial effect can be obtained with a copper ion concentration within 50–100 ppm [13
], which is significantly higher compared to the values achieved for SCNA-2Cu and SCNA-5Cu. A similar situation was observed as far as the angiogenetic effect is concerned: a clear promotion of angiogenesis was reported in vitro and in vivo when the level of copper released is on the order of a few tens of ppm [13
Detection of no mass loss at the end of the dissolution experiments for both types of scaffolds further confirms the high stability of SCNA-2Cu and SCNA-5Cu in a biological environment, which can be interesting when non-resorbable implants are needed (e.g., permanent orbital implants).
The high stability of these scaffolds in SBF and the low release of copper are dictated by the basic SCNA composition. In principle, other less-stable melt-derived glasses could be selected and doped with copper for improving the release of copper to therapeutic levels, thus eliciting acceptable antibacterial and pro-angiogenic actions. However, these glass-derived materials would undergo progressive dissolution over time, which contradicts the main requirement of orbital implants (high stability and structural integrity). Therefore, a completely different approach was investigated based on the deposition of reactive but thin glass coatings on nearly-inert glass–ceramic scaffolds.
3.2. Scaffolds Coated with Cu-Doped MBGs
In order to increase the release of copper from the scaffolds, a second approach was pursued involving the coating of SCNA scaffolds with a Cu-doped MBG layer. In fact, the mesoporous texture and high SSA of mesoporous materials are known to intensify the rate of surface reactions compared to melt-derived ones, thus leading to a faster release of therapeutic agents during glass dissolution [37
The wide-angle XRD diffraction patterns of calcined 1Cu-MBG and 5Cu-MBG are shown in Figure 6
and reveal the completely amorphous nature of both glasses, as demonstrated by the sole presence of a broad halo (2θ within 15–35°). On the contrary, in the small-angle regime (Figure 7
a,b), both glasses show the diffraction peaks typical of the scattering patterns of a two-dimensional hexagonal p6mm lattice [20
]. The d100
is about 6.8 nm for both materials and, assuming a 2D hexagonal symmetry, the cell parameter results to be 7.8 nm. The low intensities for the reflections of 1Cu-MBG is probably due to the lower order of the mesostructure compared to 5Cu-MBG. According to the available literature, the effects of copper ions on the textural properties of MBGs still are under debate. Although it was well shown that doping with copper leads to a decrease of SSA compared to mesoporous pure silica, clear relationships between the increasing content of copper and the mesostructural order, SSA and mesopore size remain to be fully understood [13
STEM images of 5Cu-MBG along the  and  directions allow the visualization of a highly-ordered hexagonal arrangement of 1D parallel channels (Figure 7
c,d), in agreement with small-angle XRD results.
adsorption-desorption measurements further confirmed the mesoporous texture of 1Cu-MBG and 5Cu-MBG as both materials exhibited a type-IV isotherm pattern (Figure 8
), which is associated to nanopores within 2–50 nm [38
]. The shape of the hysteresis loop, which can provide information about the shape of the mesopores [39
], reveals the presence of uniform mesopores of approximately cylindrical shape with MCM41-like hexagonal symmetry (H2-type loop). The textural characteristics are collected in Table 1
; these results are consistent with those assessed for the parent SiO2
ternary MBGs [20
] and suggest that the SSA can be increased by increasing the CuO content in the glass, which is in agreement with the trend observed by Wu et al. about Cu-doped MBGs [13
Pore characteristics assessed by micro-CT were in line with previous investigations on SCNA scaffolds [42
]. Interestingly, the presence of the MBG coating induced no significant variations in terms of macropore size (about 500 µm before and after the coating procedure) while the total porosity moderately increased after the surface treatment (60 vs. 50 vol. %). This difference can be explained considering that, in order to apply the MBG coating, SCNA scaffolds were immersed in a strongly acidic sol having very low value of pH (1.04 and 0.85 for 1Cu-MBG and 5Cu-MBG sols, respectively). The strong acidity of the sol may be responsible for the surface erosion of scaffold struts, which resulted in a higher porosity. This had an impact on the mechanical properties, too: in fact, the compressive strength of both Cu-MBG-SCNA scaffolds was 10.0 ± 2.0 MPa, which is about half as lower as the compressive strength obtained for uncoated SCNA samples (23.0 ± 2.0 MPa) manufactured through the same procedure. If application as orbital implant material is a goal, these values of mechanical strength are enough to allow safe manipulation during surgery as well as postoperative integrity. Comparison with commercial implants is not possible due to the lack of available data in the literature. Problems of mechanical integrity were mentioned—albeit without providing quantitative data—for bovine hydroxyapatite orbital implants [43
] that, however, are significantly more porous (80 vol. %) than the materials developed in the present work.
