CN113350486A - Pharmaceutical composition with bone defect repairing effect and application thereof - Google Patents
Pharmaceutical composition with bone defect repairing effect and application thereof Download PDFInfo
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- CN113350486A CN113350486A CN202110761675.8A CN202110761675A CN113350486A CN 113350486 A CN113350486 A CN 113350486A CN 202110761675 A CN202110761675 A CN 202110761675A CN 113350486 A CN113350486 A CN 113350486A
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- glycerophosphate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The invention relates to a combined pharmaceutical composition with bone defect repair effect and application thereof, wherein the combined pharmaceutical composition comprises recombinant protein rH-BMP2 and beta-sodium glycerophosphate. The invention creatively combines the recombinant protein rH-BMP2 and the beta-sodium glycerophosphate to be used as a combined medicine composition for repairing bone defect, can improve the bone forming effect of rH-BMP2 by tens of times on the premise of smaller dosage of rH-BMP2, and effectively solves the problem of insufficient activity of rH-BMP 2. Meanwhile, the beta-sodium glycerophosphate can also be used as a venous phosphorus supplement to meet the daily requirement of the human body on phosphorus. Thirdly, the sodium beta-glycerophosphate is used as a substrate of alkaline phosphatase, and can still cooperate with the BMP2 protein of the human body after the in vitro supplemented BMP2 fails, so that the bone defect part is repaired more quickly. In addition, the sodium beta-glycerophosphate can be used as a raw material to participate in synthesis and connection with a bone graft material, so that the rH-BMP2 can be fixed on the bone graft material and then fixed on a defect part, and the osteogenesis induction effect can be better exerted.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a pharmaceutical composition with a bone defect repairing effect and application thereof.
Background
The repair of bone defect and bone regeneration are important problems in clinical medicine, and currently, for patients with large bone defect, the bone defect is repaired and healed mainly by means of autologous bone transplantation, allogeneic bone transplantation, tissue engineering material transplantation and the like. Autologous bone grafting is a good choice but has limited sources and is difficult to repair bone defects that are greater than 1.5 times the length of the bone being grafted. Although the source of allogeneic bone transplantation is wide, the allogeneic bone transplantation has certain immunological rejection reaction and is easy to cause cross infection. Therefore, various bone substitute materials are sought to avoid the disadvantages of autologous bone and allogeneic bone, and therefore, transplantation of tissue engineering materials becomes a hot spot in current bone transplantation research. The better tissue engineering material not only has the function of bone conduction, but also has the effect of bone induction.
BMP2 is the only good bone growth stimulating factor recognized at present because of its good osteoinductive activity. However, the CHO-derived BMP2 has long production period, high cost and low yield, and the surgical application also causes great economic burden to patients. The recombinant protein rH-BMP2 avoids the above problems, so the recombinant protein rH-BMP2 is frequently used by the current domestic clinical spinal fusion operation, but the recombinant protein rH-BMP2 is generally BMP2 protein purified in a bacterial expression system, because prokaryotic cells lack a post-translational modification system, the activity of the recombinant protein rH-BMP2 is obviously lower than that of BMP2 from Chinese hamster ovary cells CHO, and if the recombinant protein rH-BMP 3578 is used in a large dose, the side reactions such as ectopic ossification, bone cyst and inflammation are easily caused. Therefore, it is necessary to develop a strategy for amplifying the effect of rH-BMP2 several times without increasing the dosage of rH-BMP 2.
Also, direct application of BMP2 has the disadvantages of being easily degradable in vivo, short half-life and not being fixed at the defect site, and therefore is often used in combination with tissue engineering materials to slow down protein degradation and flow.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a pharmaceutical composition with bone defect repair effect and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a pharmaceutical combination composition having bone defect repair efficacy, comprising recombinant protein rH-BMP2 and sodium β -glycerophosphate.
The invention creatively combines the recombinant protein rH-BMP2 and the beta-sodium glycerophosphate to be used as a combined medicine composition for repairing bone defects, the beta-sodium glycerophosphate is one of the components of osteogenesis inducing liquid, is often combined with ascorbic acid to induce in vitro bone marrow mesenchymal stem cells to differentiate into osteogenesis, and can improve the osteogenesis effect of tens of times of rH-BMP2 gene level and protein level on the premise of smaller dosage of rH-BMP2 when being combined with the rH-BMP2, thereby effectively solving the problem of insufficient activity of the rH-BMP 2. Among them, rH-BMP2 is a common bone growth stimulating factor, which can induce myoblast to differentiate into osteoblast, so that ALP activity of alkaline phosphatase of myoblast is enhanced, and β -sodium glycerophosphate is a substrate of ALP, which releases more inorganic phosphorus under ALP catalysis, and the increase of inorganic phosphorus promotes the formation of more hydroxyapatite participating in the formation of bone, thereby positively feeding back the whole osteogenesis pathway.
