Richard K. Brow

Bioactive glasses represent an emerging strategy for localized skeletal muscle repair. Here, we investigated whether an injectable borophosphate bioactive glass [cobalt oxide–time release ion matrix (CoO-TRIM)] can accelerate skeletal muscle regeneration and functional recovery following acute injury. Using a barium chloride (BaCl₂) injury model, muscle function, myogenesis, angiogenesis, and inflammation were evaluated in mouse tibialis anterior (TA) and extensor digitorum longus (EDL) muscles at 8 and 14 days post-injury (dpi). Mice were randomly assigned to Untreated, Saline-treated, and CoO-TRIM-treated groups. CoO-TRIM accelerated the regenerative process, with increased myofiber size and restoration of TA contractile function to values not significantly different from Healthy muscle by 14 dpi compared to Injured Untreated and Saline-treated muscles. Vascular endothelial growth factor (VEGF) concentrations were elevated through 14 dpi following CoO-TRIM treatment and accompanied by enhanced muscle vascularity in both TA and EDL muscles. CoO-TRIM modulated the inflammatory response, with increased inflammatory signaling during active regeneration at 8 dpi, followed by an accelerated transition toward a pro-regenerative environment characterized by increased CD163⁺ M2 macrophages and sustained IL-10 signaling. In contrast, CoO-TRIM produced no detectable effects in healthy contralateral muscles. These findings support ion-releasing bioactive glass as a cell- and growth factor-free strategy for skeletal muscle repair.

Skeletal muscle myopathy remains a significant cause of disability with limited treatment strategies. Advancements in tissue engineering have led to the development of borophosphate bioactive glasses (BPBGs) capable of enhancing skeletal muscle structure and function. Using a mouse model of severe myopathy (D2.mdx), we investigated muscle force, regeneration, angiogenesis and inflammation at 14, 70 and 140 days post-treatment (dpt). Tibialis anterior (TA) muscles of D2.mdx mice that received a single injection of cobalt oxide-doped BPBG (CoO-TRIM) particles exhibit greater active force, myofiber size, and regeneration through 70 dpt compared to control D2.mdx mice injected with Saline. Vascular endothelial growth factor (VEGF) was elevated up to 70 dpt in D2.mdx CoO-TRIM mice followed by increased muscle vascularity. As a marker of inflammation, interleukin (IL)-6 increased in D2.mdx CoO-TRIM mice compared to D2.mdx Saline controls at 14 dpt, with no differences at 70 or 140 dpt. No differences were observed in outcome measures between wild-type (WT) CoO-TRIM mice and WT Saline controls. We report that CoO-TRIM can stimulate VEGF production and promote restoration of muscle structure and function when inflammation is present. Local injection of an inorganic biomaterial alone can benefit myopathic skeletal muscle.

Introduction: Skeletal muscle injury remains a significant cause of disability with limited treatment options. Here we report accelerated skeletal muscle regeneration following injection of an inorganic biomaterial alone, cobalt oxide time-release ion matrix (CoO-TRIM). Methods: The tibialis anterior (TA) muscle of adult C57BL/6J mice was injured with 70 µL of barium chloride, with uninjured limbs serving as contralateral controls. Following the acute inflammatory phase (3 days post injury, dpi), mice were randomly separated into three groups: untreated controls, 70 µl sterile saline injection alone (vehicle control) or 70 µl CoO-TRIM (5 µg/µl) and evaluated at 8- and 14 dpi. Results: 14 dpi, injured TA muscles receiving a single injection of CoO-TRIM exhibited greater recovery of maximal force production (means ± SEM: Healthy; TRIM, 31.8 ± 0.6 N/cm ³ ; Untreated, 31.7 ± 0.8 N/cm ³ ; Saline, 31.6 ± 0.8 N/cm ³ , Injured; TRIM, 31.8 ± 0.8 N/cm ³ ; Untreated, 26.1 ± 1.1 N/cm ³ , P= vs. TRIM Injured; Saline, 26.0 ± 1.3 N/cm ³ , P= vs. TRIM Injured), increased fiber size (Healthy; TRIM, 2466 ± 128 µm ² vs. Untreated, 2351 ± 131 µm ² ; vs. Saline, 2460 ± 129 µm ² , Injured; TRIM, 2179 ± 63 µm ² vs. Untreated, 1525 ± 52 µm ² , P= vs. Saline, 1470 ± 99 µm ² , P= + fibers/mm ² vs. Untreated, 91.8 ± 11.8 eMyHC ⁺ fibers/mm ² , P= vs. Saline, 94.0 ± 9.6 eMyHC ⁺ fibers/mm ² , P= vs. Untreated, 10.3 ± 1.1 pg/mg, P= 0.04; Saline, 5.5 ± 1.2 pg/mg, P= 2 /fiber vs. Untreated, 94.9 ± 4.2 µm ² /fiber, P= 2 /fiber, P= vs. Untreated, 12.4 ± 1.5 pg/ml, P= 0.02; Saline, 11.7 ± 0.8 pg/mg, P= 0.01), with early resolution of degenerative inflammatory cytokines and elevated regenerative cytokines 14 dpi (IL-10; SEM: TRIM, 2.4 ± 0.1 pg/ml vs. Untreated, 2.1 ± 0.1 pg/ml, P= 0.25; Saline, 2.0 ± 0.1 pg/ml, P= 0.04). No differences were observed in healthy contralateral limbs following treatment with CoO-TRIM compared to healthy control mice at 8- and 14 dpi. Discussion: This work suggests CoO-TRIM enhances muscle regeneration following injury, possibly through an immune cell-mediated mechanism. Statement of Clinical Relevance: This is the first report of an injected, inorganic biomaterial alone to accelerate regeneration of injured skeletal muscle with no observed effects on healthy muscle. These findings suggest enhancements to skeletal muscle regeneration following CoO-TRIM treatment may be immune cell mediated with an increased local inflammatory response and subsequent improvements to muscle microvasculature and myogenic regulatory factors.

