CN104225676A - Application of lithium-containing natural material bioactive scaffold in osteochondral defect repair - Google Patents
Application of lithium-containing natural material bioactive scaffold in osteochondral defect repair Download PDFInfo
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Abstract
The invention discloses an application of a lithium-containing natural material bioactive scaffold in osteochondral defect repair. The lithium-containing natural material bioactive scaffold adopts a bioactive glass or silk fibroin as a carrier and lithium ions as active components. The invention provides the application of the lithium-containing natural material bioactive scaffold in osteochondral defect repair. According to the application provided by the invention, the lithium-containing natural material bioactive scaffold has the functions of promoting bones and cartilages to regenerate in the osteochondral defect repair process; the traditional materials can only meet one need of bone regeneration and cartilage regeneration, while osteochondral defect needs a material to both meet the requirements for cartilage and subchondral bone regeneration; and with the adoption of the application, a manufacturing method and the sustained release characteristics of the lithium-containing scaffold are defined, the function check in vivo is also realized, and a foundation is laid for clinical applications of the lithium-containing scaffold.
Description
(1) technical field
The present invention relates to osteochondral defect reparation, particularly a kind of containing the application of lithium nature material bioactive bracket in osteochondral defect is repaired.
(2) background technology
Osteochondral defect occurs in osteoarthritis late period, after the late period of femur head necrosis and agnogenic osteochondritis dissecans, be, with bone and cartilage, the orthopaedic disease that pathological changes changes for characteristic pathological occurs simultaneously, its definite pathogenesis it be not immediately clear and there is no effective therapeutic modality.At inherited genetic factors, cause cartilage or subchondral bone that first pathologic occurs under the independent or combined effect of biomechanics and immune factor etc. to change, then osteochondral defect is caused, morbidity as osteoarthritis is that first cartilage changes in early days, then along with bone cartilage pathological changes simultaneously appears in advancing of disease, the morbidity of femur head necrosis is that first subchondral bone changes then along with bone cartilage pathological changes simultaneously appears in advancing of disease in early days, and osteochondritis dissecans is then that the bone of reason the unknown and cartilage strip off simultaneously.Up to the present, clinically for the treatment of osteochondral defect with Autologous Chondrocyte transplanting for first-selection, although its determined curative effect, but its operative indication is limited, the patient of 80% have finally chosen joint replacement surgery, but its operation risk easily develops complications and complication with postoperative greatly, and the feature that long-term efficacy is not good.Therefore, the effective Therapeutic Method finding treatment osteochondral defect is the task of top priority.
Have the history of about 50 years containing the clinical application of lithium ion medicine as lithium carbonate and lithium chloride, have and obviously suppress mania effect, schizoid affective disorder can be improved, during therapeutic dose on normal person's ergasia without impact.In recent years, along with clinical and development that is basic research, its new purposes is found gradually.Experiment in vitro proves that lithium ion can promote propagation and the differentiation of mesenchymal stem cells MSCs; Lithium ion is worked by intracellular stable state autophagy to the improvement of nervous system disease.Owing to there is mesenchymal stem cells MSCs lazy weight in subchondral bone and bone system dysdifferentiation in the pathological process of osteochondral defect, therefore lithium ion effectively can improve the pathological process of subchondral bone; Meanwhile, in the evolution of defect, there is obvious inflammatory reaction in cartilaginous tissue, and the cell homeostasis in cartilage can effectively react by inflammation-inhibiting, and therefore lithium ion effectively can suppress the inflammatory process of cartilage.Timbering material is widely used in the basic research of osteochondral defect in recent years, but existing timbering material only can meet the Pathophysiology needs of bone or a kind of tissue of cartilage, therefore, the reparation for osteochondral defect can be needed by the Pathophysiology improving bone and cartilage containing lithium support.
(3) summary of the invention
The invention provides a kind of novelty teabag of lithium ion, namely containing the application of lithium nature material bioactive bracket in osteochondral defect is repaired, the present invention is an example with slow release lithium ion in bioactivity glass, the elution profiles of lithium ion in the physical characterization of material and material is studied, specify that containing the repair of lithium bioactivity glass to rabbit femur osteochondral defect.Existing all medicines only can meet the needs of cartilage or osteanagenesis, and the medicine namely cartilage to regeneration is harmful to osteanagenesis; To promoting that the medicine of osteanagenesis then suppresses repair of cartilage, lithium then can meet the needs of two kinds of tissue regenerations simultaneously.
