Field of the Invention

The invention relates to compositions and kits comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, and an inorganic peroxide selected from calcium peroxide (CaO2), magnesium peroxide (MgO2), and/or zinc peroxide (ZnO2) for use in the prevention or treatment of wounds, and/or pathological conditions requiring wound healing, tissue regeneration and/or bone regeneration.

In more particular, the invention relates to compositions and kits comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, and a peroxide selected from calcium peroxide (CaC ), magnesium peroxide (MgO2), and/or zinc peroxide (ZnO2) for use in the prevention or treatment of a gum disease.

Background of the Invention

Oxygen is involved in various biological processes such as cell metabolism and signal transduction. Especially during a wound healing process, the transient oxidative stress induced by oxygen is beneficial for increasing cellular activities, secretion of wound growth factors and promoting neovascularization. At sites where a chronic inflammatory response could be detected, oxygen consumption is increased and blood flow is stimulated. This local tissue p©2 change is due in part to increased oxygen consumption, including oxygen consumption by resident cells and infiltrated defense cells, and in part to decreased oxygen availability due to endothelial damage and vasoconstrictor microcirculation, as well as facultative anaerobic bacteria. These metabolic processes are found in all tissues. Hypoxic metabolic shifts are almost always found in the periodontal inflammatory process, as there is an imbalance between oxygen supply and consumption in the tissue (Gordon, H.A.; Rovin, S.; Bruckner, G. Blood flow, collagen components of oral tissue and salivary kallikrein in young to senescent, germfree and conventional rats. A study on the etiologic factors of periodontal disease. Gerontology 1978, 24, 1 -1 1 ).

A "burst release" in the physiological context of cell defense, also referred to as a "respiratory burst", describes a high and rapid increase in oxygen which produces oxygen radicals, for example for the defense against microorganisms (Gongalves, R.V.; Costa, A.M.A.; Grzeskowiak, L. Oxidative Stress and Tissue Repair: Mechanism, Biomarkers, and Therapeutics. Oxidative Medicine and Cellular Longevity 2021 , 2021 , 6204096). A burst release however is not desired for tissue regeneration. In fact, a slow, constant oxygen release is advantageous, which supplies the local microenvironment with sufficient oxygen without producing health-threatening radicals, which result in damage of tissue.

Oxygen-releasing materials have been developed in the field of tissue engineering to address the problem of insufficient oxygen supply due to lack of vascularisation of large, artificial tissue pieces (Wang, J.; Zhu, Y.; Bawa, H.K.; Ng, G.; Wu, Y.; Libera, M.; van der Mei, H.C.; Busscher, H.J.; Yu, X. Oxygen-Generating Nanofiber Cell Scaffolds with Antimicrobial Properties. ACS Applied Materials & Interfaces 2011 , 3, 67-73; Lv, X.; Li, Z.; Chen, S.; Xie, M.; Huang, J.; Peng, X.; Yang, R.; Wang, H.; Xu, Y.; Feng, C. Structural and functional evaluation of oxygenating keratin/silk fibroin scaffold and initial assessment of their potential for urethral tissue engineering. Biomaterials 2016, 84, 99-110; Park, S.; Park, K.M. Hyperbaric oxygen-generating hydrogels. Biomaterials 2018, 182, 234-244).

Calcium (Ca), zinc (Zn) and magnesium (Mg) are natural elements in many tissues and are involved in many physiological processes such as bone metabolism or wound healing (Chen, Z.; Zhang, W.; Wang, M.; Backman, L.J.; Chen, J. Effects of Zinc, Magnesium, and Iron Ions on Bone Tissue Engineering. ACS Biomater Sci Eng 2022, 8, 2321-2335). Zinc is released when zinc peroxide is broken down, and it is known to accelerate wound healing processes (Lin, P.H.; Sermersheim, M.; Li, H.; Lee, P.H.U.; Steinberg, S.M.; Ma, J. Zinc in Wound Healing Modulation. Nutrients 2017, 10). Calcium and magnesium have been shown to increase bone regeneration (Zhou, H.; Liang, B.; Jiang, H.; Deng, Z.; Yu, K. Magnesium-based biomaterials as emerging agents for bone repair and regeneration: from mechanism to application. Journal of Magnesium and Alloys 2021 , 9, 779-804; Aquino-Martinez, R.; Angelo, A.P.; Pujol, F.V. Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration. Stem Cell Research & Therapy 2017, 8, 265). Therefore, these elements have great potential for tissue engineering and tissue regeneration. However, their effects are dosedependent and higher doses can negatively affect tissue regeneration or even induce apoptosis. Very high amounts of insoluble Ca salts are known to deposit in elastic tissues such as skin as skin calcification and cause tissue damage (Pugashetti, R.; Shinkai, K.; Ruben, B.S.; Grossman, M.E.; Maldonado, J.; Fox, L.P. Calcium may preferentially deposit in areas of elastic tissue damage. J Am Acad Dermatol 2011 , 64, 296-301). Zn showed cytotoxic effects in vitro, so overdosing should also be avoided when using Zn to achieve optimal tissue engineering effects. The cytotoxic effect of zinc could be suppressed by increasing the Ca ²⁺ concentration by modulating the entry of Ca ²⁺ into the cells using channels that are also used for calcium (Borovansky, J.; Riley, P.A. Cytotoxicity of zinc in vitro. Chem Biol Interact 1989, 69, 279-291 ). Similar to Ca ²⁺ and Zn ²⁺ , Mg ²⁺ is known to cause cytotoxic effects in cells both in vivo and in vitro, and these effects are dose-dependent (Sanders, T.; Liu, Y.M.; Tchounwou, P.B. Cytotoxic, genotoxic, and neurotoxic effects of Mg, Pb, and Fe on pheochromocytoma (PC-12) cells. Environ Toxicol 2015, 30, 1445-1458).

Gingivitis, periodontitis, and peri-implantitis are the most common biofilm-associated, inflammatory gum diseases in humans or mammals. Without treatment, these gum diseases result in tooth or implant loss. Periodontitis and peri-implantitis develop as a result of dysbiosis of the bacterial population in susceptible individuals, associated with a dysregulation of the immune- inflammatory response. Dysbiosis is thus responsible for the development of microbe-related pathologies and a host-mediated breakdown of connective tissue and alveolar bone (Darveau, R.P. Periodontitis: A polymicrobial disruption of host homeostasis. Nat. Rev. Microbiol. 2010, 8,481-490.). Periodontitis and peri-implantitis also involve the destruction of the connective tissue including collagens, proteoglycans, and other components of the extracellular matrix. Gingivitis is an inflammatory response of the gingival tissues to the metabolic products and pathogenic toxins of bacteria found in oral plaque.

The transition from periodontal health to disease is associated with a dramatic shift from a symbiotic microbial community, which is composed mostly of facultative bacterial genera such as Actinomyces and Streptococci, to a dysbiotic microbial structure composed mainly of anaerobic genera from the phyla Firmicutes, Proteobacteria, Spirochaetes, Bacteroidetes and Synergistetes (Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation. Nat Rev Immunol. 2015;15(1 ):30-44. doi:10.1038/nri3785.).

A gram-negative anaerobic bacterial flora induces a cascade of immune responses and thereby promotes tissue breakdown. Soft tissue and alveolar bone are consequently degraded, and the gram-negative anaerobes can grow under optimal conditions in the periodontal pockets. However, although the etiology of periodontal and peri-implant diseases is based on a dysbiotic microbial structure, and although some well-characterized pathogens display destructive virulence factors, the pathogenesis of periodontitis and peri-implantitis is essentially mediated by the host response (Van Dyke, T.E.; Serhan, C.N. Resolution of inflammation: A new paradigm for the pathogenesis of periodontal diseases. J. Dent. Res. 2003, 82, 82-90.0).

