Update on the use of 45S5 bioactive glass in the treatment of bone defects in regenerative medicine

Table 1 Studies published over the past 10 years involving the application of 45S5 bioactive glass in bone defects

Ref.	Objective	Type of study	Composition	Methods	Outcome measures
Souza et al[26], 2020	To compare the biocompatibility of a bioactive sodium calcium silicate glass containing 2.6 mol% Nb2O5 with that of the archetypal 45S5 BG	In vitro and in vivo	A variation of 45S5 BG in which 2.6 mol% of P2O5 was replaced by 2.6 mol% of Nb2O5, resulting in the composition named BGPN2 The glass was mixed with the precursor oxides SiO2 (99.5%), CaCO3 (99.95-100.5%), Na2CO3 (≥ 99.5%), P2O5 (≥ 99.5%), and Nb2O5	Biocompatibility and genotoxicity tests Bone regeneration: rat calvarial defect (5 mm). Seventy-two rats (sham group: no defect; control group: empty defect; 45S5 BG group: filled defect; BGPN2.6 group: filled defect), with 6 rats per group for 14, 28 and 56 d Qualitative and quantitative analysis of 3D micro-CT images	BGPN2.6 glass was not cytotoxic to BM-MSCs and had no mutagenic potential Micro-CT showed that BGPN2.6 almost completely regenerated a critical-sized calvarial defect within 8 wk, surpassing the performance of standard 45S5 BG. BGPN2.6 glass demonstrated more than 90% coverage compared to 66% for 45S5 BG
Souza et al[3], 2018	To study the bioactive properties of Nb-substituted silicate glass derived from 45S5 B	In vitro and in vivo	Compositions (mol%): 45S5 BG (46.1 SiO2; 26.9 CaO; 24.4 Na2O; 2.6 P2O5; no Nb2O5) BGPN2.6 (46.1 SiO2; 26.9 CaO; 24.4 Na2O; no P2O5; 2.6 Nb2O5) BGPN1.3 (46.1 SiO2; 26.9 CaO; 24.4 Na2O; 1.3 P2O5; 1.3 Nb2O5) High purity powders SiO2, Na2CO3, CaCO3, P2O5 (> 99.9%), and Nb2O5, optical grade, > 99.5%) Glass particles between 38-53 µm in size	Compatibility and osteogenic differentiation of hESCs. Bone formation: rods composed of different glass types (BGPN1.3, BGPN2.6, and 45S5 BG) were implanted into bone defects (2 mm) in rat tibiae. Five animals per group were analyzed after 14 and 28 d	Nb-substituted BG is non-toxic to hESCs. There was a significant increase in osteogenic capacity and biocompatibility when up to 1.3 mol% Nb2O5 was added to 45S5 BG. The same increase in Nb2O5, replacing phosphorus, increased the osteostimulation of the BG
Thomas and Anbarasu[27], 2022	To evaluate cell compatibility and regenerative potential of 45S5 BG graft in critical size defects (CSD) in rat calvaria	In vitro and in vivo	45S5 BG: 45% SiO2; 24.5% Na2O; 24.5% CaO and 6% P2O5	In vitro cell viability assay of 45S5 BG using MTT assay with Novabone® and 10% DMSO as positive and negative controls, respectively, whereas cells alone served as the control Bone regeneration: 20 male rats with 6 mm diameter calvarial defects (control group: empty cavity) loaded with 2.5 mg of 45S5 BG (test group). Evaluation by CBCT after 4 and 8 wk	45S5 BG achieved a cell viability rate of > 70%, confirming cell compatibility. CBCT analysis showed a significant increase in VGi and a reduction in ROI of CSD from the fourth to the eighth weeks, showing its potential for bone regeneration
Ma et al[28], 2017	To evaluate a silicate-based composite bone cement (CSC) in a rabbit femur defect in terms of in vivo bone integration and biodegradability and compare the results with those of BG particulates and a calcium phosphate cement (CPC)	In vivo	CSC composition: tricalcium silicate (35%) and 45S5 BG (30%) with particles < 50 µm and 90-710 µm. The ratio of the two components was 1:2 (small:large); calcium sulfate (35%)	CSC cylinders molded with a 5 mm × 10 mm diameter, and CPC cylinders. Experiments conducted on 30 adult New Zealand white rabbits with femur defects. Control groups: BG particles and CPC. Analyses were conducted after 3, 6, and 12 months	The CSC underwent slower in vivo degradation compared with BG and CPC. The bone contact area at the interface between the surrounding bone and CSC gradually increased over time. CSC kept its structural integrity during in vivo implantation because of its acceptable mechanical strength