The invention provides a kind of containing the application of lithium nature material bioactive bracket in osteochondral defect is repaired, described is with bioactivity glass or fibroin albumen for carrier containing lithium nature material bioactive bracket, take lithium ion as active component.Described fibroin albumen uses with the form of silk fibroin bracket, described silk fibroin bracket preparation method is: in mould, inject the sodium chloride particle that particle diameter is 10 ~ 50 μm, again at 50 ~ 100mg/ml silk fibroin protein solution (preferred 50mg/ml) prepared by the quick implantation step (1) in sodium chloride particle surface, pushing against mold makes sodium chloride particle closely knit and discharges the gas that silk fibroin protein solution and a little sodium chloride and silk fibroin protein solution formed, mould is placed at 4 ~ 70 DEG C 2 ~ 50h to carry out being cross-linked (preferably 4 DEG C of placement 36h, room temperature (20 ~ 28 DEG C) is placed 6 hours, 60 DEG C of placements are cross-linked for 1 hour), then mould is removed, obtain cross-linked material, after cross-linked material use water cleaning postlyophilization, obtain silk fibroin bracket.
Further, of the present invention containing lithium nature material bioactive bracket take bioactivity glass as carrier time, the described lithium nature material bioactive bracket that contains is prepared as follows: join in dehydrated alcohol by poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, ethyl orthosilicate, four water-calcium nitrate, lithium chloride and triphosphoric acid ethyl ester, and by 0.5M aqueous hydrochloric acid solution adjust ph to 6.5-7.0, at room temperature stir 24h, form mixed liquor; Again aseptic polyurethane sponge is immersed in mixed liquor completely, then the polyurethane sponge after submergence is proceeded in Tissue Culture Dish, 12h is placed under room temperature (37 DEG C), 6h is sintered at 650 DEG C, obtain containing lithium bioactive bracket, namely containing lithium nature material bioactivity glass support; Described ethyl orthosilicate and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, four water-calcium nitrate and triphosphoric acid ethyl ester mass ratio are 1:0.58:0.15:0.110, described ethyl orthosilicate and lithium chloride mass ratio are 1:0.0125-0.0250 (preferred 1:0.0125), the volumetric usage of described dehydrated alcohol counts 11ml/g with ethyl orthosilicate quality, and the volumetric usage of described hydrochloric acid counts 8ml/g with ethyl orthosilicate quality.
Of the present invention containing lithium nature material bioactive bracket take fibroin albumen as carrier time, the described lithium nature material bioactive bracket that contains is prepared as follows: the preparation of (1) silk fibroin protein solution: with Bombyxmori Linnaeus silkworm silk for raw material, through coming unstuck, dissolving, dialyse, obtain trapped fluid, the concentrated solution of trapped fluid or trapped fluid to be filtered or centrifugal, get filtrate or upper strata centrifugal liquid obtains silk fibroin protein solution, and with water, silk fibroin protein solution mass concentration is adjusted to 50 ~ 100mg/ml;
(2) containing the preparation of lithium nature material bioactive bracket: inject the sodium chloride particle that particle diameter is 10 ~ 50 μm (preferably 45 ~ 50 μm) in mould, again at 50 ~ 100mg/ml (preferred 50mg/ml) silk fibroin protein solution prepared by the quick implantation step (1) in sodium chloride particle surface, pushing against mold makes sodium chloride particle closely knit and discharges the gas that silk fibroin protein solution and a little sodium chloride and silk fibroin protein solution formed, mould is placed at 4 ~ 70 DEG C 2 ~ 50h to carry out being cross-linked (preferably 4 DEG C of placement 36h, room temperature (20 ~ 28 DEG C) is placed 6 hours, 60 DEG C of placements are cross-linked for 1 hour), then mould is removed, obtain cross-linked material, after cross-linked material use water cleaning postlyophilization, (preparation of silk fibroin bracket of the present invention is see the patent of having applied for obtain silk fibroin bracket, application number 2012104113955, publication date on April 17th, 2013), silk fibroin bracket is embathed 2 ~ 24h (preferred 24h) in 10ng/ml-50ng/ml water lithium chloride solution (preferred 20ng/ml), and then dry under 30 ~ 700 DEG C of (preferably 40 DEG C) conditions, obtain containing lithium silk fibroin bracket, be containing lithium nature material bioactive bracket, the described volume ratio adding sodium chloride and silk fibroin protein solution in mould is 1:0.5 ~ 2 (preferred 1:1), described mould be one end close and sealing surface with the cylindrical shell of air-vent, the other end opening of cylindrical shell is also provided with the push rod of band piston, and the aperture of described air-vent is less than or equal to the particle diameter of sodium chloride particle.
The present invention also provides a kind of containing lithium bioactivity glass support, and described is be carrier with bioactivity glass containing lithium bioactivity glass support, take lithium ion as active component.
The present invention also provides a kind of containing lithium biological activity silk fibroin bracket, and described is be carrier with silk fibroin bracket containing lithium biological activity silk fibroin bracket, take lithium ion as active component.
