CN1163780A - Osteogenesis stimulin injection and its preparing process - Google Patents

Osteogenesis stimulin injection and its preparing process Download PDF

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Publication number
CN1163780A
CN1163780A CN 97108463 CN97108463A CN1163780A CN 1163780 A CN1163780 A CN 1163780A CN 97108463 CN97108463 CN 97108463 CN 97108463 A CN97108463 A CN 97108463A CN 1163780 A CN1163780 A CN 1163780A
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China
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growth factor
bone
bmp
osteogenesis
bfgf
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CN 97108463
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CN1068793C (en
Inventor
胡蕴玉
李亚菲
朱银善
桑宏勋
刘建
李丹
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Whole Army Inst Of Traumatology And Orthopedics Department No4 Military Medic
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Whole Army Inst Of Traumatology And Orthopedics Department No4 Military Medic
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Abstract

A bone-growth stimulant injection extracted from animal bones contains ox-bone morphogenetic protein bBMP, fibroblast growth stimulant bFGF and polyvinylpyrrolinone PVP. It is prepared through such steps as dialysis of urea, homogenizing, dialysis of bBMP, heating to dissolve PVP, dissolving bFGF, mixing, quick freezing, drying, sterilization, volatilizing and culture with aerobe or anaerobe to obtain finished product if it shows negative. Its advantages include low dosage, short course of treatment and obvious effect on promoting bone healing and malposition bone generation.

