Bioactive glass compositions and methods of treatment

Abstract

Compositions and methods are provided for improving the regeneration of soft tissue as a result of injury or disease. Various compositions are disclosed, including bioactive glass compositions resulting from calcining a reactant composition. Also disclosed is a method for treating injured or diseased skeletal muscle, comprising contacting the injured or diseased skeletal muscle with a bioactive glass composition.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 260,858, filed September 2, 2021, which is incorporated by reference in its entirety.

[0002] Compositions and methods are provided for improving the regeneration of soft tissue as a result of injury or disease. Specifically, bioactive glass compositions are described for contacting and treating tissue. [Background technology]

[0003] Acute trauma is the leading cause of death and disability in the United States. Military personnel can be injured in combat, while civilians suffer debilitating falls, vehicle accidents, and mechanical injuries. Injuries involving damage to multiple tissue components can result in complications including ischemia, denervation, and necrosis. Advances in surgical techniques have increased the prevalence of tissue regeneration, but half of affected patients still remain severely disabled seven years after surgery. Soft tissue disorders such as muscular dystrophies can also be subject to repetitive injury as a result of fragile muscle tissue.

[0004] In humans, skeletal muscles account for approximately 40% of body weight, facilitate thermoregulation, and generate force to maintain respiration and locomotion. Located throughout the body, skeletal muscles are vulnerable to injury from trauma in automobile accidents, penetrating wounds, surgical repair, and overuse. Skeletal muscles have a robust regenerative response due to a population of quiescent muscle stem cells (satellite cells) associated with mature skeletal muscle fibers, located between the sarcolemma and the basement membrane. After injury, satellite cells activate, proliferate, and differentiate into myoblasts, which then fuse into new myotubes or to the ends of damaged muscle fibers. Skeletal muscles are capable of regeneration, but with limitations. Specifically, when injuries are too severe, such as those in muscle volume loss (VML, defined as more than 20% muscle mass loss), the muscle does not regenerate, resulting instead in irreversible scarring, fibrosis, and loss of function. Advances in clinical practice have improved patient outcomes with tissue transplants, including autografts, allografts, and xenografts. Even so, regenerative strategies have limitations (immunological rejection and inflammation). In addition to VML, irreversible scarring, fibrosis, and loss of function have been observed in patients with muscular dystrophies.

[0005] For example, Duchenne muscular dystrophy (DMD) is a recessive chromosomal mutation of the gene dystrophin on the x chromosome, affecting 1 in 5,000 men. In healthy muscle, dystrophin is responsible for maintaining the integrity, flexibility, and stability of the sarcolemma by anchoring the intracellular F-actin cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex. In DMD, dystrophin deficiency leads to sarcolemma damage, for example, by contractile forces, especially eccentric (elongational) contractions (e.g., descending stairs), resulting in increased permeability of myofibrils to ions and small molecules. Therapeutic approaches have focused on two methods: 1) restoring the gene dystrophin (or a dystrophin surrogate molecule) or 2) alleviating the secondary consequences caused by dystrophin deficiency. Although FDA-approved and developmental therapies have therapeutic potential, they also suffer from many drawbacks, including disappointing increases in dystrophin protein without improvement in muscle function (less than 1% for FDA-approved gene editing drugs Vyondys and Exondys), and collateral consequences of systemic frontline medication. Mutational therapies are only approved in 15% of patients. Corticosteroids can only help slow the progression of DMD. Children with DMD show limb muscle dysfunction as early as 2 years of age due to repeated muscle tearing, which progresses to immobility by about 15 years of age as the muscles are unable to keep up with the constant injury resulting from the weakening of the myofibrillar structure. This reduced mobility is the main burden cited by families of children with DMD. Such debilitating muscle injuries and diseases have accelerated the need for biomimetic scaffolds that lead to skeletal muscle regeneration.

[0006] Therefore, novel approaches to improve soft tissue regeneration are urgently needed. Summary of the Invention

[0007] Various compositions are disclosed herein, including bioactive glass compositions resulting from calcining a reactant composition comprising about 10 wt. % to about 40 wt. % BO, about 15 wt. % to about 40 wt. % PO, about 10 wt. % to about 25 wt. % CaO, about 5 wt. % to about 20 wt. % NaO, and optionally about 2 wt. % to about 10 wt. % CoO, about 0.5 wt. % to about 2 wt. % ZnO, about 0.1 wt. % to about 1 wt. % CuO, or combinations thereof.

[0008] Also disclosed herein are various methods, including a method of treating injured or diseased skeletal muscle, comprising contacting the injured or diseased skeletal muscle with an effective amount of any of the bioactive glass compositions described herein.

[0009] The present disclosure further relates to a method of treating injured or diseased brain or neural tissue comprising contacting said injured or diseased brain or neural tissue with an effective amount of any of the bioactive glass compositions described herein.