Lithium ion of the present invention is used for the osteochondral defect occurred in osteoarthritis evolution, or the osteochondral defect that the late period of femur head necrosis occurs, or the osteochondral defect that agnogenic osteochondritis dissecans is adjoint.The physics sex character of bioactivity glass can not be changed containing lithium support and there is the sustained releasing character of continuous and effective, lithium ion containing the release of lithium support achieves the regeneration of cartilage, subchondral bone, realize reconstruction and the regeneration of the damp line between bone cartilage, thus reach the Regeneration and Repair of femur osteochondral defect.
At present, mostly concentrate on for the research of osteochondral defect and intervene osseocartilaginous pathological process realize Regeneration and Repair by discharging biochemical factors in material.Pathological changes while the subchondral bone that osteochondral defect relates to and cartilage also influences each other, and requires that the factor discharged in timbering material must meet the different needs of this two kinds of tissue characteristics simultaneously.
Semicentennial history is had for the clinical practice containing lithium medicine both at home and abroad, obtain the result with using value, be mainly manifested in and obviously suppressed mania effect and improve schizoid affective disorder, during therapeutic dose on normal person's ergasia without impact.Basic research in recent years expands the purposes of lithium salts, can improve the serial symptom of senile dementia and mesenchymal stem cells MSCs can be regulated to Osteoblast Differentiation.The improvement of the processing technology of bioactivity glass makes the sustained release profile in vivo test of lithium salts become possibility.Effective Continuous slow release containing the bioactivity glass of lithium is that experimentation of the present invention provides necessary condition.
Beneficial effect of the present invention is mainly reflected in: in prior art, the primary clinical use of lithium is nervous system degenerative disease, the invention provides a kind of containing the application of lithium nature material bioactive bracket in osteochondral defect is repaired, it has the effect promoting bone and regenerating bone or cartilage in the repair process of osteochondral defect, existing material can only meet wherein a kind of needs of bone or regenerating bone or cartilage, and osteochondral defect is the requirement needing material simultaneously to meet cartilage and subchondral bone regeneration; The present invention specify that manufacture method containing lithium support and sustained releasing character, and achieves the functional check in body, for the clinical practice containing lithium support provides the foundation.
(4) accompanying drawing explanation
Fig. 1 is bioactivity glass support scanning electron microscope (SEM) photograph, and A is low multiple (20 times), and B is high multiple (300 times);
Fig. 2 is for containing lithium bioactivity glass support scanning electron microscope (SEM) photograph, and A is low multiple (20 times), and B is high multiple (300 times);
Fig. 3 is the elution profiles containing lithium bioactivity glass support;
Fig. 4 is biological activity silk fibroin bracket scanning electron microscope (SEM) photograph, and A is low multiple (20 times), and B is high multiple (300 times);
Fig. 5 is for containing lithium biological activity silk fibroin bracket scanning electron microscope (SEM) photograph, and A is low multiple (20 times), and B is high multiple (300 times);
Fig. 6 is the elution profiles containing lithium biological activity silk fibroin bracket;
Fig. 7 is cardinal principle zoological specimens photo in embodiment 3, and a is blank group, and b is simple support group, and c is for containing lithium support group;
Fig. 8 is the blank group of cardinal principle animal in embodiment 3, and simple support group and the ICRS containing lithium support group mark;
Fig. 9 is the SO colored graph of defect section in embodiment 3, and a is blank group, and b is simple support group, and c is for containing lithium support group;
Figure 10 is the SO colored graph of defect tide line structure in embodiment 3, and a is blank group, and b is simple support group, and c is for containing lithium support group;
Figure 11 is the subchondral bone of defect in embodiment 3 and the Micro-CT scintigram of three-dimensional reconstruction, and a is blank group, and b is simple support group, and c is for containing lithium support group;
Figure 12 is embodiment 3 empty group, simple support group and structure model index (SMI) statistical analysis containing lithium support group;
Figure 13 is embodiment 3 empty group, simple support group and bone trabecula thickness (Tb.Th) statistical analysis containing lithium support group;
Figure 14 is embodiment 3 empty group, simple support group and cortical bone bone density (BMD) statistical analysis containing lithium support group;
Figure 15 is embodiment 3 empty group, simple support group and bone volume mark (BVF) statistical analysis containing lithium support group.
Figure 16 is cardinal principle zoological specimens photo in embodiment 4, and a is blank group, and b is simple fibroin support group, and c is for containing lithium fibroin support group;
Figure 17 is the blank group of cardinal principle animal in embodiment 4, and simple fibroin support group and the ICRS containing lithium fibroin support group mark;
Figure 18 is the SO colored graph of defect section in embodiment 4, and a is blank group, and b is simple fibroin support group, and c is for containing lithium fibroin support group.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment of the present invention experiment material:
Laboratory animal: Female New Zealand White Rabbit (2.5kg) SPF is provided by Zhejiang University's animal experimental center.
