Use of lactobacillus plantarum twk10 for promoting bone healing

Abstract

The present invention relates to the use of Lactobacillus plantarum TWK10 for promoting bone healing. The present invention discloses that Lactobacillus plantarum TWK10 (CGMCC NO. 13008) can be used to promote bone healing.

Description

Technical Field

[0001] This invention relates to the use of Lactobacillus plantarum TWK10 (CGMCC NO. 13008) to promote bone healing in an individual. Background Technology

[0002] When bones are injured by external forces [such as a fissure fracture], healing primarily relies on the patient's own bone healing ability. However, when the injury is more severe, causing bone defects [such as a segmental fracture], further bone implantation is required to aid healing. Sometimes, bone destruction is necessary before implanting the implant. However, the ability to heal decreases relatively with the severity of bone injury and with age. Therefore, researchers in this field are dedicated to developing methods to effectively promote bone healing.

[0003] Taiwanese patent TW I583388 B discloses that Lactobacillus plantarum BCRC910734 can be used to increase exercise performance and reduce fatigue. In an embodiment of this patent, 6-week-old mice given Lactobacillus plantarum BCRC 910734 for 6 weeks were found to have stronger forelimb grip strength and longer swimming time, and also showed a significant reduction in blood lactate and ammonia concentrations after exercise. Summary of the Invention

[0004] In this invention, the applicant unexpectedly discovered that Lactobacillus plantarum TWK10 (CGMCC NO. 13008) can achieve bone healing by promoting the activation and generation of osteoblasts and bone maturation.

[0005] Therefore, in a first aspect, the present invention provides the use of Lactobacillus plantarum TWK10 (CGMCC NO. 13008) for the preparation of a composition for promoting bone healing in an individual.

[0006] Preferably, the composition is a pharmaceutical composition.

[0007] More preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0008] More preferably, the pharmaceutical composition is in a dosage form for oral, non-enteric, or topical administration.

[0009] In a second aspect, the present invention provides a method for promoting bone healing in an individual, comprising administering to an individual in need a Lactobacillus plantarum TWK10 as described above.

[0010] Information on the Preservation of Biological Materials

[0011] Accession number: CGMCC No. 13008

[0012] Classification and nomenclature: Lactobacillus plantarum TWK10

[0013] Date of preservation: September 13, 2016

[0014] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0015] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Attached Figure Description

[0016] Figure 1 and Figure 2 The bone volume fraction (BV / TV) (%) and bone mineral density (BMD) (g / mm³) of rat femurs in control group 1 and experimental group 1 of Example 1 are shown respectively. 3 ),in" "Indicates: When compared with control group 1, p < 0.05;

[0017] Figure 3 The results of femoral tissue sections from each group of rats in Example 1 were observed by hematoxylin-eosin staining, with red arrows indicating osteoblasts;

[0018] Figure 4 The percentage of activated osteoblasts (%) was measured in femoral tissue sections from each group of rats in Example 1, where " "and" "" respectively indicate that when compared with the corresponding control group, p < 0.05 and p < 0.001;

[0019] Figure 5 The percentage (%) of layered bone area measured from femoral tissue sections of rats in Example 1 is shown, where " "and" "These respectively indicate that when compared with the corresponding control group, p < 0.01 and p < 0.001; and..."

[0020] Figure 6 The percentage (%) of SATB2 expression measured in femoral tissue sections of rats in Example 1 is shown, where " "and" "" respectively indicate that when compared with the corresponding control group, p < 0.01 and p < 0.001. Detailed Implementation

[0021] For the purposes of this instruction manual, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has the corresponding meaning.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art. Those skilled in the art will recognize many similar or equivalent methods and materials that can be used to practice the invention. Of course, the invention is by no means limited to the methods and materials described.

[0023] This invention provides the use of Lactobacillus plantarum TWK10 (CGMCC NO. 13008) for the preparation of a composition for promoting bone healing in an individual.

[0024] According to the present invention, *Lactobacillus plantarum* TWK10 can be live or dead bacteria, concentrated or non-concentrated, liquid, paste, semi-solid, or solid [e.g., pellets, granules, or powder], and can be heat-inactivated, frozen, dried, or freeze-dried [e.g., in freeze-dried or spray / fluid bed dried form]. In a preferred embodiment of the present invention, *Lactobacillus plantarum* TWK10 is present in the form of a freeze-dried powder.

[0025] As used in this article, the terms “bone healing” and “bonerepair” may be used interchangeably.