Meanwhile, the beta-sodium glycerophosphate can also be used as a venous phosphorus supplement to meet the daily requirement of the human body on phosphorus. Thirdly, the sodium beta-glycerophosphate is used as a substrate of alkaline phosphatase, and can still cooperate with the BMP2 protein of the human body after the in vitro supplemented BMP2 fails, so that the bone defect part is repaired more quickly. In addition, BMP2 has the direct disadvantages of being easily degraded in vivo, having a short half-life and not being fixed at the defect site, and sodium β -glycerophosphate can be used as a raw material to participate in synthesis and connection to a bone graft material, so rH-BMP2 can be fixed to the bone graft material and thus fixed at the defect site, and thus better bone formation induction effect can be exerted.
In the present invention, the dosage form of the pharmaceutical composition for combination includes any one of suspension, granule, capsule, powder, tablet, emulsion, solution, drop pill, injection, suppository, enema, aerosol, patch or drop.
Preferably, the pharmaceutical composition for combination further comprises pharmaceutically acceptable pharmaceutical excipients.
The combined pharmaceutical composition can be independently administered or matched with auxiliary materials to be prepared into a proper dosage form for administration.
Preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent.
The combination of at least two of the above-mentioned components, such as the combination of diluent and excipient, the combination of binder and wetting agent, the combination of emulsifier and cosolvent, etc., can be selected in any combination manner, and will not be described in detail herein.
In the present invention, the administration route of the combination drug includes oral administration, sublingual administration, rectal administration, skin mucosa administration, inhalation administration or injection administration.
Preferably, the pharmaceutical composition for combination is a single compound formulation.
Preferably, the pharmaceutical composition for combination is a combination of two separate preparations, namely a recombinant protein rH-BMP2 preparation and a sodium beta-glycerophosphate preparation.
Preferably, the two separate formulations are administered simultaneously.
Preferably, the two separate formulations are administered sequentially.
The combined pharmaceutical composition can be in a single compound preparation form, and can also be a combination of two separate preparations; when two separate preparations are combined, the administration may be simultaneous or sequential, for example, the recombinant protein rH-BMP2 may be administered first, followed by a time interval of sodium β -glycerophosphate, followed by a time interval of recombinant protein rH-BMP2, or the two preparations may be administered alternately.
In the present invention, the pharmaceutical composition for combination enhances the activity of alkaline phosphatase in myoblasts (e.g., C2C12) and osteoblast precursor cells (e.g., MC3T3E 1-14).
In the present invention, the pharmaceutical composition for combination promotes osteogenic differentiation of myoblasts (e.g., C2C12) and osteoblast precursor cells (e.g., MC3T3E 1-14).
In a second aspect, the present invention provides the use of a combination as defined in the first aspect for the manufacture of a medicament for the repair of a bone defect.
In a third aspect, the present invention provides the use of a combination as defined in the first aspect for the preparation of a bone graft material.
In a fourth aspect, the present invention provides a bone graft material comprising a base material and a combination as defined in the first aspect supported on the base material.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively combines the recombinant protein rH-BMP2 and the beta-sodium glycerophosphate to be used as a combined medicine composition for repairing bone defects, the beta-sodium glycerophosphate is one of the components of osteogenesis inducing liquid, is often combined with ascorbic acid to induce in vitro bone marrow mesenchymal stem cells to differentiate into osteogenesis, and can improve the osteogenesis effect of tens of times of rH-BMP2 gene level and protein level on the premise of smaller dosage of rH-BMP2 when being combined with the rH-BMP2, thereby effectively solving the problem of insufficient activity of the rH-BMP 2. Wherein rH-BMP2 is a common bone growth stimulating factor which can induce myoblasts and osteoblast precursor cells to differentiate into osteoblasts so as to enhance the ALP activity of alkaline phosphatase of the myoblasts and osteoblast precursor cells, and beta-sodium glycerophosphate is a substrate of ALP and releases more inorganic phosphorus under the catalysis of ALP, and the increase of the inorganic phosphorus promotes the formation of more hydroxyapatite to participate in the formation of bone, thereby positively feeding back the whole osteogenesis pathway.
