CN104667348A - Pharmaceutical composition containing sodium alginate and preparation method of pharmaceutical composition - Google Patents
Pharmaceutical composition containing sodium alginate and preparation method of pharmaceutical composition Download PDFInfo
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- CN104667348A CN104667348A CN201510058232.7A CN201510058232A CN104667348A CN 104667348 A CN104667348 A CN 104667348A CN 201510058232 A CN201510058232 A CN 201510058232A CN 104667348 A CN104667348 A CN 104667348A
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Abstract
The invention provides a pharmaceutical composition containing sodium alginate for cartilage tissue repairing. As an active ingredient of the composition, bone morphogenetic protein-4 transfected fat is derived from stem cells, sodium alginate and calcium chloride; by virtue of the composition, gaps filled between cartilage rods as well as between the cartilage rods and peripheral cartilages and subchondral bones are completely filled by regenerated tissues and mechanical properties similar to that of normal cartilage are guaranteed; full fusion of mosaic bones and cartilages with peripheral cartilages and subchondral bones is achieved, and joint function improvement and level recovery in postoperative patients are significantly improved; in addition, the invention also provides a preparation method of the pharmaceutical composition.
Description
Technical field
The present invention relates to a kind of composition and method of making the same containing sodium alginate, particularly a kind of Pharmaceutical composition containing sodium alginate promoting osteochondral tissue to repair.
Background technology
The compound damage of joint internal skeleton cartilage caused because of the disease such as wound and arthritis is very common clinically, occupies the cartilaginous tissue of critical role once destroy the normal performance just affecting function of joint largely at intraarticular.The vicious cycle caused thus causes the further disorder of intraarticular environment, Mechanics of Machinery conduction abnormalities causes the chain destruction of normal articular cartilage, finally cause the autonomic movement consequence such as limited in various degree, drastically influence the physical and mental health of extensive patients, cause white elephant also to family and society.
Because repair ability after cartilage injury is very limited, operative treatment can only be depended on, as autologous in microfrature, Drilling and bone cartilage/heteroplastic transplantation etc.But these Therapeutic Method all exist weak point more or less, can not reach desirable therapeutic effect, particularly to the treatment of large area cartilage injury, weak point is more outstanding.
At present, Mosaicplasty technology (autologous Osteochondral mosaicplasty art) is the method that clinical practice solves the reparation of large area bone cartilage complex injury comparatively widely, and it adopts autologous non-weight bearing district bone cartilage post to carry out inserted skin grafing and mending heavy burden district osteochondral defect.Between the bone cartilage post inlayed and bad with " integration " of surrounding and basilar part bone and cartilaginous tissue, thus inevitably cause the many defect gap i.e. existence in " dead band ".Therefore, the simple Mosaicplasty technology that relies on cannot obtain complete smooth, a continuous print articular surface, and the function of joint being difficult to reach better is improved and recovered.And " for district " (autologous bone cartilage Zhu Qugu district) also has poor healing, cause postoperative patient " for district " pain.Therefore, the problem solving " dead band " and " for district " the bone cartilage-derived growth reparation between bone cartilage post becomes the key for the treatment of large area osteocartilaginous and subchondralo bone injury.
Summary of the invention
For the deficiency that current Osteochondral mosaicplasty art exists in large area osteocartilaginous and subchondralo bone injury reparation application, inventor is through the experimentation of a large amount of science, provide a kind of Pharmaceutical composition containing sodium alginate that can promote osteochondral tissue Regeneration and Repair efficiently, said composition can make between the bone post of filling, and with around cartilage, gap between subchondral bone is filled and led up completely by cambium, and possess the mechanical property similar to normal cartilage, achieve and inlay bone cartilage and surrounding cartilage, the fusion completely of subchondral bone, more considerably improve improvement and the recovery level of postoperative patient function of joint.
The invention provides a kind of Pharmaceutical composition repaired for osteochondral tissue, the active component of said composition is: cell concentration is not less than 0.5 × 10
7the adipose-derived stem cells (ADSCs) of BMP-4 (BMP-4) transfection of/ml, and content is the calcium chloride of the sodium alginate and 0.3 ~ 0.5% (w/v) of 1 ~ 2% (w/v).
Said composition also can be described as a kind of compositions containing sodium alginate for osteochondral tissue reparation or a kind of Pharmaceutical composition containing sodium alginate repaired for osteochondral tissue.
