CN114191445A - Pharmaceutical composition for bone repair - Google Patents
Pharmaceutical composition for bone repair Download PDFInfo
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- CN114191445A CN114191445A CN202210012767.0A CN202210012767A CN114191445A CN 114191445 A CN114191445 A CN 114191445A CN 202210012767 A CN202210012767 A CN 202210012767A CN 114191445 A CN114191445 A CN 114191445A
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
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- 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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Abstract
The invention provides a pharmaceutical composition for bone repair, which comprises self-replicating mRNA, wherein the self-replicating mRNA comprises a bone repair gene, the pharmaceutical composition is favorable for maintaining high expression of the bone repair promoting gene for a long time, and the bone repair can be effectively performed by small-amount and small-batch injection on a bone damage position.
Description
Technical Field
The invention relates to the field of biomedicine, and particularly relates to a pharmaceutical composition for bone repair.
Background
Bone tissue defect caused by trauma, disease and other reasons is a common and difficult problem to deal with clinically, and how to promote repair of bone tissue defect is always the research focus of scholars in related fields. The clinical treatment method for the bone-cartilage full-layer defect comprises the following steps: microfractures, cartilage scraping, joint replacement, mosaic plasty, subchondral bone drilling, and prosthetic joint replacement, among others. However, these treatments have limitations such as limited donor source, lesions at the treatment site, graft loosening and prosthesis loosening, and poor durability.
With the development of biotechnology, new biotechnology is continuously applied to bone repair, for example, studies are made to directly deliver high-efficiency bone inducers to fracture, bone defect and bone nonunion parts by percutaneous injection of bone growth factors, and the method has the advantages of small wound, wide adaptation diseases and the like, and for large-section comminuted fracture patients in clinic, the bone growth factors are injected into the fracture parts after closed reduction, so that the fracture healing rate can be greatly improved; for the patients with nonunion, under the condition of perfect internal fixation or external fixation, local injection of bone growth factors is adopted, so that the application range of non-operative treatment can be further expanded, but the method needs to extract and purify the bone growth factors, the purification process is complex, meanwhile, the bone growth factors are easy to lose efficacy in the purification, storage and use processes, and the effective time is short during use, and the bone growth factors can take effect only by continuous injection. The research also utilizes the tissue engineering technology for bone repair, and the mesenchymal stem cells are compounded with the scaffold material for osteogenic induced differentiation, but the repair by the mesenchymal stem cells is often regulated by local microenvironment, inflammatory cytokines such as tumor necrosis factor, interleukin 1 and the like often inhibit the differentiation of the mesenchymal stem cells to the osteoblasts, the bone regeneration repair is influenced, and simultaneously, the tumor is easily caused by stem cell proliferation.
Disclosure of Invention
The embodiment of the invention provides a pharmaceutical composition for bone repair, which overcomes the problems described above, and the specific scheme is as follows:
a pharmaceutical composition for bone repair comprising a self-replicating mRNA, wherein the self-replicating mRNA comprises a bone repair gene.
Optionally, the bone repair gene is a BMP gene or a VEGFA gene.
Optionally, the self-replicating mRNA is BMP2 gene self-replicating mRNA, VEGFA gene self-replicating mRNA, or BMP2 and VEGFA double gene self-replicating mRNA.
Optionally, the composition further comprises a liposome, and the self-replicating mRNA is dispersed in the liposome.
Optionally, the liposome is composed of DOTAP, HSPC, Chol and PEG-DSPE.
Optionally, the liposome comprises a cationic cell-penetrating peptide.
Optionally, the composition further comprises a hydrogel, and the self-replicating mRNA and liposome are loaded on the hydrogel.
Optionally, the hydrogel is a methacrylic anhydrified gelatin hydrogel.
Optionally, in the hydrogel, the weight percentage of the methacrylic anhydrified gelatin is 3 wt% to 9 wt%.
Optionally, the sequence of the BMP2 gene self-replicating mRNA is shown in SEQ ID No.1, the sequence of the VEGFA gene self-replicating mRNA is shown in SEQ ID No.2, and the sequence of the BMP2 and VEGFA double gene self-replicating mRNA is shown in SEQ ID No. 3.
