CN102895704A - Biological activity factor composition for promoting ossification and vascularization - Google Patents
Biological activity factor composition for promoting ossification and vascularization Download PDFInfo
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Abstract
The invention discloses a biological activity factor composition for promoting ossification and vascularization, which comprises a biological active factor and carboxylated polysaccharide, the biological activity factor is one or more selected from a bone morphogenetic protein, a transforming growth factor, a blood vessel endothelial growth factor, a basic fibroblast growth factor and an ossification protein; and the carboxylated polysaccharide is one or more selected from sulfonated chitosan, sulfonated glucan, heparin, heparin sulfate and sulfate cellulose. The invention also discloses a preparation method of a composition and a transplant containing the composition. The composition provided by the invention can promote the ossification and the blood vessel generation, can be composited with a medically acceptable carrier, and can be used in the field of bone defect repair by employing an implantation mode or an injection mode.
Description
Technical field
The invention belongs to biological field, relate to a kind of promote skeletonization and the bioactie agent composition and method of making the same and the application that become blood vessel.
Background technology
The growth course of osseous tissue closely links to each other with vascularization.Blood is for being the deciding factor that guarantees activation in the engineering material of bone tissue body fully.In the process that the embryo occurs or bone injury heals of osseous tissue, good blood vessel network can provide good nutrition supply and matter transportation for tissue, and osteogenesis is unusual and knitting is bad for causing to lack good blood.During the clinical bone grafting repair deficiency, require implant bed to have abundant blood confession, and during autologous bone transplanting, also often strengthen the blood confession by vascular pedicle or band muscle flap, promote that bone graft survives in early days.Therefore, a comparatively perfect vasoganglion is very important for bone formation, and it can not only provide nutrition and oxygen, can also carry the skeletonization precursor, the required somatomedin of secretion osteoblast.
The strategy that bone tissue engineer employing three-dimensional rack and seed cell are compound, although greatly promoted the reparation of osseous tissue damage, but only depend on the periphery osmosis to absorb nutrition during its three-dimensional structure, for undersized bone reparation, rely in early days the infiltration of tissue fluid can obtain nutrition; Repair for the large scale bone, only depend on the infiltration of tissue fluid to be nowhere near, cell is many because malnutrition causes graft failure, and therefore large scale osseous tissue defect repair is faced with the vascularization deficiency at present, organizes the difficult problem of easy ischemic necrosis.How in the new osteoplastic while, promote little blood vessel grow into and cell and material composite in the reconstruction of blood confession become the key link that bone is repaired.
Blood is mainly concentrated in the following areas for the research of rebuilding in the at present relevant artificial bone: utilize microsurgical technique parcel vascular bundle, vessel pedicle or fascial flap to implant and form inherent blood vessel network, promote vascularization; And vascular endothelial cell and osteoblast are united and are implanted after cultivating support.Yang Xin is bright to wait proposition compound from body red bone marrow method construct acellular type tissue-engineered bone with the Fascial flap parcel, but first phase reparation large segmental bone defect (Chinese Tissue Engineering Study and clinical rehabilitation, 2009,13,46:9050); Pei Guoxian etc. utilize the method for Fascial flap parcel tissue engineered bone to repair the rabbit segmental radius defects, finds early stage vascularization be conducive to bone reparation speed (the Chinese experimental surgical magazine, 2007,24(6): 752).This method of implanting with blood vessel is used the vascularization that the blood vessel condition that has existed in the body promotes artificial bone, and surgical effect is clear and definite, increases the extra wound of body, is subjected to the shortcoming such as local vascular condition restriction but exist.With bone marrow stroma stem cell respectively directed differentiation be osteoblast and vascular endothelial cell the two is mutually promoted to play a role be a direction of vascularization research.Kaigler etc. find by zoopery, endotheliocyte and bone marrow stroma stem cell co-transplantation transplant more separately more be conducive to skeletonization (Kaigler D, eral, FASEB J, 2005,19(6): 665); The endotheliocyte that Tan Hongbo etc. induce rabbit bone marrow is inoculated on the decalcified bone matrix support with Fibrin Glue, confirm that the endotheliocyte that bone marrow is induced can become official jargon sample endothelium spline structure (Tan Hongbo etc. at In vitro culture in decalcification bone supporting stand, bone tissue engineering scaffold extracorporeal blood vessel preliminary study, Chongqing Medical, 2007,36(6): 499).But the cycle that cell strategy progenitor cells is cultivated is long, is unfavorable for practical clinical.