[0010] Other objects and features will be in part apparent and in part pointed out hereinafter. [Brief description of the drawings]

[0011] [Figure 1A] Microvessels analyzed after in vivo image collection are shown. The area of ​​2 mm punch injury is indicated by the black circle. Scale bar = 1 mm. Microvessels treated with CON (saline vehicle-treated / control) are shown. [Figure 1B] Microvessels analyzed after in vivo image collection are shown. The area of ​​2 mm punch injury is indicated by the black circle. Scale bar = 1 mm. Microvessels treated with TRIM (slow-release ionic matrix) (CoO) are shown. [Figure 1C]The percentage of the injury occupied by blood vessels is shown for saline vehicle-treated / control (CON) and sustained release ionic matrix (TRIM) (CoO), indicating that regeneration within the injury site does not appear to differ between CON and TRIM (CoO), as indicated by the larger mean bar. [Figure 1D] Shown is an overlap of confocal images showing fluorescent blood vessels in the gluteus maximus (GM) muscle at 21 dpi. Images were taken from the epicenter of the injury. Scale bar = 400 μM. Treatment with CON is shown. [Figure 1E] Shown is an overlap of confocal images showing fluorescent blood vessels in the gluteus maximus (GM) muscle at 21 dpi. Images were taken from the epicenter of the injury. Scale bar = 400 μM. Treatment with TRIM (CoO) is shown. [Figure 1F] Quantification of the percentage of blood vessels in the injury for CON and TRIM (CoO) is shown, revealing that the mean microcirculatory density (percentage of blood vessels in the injury) is higher at the injury site for CON than for TRIM. [Figure 2A] Figure 1 shows an overlap of confocal images showing myofibrils in GM muscle (see Figure 1A-F) at 21 dpi taken from the injury epicenter. Myofibrils in GM that received saline (CON) were sparse and disorganized. Large gaps (black) were absent from myofibers within the injury site. Bar = 400 μM. Treatment with CON is indicated. [Figure 2B] Overlap of confocal images showing myofibrils in GM muscle (see Figure 1A-F) at 21 dpi taken from the injury epicenter. Bar = 400 µM. Treatment with TRIM (CoO) is indicated. [Figure 2C] The percentage (%) of regenerated myofibrils in CON and TRIM(CoO) treatments is shown, and it is clear that mice treated with TRIM(CoO) had an increased average myofibril density compared to CON. [Figure 2D] A cross section of GM showing laminin borders and myofibrillar nuclei. The cross section contains upper and lower healthy muscle with a central injury (dotted circle). Scale bar = 200 μM. Treatment with CON is shown. [Figure 2E]A cross section of GM showing laminin borders and myofibrillar nuclei. The cross section contains upper and lower healthy muscle with a central injury (dotted circle). Scale bar = 200 μM. Treatment with TRIM (CoO) is shown. [Figure 2F] It is clear that after CON and TRIM treatment, muscle recovery in cross-section was observed, with the mean muscle thickness for TRIM approaching 1, while the mean muscle thickness for CON averaged approximately 0.75. [Figure 3A] Exemplary images of mouse GM cross sections are shown. After injury, muscles were treated with sham saline (S) or TRIMCuZn (BPCuZn) at 3 days post injury (dpi) and examined at 8 dpi. DAPI staining shows the central nuclei of regenerating myofibrils and laminin labels the borders of the cells (top). Co-staining of laminin and eMyHC is also shown (bottom). Scale bar = 100 pm and applies to all images. [Figure 3B] The percentage of eMyHC positive fibers is shown for sham saline treatment (S) at 8 dpi and for TRIMCuZn (BPCuZn) at 0 and 8 dpi. Loss of embryonic myosin heavy chain (eMyHC) indicates that TRIMCuZn accelerates myofibril maturation and improves angiogenesis. n=2-3 / group, data are reported as mean and SEM. [Figure 3C] The percentage of regenerating fibers is shown for sham saline treatment (S) at 8 dpi, and for TRIMCuZn (BPCuZn) at 0 and 8 dpi. [Figure 4A] Exemplary images and wet weights of TAs in 7-month-old mice 14 days after untreated or Dystrophix (CoO) injection are shown. The Dystrophix-treated TA had a larger mass than the three untreated TAs. [Figure 4B] Exemplary images and weights of wet EDL in 7-month-old DBA mice 14 days after untreated or Dystrophix (CoO) injection. Dystrophix-treated EDLs had greater mass than the untreated three. [Figure 4C]Exemplary images of TA cross sections from untreated and dystrophin-treated DBA mice are shown. Embryonic myosin heavy chain (eMyHC) is less abundant in dystrophin-treated muscles, which also have more central nuclei compared to untreated. Myofibril borders were labeled with laminin. Scale bar = 250 μm. [Diagram 5] The percentage of peak force after injury following treatment with saline, BP, or Dystrophix (CoO) is shown. Maximal tetanus was recorded 14 days after treatment of the left TA of mdx mice with either saline, BP, or Dystrophix (CoO). After a short break, the TA was subjected to three eccentric contractions with rest periods between them. Maximal tetanus was recorded again to measure what percentage of the initial maximum tetanic force could be elicited after eccentric injury. [Figure 6A] Exemplary images of fibers stained with CD31 for untreated 0 dpi, BPCuZn 0 dpi, 8 dpi, and BPCuZn 8 dpi samples are shown. Scale bar = 200 μm. [Figure 6B] Microvascular area / fiber is shown as a percentage of control for untreated 0 dpi, BPCuZn 0 dpi, 8 dpi, and BPCuZn 8 dpi samples, n=2-3 / group. [Figure 7A] Exemplary samples of saline and dystrophin-treated fibers stained with laminin, MyHC, and DAPI are shown. [Figure 7B] The relative frequency of myofibril cross-sectional area for saline and dystrophic treated mice is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Corresponding reference characters indicate corresponding parts throughout the drawings.

