TWI742119B - Multi-mineral supplement for improved bone density - Google Patents

Abstract

Compositions and methods are provided in which magnesium, copper, zinc, and strontium are provided to enhance osteogenesis, improve bone growth, and/or improve bone density. Magnesium, copper, zinc, and strontium are provided in a defined molar ratio providing synergistic effects that permit the use of small amounts of individual metal species, and without the need for supplemental calcium.

Description

Mineral supplements used to enhance bone growth or density

The technical field of the present invention is to provide mineral supplements for improving bone density and/or bone formation, especially mineral supplements containing two or more minerals.

Human diseases related to bone growth and development, such as osteoporosis, have caused an increasing burden on medical care costs with the aging of the population. Although some drugs have been developed to treat these conditions, these medical methods may still have disadvantages. For example, although hormone replacement therapy can effectively prevent the development of osteoporosis in menopausal women, it may increase the risk of developing certain cancers. Although more targeted compounds have been developed (for example, bisphosphonates that inhibit bone resorption), they are associated with hip and femoral fractures. Therefore, some researchers have begun to find simpler methods, such as supplementing minerals.

So far, calcium supplementation is the most common treatment for bone density loss. However, although calcium supplementation has been proven to be useful and successful, it is highly dependent on the individual's calcium absorption and utilization, which in turn may require supplementation of vitamins, hormones and other compounds. Therefore, calcium supplementation alone has limited effects in the treatment of diseases related to bone growth and absorption.

Some researchers have studied supplementing various organic compounds to treat these diseases, some of which form complexes with metals other than calcium. For example, US Patent No. 5,294,634 proposed by Yamaguchi describes the use of carnosine compounded with zinc to promote osteogenesis. All publications contained in this article are included as part of the disclosure of this article by reference, as each individual publication or patent application is specifically and individually indicated It is the same as part of the disclosure that is included in this article by reference. Where the definition or usage of a term in the reference is inconsistent with or contrary to the definition of the term provided herein, the definition of the term shall be subject to the definition provided in this document and the definition in the reference shall not apply. U.S. Patent No. 5,935,996 proposed by Yamaguchi also describes isoflavone compounded with zinc to promote bone formation, while U.S. Patent Application Publication 2008/0113038 proposed by Yamaguchi describes a compound compounded with zinc for this purpose. Use of beta-cryptoxanthin. Similarly, the US Patent Application Publication 2010/0048697 proposed by Hansen et al. describes the use of strontium salts of glutamate and alpha-ketoglutarate for the treatment of osteoporosis . The International Patent Application Publication WO 2008/147228 proposed by Cornish and Reid describes the addition of various metal salts of carboxylic acids and carboxylic acid derivatives to certain dairy products for the treatment of bone resorption or osteoblast proliferation. The use of the disease. However, the preparation of such organic composites is complicated and may have stability issues.

There have been research attempts to use devices or compositions containing metals other than calcium to treat bone growth-related disorders. For example, US Patent Application Publication 2009/0081313 filed by Aghion et al. describes the use of implants mainly made of elemental magnesium for orthopedics. However, given the known reactivity of elemental magnesium with water, it is not clear whether this method can provide magnesium in a usable form or in a controlled manner. Similarly, US Patent Application Publication 2012/0141599 proposed by Johns et al. describes the use of implants including metal-loaded zeolites for bone grafting, particularly using a combination of zinc and copper. Among related methods, the US Patent Application Publication 2012/0141429 proposed by Hass describes the use of zinc oxide-coated nanostructures to enhance bone hyperplasia. Although the International Patent Application Publication WO 2011/058443 proposed by Barralet does not directly refer to implants, it describes the application of various insoluble metal phosphates at sites that need to enhance bone mineralization. However, it is not clear whether such a method provides sufficient control over the amount of metal supplied to avoid the release of toxic and/or cytotoxic metal content.

Therefore, there is still an urgent need to propose a composition and method for providing a source of metal ions in a controlled manner to increase bone density.

The present invention relates to providing compositions and methods in which a mixture of magnesium, copper, strontium and zinc ions is provided to increase bone growth and/or density. Although calcium is a supplement commonly used to support bone growth and development, the inventors found that when magnesium, copper, strontium, and zinc ions are administered at a certain molar ratio, they can provide a synergistic effect that enhances bone growth and density. Allows the use of small amounts of various metals that do not cause cytotoxicity. Magnesium, copper, strontium, and zinc can be provided as oral administration forms, as topical preparations, or as implants.

