WO2025024551A2 - DELIVERY OF Mg FOR PERIODONTAL TREATMENT - Google Patents

Abstract

The disclosed concept includes systems and methods for the delivery of magnesium to a patient to modulate the human immune system and create an anti-inflammatory periodontal environment to support tissue regeneration. Various mechanisms for the delivery of magnesium include magnesium-polymer (PLGA) microparticles, magnesium-polymer (PLGA) electrospun fibers, and/or magnesium-polymer (PLGA) implant devices, e.g., scaffolds, constructed of the magnesium-polymer (PLGA) electrospun fibers. These mechanisms provide a controlled- and/or sustained-release of magnesium for the treatment of one or more of periodontitis, peri-implantitis, and bone defects. The magnesium-polymer (PLGA) microparticles include magnesium metal nanopowder that is coated with a fatty acid or lipid.

Description

DELIVERY OF Mg FOR PERIODONTAL TREATMENT

STATEMENT OF GOVERNMENT INTEREST

[0001] This invention was made with government support under grant # 0812348 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 63/528,477 entitled “DELIVERY OF Mg FOR PERIODONTAL TREATMENT”, filed in the U.S. Patent and Trademark Office on July 24, 2023, the contents of which is incorporated herein in its entirety.

FIELD OF THE INVENTION

[0003] The disclosed concept pertains to systems and methods for the delivery of magnesium to modulate the human immune system and create an anti-inflammatory periodontal environment to support tissue and bone regeneration. Biodegradable magnesium-loaded polymer microparticles, electrospun magnesium-incorporated polymer fibers, and/or magnesium implant devices, e.g., scaffolds, provide treatment for periodontitis, peri- implantitis, and bone defects.

BACKGROUND OF THE INVENTION

[0004] Periodontal disease (PD) is a chronic inflammatory disease and one of the most common oral diseases, affecting about 42% of adults aged 30 years or older in the United States. PD is considered one of the leading causes of tooth and alveolar bone loss. The dental plaque biofilm is a major etiologic factor in the initiation of PD and serves as a niche for dysbiotic microbiota leading to an imbalance in the host-microbe homeostasis and host immune response. The resulting aberrant immune response inevitably causes the majority of tissue destruction and bone loss. In addition to its destructive nature, the inflammatory response in the periodontium has benefits during periodontal health and induces the tissues to go through a natural resolution process of healing and repair. However, in PD, normal resolution processes are disrupted, causing an increase in the destructive phase of inflammation. The byproducts of tissue breakdown during the destructive phase provide nutrients for periodontal pathogens, leading to a vicious cycle of periodontal inflammation that results in poor repair and chronic disease. Current standards of therapy, such as scaling and root planing, and local antimicrobial delivery disregards the host response, which is responsible for the majority of the damage and disease progression. Thus, modulation of the host response is a crucial target for treating PD.

[0005] Magnesium (Mg) is an abundant intracellular cation that plays a key role in osteogenesis and immune homeostasis and, has anti-inflammatory and immunomodulatory properties promoting the polarization of M2 macrophages. Mg facilitates the process of bone repair by stimulating the local production of calcitonin gene-related peptide (CGRP) and inhibits the release of inflammatory cytokines by activated immune cells. Although Mg deficiency has been linked to impaired skeletal homeostasis in mice and increased risk of PD in humans, the exact role of Mg in periodontal health and disease has yet to be understood.

[0006] Thus, there is a need in the art to design and develop systems and methods for local Mg delivery as a potential periodontal treatment for inflammatory alveolar bone loss. The treatment of PD is based on modulating the immune response through local induction and recruitment of regulatory immune cells such as M2 macrophages which contribute to the resolution of inflammation.

[0007] The disclosed concept includes locally controlled Mg delivery systems and methods for treating periodontal tissues. The disclosed concept includes fabricating Mg-polymer sustained release microparticles (e.g., Mg-PLGA), magnesium-incorporated electrospun nanofibers and scaffolds (e.g., Mg-incorporated PLGA nanofibers and scaffolds), and translating the antiinflammatory and anti-osteoclastic properties of Mg into a therapeutic approach for treating periodontitis, peri-implantitis, and bone defects.

SUMMARY OF THE INVENTION

[0008] In one aspect, the disclosed concept includes a magnesium-polymer composite that includes a plurality of magnesium-polymer microparticles, including magnesium metal nanopowder; a lipid applied to or deposited on the magnesium metal nanopowder to form coated magnesium metal nanopowder; polymer combined with the coated magnesium metal nanopowder to form the plurality of magnesium-polymer microparticles, wherein the magnesium-polymer implant device effectively provides a controlled and/or sustained-release of magnesium for treatment of one or more of periodontitis, peri-implantitis, and bone defect. [0009] The lipid may include a saturated fatty acid. The saturated fatty acid may be selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, and combinations and mixtures thereof. The lipid may include an unsaturated fatty acid. The unsaturated fatty acid may be selected from the group consisting of myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, eicosanoic acid, erucic acid, docosahexaenoic acid, hexadecadienoic acid, and combinations and mixtures thereof.

[0010] In certain embodiments, the lipid is oleic acid. In certain embodiments, there is 2% lipid WAV, lipid/magnesium metal. In certain embodiments, there is 2% oleic acid WAV, oleic acid/magnesium metal.

[0011] The plurality of magnesium-polymer microparticles may be in the form of microspheres. In certain embodiments, the polymer comprises PLGA.

[0012] In another aspect, the disclosed concept provides a magnesium-polymer implant device, including electrospun magnesium-polymer fibers, including magnesium metal nanopowder; and polymer, wherein the magnesium-polymer implant device effectively provides a controlled and/or sustained-release of magnesium for treatment of one or more of periodontitis, peri-implantitis, and bone defect.

[0013] The electrospun magnesium-polymer fibers may further comprise calcitonin gene- related peptide. The electrospun magnesium-polymer fibers may be configured to form a sheet.

[0014] In still another aspect, the disclosed concept provides a method of preparing a magnesium-polymer implant device, including combining magnesium metal nanopowder and polymer to produce a precursor mixture; and electrospinning the precursor mixture to produce a plurality of magnesium-polymer electrospun fibers; and configuring the plurality of magnesium-polymer electrospun fibers into an implant device, wherein the magnesium- polymer implant device effectively provides a controlled and/or sustained-release of magnesium for treatment of one or more of periodontitis, peri-implantitis, and bone defect. [0015] The precursor mixture may further include calcitonin gene-related peptide [0016] The method may further include harvesting periosteum; and culturing periosteal derived cells with Mg²⁺ ions and calcitonin gene -related peptide.

[0017] In certain embodiments of the method, the polymer comprises PLGA.