For solving Osteochondral mosaicplasty art Problems existing in large area osteocartilaginous and subchondralo bone injury reparation application, inventor once attempted calcium alginate gel and this method fit applications, this cooperation of result has improvement to a certain degree to " dead band " phenomenon, but the cambium of this part non bone cartilage sample signal, like fibrous granulation tissue, its mechanical property compared with normal cartilage is mutually far short of what is expected, cannot meet the normal use of repairing posterior joint.In continuous research, improved, process, inventor surprisingly finds, the ADSCs of compound BMP-4 transfection in calcium alginate gel, bone graft cartilage and the synergy around between cartilage, subchondral bone can be improved significantly, generate hyaline cartilage sample tissue, implant part and body internal skeleton or cartilage can be made to combine together.Thus the repairing effect improved further cartilage injury, reduce the probability of secondary osteoarthritis.
Also containing pH adjusting agent in the present composition, described pH adjusting agent is that pH adjusting agent is commonly used in this area, as the hydrochloric acid, 1N sodium hydroxide etc. of 1N.
As everyone knows, calcium alginate is normally made up of water soluble algae hydrochlorate and calcium ion, calcium alginate gel thickness, poor fluidity, and the kind of its gelation speed, viscous pasty state and raw material, ratio and pH value are relevant.And gelation speed and intensity thereof concerning and application of the present invention be vital.On the one hand, the viscous pasty state of crosslinked complete calcium alginate gel make it meet to inlay that cartilage and self bone/intercartilaginous are tiny, complexity and the filling requirement of Anomalistic space.On the other hand, the nonconforming meeting of its intensity causes gel broken, can not provide the support of growth for the seed cell carrying genes of interest, is also difficult to well play the fixing of implantable bone post and supporting role, directly can has influence on surgical effect.
For taking into account the intensity of crosslinked (gel) speed, gel, and the factor such as the excessive negative effect to cell of calcium ion, inventor is through repetition test, obtain the proportioning of sodium alginate described in the present composition and calcium chloride, and the pH value range be applicable to, that is, percentage by weight is the sodium alginate of 1 ~ 2% and the calcium chloride of 0.3 ~ 0.5%, and regulates the pH adjusting agent of composition pH to 7.35 ~ 7.45.The rear gelation time of two parts mixing of this ratio, gel strength are moderate, and about 20-30 time second and crosslinkable complete.And, compositions of the present invention adopts and sodium alginate part and calcium chloride part is being mixed before use, and the mode be injected in vivo immediately is applied, about 10-15 consuming time second is completed from being mixed to injection, mixture now is still in thinner state, can realize any tiny, far-reaching gap filling object completely in this surgical wound, fills complete also without the need to deliberately waiting for, compositions has become the gel with some strength, just can sew up.Even if all gap fillings do not extend the wound open hour completely again, and gel possesses suitable intensity, and be enough to position and the shape of protecting implantation cartilage post, less external force can not cause its distortion, for the growth that cell is early stage provides support and suitable growing environment.
The ADSCs of BMP-4 transfection in vivo can long period (about 4-6 week) continuous release cytokine, stimulates its division growth, starts and accelerate reparation process, improve IA pathology environment, enhancing repairing quality; And the two-way differentiation potential of ADSCs makes it be divided into corresponding chondrocyte or osteocyte in the different microenvironments of articular cavity from subchondral bone bed.
The calcium alginate gel of the ADSCs of compound BMP-4 transfection injects " dead band ", gel is fixing, while cartilage post is implanted in protection, for the stem cell of implanting provides the three dimensional growth environment of similar physiological status, cell is uniformly distributed in gel, the exchange of nutrition and metabolism material can be carried out, adaptability to changes stimulation by gel is beneficial to hypertrophy and breaks up and form extracellular matrix, " dead band " is filled and led up by final neocartilage tissue completely, implant part is integrated in automixis, substantially increase operability and the success rate of the damage of first phase complete correction osteochondro tissue, the more important thing is the outcome significantly improving large area cartilage injury patient, improve the quality of life of such patient.
Compositions of the present invention and Osteochondral mosaicplasty art fit applications 2-3 month, cartilage post edge just can neocartilage sample tissue, and this gel section has a large amount of chondrocyte, this part with implant cartilage post and autologous cartilage is combined firmly, joint repair is respond well.In addition, the present composition is also directly applied to the filling of small size or small area cartilage injury by inventor, reaches good repairing effect equally.
It should be noted that, the adipose-derived stem cells of the BMP-4 transfection that the present invention is used is the cell in preparation 48 hours, that is, complete in before compositions uses 48 hours of transfection procedure, preferably prepared in 24 hours.
The osmotic pressure adapted with human body is possessed for making compositions of the present invention, and constant pH value, the adjuvant of above-mentioned composition also containing following content: the sodium chloride of 0.5 ~ 0.7%, and the 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) of 0.3 ~ 0.5%.
Experimental result shows, in described compositions, the content of sodium alginate directly affects the effect that cell (adipose-derived stem cells and chondrocyte) grows, and, this impact is simple direct ratio, inverse relation not, but in a specific scope, in said composition, the content of sodium alginate is preferably 1.4 ~ 1.8%.