There are different approaches for the treatment of gingivitis, periodontitis or peri-implantitis, most of them involve the application of anti-bacterial compositions (Lasserre, J.F.; Toma, S.;

Bourgeois, T.; El Khatmaoui, H.; Marichal, E.; Brecx, M.C. Influence of low direct electric currents and chlorhexidine upon human dental biofilms. Clin. Exp. Dent. Res. 2016, 2, 146-154.). In one common approach, a biodegradable chlorhexidine (CHX) chip is used as an adjunct to scaling and root planing (SRP) in the treatment of moderate to severe periodontitis patients (Medaiah S, Srinivas M, Melath A, Girish S, Polepalle T, Dasari AB. Chlorhexidine chip in the treatment of chronic periodontitis - a clinical study. J Clin Diagn Res. 2014;8(6):ZC22-ZC25. doi:10.7860/JCDR/2014/8808.4477). However, studies have shown that the use of CHX as an adjuvant after SRP has no significant positive effect on the microbiome and tissue attachment (Grisi, D.C., et al. ,2002; Carvalho, J., et al., 2007), possibly because CHX is toxic to fibroblasts and osteoblasts, and thus inhibits tissue regeneration.

Some other therapeutic products comprise a sodium perborate gel, which can be placed in the pocket adjuvantly after SRP for oxygen release. However, sodium perborates are known to be corrosive and irritating substances, which, despite their antibacterial effects, are cytotoxic and thus impair tissue regeneration. Another approach relates to compositions comprising hyaluronic acid, which have been described in the context with the treatment of gum diseases.

The treatment of gingivitis or periodontitis using a liquid composition comprising a non- biodegradable thermosensitive pharmaceutically acceptable polyalkylene oxide block copolymer, hyaluronic acid having a molecular weight of up to about 50.000 Dalton or a salt thereof, and optionally a therapeutic agent is described in WO2018/158764 A1 . Hyaluronan and its potential role in periodontal healing, in particular wound healing processes associated with periodontal diseases, has been described (MOSLEY R ET AL: “Hyaluronan and its potential role in periodontal healing”, Dental update, update publ., London, GB, vol. 29, no. 3, 1 April 2002, pages 144-148). Hyaluronic acid has also been a boon in periodontal therapy (DAHIYA PARVEEN ET AL: “Hyaluronic Acid: A Boon in periodontal Therapy”, NORTH AMERICAN JOURNAL OF MEDICALK SCIENCES, vol. 5, no. 5, 1 May 2013, pages 309-315).

DE20201602375U1 discloses hyaluronic acid in combination with the antiseptic octenidine dihydrochloride, which is suitable for the treatment and prevention of several diseases such as stomatological complications, gum injuries after calculus removal, oral mucosal wounds or tongue wounds, or of other types of wounds and injuries in the oral cavity.

EP1638582B1 discloses the use of hyaluronic acid for the preparation of compositions for the treatment of recurrent oral aphthae.

EP3056195A1 discloses a composition of hyaluronic acid and beta-glucan and its use for the treatment of, among others, periodontitis, and gingivitis. WO2018/158764A1 discloses a certain composition of a polyalkylene oxide block copolymer and hyaluronic acid and its use for the treatment of, among others, periodontitis, gingivitis, and peri- implantitis.

Furthermore, root fillings are known in which CaO2 is used as an antimicrobial agent (Malyk Yuriy (2005), "In vitro studies on the use of calcium peroxide (Ca02)-containing sealer materials in endodontic therapy", doctoral thesis, Poliklinik fur Zahnerhaltung und Parodontologie der Ludwig- Maximilians-llniversitat Munchen).

W02018/026204A1 discloses an in-situ crosslinked polymer hydrogel comprising calcium peroxide that releases a high concentration of oxygen in a sustained release form. Also described is an implant material for tissue regeneration and filling, comprising such an in-situ crosslinked sustained release oxygen-release hydrogel.

CN112826761 A and CN112870109A disclose a preparation method of a dentifrice with antibacterial effect comprising calcium peroxide.CA1334282C describes periodontal compositions and methods containing peroxide and bicarbonate.

US5616313 A describes a method for minimizing damage to gingival and periodontal tissue. The method includes delivering a first composition containing a zinc salt to a receptacle, delivering a second composition containing a bicarbonate salt to the same receptacle, and transferring within 5 minutes of delivery to the receptacle the combination into the mouth onto the gingival and periodontal tissues. However, the released oxygen is only available for a very short time and in a very onetime high concentration (burst release). High peroxide concentrations are required in order to achieve an antibacterial effect.

Although various compositions for the treatment of the various gum diseases in humans or animals are known, they do not combine a long-lasting antibacterial effect, while at the same time gum tissue regeneration is supported, making the treatment of wounds or inflammatory pathologies, in particular gingivitis, periodontitis, and peri-implantitis less efficient.

Summary of the Invention

Against this background, it is the object of the present invention to provide pharmaceutically active compositions for the prevention or treatment of wounds and inflammatory diseases that increase the amount of available oxygen, and release oxygen over a prolonged period of time. This object is solved by the subject-matter of claim 1 , in particular by a composition comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, and one or more inorganic peroxides.

The present invention is based on the surprising discovery that the amount of available oxygen is multiplied, and oxygen is released in a delayed manner when at least one inorganic peroxide is combined with hyaluronic acid in a composition. More specifically, it was found that a combination of at least one inorganic or metal peroxide and hyaluronic acid multiplies the amount of available oxygen by up to five times, depending on the conditions. In a preferred embodiment, the composition of the present invention comprises at least one inorganic peroxide selected from the group consisting of calcium oxide (CaC ), zinc peroxide (ZnO2) and/or magnesium peroxide (MgO2), and hyaluronic acid or a pharmaceutically acceptable salt thereof.

It was also found that the amount of available oxygen can be further increased or varied by the addition of MgC>2 and/or ZnC>2 and thus adapted to the needs of the individual applications and tissues. In particular, it is surprising and important that the oxygen is released over a longer period of time.

A continuous and steady release of anti-bacterial oxygen for a prolonged period makes the treatment of wounds, or inflammatory conditions more efficient and less dangerous for the treated subject, since high concentrations of peroxides that locally trigger an oxygen burst release and thus generate masses of antibacterial oxygen radicals, which damage local tissues, are avoided.

As shown herein, the compositions of the present inventions are suitable for tissue regeneration or bone regeneration, because it was surprisingly found that low concentrations of metal peroxides or inorganic peroxides exhibit unexpected properties when combined with hyaluronic acid or a pharmaceutically acceptable salt thereof. A delayed release of oxygen from a metal peroxide such as CaO2, ZnC>2 and/or Mg©2 is attributable to the matrix formed by the hyaluronic acid said composition. Furthermore, a burst release (a one-time high concentration of oxygen) and harmful concentrations of oxygen for the surrounding wound tissue can be avoided due to delayed release of oxygen from hyaluronic acid-bound inorganic peroxide, in particular calcium peroxide.

In a preferred embodiment, the composition of the present invention comprises hyaluronic acid or a pharmaceutically acceptable salt thereof, and one or more inorganic peroxide for use in the treatment of wounds, or inflammatory conditions. Examples of inflammatory conditions to be treated include, but are not limited to conditions or pathologies that require wound healing, tissue regeneration and/or bone regeneration. Preferably, the inflammatory condition is a gum disease. More preferably, the gum disease is selected from the group consisting of gingivitis, periodontitis or peri-implantitis. In a preferred embodiment, the composition of the present invention comprises hyaluronic acid or a pharmaceutically acceptable salt thereof, and one or more inorganic peroxide selected from CaO2, ZnC>2 and/or Mg©2 for use in the prevention or treatment of gingivitis, periodontitis or peri-implantitis. In a further preferred embodiment, the compositions of the present invention contain at least calcium peroxide (CaO2) and hyaluronic acid as active compounds, and are suitable for use in wound healing, tissue regeneration and/or bone regeneration. In preferred embodiments such compositions additionally contain ZnC>2 and Mg©2 to increase the amount of available oxygen.

In alternative embodiments, the composition contains two or more, in yet another embodiment the composition contains three different inorganic peroxides. Thus, in addition to the oxygen release, the ionic end products of the inorganic peroxides can also be optimized depending on the tissue, and specifically adapted for the indication.

