CN100475276C - Composite structured tissue engineering cartilage graft and preparation method thereof - Google Patents
Composite structured tissue engineering cartilage graft and preparation method thereof Download PDFInfo
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- CN100475276C CN100475276C CNB2005101347049A CN200510134704A CN100475276C CN 100475276 C CN100475276 C CN 100475276C CN B2005101347049 A CNB2005101347049 A CN B2005101347049A CN 200510134704 A CN200510134704 A CN 200510134704A CN 100475276 C CN100475276 C CN 100475276C
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Abstract
A tissue-engineered cartilage transplant with composite structure for treating the cartilage defect and surficial depressed defect is composed of the medically acceptable solid carrier with internal cavity, and the mixture of medically acceptable colloid and normal cartilage cells through filling said mixture in said cavity of biodegradable solid carrier.
Description
Technical field:
The present invention relates to medical science and biomedical engineering field, specially refer to organization engineered cartilage graft and preparation method thereof.
Background technology:
Cartilaginous tissue is a single organization of only containing chondrocyte, inner no blood vessel, and nutrition mainly relies on the infiltration of tissue fluid, is a kind of connective tissue with effects such as support, protection, dispersive stresses.On the histology, can be divided into hyaline cartilage (articular cartilage, costicartilage), elastic cartilage (credulous bone), fibrous cartilage (meniscus) three major types.Structurally, chondrocyte is positioned at cartilage lacuna, and is wrapped up by cartilage capsule on every side.Cartilage capsule is separated chondrocyte and surrounding tissue just as one barrier.So cartilaginous tissue has a little less than the antigenicity and the characteristic of light immunological rejection.Clinically, now proved the allogeneic chondrocyte justacrine substrate of can in the receptor body, surviving.
The damage of articular cartilage can produce pain, articular dyskinesia of joint area etc., and the quality and the productivity that can reduce life.Especially, the back takes place and just is difficult to thoroughly cure in the damage of articular cartilage, because its normal healing ability is very weak, and relevant with the degree of whole joint injury development.
Now, it is very common that application organizes engineering method is repaired the damaged treatment of cartilaginous tissue.Obtain chondrocyte, then chondrocyte is inoculated on the biodegradation material, be built into cell-material composite, be used for the defect repair of cartilaginous tissue.
In cartilage tissue engineered field, the chondrocyte quantity of the repairing effect at cartilage defect position and unit volume how many much relations arranged.Chondrocyte is a kind of cell that attaches growth, and itself requirement will have carrier to carry the growth of cell.And simple chondrocyte implants after the defect, owing to can not be fixed on defect, can dissociate in joint fluid, can't play the effect of repairing and treating.Therefore, need suitable carriers, in performance carrying effect, chondrocyte is played fixed effect.
Present three kinds of physical states of the chondrocyte carrier of using: 1. colloidal carrier, as collagen, fibrin clot (FC) etc.2. solid-state carrier, common as: collagen fiber net, hydroxyapatite (hydroxyapatite, HA), polylactic acid (polylactic acid, PLA), macrogol (polyethyleneglycol, PEG), polyglycolic acid (polygiycolic acid, PGA) and their copolymer, sintering bone, allogeneic decalcification bone or demineralized bone matrix, freeze drying bone, deproteinization bone, reconstituted bone xenograft and Corallium Japonicum Kishinouye bone etc.
3. complex carrier, the mixture of above-mentioned colloid and solid carrier or complex.
The complex method of colloidal carrier and chondrocyte is: directly with chondrocyte and colloidal carrier mixing; The complex method of solid-state carrier and chondrocyte is: the chondrocyte suspension is dripped plant on solid-state carrier; Or be adsorbed onto in the solid-state carrier by negative pressure-pumping, cell can be attached on the carrier after cultivation after a while.
These several bearing modes have some unfavorable: one, colloidal carrier comes off from the cartilage defect position, causes operative failure.Use colloidal carrier treatment cartilage defect now, be mostly that the applying biological albumin glue sticks to defect with carrier, to reach fixation.But the cartilage defect pilosity is the more positions of tissue fluid such as (as knee joints) in the joint, and biological fibrin glue is very easily diluted or be organized the too fast degraded of protease in the liquid, makes biogum not reach valid density, thereby viscosity degradation, causes carrier to come off.All need to open articular cavity when two, no matter being colloidal carrier or solid-state carrier reparation cartilage defect, carry out open surgery, this damage to the patient is bigger.