Therefore, still need and develop a kind of novel reconstruction biomaterials that can promote simultaneously skeletonization and vascularization in this area.
Summary of the invention
The object of the invention is to provide a kind of bioactie agent composition and method of making the same and application that can promote simultaneously skeletonization and vascularization.
A first aspect of the present invention, a kind of bioactie agent compositions is provided, described compositions comprises bioactie agent and sulfonation polysaccharide, and described bioactie agent is selected from one or more in bone morphogenetic protein (BMP), transforming growth factor (TGF), VEGF (VEGF), basic fibroblast growth factor (bFGF), the BMP (OGP); Described sulfonation polysaccharide is selected from one or more in sulfated chitosan, sulfonation glucosan, heparin, heparin sulfate, the sulfate cellulose.
In another preference, described bioactie agent is BMP-2 or BMP-7; Perhaps
Described bioactie agent is to be selected from lower group the factor and the complex of BMP-2 or BMP-7: transforming growth factor (TGF), VEGF (VEGF), basic fibroblast growth factor (bFGF), BMP (OGP) or its combination.
In another preference, the molecular weight of sulfonation polysaccharide is at 8000-350000Da, and sulfur content is 0.5%-20%.
In another preference, the molecular weight of described sulfonation polysaccharide is at 10000-300000Da, and sulfur content is 1%-20%.
In another preference, the mass ratio of described bioactie agent and sulfonation polysaccharide is 0.05 ~ 120:1.
In another preference, the mass ratio of described bioactie agent and sulfonation polysaccharide is 0.1 ~ 100:1.
In another preference,, described bioactie agent compositions also comprises water.
A second aspect of the present invention provides the preparation method of the described bioactie agent compositions of first aspect, described method to comprise bioactie agent is joined in the sulfonation polysaccharide solution, and mix homogeneously obtains the step of described bioactie agent compositions.
In another preference, described sulfonation polysaccharide solution concentration is 0.01 ~ 150 μ g/mL.
In another preference, described sulfonation polysaccharide solution concentration is 0.02 ~ 100 μ g/mL.
In another preference, described method also comprises carries out cryodesiccated step with the bioactie agent compositions.
A third aspect of the present invention provides a kind of bone implant that becomes, and described implant comprises the described bioactie agent compositions of first aspect and medically acceptable carrier.
A fourth aspect of the present invention provides the purposes of the described bioactie agent compositions of first aspect, for the preparation of repairing bone defect, treatment fracture or repair medicine or the implant of cartilage.
The invention provides a kind of bioactie agent compositions by somatomedin and sulfonation composition of Salvia polysaccharide, can promote simultaneously skeletonization and promote vascularization, and material is easy to get, cost is low, and it is convenient to use.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus consist of new or preferred technical scheme.As space is limited, this tired stating no longer one by one.
Description of drawings
Fig. 1 is the HE stained figure of the interior skeletonization of flesh bag among the embodiment 8.
Fig. 2 is the photo of skeletonization in the embodiment 8 flesh bags.
Fig. 3 is the HE stained figure of the interior skeletonization of flesh bag among the embodiment 9.
Fig. 4 is the photo of skeletonization in the embodiment 9 flesh bags.
Fig. 5 is the HE stained figure of the interior skeletonization of flesh bag among the embodiment 10.
Fig. 6 is the photo of skeletonization in the embodiment 10 flesh bags.
Fig. 7 is the HE stained figure of the interior skeletonization of flesh bag among the embodiment 11.
Fig. 8 is the photo of skeletonization in the embodiment 11 flesh bags.
Fig. 9 is the HE stained figure of the interior skeletonization of flesh bag among the embodiment 12.