Optionally, the pharmaceutical composition is an in-situ injection preparation for the bone injury part.
The pharmaceutical composition for bone repair according to the embodiment of the present invention comprises self-replicating mRNA, wherein the self-replicating mRNA contains a bone repair gene of BMP gene or VEGFA gene, and the pharmaceutical composition is advantageous for maintaining high expression of the bone repair promoting gene for a long time and can effectively repair a bone wound site by a small amount of small-batch injection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 is a microscopic observation of hydrogel spheres prepared by GelMA at concentrations of 3 wt%, 5 wt%, 7 wt%, and 9 wt% and the results of the internal characterization of tissue sections in examples;
FIG. 2 is a SEM observation morphology of a mixture of liposomes, short-chain BMP2 gene mRNA complexed with the liposomes, and self-replicating BMP2 gene mRNA complexed with the liposomes in the examples;
FIG. 3 shows the results of in vitro cell experiments on BMP4 gene self-replicating mRNA, TGFB gene self-replicating mRNA, BMP2 gene self-replicating mRNA, VEGFA gene self-replicating mRNA, and BMP2 and VEGFA double gene self-replicating mRNA in examples;
fig. 4 is a graph showing the experimental results of bone repair in practice.
Detailed Description
A specific embodiment of the present invention provides a pharmaceutical composition for bone repair comprising a self-replicating mRNA, wherein the self-replicating mRNA comprises a bone repair gene. The inventor of the invention researches and discovers that after the pharmaceutical composition is applied to a bone wound position in situ, the bone repair promotion gene can be continuously and obviously expressed in a long time, and the bone injury can be effectively repaired by small-amount and few-batch injection.
Pharmaceutical compositions of embodiments of the invention include self-replicating mRNA that includes self-replicating sequences derived from positive-strand ssRNA viruses, such as Venezuelan Equine encephalomyelitis Virus (TC83 Venezuelan Equine encephatis Virus, VEEV), sindbis Virus (Sin-dbis Virus), Chikungunya Virus (Chikungunya Virus), Eastern Equine encephalomyelitis Virus (Eastern Equine encephalii-tis Virus), Western Equine encephalomyelitis Virus (Western Equine encephalomyelitis Virus), mayalureus Virus (Mayarovirus), senlinovirus (Semliki Virus), Venezuelan Equine encephalomyelitis Virus (VEEV 83), and the like, and bone repair gene sequences, in some embodiments, self-replicating mRNA derived from positive-strand ssRNA viruses, such as Venezuelan Equine encephalomyelitis Virus (VEEV 83).
The pharmaceutical composition of embodiments of the present invention, in some embodiments, the self-replicating mRNA comprises a BMP gene or a VEGFA gene, in some embodiments, the self-replicating mRNA is a BMP2 gene self-replicating mRNA, in some embodiments, the self-replicating mRNA is a VEGFA gene self-replicating mRNA, in some embodiments, the self-replicating mRNA is a BMP2 and VEGFA double self-replicating mRNA, and the self-replicating mRNA of embodiments, particularly when the self-replicating mRNA is a BMP2 and VEGFA double self-replicating mRNA, has a more significant bone repair promoting gene expression.
In some embodiments of the pharmaceutical compositions of embodiments of the present invention, the sequence of the BMP2 gene self-replicating mRNA is shown in SEQ ID No.1, the sequence of the VEGFA gene self-replicating mRNA is shown in SEQ ID No.2, and the sequences of the BMP2 and the VEGFA double gene self-replicating mRNA are shown in SEQ ID No. 3.