[0026] According to the present invention, the individual may be an individual with a fracture, which may be caused by at least one of the following: trauma; congenital bone healing disorders, such as osteoogenesis imperfecta; cancer, such as bone metastasis; and surgery, such as osteotomies [e.g., bone lengthening and limb lengthening procedures] and tumor resection surgery [e.g., resection of primary bone tumor and resection of secondary bone cancer (also known as bone metastasis)].

[0027] According to the present invention, the fracture may include, but is not limited to: simple fracture, open fracture, transverse fracture, fissure fracture (also known as longitudinal fracture), oblique fracture, spiral fracture, compression fracture, segmental fracture, comminuted fracture, avulsion fracture, greenstick fracture, and dislocation fracture.

[0028] Alternatively, the individual may be an individual with osteonecrosis, such as osteonecrosis following a fracture, or ischemic osteonecrosis caused by osteomyelitis.

[0029] According to the present invention, the individual may be an individual who has undergone surgical intervention of bones, which may include, but is not limited to: bone replacement, such as hip joint and knee joint replacement; and bone implantation, such as tooth implantation.

[0030] Ideally, the individual is one who does not have osteoporosis.

[0031] As used in this article, the term "subject" refers to any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats.

[0032] According to the present invention, the composition may be a pharmaceutical composition.

[0033] In some specific instances, the composition is a pharmaceutical composition.

[0034] According to the present invention, the pharmaceutical composition may be in a dosage form suitable for oral administration, parenteral administration or topical administration.

[0035] According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these agents fall within the scope of expertise and routine practice of those skilled in the art.

[0036] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration using techniques well known to those skilled in the art, including, but not limited to: sterile powders, tablets, troche, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, drops, emulsions, syrups, elixirs, slurries, and the like.

[0037] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for non-enteric administration [including injection, for example, a sterile aqueous solution or dispersion] using techniques well known to those skilled in the art, and administered via a route selected from the group consisting of: intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration.

[0038] According to the present invention, the pharmaceutical composition can also be manufactured into an external preparation suitable for topical application to the skin using techniques known to those skilled in the art, including, but not limited to: emulsion, gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, serum, paste, foam, drop, suspension, salve, and bandage.

[0039] The present invention also provides a method for promoting bone healing in an individual, comprising administering to an individual in need a Lactobacillus plantarum TWK10 as described above.

[0040] As used herein, the terms “administering” and “administration” may be used interchangeably and mean introducing, providing, or delivering a predetermined active ingredient to an individual by any suitable means to perform its intended effect.

[0041] According to the present invention, the dosage and frequency of administration of *Lactobacillus plantarum* TWK10 may vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the age, physical condition, and response of the individual to be treated. Generally, *Lactobacillus plantarum* TWK10 may be administered orally or locally in a single dose or in multiple doses, without enteral administration.

[0042] The present invention will be further described with reference to the following embodiments, but it should be understood that the embodiments are for illustrative purposes only and should not be construed as limiting the implementation of the present invention.

[0043] <Example>

[0044] General experimental materials:

[0045] 1. Lactobacillus plantarum CGMCC NO. 13008:

[0046] The *Lactobacillus plantarum* CGMCC NO. 13008 (also known as *Lactobacillus plantarum* TWK10 or *Lactobacillus germinatus* TWK10) used in the following examples has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2016, in accordance with the provisions of the Budapest Treaty, and is publicly available.

[0047] In addition, the Lactobacillus plantarum TWK10 was deposited on June 30, 2016, at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (No. 331, Food Rd., Hsinchu City, Taiwan 300) under accession number BCRC 910734, and is available to the public.

[0048] 2. Laboratory animals:

[0049] The Wistar rats (6 weeks old, weighing approximately 200 to 260 g) used in the following examples were purchased from Lesco Biotechnology Co., Ltd. All experimental animals were housed in an independently air-conditioned animal room with 12 hours of light and 12 hours of darkness, a room temperature maintained at 21 ± 2°C, and a relative humidity maintained at 55 ± 10%, with adequate water and feed provided. All experimental procedures involving the experimental animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Fu Jen Catholic University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition, 2011, issued by the National Institutes of Health (NIH).

[0050] 3. Bioceramic implants:

[0051] The bioceramic implant used for bone implantation in the following examples was provided by the research laboratory of Professor Yu-Tzu Huang of the Department of Medicine, Fu Jen Catholic University. This bioceramic implant is a silica-based bioceramic scaffold made from a mixture of silica powder (Nippon LightMetal Holdings Company, Ltd.) and silica gel (Nissan Chemical America Corporation). It was fabricated using 3D printing technology, referencing Taiwan TW I421062 B.