The concentration of the adipose-derived stem cells (that is, the ADSCs of BMP-4 transfection) of described BMP-4 transfection is preferably (1 ~ 2) × 10
7/ ml.
Compositions of the present invention is (1 ~ 1.5) × 10 by cell concentration
7the adipose-derived stem cells of the BMP-4 transfection of/ml, content is the sodium alginate of 1.6 ~ 1.7%, calcium chloride, the sodium chloride of 0.5 ~ 0.7%, the 4-hydroxyethyl piperazine ethanesulfonic acid of 0.3 ~ 0.5% and the qs pH adjuster of 0.35 ~ 0.45% is made.Said composition application state is gel, and namely sodium alginate soln part, after mixing with calcium chloride solution part, cross-linking reaction occurs, and generates calcium alginate gel.
Histology and biomechanics testing result prove, the present composition coordinates with Mosaicplasty technology repairs cartilage defect, regenerating tissues does not all have difference with normal articular cartilage in organizational structure or functional (biomechanics), reach unprecedented repairing effect, perfect further large area cartilage defect repair technology.
Another object of the present invention is to, provide the preparation method of described compositions, the method comprises the following steps:
(1) extract and prepare the adipose-derived stem cells of BMP-4 transfection;
(2) prepare sodium alginate soln;
(3) prepare calcium chloride solution;
(4) in the sodium alginate soln that (2) cell suspension step (1) obtained before use obtains in step;
By step (4) with step (3) gains by volume 2:1 ratio mixing, obtain the present composition.
(1) step described in above-mentioned preparation method comprises:
A. the extracting and developing of autologous adipose tissue stem cell and cultivation, get 3 ~ 10 generation cell;
B. BMP-4 recombinant adenovirus;
C. BMP-4 recombinant adenovirus in-vitro transfection adipose-derived stem cells;
D. detect transfection, get the cell that transfection efficiency is greater than 70% and store under-65 ~-75 DEG C of environment, for subsequent use.
The extracting and developing of the Adipose Tissue in above-mentioned steps a and the method for cultivation are art technology known technology, and various common method all can use.As adopted following methods:
Take from the fatty tissue at body or allogeneic groin place.Rinse with a large amount of PBS liquid and remove surperficial blood, shred with eye scissors in the PBS and 0.1% NTx enzyme of 1:1 configuration, use in the DMEM containing 10% hyclone after 37 DEG C of digestion 30min and Digestive system, get the centrifugal 5min of 300g, discard the float containing mature fat cell, 160mM NH4Cl erythrocyte cracked liquid is added again containing in the precipitate of adipose tissue-derived stem cell, room temperature leaves standstill 10min, by its nylon mesh by 200-lm, collect filtrate, with containing 10%FBS, the dual anti-DMEM culture medium of 1% blue or green chain is spent the night at 37 DEG C/5%CO2 incubator.Primitive cell culture 4-5 days, treats its warm growth, gives to go down to posterity with 1:3.Primary cell was labeled as 0 generation, and testing cell used is 3-10 generation.The cell be separated has the differentiation to fat and osteocyte.
Restructuring in above-mentioned steps b and c and transfection method are similarly art technology known technology, and various common method all can use.Transfection method wherein can adopt following methods: third generation Adipose Tissue equivalent planted in 6 orifice plates, reach 90% converge after, abandon growth medium, get 3 hole peptic cells and count.Calculate virus (BMP-4 recombinant adenovirus) amount needed for every holes by MOI=100,250,400, and allow on ice and get not commensurability virus after the spontaneous thawing of virus that stores and be added to respectively in the DMEM liquid of 400 μ l serum-frees.By the complete culture solution reject in 6 orifice plates, PBS adds virus liquid after cleaning 2 times, and cross rocks culture plate by the abundant exposing cell of virus liquid, hatches 3 hours for 37 DEG C, and every 20 minutes of period rocked culture plate once.Draw virus liquid after 3 hours and in every hole, add 1ml complete culture solution and continue to cultivate.
For improving Chondrocyte Differentiation ability and the repair of cartilage ability of the present composition, preferably in calcium alginate gel, compound transfection efficiency is greater than the ADSCs of the BMP-4 transfection of 85%.
(2) the step of described preparation method is specially: be dissolved in the deionized water of cumulative volume 70 ~ 95% by sodium chloride, 4-hydroxyethyl piperazine ethanesulfonic acid, be heated to 50 ~ 80 DEG C, add sodium alginate, stir, obtain uniform solution, place room temperature, add pH adjusting agent adjust ph to 7.35 ~ 7.45, use deionized water standardize solution, for subsequent use.