Esfahanizadeh et al[29], 2022	To evaluate bone regeneration in critical defects of rabbit calvaria filled with magnesium- and strontium-doped BGs and compare it with standard 45S5 BG	In vivo	Standard 45S5 BG with particles of approximately 20-50 nm	Experiments on 12 male New Zealand rabbits allocated to 2 groups. Four lesions were created in each calvaria with a diameter of 8 mm spaced apart. Each lesion was filled with (1) strontium-doped BG, (2) magnesium-doped BG, (3) 45S5 BG (positive control), and (4) an empty lesion (negative control). Evaluation occurred at the end of 4 and 8 wk	At 4 wk, magnesium-doped BG showed the highest new bone formation with a mean of 11.66 ± 2.64, followed by strontium-doped BG with a mean of 11.10 ± 1.69 (P = 0.0001). At 8 wk, the highest amount of new bone was observed in the strontium-doped group with a mean of 28.22 ± 3.19, followed by the magnesium-doped group with a mean of 22.55 ± 3.43 (P = 0.0001)
Lopes et al[10], 2020	To evaluate the solubility, apatite-forming capacity, cytocompatibility, osteostimulation, and osteoinduction of Nb-containing bioactive glasses (BGNb) derived from the composition of 45S5 BG	In vitro and in vivo	Composition (mol%) of 45S5 BG and Nb-substituted 45S5 BG: 45S5 BG (46.1 SiO2; 26.9 CaO; 24.4 Na2O; 2.6 P2O5; no Nb2O5) BGSN1 (45.1 SiO2; 26.9 CaO; 24.4 Na2O; 2.6 P2O5; 1.0 Nb2O5) BGSN2.5 (43.6 SiO2; 26.9 CaO; 24.4 Na2O; 2.6 P2O5; 2.5 Nb2O5 BGSN5 (41.1 SiO2; 26.9 CaO; 24.4 Na2O; 2.6 P2O5; 5.0 Nb2O5)	In vitro: BMMSCs were isolated from the tibia and femur of adult Wistar rats. MTT assay was conducted for each of the BG compositions. Cells were cultured in complete DMEM (positive control), and cells were previously incubated in DMSO for 30 min (negative control) In vivo: glass rods (4 mm length × 2 mm diameter) composed of 45S5 BG (45S5 BG or BGSN1 groups were implanted into circular defects (2 mm diameter) in the tibia of rats (5 animals/group) Evaluated after 28 d	45S5 BG and BGSN1 developed an apatite layer on their surfaces within 3 h. Glasses with higher concentrations of Nb2O5 (2.5 and 5 mol%) required at least 12 h Nb-substituted glasses were found to be compatible with BMMSCs. BGSN1 significantly enhanced cell proliferation after 4 d of treatment. Concentrations of 1 and 2.5 mol% Nb2O5 stimulated osteogenic differentiation of BMMSCs after 21 d of treatment
Fares et al[30], 2024	To evaluate the impact of different materials for filling bone defects following anterior cruciate ligament (ACL) reconstruction surgery with bone-patellar tendon-bone (BPTB) graft	In humans	Osteopure® allograft from resected human femoral head treated by sterilization at 25 kGy Glassbone® BG, 100% synthetic, a mixture of 45% SiO2, 24.5% CaO, 25.5% Na2O, and 6% P2O5 weight%) Collapat® II, a spongy bone graft composed of a collagen structure in which hydroxyapatite granules are dispersed	A prospective, monocentric cohort study was conducted with 102 adult athletes who underwent ACL reconstruction using the same arthroscopically-assisted BPTB, with a minimum follow-up of two years. Three groups based on the type of bone substitute GB group (G1): 45S5 BG ceramic Glassbone™ (n = 36; 35.29%); CP group (G2): collagen and hydroxyapatite bone void filler in sponge-shaped Collapat® II (n = 34; 33.33%); OP group (G3) treated human bone graft Osteopure® (n = 32; 31.37%). Patients were assessed based on their ability to kneel, the presence of donor site pain, and palpation of the defect	The percentage of Glassbone™ and Collapat® patients who kneeled comfortably was significantly higher than that of Osteopure® patients (77.78% and 76.5%, vs 65.6%, respectively)