In a first variant, the composition comprises CaO2 and hyaluronic acid or a pharmaceutically acceptable salt thereof. In a second variant, the composition comprises ZnC>2 and hyaluronic acid or a pharmaceutically acceptable salt thereof. In a third variant, the composition comprises MgC>2 and hyaluronic acid or a pharmaceutically acceptable salt thereof. In a fourth variant, the composition comprises CaO2 and hyaluronic acid or a pharmaceutically acceptable salt thereof, and at least one further peroxide, including but not limited to Zn©2 or MgC>2. In a fifth variant, the composition consists of CaO2 and hyaluronic acid or a pharmaceutically acceptable salt thereof, and ZnC>2. In a sixth variant, the composition consists of CaO2 and hyaluronic acid or a pharmaceutically acceptable salt thereof, and MgC>2. In a seventh variant, the composition comprises Mg©2 and hyaluronic acid or a pharmaceutically acceptable salt thereof, and at least one further peroxide, including but not limited to CaO2 or ZnC>2. In an eight variant, the composition consists of Mg©2 and hyaluronic acid or a pharmaceutically acceptable salt thereof, and CaO2. In a nineth variant, the composition consists of Mg©2 and hyaluronic acid or a pharmaceutically acceptable salt thereof, and ZnC>2. In a further variant, the composition comprises Zn©2 and hyaluronic acid or a pharmaceutically acceptable salt thereof. In a further variant, the composition comprises Zn©2 and hyaluronic acid or a pharmaceutically acceptable salt thereof, and MgC>2 and/or CaC>2. In a preferred embodiment, the composition consists of a combination of two inorganic peroxides, selected from the group consisting of CaO2, Zn©2 and MgC>2, and hyaluronic acid or a pharmaceutically acceptable salt thereof. Preferably, such a formulation contains the two inorganic peroxides CaO2 and ZnC>2, or alternatively CaO2 and MgC>2, as well as hyaluronic acid or a pharmaceutically acceptable salt thereof. An alternative preferred formulation contains the three inorganic peroxides CaO2, ZnC>2 and MgO2, and hyaluronic acid or a pharmaceutically acceptable salt thereof. The combination of peroxides can be adjusted depending on the condition to be treated. A combination of different peroxides (CaO2, ZnC>2 and/or MgC^) increases the amount of available oxygen and makes the end products (Ca ²⁺ , Zn ²⁺ or Mg ²⁺ ) available to the tissue to be treated. Most importantly, toxic amounts of peroxides and burst release of oxygen are avoided.

In a further variant, the composition is a pharmaceutical composition, comprising at least a diluent, solvent or carrier, at least one inorganic peroxide and hyaluronic acid or a pharmaceutically acceptable salt thereof.

In one aspect, the composition of the present invention allows the treatment of different kind of wounds, including chronic wounds, such as venous leg ulcers, diabetic foot ulcers, pressure ulcers, arterial ulcers, burns, and non-healing surgical wounds. In addition, the compositions according to the present invention are also useful for treating abrasions, lacerations, minor cuts, scalds and burns, and other partial thickness wounds.

The treatment of wounds that are contaminated with harmful pathogenic microorganisms is preferred. For example, periodontal pockets provide an ideal habitat for anaerobic pathogenic germs due to their anaerobic conditions. As a consequence, inflamed tissue increases gingival crevicular fluid, the major nutritional source for subgingival microorganisms.

It is one advantage of the compositions of the present invention that the effective period of oxygen to destroy biofilms and bacteria in affected tissue, such as in the periodontal pockets, is significantly increased, so that the treatment of an inflammatory condition, such as a gum disease like gingivitis, periodontitis, or peri-implantitis, becomes more effective. As a consequence, tissue breakdown can be avoided or, at least, retarded or stopped. At the same time, the tissue regeneration is stimulated by hyaluronic acid and not adversely affected by the action of inorganic peroxide. Depending on the condition, the compositions of the present invention are used for the treatment of a disorder which is characterized by tissue breakdown or bone destruction. The compositions of the present invention a thus suitable for use in tissue regeneration, tissue engineering or bone regeneration.

A retarded release of oxygen by combining calcium peroxide with hyaluronic acid could not be expected. In fact, it would have been more likely that oxygen is released in the same manner as in water since hyaluronic acid only forms a rather coarse meshed matrix. It was hence surprising that a combination of hyaluronic acid and an inorganic peroxide such as calcium peroxide would result in a delayed release of oxygen without adversely affecting the surrounding tissue, while at the same time the antimicrobial properties of the metal peroxide are maintained. As exemplified herein, the growth of periodontal pathogenic bacteria or biofilm formation and immune inflammatory responses can be effectively inhibited. Most importantly, no toxic effects could be observed on fibroblasts or osteoblasts.

The combination of different peroxides contained in hyaluronic acid not only makes it possible to adjust the oxygen delivery to the needs of the different targets (e.g. bone, skin, cartilage). By using different peroxides, an overdose of one ion can be avoided and biomaterials can be tailored to different targets’ need for different ions.

The present invention allows that basic causes of periodontitis or peri-implantitis can be solved by (a) increasing the antimicrobial efficiency against obligate anaerobes, thus restoring a symbiotic bacterial flora, and (b) by acceleration of tissue regeneration to prevent recolonization by bacteria in periodontal pockets.

The compositions of the present invention are preferably formulated as a pharmaceutical composition. Preferably, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers, solvents, diluents, or expedients, and is preferably prepared as a formulation for topical administration in the oral cavity.

Preferably, the compositions of the present invention are formulated in form of a solution, a suspension, an emulsion, a cream, or a gel. Such formulations contain a therapeutically effective concentration of hyaluronic acid or a pharmaceutically acceptable salt thereof, and a therapeutically effective concentration of one or more inorganic peroxides, selected from CaO2, ZnC>2 or Mg©2. In a preferred embodiment, the composition of the present invention is provided as a gel, which is ready to be injected into the space between the teeth and gums with a delivery means, e.g. a syringe. Due to the inventive combination, the gel has a bactericidal effect against pathogenic periodontal germs, prevents new colonization after mechanical cleaning and accelerates tissue regeneration (e.g. bone, gums). In addition, the gel temporarily closes the gap between the teeth and the gums, in which pathogenic bacteria otherwise would multiply.

The composition of the present invention preferably includes sterile aqueous solutions, or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. The injectable solutions must be sterile and fluid to the extent that they can be injected by means of any suitable delivery means for the oral cavity, such as a syringe. The injectable solution preferably comprises a carrier, which can be any suitable solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, or vegetable oils. The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, and the severity of the condition being treated. However, in general, satisfactory results are obtained when the compounds of the invention are administered at a single dosage of up to 3mL per application.

Preferred dosage forms suitable for administration to the oral cavity comprise from about 15 mg/mL to 35 mg/mL of hyaluronic acid and from about 100 mg/L to 500 mg/L calcium peroxide in admixture with a pharmaceutically acceptable carrier. This dosage regimen may be adjusted to provide the optimal therapeutic response. The inventive combination can be administered to an oral cavity of a human or an animal suffering from a gum disease. Typically, the veterinary dosages correspond to human dosages with the amounts administered being in proportion to the weight of the animal as compared to adult humans.

For example, for administration to the oral cavity, a pharmaceutically acceptable carrier which does not adversely affect the activity of hyaluronic acid and calcium peroxide, such as a solvent or a diluent, may be contained in the pharmaceutical composition of the present invention. Such carriers include, but are not limited to agents, stabilizers, absorption delaying agents, disintegrating agents, emulsifiers, antioxidants, binders, lubricants, moisture absorbents, and the like. For example, the solvent may be selected from the group consisting of water and sucrose solutions, the diluent may be selected from the group consisting of lactose, starch, and microcrystalline cellulose, the absorption delaying agent may be selected from the group consisting of chitosan and glycosyl glycans, and the lubricant may be selected from the group consisting of magnesium stearate, talc, sodium stearyl fumarate, or combinations thereof.

In preferred embodiments of the invention, the composition of the present invention preferably further includes a carrier, a thickener, and one or more additional antimicrobial agents. In accordance with various aspects of these embodiments, the composition may include a humectant, migration enhancing agents, oils, and the like.