Meanwhile, the beta-sodium glycerophosphate can also be used as a venous phosphorus supplement to meet the daily requirement of the human body on phosphorus. Thirdly, the sodium beta-glycerophosphate is used as a substrate of alkaline phosphatase, and can still cooperate with the BMP2 protein of the human body after the in vitro supplemented BMP2 fails, so that the bone defect part is repaired more quickly. In addition, BMP2 has the direct disadvantages of being easily degraded in vivo, having a short half-life and not being fixed at the defect site, and sodium β -glycerophosphate can be used as a raw material to participate in synthesis and connection to a bone graft material, so rH-BMP2 can be fixed to the bone graft material and thus fixed at the defect site, and thus better bone formation induction effect can be exerted.
Drawings
FIG. 1 is a graph of the staining of C2C12 cells for 3 days ALP as provided in example 1 of the present invention;
FIG. 2 is a graph of the staining of C2C12 cells for 7 days ALP as provided in example 1 of the present invention;
FIG. 3 is a graph of staining of MC3T3E1-14 cells for 3 days ALP as provided in example 1 of the present invention;
FIG. 4 is a 7-day real-time fluorescent quantitative PCR (ALP) statistical chart of C2C12 cells provided in example 2 of the present invention;
FIG. 5 is a 7-day statistical chart of real-time fluorescent quantitative PCR results of C2C12 cells provided in example 2 of the present invention (RUNX 2);
FIG. 6 is a statistical chart of the 7-day real-time fluorescent quantitative PCR results of C2C12 cells provided in example 2 of the present invention (COL 1);
FIG. 7 is a 7-day statistical plot of real-time fluorescent quantitative PCR results (OCN) of C2C12 cells provided in example 2 of the present invention;
FIG. 8 is a statistical graph of the real-time fluorescent quantitative PCR results (ALP) for 7 days for MC3T3E1-14 cells as provided in example 2 of the present invention;
FIG. 9 is a 7-day statistical plot (OCN) of real-time fluorescent quantitative PCR results of MC3T3E1-14 cells provided in example 2 of the present invention;
FIG. 10 is a graph of alizarin red staining for C2C12 cells for 14 days as provided in example 3 of the present invention;
FIG. 11 is a graph of alizarin red staining for MC3T3E1-14 cells provided in example 3 of the present invention for 14 days;
FIG. 12 is a micro CT result chart of ectopic bones of each group of C57 mice;
FIG. 13 is a graph of tissue volume statistics for groups of C57 mice;
FIG. 14 is a graph of bone volume statistics for each group of C57 mice;
FIG. 15 is a micro CT result chart of skull defect of each group of SD rats;
FIG. 16 is a graph of statistical results of bone density of various groups of SD rats;
FIG. 17 is a graph of statistics of relative bone volume for groups of SD rats.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The rH-BMP2 referred to in the examples below was from R & D systems, model 355-BM-050/CF; sodium beta-glycerophosphate was obtained from sigma-aldrich and was model 35675-250 GM; the C2C12 cell is derived from Pranoxel and has the model of CL-0044; the C57BL/6J mouse is from the Guangdong province medical experimental animal center; SD rat comes from Guangdong province medical experimental animal center; medical gelatin sponge was purchased from Guangzhou kuaikang medical devices, Inc. and was type A.
Example 1
The effect of the combination on ALP activity in C2C12 and MC3T3E1-14 cells was explored:
C2C12 cells are plated on a 24-plate basis by the number of 5 multiplied by 10^5 cells per hole, and after the cells adhere to the wall, a control group is replaced by a culture medium of high-glucose DMEM, 10% FBS, 1% penicillin and streptomycin; the beta-sodium glycerophosphate group is characterized in that beta-sodium glycerophosphate is added into a culture medium, and the final concentration in the culture medium is 10 mM; recombinant BMP2 is added into the rH-BMP2 group, and the final concentration is 100 ng/mL; the synergistic group contained sodium beta-glycerophosphate and rH-BMP2 at final concentrations consistent with the simple group. After that, the culture medium is removed after changing the solution every 1 day, inducing for 3 or 7 days, washing with PBS 3 times, fixing with 4% paraformaldehyde for 10min, and washing with ddH2O 3 times. Staining was performed for 1h with BCIP/NBT alkaline phosphatase color kit. The staining solution was discarded and washed 3 times with ddH 2O. The photographs were taken under a microscope and the results are shown in FIG. 1 and FIG. 2, respectively (picture scale 500 μm).