In an embodiment of the present invention, a mineral supplement for enhancing bone growth or density is provided. The mineral supplement includes a source of magnesium, a source of copper, a source of zinc, and a source of strontium and is formulated with magnesium: strontium. The molar ratio of copper: zinc is 0.8-2.4:5.3-15.8:1:1.3-5 to provide subjects with magnesium, strontium, copper and zinc. In some embodiments, the content of these metals present in supplements can be adjusted by absorption factors (for example, oral absorption factors, local absorption factors, or implant absorption factors) to provide the required concentration and/or ratio of metals . The mineral supplement is formulated to increase bone density or bone volume compared to corresponding mineral supplements that include magnesium but do not have copper, zinc, and strontium. Magnesium, strontium, copper, and zinc can be provided as metal salts, metal complexes, and/or metal-containing compounds. In some embodiments, the mineral supplement does not include calcium. In other embodiments, the magnesium source is a magnesium salt, a magnesium complex, and/or a magnesium-containing compound. In some embodiments, the magnesium source is not an organic magnesium salt, and the copper source may be a copper salt, a copper complex, and/or a copper-containing compound. In some embodiments, the copper source is not an organic copper salt, and the zinc source may be a zinc salt, a zinc complex, and/or a zinc-containing compound. In some embodiments, the zinc source is not an organic zinc salt, and the strontium source may be a strontium salt, a strontium complex and/or a strontium-containing compound. In some embodiments, the strontium source is not an organic strontium salt.

In some embodiments, the mineral supplement is formulated to be injectable. In other embodiments, the mineral supplement is formulated for oral administration, and the content of each metal can be adjusted by the oral absorption factor to provide a molar ratio of magnesium: strontium: copper: zinc of 0.8-2.4. : 5.3-15.8:1:1.3-5. In another embodiment, the mineral supplement is formulated for topical application, and the content of each metal can be adjusted by the local absorption factor to provide absorption magnesium: strontium: copper: zinc molar ratio of 0.8-2.4 : 5.3-15.8:1:1.3-5. In yet another embodiment, the mineral supplement is formulated as an implant (which may include an absorbable carrier), and the content of each metal can be adjusted by the implant absorption factor to provide absorbed magnesium: strontium: The molar ratio of copper: zinc is 0.8-2.4:5.3-15.8:1:1.3-5. In some embodiments, the magnesium: strontium: copper: zinc of the implant The ear ratio is 1.2:7.2-7.9:1:2.5.

Another embodiment of the present invention is a method for increasing bone density or improving bone formation by providing a mineral supplement including a magnesium source, a copper source, a zinc source and a strontium source and applying the mineral supplement For individuals who need to increase bone density or improve bone formation. The mineral supplement is formulated with a molar ratio of magnesium: strontium: copper: zinc of 0.8-2.4:5.3-15.8:1:1.3-5 to provide the individual with magnesium, copper, zinc and strontium, and compare In other mineral supplements that include magnesium but do not have copper, zinc, and strontium, the mineral supplements are formulated to increase bone density or bone volume. In some embodiments, the mineral supplement does not include calcium. In other embodiments, the magnesium source is not an organic magnesium salt, the copper source is not an organic copper salt, the zinc source is not an organic zinc salt, and the strontium source is not an organic strontium salt.

The mineral supplement can be administered by injection or infusion of a solution including the mineral supplement. In other embodiments, the mineral supplements are applied through topical application of lotions, gels, suspensions or solutions including the mineral supplements, and the presence of specific metals can be adjusted by corresponding local absorption factors. Content to achieve the required ratio of magnesium: strontium: copper: zinc. In another embodiment, the mineral supplement is applied by oral administration of pills, tablets, capsules, solutions or suspensions including the mineral supplement, and the presence of the mineral supplement can be adjusted by the corresponding oral absorption factor. The content of the specific metal is required to achieve the required ratio of magnesium:strontium:copper:zinc. In yet another embodiment, the mineral supplement is applied as a surgical implant (wherein at least a part of the surgical implant includes the mineral supplement), and the presence of the mineral supplement can be adjusted by the corresponding implant absorption factor The content of the specific metal to achieve the required magnesium: strontium: copper: zinc ratio.

According to the following description of the various embodiments and the content of the drawings, the various objectives, features, implementation modes and advantages of the present invention will be more prominent, wherein similar component symbols indicate similar components.

Figures 1A to 1D: Figures 1A to 1D depict the results of cell viability studies of human mescenchymal stem cells (hMSCs) cultured with various concentrations of different metal ions. Figure 1A depicts a typical result observed with magnesium ions. Figure 1B depicts the typical results observed with copper ions. Figure 1C depicts the typical results observed with zinc ions. Figure 1D trace The typical results observed with strontium ions are described.

Figures 2A and 2B: Figures 2A and 2B depict the normalized alkaline phosphatase (ALP) activity of human mesenchymal stem cells (hMSCs) cultured with various metal ions. Figure 2A shows typical results on the 7th and 14th days after treatment with various combinations of magnesium, zinc, copper and strontium ions at specific concentrations. Figure 2B shows typical results of magnesium ions at different concentrations and time points.