Lu et al[31], 2018	To investigate the remodeling of resorbable bone cements in a stringent model of mechanically loaded tibial plateau defects in sheep	In vivo	Melt-derived 45S5 BG with fast- and slow-resorbing ceramic mini-granules (CG, 85% β-tricalcium phosphate/15% hydroxyapatite) ground to 100-300 μm diameter and biphasic PEUR composites Nanocrystalline hydroxyapatite (nHA) The resulting composite bone grafts were denoted as CG/nHA-PEUR and BGCG/nHA-PEUR CG/nHA-PEUR cement contained 55wt% CG, 24.3 wt% nHA, and 20.7 wt% PEUR, whereas BGCG/nHA-PEUR cement contained 37.5 wt% BG, 22.5 wt% CG, 21.6 wt% nHA, and 18.4 wt% PEUR	Eight sheep, with two types of bone defects in each posterior limb. The defects included a non-weight-bearing femoral plug defect on the medial and lateral distal condyles of both femurs (n = 16 per group, two defects with a 6 mm diameter and a 16 mm depth) and a weight-bearing tibial plateau slot defect (n = 8 per group) approximately 50% of the total anterior to posterior tibial depth with 6 mm height. Each sheep received both grafts (BGCG/nHA-PEUR or CG/nHA-PEUR) in separate extremities, with graft placement alternating between animals. Micro-CT analysis was conducted in the immediate postoperative period, and at 4, 8, 12, and 16 wk	CG/nHA-PEUR cements mechanically stabilized the tibial plateau defects and remodeled to form new bone at 16 wk, with early weight-bearing. Cements containing BG particles were resorbed and showed fibrous tissue filling the defect. These findings represent the first report of a settable bone cement that remodels to form new bone while providing mechanical stability in a stringent large animal model of weight-bearing bone defects near a joint
Diba et al[32], 2019	To investigate the feasibility of synthesizing novel hybrid particles by exploiting the strong interactions between alendronate and 45S5 BG	In vitro and in vivo	45S5 BG: a mean particle size of 2.0 ± 1.2 μm. Alendronic acid (4-amino-1-hydroxybutane-1,1-diphosphonic acid) powder. 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; ≥ 99.5%), and 2-(N-morpholino)ethanesulfonic acid hydrate (MES hydrate; ≥ 99.5%). Sodium hyaluronate powder (1.01-1.8 MDa) Injectable cohesive pastes: particles mixed with an aqueous solution of sodium hyaluronate (26 mg mL−1). A particle/solution ratio (g/mL) of 0.75. Final composition (wt%): HP1-7 (ALN 62.3 ± 0.6; Ca 11.4 ± 0.0; Na 12.8 ± 0.0; Si < 2; P < 1) HP2-7 (ALN 25.5 ± 9.8; Ca 16.7±0.3; Na 34.7 ± 0.0; Si 7.3 ± 0.3; P 9.9 ± 0.2)	A cylindrical defect (2.5 mm diameter and 5 mm depth) was created in the bilateral femoral condyle of osteoporotic male rats (n = 8 per experimental group) and filled with HP1-7 and HP2-7 hybrid particle pastes. Positive control: 45S5 BG	The hybrid particles released alendronate and inorganic elements (Ca, Na, Si, and P) in a controlled manner, exhibited a strong anti-osteoclastic activity in vitro, and stimulated the regeneration of osteoporotic bone in vivo