[0013] A method for in vivo regeneration using inorganic biocompatible ceramics (biocompatible glasses) in the form of powder suspended in an inert solution (e.g., sterile 0.9% saline) prior to injection into the injury site to improve local tissue scaffolding and repair response has been developed. Candidate biocompatible glass compositions have shown similar beneficial effects on skeletal muscle structure and function in healthy mice injured by needle biopsy as well as diseased dystrophic mice. Such improvements in muscle regeneration and dystrophic muscle structure and function may be primarily due to boron phosphate particles, but may also depend on other additives, such as CoO (which may improve hypoxia-inducible factor 1a), CuO (which may be angiogenic), and ZnO (which may be anti-inflammatory). Previous experiments have shown that both borate-based and phosphate-based glasses provide attachment and structural support for bone and tooth enamel through the formation of a calcium phosphate layer on the glass surface. The biocompatible glass of the present invention is made by combining borate and phosphate in a ratio that slows the dissolution rate at neutral pH without affecting the local pH. It is believed to form a calcium phosphate layer that acts as a "biomimetic microscaffold" for damaged and diseased myofibrils. This effect can be localized to the extracellular glycoprotein portion of the dystrophin-glycoprotein complex, which can substitute for dystrophin to stabilize myofibrillar structure. When injected locally into the fascial compartment, it appears to affect all muscles within the compartment, thereby serving as a therapy to maintain myofibrillar integrity and physical mobility in patients with muscle injury or muscular dystrophy.

[0014] The sustained release ionic matrix (TRIM) is a borate phosphate-based amorphous non-crystalline solid (bioactive glass) containing cobalt ions. When ground into powder, suspended in solution, and injected into damaged soft tissue (by trauma or disease), the material appears to significantly increase the rate of soft tissue regeneration. Other bioactive glass compositions have been used to treat muscle volume loss (e.g., borate aluminate glass powder). However, no other compositions have been shown to stimulate regeneration of injured skeletal muscle, dystrophic muscle, blood vessels, or peripheral nerves. It is reported herein that injured, normal (non-diseased) skeletal muscle, as well as dystrophic skeletal muscle, can improve both size and quality after TRI matrix treatment. Additional applications may include the brain and peripheral nerves to regenerate soft tissues by a hypoxia-mimicking pathway.

[0015] Various compositions are disclosed herein, including bioactive glass compositions resulting from calcining a reactant composition comprising about 10 wt. % to about 40 wt. % BO, about 15 wt. % to about 40 wt. % PO, about 10 wt. % to about 25 wt. % CaO, about 5 wt. % to about 20 wt. % NaO, and optionally about 2 wt. % to about 10 wt. % CoO, about 0.5 wt. % to about 2 wt. % ZnO, about 0.1 wt. % to about 1 wt. % CuO, or combinations thereof.

[0016] The reactant composition can include about 10% to about 40% by weight B2O3, about 15% to about 40% by weight P2O5, about 10% to about 25% by weight CaO, about 5% to about 20% by weight Na2O, and about 2% to about 10% by weight CoO.

[0017] The reactant composition can include about 33% to about 37% by weight B2O3, about 33% to about 37% by weight P2O5, about 13% to about 18% by weight CaO, about 11% to about 14% by weight Na2O, and about 3% to about 5% by weight CoO.

[0018] The reactant composition can include about 30% to about 40% by weight B2O3, about 20% to about 40% by weight P2O5, about 10% to about 20% by weight CaO, about 11% to about 18% by weight Na2O, and about 3% to about 10% by weight CoO.

[0019] The reactant composition can include about 30% to about 40% by weight B2O3, about 30% to about 40% by weight P2O5, about 10% to about 20% by weight CaO, about 10% to about 15% by weight Na2O, about 0.5% to about 2% by weight ZnO, and about 0.1% to about 1% by weight CuO.

[0020] The reactant composition can include about 33% to about 37% by weight B2O3, about 33% to about 37% by weight P2O5, about 13% to about 18% by weight CaO, about 11% to about 14% by weight Na2O, about 0.8% to about 1.2% by weight ZnO, and about 0.3% to about 0.5% by weight CuO.

[0021] The reactant composition can include about 33% to about 37% by weight B2O3, about 33% to about 37% by weight P2O5, about 13% to about 18% by weight CaO, and about 11% to about 14% by weight Na2O.

[0022] Calcination can be carried out by heating the reactant composition below the melting temperature of the reactant composition. The calcination temperature can be from about 800° C. to about 1300° C., or from about 1000° C. to about 1150° C. The reactant composition can further include phosphoric acid.

[0023] The bioactive glass composition can be used to form calcium phosphate. The bioactive glass composition can maintain a neutral pH upon decomposition, which promotes the formation of calcium triphosphate. This is in contrast to other bioactive glasses, which create an alkaline pH environment that promotes the formation of hydroxyapatite.

[0024] The present disclosure further relates to a method of treating injured or diseased skeletal muscle comprising contacting the injured or diseased skeletal muscle with an effective amount of any of the bioactive glass compositions disclosed herein. The injured or diseased skeletal muscle can have an increased average myofibril area after at least 8 days of treatment with the bioactive glass composition compared to an injured or diseased skeletal muscle receiving an otherwise similar treatment with saline. The injured or diseased skeletal muscle can have a lower embryonic myosin heavy chain (eMyHC) concentration after at least 5 days of treatment with the bioactive glass composition compared to an injured or diseased skeletal muscle receiving an otherwise similar treatment with saline. The injured or diseased skeletal muscle can have an increased muscle mass after at least 10 days of treatment with the bioactive glass composition compared to an injured or diseased skeletal muscle receiving an otherwise similar treatment with saline. The injured or diseased skeletal muscle may have an increase in muscle peak force after at least 10 days of treatment with the bioactive glass composition compared to an injured or diseased skeletal muscle receiving an otherwise similar treatment with saline.The injured or diseased skeletal muscle may have an increase in vascularization after at least 5 days of treatment with the bioactive glass composition compared to an injured or diseased skeletal muscle receiving an otherwise similar treatment with saline.