Experimental drug:
Lithium chloride Sigma-Aldrich China Branch (Sigmae Aldrich) lot number: 203637 main agents:
Ethyl orthosilicate (TEOS) Tianjin Chemical Reagents Factory No.1
The friendly chemical reagent work in triphosphoric acid ethyl ester (TEP) Shanghai three
Peng Zhen barracks chemical plant, four water-calcium nitrate (CaNT) Shanghai
P123 (EO20-PO70-EO20) Sigma-Aldrich China Branch (Sigmae Aldrich)
Experimental instrument and equipment:
Blender Jintan City Guo Wang experimental apparatus factory
Muffle furnace Shenzhen Yi Ke experimental facilities company limited
MICROW company of paraffin slicing machine Germany
OLYMPUS company of photomicrographic camera Japan
BMJ-III embedding machine and Changzhou Zhong Wei Electron equipment Co., Ltd of freezing stage China
Scanning electron microscope Hitachi
Inductive coupling plasma emission spectrograph (U.S. Thermo) Zhejiang University's energy and environment engineering experiment room
Micro-CT 3-D imaging system (SkyScan1076) Shanghai Shun hundred bio tech ltd provides technical service
Prepared by embodiment 1 support:
(1) preparation is containing lithium bioactivity glass support: by 5.8g P123,10g ethyl orthosilicate, 1.5g four water-calcium nitrate, 0.125g lithium chloride and 1.10g triphosphoric acid ethyl ester join in 110ml dehydrated alcohol, and with 0.5M salt acid for adjusting pH value to 6.5-7.0, under room temperature (37 DEG C), stir 24h.Aseptic polyurethane sponge (25ppi) is immersed 10min in above-mentioned solution completely, then proceed in Tissue Culture Dish and place 12h under room temperature (37 DEG C), repeat said process (submergence-placement) 3 times, then at 650 DEG C, sinter 6h remove organic template, thus build containing lithium bioactivity glass support.Multiple scanning electron microscope (SEM) photograph as shown in Figure 2.
(2) method of simple bioactivity glass support is the same, and only removing lithium chloride can simple bioactivity glass framework.Multiple scanning electron microscope (SEM) photograph as shown in Figure 1.
Multiple scanning electron microscope (SEM) photograph display bioactivity glass support (see in Fig. 1 a) and containing lithium bioactivity glass support (see meso-hole structure a) in Fig. 2 with 300-500um.High power lens shows that (b) empty wall construction is smooth see in Fig. 2 containing lithium bioactivity glass, and with simple bioactivity glass support (see in Fig. 1 b) without obviously distinguishing, Electronic Speculum result shows that adding of lithium ion does not affect the meso-hole structure of bioactivity glass and the structure of hole wall.
(3) elution profiles:
The bioactivity glass support of 4mm diameter and 5mm height is put into 50ml centrifuge tube, 50ml is settled to distilled water, respectively at 1d, 3d, 7d, 10d, 14d get the concentration that 20ml liquid inductive coupling plasma emission spectrograph detects lithium ion in solution, add 20ml distilled water after every sub-sampling in former pipe.
Inductive coupling plasma emission spectrograph testing result shows (see Fig. 3): the simple bioactivity glass support putting into distilled water is put at any time and all discharged without lithium ion, from 1d, lithium ion is discharged containing lithium bioactivity glass, 3d and 7d significantly increases, 10d peaks, and 14d still has lithium ion to discharge.Result thus, can infer that bioactivity glass support is put into after rabbit body can slow release general one-month period, and this support may be used for research lithium ion to the effect of osteochondral defect.
Embodiment 2 is containing lithium biological activity silk fibroin bracket
(1) preparation of silk fibroin protein solution: a) come unstuck: by 100g Bombyxmori Linnaeus silkworm silk (silk Co., Ltd of Zhejiang Huajing-Toshiba, 5A level) put into the 2M aqueous sodium carbonate of 5L, 96 DEG C of water-bath 30min, purified water is cleaned, and this process repeats 3 times, sloughs sericin, leave fibroin albumen, by fibroin albumen 50 DEG C of oven dry, obtain the dried fibroin albumen of 70g, for subsequent use; B) dissolve: above-mentioned dried fibroin albumen is dissolved in lithium bromide (LiBr) aqueous solution of 9.3M with mass volume ratio 0.2:1,60 DEG C of water-bath 90min fully dissolve to fibroin albumen, obtain the mixed liquor containing fibroin albumen and a small amount of insoluble granule composition; C) dialyse: by the dialysis of mixed liquor regenerated cellulose bag filter (molecular cut off 4000 dalton), dialyse 12 times at 3d with the axenic purification water of 10 times of mixeding liquid volumes, remove the LiBr ion in solution, obtain trapped fluid a; D) concentrated: mass concentration 50% Polyethylene Glycol (PEG, the molecular weight 7000) aqueous solution bag filter after dialysis being put into 4 times of volumes, room temperature leaves standstill concentrated 5h, gets trapped fluid b, namely obtains the concentrated solution of trapped fluid a; E) by trapped fluid b at horizontal rotor 5000g, 4 DEG C, centrifugal 10min, not dissolving in fibroin albumen and lithium bromide and may there is insoluble granule of removing bottom, gets supernatant at-80 DEG C of lyophilization 2-3 days, obtains the fibroin albumen of lyophilizing.Get the fibroin albumen 100mg after lyophilizing, dissolve 3 days at 4 DEG C with deionized water, be mixed with the silk fibroin protein solution 2ml of 50mg/ml.