Figure 3: Figure 3 depicts the position of the implant proposed by the present invention in the femur of the subject.

Figures 4A and 4B: Figures 4A and 4B depict the results of in vivo bone growth studies using metal ion-containing implants. Fig. 4A shows a typical Micro-CT image obtained from a femur implanted with an indicator implant at each time point. Figure 4B depicts the percentage change in bone volume adjacent to the implant; relative to the implant containing only magnesium, the implant containing magnesium/strontium/copper/zinc at 3, 4, and 8 weeks , The adjacent bone mass has increased significantly.

Figures 5A and 5B: Figures 5A and 5B describe the results of in vivo bone growth studies using implants containing metal ions (containing different contents of magnesium, strontium, copper, and zinc) proposed in the present disclosure. Fig. 5A shows typical Micro-CT images obtained from a femur implanted with an indicator implant at various points in time. Figure 5B depicts the percentage change in bone volume adjacent to the implant.

Figures 6A to 6C: Figures 6A, 6B and 6C describe the use of the metal ion-containing implants proposed by the present invention with different relative amounts of magnesium, strontium, copper and zinc ions for bone growth and femoral elastic modulus (elastic modulus) And the results of the research on the stiffness of the femur. Figure 6A shows typical results of changes in the volume percentage of cortical bone adjacent to the indicator implant composition at each time point. Figure 6B shows a typical result of implanting the indicator implant composition to measure the elastic coefficient of the intact femur. Fig. 6C shows a typical result of implanting the indicator implant composition to perform the hardness measurement of the intact femur.

The following discussion provides many exemplary embodiments of the present invention. Although each embodiment represents a single combination of elements of the present invention, it should still be considered that the present invention should still include all possible combinations of the disclosed elements. Therefore, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, even if it is not explicitly disclosed, the present invention should still be understood as including A, B, Other remaining combinations of C or D.

Calcium supplements (usually in the form of calcium carbonate administered orally) have long been used to treat or prevent bone growth disorders, such as menopausal and postmenopausal osteoporosis. Surprisingly, the inventors discovered that other metals (such as magnesium and other metal salts) have significant and unexpected positive effects on bone growth and density, especially when applied in combination. Although this effect occurs without calcium supplementation, the aforementioned metal or metal salt combination can also be supplemented with calcium and/or other elements (as part of a mixed metal formulation or applied as a separate formulation). Provided.

The present disclosure provides a composition and method, which provide a mixture of non-calcium metal ions (such as magnesium, copper, strontium, and zinc ions) in a certain amount and molar ratio, and compared with untreated tissue, it has The content and molar ratio of the non-calcium metal ion mixture can increase bone growth and/or density. Although calcium is generally used as a supplement to support bone growth and development, the inventors have found that when administered in a specific molar ratio range, magnesium, copper, strontium, and zinc ions can provide a synergistic effect (that is, greater than an additive effect). Enhance bone growth and density. The synergistic effect thus obtained advantageously allows individual metals to be used in non-toxic and/or non-cytotoxic concentrations while enhancing bone growth and/or density. Formulas that include magnesium, copper, strontium, and zinc in the molar ratios described herein may be supplemented or not supplemented with calcium (for example, calcium carbonate, bicarbonate, and/or chloride) and/or other elements (E.g. barium, boron, potassium and/or vanadium). These supplemental calcium or other elements can be part of a formula containing magnesium, copper, strontium, and zinc, or applied separately. The formulation proposed in this disclosure can be used as an infusion, oral administration form, as a topical preparation, and/or as an implant or artificial graft.

In the composition and method proposed in the present disclosure, a combination of four metal elements (such as magnesium (Mg), strontium (Sr), copper (Cu) and zinc (Zn)) is used to induce bone formation and/or increase Bone density. These elements can be provided in the form of ionic compounds (ie salts), complexes and/or covalent compounds. In some embodiments, local delivery (e.g., topical administration, use of implants, etc.) rather than systemic delivery (e.g., oral administration, intravenous administration, etc.) is used to provide these metal elements. Although many mineral compositions have been used for similar purposes, the inventors have found that when these metal elements are provided in a specific molar ratio range, they have a synergistic effect on bone formation and/or bone density.

It should be understood that the synergistic effect of a specific ratio of various metals in bone growth modification advantageously allows these metals to be used effectively at concentrations far lower than those that cause cytotoxicity, thereby reducing the risk to patients. At the same time retain the therapeutic effect.