Plating MC3T3E1-14 cells on 24 plates at 2.5 × 10^5 cells per well, and after the cells adhere to the plates, forming alpha-MEM + 10% FBS + 1% penicillin and streptomycin culture medium by contrast; the beta-sodium glycerophosphate group is characterized in that beta-sodium glycerophosphate is added into a culture medium, and the final concentration in the culture medium is 10 mM; recombinant BMP2 is added into the rH-BMP2 group, and the final concentration is 100 ng/mL; the synergistic group contained sodium beta-glycerophosphate and rH-BMP2 at final concentrations consistent with the simple group. After that, the culture medium is removed after changing the solution every 1 day, inducing for 3 days, washing with PBS 3 times, fixing with 4% paraformaldehyde for 10min, and washing with ddH2O 3 times. Staining was performed for 1h with BCIP/NBT alkaline phosphatase color kit. The staining solution was discarded and washed 3 times with ddH 2O. The photographs were taken under a microscope, and the results are shown in FIG. 3 (picture scale 500 μm).
As can be seen from fig. 1-3: at 3 days, the ALP staining results of the beta-sodium glycerophosphate group and the blank control group are the same, the beta-sodium glycerophosphate does not show osteogenesis effect on C2C12 cells for 3 days, and the weak enhancement effect is shown only at 7 days; rH-BMP2 has the function of inducing C2C12 cells to differentiate into osteoblasts and shows that ALP staining is deepened; the synergistic rH-BMP2+ beta-sodium glycerophosphate is obviously darker than the ALP staining of the rH-BMP2 group, and shows strong capability of enhancing the ALP activity of C2C12 cells. Therefore, rH-BMP2 can enhance the capability of the C2C12 cells to differentiate into osteogenesis in cooperation with sodium beta-glycerophosphate. The same induction effect as the trend of the C2C12 cell was shown on the osteoblast precursor cell MC3T3E1-14 cell, so that the recombinant BMP2 and the beta-sodium glycerophosphate can synergistically promote the osteogenic ALP activity of different cells.
Example 2
The influence of the combined pharmaceutical composition on the expression level of the osteogenesis related genes is explored:
C2C12 cells are plated on a 24-plate basis by the number of 5 multiplied by 10^5 cells per hole, and after the cells adhere to the wall, a control group is changed into high-glucose DMEM + 10% FBS + 1% penicillin and streptomycin; adding beta-sodium glycerophosphate into the culture medium to a final concentration of 10 mM; recombinant BMP2 is added into the rH-BMP2 group, and the final concentration is 100 ng/mL; the synergistic group contained sodium beta-glycerophosphate and rH-BMP2 at final concentrations consistent with the simple group. Changing the liquid every other 1 day, inducing for 7 days, removing the culture medium, washing with PBS for 3 times, adding 1mL TRIZOL lysed cells into each hole, adding 200 μ L chloroform into the lysate, shaking thoroughly, standing for 10min, and centrifuging at 12000rpm for 15 min; adding isopropanol into the supernatant, mixing, standing for 10min, and centrifuging at 12000rpm for 10 min; discarding the supernatant, washing the precipitate with 75% alcohol solution prepared with DEPC water for 1 time, and centrifuging at 12000rpm for 5 min; the supernatant was discarded, air dried for 10min, 20. mu.L of DEPC water was added to dissolve RNA, and the RNA concentration was determined in NanoDrop 2000. After reverse transcription was performed on each sample at 500ng RNA/10. mu.L reaction system, 5-fold DEPC water was added to dilute the cDNA, and real-time fluorescent quantitative PCR was performed, the results of which are shown in FIGS. 4 to 7.
Plating MC3T3E1-14 cells at 2.5 × 10^5 cells per well on 24 plates, and after the cells adhere to the wall, replacing the control group with alpha-MEM + 10% FBS + 1% penicillin and streptomycin culture medium; adding beta-sodium glycerophosphate into the culture medium to a final concentration of 10 mM; recombinant BMP2 is added into the rH-BMP2 group, and the final concentration is 100 ng/mL; the synergistic group contained sodium beta-glycerophosphate and rH-BMP2 at final concentrations consistent with the simple group. Changing the liquid every other 1 day, inducing for 7 days, removing the culture medium, washing with PBS for 3 times, adding 1mL TRIZOL lysed cells into each hole, adding 200 μ L chloroform into the lysate, shaking thoroughly, standing for 10min, and centrifuging at 12000rpm for 15 min; adding isopropanol into the supernatant, mixing, standing for 10min, and centrifuging at 12000rpm for 10 min; discarding the supernatant, washing the precipitate with 75% alcohol solution prepared with DEPC water for 1 time, and centrifuging at 12000rpm for 5 min; the supernatant was discarded, air dried for 10min, 20. mu.L of DEPC water was added to dissolve RNA, and the RNA concentration was determined in NanoDrop 2000. After reverse transcription was performed on each sample at 500ng RNA/10. mu.L reaction system, 5-fold DEPC water was added to dilute the cDNA, and real-time fluorescent quantitative PCR was performed, the results of which are shown in FIGS. 8 to 9.