(2) Mold Making: with 1ml disposable volume syringe 5 (external diameter is 6.5mm), comprises the cylindrical shell of syringe and the push rod with piston, as mould; And this one end of cylindrical shell needle stand of disposable molten amount syringe sintering is closed and dig on sealing surface have through hole make described in mould cylinder, aperture and sodium chloride particle quite (namely 45 ~ 50 μm), pitch-row 2mm.
(3) Material compression: be first that the NaCl granule 0.28g (0.2ml) of 45 ~ 50 μm pours mould into by the granular size ground in advance, then add 0.2ml step (1) silk fibroin protein solution.Push solution fast with the propelling speed of 1cm/s, push immediately, water and a small amount of gas are discharged from the aperture of the other end, till no longer including aqueous vapor discharge.
(4) crosslinked: the mould of step (3) is placed 36h at 4 DEG C successively, room temperature (20 ~ 28 DEG C) is placed 6 hours, 60 DEG C of placements are cross-linked for 1 hour, remove the small bore end of mould, cross-linked material is pushed in deionized water and soak 2 days, period changes water 5 times, and cross-linked material is 1:100 with single volume ratio of steaming water.Take out cross-linked material at-110 DEG C of lyophilizing 12h, obtain silk fibroin bracket, thickness 8mm, diameter 4.5mm.
(5) containing lithium bioactive bracket: after the silk fibroin bracket water cleaning that step (4) is obtained,-110 DEG C of lyophilizations are put into after-20 DEG C of freezing 2h, 24h is embathed in 20ng/ml water lithium chloride solution, and then dry under 40 DEG C of conditions, obtain containing lithium silk fibroin bracket, be containing the biological fibroin albumen active scaffold of lithium.Multiple scanning electron microscope (SEM) photograph as shown in Figure 4.
Under similarity condition, the step of embathing in water lithium chloride solution in step (5) is removed, obtains blank silk fibroin bracket, in contrast.Multiple scanning electron microscope (SEM) photograph as shown in Figure 5.
The preparation of elution profiles, with embodiment 2, the results are shown in Figure shown in 6.The simple silk fibroin bracket putting into distilled water is put all without lithium ion release at any time, and from 1d, discharge lithium ion containing the biological fibroin albumen active scaffold of lithium, 3d and 7d significantly increases, and 18d peaks, and 21d still has lithium ion to discharge.Result thus, can infer that to put into after rabbit body containing the biological fibroin albumen active scaffold of lithium can slow release general one-month period, this support may be used for research lithium ion to the effect of osteochondral defect.
Embodiment 3
1, Animal Model and stenter to implant
1. anaesthetize: calculate, with 1% pentobarbital sodium intraperitoneal anesthesia by 40mg/kg.
2. row rabbit knee joint Skin sensitization test in an aseptic environment: use 2% iodophor disinfection, the otch of about 1cm size is cut along patellar ligament medial border, successively cut and expose knee joint, drill through the osteochondral defect of 4mm diameter and the 5mm degree of depth with hand traverse with a scale at femoral bone pulley, complete modeling.
2, zoopery grouping:
18 new zealand white rabbits all carry out modeling, manufacture after defect and are divided into 3 groups at random: blank group, simple support group and containing lithium support group, each 6.
3, specimen is taked:
Modeling rabbit in step 1 is divided into three groups, namely blank group, simple support group and containing lithium support group, bioactivity glass support (prepared by embodiment 1) is implanted damp line with upper/lower positions by simple support group, namely the upper surface of support and damp line are point-blank, identical method is adopted to implant containing lithium bioactivity glass support, sterilize immediately, sew up.Disinfection and stitching after blank group exposure knee joint generation defect.
Within after experiment 16 weeks, put to death all rabbits, get rabbit knee joint femoral, after being placed in the fixing 48h of 10% formalin immersion, carry out gross examination of skeletal muscle, ICRS scoring and Micro-CT scanning, then through mass concentration 4% ethylenediaminetetraacetic acid (EDTA) aqueous solution decalcification 8 weeks, through dehydration, specimens paraffin embedding slices, sarranine-alcian blue (SO) dyeing routinely, carries out liver histopathological analysis.