[0018] In yet another aspect, the disclosed concept includes a method of preparing a magnesium-polymer composite, including forming a plurality of magnesium-polymer microparticles, including obtaining magnesium metal nanopowder; applying or depositing a lipid onto the magnesium metal nanopowder to form coated magnesium metal nanopowder; mixing the coated magnesium metal nanopowder with polymer to form the magnesiumpolymer microparticles; and locally delivering the magnesium-polymer microparticles to a target location in a patient, wherein the magnesium-polymer microparticles effectively provide a controlled and/or sustained-release of magnesium for treatment of one or more of periodontitis, peri-implantitis, and bone defect.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1 and 2 are scanning electron microscopy images of Mg-PLGA microparticles, in accordance with certain embodiments of the disclosed concept.

[0020] FIG. 3 is a plot that illustrates the Mg2+ release curve measured by ICP-MS, in accordance with certain embodiments of the disclosed concept.

DETAILED DESCRIPTION:

[0021] As used herein, the singular form of “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0022] As used herein, the statement that two or more parts or components are “coupled” shall mean that the parts are joined or operate together either directly or indirectly, i.e., through one or more intermediate parts or components, so long as a link occurs.

[0023] As used herein, the term “number” shall mean one or an integer greater than one (i.e., a plurality).

[0024] Directional phrases used herein, such as, for example and without limitation, top, bottom, left, right, upper, lower, front, back, and derivatives thereof, relate to the orientation of the elements shown in the drawings and are not limiting upon the claims unless expressly recited therein.

[0025] The disclosed concept will now be described, for purposes of explanation, in connection with numerous specific details in order to provide a thorough understanding of the subject invention. It will be evident, however, that the disclosed concept can be practiced without these specific details without departing from the spirit and scope of this innovation. The disclosed concept includes systems and methods for the delivery of magnesium (Mg) for periodontal-related treatments to modulate the human immune system and create an antiinflammatory periodontal environment to support tissue regeneration. Mg in the form of microparticles, electrospun fibers, and implant devices, e.g., scaffolds, provide one or more of periodontitis, peri-implantitis and bone defect treatments. [0026] Mg is an important anti-inflammatory and immunomodulatory intracellular cation that has anti-inflammatory and immunomodulatory properties promoting the polarization of M2 macrophages, and its deficiency can result in a dysregulated immune response and impaired tissue turnover. There is a close relationship between Mg and the immune system, especially the regulation of the inflammatory response. In general, Mg modulates the immune system by polarizing macrophages to a M2-like phenotype creating an anti-inflammatory environment supporting tissue regeneration. It has been found that periosteal cells exposed to Mg2+ concentration facilitates the process of fracture healing by stimulating local production of CGRP. Alkaline phosphatase activity is regulated by CGRP and Mg2+.

[0027] The polymer for use in the disclosed concept is selected from polymers known in the art to exhibit both biocompatability and biodegradability, such as, PLGA. For ease of description, “PLGA” is identified in various embodiments herein. However, it is contemplated and understood that “PLGA” is an example of a polymer suitable for use in the disclosed concept and therefore, recitation of “polymer” in the disclosed concept is not limited to PLGA. PLGA is a widely used degradable polymer that has been approved by the U.S. Food and Drug Administration (FDA) for clinical use, and is applied mainly for drug delivery and implantable devices. The degradation rate of PLGA can be tailored specifically for the intended applications, by adjusting the ratio between lactic and glycolic acid and its molecular weight. PLGA degrades slower with higher lactic acid content and molecular weight.

[0028] The disclosed concept includes (i) Mg metal nanopowder in biodegradable polymer microparticles (Mg-PLGA microparticles), (ii) Mg metal nanopowder electrospun into polymer (PLGA) fibers, and (iii) polymer (PLGA), Mg, and CGRP implant devices, e.g., scaffolds.

[0029] In certain embodiments, the Mg-PLGA microparticles are locally delivered to a target location in a patient to effectively provide a controlled- and/or sustained-release of Mg for prevention therapy and/or interventional therapy and/or reparative therapy of one or more of periodontitis and peri-implantitis defects.

[0030] In certain embodiments, the Mg-PLGA microparticles are locally delivered to a target location in a patient to effectively provide a controlled- and/or sustained-release of Mg for calvarial bone defects.

[0031] The Mg-PLGA microparticles are effective to ameliorate alveolar bone loss. Without being bound by any particular theory, this local Mg treatment upregulates the mRNA expression of M2 macrophage markers and downregulates the expression of pro-inflammatory markers.

[0032] Preventive therapy for one or more of periodontitis and peri-implantitis, includes administration of Mg to prevent its initiation (or maintenance). The Mg-PLGA microparticles are locally delivered to target site of a patient that is at risk for one or more of periodontitis and peri-implantitis. In certain embodiments, the preventive therapy includes local delivery, such as by injection (e.g., locally injected in the buccal and palatal gingiva of the diseased teeth) of the Mg-PLGA microparticles. In the preventative therapy, the Mg-PGLA microparticles are effective to ameliorate initiation and induce an anti-inflammatory gene expression profile in gingival tissues.

[0033] Interventional therapy for one or more of periodontitis and peri-implantitis, e.g., includes administration of Mg to the diseased site (already inflammation is present) to reduce or preclude its progression. The Mg-PLGA microparticles are locally delivered to a target site of a patient with one or more of periodontitis and peri-implantitis. In certain embodiments, the interventional therapy includes local delivery, such as by injection (e.g., locally injected in both buccal and palatal gingiva of the diseased teeth) of the Mg-PLGA microparticles. In the interventional therapy, the Mg-PGLA microparticles are effective to ameliorate progression and induce an anti-inflammatory gene expression profile in gingival tissues.

[0034] Reparative therapy for periodontitis and/or peri-implantitis, includes the local delivery to a target site of a patient, such as by injection (e.g., locally injected in both buccal and palatal gingiva of the diseased teeth) of the Mg-PLGA microparticles.

[0035] The Mg-PLGA microparticles include Mg metal nanopowder embedded or encapsulated in PLGA. The Mg metal nanopowder is treated with a fatty acid or lipid. In certain embodiments, a coating including a fatty acid, e.g., a lipid, is applied to or deposited on the exterior surface of the Mg metal nanopowder. The feature of the Mg metal being embedded and/or encapsulated into the PLGA and the Mg metal including the applied or deposited fatty acid or lipid coating, provides the capability for controlled- and/or sustained-release of the Mg ion. A wide variety of suitable fatty acids and lipids are known in the art. In certain embodiments, the lipid is a saturated fatty acid or an unsaturated fatty acid. Suitable saturated fatty acids include, but are not limited to, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, and combinations and mixtures thereof. Suitable unsaturated fatty acids include, but are not limited to, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, eicosanoic acid, erucic acid, docosahexaenoic acid and hexadecadienoic acid. In certain embodiments, the lipid is oleic acid. In further embodiments, the coating composition includes 2% fatty acid or lipid, e.g., oleic acid (WAV, lipid/Mg metal).