Therefore, this area presses for to develop and damages organization engineered cartilage graft little, safe, strong operability.
Summary of the invention:
The purpose of this invention is to provide a kind of new safe, easy, workable organization engineered cartilage graft of method for making and preparation method thereof.
In order to achieve the above object, the present invention adopts following technical scheme: the through engineering approaches cartilage graft of this composite construction be by medically acceptable material as solid carrier, leave cavity in this solid carrier; Be built-in with medically acceptable colloid and have the mixture that the chondrocyte of normal function mixes equably at the cavity of this solid carrier, mixture is to be 200-600IU/ml, Ca by containing concentration of thrombin
2+Concentration is the solution of 20-60mmol/L and be that 20-100mg/ml, XIII factor concentration are that the solution of 5-75U/ml mixes with fibrinogen concentration again after the chondrocyte with normal function mixes; The content of chondrocyte is not less than 0.5 * 10 in the mixture after making
7Individual/ml.
Medically acceptable colloidal carrier described in the above technical scheme can be a biological fibrin glue, and it is selected from the colloid of natural or synthetic such as alginic acid, Fibrin Glue (FG), gelatin collagen; Chondrocyte is to derive from from body, allochthonous chondrocyte and by what other cell inductions transformed to can be used for the local organization repair cell; The diameter of solid carrier is that 3-10mm, length are 5-15mm, and its internal cavities adopts network.
Medically acceptable biodegradation material is by collagen fiber net, hydroxyapatite (hydroxyapatite, HA), polylactic acid (polylactic acid, PLA), macrogol (polyethyleneglycol, PEG), polyglycolic acid (polygiycolic acid, PGA) and their copolymer, sintering bone, allogeneic decalcification bone or demineralized bone matrix, freeze drying bone, deproteinization bone, reconstituted bone xenograft, Corallium Japonicum Kishinouye bone tricalcium phosphate, calcium sulfate etc. are natural or the material of synthetic.
The mixture of described chondrocyte and colloidal carrier has a fixed structure and intensity, and it can be shapes such as cylindrical, spherical, oval, square, rectangle, taper.
Described medically acceptable biodegradation material has definite shape, and it can be shapes such as cylindrical, spherical, oval, square, rectangle, taper.
Described organization engineered cartilage graft is the mixture of above-mentioned chondrocyte and colloidal carrier and the assembly of above-mentioned medically acceptable biodegradation material, therefore, its shape is the assembly of the mixture shape and the above-mentioned medically acceptable biodegradation material shape of above-mentioned chondrocyte and colloidal carrier.
In order to make the organization engineered cartilage graft, the invention provides a kind of method for preparing this organization engineered cartilage graft, its step is as follows:
A. under gnotobasis, with chondrocyte with contain thrombin and sterilized Ca
2+The solution mix homogeneously, be prepared into solution 4, suck in the needle applicators solution 4 standby;
B. under gnotobasis, be that main component is prepared into solution 5, solution 5 is sucked in another needle applicator, suck volume and equate with solution 4 with the Fibrinogen and the XIII factor;
C. under gnotobasis, with diameter 3-10mm, the solid carrier of length 5-15mm is uprightly placed, and after the syringe needle of solution 4 with the syringe of solution 5 supports the top of solid carrier solution 4, solution 5 is slowly squeezed into solid carrier containing;
D. wait for 4-8 minute, treat that 5 mixed liquor coagulations become colloid to solution 4 with solution, at this moment, the colloidal carrier that solution 4 and solution 5 mixed liquor coagulations form contains a large amount of chondrocytes, and is fixed in the solid carrier firmly.
Chondrocyte concentration is not less than 1 * 10 in solution 4 liquid among the above-mentioned steps a
7Individual/ml; With solution before chondrocyte mixes in the content of thrombin at 200-600IU/ml, Ca
2+Concentration be 20-60mmol/L.
Composition among the above-mentioned steps b in the solution 5 comprises Fibrinogen, the XIII factor, alginic acid, gelatin collagen, and wherein fibrinogenic content is at 20-100mg/ml; The content of the XIII factor is at 5-75U/ml.