(3) the step of described preparation method is specially: get calcium chloride and 4-hydroxyethyl piperazine ethanesulfonic acid, adds 50 ~ 95% deionized waters and makes solution, adjust ph to 7.35 ~ 7.45, deionized water standardize solution, and 0.22 μm of membrane filtration is for subsequent use.
Wherein, in the calcium chloride solution of the described step (2) sodium alginate soln of gained and step (3) gained, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is about 2:1.
The step cell suspension method (4) of described preparation method is conventional method, makes cell concentration reach 0.75 × 10
7/ more than ml.
Obtain the present composition according to after described step (5) described method mix homogeneously, after mixing, alginate start to be cross-linked immediately, at once mixture should be injected into sutura, fill complete can stitching.
Present invention also offers described compositions and prepare the application in osteochondral tissue repair medicine and/or medical material.
Inventor verifies the functional effect of product of the present invention further by specific experiment.
Reiterate: following experiment is illustrative experiment in numerous experiment in development process of the present invention, and do not contain all experiments done for the present invention with limit invention people, object is only the beneficial effect of setting forth product of the present invention by those data.
Experimental technique: be object of study with miniature pig, in femur, a diameter 8mm is in condyle heavy burden district, dark 5mm osteochondral defect; Get 3 diameter 3.5mm long 5mm bone cartilage posts in nonfunctional area, femoral bone pulley edge respectively and fill most of defect area with Mosaicplasty technology.Experiment stochastic averagina is divided into 3 groups to repair.12 weeks after surgery respectively, within 24 weeks, carry out scanning electron microscope, histological score and Study on biomechanics.Quantitative result mean ± standard deviation represents, statistical analysis uses SPSS software to adopt Mann – Whitney inspection, and P<0.05 is for there being statistical significance
A group: simple Mosaicplasty group;
B group: Mosaicplasty+ calcium alginate gel group;
C group: calcium alginate gel (described in the embodiment 1 present composition) group of the ADSCs of Mosaicplasty+ compound BMP-4 transfection.
1, scanning electronic microscope examination
Inspection method: after sacrifice of animal, takes off rapidly cartilage defect repair place, normal saline flushing.2% glutaraldehyde solution dropping into pre-cooling fixes 8 hours.Distilled water fully cleans, and adds 1% Osmic acid. and fixes 1 hour.Sucking-off fixative, adds distilled water and fully cleans 1 hour, within every 15 minutes, changes first water.Sucking-off distilled water, ethanol is serial dehydration step by step, sucking-off ethanol, adds isoamyl acetate alcohol mixeding liquid (isoamyl acetate and proportion of ethanol are 1:1) after 20 minutes, sucks liquid, adds pure isoamyl acetate 30 minutes.Filter paper blots sample surfaces isoamyl acetate, sample is proceeded to critical point drying instrument, dry under 31 DEG C, 72.8 atmospheric critical states.Ion sputtering process surface gold-plating.Scanning electron microscopic observation.
Check result: (see accompanying drawing 1)
When 12 weeks, a group visible bone graft cartilage post and surrounding sharpness of border, the distortion of bone cartilage post is obviously (Fig. 1-a1); The distortion of b group visible bone cartilage post is less, and there is cambium at injection calcium alginate gel place, but tissue fibers arranges unordered disorder, and bone cartilage post and surrounding still exist crack (Fig. 1-b1); The distortion of c group visible bone cartilage post is little, and there is neocartilage sample tissue at cartilage post edge, and cambium surface is similar with cartilage post surface.Do not merge completely between cartilage post, but crack is significantly less than first two groups (Fig. 1-c1);
When 24 weeks, between a group bone graft cartilage post and and surrounding normal cartilage tissue between crack clear, the distortion of bone cartilage post is obviously (Fig. 1-a2); Between b group visible bone graft cartilage post and and surrounding normal cartilage tissue between have part crack to be reproduced to organize and substantially to fill and lead up, but surface irregularity, fiber alignment is irregular, has zanjon (Fig. 1-b2).The distortion of c group visible bone cartilage post is little, and cartilage intercolumniation crack disappears, and merges completely between osteochondral tissue, and surface is continuous whole, and regenerating tissues surface is homogeneous threadiness, more smooth, and fiber alignment is neat, similar to normal cartilage.(Fig. 1-c2).
The above results shows: simple employing a prescription method, and object of study cannot complete implant part and the fusion growth between self voluntarily, and injection unit divides the distortion that is easily shifted, and surgical effect is very undesirable; Although b prescription method comparatively a group effect makes moderate progress, merge poor, poor stability, the stimulation that normal activity brings can not be met; And adopt the c group of the present composition to regenerate chondroid tissue, achieve bone cartilage intercolumniation and merge completely.