[0025] The injured or diseased skeletal muscle can be an injured skeletal muscle. The injured skeletal muscle can be a pulled muscle, a traumatically injured muscle, a torn muscle, an injured muscle resulting from muscle overuse or misuse, or a combination thereof. An injured muscle resulting from muscle overuse or misuse may be the result of a sports injury.

[0026] The injured or diseased skeletal muscle can be diseased skeletal muscle. The diseased skeletal muscle can be dystrophic skeletal muscle, cachectic skeletal muscle, sarcopenic skeletal muscle, or a combination thereof.

[0027] The present disclosure further relates to a method of treating injured or diseased brain or neural tissue comprising contacting said injured or diseased brain or neural tissue with an effective amount of any of the bioactive glass compositions disclosed herein.

[0028] For any of the methods disclosed herein, the bioactive glass composition is capable of maintaining a neutral pH upon decomposition.

[0029] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims. EXAMPLES

[0030] To further illustrate the present invention, the following non-limiting examples are provided.

[0031] overview Skeletal muscles are subject to injury from trauma in car accidents, penetrating wounds, surgical repair, and overuse. Skeletal muscles are capable of regenerating, but there are limitations. Specifically, when injuries are too severe, such as in muscle volume loss (VML, defined as more than 20% muscle mass loss), the muscle does not regenerate, resulting instead in irreversible scarring, fibrosis, and loss of function. Furthermore, Duchenne muscular dystrophy (DMD) results in loss of muscle regenerative capacity due to repeated myofibril ruptures. Biomaterials have shown promising muscle regeneration promotion after VML.

[0032] Experiments were performed to test whether a biomaterial sustained-release ionic matrix (TRIM) enhances skeletal muscle microvascular and myofibrillar regeneration after chemical injury, VML, and in conditions of DMD. Chemical injury was induced using a biopsy punch (2 mm diameter) to induce VML and BaCl2, both in the left gluteus maximus (GM) of female C57BI / 6 and Cdh5-mTmG mice (4-5 months old). Dystrophic mice [n=2, mdx + / +Mice (3-5 months old) were used for eccentric injury experiments after a single injection of TRIM or vehicle into the left tibialis anterior (TA) muscle, whereas DBA mdx Mice [n=1, (7 months old)] were used as a model of severe dystrophy to determine whether TRIM can restore TA muscle mass. Mice were divided into two groups: saline vehicle-treated (CON) or TRIM-treated. For treated mice, 250 μg of TRIM powder was suspended in 0.9% sterile saline (5 μg / pL) and 70 μL of TRIM solution was injected under the GM 3 days post-injury (dpi) for BaCl2 injury and 7 dpi for VML injury, as well as 50 μL injected into the TA of dystrophic mice 10 days prior to data collection. For CON mice, 70 μL of 0.9% saline (no TRIM) was injected into the GM as a negative control at 7 dpi, while 50 μL was injected into the TA of dystrophic mice. Muscles were evaluated by intravital microscopy, confocal microscopy, and histological sections. Intravital microscopy revealed no differences between TRIM or CON with respect to the area of ​​injury occupied by blood vessels, but confocal z-stacks suggest that TRIM reduced vascular density within the injured area. In contrast, both confocal z-stacks and muscle cross-sections suggest that TRIM improved myofibrillar regeneration in all treated mice, as well as the resistance of dystrophic muscle to injury. This finding suggests that TRIM treatment may be beneficial for myofibrillar regeneration after chemical injury of skeletal muscle, VML injury, and in muscular dystrophic conditions.

[0033] Example 1: Materials and Methods The following materials and methods were used in all remaining examples.

[0034] animal Mice were chosen because skeletal muscle structure and function are conserved across species, and the invasive nature of these experiments precludes experimentation in humans. All experiments were approved by the University of Missouri Animal Care and Use Committee. Male C57BI / 6 mice (n=3, approximately 4 months old) were selected for chemical injury and TRIM treatment. Female Cdh5-mTmG (endothelial cell green fluorescent protein (GFP) reporter, approximately 4 months old) were used to visualize microvascular regeneration after muscle volume loss injury (VML). An endothelial-specific cre recombinase Cdh5 Cre-ERT2 (Wang, Y., et al. (2010). Nature, 465 (7297)) was selected and crossed with the mTmG reporter to generate a valid endothelial cell (EC) reporter mouse (Muzumdar, D., et al. (2007). Genesis, 45 (9), 593-605.). Mice were divided into two groups: saline vehicle-treated (CON) (n=3) or biocompatible ceramic [sustained release ionic matrix (TRIM)]-treated (n=2). Mice were maintained under a 12:12-h light / dark cycle, housed in bed cubes, and allowed free access to food and water.

[0035] Two strains of dystrophic mice were used to assess the effect of TRIM on dystrophic muscle: C57BI / 6 mdx+ / + mice (n=2, approximately 4 months old) were used to assess the effect of TRIM on the resistance of dystrophic muscle to injury, while D2.B10 Dmdmdx mice (n=1, 7 months old), which exhibit a more severe muscle phenotype, were used to determine the ability of TRIM to restore dystrophic muscle quality and quantity.