[0036] The shape of the Mg-PLGA microparticles vary and in certain embodiments, the Mg- PLGA microparticles are in the shape of microspheres. The Mg-PLGA microparticles, e.g., microspheres, are prepared using an oil-water single emulsion procedure. In certain embodiments, Mg metal nanopowder (800nm, 99%) is coated with a fatty acid or lipid, e.g., 2% oleic acid (W/W). The coating thickness can vary and in certain embodiments, is about 20 nm. The PLGA microparticles are prepared by mixing oleic acid-coated Mg metal nanopowder (e.g., about 50 mg) with PLGA (e.g., about 200 mg) dissolved in dichloromethane (e.g., about 4 mL). The mixture is vortexed (e.g., for about 10 seconds and sonicated (e.g., for about 10 seconds) if there is a large sized magnesium aggregate. This solution is transferred to an aqueous solution of 2% polyvinyl alcohol and homogenized (e.g., for 60 seconds at 1000 RPM). This solution is then mixed with pre-cooled 1% polyvinyl alcohol and stirred to allow evaporation of dichloromethane. The microparticles are then collected by centrifugation and washed with DI water to remove residual polyvinyl alcohol, before being re-suspended in DI water, frozen, and lyophilized.

[0037] In certain embodiments, the Mg-PLGA microparticles are used to treat periodontitis or peri-implantitis. In other embodiments, the Mg-PLGA microparticles are used to treat bone defects, such as calvaria defects.

[0038] The disclosed concept further includes Mg metal nanopowder/PLGA electropsun fibers. The Mg/PLGA fibers exhibit the ability of Mg to regenerate bone by modulating the immune response thereby providing treatment. Electrospinning, an electrostatic fiber fabrication technique, is used to shape the electrospinning solution including PLGA supplemented with Mg metal nanopowder, and optionally CGRP peptide, into uniform 3D nanofibers. The overlaying structure of the Mg/PLGA electrospun fibers closely mimics the extracellular matrix, providing tunable porosity, high surface area to volume ratio, better support to the cells, and stronger cell adhesion.

[0039] The release profile of Mg ion, and optionally CGRP, from the PLGA nanofibers is controlled or adjusted, such that the implanted scaffold in a bone defect is effective for bone regeneration. In certain embodiments, the Mg/PLGA electrospun fibers are used to treat bone defects, such as calvaria defects. In other embodiments, a combination, mixture or blend of Mg/PLGA electrospun fibers and Mg/PLGA microparticles are used to treat bone defects, such as calvaria defects.

[0040] In other embodiments, the Mg/PLGA electrospun fibers are used to treat periodontitis. In certain of these embodiments for periodontitis, the Mg/PLGA electrospun fibers are in the form of a sheet. The disclosed concept also includes biodegradable implant devices constructed from the electrospun Mg metal nanopowder/PLGA fibers. The implant device, e.g., scaffold, includes PLGA, Mg, i.e., Mg²⁺ ions, and optionally CGRP. In certain embodiments, periosteal-derived cells (PDCs) are cultured with varying concentrations of Mg and optionally CGRP. The electrospinning solution includes PLGA supplemented with Mg metal nanopowder and optionally CGRP.

[0041] Electrospinning is used to shape the PLGA, supplemented with Mg metal nanopowder and optionally CGRP, into electrospun nanofibers. The electrospun nanofibers form a PLGA scaffold that is implanted into a bone defect of a patient for the sustained-release of Mg²⁺ ions and optionally CGRP into the periosteum for bone generation, e.g., the formation of new bone adjacent to the Mg bone implant.

[0042] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word “comprising” or “including” does not exclude the presence of elements or steps other than those listed in a claim. In a device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The word “a” or “an” preceding an element does not exclude the presence of a plurality of such elements. In any device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain elements are recited in mutually different dependent claims does not indicate that these elements cannot be used in combination.

[0043] Although the invention has been described in detail for the purpose of illustration based on what is currently considered to be the most practical and preferred embodiments, it is to be understood that such detail is solely for that purpose and that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover modifications and equivalent arrangements that are within the spirit and scope of the appended claims. For example, it is to be understood that the present invention contemplates that, to the extent possible, one or more features of any embodiment can be combined with one or more features of any other embodiment. EXAMPLES

[0044] A locally controlled Mg delivery strategy was developed for periodontal tissues and their therapeutic efficacy was evaluated. The aim was fabricating Mg-PLGA sustained-release microparticles and translating the anti-inflammatory and anti-osteoclastic properties of Mg into a therapeutic approach for PD.

EXAMPLE 1

1. Materials and Methods

1.1 Fabrication and Characterization of Poly (lactic-co-glycolic) acid -Magnesium Metal Microparticles (PLGA-Mg)

[0045] PLGA microspheres containing magnesium (Mg) metal were prepared using an oilwater single emulsion procedure. Mg nano-powder (800nm, 99%) was from US Research Nanomaterial, Inc. and was coated with 2% Oleic acid (W/W). The coating thickness was 20 nm by calculation. Briefly, the PLGA (RG502H, Sigma) microspheres were prepared by mixing 50 mg of Oleic acid coated Mg with 200 mg of polymer dissolved in 4 mL of dichloromethane, vortexed for 10 seconds and sonicated for 10 seconds if there is a large sized magnesium aggregate, continually stir at 125 rpm at the room temperature for at least 1 hour. This solution was quickly transferred to 60 mL an aqueous solution of 2% polyvinyl alcohol (pre-cooled) (M.W. ~25,000, 98 mol. % Hydrolyzed, PolySciences, Warrington, PA) and homogenized for 60 seconds at 1000 RPM. This solution was then mixed with pre-cooled 1% polyvinyl alcohol and placed on a stir plate agitator (with ice) for 3 hours to allow the dichloromethane to evaporate. The microspheres were then collected and washed 3 times in pre-cooled deionized (DI) water, to remove residual polyvinyl alcohol, before being resuspended in 5 mL of DI water, frozen, and lyophilized for 72 hours. Blank PLGA microspheres containing 1 mg of oleic acid were used as the control and were fabricated in the same manner (50 mg of Oleic acid coated Mg contains 1 mg of Oleic acid by calculation).

[0046] PLGA-Mg particles were gold-coated and examined on scanning electron microscope (JSM 6335F, CBI) operating at 3 KV/5KV. Mg ion release from microparticles was determined by suspending 4 mg of microspheres in 1 mL of phosphate buffered saline (PBS without Mg & Ca) placed on a Fisher Mini Tube Rotator at 37°C (18 rpm). Mg ion release sampling was conducted at various time intervals (0, 4 hr, and days at 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 25 and 30) by centrifuging microspheres and removing the supernatant for Mg ion quantification using Inductively Coupled Plasma (ICP) analysis. For the ICP assay, the 50 ul of collected supernatants were mixed with 50 pl of the distilled HN03 (67 to 70%) and incubated at 65 °C overnight; the samples were centrifuged at 15000 x g for 10 min, then the supernatant of samples were mixed with 5 mL of 2% distilled HN03 at room temperature in a 15 ml falcon tube. The samples were vigorously mixed three times (15 s each time) with 40pl of an internal standard (provided by Department of Geology at University of Pittsburgh) in a Vortex-mixer, the mixture was ready for ICP-MS isotope analysis (NexION 300).