We report on the electrical conductivity of barium iron phosphate glass, a candidate for radioactive waste encapsulating glass. The glass composition has been optimized for slow dissolution in hot water because the precipitation of a stable hydration layer of FeO(OH) on the glass surface enhances the water durability of the glass. The mechanism of electrical conductivity of the glass is assumed to be an electron hopping between the electronic energy levels of Fe (II) and Fe (III) ions, obeying the electron carrier generation of Fe (II) → Fe (III) + e ⁻ . The electrical conductivity of the base glass was ∼3.2 × 10 ⁻⁷ to ∼5.0 × 10 ⁻⁴ S/cm, from 200 to 550°C, whereas samples with surface hydration layers after the water durability tests had conductivities one order of magnitude lower than the base glass sample. The electrical conductivity of a crystallized sample was ∼0.8 × 10 ⁻⁷ S/cm at 400°C. X‐ray photoelectron spectroscopy revealed the existence of Fe (II) and Fe (III), and P−O−Fe (Ba) and P−O−P bonds, and it was found that electrical conductivity increased in the order of increasing Fe (II)/Fe (III) ratio for the bulk, the corroded, and the crystallized samples.

The atomic structures of Zn and Na borophosphate glasses were studied using X-ray and neutron scattering techniques. Peaks assigned to the B−O, P−O, and O−O distances confirm that only BO₄ units co-exist with the PO₄ tetrahedra. The Zn−O and Na−O coordination numbers are found to be a little larger than four. The narrowest peaks of the Zn−O first-neighbor distances exist for the glasses along a line connecting the Zn(PO₃)₂ and BPO₄ compositions (50 mol% P₂O₅), which is explained by networks of ZnO₄, BO₄, and PO₄ tetrahedra with twofold coordinated oxygens. The calculated amounts of available oxygen support this interpretation. Broadened peaks occur for glasses with lower P₂O₅ contents, which is consistent with the presence of threefold coordinated oxygens. The two distinct P−O peak components of the Zn and Na borophosphate glasses differ in their relative abundances. This is interpreted as follows: Na⁺ cations coordinate oxygens in some P−O−B bridges, which is something not seen for the Zn²⁺ ions.

The two-point bend (TPB) technique was used to measure the failure strains of freshly-drawn xNa2O.(100−x)B2O3 glass fibres under liquid nitrogen. In such experiments, the effects of extrinsic flaws and environmental fatigue are minimised so that intrinsic failure characteristics might be determined and related to glass composition and structure. The inert failure strain for pure B2O3 glass ranges between 29 and 36%; to our knowledge this is the largest inert failure strain ever reported for an oxide glass in a TPB experiment. Failure strains decrease systematically with increasing Na2O content. The addition of Na2O increases the dimensionality and connectivity of the borate glass structure, increasing Young’s elastic modulus and hence increasing resistance to deformation before failure. Similar correlations between inert failure strain and network connectivity have been reported for silicate and aluminosilicate glasses, and may be related to the resistance to crack initiation of the glass network.