Figure 10 is the photo of skeletonization in the embodiment 12 flesh bags.
Figure 11 is the HE stained figure of the interior skeletonization of flesh bag among the embodiment 13.
Figure 12 is the photo of skeletonization in the embodiment 13 flesh bags.
Figure 13 is the HE stained figure of the interior skeletonization of flesh bag among the embodiment 14.
Figure 14 is the photo of skeletonization in the embodiment 14 flesh bags.
Figure 15 is that the interior graft area of flesh bag is organized HE stained figure in the Comparative Examples 1.
Figure 16 is the photo of skeletonization in the Comparative Examples 2 flesh bags.
The specific embodiment
The present inventor is through extensive and deep research, and the unexpected bioactie agent compositions that comprises bioactie agent and sulfonation polysaccharide of finding can promote angiogenesis when promoting skeletonization.On this basis, finished the present invention.
Bioactie agent
So-called bioactie agent, refer to from the organism to the influential trace of biosis tool or a small amount of material, mainly be biologically active polypeptide and cytokine two large classes.
Biologically active peptide (biologicalactivepeptides, bap) refers to the molecule amount less than 6000d, has the polypeptide of various biological function.Its molecular structure complexity differs, can be from simple dipeptides to annular macromole polypeptide, and also these polypeptide can be modified by phosphorylation, glycosylation or acyl group.
Cytokine (cytokine, be called for short ck) refer to can regulate the cell physiological function by activating immune cell or nonimmune cell (comprising stromal cell in bone marrow, the thymus, vascular endothelial cell, fibroblast etc.) synthesis secretion, the micromolecule polypeptide of the various biological effects such as inducing inflammatory reaction, participation immunne response and tissue repair, their belong to secreting type immune molecules the same as immunoglobulin and complement.
Somatomedin belongs to cytokine, is the extracellular polypeptide signal molecule of a class stimulating cellular growth and propagation.
Bone morphogenetic protein (bone morphogenetic proteins, BMP) be a kind of biomaterial that can pass through the induced osteogenesis repairing bone defect, it is a class growth and differentiation factor family, comprise BMP-1, BMP-2, BMP-3, BMP-4, BMP-7 etc., effect has concentration dependent, the meeting of being combined with corresponding sensor produces the signal representation of a series of bone morphogenetic protein response genes and transcribes, and the induced interstitial cell differentiation is bone or cartilage.In nearly 20 years, people not only separate from the many animals osseous tissue and are purified into natural BMP-1, BMP-2, BMP-3 and BMP-4, but also have further gone out the bone morphogenetic protein (rh BMPs) of human gene's restructuring at oocyte and the expression in escherichia coli of Chinese hamster by gene recombination technology.At present existingly surpass that 15 kinds of bone morphogenetic proteins are cloned and at people and Mus expression in vivo.
Transforming growth factor (transforming growth factor, TGF) is the somatomedin that can act on Growth of Cells, conversion etc.Comprise transforminggrowthfactor-α (TGF-α) and transforming growth factor-beta (TGF-β) two classes.TGF-α is by macrophage, and brain cell and epidermis cell produce, and can induce epithelial development.TGF-β is a multifunctional protein, can affect the growth of various kinds of cell, the functions such as differentiation, apoptosis and immunomodulating.TGF-β can be incorporated into the TGF-beta receptor combination of cell surface and activates its receptor.The TGF-beta receptor is the serine/threonine kinase receptor.Its signal transmission can be passed through SMAD signal path and/or DAXX signal path.
VEGF (vascular endothelial growth factor, VEGF) somatomedin of genus platelet-derived growth factor family, stimulate the mitosis of vascular endothelial cell and the generation of blood vessel, improve the permeability of monolayer endothelium, can form heterodimer with placental growth factor.VEGF is the specific heparin binding growth factor of vascular endothelial cell, and induction of vascular is newborn in vivo.