2, histological examination
Inspection method: after Animal Anesthesia, takes out specimen from implantation respectively, the soft tissue that careful rejecting is unnecessary, immerses in the paraformaldehyde of 4% and fix 48 hours after PBS rinses.Specimen is taken out, tap water 20min, distillation washing 2 times from fixative.Ascending gradient ethanol dewaters successively, enters the transparent rear waxdip of dimethylbenzene, embedding is made wax stone and cuts into slices, and the dyeing dyeing of H.E, toluidine blue and the dyeing of II Collagen Type VI are observed.
Check result: (see accompanying drawing 2)
When 12 weeks: a group, visible bone graft cartilage post intersection is without neocartilage, and minute quantity fibroid tissue fills subchondral bone, transplants cartilage intercolumniation crack and exists, and fails to merge (Fig. 2-a1); B group, visible cartilage intercolumniation subchondral bone of transplanting heals better, is still existed (Fig. 2-b1) between cartilaginous tissue by fibroid tissue filled opening.Toluidine blue and the dyeing of II Collagen Type VI are feminine gender.C group visible bone graft cartilage post intersection is repaired by chondroid tissue, and surface is more smooth, inside has a large amount of chondrocyte, is positioned at cartilage lacuna, is arranged in column or tufted; Visible endochondral ossification in deep zone cartilage, subchondral bone partial reconstitution; Be with acellular disappearance of surrounding normal cartilage intersection, but boundary line obviously (Fig. 2-c1).Toluidine blue and the dyeing of II Collagen Type VI are the positive.
When 24 weeks: a group, visible bone graft cartilage post intersection crack narrows and to be filled by a small amount of fibrous tissue, and crack still exists (Fig. 2-a2), toluidine blue dye and II Collagen Type VI negative staining.B group, visible bone graft cartilage post intersection crack is mixed by fibrous cartilage sample tissue repairs, and surface irregularity, inside has chondrocyte and fibrocyte, is scatteredly distributed in (Fig. 2-b2) in regenerating tissues.The weak positive that toluidine blue, masson dye and II Collagen Type VI dyes.C group, visible bone graft cartilage post intersection is repaired by cartilaginous tissue, surfacing, and inside have a large amount of chondrocyte, cells of superficial layer is parallel to surface, and deep layer cells becomes columnar arrangement, is tending towards normal, damp line under visible cartilage; Defect band acellular with surrounding normal cartilage intersection, boundary line unclear (Fig. 2-c2).Toluidine blue staining, masson dyeing and the dyeing of II Collagen Type VI are the positive.
The above results shows: only have the newborn repair tissue of C group to have the histological characteristic of normal cartilage.
Histological score adopts O ' Driscol standards of grading, and the higher explanation repairing effect of mark is better, and full marks are 24 points.Wherein 12 weeks time, a number of components is 8.8 ± 3.12 at 5.2 ± 1.60, b number of components, and c number of components just reaches 16.2 ± 2.63; When 24 weeks, a number of components is 12 ± 4.56 at 7.2 ± 1.32, b number of components, and c number of components is up to 21.4 ± 1.35, close to full marks.
The above results shows: the repair tissue of c group new life is histologically close to normal structure.
3, biomechanics detects
Inspection method: nano impress biological mechanics determining
Main employing three indexs evaluate the biomechanics characteristic of repair tissue, comprise contact stiffness (contact stiffness), reduced modulus (elastic modelling quantity), hardness (hardness).Contact stiffness is mainly used to the ability evaluating the deformation that repair tissue opposing external force causes.Elastic modelling quantity is mainly used to the elasticity evaluating repair tissue.Hardness is the index of the hardness evaluating repair tissue.
Experimental result display (see accompanying drawing 3-6), the mechanical property of c group repair tissue is all obviously better than other two groups, close to normal cartilage in these three indexs.Though b repair tissue mechanical property is better than a group, comparatively c group differs greatly.The biomechanical property of the repair tissue that a, b are two groups reaches far away normal cartilage and fill area, cannot meet repair of cartilage demand.Illustrate, the sustainable induction of the present composition, promotion regenerating tissues, to bone cartilage differentiation, realize histology and the function assessment characteristic of recovering osteochondral defect district in a short time.
In order to verify the functional effect of the present composition, inventor carrying out repeatability experiment, with above-mentioned experiment unlike, each amounts of components, cell concentration difference (comprising embodiment 2-6), the parameter in preparation method is finely tuned.Wherein, the domain of walker of described each amounts of components (w/v) is: the sodium alginate of 1 ~ 2%, the calcium chloride of 0.3 ~ 0.5%, 0.5 ~ 0.7% sodium chloride, and the 4-hydroxyethyl piperazine ethanesulfonic acid of 0.3 ~ 0.5%; The concentration range of the ADSCs of described BMP-4 transfection is (0.5 ~ 2) × 10
7/ ml.