Although the specific salts of magnesium, strontium, copper, and zinc used in this disclosure are described below, it should be understood that any suitable inorganic salts of these metals can also be used. For example, suitable anions of the aforementioned magnesium, strontium, copper and/or zinc salts include chloride, fluoride, carbonate, bicarbonate, sulfate, phosphate, borate and/or nitric acid. Root ion. In some embodiments, magnesium, strontium, copper, and/or zinc may be provided in the form of oxides. It should be understood that different ratios of metals can be provided in the form of metal salts and/or oxides. For example, relatively insoluble magnesium, strontium, copper, and zinc salts (such as phosphate and/or sulfate) can be selected in situations where long-term effects are required (such as implants). Alternatively, relatively soluble magnesium, strontium, copper, and zinc salts (such as nitrates and/or chlorides) can be selected when immediate effects are required (such as infusion).

In some embodiments, the numbers used to describe and claim certain embodiments of the present invention indicating the amounts and characteristics of ingredients (such as concentration, reaction conditions, etc.) should be understood as modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters described in this specification and the scope of the appended patent application are approximate values, which can be changed according to the desired characteristics that the specific embodiment is trying to obtain. In some embodiments, it should be The numerical parameters are explained based on the number of significant digits recorded and the application of general numerical simplification techniques. Although the wide-ranging numerical ranges and numerical parameters described in some embodiments of the present invention are approximate, the numerical values proposed in the specific examples are It is recorded as accurately as possible. The values recorded in some embodiments of the present invention may contain certain errors, which are inevitably caused by the standard deviations existing in their respective test measurements.

As in the description used herein and the entire claimed content, unless the context clearly dictates otherwise, "a", "their" and "the" include plural meanings. In addition, as used in the description in this specification, unless the context clearly dictates otherwise, the meaning of "在" includes "in" and "on".

Unless the context dictates otherwise, all ranges presented herein should be interpreted as including their endpoints, and open-ended ranges should be interpreted as including commercially feasible values. Similarly, Unless the context dictates otherwise, all lists of values shall be considered to include the values in between.

The description of the numerical range in the present invention is only intended as a shorthand method for referring to each individual numerical value falling within the range. Unless otherwise stated, each individual value within the range is included as part of the disclosure of this specification, as if it were referred to one by one in this document. Unless otherwise stated or clearly contradictory to the context, all methods described in the present invention can be performed in any suitable order. Any and all examples or exemplary terms (such as "for example") used in certain embodiments of the present invention are only intended to describe the present invention more clearly, and are not intended to limit the scope of protection claimed by the present invention. Any words in the specification should not be interpreted as not found in the scope of the patent application but are essential elements for implementing the present invention.

The groups of alternative elements or embodiments disclosed herein should not be construed as limitations of the present invention. Each group member may be mentioned and claimed individually, or may be mentioned and claimed in any combination with other members in the group or other elements in this document. For reasons of convenience and/or patentability, one or more members of the group can be included in the group or deleted from the group. When any of the aforementioned inclusions or deletions occur, the specification should be deemed to include the revised group, so as to satisfy all the written description requirements of the Markussi-style group used in the scope of the attached patent application.

The inventors found that cytotoxicity can be induced by magnesium, strontium, copper and/or zinc ions. Tetramethylthiazol salt (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) colorimetric method provides an intuitive and quantifiable method to determine various cell types Cell survival rate. MTT colorimetry was used to determine the cytotoxicity of magnesium, strontium, copper and/or zinc ions in human mesenchymal stem cells. In a general test, 2.8×10 ⁴ cells/cm 2 (cm ² ) of human mesenchymal stem cells (hMSC) were cultured in 96-well tissue culture dishes. The culture medium was low glucose DMEM medium with 10% (v/v) Fetal bovine serum (FBS, Biowest, France), antibiotics (100U/ml penicillin and 100μg/ml streptomycin) and 2mM L-glutamine . After the cells were cultured for one day, test solutions containing different potential ion concentration ranges (as shown in Table 1) were added to each well. Phosphate buffered saline (PBS, OXOID Limited, UK) was added with thiazolyl blue tetrazolium bromide powder to prepare MTT solution, and 10 μL of 5 mg/mL MTT solution was added every other day. After incubating for 4 hours, add 100μL of 10% sodium dodecyl sulfate (SDS, Sigma, USA) to each well, and incubate at 37°C, 5% CO ₂ and 95% air. Incubate in the environment until the next day. Finally, the absorbance values of 570nm wavelength and 640nm reference wavelength are recorded through a full-wavelength multifunctional reader, and the cell survival rate is quantified from the absorbance value.