Basic fibroblast growth factor (basic fibroblast growth factor, bFGF) is one and transmits the growth signal, can promote the fissional polypeptide of mesoderm and neuroderm.Has strong angiogenesis function.External, can stimulate cellular proliferation, move, induce activator of plasminogen and collagenase activity, be the cell mitogen that high-affinity is arranged with heparin.
BMP (osteogenin protein, OGP) is to induce osteoplastic protein (less than 50kDa), with extracellular matrix and Heparin-binding, is multifunctional cytokine, belongs to the transforming growth factor superfamily.
The sulfonation polysaccharide
Polysaccharide (polysaccharide) is the sugar chain by the glycosidic bond combination, will surpass the polymerization sugar polymeric carbohydrate that the monosaccharide more than 10 forms at least.Comprise chitosan, starch, aminoglycan and cellulose etc.
The sulfonation polysaccharide is the product that polysaccharide obtains through sulfonating reaction.
Sulfonation is a kind ofly to introduce sulfonic group (-SO in organic molecule
3H) or sulfuryl chlorio (-SO
3Cl) course of reaction.Used sulfonating agent has sulfur trioxide, concentrated sulphuric acid, oleum etc. usually.Sometimes also add chlorine, sulfur dioxide oxygenation and sodium sulfite etc. as sulfonating agent with chlorosulfonic acid, sulfur dioxide.Method of sulfonating has liquid phase sulfonation method and gas phase sulfonation method.Hydrogen in the sulfonation process on the sulfonic group alternate c atoms is called Direct Sulfonation; Halogen on the sulfonic group alternate c atoms or nitro are called indirect sulfonation.
Sulfonation polysaccharide among the present invention is selected from one or more in sulfated chitosan, sulfonation glucosan, heparin, heparin sulfate, the sulfate cellulose.Adopt methods known in the art take chitosan, glucosan etc. as raw material, after sulfonation, make.As the standby sulfated chitosan of reference chlorosulfonic acid sulfonation method or concentrated acid sulfonation legal system and sulfonation glucosan (the Jilin Institute of Chemical Technology journal, 2007,24 (2): 20. China West Normal University's journals, 2006,27(1): 95).
The sulfur content of sulfonation polysaccharide is measured (Elementar, German Vario Macro company) by elemental analyser.
Bioactie agent compositions and preparation thereof
Bioactie agent compositions of the present invention, comprise bioactie agent and sulfonation polysaccharide, described bioactie agent is selected from one or more in bone morphogenetic protein (BMP), transforming growth factor (TGF), VEGF (VEGF), basic fibroblast growth factor (bFGF), the BMP (OGP); Described sulfonation polysaccharide is selected from one or more in sulfated chitosan, sulfonation glucosan, heparin, heparin sulfate, the sulfate cellulose.
The source of described bioactie agent can be people's gene restructuring, through fermentation, separate and obtain, also can be directly extracts in the animal body and obtains.
In another preference, described bioactie agent is BMP-2.
In another preference, described bioactie agent is BMP-7.
In another preference, described bioactie agent is BMP-2 and the complex that is selected from lower group the factor: transforming growth factor (TGF), VEGF (VEGF), basic fibroblast growth factor (bFGF), BMP (OGP) or its combination.
In another preference, described bioactie agent is BMP-7 and the complex that is selected from lower group the factor: transforming growth factor (TGF), VEGF (VEGF), basic fibroblast growth factor (bFGF), BMP (OGP) or its combination.
The molecular weight of sulfonation polysaccharide is at 8000-350000Da, and sulfur content is 0.5%-20%.
In another preference, the molecular weight of described sulfonation polysaccharide is at 10000-300000Da, and sulfur content is 1%-20%.
The mass ratio of described bioactie agent and sulfonation polysaccharide is 0.05 ~ 120:1.
In another preference, the mass ratio of described bioactie agent and sulfonation polysaccharide is 0.1 ~ 100:1.
Described bioactie agent compositions can be lyophilized formulations, exists with solid form; Perhaps soluble in water, exist with the solution form.
The preparation method of described bioactie agent compositions comprises bioactie agent is joined in the sulfonation polysaccharide solution that mix homogeneously obtains the step of described bioactie agent compositions.