Preferably, the pharmaceutically acceptable salts of hyaluronic acid are hyaluronan salts and include, but are not limited to alkaline metal or alkaline earth metal salts of hyaluronan, e.g. sodium hyaluronate, potassium hyaluronate and calcium hyaluronate. In one embodiment, the pharmaceutically acceptable salt of hyaluronic acid is sodium hyaluronate. In an alternative embodiment, the pharmaceutically acceptable salt of hyaluronic acid is potassium hyaluronate or calcium hyaluronate. The composition of the invention may also comprise a combination of sodium hyaluronate, potassium hyaluronate and/or calcium hyaluronate.

Preferably, in the compositions of the present invention, the hyaluronic acid or pharmaceutically active salt thereof is provided in form of a matrix to allow binding of calcium peroxide. The hyaluronic acid matrix according to the present invention promotes tissue regeneration and viability of fibroblasts. In addition, the hyaluronic acid matrix prevents calcium peroxide going into solution, and reduces the risk of further depositions of new, potentially pathogenic, germs. When calcium peroxide is bound to the hyaluronic acid matrix, oxygen is released evenly from the calcium peroxide component in the composition, and thereby inhibits the growth of periodontal pathogenic organisms over a long period of time. Example of such periodontal pathogenic organisms include obligate anaerobic germs, in particular periopathogens, which include, but are not limited to Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, or grampositive aerobes composed of Streptococcus spp. such as Streptococcus oralis, and Actinomyces spp.

The hyaluronic acid or salt thereof used in the composition of the present invention can be either a high-molecular weight hyaluronic acid, or a low-molecular weight hyaluronic acid, or salts thereof. The high-molecular weight hyaluronic acid is preferred in the context of the present invention. The high-molecular weight hyaluronic acid or salt thereof preferably has an average molecular weight of about 300,000 to about 6 million Daltons (Da). Most preferably, it has an average molecular weight of from about 500,000 to about 3 million Daltons. The low-molecular weight hyaluronic acid or salt thereof comprised within the composition of the present invention has a molecular weight of up to about 50,000 Da, and is optionally hydrolyzed or partially hydrolyzed. In certain embodiments, the low-molecular weight hyaluronic acid has a molecular weight of from about 3,000 to about 50,000 Da, from about 4,000 to about 20,000 Da, or from about 6,000 to about 10,000 Da.

In a preferred embodiment, the hyaluronic acid or the pharmaceutically acceptable salt thereof comprised with the composition of the present invention is present in an amount of from about 1 mg/mL to about 100 mg/mL, preferably from about 5 mg/mL to about 80 mg/mL, from about 5 mg/mL to about 50 mg/mL, or from about 10 mg/mL to about 40 mg/mL, more preferably from about 10 mg/mL to about 30 mg/mL, most preferably 20 mg/mL to about 25 mg/mL ,e.g. in an amount of 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL or 25 mg/mL. Preferably, the hyaluronic acid is present in the composition at the indicated concentrations as sodium hyaluronate. The inorganic peroxide component of the composition of the present invention is included in an amount sufficient to allow even release of oxygen when the composition is applied to the oral cavity. Typically, the peroxide component can be employed in the composition of the present invention in amounts so that at least about 1 mg/L of the composition comprises an inorganic peroxide, selected from CaO2, ZnC>2 or MgCL.

In a preferred embodiment, the calcium peroxide is present in the composition in an amount of about 1 mg/L to 5000 mg/L (resp. 1 .4*10 ^-5 to 0.07 mol/L). In a further preferred embodiment, the calcium peroxide comprised with the composition of the present invention is present in an amount of about 10 mg/L to 1000 mg/L (resp. 1 .4*10 ^-4 to 0.014 mol/L), preferably from about 10 mg/L to about 800 mg/L (resp. 1 .4*10 ^-4 to 0.011 mol/L), from about 100 mg/L to about 800 mg/L (resp. 1 .4*10 ^-3 to 0.011 mol/L), or from about 100 mg/L to about 500 mg/L (resp. 1 .4*10 ^-3 to 0.0069 mol/L), more preferably from about 200 mg/L to about 500 mg/L (resp. 0.0028 to 0.0069 mol/L), e.g. in an amount of 200 mg/L, 250mg/L, 300 mg/L, 400 mg/L or 500 mg/L.

The concentration of zinc peroxide is preferably in the range of 1 mg/L to 10000 mg/L (corresponding to 1.02*10 ^-5 to 0.103 mol/L).

The concentration of magnesium peroxide is preferably in the range of 1 mg/L to 15000 mg/L (corresponding to 1.77*10 ^-5 to 0.266 mol/L).

A further aspect of the present invention relates to additional components that are preferably present in the composition to increase its efficiency or therapeutic effectivity. In a first aspect of the present invention, the composition comprises one or more additives, which include, but are not limited to plant extracts, calcium hydroxyapatite, and/or collagen. For example, the composition of the invention preferably additionally comprises a preparation of one or more plant extracts, preferably derived from the plant variety selected from the group consisting of eucalyptus, rhubarb root, clove oil, aloe vera, and/or cumin. The presence of plant extracts in the composition of the present invention inhibits the growth of the germs and constitutes a feasible alternative to antibiotics. As such, the plant extracts enhance the antibacterial effect of CaO2, ZnC>2 or MgC>2 when present in the composition of the present invention.

In a further preferred embodiment of the present invention, the additive in the inventive composition is calcium hydroxyapatite, which was found to induce the formation and storage of natural collagen. Furthermore, calcium hydroxyapatite promotes the reconstruction of the teeth and bone material. In a further preferred embodiment of the present invention, the additive in the inventive composition is collagen. Collagen supports the adhesion of the gums to the teeth when present in the composition of the present invention.

When the composition comprises one or more additives selected from the group consisting of plant extracts, calcium hydroxy apatite, or collagen, the plant extract is present in the composition of the present invention in an amount of from about 0.1% to about 10% by weight, more preferably in an amount of from about 0.3% to about 3% by weight, most preferably in a concentration of 0.5% by weight.

In a further preferred embodiment, the calcium hydroxyapatite is present in the composition of the present invention in a ratio of 0.8 to 20 for calcium/phosphate and an amount from about 1% to about 30% by weight, more preferably in an amount of from about 5% to about 20% by weight, most preferably in a concentration of 10% by weight.

In a further preferred embodiment of the present invention, the collagen is present in the composition of the present invention in an amount of from about 0.9% to about 25% by weight, more preferably in an amount of from about 5% to about 20% by weight, most preferably in a concentration of 15% by weight.

The invention also comprises variants or modified derivatives of the compounds described herein, in particular chemically or biologically modified forms of hyaluronic acid, or its salts, calcium peroxide, plant extracts, calcium hydroxy apatite or collagen.

A pH value of the composition may vary in accordance with a particular application. In accordance with various embodiments of the invention, the pH value is between about 4 and about 9, preferably the pH is between about 6 and 8.5, most preferably between 6.5 and 8.

The compositions of the present invention do not only exhibit a bactericidal effect on the periodontal pathogenic germs and temporarily prevent their resettlement, but they also actively support tissue regeneration. In particular, the matrix of hyaluronic acid enables a promoted release of oxygen over a prolonged period of time with a simultaneous positive effect on tissue regeneration. Thereby, the compositions of the present invention result in a higher success rate for the treatment of a gum disease, and reduce the likelihood of a re-treatment. It is apparent for a person skilled in the art that due to the general properties, the compositions of the present invention may be suitable for the treatment of any gum disease but is in particular useful for the treatment or prevention of gingivitis, periodontitis or peri-implantitis. In a preferred embodiment of the invention, the amount of available oxygen can be increased by applying a special processing in the production of the formulation. The processing to obtain the inventive combination comprises the following steps:

1 . Dissolving a preservative in a liquid matrix;

2. Dissolving optional soluble components;

3. Suspending insoluble components;

4. Inserting the hyaluronic acid under strong stirring.

An example of a preservative used in the context of the present invention is methyl-4- hydroxybenzoate (MHB).

A preferred liquid matrix is PBS buffer or any other suitable solution. Soluble components comprise the additives such as plant extracts. Preferred insoluble components include, but are not limited to peroxides, calcium hydroxy apatite, or other kinds of plant extracts. By applying this procedure, the amount of oxygen available can be significantly increased as compared to a simple mixing of the compounds and subsequent dissolving and suspending.