The through engineering approaches cartilage graft of this composite construction, be converted into fibrin monomer at the external Fibrinogen of promptly having finished, and the spontaneous continuous polymeric colloid process of bridged bond that is gathered into of monomer, the bioprotein colloid that makes formation can be not diluted the also not interference of various protease in the receptor, colloid is firm.Simultaneously, because biological fibrin glue coagulation process is to carry out in internal communication and solid carrier inside with network, thereby colloid and solid carrier are also bonded quite firm, and colloid can not come off from solid carrier inside under the external force of general operation technique.In operation process, only need arthroscope after the boring of cartilage defect position, graft is got final product by the arthroscopic cannula insertion, easy and simple to handle.
In brief, its advantage is: carrier is tight in the combination of cartilage defect position, difficult drop-off; Chondrocyte all is fixed on the corresponding cartilage layers that needs reparation, has saved a large amount of chondrocytes; Use Minimally Invasive Surgical Technology, the patient is carried out Minimally Invasive Surgery, reduce patient's misery, operation technique is simple.
Description of drawings:
Fig. 1 is the cartilage tissue engineered graft structure sketch map of composite construction of the present invention.
Fig. 2 is the preparation process sketch map of cartilage tissue engineered graft.
The specific embodiment:
Preparation, the molding of embodiment 1 allogeneic demineralized bone:
1. the spongy bone of homogeneous allogenic bone is partly made diameter 4mm, the cylinder of length 6mm;
2. the cylinder allograph bone defat fat, the protein Preparation that prepare are removed the antigen bone: take off cell with the surfactant defat, a kind of biogenic artificial bone that is used for of concrete grammar referenced patent goes antigenic preparation method;
3. the cylinder allosome is removed the hydrochloric acid of antigen bone with 0.1-2.0mol/L concentration, weight ratio is that 50: 1 ratio is carried out demineralization, and the demineralization time is 0.5-3 hour.The concentration of hydrochloric acid is 0.5mol/L in the present embodiment, and the weight ratio of hydrochloric acid and artificial bone is 50: 1, and the demineralization time is 1 hour;
4. homogeneous allogenic bone is through taking off the network that forms internal communication after cell, defat, the demineralization;
5. homogeneous allogenic bone lyophilizing;
6. cylinder removes antigen allosome demineralized bone cobalt 60 radiation sterilizations, and is standby.
Obtaining of embodiment 2 chondrocytes:
We are experimental subject with the Canis familiaris L..The left back lower limb knee joint of Canis familiaris L. is exposed, and the non-bearing district cartilage of getting its distal part of femur is some, and articular cavity is sewed up.The cartilaginous tissue that takes off with the ratio of 200mg cartilage/50ml PBS be soaked in contain gentamycin (among the aseptic PBS (concentration 0.01mol/L, PH 7.4 ± 0.1) of 40 units/ml) 45 minutes-60 minutes, afterwards with the manual fritter that is cut into 1mm * 1mm * 1mm size of cartilaginous tissue; The cartilaginous tissue piece of pulverizing is inserted in the 0.2%mg/mlII Collagen Type VI enzymatic solution, place in 37 ℃ ± 0.5 ℃ constant temperature oscillator to digest 8-12 hour, filter, centrifugal; Sedimentation cell detects cell viability with aseptic PBS (concentration 0.01mol/L, PH 7.4 ± 0.1) washed twice, trypan blue dyeing, and vigor＞60% is standby.
The preparation of embodiment 3 cartilage tissue engineered grafts: (following steps all require the sterile working)
1. with 2.5 * 10
5Individual chondrocyte and 25 μ l contain thrombin and Ca
2+Solution (sterilizing) mix homogeneously, it is standby to be prepared into solution 4;
2. the solution that with the main component is the Fibrinogen and the XIII factor is solution 5, and solution 5 is sucked in another needle applicator, sucks volume and equates with solution 4;
3. with diameter 4mm, the cylindrical solid carrier of length 6mm is uprightly placed, and solution 4, solution 5 is slowly squeezed into cylindrical solid carrier prop up the top of cylindrical solid carrier with the syringe needle of the syringe that contains solution 4 and solution 5 after;
4. left standstill 5 minutes, and treated that solution 4 and solution 5 closed the liquid coagulation.At this moment, the colloidal carrier that forms of solution 4 and solution 5 mixed liquor coagulations contains a large amount of chondrocytes and is fixed on firmly in the cylindrical solid carrier.