In another preference, described sulfonation polysaccharide solution concentration is 0.01 ~ 150 μ g/mL, and preferably, described sulfonation polysaccharide solution concentration is 0.02 ~ 100 μ g/mL.
In another preference, the mass ratio of described bioactie agent and sulfonation polysaccharide is 0.05 ~ 120:1, and preferably, the mass ratio of described bioactie agent and sulfonation polysaccharide is 0.1 ~ 100:1.
In another preference, described method also comprises carries out cryodesiccated step with the bioactie agent compositions.
Lyophilization claims again sublimation drying.Water-containing materials is freezing to below freezing, make water change ice into, then under high vacuum, change ice into steam and the drying means removed, be the usual method for preparing lyophilized formulations of using in this area.Have no particular limits among the present invention, can adopt conventional operating condition to carry out lyophilizing, as with solution-20 ℃ freezing, water changes ice into and is placed on that drying obtains lyophilized formulations in the freezer dryer.
Can become bone implant
In the present invention, can become bone implant to refer to comprise the graft of above-mentioned bioactie agent compositions and medically acceptable carrier, be used for repairing bone defect, treatment fracture or repair cartilage.
Bioactie agent compositions of the present invention comprises bioactie agent and sulfonation polysaccharide, compares with simple use bioactie agent, has the potentiation synergism, when namely promoting skeletonization, promotes the generation of blood vessel.Because promote that angiogenesis function is remarkable, the generation of blood vessel also promotes the generation of bone simultaneously.
Can be used for that of the present invention to become the medically acceptable carrier of bone implant be medically acceptable biomaterial, include, but is not limited to:
(1) inorganic material, such as hydroxyapatite, tricalcium phosphate, calcium phosphate bone cement, bio-vitric, calcium silicates, calcium sulfate etc.;
(2) synthetic high polymer, such as poly-'alpha '-hydroxy acids (such as polylactic acid PLA, polyglycolic acid PGA, poly butyric PHB etc.), poly-anhydride, Polyethylene Glycol, polycaprolactone etc.;
(3) natural polymer, for example collagen, gelatin, glycosaminoglycans (glycosaminoglycan, GAGs), chitosan (chitosan), chitin (chitin), alginic acid, glucosan etc.;
(4) mixture of above-mentioned material or composite.
Bioactie agent compositions of the present invention placed on the medically acceptable carrier to consist of can become bone implant, can become bone implant to be implanted in the body, bioactie agent and sulfonation polysaccharide synergism promote skeletonization and angiogenesis.
Medically acceptable carrier can be the degradable biological compatibility material, and gradually degraded discharges growth factor combination simultaneously in vivo, promotes the formation of new bone.Along with the progressively degraded of material, thereby area of new bone replaces the vivo degradation process of original material accelerated material gradually.
When medically acceptable carrier of the present invention is syringeability material (such as capable of injecting bone cement), this fluent material is mixed with bioactie agent compositions of the present invention, be expelled to the bone injury position, carry out the original position skeletonization.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can combination in any.All features that this case description discloses can with any composition forms and usefulness, each feature that discloses in the description, the alternative characteristics that can anyly be provided identical, impartial or similar purpose replaces.Therefore except special instruction is arranged, the feature that discloses only is the general example of equalization or similar features.
The invention has the advantages that:
(1) the invention provides a kind of bioactie agent compositions by somatomedin and sulfonation composition of Salvia polysaccharide, its effect is can promote simultaneously skeletonization and promote vascularization, and the wound that produces is implanted in potential risk and the use having avoided using multiple-factor to bring with blood vessel.
(2) material of the present invention is easy to get, and preparation condition is gentle, and prepared bioactie agent compositions can join easily in the medically acceptable carrier and together use, and can adopt implantation or injecting method to insert in the body, and it is convenient to use.
(3) the present invention is applied widely, can adapt to difform defect repair and reconstruction.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
Embodiment 1
Be 100,000 with molecular weight, sulfur content is that 8% sulfated chitosan is dissolved in the deionized water, is mixed with 10 μ g/mL sulfated chitosan aqueous solutions.The mass ratio 1:1 that presses bioactie agent and sulfonation polysaccharide adds human bone morphogenesis protein-2 (BMP-2), and mix homogeneously namely obtains the bioactie agent compositions.
