Background technology
Epiphyseal plate (claiming growth plate again) is the hyaline cartilage that one deck has the longitudinal growth ability between long bone epiphysis and metaphysis.Be responsible for the growth of bone, its organizational structure and substrate composition are step to be changed.Physiological activity and biological behaviour complexity, be subjected to the regulation and control of whole body secretion (comprising hormone, somatomedin) and local paracrine and autocrine (multiple somatomedin), dyshormonia, stromatin gene mutation, the Nutrition and Metabolism obstacle, toxicant, physical factor etc. all can cause multiple epiphyseal plate growth promoter disease, as dyschondroplasia, dwarfism etc., epiphyseal plate damage and infection can cause epiphyseal plate partly or entirely early to close, and anamorphosis has a strong impact on limb function and labour force.
Growth plate cartilage is the most active position of osteogenesis, and its growth is directly connected to the growth promoter of long bone.Just often the removing of propagation, hypertrophy, substrate calcification and the osteoclast that the cartilage internalization must be by chondrocyte absorbs.Along with the intrusion formation class osseous tissue of blood vessel and osteoblast, and then the ossified osseous tissue that forms of calcification.
Epiphyseal plate is divided into three parts from dissecting: 1.. the cartilage epiphyseal plate of forming by different chondrocytes; 2.. ossified metaphysis; 3.. the Ranvier perichondrial ossification ditch around the epiphyseal plate, wherein the cartilage epiphyseal plate divides three layers, is followed successively by storage belt from the epiphysis end, propagation band and mastocyte band.The propagation band is the place of epiphyseal plate longitudinal growth, Ranvier perichondrial ossification ditch then is the place of epiphyseal plate cross growth, epiphyseal plate is that blood is for abundant tissue, arrive the neonatal period epiphyseal plate and just formed blood fortune barrier gradually, if owing to reasons such as fracture, inflammation damage cartilage epiphyseal plate, the destruction that finally causes blood fortune barrier, this moment, epiphysis and metaphysis blood fortune was invaded epiphyseal plate.Identical traffic, this is the basic condition that hone lamella forms, and what at first form is the fiber vessel bridges, slowly ossified then, and if the epiphyseal plate damage is enough greatly, allowing down is the deposition of bone, and the bone bridge finally forms.
Epiphyseal plate damage is very serious a kind of in children's's bone injury, owing to impaired limbs the carrying out property growth retardation and the deformity of causing of growth plate.Epiphyseal plate is the basis of child's skeleton development, has special 26S Proteasome Structure and Function, and multiple reason can cause the part or all of closure in advance of epiphyseal plate.Make limbs appearance deformity and cause corresponding joint function disturbance, and along with growth of children, deformity can increase the weight of gradually.The method that treatment epiphyseal plate damaged portion is early closed is a lot, but each method all has the no thoroughness of its limitation and treatment, the implant of using at present clinical has autologous fat, bone is cured, cartilage, bone cement, medical silica-gel, gelfoam and from body epiphyseal plate (comprising free vascular pedicle and band muscle flap person) though and the epiphyseal plate extracellular is cultivated and is transplanted and might address this problem, still have many problems at present.For example: best seed cell, between biotic environment and the cartilage cells of epiphyseal plate to concern that problem etc. all has to be solved.Current molecular biology and engineered fast development, induce research for bone wide platform is provided, the research of bone morphogenetic protein and somatomedin is deep day by day, for the thinking that provides new is early closed in the damage of treatment epiphyseal plate, adult's bone marrow stroma stem cell has differentiation potential, and is the focus that people competitively study.The epiphyseal plate damage is early closed and is caused serious deformity of limbs and dysfunction, it is common children's's bone injury disease, still lack at present the ideal material that can repair damage epiphyseal plate organizational structure and its growth function of recovery, the microencapsulated cell technology is closed the treatment means that provides new damaged morning for epiphyseal plate.