The invention furthermore comprises a kit for delivering a composition of the invention to a target. In a preferred embodiment, the invention relates to a kit for wound healing, tissue regeneration and/or bone regeneration, comprising a composition according to the invention, and a delivery means for applying said composition into said wound, tissue or bone.

In a preferred embodiment, the kit of the present invention is configured to delivering a composition into a gingival pocket, a periodontal pocket or a pocket resulting from peri-implantitis, said kit comprising a composition according to the invention, and a delivery means for topically applying said composition into said gingival pocket, periodontal pocket or pocket resulting from peri-implantitis.

In an alternative preferred embodiment, the kit of the present invention is configured to applying said composition on or into the oral mucosa, said kit comprising a composition according to the invention, and a delivery means for topically applying said composition on or into said oral mucosa.

In a preferred embodiment, in the kit of the present invention, the composition is provided in form of a gel which is preferably applied to the oral cavity with a syringe. Preferably, the gel is provided in a gingival pocket, a periodontal pocket, or a pocket that has been formed as a result of peri- implantitis. In a preferred embodiment, the delivery means for the composition in the kit is a syringe. In an alternative embodiment, the composition of the invention is applied on or into the oral mucosa. The composition in the kit of the invention allows a sustained release dosage form that can be administered to an oral cavity of an animal or human.

Taken together, a combination of peroxides can optimally supply various tissues with oxygen and ions without becoming cytotoxic. As shown herein, the compositions and kits of the present invention allow a delayed release of oxygen from antimicrobial calcium peroxide, whereby infectious or inflammatory diseases such as gum diseases, including but not limited to gingivitis, periodontitis and peri-implantitis can be successfully treated without adversely affecting the surrounding tissue, such as tooth or implant tissue.

The invention is explained in more detail in the following examples, which, however, are intended to be of illustrative nature only. By no means shall the herein described examples and embodiments limit the scope of the invention. The invention also comprises any variation, modification, and/or combination of components described herein.

Brief description of the Figures

Figure 1 shows the cytotoxicity of hyaluronic acid, calcium peroxide, and a combination thereof in human primary fibroblasts in vitro.

Figure 2 shows the cytotoxicity of hyaluronic acid, calcium peroxide, and a combination thereof in human primary osteoblasts in vitro.

Figure 3 shows the cytotoxicity of hyaluronic acid, calcium peroxide, and a combination thereof in human primary umbilical vein endothelial cells (HLIVEC) in vitro.

Figure 4 shows the biocompatibility of hyaluronic acid, calcium peroxide, and a combination thereof in a chorioallantoic membrane assay (Figure 4A: Vascularized Surface of the Sponge; Figure 4B: Degradation of the Sponge).

Figure 5 shows the effect of calcium peroxide on planktonic bacteria colonies of Streptococcus oralis (S. oralis) and Porphyromonas gingivalis (P. gingivalis). Figure 6 shows the effect of hyaluronic acid and calcium peroxide on mature P. gingivalis biofilms.

Figure 7 shows the effect of hyaluronic acid and calcium peroxide on mature S. oralis biofilms.

Figure 8 shows the slow oxygen release from a combination of hyaluronic acid and calcium peroxide compared to calcium peroxide alone and hyaluronic acid alone.

Figure 9 shows the oxygen content of formulations with and without hyaluronic acid. The formulations include a combination of hyaluronic acid and calcium peroxide, zinc peroxide and magnesium peroxide.

Figure 10 shows the effect if the components are merely mixed but not processed correctly in a method of producing a formulation comprising hyaluronic acid and calcium peroxide.

Modes for carrvinq out the invention:

Suitable formulations for application into the oral

The following non-limiting examples describe formulations that are suitable for the treatment of a number of gum diseases, including but not limited to gingivitis, periodontitis and peri-implantitis, or other gum disease-related inflammatory diseases. The formulations can further comprise any suitable carrier or excipient known to the person skilled in the art. The dosage regimens may be adjusted to provide the optimal therapeutic response. All formulations exhibit the desired therapeutic effect.

Example 1 :

The formulation comprises 256 mg/L calcium peroxide suspended in phosphate buffered saline (PBS) to stabilize the pH in the range of 7 and 7.5. Then, 23 mg/mL hyaluronic acid or salt thereof is added under vigorous stirring until a homogenous gel is produced.

Example 2:

The formulation comprises 256 mg/L calcium peroxide suspended in phosphate buffered saline (PBS) to stabilize the pH in the range of 7 and 7.5. Then 23 mg/mL hyaluronic acid or salt thereof and 4 mg/L Rheum palmatum root extract is added under vigorous stirring until a homogenous gel is produced. Example 3:

The formulation comprises 256 mg/L calcium peroxide suspended in phosphate buffered saline (PBS) to stabilize the pH in the range of 7 and 7.5. Then 23 mg/mL hyaluronic acid or salt thereof and 100 mg/L calcium hydroxy apatite is added under vigorous stirring until a homogenous gel is produced.

Example 4:

The formulation comprises 500 mg/L calcium peroxide suspended in phosphate buffered saline (PBS) to stabilize the pH in the range of 7 and 7.5. Then 30 mg/mL hyaluronic acid or salt thereof and 100 mg/L calcium hydroxy apatite is added under vigorous stirring until a homogenous gel is produced.

Example 5:

The formulation comprises 256 mg/L calcium peroxide suspended in phosphate buffered saline (PBS) to stabilize the pH in the range of 7 and 7.5. Then 23 mg/mL hyaluronic acid or salt thereof and 10 mg/L collagen is added under vigorous stirring until a homogenous gel is produced.

Example 6:

The formulation comprises 115 mg sodium hyaluronate and 50 mg CaO ₂ diluted in 5 mL H ₂ O.

Example 7:

The formulation comprises 250 mg sodium hyaluronate and 50 mg CaO2, 250 mg MgO ₂ and 200 mg ZnO ₂ diluted in 5 mL PBS buffer.

Examination of the tissue compatibility of the substances used in the invention

To investigate the cytotoxicity of calcium peroxide, hyaluronic acid, and a combination thereof on cells, the substances were given to human primary fibroblasts, osteoblasts, and human umbilical vein endothelial cells (HUVEC) in vitro and compared to untreated cells and a dead control. For this purpose, cytotoxicity tests were performed according to ISO 10993-5 by measuring the cell viability quantitatively and calorimetrically. Additionally, biocompatibility tests were performed in the chorioallantoic membrane assay (CAM assay) in fertilized hen's eggs. All tests were repeated at least three times independently. In all tests 30 mg/mL hyaluronic acid and 0.256 mg/mL CaO ₂ were used. According to ISO 10993-5, cytotoxicity is defined by more than 30% reduction of the viable cell number by the substance. The results are shown in Figures 1 to 4. Figure 1 shows the cellular viability of human primary fibroblasts obtained from human oral mucosa. Fibroblasts were treated with hyaluronic acid, or a combination of calcium peroxide and hyaluronic acid. Both treatments hardly showed a reduction of the cellular viability in comparison to untreated fibroblasts (=100% cell viability). This indicates that neither hyaluronic acid alone nor in combination with calcium peroxide is cytotoxic. By contrast, fibroblasts treated with 0.256 mg/mL calcium peroxide showed a reduction in cell viability by more than 30%, indicating the cytotoxicity of calcium peroxide as single substance.

Figure 2 shows the cellular viability of human primary osteoblasts. Osteoblasts were treated with hyaluronic acid, calcium peroxide, or a combination thereof. Osteoblasts hardly showed a reduction of their viability after treatment in comparison to untreated osteoblasts (=100% cell viability). This indicates that both substances, as single substances and in combination, have no cytotoxic effect on osteoblasts.

Figure 3 shows the cellular viability of human primary umbilical vein endothelial cells (HLIVECs). Both substances, even in combination, had no cytotoxic effects on HLIVECs. The viability of HUVECs was slightly increased by the combination of both substances in comparison to treatment with hyaluronic acid as single substance.