Embodiment 2
Be 300,000 with molecular weight, sulfur content is that 5% sulfated chitosan is dissolved in the deionized water, is mixed with 50 μ g/mL sulfated chitosan aqueous solutions.The mass ratio 2:1 that presses bioactie agent and sulfonation polysaccharide adds human bone morphogenesis protein-7 (BMP-7), and mix homogeneously namely obtains the bioactie agent compositions.
Embodiment 3
Be 200,000 with molecular weight, sulfur content is that 15% sulfonation glucosan is dissolved in the deionized water, is mixed with 0.02 μ g/mL sulfated chitosan aqueous solution.The mass ratio 1:10 that presses bioactie agent and sulfonation polysaccharide adds human bone morphogenesis protein-2 (BMP-2), and mix homogeneously namely obtains the bioactie agent compositions.
Embodiment 4
Be 10,000 with molecular weight, sulfur content is that 12% sulphation heparin is dissolved in the deionized water, is mixed with 100 μ g/mL sulfated chitosan aqueous solutions.The mass ratio 100:1 that presses bioactie agent and sulfonation polysaccharide adds bone morphogenesis protein-2 (BMP-2) and the transforming growth factor (TGF) of quality equalization, and mix homogeneously namely obtains the bioactie agent compositions.
Embodiment 5
Be 120,000 with molecular weight, sulfur content is that 1% sulfated chitosan is dissolved in the deionized water, is mixed with 20 μ g/mL sulfated chitosan aqueous solutions.The mass ratio 1:1 that presses bioactie agent and sulfonation polysaccharide adds bone morphogenesis protein-7 (BMP-7) and the VEGF (VEGF) of quality equalization, and mix homogeneously namely obtains the bioactie agent compositions.
Embodiment 6
Be 100,000 with molecular weight, sulfur content is that 10% sulfonation glucosan is dissolved in the deionized water, is mixed with 50 μ g/mL sulfated chitosan aqueous solutions.The mass ratio 1:2 that presses bioactie agent and sulfonation polysaccharide adds bone morphogenesis protein-2 (BMP-2) and the BMP (OGP) of quality equalization, and mix homogeneously namely obtains the bioactie agent compositions.
Embodiment 7
Be 100,000 with molecular weight, sulfur content is that 11% sulfate cellulose is dissolved in the deionized water, is mixed with 150 μ g/mL sulfate cellulose aqueous solutions.The mass ratio 50:1 that presses bioactie agent and sulfonation polysaccharide adds bone morphogenesis protein-7 (BMP-7) and the basic fibroblast growth factor (bFGF) of quality equalization, and mix homogeneously namely obtains the bioactie agent compositions.
Embodiment 8
The bioactie agent compositions (embodiment 1) that will contain 10 μ g growth factor B MP-2 is added drop-wise on the sponeostan, lyophilizing, and encapsulation, stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The bioactie agent compositions that contains 10 μ g somatomedin of as above preparation is implanted in the right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out the histological observation (see figure 1), find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.More sturdy blood capillary appears around the osseous tissue.All the other mices are dissected its right lower limb and take out new long bone (see figure 2), and the mean fresh of new bone is 160mg; New bone is placed on 600 ℃ of lower calcining 6h in the high temperature furnace, behind the furnace cooling, takes out and claim that its ash average weight is 100mg, confirm that the bioactie agent compositions has a good induced osteogenesis active, and be conducive to the vascularization of osteogenetic process.