Description

Osteogenesis stimulin injection and preparation method thereof
The invention belongs to the medicinal preparation technical field, relate to a kind of bone growth factor injection of from the natural animal bone material, extracting and preparation method thereof.
At Osteopathic Medicine circle, traditional bone conduction theory is subjected to bone in recent years and induces theoretical impact.Orthopaedic diseases such as, bone does not connect damaged with skeletal growth factor treatment fracture, bone are the very active both at home and abroad research topics of competitively carrying out.A large amount of research reports is verified, bone morphogenetic protein BONE MORPHONETIC PROTEIN (hereinafter to be referred as BMP) but bone that the mesenchymal cell that moves about around the induction of vascular is converted into irreversibility is born of the same parents, thereby can produce cartilage and osseous tissue in the position beyond skeleton or skeleton, this mechanism is generally acknowledged by domestic and international Osteopathic Medicine circle.In the domestic and international prior art before the present invention, with the research work of every induced osteogenesis on basis, BMP position carry out a lot, for example: to the research of bone morphogenetic protein (BMP) immunity principle and regulatory function; Experimentation to endogenous BMP and guided bone regeneration; Multiple to BMP and other factor and use research; The research of gene recombinaton BMP; Carrier is to research of BMP activity influence or the like.Existing experiment showed, that the partially purified BMP of natural extract has good bone-inducting active, contain the bone-inducting active height of the BMP of several molecular weight, but when cost height, manufacturing cost, can not satisfy the demand than the purification unimodal molecular weight.The active relation of carrier and BMP is an inevitable problem in the experimentation of BMP and the clinical practice, because the content of BMP in bone seldom, extracted amount is limited, uses the BMP of high purification that the danger of being taken away by local body fluid is arranged again.Therefore, the application of BMP must consider to adopt the problem of which kind of optimum carrier simultaneously.Carrier commonly used in the prior art has decalcified bone matrix (DBM), porous calcium phosphate (TCP), hydroxyapatite (HAP), bioactivity glass magnetic and fibrin paste etc., but all there is such or such problem more or less in these carriers.In sum, though in recent years, formed climax based on the research of the bone induced osteogenesis of BMP, some work also has innovation very much, up to now, belongs to very sophisticated, to can be used in clinical BMP medicinal preparation actually rare.Induce in the different another kind of prior aries of mechanism with above-mentioned bone, Chinese CN89105016.7 patent disclosure a kind of " cell growth stimulant and manufacture method thereof ".Main component in this medicine is aseptic plasma-coagulase, and contains protein, polypeptide and several amino acids, is the extracting solution of thanking to the golden staphylococci metabolite that pig myocardium peptone and sodium-chloride water solution are made training oxygen base.This injection that impels union of fracture and treatment ulcer, what emphasize on the mechanism of curing the disease is the synergism of multiple bioactive substance, do not need to consider effective ingredient or the effective site mechanism of action and separation problem, therefore, this medicine consumption in actual use is also very big, need carry out just produce effects fruit of multiple injection.At present, this medicine several years that appeared on the market, but so far its medical mechanism and actual efficacy are not had clear and definite saying.Induce skeletal growth factor treatment mechanism under the theoretical direction that difference on the level is arranged with above-mentioned bone.
At above-mentioned prior art situation, the objective of the invention is to: provide a kind of and have based on the compound skeletal growth factor of the partially purified BMP of natural extract and have the injection type medicinal preparation and the preparation method of the best sensitization carrier of effective slow releasing function.And make every effort to make that its effect is remarkable, expense is few, be easy to preserve, easy to use.
Now design of the present invention and technical solution are described below:
Induced osteogenesis theory based on BMP is thought: induced osteogenesis must possess three conditions, that is: (1) induces stimulus object (as skeletal growth factor); (2) mesenchymal cell; (3) be beneficial to the blood supply environment of osteogenesis.The present invention to the associating topical application of bone morphogenetic protein (BMP) with angiogenesis factor (FGF), has done deep research on the basis of a large amount of experiments.Experimental result confirms, except bone morphogenetic protein (BMP) but the bone that the mesenchymal cell that moves about is converted into irreversibility around the induction of vascular is born of the same parents, the position produces outside cartilage and the osseous tissue beyond skeleton or skeleton, alkalescence becomes cell fibroblast growth factor (FGF), there is the chondrocyte of stimulation to increase the matter effect, be a kind of powerful capillary proliferation stimulant, and the formation of endochondral ossification and blood capillary is the important step behind the bone induced osteogenesis.Experiment is proof also, polyethylene pyrrolinone (PVP) has the good adsorption effect to the protein high molecular material, to skeletal growth factor---the sensitization that is bone morphogenetic protein and fibroblast growth factor is fairly obvious, and it is fine with the intermiscibility of the two, can form suspension completely, be a kind of effective slow-released carrier.In view of the above, the present invention at first provides a kind of osteogenesis stimulin medicinal preparation, it is characterized in that: the exogenous skeletal growth factor and the carrier that contain more than one; Exogenous skeletal growth factor is Bovine Bone Morphoge Protnetin (b BMP) or human bone morphogenesis protein (hBMP) and fibroblast growth factor (bFGF) or gene recombinaton human fibroblastic growth factor; The sensitization carrier is polyethylene pyrrolinone (PVP); Exogenous skeletal growth factor Bovine Bone Morphoge Protnetin (b BMP) or human bone morphogenesis protein (hBMP) are 3: 10 with the weight ratio of sensitization carrier polyethylene pyrrolinone (PVP); Under this conditions of mixture ratios, need to add the fibroblast growth factor (bFGF) or the gene recombinaton human fibroblastic growth factor of 80000IU unit.Secondly, the present invention also provides the preparation method of osteogenesis stimulin injection, it is characterized in that, the preparation process of this preparation is:
(1), take by weighing BMP1.5 gram, pulverize, ooze with the urea element of 20 milliliters of 4M and separate, place in 4 ℃ of refrigerators and use homogenizer homogenate after 12 hours;
(2), the good BMP of homogenate installs the back packing with dialyzer, under 4 ℃ of conditions, with 4000 ml distilled waters dialysis 5~10 times, each interval 5~6 hours; After the dialysis fully, open and wrap homogenate once more, stand-by;
(3), 5 gram PVP are added in 40 mL of saline, heating for dissolving is put cold after-filtration, and transfers PH to 7 with the NaOH of 2M, and 2, dilute the bFGF of 80000IU unit then with this liquid, stand-by;
(4), with (2) liquid and (3) liquid mix homogeneously, transfer to 100ml with Sterile Saline, go into hundred clean, be distributed into bottle; Put-75 ℃ of refrigerator quick-freezings 5 minutes, put again in-30 ℃ of refrigerators and froze 20 minutes, send lyophilizing in the freeze dryer.
(5), take out sample, put in the drying tower, and use oxirane disinfection; And then put volatilization in hundred clean, do aerobe, anaerobic culture simultaneously;
(6), if aerobic, anaerobic culture is negative, be finished product, gland, the rearmounted 4 ℃ of refrigerators of labelling are preserved.
Below, be combined into fibroblast growth factor (bFGF) and strengthen Bovine Bone Morphoge Protnetin (b BMP) in the intravital experiment of mice, the osteogenesis stimulation of medicinal preparation of the present invention is described further:
1, material and method: the method for pressing reported in literature is extracted partially purified bMP from the fresh calf bone, goes into experiment through mice flesh pocket planting, confirms its bone-inducting active.The bMP of 96mg is dissolved in the urea cellulose solution of 24ml6M, and to water, dialysis is 24 hours in the time of 4 ℃, bBMP suspension 24 ampoules of on average packing into, lyophilizing is sealed standby.BFGF is a dried frozen aquatic products.48 kunming mices, male, body weight 25g is divided into 4 groups at random.A group: inject the ampoule that contains bBMP4mg with PBS liquid (phosphoric acid and the phosphatic buffer solution) 0.3ml that contains bBMP100ng, make into suspension, suck the skin test syringe, inject in the mice muscle of thigh.The B group: bBMP4mg/PBS liquid 0.3ml suspension injects in the mice muscle of thigh.C group: bFGF100mg/PBS liquid 0.3ml.D group: PBS liquid 0.3ml.All inject with method.Remove dead mice in the time of 21 days, cut the tissue of injection site, fix with 10% neutral formalin, hydrochloric acid decalcification 24 hours, washing, decalcification, paraffin embedding, section, HE dyeing, light microscopic is observed down.Each is organized all the other specimen and measures as calcium content.Remove specimen surface muscle, homogenate, centrifugal with 10000rpm, precipitate digests with hydrochloric acid, in the atomic absorption spectrophotometer calcium content, as the quantitative index of skeletonization.
2, a bone scleroma can be touched in result: A group mice intramuscular injection position, and as seen lamellar bone is arranged under the histological examination mirror, and bone trabecula and red bone marrow form, and fibrous tissue is arranged.The B group also has new bone formation, but does not see new capillary vessel.C group and D group are not seen new bone.Calcium content is measured, and the A group is 3 times of B group, is 8 times of C group; The B group is greater than C group and D group.
3, conclusion: contain multiple bone-inducing factor and in orthopaedics is clinical, have broad prospects with suitable carrier-bound medicinal preparation.
The present invention compares with prior art, and using dosage is little, number of times is few, to promoting knitting and dystopy skeletonization effect obvious, can be used as sophisticated medical medicine producing and has been used for clinical.