4, index determining method:
(1) International Cartilage Repair Society cartilage injury hierarchy system (ICRS): defect repair degree: repairing completely is 4 points, 75% reparation is 3 points, and 50% reparation is 2 points, and 25% reparation is 1 point, and 0% reparation is 0 point; With the combination degree of surrounding normal cartilage: be fused to 4 points completely with normal articular cartilage, being less than 1mm with the distance on border is 3 points, 3/4 to be connected with surrounding tissue but the tissue of 1/4 is greater than 1mm thickness is 2 points, 1/2 tissue is connected with surrounding tissue but the tissue of 1/2 is greater than 1mm thickness is 1 point, is not connected to 0 point with surrounding tissue; Gross examination of skeletal muscle: complete surface texture is 4 points, Fibrotic surface is 3 points, little and tissue that is dispersion entrusts 2 points, and large defect is 1 point, entrusts 0 point without any tissue.Overall scoring: one-level is 12 points; Secondary is that 11-8 divides; Three grades are divided for 7-4; Level Four is that 3-1 divides.
(3) tissue slice SO dyes: tissue slice is through dewaxing, and contaminate 8min with 0.02%Fastgreen after aquation, then tap water washes away unnecessary dyestuff, color separation in 1% acetic acid, washing, 0.1%Safrain-O contaminates 2min, decolouring, transparent, mounting is used for observing.
(4) subchondral bone Micro-CT scans and three-dimensional reconstruction: the specimen after fixing is used for Micro-CT scanning and heavily adds.Scanning association instrument: 44mltube-21um-150min-ss.Sweep parameter: scanning resolution 21um, the anglec of rotation 360 °, rotation angle increment 0.4 °, voltage 80kV, electric current 80 μ A, time of exposure 3000ms, frame average is 4, and combination of pixels is 1 × 1.After having scanned, Mic-view V2.1.2 software is used to carry out three-dimensional reconstruction process.The special bone analysis software of ABA tests following parameter: BVF, SMI, Tb.Th and cortical bone BMD.
5, statistical method
Statistical procedures: continuous data all represents with x ± s, adopts SPSS13.0 statistical analysis software to process.
Compare between many groups and compare employing variance analysis between any two.P<0.01 thinks there is remarkable significant difference between group between two, and P<0.05 thinks there is significant difference between group between two.
6, experimental result
The gross examination of skeletal muscle of 6.1 defect and ICRS scoring
(a) can observe obvious cartilage defect see in Fig. 7, with surrounding tissue in conjunction with bad, the tissue surface structure of defect is imperfect for blank group.Simple support group (b) reaches 75% reparation see in Fig. 7, is less than 1mm and complete surface texture with the distance on border, and (be better than simple support group see repair tissue c) in Fig. 7 and close to normal structure containing lithium support group.
ICRS appraisal result (see Fig. 8) display is carried out to cardinal principle Histological results: blank group is divided into level Four close to 3, and simple support group about 10 points is secondary, be about 12 points is one-level containing lithium support group.Simple support group or containing lithium support group compared with blank group, there is significant difference (P<0.01); Significant difference (P<0.05) is had compared with simple support group containing lithium support group.
The cartilage SO coloration result of 6.2 regeneration:
Blank group (see in Fig. 9 a) display of dyeing there is no the reparation of class chondrocyte, but become fibrous tissue on a small quantity as seen; Simple support group (see in Fig. 9 b) dyeing show to there is proteoglycan and II expression of collagen, but dysplasia.Containing lithium support group (see in Fig. 9 c) display there is proteoglycan and II expression of collagen and structure is better than simple support group, neocartilage and cartilage undertissue are close to normal organization.SO coloration result shows to have good repair of cartilage effect containing lithium support, owing to only adding lithium ion than simple support containing lithium support, therefore can think that the regenerative process of lithium ion to cartilage discharged in support has facilitation more.
6.3 the damp line structure SO coloration result of regeneration: damp line is a kind of clear and definite histological structure, between the emitting layer and calcified cartilage of articular cartilage, be generally slightly wavy straight line, its wide footpath is 2-5 micron, if there is damp line or class tide line structure in repair tissue, shows that the cartilage that regenerates and subchondral bone have integrity functionally.(although a) there is regeneration tissue but nodal line structure between cartilage and subchondral bone see in Figure 10 in blank group; (b) between cartilage and subchondral bone, regenerating cartilage tissue is there is and has incomplete damp line structure in simple support group see in Figure 10; (c) between cartilage and subchondral bone, there is regenerating cartilage tissue and have complete damp line structure see in Figure 10 containing lithium support group.
The subchondral bone Micro-CT of 6.4 regeneration scan and three-dimensional reconstruction and statistical analysis: blank group (see in Figure 11 a) almost without "dead" development, simple support group (b) has stronger lonizing radiation development see in Figure 11, differentiation-inducing tissue is cylindric, but in cartilage lower portion almost distribution-free, therefore good supporting role can not be carried out to the cartilaginous tissue of new life, and (c) all have stronger lonizing radiation development and under development is mainly distributed in cartilage, therefore supporting function is played to the regeneration of cartilage see in 11 containing lithium support group.