Embodiment 9
The bioactie agent compositions (embodiment 2) that will contain 10 μ g somatomedin is added drop-wise on the calcium phosphate support, lyophilizing, and encapsulation, stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The bioactie agent compositions that contains 10 μ g somatomedin of as above preparation is implanted in the right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out the histological observation (see figure 3), find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.More sturdy blood capillary appears around the osseous tissue.All the other mices are dissected its right lower limb and take out new long bone (see figure 4), and the mean fresh of new bone is 150mg; New bone is placed on 600 ℃ of lower calcining 6h in the high temperature furnace, behind the furnace cooling, takes out and claim that its ash average weight is 95mg, confirms that prepared bioactie agent compositions has good induced osteogenesis activity, and be conducive to the vascularization of osteogenetic process.
Embodiment 10
The bioactie agent compositions (embodiment 3) that will contain 10 μ g somatomedin is added drop-wise in the injectable chitosan gel rubber, lyophilizing, and encapsulation, stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The bioactie agent compositions that contains 10 μ g somatomedin of as above preparation is injected right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out the histological observation (see figure 5), find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.More sturdy blood capillary appears around the osseous tissue.All the other mices are dissected its right lower limb and take out new long bone (see figure 6), and the mean fresh of new bone is 162mg; New bone is placed on 600 ℃ of lower calcining 6h in the high temperature furnace, and behind the furnace cooling, taking out its ash average weight of title is 150mg, and it is active that the bioactie agent compositions of preparation has good induced osteogenesis, and be conducive to the vascularization of osteogenetic process.
Embodiment 11
The bioactie agent compositions (embodiment 4) that will contain 10 μ g somatomedin is added drop-wise on the sponeostan, lyophilizing, and encapsulation, stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The bioactie agent compositions that contains 10 μ g somatomedin of as above preparation is implanted in the right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out the histological observation (see figure 7), find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.More sturdy blood capillary appears around the osseous tissue.All the other mices are dissected its right lower limb and take out new long bone (see figure 8), and the mean fresh of new bone is 130mg; New bone is placed on 600 ℃ of lower calcining 6h in the high temperature furnace, and behind the furnace cooling, taking out its ash average weight of title is 90mg, and it is active that the bioactie agent compositions of preparation has good induced osteogenesis, and be conducive to the vascularization of osteogenetic process.
Embodiment 12
The bioactie agent compositions (embodiment 5) that will contain 10 μ g somatomedin is added drop-wise in the calcium phosphate support, lyophilizing, and encapsulation, stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The bioactie agent compositions that contains 10 μ g somatomedin of as above preparation is implanted in the right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out the histological observation (see figure 9), find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.More sturdy blood capillary appears around the osseous tissue.All the other mices are dissected its right lower limb and take out new long bone (see figure 10), and the mean fresh of new bone is 123mg; New bone is placed on 600 ℃ of lower calcining 6h in the high temperature furnace, and behind the furnace cooling, taking out its ash average weight of title is 85mg, and it is active that the bioactie agent compositions of preparation has good induced osteogenesis, and be conducive to the vascularization of osteogenetic process.
Embodiment 13
The bioactie agent compositions (embodiment 6) that will contain 10 μ g somatomedin is added drop-wise in the silicon based xerogel, lyophilizing, and encapsulation, stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The bioactie agent compositions that contains 10 μ g somatomedin of as above preparation is implanted in the right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out histological observation (seeing Figure 11), find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.More sturdy blood capillary appears around the osseous tissue.All the other mices are dissected its right lower limb and take out new long bone (seeing Figure 12), and the mean fresh of new bone is 142mg; New bone is placed on 600 ℃ of lower calcining 6h in the high temperature furnace, and behind the furnace cooling, taking out its ash average weight of title is 110mg, and it is active that the bioactie agent compositions of preparation has good induced osteogenesis, and be conducive to the vascularization of osteogenetic process.
Embodiment 14
The bioactie agent compositions (embodiment 7) that will contain 10 μ g somatomedin is added drop-wise in the injectable chitosan gel rubber, lyophilizing, and encapsulation, stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The bioactie agent compositions that contains 10 μ g somatomedin of as above preparation is injected right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out histological observation (seeing Figure 13), find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.More sturdy blood capillary appears around the osseous tissue.All the other mices are dissected its right lower limb and take out new long bone (seeing Figure 14), and the mean fresh of new bone is 127mg; New bone is placed on 600 ℃ of lower calcining 6h in the high temperature furnace, and behind the furnace cooling, taking out its ash average weight of title is 108mg, and it is active that the bioactie agent compositions of preparation has good induced osteogenesis, and be conducive to the vascularization of osteogenetic process.