1.2 Murine ligature-induced periodontal disease

[0047] Mice were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg) and periodontal disease was induced in 8-10-week-old male BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) (n=6) weighing 20-25 grams, by inserting 6.0 silk sutures (Henry Schein) between the maxillary second molar with the knot placed at the palatal side to induce plaque buildup. The contralateral side was left non-ligated to be used for the normalization of measurements. For the preventive experiment model, a ligature was placed on day 0 and left in place for 7 days; PLGA MPs delivery was on day 0, and sacrifice was on day 7. For the interventional experiment model, a ligature was placed on day 0 and left in place for days; PLGA MPs delivery was on day 4, and sacrifice on day 10. This study was approved by Institutional Animal Care and Use Committee of the University of Pittsburgh (protocol identification number 15053781).

1.3 Mg Microparticle delivery and sample collection

[0048] Following ligature placement, mice were divided into three groups: 1) Untreated (ligature placement without MPs injection), 2) Mg MPs (ligature placement and Mg MPs injection on same day or after 4 days) and 3) Blank MPs (ligature placement and blank MPs injection on same day or after 4 days). 50 pl of either Mg or blank MP suspended in 2% carbo xymethylcellulose in PBS (10 mg MP/ml) were locally injected in the buccal and palatal gingiva of the ligated maxillary second molar. 20 pl were injected in the mid-buccal aspect and the remaining 30 pl were distributed on the mesial, middle, and distal palatal aspects of the ligated maxillary second molar. Mg and blank MPs were injected into gingival tissues as preventive therapy on the same day as ligature placement and mice were sacrificed on Day 7 for the preventive model. Mg and blank MPs were injected into gingival tissues as an interventional therapy after 4 days of ligature placement and mice were sacrificed on Day 10 for the interventional model. The contralateral side was used for healthy control. 1.4 Alveolar bone loss quantification

[0049] Mice maxillae were harvested and fixed for 24 hours in 10% formalin, then transferred to 70% ethanol. Following formalin fixation, maxillae were scanned using micro-computed tomography system (pCT) (Scanco pCT 50, Scanco Medical) for quantification of alveolar bone loss. A resolution of 10pm voxel size, 55KVp, 0.36 degrees rotation step (180 degrees angular range) and a 1200 ms exposure per view were used. All scans were reoriented with DataViewer software (GE Healthcare) to a standardized orientation guided by pre-defined anatomical landmarks. For alveolar bone loss evaluation, reoriented images were used to measure the distance between the cementoenamel junction (CEJ) of the maxillary second molar and the level of the alveolar bone crest (ABC) on the mesial, distal, buccal and palatal aspects using CTAN software (Broker). For mesial and distal measurements, the CEJ to ABC distance were measured on 10 measurement slices with a distance interval of 30 pm in between. For the buccal and palatal measurements, the CEJ to ABC distance were measured on 10 measurement slices with a distance interval of 30 pm in between. The average measurements on each aspect on the ligated side were normalized to the corresponding averages on the healthy side of the same maxilla. 3D reconstruction and viewing of images were conducted on Scanco Micro-CT software (HP, DEC windows Motif 1.6).

1.5 Quantitative polymerase chain reaction (qPCR) analysis

[0050] For the gene expression analysis, a second cohort of mice were sacrificed to harvest gingival tissues which RNA was extracted for real-time PCR (qPCR). Mice were sacrificed and placed on ice immediately. The maxillary gingival tissues around the molars were dissected using a microsurgical blade and the dissected tissues were stored in RNA later solution. Next, stored tissues were homogenized using a tissue homogenizer. RNA was extracted from homogenized tissue samples using RNA easy Mini-Kit. After assessing the quality and concentration of extracted RNA, qPCR was conducted to analyze the expression of pro- inflammatory (IL-6, IL-17a and Nos2) and anti-inflammatory (Argl, CCL8, IL-10, IL-1RN and CCL19) markers by qPCR using TaqMan probes (Applied Biosystems, Foster City, CA). qPCR was performed using QuantStudioTM 6 Flex System (Thermo Fisher Scientific). Data were analyzed using the 2“^AACT method and presented as the mean fold change normalized to the healthy control group. 1 .6 Statistical analysis

[0051] Statistical analysis was conducted in GraphPad Prism v9. One-way ANOVA was used to compare experimental groups followed by a Tukey post-hoc test. Results were presented as mean ±SD and statistical significance was considered at p <0.05. The sample size was calculated using G*Power 3. 1 software. A power level of 0.8 and an alpha of 0.05 was used in estimating the sample size.

2. Results

2.1 Sustained release of Mg from microparticles

[0052] SEM scanning of the Mg MPs showed uniform spheres with different degrees of surface porosity (FIGS. 1 and 2). The Mg ion release curve (FIG. 3) started with a burst release of encapsulated Mg followed by a gradual release observed at 5 days increments up to 30 days.

2.2 Local sustained release of Mg ameliorated inflammatory alveolar bone loss

[0053] The pCT results from the preventive model showed that local delivery of Mg MPs prior to PD induction significantly prevented interdental, buccal and palatal bone loss compared to untreated or blank MPs groups.

[0054] It was then evaluated if the activity of PD following Mg delivery affected the protective effect against bone loss. pCT analysis from the interventional model revealed that local delivery of Mg MPs after 4 days from the start of PD induction (ligature placement) for 10 days, inhibited further interdental, buccal and palatal bone loss when delivered in the presence of active inflammation in mice (day 4) compared to untreated and blank MPs groups. These results indicate that the ameliorating effect of local sustained Mg release against inflammatory bone loss is effective in the presence of active PD.

2.3 Mg induced an anti-inflammatory cytokine expression profile in gingival tissues

[0055] Generated qPCR data showed gingival tissues that received Mg MPs as preventive therapy expressed lower levels of pro-inflammatory (IL-6 and IL-17a) and Ml-like macrophage (Nos2) markers and higher levels of anti-inflammatory (CCL8 and IL- 10) and M2-like macrophage markers compared to untreated (ligature only) mice or mice that received blank MPs.

[0056] Similar to the preventive model, there was assessed the expression of gingival inflammatory markers by qPCR. Mg MPs were locally delivered 4 days after the initiation of murine ligature PD induction for 10 days (Interventional model). Interventional Mg MPs delivery resulted in higher expression of anti-inflammatory and M2-like macrophage markers and lower expression of pro-inflammatory and Ml -like macrophage markers compared to untreated (ligature only) mice or mice that received blank MPs.