Prado Ferraz et al[33], 2017	To evaluate the in vitro osteogenic and osteoinductive potentials of BioS-2P and its ability to promote in vivo bone repair	In vitro and in vivo	Biosilicate®: 23.75 Na2O; 23.75 CaO; 48.5 SiO2; 4 P2O5 (wt%), containing two crystalline phases (BioS-2P) Composition (mol%): BioS-2P (23.3 Na2O; 25.8 CaO; 49.2 SiO2; 1.7 P2O5) 45S5 BG (24.4 Na2O; 26.9 CaO; 46.1 SiO2; 2.6 P2O5)	BioS-2P and 45S5 BG were cut into 3 mm thick discs and ground with silicon carbide paper to a grit of 400 (~35 μm). MSCs were obtained from the femur of two male Wistar rats and cultured on both types of discs and on polystyrene (control group). CSDs with a 5 mm diameter were created in 15 male Wistar rats and implanted with scaffolds. Evaluation occurred at 4, 8, and 12 wk (n = 5 per period). BioS-2P scaffolds seeded with unlabeled MSCs were implanted into calvarial defects and evaluated 8 wk later	Extracellular matrix mineralization increased in cells cultured on BioS-2P compared with 45S5 BG (P = 0.029)
Zhang et al[34], 2017	To compare the osteogenic capacity and effects of 45S5 BG scaffolds reinforced with ZnO/B2O3 (ZB), called BG-ZB, with pure 45S5 BG.	In vivo	BG-ZB: 30 SiO2; 28 CaO; 2 P2O5; 30 B2O3; 10 ZnO). 45S5 BG containing 4% BG-ZB 45S5/ZBx powders were homogeneously mixed with paraffin microspheres (porogen) of ~350 and ~500 μm diameter. BGs scaffolds manufactured with different porogens: 45S5/ZB0-350, 45S5/ZB4-350, and 45S5/ZB4-500	Thirty-six adult male rabbits were randomly separated into three groups according to the scaffolds (45S5/ZB0-350, 45S5/ZB4-350, and 45S5/ZB4-500). Each animal underwent surgery for a CSD (Ø 6 × 10 mm) in the bilateral distal femur, with two different implants inserted into the right and left femurs	Open porosity decreased with the addition of 4% ZB, but the percentage of interconnected pores (> 50 μm) increased with increasing porogen size from 350 to 500 μm. Stronger scaffolds containing 4% ZB and 500 μm porogen were beneficial for osteogenic capacity. In contrast, both scaffolds with smaller pore sizes exhibited a low level of new bone growth (< 32%) after 6-12 wk of implantation
Westhauser et al[35], 2019	To evaluate the effects of 0106-B1-BG and 45S5 BG on osteogenic differentiation, viability, and proliferation of MSCs in vitro and in vivo in severe combined immunodeficient (SCID) mice	In vitro and in vivo	Borosilicate glass (0106-B1-BG) (wt%): 37.5% SiO2, 22.6% CaO, 5.9% Na2O, 4% P2O5, 12% K2O, 5.5% MgO, 12.5% B2O3) 45S5 BG (wt%): 45%SiO2, 24.5% CaO, 24.5% Na2O, 6% P2O5)	Ten scaffolds per BG type were seeded with MSCs. Two scaffolds per BG type were implanted without MSCs as a control (total of 24 scaffolds). Four scaffolds were implanted per animal (female SCID mice), with two subcutaneous pockets created on the forelimbs and two on the hindlimbs Evaluation occurred after 10 wk	In vitro: both 45S5 BG and 0106-B1-BG were comparable in terms of MSC proliferation, viability, and osteogenic differentiation In vivo: 0106-B1-BG scaffolds were significantly superior to 45S5 BG in terms of osteoid quantity and maturation and angiogenic gene expression patterns
Jing et al[36], 2018	To investigate the relationship between icariin-doped 45S5 BG seeded with ASCs and angiogenesis of rat EPCs, in rat calvarial bone defect	In vitro and in vivo	45S5 BG (wt%): 45% SiO2, 24.5% Na2O, 24.5% CaO, and 6% P2O5, in a cubic and porous format with a volume of 5 × 5 × 5 mm3 loaded with 30 μL of icariin at a concentration of 5 × 10-3 mol/L Pure 45S5 BG scaffolds were used for comparison	A 8 mm diameter calvarial defect was created in the dorsal portion of the parietal bone in twenty male Sprague-Dawley rats, which were allocated into four groups: Group A (control, no implant), Group B (45S5 BG), Group C (45S5 BG/ASCs, 45S5 BG seeded with ASCs), and Group D (icariin/45S5 BG/ASCs, icariin/45S5 BG seeded with ASCs). Evaluation after 12 wk	Treatment with icariin was optimal in promoting VEGF secretion from ASCs, and it was hypothesized to promote angiogenesis of rat EPCs. This suggests a paracrine role for VEGF in mediating the interaction between icariin-induced ASCs and EPCs