The cytotoxicity of magnesium, strontium, copper and/or zinc is evaluated in the form of ions, and independent tests are carried out to find out the effective concentration range that will not produce cytotoxic effects. The results are shown in Figures 1A to 1D. Figure 1A shows a typical result of a study using magnesium ions. Figure 1B shows a typical result of a study using copper ions. Figure 1C shows a typical result of a study using zinc ions. Figure 1D shows a typical result of a study using strontium ions. Generally, when the magnesium ion concentration is 50 to 200 ppm, the maximum survival rate of human mesenchymal stem cells (hMSC) can be observed; when the concentration exceeds 400 ppm, the survival rate begins to decrease. When the magnesium ion concentration exceeds 1000 ppm, most of the cells die. Human mesenchymal stem cells can only survive in copper ions at a concentration of 5 to 10 ppm; when the concentration is 25 ppm or higher, the survival rate decreases. Surprisingly, human mesenchymal stem cells (hMSC) are particularly sensitive to zinc ions. When the concentration of zinc ions exceeds 1 ppm, significant cell death is found. On the other hand, human mesenchymal stem cells have been found to be tolerant to strontium ions, because cells treated with up to 1000 ppm strontium+ still have a survival rate close to 100%. Surprisingly, the experimental results show that the survival rate of human mesenchymal stem cells usually increases to 115% (relative to the control cells) at a strontium ion concentration of 100 to 400 ppm.

The effect of magnesium, strontium, copper and/or zinc on the osteogenic differentiation of human mesenchymal stem cells is measured by using the alkaline phosphatase (ALP) activity of the cells as an indicator of differentiation. Therefore, the osteogenic differentiation properties of magnesium, strontium, copper, and/or zinc, either individually or in combination, can be tested using the ALP assay. In a typical ALP measurement, 1×10 ⁴ hMSC cells/cm 2 (cm ² ) were cultured in a 10% (v/v) fetal bovine serum (FBS, Biowest, France), antibiotics (100U/cm 2) on the first day. ml penicillin and 100μg/ml streptomycin) and 2mM L-glutamic acid in low glucose DMEM medium. On the second day, replace the culture medium in each well with differentiated DMEM (differentiation DMEM) medium. The differentiated DMEM medium contains 50μg/ml ascorbic acid (Sigma, USA) and 10mM β-glycerol phosphate (MP Biomedicals). , France) and 0.1μM Decortisol (dexamethasone, Sigma, USA) and different combinations of magnesium, strontium, copper and/or zinc ions, and _{incubated at 37℃, 5% CO 2} for 3, 7 and 14 days . The test concentrations of each ion and test group are shown in Table 2 and Table 3, respectively. These culture fluids are replaced every 3 days. After incubation, the cells were washed 3 times with phosphate buffered saline (PBS) and lysed with 0.1% Triton X-100 at 4°C for 30 minutes. The cell lysate was centrifuged at 574 gravitational acceleration (g) and 4°C for 10 minutes (2-5 Sartorius, Sigma, USA). The supernatant (10 μl) of each sample was transferred to a 96-well tissue culture dish. The ALP activity was determined by colorimetry; it was performed using an ALP reagent (Stanbio, USA) containing p-nitrophenyl phosphate (p-NPP) as a substrate. Record the absorbance at a wavelength of 405nm through the Beckman Coulter DTX 880, a full-wavelength multi-function reader. The observed ALP activity was normalized to the total protein concentration of the sample, and the total protein concentration was measured using the Bio-Rad protein assay (Bio-Rad, USA) method.

Typical results of osteogenic differentiation studies are shown in Figures 2A and 2B. Figure 2A shows typical normalized ALP activities measured from cells treated with different combinations and concentrations of copper, zinc, strontium, and magnesium ions. Figure 2B shows typical results of cells treated with magnesium ions. Surprisingly, up to 14 days after the start of the treatment, compared with the control cells (an indicator of osteogenic differentiation), the ALP activity of the cells was still increased when treated with a combination of copper, zinc, strontium, and magnesium ions. The foregoing result is that copper, zinc, strontium, and magnesium are provided in the form of simple salts, and there are no organic salts of these metals in the culture environment (for example, copper, zinc, strontium containing organic compounds that are effective for osteogenesis) And magnesium organic salts). The applicant believes that this differentiation result will lead to an increase in bone density and/or bone content in the body. However, other treatments with a combination of two or three of the aforementioned ions showed no increase in osteogenic differentiation. This means that a combination of copper, zinc, strontium, and magnesium is necessary. In addition, in the group that was treated with the combination of the aforementioned four ions and produced effective results, the concentration of individual ions was much lower than the concentration that would cause cytotoxicity. It is worth noting that the results show that it does not require calcium (Ca) and/or phosphate supplementation. The results also show that there is no need for complex organic compounds.

The inventors have determined that similar combinations of metal elements are also effective when delivered/administered locally in the body. In order to deliver the combination of these metal elements in the body, the salts of copper, zinc, strontium, and magnesium can be combined with a biocompatible carrier (for example, polycaprolactone (PCL)). In typical applications, the selected metal salt can be mixed with polycaprolactone in a specific content. In a typical study, the implant is made of the aforementioned material with a rod with a diameter of 2 mm and a length of 6 mm. The metal content of the implants used in the preliminary study is shown in Table 4.