EXAMPLE 2

1. Materials and Methods

1.1 Experimental Treatment and Western Blot Analysis of Mouse and Human PDCs

[0057] Varying concentrations of MgSO4 and Na2SC>4 (sulfate control) (1.2 mM, 5 mM, 10 mM, and 15 mM) were added in osteogenic induction medium, which is Eagle’s minimal essential medium (DMEM) supplemented with 10% FBS, 1 nM dexamethasone, 50 pM ascorbic acid, and 20 mM [3-glycerol phosphate.

[0058] Mouse and human PDCs were plated in 6- well plates in a density of 3.5x10⁵ cells/well. After 24 hr, cells were treated with various concentrations of MgSO4 and NazSCH for 3 days. At the end of the treatment, cells were harvested for Western blotting analysis. Antibodies against human SP7/Osterix (R&D Systems, MAB7547) and Runx2 (Santa Cruz, sc-390351) were diluted in 1:500 in blocking buffer for incubation of the membranes overnight at 4°C, followed by 1 hr incubation of HRP-conjugated secondary antibody (dilution of 1 :2,000 in blocking buffer). After washing the membranes with TBST, HRP signals were detected using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, IL, USA).

1.2 Alkaline phosphatase (ALP) assay

[0059] Alkaline phosphatase (ALP) is a marker of osteogenic activity. Its expression has been associated with osteoblast differentiation. Quantitatively measured was the concentration of ALP expression using the SensoLyte pNPP ALP assay kit (AnaSpec, California, USA). In brief, PDCs were plated in 12-well plates in a density of IxlO⁵ cells/well. After 1 and 2 weeks of treatment in 12-well plates with biological triplicates, PDCs were first washed twice with dilution buffer before lysed. Cell suspension was incubated at 4°C for 10 min under agitation and centrifuged at 2500 g for 10 min. 25 l of standards and samples were first pipetted into a 96-well plate. Immediately after, 25 pl of pNPP substrate was added into each well containing standards and samples. Mixture was incubated in a dark room for 30 min before reading the absorbance with a plate reader at a wavelength of 405 nm. Total concentrations of protein from the cell lysate were also determined using the hicinchoninic acid (BCA) assay (Pierce BCA protein assay kit, ThermoFisher Scientific, MA) to normalize the ALP readings.

1 .3 Quantitative polymerase chain reaction (qPCR) analysis

[0060] Mouse and human PDCs were plated in 6-well plates at a density of IxlO⁶ cells/well and treated with osteogenic induction medium, 1.2 mM and 10 mM MgSO4 supplemented in osteogenic induction medium. Total RNA extraction and purification were performed using RNeasy Mini Kit (Qiagen, Valenia, CA) following manufacturer’s instructions. The quantity and quality of the extracted RNAs were measured using NanoPhotometer (Implen, p330). Samples were used only if the quality of absorbance readings 260/280 were higher than 1.5, and 260/230 were between 1.8 and 2.0. The extracted RNAs were reverse transcribed to cDNA and amplified using TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems, 4392938). qPCR was performed using StepOnePlus Real-Time PCR Systems (Applied Biosystems) under repeated thermal cycling conditions consisting of 95°C denaturing for 15 sec and 60°C annealing for 60 sec. Eukaryotic 18S (Applied Biosystems, 4352930E) was selected to be the endogenous control. Samples from each group were triplicated. Following the RNA extraction, a technical triplicate was performed for the qPCR analysis.

1.4 Alizarin Red S staining

[0061] Mouse and human PDCs were plated in a 6-well plate at a density of 3.5 x 10⁵ cells/well in growth medium. 24 hr after plating, the medium was switched to osteogenic induction medium with the addition of various MgSO4 and Na2SO4 concentrations and, IO ¹⁰ M CGRP with three biological replicates per group. After 21 days of treatment, cells were fixed in 10% formalin for 1 hr and washed with deionized and distilled water. A solution of 2% Alizarin Red S (Electron Microscopy Sciences, PA, USA) dissolved in 2% ethanol was adjusted to pH of 4.2 and used to detect the extracellular calcium deposits produced by the PDCs. Cells were stained with 2% Alizarin Red S solution for 20 min. The stain was removed and washed with distilled water repeatedly after incubation.

1.5 Electrospinning of Mg metal nanopowder and CGRP solution in PLGA fibers

[0062] High molecular weight (54,000- 69,000) poly (lactic-co-glycolic acid) (PLGA, Sigma- Aldrich, MO, USA) was dissolved in hexafluoroisopropanol (HFIP) to make 16% PLGA solution. After rotating on a rotator at room temperature overnight, 100 mg of Mg metal nanopowder (800 nm, 99% metal basis, US Research Nanomaterials, Inc. TX, USA) was added into 1 mL of PLGA solution. The mixture was sonicated for 30 min before rotating for another 4 hr before electrospinning. Similarly, 10"⁸ M CGRP mixed in water was added to 1 mL of PLGA solution, rotated in 4°C overnight, and sonicated for 15 min before spinning. The apparatus for electrospinning consists of a syringe pump with the clamp and the ground collector for the deposited 1'ibers. A high voltage source connects the needle tip and the collector. 1 mL of Mg nanopowder/PLGA solution was dispensed into a syringe of 12.45 mm diameter and clamped onto the pump, which was set with 18 kV voltage and 1 mL/hr pumping rate. The distance between the tip of the needle and the ground collector was adjusted to 15 cm. PLGA scaffolds were stored in vacuum overnight to allow evaporation of HF1P before further processing.

1.5.1 In vitro release study of the electrospun scaffold

[0063] Electrospun PLGA fiber sheets were punched into 5 mm diameter circular scaffolds using a multi-size hole punch (McMaster-Carr, Elmhurst, IL). The scaffolds including either Mg metal nanopowder or CGRP solution were placed in 1 mL PBS solution in a 1.5 mL microcentrifuge tube and rotated on a rotator in 37°C incubator. Samples were collected after first hour and once daily until complete degradation of the scaffold. At each time point, 500 pl of the PBS solution was collected from each tube and replaced with 500 pl fresh PBS. For scaffolds containing Mg metal nanopowder, 100 pl of samples were 50x diluted in 2% filtered nitric acid and measured for Mg ion concentration using an inductively coupled plasma mass spectrometry (ICP-MS) machine (NexION 300X, PerkinElmer, MA). For scaffolds containing CGRP solution, protein concentration was analyzed using enzyme-linked immunosorbent assay (CGRP EIA kit, Cayman Chemical, MI). Sample size was three for each group.