Pharmaceutical compositions of embodiments of the invention, to increase the biological activity of the self-replicating mRNA, in some embodiments, the compositions further comprise liposomes through which the self-replicating mRNA is dispersed as a delivery vehicle for the pharmaceutical composition. In some embodiments, the lipid of the liposome is a phospholipid, such as hydrogenated soy lecithin (HSPC), lecithin, phosphatidylethanolamine, sphingomyelin, cephalin, cardiolipin, and in some embodiments the lipid further includes cationic lipids, such as 1, 2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1, 2-dioleyloxy-3- (trimethylamino) propane (DOTAP), N- [1- (2, 3-ditetradecyloxy)) propyl ] -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N- [1- (2, 3-dioleyloxy) propyl ] -N, N-dimethyl-N-hydroxyethyl-ammonium bromide (DORIE), N- [1- (2, 3-dioleyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA), 3 β [ N- (N ', N' -dimethylaminoethane) carbamoyl ] cholesterol (DC-Chol) and dimethyl-dioctadecyl ammonium bromide (DDAB), in some embodiments the lipids also include sterols, such as cholesterol (Chol), cholesterol hemisuccinate, cholesterol sulfate or any other derivative of cholesterol, in some embodiments the liposomes also include lipopolymers, such as PEG-DSPE (distearoylphosphatidylethanolamine). As a more efficient delivery liposome, in one embodiment, the liposome is composed of DOTAP, HSPC, Chol and PEG-DSPE, and in some embodiments, the liposome further comprises cationic cell-penetrating peptides (CPPs) in order to further improve the delivery efficiency.
The pharmaceutical composition of embodiments of the present invention, in order to achieve loading and slow release of the drug, in some embodiments, the composition further comprises a hydrogel, the self-replicating mRNA and the liposome are loaded on the hydrogel, the hydrogel has a hydrophilic polymer chain, in some embodiments, the hydrogel has compatibility with the cell, such as gelatin, methacrylic acid anhydrified gelatin, sodium alginate, fibroin, chitosan, collagen, and the like, in some embodiments, the hydrogel is methacrylic acid anhydrified gelatin hydrogel, which is a hydrogel that combines a small collapse rate of the hydrogel sphere and a large adsorption ratio of the liposome, and in some embodiments, the weight percentage of the methacrylic acid anhydrified gelatin in the hydrogel is 3 wt% to 9 wt%.
The pharmaceutical composition of the embodiment of the invention is suitable for bone tissue defects caused by various wounds or diseases, such as bone fractures, bone defects or bone nonunions and the like.
The pharmaceutical composition of the present embodiment, the bone repair comprises regeneration or formation of bone.
The pharmaceutical composition of the embodiments of the present invention, in some embodiments, is an in situ injection preparation for a bone injury site, and when in use, the pharmaceutical composition is injected into the bone injury site in situ.
The present invention is further illustrated by the following specific examples.
Examples
Hydrogel preparation
20g of gelatin was weighed, dispersed in 200mL of PBS (phosphate buffered saline) (0.01M), placed in a 500mL flask or beaker, and heated and stirred in a water bath to bring the gelatin temperature to 60 ℃ and dissolved to be clear (about 30 min). 16ml of MA (methacrylic anhydride) was removed by aspiration with a syringe, and slowly added to the gelatin at a rate of 0.25ml/min using a micro syringe pump, taking care to keep out light. After the completion of MA addition, the reaction was continued for 2 hours in a water bath, and then 200ml of PBS was added to terminate the reaction. After the reaction is terminated for 15min, GelMA (methacrylic acid anhydridized gelatin) is subpackaged in a dialysis bag (MWCO 3500), dialyzed at 38 ℃ overnight, centrifuged at 7000rpm for 15min to remove insoluble substances, and the supernatant is taken out to be continuously dialyzed for 2-3 days. Filtering with vacuum pump to remove macromolecules with the aperture of the ultrafiltration membrane (0.22 micron). And (4) subpackaging the filtered GelMA into a plurality of 10cm plates, and placing the plates in a minus 80 refrigerator for freezing and storing. And freeze-drying at low temperature to obtain the product GelMA.