Claims (3)

1, a kind of osteogenesis stimulin injection has the skeletal growth factor that extracts from the natural animal bone material, it is characterized in that: the exogenous skeletal growth factor and the sensitization carrier that contain more than one; Exogenous skeletal growth factor is Bovine Bone Morphoge Protnetin (b BMP) or human bone morphogenesis protein (hBMP) and fibroblast growth factor (bFGF) or gene recombinaton human fibroblastic growth factor; The sensitization carrier is polyethylene pyrrolinone (PVP).
2, osteogenesis stimulin injection according to claim 1 is characterized in that: exogenous skeletal growth factor Bovine Bone Morphoge Protnetin (b BMP) or human bone morphogenesis protein (hBMP) are 3: 10 with the weight ratio of sensitization carrier polyethylene pyrrolinone (PVP); Under this conditions of mixture ratios, need to add the fibroblast growth factor (bFGF) or the gene recombinaton human fibroblastic growth factor of 80000IU unit.
3, a kind of preparation method of osteogenesis stimulin injection according to claim 1 is characterized in that:
The preparation process of this preparation is:
(1), take by weighing BMP1.5 gram, pulverize, ooze with the urea element of 20 milliliters of 4M and separate, place in 4 ℃ of refrigerators and use homogenizer homogenate after 12 hours;
(2), the good BMP of homogenate installs the back packing with dialyzer, under 4 ℃ of conditions, with 4000 ml distilled waters dialysis 5~10 times, each interval 5~6 hours; After the dialysis fully, open and wrap homogenate once more, stand-by;
(3), 5 gram PVP are added in 40 mL of saline, heating for dissolving is put cold after-filtration, and transfers PH to 7 with the NaOH of 2M, and 2, dilute the bFGF of 80000IU unit then with this liquid, stand-by;
(4), with (2) liquid and (3) liquid mix homogeneously, transfer to 100ml with Sterile Saline, go into hundred clean, be distributed into bottle; Put-75 ℃ of refrigerator quick-freezings 5 minutes, put again in-30 ℃ of refrigerators and froze 20 minutes, send the lyophilizing of lyophilizing machine.
(5), take out sample, put in the drying tower, and use oxirane disinfection; And then put volatilization in hundred clean, do aerobe, anaerobic culture simultaneously;
(6), if aerobic, anaerobic culture is negative, be finished product, gland, the rearmounted 4 ℃ of refrigerators of labelling are preserved.
CN97108463A 1997-04-17 1997-04-17 Osteogenesis stimulin injection and its preparing process Expired - Fee Related CN1068793C (en)