To investigate the biocompatibility of hyaluronic acid, calcium peroxide, and a combination thereof, a CAM assay was performed (Figure 4). The data of Figure 4A and 4B demonstrates that none of the tested substances had a cytotoxic effect on the CAM. The degradation of the sponge on which the different substances were applied was not different from the degradation of an empty sponge and no adverse tissue reactions to the substances were seen on the CAM. Angiogenesis, measurable in the difference of the area of the sponge that is covered by blood vessels was not significantly different in the CAM tissue treated with hyaluronic acid, calcium peroxide, or a combination thereof compared to untreated CAM tissue.

In summary, the combination according to the invention has been shown to be non-cytotoxic in all investigated cell types and in the CAM assay it proved to be biocompatible. ic bacteria

To demonstrate the inhibitory efficacy of calcium peroxide on planktonic bacteria, a selection of calcium peroxide concentrations was utilized. According to Figure 5, different concentrations of calcium peroxide were applied to mature biofilms in combination with hyaluronic acid. Calcium peroxide was added to planktonic bacterial strains S. oralis and P. gingivalis in concentrations ranging from 0 mg/L (control) to 500 mg/L with hyaluronic acid being present in a concentration of 11 .550 mg/L. Shown in each case are the optical density at 600 nm, a parameter for the growth of a bacterial culture, and the relative metabolic activity. At concentrations above 125 mg/L, the optical density decreased, showing that the growth of the periodontitis-causing bacterium P. gingivalis was significantly reduced.

In the Figure 5, upper left part, there is shown that the optical density of a planktonic bacterium of the physiological oral flora, S. oralis, decreased significantly at calcium peroxide concentrations of 250 mg/L and 500 mg/L compared to the control. In the lower left part, it is shown that the relative metabolic activity of S. oralis was unchanged compared to the control. In the Figure 5, top right part, it is shown that the optical density of the periodontitis-causing bacterium P. gingivalis significantly decreased at calcium peroxide concentrations of 125 mg/L, 250 mg/L, and 500 mg/L compared to the control. In the lower right part, it is shown that the relative metabolic activity of P. gingivalis significantly decreased at calcium peroxide concentrations of 125 mg/L, 250 mg/L, and 500 mg/L compared to the control.

These results show that a calcium peroxide concentration of 125 mg/L has a statistically significant inhibitory effect on the periodontitis-causing bacterium P. gingivalis, but not on S. oralis, a gram-positive, facultatively anaerobic bacterium of the streptococcus family which is found in the physiological oral flora. At concentrations of around 500 mg/L a stress response is induced in S. oralis by increasing concentrations of calcium peroxide, whereas a stress response in periodontal pathogenic bacterium P. gingivalis is already induced at significantly lower concentrations. However, at concentrations of greater than 125 mg/L, the oxygen showed a toxic effect regarding P. gingivalis. On the other hand, the growth of bacteria of the commensal flora, as presented by S. oralis, is only very slightly inhibited by concentrations of 125 mg/L. The data indicate a selective inhibition of the harmful bacteria, while the physiological flora remains largely unaffected. of calcium peroxide on biofilms

To show the effect of a combination of calcium peroxide and hyaluronic acid on biofilms, different concentrations were utilized and the biofilm volume, percentage of alive and dead cells as well as the relative metabolic activity of the periodontitis-causing bacterium P. gingivalis and the physiological bacterium of the oral flora S. oralis were measured. The results are shown in Figure 6 and 7. Figure 6 shows the effect of hyaluronic acid and calcium peroxide on mature P. gingivalis biofilms. In the Figure 6, left part, it is shown that the biofilm volume [pm ³ ] decreased significantly at calcium peroxide concentrations of 3.9 mg/L, 125 mg/L, and 500 mg/L compared to the control. In Figure 6, top right part, it is shown that the percentage of dead bacteria increased significantly at calcium peroxide concentrations of 3.9 mg/L, 125 mg/L, and 500 mg/L compared to the control. In addition, the percentage of dead bacteria increased significantly at calcium peroxide concentration of 500 mg/L compared with 3.9 mg/L and 125 mg/L. In the figure below right, it is shown that metabolic activity shows no significant difference.

Figure 7 shows the effect of hyaluronic acid and calcium peroxide on mature S. oralis biofilms. In the Figure 7, left part, it is shown that the biofilm volume [pm ³ ] increased significantly at a calcium peroxide concentration of 125 mg/L compared to the control.

In the Figure 7, top right part, it is shown that the percentage of dead bacteria increased significantly at calcium peroxide concentrations of 3.9 mg/L, 125 mg/L, and 500 mg/L compared to the control. In the Figure 7, lower right part, it is shown that the metabolic activity increased significantly at a calcium peroxide concentration of 500 mg/L compared to the control.

The results indicate that application to biofilms according to the invention leads to a significant inhibition of the growth of the anaerobic periodontitis-causing bacterium P. gingivalis by calcium peroxide concentrations of 3.9, 125 and 500 mg/L in hyaluronic acid. In contrast, aerobic bacteria of the physiological oral flora, such as S. oralis, are merely affected by the oxygen released from the matrix.

Figure 8 shows the slow oxygen release effect of the combination of hyaluronic acid and calcium peroxide compared to calcium peroxide or hyaluronic acid only. It is shown that the release of oxygen increased significantly if calcium peroxide is enclosed in a hyaluronic acid matrix. Furthermore, the combination of hyaluronic acid and calcium peroxide increased the amount of available oxygen as indicated by a larger area under the curve (AUC). The experiments were conducted at different pH levels (Fig. 8A at pH 2, Fig. 8B at pH 6, and Fig. 8C at pH 8) with either 50 mg calcium peroxide solved in 5 mL aqua (H ₂ O) or 5 mL hyaluronic acid matrix compared to baseline over a time period of 850 min.

Figure 9 shows the oxygen content of different biomaterial combinations with and without hyaluronic acid. Baseline: Development of the normalized dissolved oxygen content (norm. DO) as a function of time in 200 mL 0.1 M citric acid in 0.9w% NaCI solution at 34.1 °C. Blank curve composition I: Addition of 5 mL H ₂ O with 50 mg CaO ₂ . Composition I: 5 mL H ₂ O with 115 mg sodium hyaluronate and 50 mg CaO2. Blank curve composition II: Addition of 5 mL PBS buffer with 10 mg MHB, 50 mg CaO2, 250 mg Mg©2 and 200 mg ZnC>2. Composition II: Addition of 5 mL PBS buffer containing 10 mg MHB with 250 mg sodium hyaluronate, 50 mg CaO2, 250 mg Mg©2 and 200 mg ZnC>2. MHB: Preservative (methyl 4-hydroxybenzoate).

The data exemplify that the amount of available oxygen is multiplied by up to 5 times when inorganic peroxides are combined with hyaluronic acid or a derivative thereof. The amount of available oxygen (corresponding to the area below the curves in Fig. 9) can be further increased or varied by using Mg©2 and/or Zn©2 and thus adapted to the needs of the individual applications and tissues. In particular, it was a surprising discovery that oxygen is released over a longer period of time when inorganic peroxides are combined with hyaluronic acid or a derivative thereof.

Figure 10 shows that a special treatment during the process of production can increase the efficiency of the formulation to provide an increased oxygen concentration over a prolonged period of time. If calcium peroxide and hyaluronic acid components of the composition are simply mixed and then dissolved/suspended, the amount of oxygen available is significantly lower. The oxygen concentration and oxygen release over a prolonged period of time can be increased by first dissolving the preservative in the liquid matrix (e.g. PBS buffer), then dissolving the soluble components (e.g. plant extracts), followed by suspension of the insoluble components (e.g. peroxides, hydroxyapatite, plant extracts, etc.) and finally insertion of the hyaluronic acid under strong stirring.

In summary, the results indicate that the combination of one or more inorganic peroxide and hyaluronic acid effectively minimizes the growth of periodontic anaerobes without affecting the physiological flora. Because inorganic peroxide (as exemplified by CaO2) is trapped in the hyaluronic acid matrix, oxygen is released in a delayed manner, such that only non-harmful concentration oxygen reaches the cells through the hyaluronic acid matrix. At the same time, tissue regeneration is promoted and not adversely affected.