Embodiment 4 cartilage tissue engineered grafts return plants in the animal body:
The right rear leg articular cavity of same Canis familiaris L. is exposed, and in the cartilage load-bearing district of its distal part of femur, causing diameter is 2 of the circular cartilage defects of 4mm.Any reparation is not done as blank in a hole.The cartilage tissue engineered graft reparation that the hole is prepared into this method.Put to death with 4 weeks, 12 weeks, 27 weeks respectively, draw materials every group of 4 Canis familiaris L.s.
Claims (7)
1, a kind of organization engineered cartilage graft of composite construction is characterized in that: the organization engineered cartilage graft of this composite construction is as solid carrier (1), this solid carrier by medically acceptable material
(1) leave cavity in, be built-in with medically acceptable colloid (2) and have the mixture (6) that the chondrocyte (3) of normal function mixes equably at cavity, this mixture (6) is to be 200-600IU/ml, Ca by containing concentration of thrombin
2+Concentration is the solution of 20-60mmol/L and be that 20-100mg/ml, XIII factor concentration are that the solution of 5-75U/ml mixes with fibrinogen concentration again after chondrocyte (3) mixes.
2, the organization engineered cartilage graft of composite construction according to claim 1 is characterized in that: the content of chondrocyte (3) is not less than 0.5 * 10 in this mixture (6)
7Individual/ml.
3, the organization engineered cartilage graft of composite construction according to claim 1 is characterized in that: the colloid that this colloid (2) is made by biological fibrin glue.
4, the organization engineered cartilage graft of composite construction according to claim 1 is characterized in that: this solid carrier (1) is made by medically acceptable biodegradation material, and its diameter is 3-10mm, and length is 5-15mm.
5, the organization engineered cartilage graft of composite construction according to claim 1 is characterized in that: the internal cavities of this solid carrier (1) adopts network.
6, a kind of preparation method of making the through engineering approaches cartilage graft of the described composite construction of claim 1, its step is as follows:
A. under gnotobasis, with chondrocyte with contain thrombin and sterilized Ca
2+The solution mix homogeneously, be prepared into solution (4), suck in the needle applicator solution (4) standby;
B. under gnotobasis, be that main component is prepared into solution (5), solution (5) is sucked in another needle applicator, suck volume and equate with solution (4) with the Fibrinogen and the XIII factor;
C. under gnotobasis, with diameter 3-10mm, the solid carrier of length 5-15mm is uprightly placed, and solution (4), solution (5) is slowly squeezed into solid carrier after the syringe needle of the syringe that contains solution (4) and solution (5) being propped up the top of solid carrier;
D. wait for 4-8 minute, treat that the mixed liquor coagulation becomes colloid to solution (4) with solution (5), at this moment, the colloidal carrier that solution (4) and solution (5) mixed liquor coagulation form contains a large amount of chondrocytes, and is fixed in the solid carrier firmly.
7, according to the preparation method of the through engineering approaches cartilage graft of the described composite construction of claim 6, it is characterized in that: the chondrocyte among this step a is to derive from from body, allochthonous chondrocyte and by what other cell inductions transformed to can be used for the local organization repair cell, and chondrocyte concentration is not less than 1 * 10 in solution (5)
7Individual/ml.
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| CNB2005101347049A CN100475276C (en) | 2005-12-19 | 2005-12-19 | Composite structured tissue engineering cartilage graft and preparation method thereof |
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| CNB2005101347049A CN100475276C (en) | 2005-12-19 | 2005-12-19 | Composite structured tissue engineering cartilage graft and preparation method thereof |
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| CN100475276C true CN100475276C (en) | 2009-04-08 |
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| CN102634978A (en) * | 2011-02-11 | 2012-08-15 | 傅亚 | Preparation method of fibrin glue reinforced polylactic acid fiber |
| CN110101914A (en) * | 2019-05-04 | 2019-08-09 | 西北工业大学 | A kind of Prevascularized two-phase artificial bone scaffold and preparation method thereof |
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