[0036] Tamoxifen injection To induce Cre-ERT2 recombination and eGFP expression in the endothelium, mice were restrained by trained personnel and injected intraperitoneally with 100 μL of tamoxifen solution (1 mg tamoxifen + 5% ethanol in corn oil) at 27 gauge for three consecutive days as reported (Biomimetic Bioactive Biomaterials: The Next Generation of Implantable Devices. (2017). ACS Biomaterials Science & Engineering, 3(7), 1172-1174.). All mice were tested 7 days after the first tamoxifen injection.

[0037] Formation of sustained release ionic matrix (TRIM) TRIM is produced by mixing the dry, powdered components and placing them in a platinum crucible.

[0038] For these experiments, three different compositions of TRIM were used.

[0039] The compositions of the candidate materials for the biocompatible glass particles are as follows (values ​​are in weight percent):

[0040] CoO:34.6% B2O3, 35.3% P2O5, 14.0% CaO, 12.3% Na2O, 3.8% CoO

[0041] BPCuZn: 35% B2O3, 35.6% P2O5, 16.2% CaO, 11.8% Na2O, 1% ZnO, 0.4% CuO

[0042] BP:34.9% B2O3, 35.8% P2O5, 16.9% CaO, 12.4% Na2O

[0043] If phosphoric acid was required, it was then slowly stirred into the dry ingredients. The batch was calcined overnight to evaporate the water, then melted (1000-1150°C) for 60 minutes, then stirred with a platinum bar for 30 minutes. The molten TRIM mixture was milled using a Spex mill to form particles smaller than 20 μm. A solution of TRIM particles was made (5 mg / mL in 0.9% sterile saline) and injected as described below.

[0044] BaCl2 for muscle injury and TRIM treatment To induce chemically induced muscle injury in vivo, mice were anesthetized with ketamine and xylazine (100 mg / kg and 10 mg / kg, respectively, intraperitoneal injection), the skin overlying the target muscle was shaved, and then 1.2% BaCl2 was injected unilaterally in the TA [50 μL, (Hench, LL, & Thompson, I. (2010). Journal of The Royal Society Interface, 7(suppl_4), S379-S391.)] or under the GM [75 μL, (Hench, LL, & Polak, JM (2002). Science, 295(5557), 1014)] as described. Mice were kept warm during recovery and then returned to their cages.

[0045] Punch biopsy wounds for VML and TRIM procedures Mice were anesthetized with ketamine / xylazine (100 mg / kg and 10 mg / kg, respectively) IP and kept on an aluminum warming plate to maintain body temperature at 37°C. Additional injections (approximately 20% of the initial) were given as necessary to maintain a steady state of anesthesia, confirmed by the absence of tail or toe withdrawal to pinch, every 15 min. The skin overlying the left GM was shaved and sterilized by swabbing with Betadine (10% povidone-iodine topical solution) followed by three alcohol wipes. Under stereomicroscope guidance, the mouse was placed on its abdomen and an approximately 5 mm incision was made in the overlying skin to access the GM. The exposed tissue was continuously irrigated with physiological saline (PSS). Care was taken not to injure the GM blood vessels. A sterile 2 mm diameter punch biopsy (Anderson, S., et al. (2019). Tissue Engineering Part C: Methods, 25(2), 59-70) was used to inflict local injury on the GM. 2 mm was chosen to represent a model of VML below the critical threshold of non-regenerative muscle loss (Anderson, S., et al. (2019). Tissue Engineering Part C: Methods, 25(2), 59-70). To keep the location of the punch injury constant, a custom-made measuring device, 1 cm long and 0.5 cm wide, was placed along the lumbar spine to provide a reference point on the GM. For VML mice treated with TRIM, 250 μg of powder was suspended in 0.9% sterile saline and then injected under the GM. For CON mice, 7 days after injury (dpi), 70 μL of 0.9% saline (solution) was injected under the muscle to mimic the procedure. The skin incision was closed with 4-5 discontinuous sutures across the skin using sterile 6-0 nylon sutures. For dystrophic mice, 250 μg of powder was suspended in 0.9% sterile saline and then injected into the left tibialis anterior (TA) muscle while the mice were anesthetized and immobilized. Mice were kept warm and monitored until they regained ambulation (2-3 hours), after which they were returned to their cages and observed daily. After data collection at 21 dpi for VML and 14 days for dystrophic mice, mice were killed by cervical dislocation under anesthesia.

[0046] Intravital microscopy of GM To prepare the GM for biomicroscopy (in vivo imaging of the microcirculation) while maintaining the integrity of the vascular supply, mice were anesthetized as described (Fernando, CA, et al. (2019). The Journal of Physiology, 597(5), 1401-1417.). Ketamine / xylazine was injected IP to shave the skin overlying the GM and remove the hair. The mouse was transferred to a warming plate at a temperature of 37°C to maintain body temperature. An incision was made in the overlying skin along the spinal cord with a stereomicroscope. Excess connective tissue and fat were removed using microdissection, avoiding major blood vessels. The exposed GM was continuously irrigated with PSS. The GM was then dissected from its origin along the lumbar fascia, sacrum, and iliac crest and reflected from the body to expose the vascular supply. The GM was then spread on the surface of a clear rubber pedestal and pinned at the edges, approximating its native dimensions. Spreading and fixing the tissue on the pedestal produced a thin, flat preparation suitable for high-resolution imaging of the microvasculature. Saran wrap was used to cover any other exposed tissue to prevent dehydration during in vivo imaging.