GelMA with the concentration of 3 wt%, 5 wt%, 7 wt% and 9 wt% is dissolved in double distilled water and heated at 37 ℃, microspheres are prepared by using a microfluidic device, and the microspheres are frozen at-30 ℃ and then irradiated with blue light for 5 min. As can be seen from the observation of the microscope in FIG. 1, when the weight percentage of the methacrylic anhydrified gelatin is 3 to 9 wt%, both the small collapse rate and the large liposome adsorption ratio of the hydrogel sphere can be ensured, and particularly, when the weight percentage is 5 wt%, the maximum liposome adsorption ratio of the hydrogel sphere can be ensured under the condition of the minimum collapse rate.
Liposome preparation
DOTAP (SigmaAldrich), HSPC (SigmaAldrich), Cholesterol (SigmaAldrich) and mPEG2000-DSPE (Shanghai, Biotech Co., Ltd.) were precisely measured with an electronic analytical balance, 289w (cationic cell-penetrating peptide) (reference Acta BIOMATERIALIA 63(2017)123-134 dispersing the composition of permeation for permeation from corn recovery), each of which was dissolved in chloroform to a constant volume, and 1.5mL of 10mg/mL solutions were prepared in the following amounts: DOTAP (MW 698.5):0.706mg/70.6uL, HSPC (MW 78.3):10mg/mL,7.92mg/792uL, Chol (MW 386):3.52mg/352uL, DSPE.PEG2000(MW 2750):1.667mg/166.7uL, 289w 2.701mg/270.1uL were added to the above solutions into a 25mL flask, followed by a rotary evaporator, clamped using a clamp, water bath 38 ℃, rotary switch was turned on, vacuum pump was turned on, vacuum valve was adjusted, and vacuum was slowly pulled. Measuring for 20min after a lipid membrane is formed at the bottom of the flask, closing a rotary switch, adjusting the position of the flask, wiping off water outside the bottom of the flask, opening a vacuum valve, closing a vacuum pump, slowly dripping 1.5mL of pure water into the bottom of the flask by a pipettor, opening an ultrasonic cell disruptor, adjusting the power of parameters to be 20-40%, working for 3min for 2s and stopping for 1s, cleaning an ultrasonic head, enabling the ultrasonic head to be submerged in the liquid level at the bottom of the flask for hydration, observing the hydration condition of the lipid membrane at the bottom, pumping the solution by using a 1mL needle tube, respectively filtering the solution into a 2mL EP tube through 0.45 mu m and 0.22 mu m filters, sealing the membrane, and storing at 4 ℃.
Self-replicating mRNA preparation
Plasmid templates for expressing BMP4 gene, TGFB gene, BMP2 gene, VEGFA gene and BMP2 and VEGFA double genes are respectively constructed by using Simplicon RNA plasmid. And (3) carrying out sequencing verification on the constructed plasmid template (carrying out gene sequence sequencing by using a second-generation sequencer), amplifying the verified plasmid template by using a QRT-PCR (quantitative polymerase chain reaction) method, carrying out linearization treatment on 60ug of BMP4, TGFB, BMP2, VEGFA, BMP2 and VEGFA gene plasmids by using restriction endonuclease respectively, and precipitating and purifying to obtain 30-50ug of linearized BMP4, TGFB, BMP2, VEGFA, BMP2 and VEGFA gene templates. The method comprises the steps of utilizing a linearized template to perform T7 promoter in-vitro transcription by using an in-vitro transcription kit, transcribing for 1-2 hours at the constant temperature of 37 ℃, then performing 5 'end capping and 3' end tailing modification at the constant temperature of 37 ℃, purifying the modified mRNA by using a transcription purification kit, and finally eluting by using deionized water to obtain BMP4, TGFB, BMP2, VEGFA, BMP2 and VEGFA double-gene Self-replicating mRNA (Self-replicating mRNA) solution.
Self-replicating mRNA and liposome composite preparation
500ul (concentration 1ug/ul) of the self-replicating mRNA solution obtained by the above preparation was further dissolved in 500ul of serum-free medium, 500ul of liposome solution (concentration 10mg/ml) was dissolved in 500ul of serum-free medium, and the ratio of the self-replicating mRNA and liposome mixture was adjusted to 1: 1 ratio incubation for 10min resulted in a self-replicating mRNA/liposome mixture.