The result display of completed embodiment 1-3, after each amounts of components, cell concentration adjust within the specific limits, still possesses and well promotes osteochondral tissue repairing effect.Carry out the data display of statistical analysis on this basis, the compositions in above-mentioned domain of walker is for promoting that the effect of osteochondral tissue reparation has similarity.
Because length limit, the method for above-mentioned experiment, step and related data do not repeat them here.
Accompanying drawing explanation
Fig. 1: scanning electronic microscope examination collection of illustrative plates, wherein, a, b, c represent group respectively, and first row is 12 weeks zoological specimens, and second row is 24 weeks zoological specimens;
Fig. 2: histological examination HE dyes collection of illustrative plates, and wherein, a, b, c represent group respectively, and first row is 12 weeks zoological specimens, and second row is 24 weeks zoological specimens; N is normal cartilage, and R is neocartilage;
Fig. 3: a group biomechanics inspection, Normal is normal cartilage district, and Mosaic is the bone cartilage area of transplanting, defective region when blank is operation;
Fig. 4: b group biomechanics inspection, Normal is normal cartilage district, and Mosaic is the bone cartilage area of transplanting, and Alginate fills calcium alginate gel district for during operation;
Fig. 5: c group biomechanics inspection, Normal is normal cartilage district, and Mosaic is the bone cartilage area of transplanting, and Alginate+ADSC fills present composition district for during operation.
Biomechanics between Fig. 6: three groups compares.
Detailed description of the invention
Embodiment 1:
150ml composite formula:
Preparation method:
(1) extract and prepare the adipose-derived stem cells of BMP-4 transfection;
Take from the fatty tissue at body groin place.Rinse with a large amount of PBS liquid and remove surperficial blood, shred with eye scissors in the PBS and 0.1% NTx enzyme of 1:1 configuration, use in the DMEM containing 10% hyclone after 37 DEG C of digestion 30min and Digestive system, get the centrifugal 5min of 300g, discard the float containing mature fat cell, 160mM NH4Cl erythrocyte cracked liquid is added again containing in the precipitate of adipose tissue-derived stem cell, room temperature leaves standstill 10min, by its nylon mesh by 200-lm, collect filtrate, with containing 10%FBS, the dual anti-DMEM culture medium of 1% blue or green chain is at 37 DEG C/5%CO
2incubator spends the night.Primitive cell culture 4-5 days, treats its warm growth, gives to go down to posterity with 1:3.Primary cell was labeled as 0 generation, and cell used was the 3rd generation.
Transfection method (compositions use first 24 hours in operation): the 3rd fat subsitutes tissue stem cell equivalent is planted in 6 orifice plates, reach 90% converge after, abandon growth medium, get 3 hole peptic cells and count.Calculate virus (BMP-4 recombinant adenovirus) amount needed for every holes by MOI=100,250,400, and allow on ice and get not commensurability virus after the spontaneous thawing of virus that stores and be added to respectively in the DMEM liquid of 400 μ l serum-frees.By the complete culture solution reject in 6 orifice plates, PBS adds virus liquid after cleaning 2 times, and cross rocks culture plate by the abundant exposing cell of virus liquid, hatches 3 hours for 37 DEG C, and every 20 minutes of period rocked culture plate once.Inhale after 3 hours to abandon virus liquid and in every hole, add 1ml complete culture solution and continue to cultivate.
Get the BMP-4 transfection ADSCs that transfection efficiency is greater than 85% to store under about-70 DEG C of environment, for subsequent use.
(2) prepare sodium alginate soln: (100ml)
Take HEPES 0.48g, sodium chloride 0.88g, be dissolved in 80ml deionized water, be heated to 60 DEG C, insulation, adds 2.5g sodium alginate, Keep agitation, until solution mixing, after cool to room temperature, drip the hydrochloric acid adjustment pH 7.4 that concentration is 1N, supplement deionized water to 100ml, for subsequent use.
(3) prepare calcium chloride solution: 50ml
Take the calcium chloride of 0.57g and the HEPES of 0.12g, be dissolved in 40ml deionized water, the hydrochloric acid dripping 1N makes pH value be 7.4, supplements deionized water to 50ml, 0.22 μm of membrane filtration, for subsequent use.
(4) in the sodium alginate soln that (2) cell suspension step (1) obtained before use obtains in step.Specifically: digested from culture dish by cell pancreatin, with in serum and pancreatin, centrifugal, its supernatant, cultivates the cell of the basic weight whiz heart, and concrete steps are with reference to cell culture; Cell concentration is made to reach 2 × 10
7/ mL.