As can be seen from fig. 4-9: from the gene expression of ALP, the sodium beta-glycerophosphate has weak ALP activity promoting C2C12 cells, and the synergistic group enhances the ALP activity by 20 times compared with the single rH-BMP2 group and enhances the ALP activity by hundreds of times compared with the single sodium beta-glycerophosphate group. The synergistic group has an osteogenesis inducing effect on C2C12 cells, and is shown to enhance the expression level of osteogenesis related genes such as ALP, RUNX2, COL1 and OPN. Meanwhile, MC3T3E1-14 cells, which are osteoblast precursor cells, showed that the synergistic group was able to enhance the gene expression of ALP and OCN of recombinant BMP2 by 3 times. Thus, the recombinant BMP2 and the sodium beta-glycerophosphate are proved to be capable of synergistically promoting osteogenic differentiation of different cells. Significant differences in the figures are represented by a, b and c, and p values are less than 0.05, which respectively represent significant differences in the control group, the sodium beta-glycerophosphate and the recombinant BMP 2. The method adopted for significance analysis is one-way analysis of variance.
Example 3
The capacity of the combined pharmaceutical composition to mineralize C2C12 and MC3T3E1-14 cells was explored:
C2C12 cells are plated in 24 plates by the number of 5 multiplied by 10^5 cells per hole or MC3T3E1-14 cells by the number of 2.5 multiplied by 10^5 cells per hole, and after the cells are attached to the wall, a control group is replaced by high-sugar DMEM + 10% FBS + 1% penicillin and streptomycin culture medium; the beta-sodium glycerophosphate group is characterized in that beta-sodium glycerophosphate is added into a culture medium, and the final concentration in the culture medium is 10 mM; recombinant BMP2 is added into the rH-BMP2 group, and the final concentration is 100 ng/mL; the synergistic group contained sodium beta-glycerophosphate and rH-BMP2 at final concentrations consistent with the simple group. After 1 day, the culture medium is removed after the liquid is changed and induced for 14 days, the culture medium is washed 3 times by PBS, fixed for 10min by 4 percent paraformaldehyde and washed 3 times by ddH 2O. Staining with 40mM alizarin red for 15 min. The staining solution was discarded, washed 3 times with PBS, and left for 2min each time. The photographs were then taken under a microscope and the results are shown in FIGS. 10-11, respectively.
Example 4
The research on the combination of the medicinal composition for promoting the ectopic osteogenesis of the calf muscles of the mice:
preparing materials, namely implanting medical gelatin sponge used as a material of calf muscles, cutting gelatin sponge blocks into uniform square small blocks, just placing the small blocks in a 96-well plate, and taking gelatin sponge as a control group; the amount of 0.2mM substance is dropped into each small block of the beta sodium glycerophosphate group; 2.5 mu g of protein is dripped into each small block of the recombinant BMP2 group; the synergistic group was added dropwise with 0.2mM BGP and 2.5. mu.g rH-BMP2, and after aspiration into gelatin sponge, frozen at-80 ℃ for 2h, and then lyophilized.
Preoperative preparation, 24C 57BL/6J mice were divided into 4 groups of 6 mice per group by the upper speed. During operation, after a mouse is anesthetized by 1% sodium pentobarbital, leg hair on the muscle side of the calf on the two sides of the mouse is shaved off, the muscle is cut, the material is inserted into the muscle gap, and the suture is sewn. Two weeks after surgery, the formed ectopic bone in the mouse calf was removed and fixed with 4% paraformaldehyde. After the fixed tissue was photographed with a brookficro CT SkyScan1272 (as shown in fig. 12), the corresponding SkyScan1272 software was used to reconstruct and analyze, and the tissue volume and bone volume of each group were calculated, and the statistical results are shown in fig. 13 and fig. 14, respectively.