[0047] After completion of surgery, the mouse preparation was transferred to the stage of a Nikon 600fn intravital microscope and continuously irrigated with PSS equilibrated with 5% CO2 / 95% N2. Digital images were acquired by imaging the entire punch injury with Piper software using a low-light CMOS FP-Lucy camera (Stanford Photonics) and Long Working Distance (LWD) 4x and 10x objectives (Nikon). After intravital microscopy, the GM was dissected and trimmed to include the area containing the punch injury for confocal imaging, then frozen and sectioned for histology.

[0048] TA muscle strength measurement, and TA and EDL muscle mass The TA was prepared for in situ measurements as described (Wang, Y., et al. (2010). Nature, 465 (7297), 483-486). Briefly, in anesthetized mice, a 2-0 suture was placed around the left patellar tendon. The sciatic nerve was isolated and cut close to the TA for muscle force stimulation via electrodes with a Grass™ stimulator. The distal TA tendon was isolated and secured with a 2-0 suture, then severed from its insertion. Mice were placed prone on a plexiglass plate, and the patellar tendon was secured to a vertical metal peg fixed to the plate. The distal TA tendon was strapped to a load beam (LCL-113G, Omega, Stamford, CT, USA) connected to a Transbridge amplifier (TBM-4, World Precision Instruments, Sarasota, FL, USA). The load beam was attached to a micrometer to adjust the optimal length (Lo) measured during a twitch at 1 Hz (Hench, LL, & Polak, JM (2002). Science, 295(5557), 1014). A piece of KimWipe® was wrapped around the TA, and physiological saline was irrigated into the TA (3 mL min-1), and maximal force was assessed at 120 Hz before and after eccentric contraction injury by Power Lab acquisition software (ADlnstruments, Colorado Springs, CO, USA). Maximal tetanus was assessed after establishing optimal muscle length and performing three warm-up contractions, after which the muscle was stretched approximately 40% during three maximal contraction conditions as described (Muzumdar, D., et al. (2007). Genesis, 45(9), 593-605). After a 2-minute rest, maximal tetanus was obtained again to measure the percentage loss of force after eccentric injury. After data collection, muscle length was measured and both the TA and EDL muscles were removed to assess muscle mass.

[0049] Histochemistry (imaging and analysis) GM specimens were transferred to the stage of a laser scanning confocal microscope to image microvessels and myofibrils. After confocal image collection, optimal cutting temperature (OCT) compound was poured into a shallow cryomold and the excised GM was laid flat and oriented centrally. A 2 mm long silk suture was placed next to the GM in the cryomold to indicate the location of the VML injury and frozen in liquid nitrogen-cooled isopentane. The frozen GM was wrapped in foil, labeled for reference, and stored at -80°C until processed for sectioning.

[0050] Staining of frozen sections Frozen GM and TA sections were cut at 10 μm thickness on a cryostat (HM 550 Cryostat, Thermo Scientific, Waltham, MA), stained for laminin (Thermo Scientific #RB-9024-R7), myosin heavy chain and embryonic myosin (Hybridoma Bank A4.840 s IgM 1:15) as described (Morton, AB, et al. (2019). Redox Biology, 20, 402-413) and fixed with Prolong Gold containing DAPI (Thermo Fisher).

[0051] ImageJ analysis For in vivo imaging, blood vessels were analyzed to assess the amount of microvascular regeneration. For confocal images, both blood vessels and myofibrils were analyzed separately to quantify each component. Images of muscle cross sections were acquired to assess muscle thickness as described below.

[0052] Images acquired with a Nikon 600fn intravital microscope were analyzed with the open access software ImageJ and Microsoft PowerPoint. A reference image of the punch biopsy injury was used to confirm the original size when analyzing both CON and TRIM treated GM. Using PowerPoint, the specimen image was overlaid on the reference image to define the injured area. These images were merged and saved as one TIFF file, including a circle from the PowerPoint tool to outline the injured area (Figure 1A and Figure 1B). After importing into ImageJ, the merged image was converted to 32-bit grayscale. To identify blood vessels within the injured area (A01), a background threshold was established and the images were binary converted, where the background (non-vasculature) was colored white and the tissue components of interest (blood vessels) were colored black. These images were used to measure the extent of myofibrillar regeneration and revascularization, defined as the percentage of total A01 occupied by microvessels.

[0053] Confocal images were acquired with a 10x objective with x0.75 digital zoom on an inverted laser scanning confocal microscope (TCS SP8, Leica Microsystems Buffalo Grove, IL, USA) using Leica LAX software. Image stacks (approximately 70 pm thick) were used to resolve VML morphology using ImageJ software (NIH, open access) (Morton, AB, et al. (2019). Skeletal Muscle, 9(1), 27). After importing into ImageJ, confocal Z-stacks acquired in two color channels were separated into GFP (EC) and TD (Tomato) (myofibrils). Each color channel image was converted to 32-bit grayscale using the thresholding guidelines described above. Areas occupied in black (vessels or myofibrils) were expressed as a percentage of the total A01. Images of vessels and muscles were analyzed separately.

[0054] For confocal image analysis, % vascular area and % muscle area were compared between treatments and across time points. For both analyses, the experimenter was blinded to the experimental groups. The coefficient of variation for the collected data was less than 5%.