In a typical study, the aforementioned implants were implanted into 2-month-old female SD rats (Sprague-Dawley rats, the experimental animal unit of the University of Hong Kong, with an average weight of 200-250g), and the selected surgical site was outside. Epicondyle (lateral epicondyle). Each rat was implanted with ion combination (magnesium/strontium/copper/zinc)/polycaprolactone or magnesium/polycaprolactone complex on the right lateral epicondyle. In order to monitor the formation of new bone around the implant, continuous monitoring was performed at 1, 2, 3, 4, and 8 weeks. The magnesium/polycaprolactone complex was used as a control group.

Rats were anesthetized by intraperitoneal injection of ketamine (67mg/kg) and xylazine (6mg/kg). The surgical site of the rat was shaved and peeled. Using a minimally invasive method, a hand drill was used to make a hole with a diameter of 2mm and a depth of 6mm at the lateral epicondyle. Subsequently, the sample was implanted in the hole drilled in the right femur of the rat in the aforementioned manner. Then the wound is sutured layer by layer, and an appropriate dressing is applied to the incision. After the operation, rats were injected subcutaneously with 1 mg/kg terramycin (antibiotic) and 0.5 mg/kg ketoprofen. The rats were euthanized 8 weeks after surgery.

At various time points (ie 1, 2, 3, 4, and 8 weeks), micro-computed tomography (micro-CT) (SKYSCAN 1076, Skyscan Company) was performed on the surgical site to monitor the healing process and detect implantation The formation of new bone around the object. Use NRecon (Skyscan Company) to reconstruct the two-dimensional plane.

In some studies, in order to further evaluate the effect of released ions on osteogenesis, in addition to monitoring the formation of new bone around the implant, the thickness of cortical bone was also measured. The limbs without implants were used as the control group. Microcomputer tomography was performed in the 4th and 8th weeks to monitor the formation of new bone and the thickness of cortical bone. At these two time points, the rats were scanned with a microcomputer tomography device (SKYSCAN 1076, Skyscan Company), and the bone volume was analyzed by CTAn software (Skyscan Company).

In other studies, the bending hardness and strength properties of the obtained femur were measured by a 3-point bending test (MTS 858.02 Mini Bionix) based on the ASTM D7264-01 standard protocol. The test speed is 1mm/min.

Typical results of preliminary studies using a single concentration combination (as described in Table 4) of magnesium, strontium, copper, and zinc are shown in Figures 4A and 4B. Figure 4A shows the implanted sample from the rat A typical micro-CT image of his femur from day 0 to the 8th week after surgery. Figure 4B shows the percentage change in bone volume adjacent to the sample implant. An increase of 40%-87% in bone volume adjacent to the magnesium/strontium/copper/zinc implant (relative to the magnesium-only control group) is usually observed at 2, 4, and 8 weeks after surgery. This means that the combination of magnesium, strontium, copper and zinc applied locally is very effective in inducing new bone formation. It is worth noting that the aforementioned results occurred without providing calcium or phosphate supplements. In addition, when simple chemical salts (compared to metal salts formed with pharmacologically active organic compounds) are used to supplement copper, zinc, magnesium, and strontium, they can still produce effective effects.

A similar research method is used to determine the maximum dose of metal ions. Sample preparation, surgical procedures, and post-operative Micro-CT scans are roughly the same as the studies outlined in Figures 4A and 4B. The metal salt content of the implant used is shown in Tables 5A and 5B, which show the weight and proportion of metal elements, respectively.

The content of each metal in the first and second groups (namely magnesium and magnesium/strontium/copper/zinc) used here is the same as that used in the previous study, and the third group (namely (magnesium/strontium/copper/zinc) +) and the fourth group (ie (magnesium/strontium/copper/zinc)++) increased the concentration of certain metal ions (as shown in Table 5), and the increased concentration ranged from 2 times to 10 times. Polycaprolactone without metal ions was used as a control group. Generally, the magnesium/strontium/copper/zinc group is compared with magnesium/polycaprolactone and polycaprolactone-only group during the entire implantation period. With implants, the bone content increases by at least 30% to 50% (see Figure 5). However, when using (magnesium/strontium/copper/zinc)+ and (magnesium/strontium/copper/zinc)++ implants, bone loss of 25% or more was found one week after surgery. Although bone loss was reduced at a later point in time, no new bone formation was found in the experimental results of the group implanted with the aforementioned implants. This result indicates that new bone formation will be inhibited by higher concentrations of metal ions.