From FIGS. 12-14, it can be seen that: the control group of pure gelatin sponge did not form ectopic bone and thus the value was 0; sodium beta-glycerophosphate alone does not form ectopic bone either; the single protein group and the synergistic group form ectopic bones, and the synergistic group has significant difference in bone generation amount compared with the rH-BMP2 group, which indicates that the beta-sodium glycerophosphate promotes the bone regeneration effect of the rH-BMP 2. Significant differences in the figures are represented by a, b and c, and p values are less than 0.05, which respectively represent significant differences in the control group, the sodium beta-glycerophosphate and the recombinant BMP 2. The method adopted for significance analysis is one-way analysis of variance.
Example 5
The repairing effect of the combined medicinal composition on the skull defects of rats is explored:
preparing materials, namely cutting the medical gelatin sponge blocks into required sizes, wherein the blank material group is gelatin sponge; the amount of 0.2mM substance is dropped into each small block of the beta sodium glycerophosphate group; 2.5 mu g of protein is dripped into each small block of the recombinant BMP2 group; the synergistic group was added dropwise with 0.2mM BGP and 2.5. mu.g rH-BMP2, and after aspiration into gelatin sponge, frozen at-80 ℃ for 2h, and then lyophilized.
Preoperative preparation, 35 SD rats were divided into 5 groups of 7 rats each, blank control group, blank material group, sodium beta-glycerophosphate group, recombinant BMP2 group and synergistic group, respectively, with no material added to the blank control group.
During operation, rats are anesthetized by 2% sodium pentobarbital, skull skin is cut, round holes with the diameter of 2mm are drilled on the two sides of the skull by skull drills respectively, the skull is just filled with prepared materials, and the skull is sutured. The skull around the skull defect of the SD rat is taken out after 4 weeks of operation and fixed in 4% paraformaldehyde. After micro CT was taken with SkyScan1272 instrument (as shown in fig. 15), bone density and relative bone volume were calculated corresponding to software reconstruction and analysis, as shown in fig. 16 and 17, respectively.
As can be seen from FIGS. 15-17: the bone defect repairing effect is that the synergistic group is beta-sodium glycerophosphate group, rH-BMP2 group and blank group. The two components have obvious effects when used independently, the effect of the sodium beta-glycerophosphate group is better than that of the single rH-BMP2 group, and the synergistic effect is the best. Therefore, the invention has great clinical significance if being used for repairing bone defects. The significant differences in the figures are represented by x, a, b and c, and the p value is less than 0.05, which respectively represents the significant differences in the blank control group, the material control group, the sodium beta-glycerophosphate and the recombinant BMP 2. The method adopted for significance analysis is one-way analysis of variance.
The applicant states that the present invention is illustrated by the above examples to provide a pharmaceutical composition with bone defect repair effect and its application, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A pharmaceutical combination composition having bone defect repair efficacy, comprising recombinant protein rH-BMP2 and sodium β -glycerophosphate.
2. The combination according to claim 1, wherein the dosage form of the combination comprises any one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, dripping pills, injections, suppositories, enemas, aerosols, patches or drops.
3. The combination composition of claim 1, further comprising a pharmaceutically acceptable pharmaceutical excipient;
preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent.
4. The combination composition of claim 1, wherein the route of administration of the combination comprises oral, sublingual, rectal, mucocutaneous, inhalation, or injection.
5. The combination pharmaceutical composition of claim 1, wherein the combination pharmaceutical composition is a single, combination formulation.
6. The combination pharmaceutical composition of claim 1, wherein the combination pharmaceutical composition is a combination of two separate formulations of recombinant protein rH-BMP2 and a formulation of sodium β -glycerophosphate;
preferably, the two separate formulations are administered simultaneously;
preferably, the two separate formulations are administered sequentially.
7. The combination pharmaceutical composition of claim 1, wherein the combination pharmaceutical composition enhances the activity of myoblast and osteoblast precursor cell alkaline phosphatase;
preferably, the pharmaceutical composition promotes osteogenic differentiation of myoblasts C2C12 and osteoblast precursor cells MC3T3E 1-14.
8. Use of a combination pharmaceutical composition according to any one of claims 1 to 7 for the preparation of a medicament for the repair of a bone defect.
9. Use of a combination pharmaceutical composition according to any one of claims 1 to 7 for the preparation of a bone graft material.
10. A bone graft material comprising a base material and, supported thereon, the combination pharmaceutical composition according to any one of claims 1 to 7.
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