Westhauser et al[37], 2016	To evaluate the bone formation potential of three different types of hBMSC-seeded polymer-coated 45S5 BG scaffolds in 3D using standardized protocols	In vitro and in vivo	Three types of 3D-polymer coated 45S5 BG scaffolds: Group A - scaffold coated in 5% w/v gelatin solution, (50 °C). Group B - scaffold coated in 5% w/v cross-linked gelatin-genipin (99:1) solution (50 ºC) Group C - scaffold coated in 5% w/v PHBV solution (room temperature)	Each group (A-C) had four identical scaffolds differing only in the type of polymer coating. Scaffolds had a nominal size of 5 × 5 × 5 mm and were implanted subcutaneously on the back above the upper and lower extremities of three female SCID mice. Evaluated 8 wk after surgery. hBMSCs from human bone marrow aspirate were seeded onto each scaffold	All groups exhibited bone formation and good infiltration of connective tissue cells, as well as a dense vascularization network. A-group showed a greater amount of bone. C-group, and especially B-group, exhibited a high dissolution. Both B- and C-groups showed more singular bone formation with no signs of interconnectivity
Moreira et al[38], 2018	To evaluate the effect of low-intensity laser therapy (LLLT) on the healing of bone defects filled with autogenous bone or 45S5 BG	In vivo	45S5 BG Biogran® Biomet 3i	A 5 mm diameter CSD was created on the calvaria of sixty adult male rats were divided into six groups (n = 10): group C (control, blood clot); group LLLT (LLLT-GaAlAs, wavelength of 780 nm, power of 100mW, energy density of 210 J/cm2 per point for 60 seconds/point, in five points, only once, after creation of the surgical defect); group AB (autogenous bone); group AB+LLLT (autogenous bone + LLLT); group BG (45S5 BG); group BG+LLLT (45S5 BG + LLLT). Evaluation after 30 d	The highest ANFB was recorded in the LLLT group (47.67% ± 8.66%), followed by the AB+LLLT (30.98% ± 16.59%) and BG+LLLT (31.13% ± 16.98%) groups. There was a statistically significant difference in ANFB values between group C and the other groups, except for the BG group (P > 0.05). There was no statistically significant difference in ANFB values between group AB and the other groups, between group AB+LLLT and groups BG and BG+LLLT, and between groups BG and BG+LLLT. The highest area of remaining particles was found in the BG group (25.15% ± 4.82%), followed by the BG+LLLT group (17.06% ± 9.01%), and there was no significant difference between the groups

Nb₂O₅: Niobium pentoxide; 3D: Three-dimensional; micro-CT: Micro-computed tomography; BM-MSCs: Bone marrow-derived mesenchymal stem cells; hESCs: Human embryonic stem cells; CSD: Critical size defects; DMSO: Dimethyl sulfoxide; CBCT: Cone-beam computed tomography; VGi: Grayscale value in; ROI: Region of interest; CSC: Composite bone cement; CPC: Calcium phosphate cement; BGNb: Nb-containing bioactive glasses; BMMSCs: Bone marrow-derived mesenchymal stem cells; ACL: Anterior cruciate ligament; BPTB: Bone-patellar tendon-bone; PEUR: Poly(ester urethane); nHA: Nanocrystalline hydroxyapatite; BioS-2P: Biosilicate^® containing two crystalline phases; MSCs: Mesenchymal stromal cells; hMSC: Human mesenchymal stem cells; MTT: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; SCID: Severe combined immunodeficiency; ASCs: Adipose-derived stem cells; EPCs: Endothelial progenitor cells; VEGF: Vascular endothelial growth factor; PHBV: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); LLLT: Low-intensity laser therapy; ANFB: Newly formed bone; GaAlAs: Gallium-aluminum-arsenide.