Silicate and borate bioactive glasses have been reported to create alkaline conditions by rapidly releasing ions when reacting in aqueous solution. At certain levels, this alkaline solution can negatively affect cell viability. Adding phosphate ions to the glass composition can control the degradation rate of bioactive glasses and create a neutral pH environment. This study evaluated a series of borophosphate bioactive glasses (BPBGs) with nominal molar compositions 16Na₂O-24CaO-xB₂O₃-(60-x)P₂O₅, where x = 0, 40, or 60. The phosphate (X0) glass (PBG) produced an acidic solution when dissolved in water; the borate (X60) glass (BBG) produced an alkaline solution, and the BPBG glass produced a pH-neutral solution. These three glasses were evaluated using adipose stem cells (ASCs), a cell population known for their therapeutic abilities. The effects of each glass on the pH of cell culture, ions released during degradation, and on ASC functions, including viability, migration, angiogenic ability, differentiation, and protein secretions, were evaluated. The X40 BPBG created a physiologically neutral pH in cell culture media after 24 h. The X0 phosphate glass promoted ASC migration, while the highly alkaline X60 borate increased the angiogenic ability of ASCs. These results indicate that BPBG can be used safely in cell culture studies and customized for specific biomedical applications.

The atomic structures of Zn and Na borophosphate glasses were studied by X-ray and neutron diffraction. Peaks assigned to the B−O, P−O, and O−O distances confirm that only BO4 units co-exist with the PO4 tetrahedra. The Zn−O and Na−O coordination numbers are found to be a little larger than four. The narrowest peaks of the Zn−O first-neighbor distances exist for the glasses along a line connecting the Zn(PO3)2 and BPO4 compositions (50 mol% P2O5) which is explained by networks of ZnO4, BO4, and PO4 tetrahedra with twofold coordinated oxygens. Calculated amounts of the available oxygen support this interpretation. Broadened peaks occur for glasses with lower P2O5 contents, consistent with the presence of threefold coordinated oxygens. The two distinct P−O peak components of the Zn and Na borophosphate glasses differ in their relative abundances which is interpreted by Na+ cations that coordinate oxygens in some P−O−B bridges, something not seen for the Zn2+ ions.

Borate bioactive glasses have gained attention in recent years due to their therapeutic and regenerative effects in vivo. However, borate bioactive glasses release alkaline ions, increasing the local pH and creating a toxic environment for cell culture studies. A partial compositional substitution of phosphate for borate can create a pH‐neutral glass that does not significantly affect the local pH while still releasing therapeutic ions. In the present study, a series of Na‐Ca‐borophosphate bioactive glasses with different borate‐to‐phosphate ratios was evaluated in vitro and in vivo for cytotoxicity and angiogenic effects. Compared to more basic borate glasses, the pH‐neutral glasses supported endothelial cell migration and stimulated greater blood vessel formation in a chick chorioallantoic membrane model. The results from this study indicate that these pH‐neutral glasses are promising angiogenic biomaterials for use in tissue engineering and regenerative medicine.

Hollow biphasic calcium phosphate (BCP) microspheres with different fractions of nanocrystalline hydroxyapatite (HA) and β‐tricalcium phosphate (β‐TCP) were prepared, and their in vitro and in vivo reactivity were assessed. The microspheres were prepared by reacting soluble borate glass microspheres in phosphate solutions (0.1‐ or 0.25‐M K ₂ HPO ₄ ) with different values of pH (7–12) to produce hollow calcium‐deficient hydroxyapatite (CDHA) microspheres with Ca/P ratios that increased with solution pH. Heating the hollow CDHA microspheres to 800°C produced hollow, nanoporous BCP microspheres with ratios of nanocrystalline HA and β‐TCP that correspond to the Ca/P ratio of the CDHA precursors. Hollow BCP microspheres with a greater fraction of β‐TCP released calcium ions most quickly in a K‐acetate acidic buffer solution, consistent with their greater bioactivity in the in vivo tests. CDHA and BCP microspheres converted to nanocrystalline carbonated HA when immersed in 37°C simulated body fluid. Hollow CDHA and BCP microspheres were implanted in rat calvarial defects, and after 8 weeks, the BCP microspheres with a β‐TCP/HA ratio of 70/30 (by weight) promoted a greater fraction of new bone formation (33% ± 7%) compared to BCP microspheres with a β‐TCP/HA ratio of 44/56 (20% ± 9%) and compared to the respective CDHA precursor microspheres (17% ± 4%, 17% ± 5%). Bone formation was not observed in the subcutaneous implants of any types of these hollow microspheres, after 8 and 12 weeks, and the more reactive BCP microspheres (β‐TCP/HA = 70/30) were associated with significantly more blood vessel formation in the subcutaneous implants. These results indicate that the properties of hollow BCP microspheres can be tailored by their processing conditions and are useful as scaffold materials for tissue regeneration.