[0052] General experimental methods:

[0053] 1. Statistical analysis:

[0054] In the examples below, experimental data for each group are expressed as mean ± standard error (SE). All data were analyzed using the Mann-Whitney U test to assess differences between groups. A p-value < 0.05 was considered statistically significant.

[0055] Example 1. Evaluation of the efficacy of Lactobacillus plantarum TWK10 in promoting bone healing.

[0056] Experimental methods:

[0057] A. Preparation of freeze-dried Lactobacillus plantarum TWK10 bacterial powder:

[0058] First, *Lactobacillus plantarum* TWK10 was inoculated into MRS medium and cultured at 37°C for 16-18 hours. The resulting culture was then centrifuged at 5,000 rpm for 10 minutes at 4°C. The supernatant was removed, and the pellets were washed with an appropriate amount of physiological saline. After bacterial counting on plate count medium, the culture was freeze-dried and then mixed with an appropriate amount of maltodextrin to obtain a bacterial concentration of 10⁻⁶. 10 Freeze-dried Lactobacillus plantarum TWK10 bacterial powder, CFU / g.

[0059] B. Administration of Lactobacillus plantarum TWK10, bone destruction, and bone grafting:

[0060] First, Wistar rats were randomly divided into 4 groups (n=4 in each group), including 2 control groups (control group 1 and control group 2) and 2 experimental groups (experimental group 1 and experimental group 2). Next, each experimental group was administered freeze-dried Lactobacillus plantarum TWK10 powder (at a dose of 10g) via oral gavage, according to item A above. 9 The rats in each control group were given an equal weight of maltodextrin (prepared in ddH2O) and the rats in each experimental group were given the drug 5 times a week for a total of 5 weeks. The rats in the control group and experimental group were given the drug 2 times a week for a total of 13 weeks.

[0061] At the end of the first week after drug administration, the rats in each group underwent bone destruction and bone grafting as described below: First, the rats in each group were anesthetized by inhalation of isoflurane. Next, the abdomen of each group of rats was shaving, and then an incision was made in the skin layer using sterile surgical scissors. Then, the muscles beneath the skin layer were cut using a sterile scalpel to expose the femur. Subsequently, the femur was destroyed using a drill, creating a 2 mm diameter borehole. Finally, a bioceramic implant was inserted into the borehole and the area was sutured.

[0062] At the end of week 5 after drug administration, rats in both control group 1 and experimental group 1 were sacrificed using CO2, and their femurs were then removed and used for experiments C through E below. Similarly, at the end of week 13 after drug administration, rats in both control group 2 and experimental group 2 were sacrificed using CO2, and their femurs were then removed and used for experiments D through E below.

[0063] C. Analysis of bone quality parameters:

[0064] The femur of rats was photographed using a micro-computed tomography (micro-CT) system (SKYSCAN 1176, Bruker Corporation) at 400x magnification. Five areas were then randomly selected around the bioceramic implant to measure bone volume (BV), tissue volume (TV), and bone mass density (BMD) (g / mm²). 3 ).

[0065] Bone volume fraction (BV / TV) (%) is calculated by substituting the measured bone volume and tissue volume into the following formula (1):

[0066] Formula (1): A = (B / C) × 100

[0067] Where: A = Bone volume fraction (BV / TV) (%)

[0068] B = Bone volume (mm²)3 )

[0069] C = Tissue volume (mm²) 3 )

[0070] Then, the obtained experimental data were analyzed according to the method described in item 1, "Statistical Analysis," of the "General Experimental Methods" section above.

[0071] D. Histopathological analysis:

[0072] Rat femurs were fixed overnight using a 10% formaldehyde solution, followed by decalcification with 3% hydrochloric acid for 5 hours. The fixed and decalcified femurs were then embedded in paraffin and sectioned to obtain tissue sections with a thickness of 4 μm.

[0073] Subsequently, a portion of the tissue sections were stained using hematoxylin-eosin and according to techniques well known and commonly used by those skilled in the art. The stained tissue sections were observed and photographed using an optical microscope (Olympus BX51, Japan) at 400x magnification, and the number of active osteoblasts and inactive osteoblasts were counted. The active osteoblast ratio (%) was calculated by substituting the obtained number of active and inactive osteoblasts into the following formula (2):

[0074] Formula (2): D=[E / (E+F)]×100

[0075] Where: D = percentage of activated osteoblasts (%)

[0076] E = Number of activated osteoblasts (cells)

[0077] F = Number of unactivated osteoblasts (individuals)

[0078] In addition, lamellar bone formation was observed and photographed under polarized light view, and analyzed using ImageJ Imaging Software (version 1.48) to measure the lamellar bone area and total tissue area surrounding the bioceramic implant. Lamellar bone formation represents bone maturation. The lamellar bone area percentage (%) was calculated by substituting the measured lamellar bone area and total tissue area into the following formula (3):

[0079] Formula (3): G = (H / I) × 100

[0080] Where: G = percentage of layered bone area (%)

[0081] H = Layered bone area (mm²) 2 )

[0082] I = Total tissue area (mm²) 2 )

[0083] Then, the obtained experimental data were analyzed according to the method described in item 1, "Statistical Analysis," of the "General Experimental Methods" section above.