A similar research method was used to further optimize the concentration of the metal salt content of the implant. The metal salt composition of the implants used in these studies is shown in Tables 6A and 6B. Table 6A shows the weight of salt in the implant, and Table 6B shows the ratio of metal elements to each other in the implant. In this study, the implant has a diameter of 1.3 mm, a length of 2 cm, and a rod-like shape. In addition, the implantation site is located in the femoral bone marrow instead of the lateral epicondyle to test the mechanical properties of the entire femur.

The surgical operation is as after. Expose the distal femur until you see the patella, then move the patellar tendon to the lateral epicondyle, drill through the lateral epicondyle and drill a hole with a diameter of 0.5mm and a length of 3mm on the distal end of the right femur, and then implant the sample into the In the hole. The left side (without implant) served as a control group. Then, the wound is sutured layer by layer, and an appropriate dressing is applied to the incision. After the operation, the rats were injected subcutaneously with 1 mg/kg oxytetracycline (antibiotic) and 0.5 mg/kg ketoprofen. The rats were euthanized 8 weeks after surgery.

Since magnesium and strontium ions are abundantly present in the body, the tolerance of these ions can be considered relatively high compared with copper and zinc ions. The inventor considers that the concentration of copper and zinc may be crucial, therefore, further optimization research focuses on the optimization of copper and/or zinc. Implants (310) prepared with various contents of magnesium, strontium, copper and zinc were implanted into the intramedullary canal of the femur (as shown in Figure 3) by the aforementioned surgical method to study the combination of metal ions The effect on the thickness of cortical bone. The femur without implant was used as the control group. Typical results are shown in Figures 6A, 6B and 6C. Figure 6A shows the percentage change in cortical bone volume, and shows that all magnesium, strontium, copper, and zinc preparations tested have an increase in average cortical bone volume relative to the control group without implants and containing only magnesium. Surprisingly, compared to the control group, all tested combinations of magnesium, strontium, copper and zinc had an increase in average bone volume. As shown in Table 6B, the molar ratios of magnesium, strontium, and zinc relative to the copper content can range from 0.8 to 2.4, 5.3 to 15.8, 1.2 to 5, respectively, and can provide the aforementioned Effect. In particular, compared with the magnesium/polycaprolactone control group, in the implant preparations MSCZ 2, MSCZ 3 and MSCZ 8, the results showed that they had significantly higher cortical bone volume. In 4 weeks, it increased by 4-8%, and in the 8th week after surgery, it increased by 6-11%. In addition, the implant preparation MSCZ 7 also showed a significantly higher cortical bone thickness than the magnesium/polycaprolactone control group, about 9% after 8 weeks of implantation. Fig. 6B and Fig. 6C show the elastic modulus and stiffness of the implanted and non-implanted femur, respectively. Compared with the control group, in the group implanted with the MSCZ 3 implant, the results showed a significantly higher coefficient of elasticity and stiffness. Generally, compared to the case where the femur is not surgically implanted, the result shows a 16% increase in elastic modulus and a 14% increase in stiffness in the femur implanted with the MSCZ 3 implant. Therefore, MSCZ2 (magnesium:strontium:copper:zinc=2.4:15.8:1:5), MSCZ3 (magnesium:strontium:copper:zinc=1.0:6.3:1:2), MSCZ 7 (magnesium:strontium:copper: Zinc=1.2:7.9:1:3.1) and MSCZ 8 (magnesium:strontium:copper:zinc=1.2:7.9:1:3.8) implants are particularly effective for bone formation.

Although the above-mentioned embodiments utilize implants, other embodiments proposed in this disclosure include methods, devices, and compositions that provide local delivery of magnesium, strontium, copper, and/or zinc instead of using implants. In this embodiment, magnesium, strontium, copper, and/or zinc can be provided in a flowable dosage form, such as a gel or other semi-solid, which can be applied at or near the site where it is desired to increase or enhance bone production. For example, such gels or semi-solids can be introduced by injection or minimally invasive surgery. In some embodiments, the flowable formulation can be cured after application to enhance positioning. In a preferred embodiment, the flowable formulation is absorbable by the individual.

In other embodiments of the present disclosure, magnesium, strontium, copper, and/or zinc may be provided systemically. Examples of suitable systemic preparations include injections suitable for parenteral administration and preparations that can be taken orally. Suitable oral formulations can be liquid, solid or semi-solid. In the oral preparation, the amount and/or ratio of magnesium, strontium, copper, and/or zinc provided can be adjusted to the required content and/or molar ratio of these ions by using absorption factors. For example, the absorption factor of water-soluble magnesium salts used for oral administration is about 30%. Similarly, the absorption factor of strontium applied to oral administration may be about 15% to about 30%. The absorption factor of zinc applied to oral administration may be about 20% to 40%. In some embodiments, the absorption factor may vary with the content of magnesium, strontium, copper, and/or zinc provided. For example, when the copper content is reduced, the absorption factor of the orally ingested copper may be in the range of about 30% to about 65%. In some embodiments, absorption can vary depending on the specific magnesium, strontium, copper, and/or zinc compounds used in the formulation. For topical preparations and implant-type preparations, it can be considered that there are nearly Similar absorption factors and applied to these preparations to provide the required molar ratio of copper: zinc: strontium: magnesium.