For comparison, a mixture of short chain BMP2 gene mRNA complexed with liposomes was prepared in the above manner.
The shape of the compound formed by compounding the liposome, the short-chain BMP2 gene mRNA and the liposome and the compound formed by compounding the self-replicating BMP2 gene mRNA and the liposome are shown in figure 2 through SEM observation, and as shown in figure 2, the shape of the compound formed by compounding the liposome and the self-replicating BMP2 gene mRNA is basically consistent with that of the liposome before mixing, so that the liposome has an excellent loading effect on the self-replicating BMP2 gene mRNA.
Hydrogel loading preparation of self-replicating mRNA and liposome complexes
Placing more than 2000ul of the compound mixture of the self-replicating mRNA and the liposome in a 15ml centrifuge tube A, placing 2000ul of the prepared 5 wt% GelMA hydrogel product in a 15ml centrifuge tube B, adding the compound mixture of the self-replicating mRNA and the liposome in the centrifuge tube A into the centrifuge tube B, and incubating for 30min on ice.
Cell experiments
Bone marrow mesenchymal stem cells (BMSC) are cultured in vitro in six-well culture dishes, GFP mRNA is set to be treated as a control group, hydrogel-loaded BMP4 gene self-replicating mRNA (Sa-BMP4), TGFB gene self-replicating mRNA (Sa-TGFB), BMP2 gene self-replicating mRNA (Sa-BMP2), VEGFA gene self-replicating mRNA (Sa-VEGF), BMP2 and VEGFA double gene self-replicating mRNA (Sa-BMP2+ VEGF) obtained as above are used as an experimental group, three repeated experimental wells are set for each group, 1ug of corresponding mRNA is transferred into each well, expression conditions of main bone repair indexes Runx2, OPN and OCN are observed by qRT-PCR, expression conditions of the fifth day are selected as shown in FIG. 3, and results of BMP2 gene self-replicating mRNA and BMP4 and TGFB gene self-replicating mRNA have slight improvements in expression of Runx2, OPN and OCN on the fifth day relative to blank samples, BMP2 and VEGFA double-gene self-replicating mRNA all have obvious expression of Runx2, OPN and OCN on the fifth day, and particularly BMP2 and VEGFA double-gene self-replicating mRNA have the most obvious expression.
Bone repair experiment
1) Establishing rat femur distal bone defect model
According to the weight of each experimental animal, pentobarbital sodium and isoflurane inhalation are used for compound anesthesia. The rat is fixed on the operation table in the supine position, the skin is prepared around the inner side of the right hind limb knee joint, and the rat is disinfected and paved by a conventional method. Taking longitudinal incisions on the inner side of the knee joint, entering layer by layer, exposing the distal end of the femur, horizontally inserting a needle from the inner side surface of the distal end of the femur by using a Kirschner wire with the diameter of 3mm, constructing a bone tunnel with the diameter of 3mm, injecting physiological saline or different materials according to groups, and sealing the tunnel opening by using bone wax. The incision is closed layer by layer after repeated flushing. And (5) putting back to a single cage for feeding after the operation.
2) Preparation of injection sample
And (3) sham: physiological saline
Sa-mRNA/Lip @ GelMA hydrogel loaded BMP2 and VEGFA double-gene self-replicating mRNA and liposome complex prepared by the hydrogel loading preparation method of the self-replicating mRNA and liposome complex;
lip @ GelMA the hydrogel-loaded preparation method of the complex of the self-replicating mRNA and the liposome, wherein the self-replicating mRNA is not mixed in the liposome, and the prepared hydrogel loads the liposome;
the hydrogel prepared by the hydrogel loading preparation method of the self-replicating mRNA and liposome complex loads the short-chain mRNA and the liposome complex, wherein the self-replicating mRNA is replaced by the short-chain BMP2 gene mRNA and is mixed with the short-chain VEGFA gene mRNA (1mol:1 mol).
The hydrogel prepared according to the hydrogel loading preparation method of the self-replicating mRNA and liposome compound loads BMP2 and VEGFA double-gene self-replicating mRNA and liposome compound, but the liposome does not contain cationic Cell Penetrating Peptides (CPPs).