[0055] To assess muscle thickness, muscle cross sections were analyzed at five different locations: the edge of the section (thickest, uninjured), the center of the section, and the injured area midway between the center and the edge of the section. The thicknesses of the uninjured edges of the sections were averaged (UT), followed by the average thickness of the injured sections (IT). Final values ​​were calculated using the following formula: IT / UT=muscle recovery. A ratio of 1 indicated recovery of muscle thickness throughout the injured area, while a ratio less than 1 indicated less regeneration.

[0056] Example 2: TRIM does not appear to increase vascular density in the GM after VML Following muscle injury to the gluteus maximus (GM) with a 2 mm biopsy punch, the microcirculation and muscle within the injury site were removed. To nourish and maintain myofibrils, the microcirculation must regenerate after injury. Intravital imaging shows endothelial cell green fluorescent protein (GFP) (Figure 1A and B) at 21 days post injury (dpi). Addition of TRIM at 7 dpi did not appear to alter vascular density in the injured area by day 21, as assessed by fluorescent image analysis (CON=19.5% of A01, TRIM-treated=19%, Figure 1C).

[0057] Vascular density at 21 dpi was further resolved in confocal microscopy images taken from the center of the VML injury site (Figure 1D and Figure 1E). Vascular density in TRIM-treated GM (15% of A01) appeared to be lower than in CON (30.2%) (Figure 1F).

[0058] Example 3: TRIM improves myofibril regeneration after VML Confocal images show myofibril regeneration after injury at 21 dpi (Figures 2A and 2B). The addition of TRIM appeared to enhance myofibril regeneration in the injured area. Myofibrils in TRIM-treated GM (Figure 2B) were organized in parallel and more evenly distributed throughout the injury compared to saline-treated controls (CON). Myofibril density appeared to be increased in A01 TRIM (87%) compared to CON (CON=78%) (Figure 2C).

[0059] Frozen GM sections were sliced ​​and stained for laminin and myosin heavy chain. Analysis of muscle cross-sectional images revealed that TRIM could improve myofibrillar regeneration as measured by muscle thickness (Figure 2D and Figure 2E). Saline-treated GM (Figure 2D) had reduced thickness at the center of the injury. In contrast, TRIM-treated GM (Figure 2E) had similar thickness throughout both uninjured and injured muscles. Thus, at 21 days of recovery, the average muscle thickness was greater in TRIM than in CON. Expressed as a ratio of IT / UT, the average of CON GM was 0.75, while the average of TRIM was 1.0 (Figure 2F).

[0060] Example 4: TRIM improves myofibril regeneration after chemical injury Uninjured BPCuZn-treated fibers, saline-treated fibers (8 dpi), and BPCuZn-treated fibers (8 dpi) were stained for eMyHC, laminin, and DAPI (Figure 3A). Compared to saline-treated 8 dpi fibers, BPCuZn-treated 8 dpi fibers had a lower percentage of eMyHC-positive fibers (Figure 3B). Compared to saline-treated 8 dpi fibers and uninjured BPCuZn-treated fibers, BPCuZn-treated 8 dpi fibers also had a higher percentage of regenerating fibers (Figure 3C).

[0061] Example 5: TRIM Improves Mass in Severely Dystrophic Muscles After a single injection of Dystrophix, muscle mass increased by approximately 20-30% compared to untreated TA and EDL, indicating that the muscles of the anterior fascial compartment in the leg respond 14 days after a single injection (Figures 4A-4C). Also, because only the left leg was treated, TA and EDL from the contralateral right leg were also harvested, indicating that Dystrophix does not have a systemic effect (Figures 4A-4C, Dystrophix-treated TA and EDL are shown along with the contralateral untreated leg). To determine whether the increase in muscle mass was due to myofibril regeneration, muscle cross sections were obtained in 10 μm pressure sections and labeled with laminin to identify myofibril boundaries, embryonic myosin heavy chain (eMyHC) as a marker for regenerating myofibrils, and DAPI to visualize nuclei. Compared to untreated samples, dystrophix-treated samples exhibited more centrally located nuclei with less fibrosis and eMyHC (a marker of regenerating myofibrils) (Figure 4), indicating increased effective muscle regeneration with more mature myofibrils.

[0062] Example 6: TRIM improves dystrophic muscle structure TAs of dystrophic mice receiving a single Dystrophin injection exhibited greater force (i.e., were more resistant to eccentric muscle injury) than saline controls (Figure 5). When comparing the muscle force produced after eccentric contraction-induced injury to the muscle force produced before injury, Dystrophin-treated dystrophic muscles maintained approximately 90% of their pre-injury values ​​compared to saline controls, which maintained approximately 65% ​​of their pre-injury values.

[0063] Example 7: TRIM enhances blood vessel growth Mice at 0 dpi (days post injury) received no treatment. Mice at 8 dpi were injected with BaCl2 to induce chemical injury and analyzed at 8 dpi. BPCuZn 0 dpi mice were injected with 10 μg / g body weight BpCuZn and analyzed at 3 dpi. BPCuZn 8 dpi mice were injected with BaCl2 to induce chemical injury. They were then injected with 10 μg / g body weight BpCuZn at 3 dpi and analyzed at 8 dpi.

[0064] CD31 staining indicates vascular differentiation (Figure 6A). The relative amount of microvascular area / fibers is slightly higher in BPCuZn mice at 8 dpi compared to 8 dpi mice (Figure 6B). These results suggest that BPCuZn enhances vascular growth.

[0065] Example 8: TRIM increases fiber size Fibers from saline- and dystrophin-treated mice were stained for laminin, MyHC, and DAPI (Figure 7A). In quantification of myofibril cross-sectional area frequency, treatment with dystrophin caused a rightward shift compared to saline controls (Figure 7B), indicating that dystrophin treatment increases fiber size.