[0084] E. Immunohistochemistry staining:

[0085] The tissue sections obtained in section D above were collected, and then immunohistochemical staining was performed using a special AT-rich sequence-binding protein 2 (SATB2) antibody (Abcam, catalog number ab92446) as a primary antibody, and a rabbit-specific HRP / DAB (ABC) Detection IHC Kit (Abcam, catalog number ab64261) according to techniques known and commonly used by those skilled in the art. The stained tissue sections were observed and photographed using an optical microscope (Olympus BX51, Japan) at 400x magnification, and then analyzed using ImageJ imaging software (version 1.48) to measure the area of ​​SATB2-expressing tissue and the total tissue area surrounding the implantation of the bioceramic implant, where SATB2 expression represents osteoblastogenesis. The SATB2 expression percentage (%) is calculated by substituting the measured area of ​​tissues with SATB2 expression and the total tissue area into the following formula (4):

[0086] Formula (4): J = (K / L) × 100

[0087] Where: J = SATB2 expression percentage (%)

[0088] K = Tissue area with SATB2 expression (mm²) 2 )

[0089] L = Total tissue area (mm²) 2 )

[0090] Then, the obtained experimental data were analyzed according to the method described in item 1, "Statistical Analysis," of the "General Experimental Methods" section above.

[0091] result:

[0092] A. Analysis of bone parameters:

[0093] Figure 1 and Figure 2 The values ​​of BV / TV and BMD, measured in the femur of rats in control group 1 and experimental group 1, are shown at the end of week 5 after the start of drug administration. Figure 1 and Figure 2It can be seen that, compared with control group 1, the measured BV / TV and BMD in experimental group 1 were significantly increased.

[0094] B. Histopathological analysis:

[0095] Figure 3 The results are shown in the femoral tissue sections of rats from each group, obtained by hematoxylin-eosin staining at the end of week 5 or week 13 after the start of drug administration. Figure 3 It is evident that, compared to control group 1, the femoral tissue of experimental group 1 has significantly more osteoblasts. Conversely, compared to control group 2, thicker bone formation can be observed in the femoral tissue of experimental group 2.

[0096] Figure 4 The percentage of activated osteoblasts was shown in femoral tissue sections from rats in each group at the end of week 5 or week 13 after the start of drug administration. Figure 4 It is evident that the proportion of activated osteoblasts measured in each experimental group was significantly higher than that in the corresponding control group.

[0097] Figure 5 This shows the percentage (%) of layered bone area measured in femoral tissue sections from rats in each group at the end of week 5 or week 13 after the start of drug administration. Figure 5 It is evident that the percentage of layered bone area measured in each experimental group was significantly higher than that in the corresponding control group.

[0098] C. Immunohistochemical staining:

[0099] Figure 6 The percentage (%) of SATB2 expression measured in femoral tissue sections from rats in each group was shown at the end of week 5 or week 13 after the start of drug administration. Figure 6 It is evident that the percentage of SATB2 expression in each experimental group was significantly higher than that in the corresponding control group.

[0100] Based on the above experimental results, it is clear that regardless of whether the treatment duration is 5 weeks or 13 weeks, *Lactobacillus plantarum* TWK10 can effectively promote osteoblast activation and generation, as well as bone maturation. Therefore, the applicant believes that *Lactobacillus plantarum* TWK10 has high potential for promoting bone healing.

[0101] All patents and documents cited in this specification are incorporated herein by reference in their entirety. In the event of any conflict, the detailed description herein (including its definitions) shall prevail.

[0102] Although the invention has been described with reference to the specific examples described above, it is apparent that many modifications and variations can be made without departing from the scope and spirit of the invention. Therefore, it is intended that the invention be limited only to those shown in the appended claims.

Claims

1. Use of Lactobacillus plantarum TWK10, which is deposited with the China General Microbiological Culture Collection Center under accession number CGMCC NO. 13008, for preparing a pharmaceutical composition for promoting bone healing in an individual without osteoporosis.

2. Use according to claim 1, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

3. Use according to claim 1, characterized in that: The pharmaceutical composition is in a dosage form for oral administration, parenteral administration or topical administration.

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