Structure model index (SMI) is the index being described bone trabecula tabular and shaft-like degree by the curvature of calculating bone surface, desirable tabular bone trabecula and shaft-like bone trabecular SMI numerical value are respectively 0 and 3, when there is osteoporosis, bone trabecula is from tabular to shaft-like transformation, and SMI numerical value increases.The mark of simple support group is greater than blank group, and containing lithium support group between therebetween, shows that the subchondral bone tissue repaired containing lithium support group is between tabular bone trabecula and baculum girder, namely closer to normal osseous tissue (see Figure 12).
Bone trabecula thickness (Tb.Th) refers to bone trabecular average thickness, and when there is osteoporosis, Tb.Th value reduces.Measurement result (see Figure 13) has containing lithium support group and all has remarkable statistical significance compared with simple support group or blank group, shows that the bone trabecula thickness formed containing lithium support group is closer to normal bone tissues.
Cortical bone bone density (BMD) measurement result (see Figure 14) shows between each group and compares not statistically significant, because normal subchondral bone is spongy bone, does not therefore need to form cortical bone constructs.
The new bone that bone volume mark (BVF) measurement result (see Figure 15) generates containing lithium support is maximum, and has significant difference compared with blank group or bare stent group.
Subchondral bone Micro-CT scans and three-dimensional reconstruction and statistical analysis show to contain the subchondral bone tissue that lithium support defines relative good, the strong regeneration supporting cartilage.
Embodiment 4
By in embodiment 3 containing lithium bioactivity glass support replace with prepared by embodiment 2 method containing lithium biological activity silk fibroin bracket, simultaneously with blank biological activity silk fibroin bracket for contrast, 18 new zealand white rabbits are all carried out modeling, 3 groups are divided at random: blank group after manufacturing defect, simple fibroin support group and containing lithium fibroin support group, each 6.Other operation, with embodiment 3, the results are shown in Figure shown in 16-Figure 18.
In Figure 16, blank group (a) can observe obvious cartilage defect see in Figure 16, with surrounding tissue in conjunction with bad, the tissue surface structure of defect is imperfect.Simple fibroin support group (b) reaches 80% reparation see in Figure 16, be less than 1mm and complete surface texture with the distance on border, and (be better than simple fibroin support group see repair tissue c) in Figure 16 and close to normal structure containing lithium fibroin support group.
ICRS appraisal result (see Figure 17) display is carried out to cardinal principle Histological results: blank group is divided into level Four close to 2, and simple fibroin support group about 5 points is three grades, be about 11 points is secondary containing lithium fibroin support group.Simple fibroin support group or containing lithium fibroin support group compared with blank group, there is significant difference (P<0.01); Significant difference (P<0.05) is had compared with simple fibroin support group containing lithium fibroin support group.
The cartilage SO coloration result of regeneration as shown in Figure 18, blank group (see in Figure 18 a) display of dyeing there is no the reparation of class chondrocyte, but become fibrous tissue on a small quantity as seen; Simple fibroin support group (see in Figure 18 b) dyeing show to there is proteoglycan and II expression of collagen, but dysplasia.Containing lithium fibroin support group (see in Figure 18 c) display there is proteoglycan and II expression of collagen and structure is better than simple fibroin support group, neocartilage and cartilage undertissue are close to normal organization.SO coloration result shows to have good repair of cartilage effect containing lithium fibroin support, owing to only adding lithium ion than simple fibroin support containing lithium fibroin support, therefore can think that the regenerative process of lithium ion to cartilage discharged in support has facilitation more.
Claims (5)
1. containing the application of lithium nature material bioactive bracket in osteochondral defect is repaired, it is characterized in that described containing lithium nature material bioactive bracket with bioactivity glass or fibroin albumen for carrier, take lithium ion as active component.
2. apply as claimed in claim 1, when taking bioactivity glass as carrier containing lithium nature material bioactive bracket described in it is characterized in that, described prepares as follows containing lithium nature material bioactive bracket: join in dehydrated alcohol by poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, ethyl orthosilicate, four water-calcium nitrate, lithium chloride and triphosphoric acid ethyl ester, and by 0.5M aqueous hydrochloric acid solution adjust ph to 6.5-7.0, at room temperature stir 24h, form mixed liquor; Again aseptic polyurethane sponge is immersed in mixed liquor completely, then the polyurethane sponge after submergence is at room temperature placed 12h, then sinter 6h at 650 DEG C, obtain containing lithium nature material bioactive bracket; Described ethyl orthosilicate and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, four water-calcium nitrate and triphosphoric acid ethyl ester mass ratio are 1:0.58:0.15:0.110, described ethyl orthosilicate and lithium chloride mass ratio are 1:0.0125-0.0250, and the volumetric usage of described dehydrated alcohol counts 11ml/g with ethyl orthosilicate quality.