Exposed cell transplantation goes into to run into intensive immunity of organism rejection in the body again; microcapsule then can rely on the insulation blocking function of film and select permeation; guarantee that bioactive substance diffuses through; antibody or immunocyte are carried stays isolation; thereby on physiology, avoided individual immunologic rejection; become the desirable means that solve cell transplantation process immunologic rejection problem, thereby cause the interest of numerous researcheres.1997, people such as Japan Okada propose " cell preparation " speech, promptly utilize microencapsulated cell can resist immune attack in vivo, keep normal physiological function, release continuously has the cellular products of therapeutical effect---bioactie agent, can form long-term drug delivery system in the cell gonosome.So far, microencapsulated cell at first proposes as the drug delivery system notion aspect pharmacy.
Sodium alginate gel has and the similar structure of cartilage matrix composition proteoglycan, chondrocyte is grown in a kind of environment of similar physiological status, gel networks can be wrapped up chondrocyte fully simultaneously, be in three dimensional structure, to grow fully, helping cell and keep phenotype stable and secrete a large amount of substrate, is comparatively ideal cultured chondrocytes material.Make microcapsule with semipermeable membrane function with nontoxic high molecular polymer, specific cell is wrapped in the microcapsule, its principle is: the semi permeability of utilizing microcapsule membrane, micromolecule such as oxygen, nutrient substance can see through film and supply with cell, make it to keep physiologically active, and the bioactive product of emiocytosis can appear from microcapsule also, play specific function, have immunocompetent macromolecular substances simultaneously, cell etc. can not act on the capsule inner cell by microcapsule membrane, thereby play immune isolated effect.
A series of closely-related extracellular matrix proteins in the collagen protein family, extensively be present in the mammalian body, account for 25.33% of human body protein total amount, collagen is the basic structure material of organs such as skeleton, tendon, interosseous membrane, skin, cartilage and ligament, make it have very high tensile strength, and same cell interaction is for the adhering to of cell, transplanting, propagation, differentiation and existence provide signal of interest.Tropocollagen molecule has particular structure and aminoacid sequence to make it have two distinguishing features: (1) per 3 amino acid residues just have a glycine residue to form the Gly-X-Y repetitive sequence, and (2) high-load food in one's mouth propylhomoserin and hydroxyl are fed the propylhomoserin residue.Collagen is a kind of favorable tissue engineering scaffold material as a kind of n cell epimatrix biopolymer, collagen has many advantages: (1) wide material sources, be easy to separation and purification from living tissue, so the price of collagen goods is relatively cheap, and supply is stable; (2) absorbed by body easily, and have very low antigenicity; (3) good mechanical performance, natural collagen fiber have intensity and viscoelasticity preferably, the most important thing is, and as a kind of biomaterial, it has excellent biological compatibility and avirulence, and good biological plasticity and anti-fatigue ability are the essential compositions of cartilage.
Therefore, how sodium alginate and collagen protein are just become the technical barrier that this technical field urgent need will solve as carrier parcel cartilage cells of epiphyseal plate jointly.
Summary of the invention
One of purpose of the present invention is to provide a kind of good biocompatibility, the mechanical strength height, and toxic and side effects is little, has to fill and the secretion bioactie agent, discharges continuously to have the cellular products (microencapsulated cell preparation) of therapeutical effect.
Above-mentioned purpose of the present invention reaches by the following technical programs:
A kind of cartilage cells of epiphyseal plate (or other chondrocytes) compound microcapsule preparation that contains is characterized in that: comprise fresh cartilage cells of epiphyseal plate or immortalization cartilage cells of epiphyseal plate (or other chondrocytes); Comprise mixed carrier alginate-collagen protein-polylysine; Comprise the liquefier sodium citrate; Comprise firming agent calcium chloride.
Describedly contain fresh cartilage cells of epiphyseal plate or immortalization cartilage cells of epiphyseal plate (or other chondrocytes) number is: 2～8 * 106
Consisting of of described mixed carrier: alginate 50 to 600 weight portions, collagen protein 10 to 100 weight portions, polylysine 1 to 10 weight portion.
Consisting of of described liquefier sodium citrate: sodium citrate 2 to 20 weight portions.