Most importantly, the beneficial effect of a combination of CaO2 and hyaluronic acid can be increased if an additional inorganic peroxide is present in the composition, i.e. Mg©2 and/or ZnC>2. In other words: not only the amount of available oxygen can be further increased or varied (Figure 9), but also the amount of end products of the utilized inorganic peroxides available to the tissue (Ca ²⁺ , Zn ²⁺ or Mg ²⁺ ) can be adapted in accordance with the tissue requirements.

Material and Methods Preparations of inorganic peroxide and hyaluronic acid

Calcium peroxide (Sigma Aldrich, St. Louis, MO, US), zinc peroxide and/or magnesium peroxide was suspended in water or buffered aqueous solution. The desired amount of hyaluronic acid sodium salt (HERRLAN-PSM e.K.) was added during vigorous stirring.

Preparations of inorganic peroxide and hyaluronic acid for bacterial assays

The autoclaved calcium peroxide (CaO2) (Sigma Aldrich, St. Louis, MO, US), ZnO2 and/or MgO2 was dissolved in sterile filtered, deionized, and autoclaved water. By serial dilutions with water (for planktonic experiments) or BHI/VitK (for biofilm experiments) the solutions were adjusted to concentration of 3.9 - 500 mg/l Ca02for bacterial experiments. All solutions were freshly prepared for each experiment.

Cell Isolation

Primary human cells were obtained from patients who underwent surgery at the University Medical Center Mainz, Germany. The use of residual materials was approved by the ethics committee of the Landesarztekammer Rheinland-Pfalz in accordance with the principles expressed in the Declaration of Helsinki and the International Harmonized Guidelines for good clinical practice (ICH-GCP). All patients provided written consent.

Fibroblasts were obtained from human oral mucosa. Tissue samples were cut into small pieces of approximately 2x2 mm with a sterile disposable scalpel. Prior to cell isolation, the tissue pieces were stepwise sterilized in 70% ethanol, in sterillium® classic pure (Bode Chemie GmbH, Hamburg, Germany), and again in 70% ethanol. Then they were transferred to 5-10 mL (depending on the amount of tissue) 0.5% protease solution (P6141 , Sigma-Aldrich, St. Louis, MO, US) in phosphate buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, US) and incubated overnight at 4°C. The next day, the protease solution was incubated while shaking for further 15 min at 37°C. The sample was then passed through a cell sieve (EASYstrainerTM 70 pm sterile, Greiner bio-one, Kremsmunster, Austria) with the help of a cell scraper (Falcon®, Corning, NY, US). Cells were pelleted by centrifugation (1 ,500 rpm, 5 min), were transferred to cell culture medium, and seeded into small cell culture flasks with a grow area of 25 cm ² . Cells were characterized morphologically and were used at most until passage 10 to ensure primary identity. Cells were maintained in DMEM/Ham's F12 (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum and antibiotics (10,000 U/mL penicillin and 10 mg/mL streptomycin; Sigma-Aldrich, St. Louis, MO, USA) at 37°C in 5% CO2. Primary human osteoblasts were isolated according to the following protocol. Human bone specimens were obtained during hip or knee joint replacement surgeries. The use of residual materials was approved by the ethics committee of the Landesarztekammer Rheinland-Pfalz in accordance with the principles expressed in the Declaration of Helsinki and the International Harmonized Guidelines for good clinical practice (ICH-GCP). All patients provided written consent.

Cancellous bone fragments were removed with bone rongeurs from bone specimens. The isolated fragments were washed several times with PBS (Sigma-Aldrich, St. Louis, MO, USA) until a clear supernatant was achieved. The supernatant was discarded and 15 mL collagenase type I solution (1 mg/mL in medium 199) was added. Collagenase digestion was carried out under mechanical stirring in a water bath at 37 °C. After 45 min, the fragments were washed again several times with PBS (Sigma-Aldrich). The washed bone pieces were transferred into 6- well tissue culture plates with sterile forceps, followed by addition of DMEM/F-12 medium supplemented with 20% fetal calf serum (FCS) and 1% penicillin/streptomycin (PS).

After the first passage, human osteoblasts were cultured in DMEM/Ham's F12 (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum and antibiotics (10,000 U/mL penicillin and 10 mg/mL streptomycin; Sigma-Aldrich, St. Louis, MO, USA). The medium was changed twice a week. For osteoblast differentiation, the medium was supplemented with 10 nM dexamethasone, 3.5 mM b-glycerophosphate, and 50 pg/mL ascorbic acid.

Primary Human Umbilical Vein Endothelial Cells (HUVEC) were isolated from the vein of the umbilical cord. The use of residual materials was approved by the ethics committee of the Landesarztekammer Rheinland-Pfalz in accordance with the principles expressed in the Declaration of Helsinki and the International Harmonized Guidelines for good clinical practice (ICH-GCP). All patients provided written consent.

The umbilical cord was flushed with PBS (Sigma-Aldrich) until the buffer was clear and blood clots in the vein were removed. Then, a collagenase solution was injected into the umbilical cord, and it was placed in warm PBS and incubated for 12 min at 37 °C. After incubation, the collagenase solution containing the endothelial cells was flushed from the cord perfusing the vein with PBS (Sigma-Aldrich). The effluent was collected and centrifuged for 10 min at room temperature with max. 420 g. Cells were kept in culture dishes coated with 3 mL 0.1% gelatin at 37°C for the first cell passages. Cells were incubated overnight at 37°C and washed with PBS (Sigma-Aldrich) the next day. Endopan 3 with 3% fetal blood serum (PAN Biotech, Aidenbach, Germany) was used as culture cell medium. Cellular Viability Assays

Cells were seeded into a 24-well-plate and they were given time to adhere overnight. The cell number per well was 50.000 for fibroblasts and HUVECs and 40.000 for osteoblasts each with respectively 1 .5 mL medium per well. After 24 h, cell culture medium was replaced by 400 pl fresh medium. Cells were treated with 0.256 mg/mL CaC>2 (Sigma Aldrich, St. Louis, MO, USA), or 30 mg/mL hyaluronic acid (Herrlan PSM e.K.) or a combination thereof. Untreated cells served as control. The substances were applied into inserts with a 10 pm thick translucent polycarbonate membrane (Corning Inc., New York, USA) with 0.4 pm pores. Those inserts then were inserted in the 24-well-plate and were thus in direct contact with the cell culture medium of the cells and incubated overnight. The inserts were removed after 24 h, the cell culture medium was exchanged for 1 mL of medium with 10% AlamarBlue™ Cell Viability Reagent (ThermoFisher Scientific, Waltham, MA, USA) and the cells were incubated for 4 h at 37°C. After 4 h, the liquid (200 pl per well) was transferred from the 24-well-plate to a black 96-well-plate (Greiner bio-one GmbH, Frickenhausen) for measurements. The AlamarBlue assay is based on the change of the blue color of the non-fluorescent indicator dye (resazurin) after acceptance of electrons, which, passing from the oxidized state to the reduced state, becomes a fluorescent pink compound. Fluorescence was measured on a Fluorescence Microplate Reader (Fluoroskan Ascent Microplate reader, ThermoFisher Scientific, Waltham, MA, USA). Results were given as relative fluorescence using a 538 nm excitation filter and a 600 nm emission filter, normalized to untreated control.