3. apply as claimed in claim 1, when taking fibroin albumen as carrier containing lithium nature material bioactive bracket described in it is characterized in that, described prepares as follows containing lithium bioactive bracket: the preparation of (1) silk fibroin protein solution: with Bombyxmori Linnaeus silkworm silk for raw material, through coming unstuck, dissolving, dialyse, obtain trapped fluid, the concentrated solution of trapped fluid or trapped fluid to be filtered or centrifugal, get filtrate or upper strata centrifugal liquid obtains silk fibroin protein solution, and with water, silk fibroin protein solution mass concentration is adjusted to 50 ~ 100mg/ml;
(2) containing the preparation of lithium bioactive bracket: inject the sodium chloride particle that particle diameter is 10 ~ 50 μm in mould, again at 50 ~ 100mg/ml silk fibroin protein solution prepared by the quick implantation step (1) in sodium chloride particle surface, pushing against mold makes sodium chloride particle closely knit and discharges the gas that silk fibroin protein solution and a little sodium chloride and silk fibroin protein solution formed, mould is placed at 4 ~ 70 DEG C 2 ~ 50h to be cross-linked, then remove mould, obtain cross-linked material; Cross-linked material water is cleaned postlyophilization, obtains silk fibroin bracket; Silk fibroin bracket is embathed 2 ~ 24h in 10ng/ml-50ng/ml water lithium chloride solution, and then dries under 30 ~ 700 DEG C of conditions, obtain containing lithium silk fibroin bracket, be containing lithium nature material bioactive bracket; The described volume ratio adding sodium chloride and silk fibroin protein solution in mould is 1:0.5 ~ 2; Described mould be one end close and sealing surface with the cylindrical shell of air-vent, the other end opening of cylindrical shell is also provided with the push rod of band piston, and the aperture of described air-vent is less than or equal to the particle diameter of sodium chloride particle.
4. one kind contains lithium nature material bioactivity glass support, it is characterized in that the described lithium nature material bioactivity glass support that contains is prepared as follows: join in dehydrated alcohol by poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, ethyl orthosilicate, four water-calcium nitrate, lithium chloride and triphosphoric acid ethyl ester, and be 6.5-7.0 with 0.5M salt acid for adjusting pH value, at room temperature stir 24h, form mixed liquor; Again aseptic polyurethane sponge is immersed in mixed liquor completely, then the polyurethane sponge after submergence is proceeded in Tissue Culture Dish, at room temperature place 12h, at 650 DEG C, sinter 6h, obtain containing lithium nature material bioactivity glass support; Described ethyl orthosilicate and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, four water-calcium nitrate and triphosphoric acid ethyl ester mass ratio are 1:0.58:0.15:0.110, described ethyl orthosilicate and lithium chloride mass ratio are 1:0.0125-0.025, and the volumetric usage of described dehydrated alcohol counts 11ml/g with ethyl orthosilicate quality.
5. one kind contains lithium nature material biological activity silk fibroin bracket, it is characterized in that the described lithium nature material biological activity silk fibroin bracket that contains is prepared as follows: the preparation of (1) silk fibroin protein solution: with Bombyxmori Linnaeus silkworm silk for raw material, through coming unstuck, dissolving, dialyse, obtain trapped fluid, the concentrated solution of trapped fluid or trapped fluid to be filtered or centrifugal, get filtrate or upper strata centrifugal liquid obtains silk fibroin protein solution, and with water, silk fibroin protein solution mass concentration is adjusted to 50 ~ 100mg/ml;
(2) containing the preparation of lithium nature material bioactive bracket: inject the sodium chloride particle that particle diameter is 10 ~ 50 μm in mould, again at 50 ~ 100mg/ml silk fibroin protein solution prepared by the quick implantation step (1) in sodium chloride particle surface, pushing against mold makes sodium chloride particle closely knit and discharges the gas that silk fibroin protein solution and a little sodium chloride and silk fibroin protein solution formed, mould is placed at 4 ~ 70 DEG C 2 ~ 50h to be cross-linked, then remove mould, obtain cross-linked material; After cross-linked material use water cleaning postlyophilization, obtain silk fibroin bracket, silk fibroin bracket is embathed 2 ~ 24h in 10ng/ml-50ng/ml water lithium chloride solution, and then dry under 30 ~ 700 DEG C of conditions, obtain containing lithium silk fibroin bracket, be containing lithium nature material bioactive bracket; The described volume ratio adding sodium chloride and silk fibroin protein solution in mould is 1:0.5 ~ 2; Described mould be one end close and sealing surface with the cylindrical shell of air-vent, the other end opening of cylindrical shell is also provided with the push rod of band piston, and the aperture of described air-vent is less than or equal to the particle diameter of sodium chloride particle.
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