1 .6 Rat calvaria defects implantation

[0064] Sprague-Dawley rats (Charles River, USA), 16 male and 14 female, with 7-9 weeks of age were used in this study. Two rats were housed in each cage in a controlled environment. Rats were randomly assigned into 8 testing groups: empty defect controls +/- periosteum (abbreviated as Empty -, Empty +), PLGA scaffolds +/- periosteum (PLGA -, PLGA +), PLGA scaffolds electrospun with Mg metal nanopowder +/- periosteum (Mg -, Mg +), PLGA scaffolds electrospun with CGRP solution +/- periosteum (CGRP -, CGRP +). Two samples were implanted in each rat. Each control group has seven samples (three in female and four in male rats), and each experimental group has eight samples (four in female and four in male rats). Rats were anesthetized with isoflurane through a nosecone. A 2 cm incision on the skin and the fascia underneath was created by blunt dissection. Periosteum above the bone was carefully retracted. Two circular defects, each 5 mm in diameter, were cut by a trephine in parallel on either side of the sagittal suture in the bone of the cranium. The scored calvaria was removed by lifting from the edge by an elevator. Sample was filled into each empty defect. Periosteum was sutured on top of the implant in groups containing the membrane and removed in groups without periosteum. The fascia and incision were closed with sutures. After 1 month, rats were anaesthetized and euthanized. Calvaria and the soft tissue around the defects was explanted and fixed in buffered 10% formalin.

1.7 High resolution micro-computed tomography (pCT) analysis

[0065] Explants were immersed in 70% ethanol before placing in a 34 mm diameter tube and scanned in high resolution pCT 50 compact cabinet microCT scanner (Scanco Medical, Briittisellen, Switzerland) with voltage set at 55 kVp and current at 145 mA. 17.2 m voxel size, 0.36 degrees rotation step (180 degrees angular range) and an 800 ms exposure per view were used for the scans which were performed in 70% ethanol. Scanco pCT software (HP, DEC windows Motif 1.6) operating in an Open VMS environment was used for reconstruction of the 3D volume, viewing of the images, and creating 3D renderings. Newly formed bone volume in the rat calvaria defect was evaluated using the CTAn (Bruker-Skyscan, Aartselaar, Belgium) 3D analysis software after directly importing the calibrated for mineral density .isq format files from the Scanco software, using an available in CTAn function for the purpose. A 5 mm diameter circular region of interest (ROI) was drawn in the defect area on sequential slices. Volumes were segmented using a global threshold corresponding to 0.5 g HA/cc.

1.8 Histology assessment

[0066] Mg device and the surrounding tissues were fixed in 10% formalin after harvesting, and before being embedded and polymerized in Osteo-Bed Plus (Poly sciences, PA, USA) following manufacturer’s instructions. All blocks were cut into sections of 5 pm thickness, which was attached on Tesa tapes (Beiersdorf, Germany), using a RM2255 microtome (Leica, Benshiem, Germany) with a tungsten carbide blade (ThermoFisher Scientific, USA) and placed in a tissue embedding cassette. Tissue sections on tapes were stained directly. Before each staining, sections were deacrylated in xylene twice, and 2-methoxyethylacetate for 20 min, followed by a series of decreasing percentages of ethanol and finally water for rehydration.

1.8.1 Goldner’s trichrome staining [0067] Goldner’s trichrome staining was used to visualize new bone formation in the calvaria defect. All reagents for Goldner’s trichrome staining were purchased from Electron Microscopy Sciences (Hatfield, PA). In brief, rehydrated samples were placed in hematoxylin for 20 min, rinsed in distilled water before being immersed in Ponceau-acid fusion for 5 min, followed by 1 % acetic acid. Samples were transferred into orange G-phospho solution for 2 min before being rinsed with 1% acetic acid. Samples were then stained in light green solution for 20 min for mineralized bone, and then rinsed in 1% acetic acid. Finally, samples were dehydrated in absolute alcohol, cleaned in xylene, and mounted with mounting medium.

1.9 Statistical analysis

[0068] A mean value and standard deviation were calculated from three female and four male rats in control groups (empty and PLGA only), and four female and four male rats in experimental groups (Mg and CGRP). Statistical analysis was conducted using Graph Pad Prism 6 software. Differences between groups were tested with two-tailed t-test and one-way analysis of variance (ANOVA) at a 95% confidence interval, followed by Tukey multiple comparisons and post-hoc test. Statistical significance was set at P<0.05.

2. Results

2. 1 Periosteal cells characterization

[0069] The FACS, based on the strategy described in Materials and Methods, showed that CD34-APC stained mouse PDCs plotted against CD45-pacific blue to be 98.9% negative and CD90-PE-Cy7 stained mouse PDCs plotted against CD45-pacific blue to be 99.6% positive. Human PDCs were also analyzed using FACS: CD34-PE and CD45- PE-Cyanine5 were stained 97.7% negative, and 99.1% CD90-Alexa Fluor 700 positive for human PDCs. As expected, CD105 and periostin were detected around 70 KDa and 100 KDa, respectively, from mouse and human PDCs lysates by Western blotting. Immunostaining also showed CD105 expression from both mouse and human PDCs. These findings are consistent with previous research confirming that these cells are derived from the periosteum.

2.2 Alkaline phosphatase (ALP) activities elevated significantly by different concentrations of Mg2+ and CGRP in medium

[0070] ALP acts as an early indicator of osteoblast differentiation. ALP activities measured from mouse PDCs increased after being treated with 10 ¹⁰ M and 10‘⁸ M of CGRP for 1 week but decreased by the end of 2 weeks. Similarly, ALP activities measured from human PDCs increased significantly after treated with 10'¹² M and IO^-10 M of CGRP for 1 week and continued to increase even after 2 weeks of treatment.

[0071] The effect of Mg²⁺ concentrations on ALP levels of mouse PDCs were assessed. Compared to osteogenic control, at 1 week, 1.2 mM and 15 mM Mg²⁺ increased the ALP levels significantly in mouse PDCs. At 2 weeks, increasing Mg²⁺ in osteogenic medium induced continued increase of ALP in mouse PDCs, compared to osteogenic control.

[0072] The effect of Mg²⁺ concentrations on ALP levels of human PDCs were assessed. ALP levels increased after both time points compared to osteogenic control. However, the increase became smaller after 2 weeks, except from 15 mM Mg²⁺ treatment.

2.3 Runx2 and Osterix protein expressions enhanced by Mg²⁺ and CGRP in medium

[0073] Runt related transcription factor 2 (Runx2) and Osterix (also known as SP7) are two transcription factors that play a crucial role in the early stage of osteoblast differentiation. Western blot results revealed that after 3 days, Runx2 and Osterix expressions were increased by Mg²⁺ in a concentration-dependent manner. Levels of expression from mouse PDCs (left) increased gradually and reached the maximum at 5 mM MgSO4, then decreased as Mg²⁺ increased. However, Runx2 and Osterix expressions from human PDCs (right) continued to increase as Mg²⁺ concentrations increased, reaching the maximum at 15mM. Moreover, human PDCs expressed higher levels of both osteogenic proteins at higher CGRP concentration, while mouse PDCs did not show noticeable change in response to all CGRP concentrations.