Chorioallantoic membrane assay (CAM Assay)

Fertilized white Leghorn chicken eggs (LSL Rhein-Main GmbH, Dieburg, Germany) were incubated at 38°C with constant humidity of 55 rH in an incubator (Type 3000 digital and fully automatic, Siepmann GmbH, Herdecke, Germany). For the first three days, eggs were placed horizontally on one side to ensure that the CAM would detach from the upwards pointing eggshell. On the embryonic development day (EDD) 3, eggs were prepared by removing 5-6 mL of the albumen in order to enlarge the space between eggshell and CAM. A small window of 3x2 cm was cut into the upwards pointing part of the eggshell. The window was covered with Parafilm® (Sigma-Aldrich, St. Louis, MO, USA) to prevent evaporation. On EDD-8, Gelaspon Strips (Bausch & Lomb Inc.; New York, USA) were cut in slices of 1x0.5x2 mm. 20 pl of each substance (512 mg/L CaO2, 10 mg/mL sodium hyaluronate, combination thereof) were added to the sponge. 20 pl of water was used as control. The further assessment of potential tissue adverse events was performed blinded. After 3 h, on EDD-10, and EDD-12, pictures were taken with a digital intravital fluorescence microscope (Olympus BXFM, Olympus Deutschland GmbH, Hamburg, Germany) at a 100-fold magnification using the cellSens Dimension software package. Microorganisms and culture conditions

Streptococcus oralis ATCC 981 1 was obtained from the American Type Culture Collection (ATCC) and Porphyromonas gingivalis DSM 20709 derived from the German Collection of Microorganisms and Cell Cultures (DSMZ). The strains were grown under anaerobic conditions (80% N ₂ , 10% H ₂ , 10% CO ₂ ) at 37°C for 24 h in 50 mL screw cap tube (Sarstedt, Numbrecht, Germany) with Brain Heart Infusion Medium (BHI; Oxoid, Wesel, Germany) containing 10 pg/mL vitamin K (Roth, Karlsruhe, Germany; BHI/VitK) to obtain 24-hour-old precultures for further processing.

Investigation of the planktonic bacterial growth and metabolic activity of S. oralis and

P. gingivalis under the influence of CaO ₂

In order to determine the influence of CaO ₂ on planktonic S. oralis and P. gingivalis, the 24-hour- old precultures were pelleted by centrifugation at 4,000 g for 15 min at 4°C, the supernatant was discarded and the bacterial pellets were resuspended in fresh double concentrated BHI/VitK medium (2x BH/VitK). The optical density was measured at 600 nm (OD ₆ oo; BioPhotometer, Eppendorf, Hamburg, Germany), adjusted to 0.2 and dilute 1 :10 with 2x BHI/VitK to 0.02. The freshly prepared CaO ₂ solution was mixed equally with the bacterial cultures (OD ₆ oo = 0.02) to a final OD ₆ OO = 0.01 and following final concentrations for CaO ₂ :

As positive controls, bacterial cultures (OD ₆ oo = 0.02) were mixed 1 :2 with sterile filtered, deionized and autoclaved water (growth controls). As negative controls, 2x BHI/VitK was mixed equally with sterile filtered, deionized and autoclaved water (medium control) and the CaO ₂ solution was mixed 1 :2 with 2x BHI/VitK (CaO ₂ solution control). Finally, 150 pL of each suspension was transferred into each well of a 96-well plate and cultivated for 24 h at 37°C rotating (180 rpm) under anaerobic conditions (Anaerobic Jar and AnaeroGen; Oxoid, Wesel, Germany). All experiments were carried out in three biological and two technical replicates.

To evaluate the effect of CaO ₂ on the growth of planktonic S. oralis and P. gingivalis cultures, the 24-h-old bacterial cultures were mixed and the optical density (OD ₆ oo) was measured with a plate reader (Tecan, Mennedorf, Switzerland). Metabolic activity was measured using the BacTiter- Glo™ Microbial Viability Assay (Promega, Mannheim, Germany). 50 pL BacTiter-Glo™ reagent and 50 pL of the well-mixed bacterial cultures were added to opaque 96-well plates. After 5 min of incubation at room temperature under light protection, the samples were mixed again and the amount of adenosine triphosphate (ATP) was determined by measuring the luminescence using the plate reader. All results were normalized to the medium or CaO ₂ solution controls. Investigation of CaO2 effect on S. oralis and P. gingivalis biofilms

To evaluate the effects of CaO2 on biofilms of S. oralis and P. gingivalis, 24-h-old pre-cultures were processed as described for the planktonic experiments, except that 1x BHI/VitK medium was used as well as OD ₆ oowas adjusted to 0.1 and diluted to 0.01 . A volume of 2 mL of each bacterial culture (OD ₆ oo = 0.01 ) was added to each well of a 6-well plate and cultivated for 24 h at 37°C under anaerobic conditions (Anaerobic Jar and AnaeroGen; Oxoid, Wesel, Germany). All experiments were carried out in three biological and two technical replicates.

After 24 h of growth, pH value was measured of all samples and controls with indicator sticks (PH-FIX indicator sticks pH 0-14; Macherey-Nagel, Duren, Germany). Supernatants were removed, 2 mL fresh BHI/VitK was added to the controls, and 2 mL of CaO2 solution was added to the biofilms that should be treated with following concentrations:

After two hours of incubation under anaerobic conditions (Anaerobic Jar and AnaeroGen; Oxoid, Wesel, Germany) at 37°C, the pH values of all samples and controls were measured again. The metabolic activity was determined using the BacTiter-Glo Microbial Viability Assay (Promega, Mannheim, Germany). Biofilms were rinsed twice with Phosphate Buffered Saline (PBS; Biochrom GmbH, Berlin, Germany) and 1 mL BacTiter-Glo reagent was added to each biofilm. The biofilms rinsed off with the reagent by pipetting up and down several times. After 5 min of incubation under rotation (180 rpm) and light protection, 100 pL of the solutions were added to opaque 96-well plates. Samples were mixed again and the luminescence was measured with the plate reader (Tecan, Mennedorf, Switzerland). In order to analyze the effect of CaO2 on biofilm volume and live/death distribution, the LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies, Carlsbad, California, USA) was used to stain the biofilms with SYTO 9 and propidium iodide according to the manufacturer's recommendations. Subsequently, the biofilms were rinsed twice with PBS and fixed with 2.5% glutardialdehyde (Roth, Karlsruhe, Germany) in PBS for 30 min. After fixation, the biofilms were washed twice and covered with 3 mL PBS. They were microscopically analyzed using a confocal laser scanning microscope (CLSM; Leica TCS SP8, Leica Microsystems, Mannheim, Germany) with excitation and emission wavelengths for SYTO 9 of 488 nm and 500-550 nm and for propidium iodide of 552 nm and 675-750 nm. Three images were taken from each biofilm using a z-step of 2 pm. 3D image processing and analysis of the biofilm volumes and proportions of dead and viable bacteria was performed using Imaris x64 software (version 8.4.1 , Bitplane AG, Zurich, Switzerland).

Oxygen Release Measurements 50 mg CaO2 (75% 200 mesh, Sigma Aldrich) was suspended in 5 mL deionized water (millipore grade). 115 mg of hyaluronic acid sodium salt (1 ,5 - 2,5MDa: Herrlan-PSM, Alpen, Germany) were added under vigorous stirring. The mixture was stirred for at least 4 h and filled without bubbles into a syringe.

The concentration profile of dissolved oxygen was determined by means of a HI 5421 (Hanna Instruments, Vohringen, Germany). The sensor was placed in a thermostated beaker right above a magnetic stirring bar. The beaker was filled with 200 mL of 0.1 M citric acid (>99,5%: Carl Roth, Karlsruhe, Germany) in 0.9wt% NaCI aqueous solution. The pH was adjusted by means of 1 M NaOH (Carl Roth). A cellulose filter bag was placed into the solution to enable free migration of dissolved substance but avoiding the free migration of the hyaluronic acid matrix or undissolved CaO2 particles.

The detector started to detect the concentration of dissolved oxygen (DO mode) and the baseline was detected for ca. 50 min. Then 5 mL of the test solution were added into the cellulose filter bag within 30 s. The concentration profile of the dissolved oxygen was detected for several hours at base line and with 50 mg CaO2 in 5 mL water either with or without hyaluronic acid sodium salt. The concentration of the dissolved oxygen was normalized to the concentration of the dissolved oxygen at the time when samples was added.

Statistical analysis

Graphic processing and statistical analysis were performed using the Graph Pad Prism software 8.4 (GraphPad Software Inc., La Jolla, USA). To determine whether the data are normally distributed, the Kolmogorov-Smirnov normality test was applied. If data were normally distributed, Ordinary One-Way ANOVA with Dunnett's correction for multiple comparisons was used for to determine statistically significant differences of treated samples compared to the controls. If data failed normal distribution assumption, Kruskal-Wallis test with Dunn's correction for multiple comparisons was used. The significance level was set to p < 0.05 for all comparisons.