shows SEM images at different magnifications of 5Cu-MBG-SCNA. In Figure 9
a, the 5Cu-MBG layer is shown to coat quite uniformly the pore walls of the scaffold. As displayed in Figure 9
b, the MBG coating appears to cover all the surface of the single struts lying underneath; however, some defects and cracks can be observed in the coating, which suggest the need for further optimization of the deposition procedure and/or calcination treatment to produce flawless glass layers.
The pore/strut architecture of a given scaffold clearly dictates the potential suitability for a specific application. The total porosity and macropore size of MBG-coated materials are adequate for use in the field of anopthalmic socket surgery, being comparable to those of commercially-available and clinically-used orbital implants (see the considerations already presented for SCNA-2Cu and SCNA-5Cu scaffolds in the Section 3.1
The release of copper ions from 1Cu-MBG-SCNA and 5Cu-MBG-SCNA scaffolds as a function of immersion time in SBF is shown in Figure 10
. The trend of copper ion release can be split into three parts: (i) initial increase in the first hours of immersion; (ii) drop observed around 48 h; and (iii) a plateau stage (1Cu-MBG-SCNA) or a slight increase till 2 weeks (5Cu-MBG-SCNA). The drop of copper levels might be due to the precipitations of copper in the hydroxyapatite layer that is formed on the surface of both scaffolds upon immersion in SBF. In fact, it is known that hydroxyapatite can act as a cation exchanger and may incorporate metallic ions such as Zn, Co and Cu in its structure [44
Interestingly, weight measurements of scaffolds before and after in vitro tests (2 weeks) revealed no mass variation during immersion in SBF. Furthermore, pH measurements performed on the testing solution just revealed a slight variation towards alkalinity (from 7.4 to 7.6).
From these results it is clear that the samples are able to deliver copper ions and that this release depends on the initial concentration of copper in the MBG formulation. Altogether, the release of copper ions from 5Cu-MBG-SCNA scaffolds is one order of magnitude larger than that from melt-derived SCNA-2Cu and SCNA-5Cu at each time point (Figure 10
vs. Figure 5
). On the contrary, when results from 1Cu-MBG-SCNA are compared with those from melt-derived SCNA-2Cu and SCNA-5Cu, copper ion release appears larger at each time point, yet the values are of the same order of magnitude. This further confirms that the high SSA of the mesoporous coating facilitates the delivery capacity of the therapeutic metallic agent. The concentrations of copper released from 5Cu-MBG-SCNA could be potentially effective to elicit antiseptic and angiogenetic effects [13
], thereby encouraging further biological investigation on this material.
shows exemplary SEM images of a 5Cu-MBG-SCNA scaffold after 15 days of immersion in SBF at different magnifications. The pore walls and struts of the scaffolds are coated by a newly formed phase characterized by a cauliflower morphology, i.e., globular agglomerates formed by needle-like nanocrystals. Compositional analysis reveals that this phase is a calcium phosphate with a Ca-to-P atomic ratio of 1.68, which is very close to that of stoichiometric hydroxyapatite (1.67). It is worth underlining that, during hydroxyapatite formation, just the scaffold surface (i.e., the thin surface layer of MBG) reacts with the biological fluids, while the SCNA skeleton exhibits nearly inert behavior [45
]; this fulfils the primary requirement of orbital implant materials, i.e., the stability over time to allow orbital volume filling and adequate support to surrounding orbital structures.
If apatite-forming ability is unnecessary for orbital implant materials, on the other hand the bioactivity of the Cu-doped MBG-SCNA scaffold would be key for applications in other biomedical fields, such as bone tissue repair. Therefore, Cu-doped MBG-SCNA scaffolds show promise for use in contact with hard tissues to regenerate bone, ensuring structural support at the defect site (due to the strong SCNA skeleton) as well as promoting bone in-growth and safe anchorage to surrounding host tissues (due to the MBG layer).