By step (4) with step (3) gains by volume 2:1 ratio mixing, obtain the present composition after mixing.The present composition in actual applications, should be injected into fill area in body upon mixing immediately, has injected crosslinked substantially completing, in gel state.
Embodiment 2:
150ml composite formula:
Preparation method:
Difference from Example 1 is:
(1) extract and prepare the adipose-derived stem cells of BMP-4 transfection; Primary cell was labeled as 0 generation, and cell used was the 5th generation; Get the BMP-4 transfection ADSCs that transfection efficiency is greater than 90% to store under about-65 DEG C of environment (transfection procedure carries out in first 48 hours in compositions application), for subsequent use.
(2) prepare sodium alginate soln: (100ml)
Take HEPES 0.6g, sodium chloride 0.75g, be dissolved in 70ml deionized water, be heated to 70 DEG C, insulation, add 2.1g sodium alginate, Keep agitation, until solution mixing, after cool to room temperature, drip the hydrochloric acid of 1N, adjustment pH 7.35, supplement deionized water to 100ml, for subsequent use.
(3) prepare calcium chloride solution: 50ml
Take the calcium chloride of 0.45g and the HEPES of 0.15g, be dissolved in 47.5ml deionized water, the hydrochloric acid dripping 1N makes pH value be 7.35, supplements deionized water to 50ml, 0.22 μm of membrane filtration, for subsequent use.
(4) in the sodium alginate soln that (2) cell suspension step (1) obtained before use obtains in step; Cell concentration is made to reach 1.5 × 10
7/ mL.
Embodiment 3:
150ml composite formula:
Preparation method:
Difference from Example 1 is:
(1) extract and prepare the adipose-derived stem cells of BMP-4 transfection; Primary cell was labeled as 0 generation, and cell used was the 7th generation; Get the BMP-4 transfection ADSCs that transfection efficiency is greater than 70% to store under about-75 DEG C of environment, for subsequent use
(2) prepare sodium alginate soln: (100ml)
Take HEPES 0.36g, sodium chloride 1.05g, be dissolved in 95ml deionized water, be heated to 50 DEG C, insulation, adds 2.7g sodium alginate, Keep agitation, until solution mixing, after cool to room temperature, drip the hydrochloric acid adjustment pH 7.45 of 1N, supplement deionized water to 100ml, for subsequent use.
(3) prepare calcium chloride solution: 50ml
Take the calcium chloride of 0.75g and the HEPES of 0.09g, be dissolved in 25ml deionized water, the hydrochloric acid dripping 1N makes pH value be 7.45, supplements deionized water to 50ml, 0.22 μm of membrane filtration, for subsequent use.
(4) in the sodium alginate soln that (2) cell suspension step (1) obtained before use obtains in step; Cell concentration is made to reach 0.75 × 10
7/ mL.
Embodiment 4:
150ml composite formula:
Preparation method difference from Example 1 is:
(1) extract and prepare the adipose-derived stem cells of BMP-4 transfection; Primary cell was labeled as 0 generation, and cell used was the 10th generation; Get the BMP-4 transfection ADSCs that transfection efficiency is greater than 80% to store under about-70 DEG C of environment, for subsequent use
(2) prepare sodium alginate soln: (100ml)
Take HEPES 0.46g, sodium chloride 0.9g, be dissolved in 90ml deionized water, be heated to 80 DEG C, insulation, adds 2.4g sodium alginate, Keep agitation, until solution mixing, after cool to room temperature, drip hydrochloric acid adjusted to ph to 7.4, supplement deionized water to 100ml, for subsequent use.
(3) prepare calcium chloride solution: 50ml
Take the calcium chloride of 0.6g and the HEPES of 0.12g, be dissolved in 35ml deionized water, drip hydrochloric acid and make pH value be 7.4, supplement deionized water to 50ml, 0.22 μm of membrane filtration, for subsequent use.
(4) in the sodium alginate soln that (2) cell suspension step (1) obtained before use obtains in step; Cell concentration is made to reach 3 × 10
7/ mL.
Embodiment 5:
150ml composite formula:
Preparation method:
Difference from Example 1 is:
(1) extract and prepare the adipose-derived stem cells of BMP-4 transfection; Primary cell was labeled as 0 generation, and cell used was the 4th generation; Get the BMP-4 transfection ADSCs that transfection efficiency is greater than 90% to store under about-70 DEG C of environment, for subsequent use
(4) in the sodium alginate soln that (2) cell suspension step (1) obtained before use obtains in step; Cell concentration is made to reach 2.25 × 10
7/ mL.
Embodiment 6:
150ml composite formula:
Preparation method is with embodiment 1.