3) Bone repair analysis
Extracting 150ul of the injection sample in the step 2), performing joint injury in-situ injection on the rat femur distal bone defect model in the step 1), performing secondary injection after 2 weeks, obtaining mouse joints at time points after 2 weeks and 4 weeks for tissue fixation, and performing sampling and micro-CT analysis, wherein the result is shown in figure 4. According to the results shown in FIG. 4, the sham-like and Lip @ GelMA-like products have no bone repair effect, and the mRNA/Lip (-CPPs) @ GelMA-like and mRNA/Lip @ GelMA-like products still have cavities, while the Sa-mRNA/Lip @ GelMA-like products have the best bone healing at the epiphyseal part and compact bone hyperplasia, which indicates that the BMP2 and VEGFA double-gene self-prepared mRNA is loaded by liposome containing cationic transmembrane peptide and has very excellent bone repair effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shanghai Weike Biotechnology Limited
<120> a pharmaceutical composition for bone repair
<130> MP21018097
<141> 2021-11-11
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Claims (11)
1. A pharmaceutical composition for bone repair, comprising a self-replicating mRNA, wherein the self-replicating mRNA comprises a bone repair gene.
2. The pharmaceutical composition of claim 1, wherein the bone repair gene is a BMP gene or a VEGFA gene.
3. The pharmaceutical composition of claim 2, wherein the self-replicating mRNA is BMP2 gene self-replicating mRNA, VEGFA gene self-replicating mRNA, or BMP2 and VEGFA double gene self-replicating mRNA.
4. The pharmaceutical composition of claim 1, wherein the composition further comprises a liposome, and wherein the self-replicating mRNA is dispersed in the liposome.
5. The pharmaceutical composition of claim 1, wherein the liposome is comprised of DOTAP, HSPC, Chol, and PEG-DSPE.
6. The pharmaceutical composition of claim 5, wherein the liposome comprises a cationic cell-penetrating peptide.
7. The pharmaceutical composition of claim 4, wherein the composition further comprises a hydrogel, and the self-replicating mRNA and liposome are loaded on the hydrogel.
8. The pharmaceutical composition of claim 7, wherein the hydrogel is a methacrylic anhydrified gelatin hydrogel.
9. The pharmaceutical composition of claim 6, wherein the hydrogel comprises 3 to 9 weight percent methacrylic anhydrified gelatin.
10. The pharmaceutical composition of claim 3, wherein the BMP2 gene self-replicating mRNA has the sequence shown in SEQ ID NO.1, the VEGFA gene self-replicating mRNA has the sequence shown in SEQ ID NO.2, and the BMP2 and VEGFA double gene self-replicating mRNA has the sequence shown in SEQ ID NO. 3.
11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition is an in situ injection preparation for the bone injury.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210012767.0A CN114191445B (en) | 2022-01-06 | 2022-01-06 | Pharmaceutical composition for bone repair |
| PCT/CN2022/140988 WO2023130973A1 (en) | 2022-01-06 | 2022-12-30 | Pharmaceutical composition for bone repair |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116098856A (en) * | 2023-01-16 | 2023-05-12 | 四川大学华西医院 | Photocurable hydrogel composite nucleic acid delivery system and medicine for treating spinal cord injury |
| WO2023130973A1 (en) * | 2022-01-06 | 2023-07-13 | 上海唯可生物科技有限公司 | Pharmaceutical composition for bone repair |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101068509A (en) * | 2004-09-01 | 2007-11-07 | 华沙整形外科股份有限公司 | A polymeric wrap for in vivo delivery of osteoinductive formulations |
| CN102430129A (en) * | 2011-12-17 | 2012-05-02 | 浙江大学 | BMP-2/bFGF dual-gene chitosan nano-microcapsule and application thereof |