[0066] When introducing elements of the invention or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0067] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[0068] Because various changes can be made in the above compositions and processes without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

Claims

1. A bioactive glass composition obtained by calcining a reactant composition comprising: about 10% to about 40% by weight of B ２ O ３ , about 15% to about 40% by weight of P ２ O ５ , about 10 wt. % to about 25 wt. % CaO; About 5% to about 20% by weight of Na ２ O, and Optionally, about 2 wt. % to about 10 wt. % CoO, about 0.5 wt. % to about 2 wt. % ZnO; About 0.1% to about 1% by weight of CuO, or a combination thereof.

2. The reactant composition comprises: about 10% to about 40% by weight of B ２ O ３ , about 15% to about 40% by weight of P ２ O ５ , about 10 wt. % to about 25 wt. % CaO; About 5% to about 20% by weight of Na ２ O, and about 2% to about 10% by weight of CoO The bioactive glass composition of claim 1 , comprising:

3. The reactant composition comprises: about 33% to about 37% by weight of B ２ O ３ , about 33% to about 37% by weight of P ２ O ５ , about 13 wt. % to about 18 wt. % CaO; about 11% to about 14% by weight of Na ２ O, and about 3% to about 5% by weight CoO The bioactive glass composition of claim 1 , comprising:

4. The reactant composition comprises: about 30% to about 40% by weight of B ２ O ３ , about 20% to about 40% by weight of P ２ O ５ , about 10 wt. % to about 20 wt. % CaO; about 11% to about 18% by weight of Na ２ O, and about 3% to about 10% by weight of CoO The bioactive glass composition of claim 1 , comprising:

5. The reactant composition comprises: about 30% to about 40% by weight of B ２ O ３ , about 30% to about 40% by weight of P ２ O ５ , about 10 wt. % to about 20 wt. % CaO; About 10% to about 15% by weight of Na ２ O. about 0.5 wt. % to about 2 wt. % ZnO, and about 0.1% to about 1% by weight CuO The bioactive glass composition of claim 1 , comprising:

6. The reactant composition comprises: about 33% to about 37% by weight of B ２ O ３ , about 33% to about 37% by weight of P ２ O ５ , about 13 wt. % to about 18 wt. % CaO; about 11% to about 14% by weight of Na ２ O. about 0.8 wt. % to about 1.2 wt. % ZnO, and about 0.3 wt. % to about 0.5 wt. % CuO The bioactive glass composition of claim 1 , comprising:

7. The reactant composition comprises: about 33% to about 37% by weight of B ２ O ３ , about 33% to about 37% by weight of P ２ O ５ , about 13% to about 18% by weight of CaO, and about 11% to about 14% by weight of Na ２ O The bioactive glass composition of claim 1 , comprising:

8. The calcining was performed by heating the reactant composition at a temperature below the melting temperature of the reactant composition; or The temperature for said calcination was from about 900°C to about 1150°C; or The temperature for the calcination was from about 1000°C to about 1150°C. The bioactive glass composition of claim 1 .

9. the reactant composition further comprises phosphoric acid; or using the composition to form calcium phosphate; Alternatively, the bioactive glass composition maintains a neutral pH upon decomposition. The bioactive glass composition of claim 1 .

10. 10. A medicament for treating injured or diseased skeletal muscle, comprising a bioactive glass composition according to any one of claims 1 to 9, wherein said bioactive glass composition is contacted with said injured or diseased skeletal muscle.

11. The injured or diseased skeletal muscle has an increase in average myofibril area after at least 8 days of treatment with the bioactive glass composition compared to an otherwise similarly treated injured or diseased skeletal muscle with saline; or The injured or diseased skeletal muscle has a lower embryonic myosin heavy chain (eMyHC) concentration after at least 5 days of treatment with the bioactive glass composition compared to an otherwise similarly treated injured or diseased skeletal muscle with saline; or The injured or diseased skeletal muscle has an increase in muscle mass after at least 10 days of treatment with the bioactive glass composition compared to an otherwise similarly treated injured or diseased skeletal muscle with saline; or The injured or diseased skeletal muscle has an increase in muscle peak force at least 10 days after treatment with the bioactive glass composition compared to an otherwise similarly treated injured or diseased skeletal muscle with saline; or 11. The method of claim 10, wherein the injured or diseased skeletal muscle has increased vascularization at least 5 days after treatment with the bioactive glass composition compared to an otherwise similarly treated injured or diseased skeletal muscle with saline.

12. The method of claim 10, wherein the injured or diseased skeletal muscle is an injured skeletal muscle.

13. The injured skeletal muscle is a pulled muscle, a traumatically injured muscle, a torn muscle, an injured muscle resulting from muscle overuse or misuse, or a combination thereof; or the injured muscle resulting from muscle overuse or misuse is the result of a sports injury. The pharmaceutical composition according to claim 12.

14. The pharmaceutical composition of claim 10, wherein the injured or diseased skeletal muscle is diseased skeletal muscle, and the diseased skeletal muscle is dystrophic skeletal muscle, cachectic skeletal muscle, sarcopenic skeletal muscle, or a combination thereof.

15. 10. A medicament for treating injured or diseased brain or nervous tissue, comprising a bioactive glass composition according to any one of claims 1 to 9, wherein said bioactive glass composition is brought into contact with said injured or diseased brain or nervous tissue.

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