In some embodiments of the present disclosure, magnesium, strontium, copper, and/or zinc may be provided together with additional elements that can support bone formation and/or inhibit bone resorption. These additional elements include barium, boron, calcium, potassium and/or vanadium. These additional elements can be provided in the form of organic or inorganic salts, complexes or compounds. For example, barium, boron, calcium, potassium, and/or vanadium can be provided in the form of cations in salts. The aforementioned salts may include anions such as chloride ion, fluoride ion, carbonate ion, bicarbonate ion, sulfate ion, phosphate ion, borate ion or nitrate ion. In another embodiment, boron may be provided in the form of borate and/or boron/sugar (carbohydrate) complexes.

Those with ordinary knowledge in the technical field of the present invention should understand that in addition to the previously described embodiments, there may be other modifications that do not deviate from the concept of the present invention. Therefore, any equivalent modifications or changes that do not depart from the spirit and scope of the present invention should be included in the scope of the appended patent application. In addition, when interpreting both the specification and the scope of the patent application, all technical terms should be interpreted in the broadest possible way consistent with the context. In particular, the terms "including" and "including" should be interpreted as referring to elements, components, or steps in a non-exclusive form, which means that the referred elements, ingredients, or steps can be combined with other elements, components, or steps that are not explicitly mentioned. The ingredients or steps are present, used or combined together. When it is mentioned in the specification or the scope of patent application that at least one of something is selected from the group consisting of A, B, C... and N, the content should be interpreted as having one element in the group. It is not necessary to have A plus N, or B plus N, and so on.

references

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Claims (10)

A mineral supplement for enhancing bone growth or density, comprising: a first mineral source, which contains magnesium; a second mineral source, which contains copper; a third mineral source, which contains zinc; and A fourth mineral source comprising strontium, wherein the first, second, third and fourth mineral sources have a molar ratio of magnesium: strontium: copper: zinc of 0.8-2.4:5.3-15.8:1: The presence of 1.3-5 provides that, compared to a mineral supplement that includes magnesium but does not have copper, zinc, and strontium, the mineral supplement is formulated to increase bone density or bone volume. The mineral supplement described in item 1 of the scope of patent application, wherein the mineral supplement does not include calcium. The mineral supplement according to item 1 of the scope of the patent application, wherein the mineral supplement is formulated as an injectable, for oral administration, for topical administration, or a surgical implant. The mineral supplement described in item 1 of the scope of patent application further includes an element selected from the group consisting of barium, boron, calcium, potassium and vanadium. A method for formulating the mineral supplement described in item 3 of the scope of patent application for oral administration, comprising: determining oral absorption factors for magnesium, strontium, copper, and zinc; and using the oral absorption factors Adjust the individual sources of the first, second, third and fourth minerals to provide a molar ratio of 0.8-2.4:5.3-15.8:1:1.3-5 when the mineral supplement is administered orally. The magnesium: strontium: copper: zinc. A method for formulating a mineral supplement as described in item 3 of the scope of the patent application, which comprises: determining the local absorption factors for magnesium, strontium, copper and zinc; and adjusting the individual first, second, third and The fourth mineral source provides absorbed magnesium: strontium: copper: zinc with a molar ratio of 0.8-2.4:5.3-15.8:1:1.3-5 when the mineral supplement is administered locally. A formulation for the mineral supplement as described in item 3 of the scope of the patent application for administration as an implant The method of treatment includes: determining the implant absorption factors for magnesium, strontium, copper, and zinc; and adjusting the content of individual first, second, third, and fourth mineral sources. When the mineral supplement is administered, it provides absorbed magnesium: strontium: copper: zinc with a molar ratio of 0.8-2.4:5.3-15.8:1:1.3-5. A use of the mineral supplement described in any one of items 1 to 4 in the scope of the patent application for the manufacture of medicines suitable for increasing bone density or improving bone formation, wherein compared with other products that include magnesium but not A mineral supplement of copper, zinc, and strontium that is formulated to increase bone density or bone volume. The use described in item 8 of the scope of patent application, wherein the mineral supplement does not include calcium. The use described in item 8 of the scope of patent application, wherein the magnesium source is not an organic magnesium salt, the copper source is not an organic copper salt, the zinc source is not an organic zinc salt, and the strontium source is not an organic strontium salt.

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