2.4 Effect of Mg2+ on osteogenic gene expressions of mouse and human PDCs

[0074] The expression of osteogenic genes, including osteocalcin (BGLAP), collagen I (COL1A1), and BSP (IBSP), were determined using qPCR. One week after the treatment, osteocalcin gene expression measured from mouse PDCs was upregulated significantly by 15 mM MgSO4, compared to the cells cultured in osteogenic induction medium as osteogenic control, but decreased significantly for human PDCs. Interestingly, collagen I and BSP genes were downregulated after a week from both mouse and human PDCs when treated with 15 mM MgSO4 compared to osteogenic controls. However, two weeks after treated in 1.2 mM and 10 mM MgSO4, gene expressions of osteocalcin and BSP in mouse PDCs were significantly upregulated, and collagen I was increased as well. However, osteocalcin expression in human PDCs was further downregulated, while collagen level increased. BSP gene could not be determined at both time points. 2.5 Influence of Mg²⁺ on extracellular mineralization is concentration-dependent

[0075] After 3 weeks of treatment in different concentrations of MgSCU and CGRP, Alizarin Red S staining detected extracellular mineralization from both mouse and human PDCs cultured in osteogenic induction medium, as well as from the cells cultured in IO^-10 M CGRP in osteogenic induction medium. However, fewer mineral nodules were detected when concentrations of MgSO4 were increased in the medium. MgSO4 concentrations at and above 5 mM completely inhibited the mineralization at 3 weeks.

2.6 Release of Mg ions and CGRP from PLGA scaffold

[0076] The final product of the PLGA scaffolds containing Mg metal nanopowder (left) and CGRP (right), were each 5mm in diameter. The SEM image of Mg metal nanopowder appeared as dark spots under the SEM, in the PLGA scaffold. Mg powder was homogenously dispersed in the electrospun PLGA fibers, and overlapped into multiple layers. Mg²⁺ accumulative release profile obtained by ICP-MS showed that the Mg²⁺ could be detected at 1 hr after placed in the solution. Mg²⁺ in the solution continued to increase quickly until day 7. At day 12, almost all Mg nanopowder had been released into the solution, reaching a plateau of approximately 120 mg/L. Similarly, CGRP was also electrospun into PLGA scaffold, as shown in SEM image. The accumulative CGRP release profile showed that about 2 pg/mL of CGRP was detected by ELISA in the solution starting at day 2. At week 7, total released CGRP was measured to be a total of 8.5 pg/mL.

2.7 Mg and CGRP stimulated new bone growth from both male and female rat calvaria defect [0077] The 3D rendering of the representative images of new bone formation from rat calvaria defect after 1 month of implantation showed, in general, more new bone was generated when periosteum still covered the defect, compared to the groups without periosteum. This difference is especially striking in Mg and CGRP groups.

[0078] New bone volume from female and male rats was analyzed separately using high resolution yCT. Except for the PLGA (both female and male) and empty (male) groups, the presence of periosteum had a significant impact on bone regeneration: new bone volume in groups containing periosteum was significantly higher than those without periosteum. Analysis from female rat calvaria measured the highest new bone volume to be from the CGRP+ periosteum group, followed by the Mg-i- periosteum group. However, Mg induced higher new bone volume from periosteum than did CGRP groups in male rat calvaria. In general, female rats had more new bone growth compared to male rats. [0079] Goldner’s trichrome staining on calvaria samples was also used to visualize new bone, which was almost completely bridging the calvaria after 1 month implanted with scaffold containing Mg or CGRP covered by the periosteum.

3. Discussion

[0080] Mg has been shown to influence the osteogenic activity of many types of cells, including bone marrow stromal cells, osteoblast, osteoclast, and even vascular smooth muscle cells. The study explored the direct effect of Mg on the periosteum by exposing various mouse and human PDCs to varying concentrations of MgSCh and CGRP.

[0081] An initial finding was that exposure of mouse and human PDCs to CGRP and Mg²⁺, respectively, had differing effects on ALP activity. When mouse PDCs were exposed to CGRP, ALP expression increased at week 1 , but decreased by the end of week 2 when mineral nodules could be visually detected in culture. By comparison, addition of CGRP to human PDCs resulted in ALP expression continuing to increase through week 2. This difference in response between mouse and human cells suggests that under the same CGRP treatment, mouse PDCs undergo an earlier switch to differentiation than human PDCs.

[0082] The addition of Mg²⁺ to mouse and human PDCs resulted in ALP expression continuing to increase beyond 2 weeks. This extended duration of ALP expression subsequent to Mg²⁺ exposure, in contrast to CGRP exposure suggests that CGRP accelerates the osteogenic differentiation of mouse PDCs compared to Mg²⁺. Bone mesenchymal stem cells transfected with CGRP showed enhanced osteogenic differentiation capacity when compared to control cells. The cross-species finding that PDCs exposed to Mg²⁺ demonstrated increased expression of ALP relative to cells exposed to CGRP suggests that Mg²⁺ has an additional and direct osteogenic effect on PDCs that is independent of the CGRP effect.

[0083] A 3-day in vitro treatment of mouse and human PDCs with Mg²⁺ allowed examination of osteogenic differentiation at early stage. Runx2 is the earliest marker of the osteoblast lineage, expressed as early as the differentiation of mesenchymal cells into preosteoblasts. Similarly, Osterix is downstream of Runx2 and expressed early when cells become committed to osteoblast lineage. Results from Western blots indicated that higher concentrations of Mg²⁺ (up to 15 mM) caused human PDCs to commit early to osteoblast lineage, whereas lower concentrations of Mg²⁺ (1.2 mM and 5 mM) led to early response of mouse PDCs. Compared to osteogenic control, higher Runx2 and Osterix expressions were observed from both mouse and human PDCs treated with varying concentrations of CGRP. This finding confirmed the results from ALP that CGRP can stimulate osteogenic differentiation, and also indicated that such stimulation can be observed very early.

[0084] In the next phase of the study, qPCR was used to examine late osteogenic markers of mature osteoblasts, including BGLAP (coded for osteocalcin), BSP (bone sialoprotein), and COL1A1 (collagen I), a main component of bone extracellular matrix. Gene expressions of COL1A1 and BSP from both species were decreased after one week exposure to high Mg²⁺ concentration (15 mM). By comparison, osteocalcin decreased in human PDCs, but increased in mouse PDCs. This finding was unexpected, as one week is a relatively early time point for osteoprogenitor cells to express late-stage osteogenic markers. After two weeks, cells were treated with both low (1.2 mM) and high (10 mM) concentrations of Mg²⁺. As expected, gene expression in mouse PDCs was upregulated, thus implying that the cells had progressed to immature osteoblast. Surprisingly, osteocalcin expression in human PDCs was still decreased. The finding of species -specific effects of CGRP on ALP expression described above raises the possibility that mouse PDCs can undergo an earlier switch to osteogenic differentiation. Given these data and the qPCR findings, it seems reasonable to infer that two weeks maybe sufficient for mouse PDCs to differentiate, while still being too early for human PDCs to undergo the same activities.