Comparative Examples 1
The sulfated chitosan aqueous solution is added drop-wise in the sponeostan, and lyophilizing encapsulates, and obtains containing the graft of 10 μ g sulfated chitosans, and is stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The graft that contains 10 μ g sulfated chitosans of as above preparation is implanted in the right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out histological observation (seeing Figure 15), find to have no foreign body and inflammatory reaction in the striated muscle tissue, have no simultaneously osseous tissue, bone trabecula and bone marrow.All the other mices are dissected its right lower limb and take out transplantation site, are showed no osseous tissue and form.Simple sulfonation polysaccharide can not be induced new osteogenesis.
Comparative Examples 2
Bone morphogenesis protein-2 (BMP-2) aqueous solution is added drop-wise in the sponeostan, lyophilizing, encapsulation, the graft that obtains containing 10 μ g bone morphogenesis protein-2s is stand-by in-20 ℃ of storages.
The Kunming mouse in 2 weeks of growth is carried out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh.The graft that contains 10 μ g bone morphogenesis protein-2s of as above preparation is implanted in the right thigh flesh bag.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice after operation, wherein 1 mice is taken out rapidly embedded material and surrounding soft tissue thereof and immerses in 10% the formalin solution fixing, section, hematoxylin-eosin staining is observed the skeletonization situation and is carried out histological observation, find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.More sturdy blood capillary does not appear around the osseous tissue.All the other mices are dissected its right lower limb and take out new long bone (seeing Figure 16), and the mean fresh of new bone is 105mg; New bone is placed on 600 ℃ of lower calcining 6h in the high temperature furnace, behind the furnace cooling, takes out and claim that its ash average weight is 60mg, the result shows, single somatomedin can't be taken into account simultaneously skeletonization and become vascular function, does not have preferably angiogenesis in the cambium.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. bioactie agent compositions, it is characterized in that, described compositions comprises bioactie agent and sulfonation polysaccharide, and described bioactie agent is selected from one or more in bone morphogenetic protein, transforming growth factor, VEGF, basic fibroblast growth factor, the BMP; Described sulfonation polysaccharide is selected from one or more in sulfated chitosan, sulfonation glucosan, heparin, heparin sulfate, the sulfate cellulose.
2. bioactie agent compositions according to claim 1 is characterized in that, described bioactie agent is BMP-2 or BMP-7; Perhaps
Described bioactie agent is to be selected from lower group the factor and the complex of BMP-2 or BMP-7: transforming growth factor, VEGF, basic fibroblast growth factor, BMP or its combination.
3. bioactie agent compositions according to claim 1 is characterized in that, the molecular weight of sulfonation polysaccharide is at 8000-350000Da, and sulfur content is 0.5%-20%.
4. bioactie agent compositions according to claim 1 is characterized in that, the mass ratio of described bioactie agent and sulfonation polysaccharide is 0.05 ~ 120:1.
5. bioactie agent compositions according to claim 1 is characterized in that, described bioactie agent compositions also comprises water.
6. the preparation method of each described bioactie agent compositions according to claim 1 ~ 5, it is characterized in that, described method comprises bioactie agent joined in the sulfonation polysaccharide solution, and mix homogeneously obtains the step of described bioactie agent compositions.
7. preparation method according to claim 6 is characterized in that, described sulfonation polysaccharide solution concentration is 0.01 ~ 150 μ g/mL.
8. preparation method according to claim 6 is characterized in that, described method also comprises carries out cryodesiccated step with the bioactie agent compositions.
9. one kind can become bone implant, it is characterized in that, described implant comprises bioactie agent compositions claimed in claim 1 and medically acceptable carrier.
10. the purposes of bioactie agent compositions as claimed in claim 1 is characterized in that, for the preparation of medicine or the implant of repairing bone defect, treatment fracture or reparation cartilage.
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