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CN1068793C CN1068793C (en) 2001-07-25

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003060076A3 (en) * 2001-12-21 2004-03-04 Ct Pulse Biolog Inc Povidone-containing carriers for polypeptide growth factors
WO2006007780A1 (en) * 2004-07-22 2006-01-26 Fang Xu Injectable bone-repairing bioactive material capable of forming gel and its preparation method
US7087577B2 (en) * 1998-10-16 2006-08-08 Zimmer Orthobiologies, Inc. Method of promoting natural bypass
USRE41286E1 (en) 1997-08-14 2010-04-27 Zimmer Orthobiologics, Inc. Compositions for regeneration and repair of cartilage lesions
CN101816634B (en) * 2010-02-05 2012-05-23 深圳兰度生物材料有限公司 Technology for preparing bone growth factor carrying microsphere by using ultrasonic atomization method
CN103990181A (en) * 2014-05-26 2014-08-20 中国人民解放军第四军医大学 Preparation method of microcarrier-cell compound with induction activity and application of compound
US8829166B2 (en) 2002-06-26 2014-09-09 Zimmer Orthobiologics, Inc. Rapid isolation of osteoinductive protein mixtures from mammalian bone tissue
CN111714285A (en) * 2020-06-29 2020-09-29 温州大学 Layer-by-layer self-assembled film and preparation method thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5364839A (en) * 1990-06-18 1994-11-15 Genetics Institute, Inc. Osteoinductive pharmaceutical formulations
DK0831884T3 (en) * 1995-06-05 2003-11-03 Inst Genetics Llc Use of bone morphogenetic proteins for healing and repair of connective tissue binding

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE41286E1 (en) 1997-08-14 2010-04-27 Zimmer Orthobiologics, Inc. Compositions for regeneration and repair of cartilage lesions
US7087577B2 (en) * 1998-10-16 2006-08-08 Zimmer Orthobiologies, Inc. Method of promoting natural bypass
US6992066B2 (en) * 1998-10-16 2006-01-31 Zimmer Orthobiologics, Inc. Povidone-containing carriers for polypeptide growth factors
US7341999B2 (en) 1998-10-16 2008-03-11 Zimmer Orthobiologics, Inc. Povidone-containing carriers for polypeptide growth factors
WO2003060076A3 (en) * 2001-12-21 2004-03-04 Ct Pulse Biolog Inc Povidone-containing carriers for polypeptide growth factors
EP1465652A4 (en) * 2001-12-21 2009-06-17 Zimmer Orthobiologics Inc Povidone-containing carriers for polypeptide growth factors
US8829166B2 (en) 2002-06-26 2014-09-09 Zimmer Orthobiologics, Inc. Rapid isolation of osteoinductive protein mixtures from mammalian bone tissue
WO2006007780A1 (en) * 2004-07-22 2006-01-26 Fang Xu Injectable bone-repairing bioactive material capable of forming gel and its preparation method
CN101816634B (en) * 2010-02-05 2012-05-23 深圳兰度生物材料有限公司 Technology for preparing bone growth factor carrying microsphere by using ultrasonic atomization method
CN103990181A (en) * 2014-05-26 2014-08-20 中国人民解放军第四军医大学 Preparation method of microcarrier-cell compound with induction activity and application of compound
CN103990181B (en) * 2014-05-26 2015-07-08 中国人民解放军第四军医大学 Preparation method of microcarrier-cell compound with induction activity and application of compound
CN111714285A (en) * 2020-06-29 2020-09-29 温州大学 Layer-by-layer self-assembled film and preparation method thereof
CN111714285B (en) * 2020-06-29 2022-08-19 温州大学 Layer-by-layer self-assembled film and preparation method thereof

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