[0085] Alizarin Red S staining provided further insight into extracellular mineralization in both mouse and human PDCs. After 3 weeks, exposure to 10"¹⁰ M CGRP in culture led to mineral formation in both mouse and human PDCs. However, exposure to Mg concentrations higher than 1.2 mM inhibited mineral matrix formation in PDCs from both species. The treatment time point was extended to 5 weeks but obtained the same results.

[0086] The pCT analysis on rat calvaria defect showed that periosteum plays a critical role in bone regeneration. The data shows that higher new bone volume has been observed when periosteum was sutured above the defect in most groups, except in PLGA groups. The pCT results showed that most bone growth was observed from Mg-i- groups in male rat calvaria, but CGRP was able to induce higher bone volume in female rat calvaria. This difference might be dependent upon sex, as the influence of sex on bone healing has been previously reported. It has been shown that the osteogenic potential could be different between periosteum locations. Periosteum at the calvaria, also named pericranium, has lower osteogenic potential compared to that from long bones. Moreover, the sensory nerve fibers at the periosteum of the calvaria bone may not produce as much CGRP as those from the long bones due to anatomical differences. Furthermore, the release of Mg²⁺ could have a more pronounced direct and indirect (via CGRP production) effect on periosteal cells in female rats compared to male rats. Taken together, these data from pCT reveal that our Mg or CGRP containing PLGA scaffolds are able to promote new bone formation in both a female and a male rat calvaria defect.

4. Conclusion

[0087] In the present study the osteogenic effect of Mg and CGRP on mouse and human periosteal derived cells, and rat calvaria defects was assessed. It was demonstrated that the osteogenic differentiation of the periosteal cells depends on the concentration and time point and can be species dependent. It was also shown that periosteum is critical in new bone regeneration. Mg and CGRP released from electrospun PLGA scaffold effectively induced new bone formation from the periosteum in rat calvaria defect. The results also suggest that new bone formation can be sex-dependent, as higher new bone volume in general from female compared to male rats was observed. Furthermore, the novel, Mg- or CGRP-containing electrospun PLGA scaffolds promote the osteogenic differentiation of the cells in rat calvaria periosteum, suggesting the therapeutic potential of this biomaterial to facilitate the repair of cranial injury.

Claims

What is claimed is:

1. A magnesium-polymer composite, comprising: a plurality of magnesium-polymer microparticles, comprising: magnesium metal nanopowder; a lipid applied to or deposited on the magnesium metal nanopowder to form coated magnesium metal nanopowder; polymer combined with the coated magnesium metal nanopowder to form the plurality of magnesium-polymer microparticles, wherein the magnesium-polymer implant device effectively provides a controlled and/or sustained-release of magnesium for treatment of one or more of periodontitis, peri-implantitis, and bone defect.

2. The magnesium-polymer composite of claim 1 , wherein the lipid is selected from the group consisting of saturated fatty acid.

3. The magnesium-polymer composite of claim 2, wherein the saturated fatty acid is selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, and combinations and mixtures thereof.

4. The magnesium-polymer composite of claim 1, wherein the lipid is selected from the group consisting of unsaturated fatty acid.

5. The magnesium-polymer composite of claim 4, wherein the unsaturated fatty acid is selected from the group consisting of myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, eicosanoic acid, erucic acid, docosahexaenoic acid, hexadecadienoic acid, and combinations and mixtures thereof.

6. The magnesium-polymer composite of claim 5, wherein the lipid is oleic acid.

7. The magnesium-poly mer composite of claim 1, wherein there is 2% lipid WAV, lipid/magnesium metal.

8. The magnesium-polymer composite of claim 6, wherein there is 2% oleic acid WAV, oleic acid/magnesium metal.

9. The magnesium-polymer composite of claim 1, wherein the plurality of magnesium-polymer microparticles are in the form of microspheres.

10. The magnesium polymer composite of claim 1, wherein the polymer comprises PLGA.

11. A magnesium-polymer implant device, comprising: electrospun magnesium-polymer fibers, comprising: magnesium metal nanopowder; and polymer, wherein the magnesium-polymer implant device effectively provides a controlled and/or sustained-release of magnesium for treatment of one or more of periodontitis, peri-implantitis, and bone defect.

12. The magnesium-polymer implant device of claim 11 , wherein the electrospun magnesium-polymer fibers further comprise calcitonin gene- related peptide.

13. The magnesium-polymer implant device of claim 11, wherein the electrospun magnesium-polymer fibers are configured to form a sheet.

14. The magnesium-polymer implant device of claim 11, further comprising a plurality of magnesium-polymer microparticles.

15. A method of preparing a magnesium-polymer implant device, comprising: combining magnesium metal nanopowder and polymer to produce a precursor mixture; and electrospinning the precursor mixture to produce a plurality of magnesium-polymer electrospun fibers; and configuring the plurality of magnesium-polymer electrospun fibers into an implant device, wherein the magnesium-polymer implant device effectively provides a controlled and/or sustained-release of magnesium for treatment of one or more of periodontitis, peri-implantitis, and bone defect.

16. The method of claim 15, wherein the precursor mixture further comprises calcitonin gene-related peptide.

17. The method of claim 15, further comprising: harvesting periosteum; and culturing periosteal derived cells with Mg²⁺ ions and calcitonin gene-related peptide.

18. The method of claim 15, wherein the polymer comprises PLGA.

19. A method of preparing a magnesium-polymer composite, comprising: forming a plurality of magnesium-polymer microparticles, comprising: obtaining magnesium metal nanopowder; applying or depositing a lipid onto the magnesium metal nanopowder to form coated magnesium metal nanopowder; mixing the coated magnesium metal nanopowder with polymer to form the magnesium-polymer microparticles; and locally delivering the magnesium-polymer microparticles to a target location in a patient, wherein the magnesium-polymer microparticles effectively provide a controlled and/or sustained-release of magnesium for treatment of one or more of periodontitis, peri-implantitis, and bone defect.

20. The method of claim 19, wherein the lipid is selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, eicosanoic acid, erucic acid, docosahexaenoic acid, hexadecadienoic acid, and combinations and mixtures thereof.

21. The method of claim 19, wherein there is 2% lipid WAV, lipid/magnesium metal.

22. The method of claim 19, wherein the lipid is oleic acid and there is 2% oleic acid WAV, oleic acid/magnesium metal.

23. The method of claim 19, wherein the polymer comprises PLGA.