The present invention is not limited to above-mentioned embodiment, anyone other any or akin products identical with the present invention drawn under enlightenment of the present invention, is all not precluded within outside protection scope of the present invention.
Claims (10)
1. contain a Pharmaceutical composition for sodium alginate for osteochondral tissue reparation, it is characterized in that, the active component of said composition is: cell concentration is not less than 0.5 × 10
7the adipose-derived stem cells of the BMP-4 transfection of/ml, and content is the calcium chloride of the sodium alginate and 0.3 ~ 0.5% (w/v) of 1 ~ 2% (w/v).
2. compositions as claimed in claim 1, it is characterized in that, this compound also comprises the adjuvant of following content: the sodium chloride of 0.5 ~ 0.7% (w/v), and the 4-hydroxyethyl piperazine ethanesulfonic acid of 0.3 ~ 0.5% (w/v).
3. compositions as claimed in claim 1 or 2, it is characterized in that, the content of described sodium alginate is 1.4 ~ 1.8%.
4. compositions as claimed in claim 1 or 2, it is characterized in that, the concentration of the adipose-derived stem cells of described BMP-4 transfection is (1 ~ 2) × 10
7/ ml.
5. the compositions as described in claim 1-4 arbitrary, is characterized in that, said composition is (1 ~ 1.5) × 10 by cell concentration
7the adipose-derived stem cells of the BMP-4 transfection of/ml, content is the sodium alginate of 1.6 ~ 1.7%, calcium chloride, the sodium chloride of 0.5 ~ 0.7%, the 4-hydroxyethyl piperazine ethanesulfonic acid of 0.3 ~ 0.5% and the qs pH adjuster of 0.35 ~ 0.45% is made.
6. the preparation method of compositions according to claim 1, is characterized in that, the method comprises the following steps:
(1) extract and prepare the adipose-derived stem cells of BMP-4 transfection;
(2) prepare sodium alginate soln;
(3) prepare calcium chloride solution;
(4) in the sodium alginate soln that (2) cell suspension step (1) obtained before use obtains in step;
By step (4) with the ratio mix homogeneously of step (3) gains 2:1 by volume, obtain the present composition.
7. preparation method as claimed in claim 6, it is characterized in that, (1) described step comprises:
A. the extracting and developing of Adipose Tissue and cultivation, get 3 ~ 10 generation cell;
B. BMP-4 recombinant adenovirus;
C. BMP-4 recombinant adenovirus in-vitro transfection adipose-derived stem cells;
D. detect transfection, get the cell that transfection efficiency is greater than 70% and store under-65 ~-75 DEG C of environment, for subsequent use.
8. preparation method as claimed in claim 6, it is characterized in that, (2) described step is specially: be dissolved in the deionized water of 70 ~ 95% by sodium chloride, 4-hydroxyethyl piperazine ethanesulfonic acid, be heated to 50 ~ 80 DEG C, insulation, add sodium alginate, stir, obtain uniform solution, place room temperature, add pH adjusting agent adjust ph to 7.35 ~ 7.45, use deionized water standardize solution, for subsequent use.
9. preparation method as claimed in claim 6, it is characterized in that, (3) described step is specially: get calcium chloride and 4-hydroxyethyl piperazine ethanesulfonic acid, add 50 ~ 95% deionized waters and make solution, adjust ph to 7.35 ~ 7.45, deionized water standardize solution, 0.22 μm of membrane filtration, for subsequent use.
10. compositions according to claim 1 is preparing the application in osteochondral tissue repair medicine and/or medical material.
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| CN106267357A (en) * | 2016-08-09 | 2017-01-04 | 上海交通大学 | A kind of repair the two-layer compound hydrogel of osteochondral tissue, preparation method and application |
| CN106474558A (en) * | 2016-11-10 | 2017-03-08 | 广东泰宝医疗科技股份有限公司 | A kind of cartilage repair material with biologically active and preparation method thereof |
| CN107854730A (en) * | 2017-10-31 | 2018-03-30 | 暨南大学 | It is crosslinked the preparation method and applications of CGA sodium alginate gelatin cross-blend syndesis sticking patch |
| CN108135928A (en) * | 2015-09-07 | 2018-06-08 | 持田制药株式会社 | The freeze-dried preparation of alginic acid |
| JP7189659B2 (en) | 2017-06-30 | 2022-12-14 | ロート製薬株式会社 | Cell pharmaceutical composition for treating disease, kit for treating disease, method for preparing cell pharmaceutical composition |
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| CN115531297A (en) * | 2022-10-28 | 2022-12-30 | 东莞市东南部中心医院 | Injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and preparation method and application thereof |
| CN115531297B (en) * | 2022-10-28 | 2024-09-17 | 东莞市东南部中心医院 | Injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and preparation method and application thereof |
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