| CN102824647A (en) * | 2011-06-13 | 2012-12-19 | 香港中文大学 | Bone-targeted delivery system for osteogenesis treatment based on small nucleic acid medicine, and preparation method thereof |
| CN109568671A (en) * | 2018-12-24 | 2019-04-05 | 四川大学华西医院 | 3D bone repair scaffold with hydrogel loaded with cells and preparation method thereof |
| CN111569148A (en) * | 2020-04-14 | 2020-08-25 | 杭州医学院 | Composite hydrogel for promoting bone repair and preparation method and application thereof |
| US20210052776A1 (en) * | 2019-07-02 | 2021-02-25 | Nevin Witman | Mesenchymal stem cells or stromal cells harboring modified rnas encoding vegf and bmp polypeptides |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107441506A (en) * | 2016-05-30 | 2017-12-08 | 上海交通大学 | Gene delivery carrier and its preparation and application |
| CN111087628A (en) * | 2019-12-31 | 2020-05-01 | 杭州彗搏科技有限公司 | Hydrogel for bone repair and preparation method thereof |
| CN114191445B (en) * | 2022-01-06 | 2024-07-02 | 上海唯可生物科技有限公司 | Pharmaceutical composition for bone repair |
-
2022
- 2022-01-06 CN CN202210012767.0A patent/CN114191445B/en active Active
- 2022-12-30 WO PCT/CN2022/140988 patent/WO2023130973A1/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101068509A (en) * | 2004-09-01 | 2007-11-07 | 华沙整形外科股份有限公司 | A polymeric wrap for in vivo delivery of osteoinductive formulations |
| CN102824647A (en) * | 2011-06-13 | 2012-12-19 | 香港中文大学 | Bone-targeted delivery system for osteogenesis treatment based on small nucleic acid medicine, and preparation method thereof |
| CN102430129A (en) * | 2011-12-17 | 2012-05-02 | 浙江大学 | BMP-2/bFGF dual-gene chitosan nano-microcapsule and application thereof |
| CN109568671A (en) * | 2018-12-24 | 2019-04-05 | 四川大学华西医院 | 3D bone repair scaffold with hydrogel loaded with cells and preparation method thereof |
| US20210052776A1 (en) * | 2019-07-02 | 2021-02-25 | Nevin Witman | Mesenchymal stem cells or stromal cells harboring modified rnas encoding vegf and bmp polypeptides |
| CN111569148A (en) * | 2020-04-14 | 2020-08-25 | 杭州医学院 | Composite hydrogel for promoting bone repair and preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| CAROLINE M ET AL.: "Combinatorial Gene Therapy Accelerates Bone Regeneration: Non-Viral Dual Delivery of VEGF and BMP2 in a Collagen-Nanohydroxyapatite Scaffold", 《ADVANCED HEALTHCARE MATERIALS》, vol. 4, no. 2, pages 223 - 227 * |
| N/A: "Rattus norvegicus bone morphogenetic protein 2, mRNA", Retrieved from the Internet <URL:ncbi.nlm.nih.gov/nucleotide/NM_017178.2> * |
| N/A: "Rattus norvegicus vascular endothelial growth factor A, transcript variant 2, mRNA", Retrieved from the Internet <URL:ncbi.nlm.nih.gov/nucleotide/NM_001110333.2> * |
| 程若昱 等: "双效骨诱导性的有机-无机水凝胶的构建及其骨再生研究", 上海交通大学学报（医学版）, vol. 38, no. 8, pages 901 - 909 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023130973A1 (en) * | 2022-01-06 | 2023-07-13 | 上海唯可生物科技有限公司 | Pharmaceutical composition for bone repair |
| CN116098856A (en) * | 2023-01-16 | 2023-05-12 | 四川大学华西医院 | Photocurable hydrogel composite nucleic acid delivery system and medicine for treating spinal cord injury |
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| Publication number | Publication date |
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| CN114191445B (en) | 2024-07-02 |
| WO2023130973A1 (en) | 2023-07-13 |
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Denomination of invention: A drug combination for bone repair Granted publication date: 20240702 Pledgee: Minhang Branch of Shanghai Rural Commercial Bank Co.,Ltd. Pledgor: Shanghai Weike Biotechnology Co.,Ltd. Registration number: Y2024310001060 |