Consisting of of described firming agent calcium chloride: 1 to 10 weight portion.
Described cartilage cells of epiphyseal plate (or other chondrocytes) compound microcapsule that contains can be to be stored in the middle of the culture fluid or the microcapsule in the normal saline.
Describedly be stored in the microcapsule in the culture fluid or be stored in microcapsule particle size range in the normal saline at 100～1000 μ m.
The particle size range of described microcapsule also can be made different magnitude range as required, as 100～200 μ m; 200～300 μ m; 300～550 μ m; 500～750 μ m or 700～950 μ m etc.
The cartilage cells of epiphyseal plate of described purification is cartilage cells of epiphyseal plate or the cryopreservation resuscitation cartilage cells of epiphyseal plate or the immortalization cartilage cells of epiphyseal plate of fresh separated.
Two of purpose of the present invention provides the preparation method of a kind of cartilage cells of epiphyseal plate or other chondrocyte compound microcapsules.
A kind of preparation method that contains cartilage cells of epiphyseal plate or other chondrocyte compound microcapsule preparations, its step is as follows:
(1) sodium alginate of 50 to 600 weight portions is weighed dissolving; Get microcapsule solution;
(2) collagen protein of 10 to 100 weight portions is weighed, dissolving gets another kind of microcapsule solution;
(3) polylysine of 1 to 10 weight portion is weighed, dissolving gets another kind of microcapsule solution;
(4) sodium citrate of 2 to 20 weight portions is weighed, dissolving gets another kind of microcapsule liquefaction solution;
(5) calcium chloride or the barium chloride of 1 to 10 weight portion are weighed, dissolving gets another kind of microcapsule solid-to-liquid ratio solution;
(6) cartilage cells of epiphyseal plate of purification is pressed 2～8 * 106Quantity gets Cell sap;
(7) with gained Cell sap and sodium alginate and collagen solution mixing, and by high-pressure electrostatic bull microcapsule drop generating device and the reaction of described consolidation liquid, be solidified into the micro gel bead of circle or similar round, the micro gel bead that micro gel bead must be strengthened in step (3) gained polylysine solution effects, micro gel bead in the effect of step (4) gained sodium citrate, must contain cartilage cells of epiphyseal plate compound microcapsule preparation again after the liquefaction.
Described high-pressure electrostatic bull microcapsule drop generating device comprises: an electrostatic generator, on the described electrostatic generator positive and negative polarities are arranged, positive pole links to each other with the syringe needle of microinjection apparatus, negative pole is connected with rustless steel steel ring in being immersed in described consolidation liquid, mixed carrier solution and cartilage cells of epiphyseal plate are housed in the injection device, splash in the described consolidation liquid and form micro gel bead, gained cartilage cells of epiphyseal plate compound recipe micro gel bead is deposited to the bottom (each step is all in aseptic operation down) of container, must contain cartilage cells of epiphyseal plate (or other chondrocytes) compound microcapsule again after the liquefaction of polylysine curing-sodium citrate solution, can be to be stored in the middle of the culture fluid or the microcapsule in the normal saline.
The cartilage cells of epiphyseal plate of described purification is cartilage cells of epiphyseal plate or the cryopreservation resuscitation cartilage cells of epiphyseal plate or the immortalization cartilage cells of epiphyseal plate of fresh separated.
The purification (amplification) of described cartilage cells of epiphyseal plate (or other chondrocytes): take from aspiration birth control person 4 monthly age induced labor foetus (or childhood rabbit), under aseptic condition, get distal femur growth plate cartilage piece, be cut into the piece of 1mm * 1mm * 1mm size, after one's mother's sister's enzymic digestion, centrifugal, washing, the hyaluronic acid enzymic digestion; Centrifugal, washing, the type i collagen enzymic digestion; Centrifugal, washing are extracted in the cartilage cells of epiphyseal plate .DMEM culture medium and are carried out primitive cell culture, with one's mother's sister's enzyme primary cell are digested again, cultivate and make cell confluency about 70%, use the SV40LTAg gene transfecting cell, and positive cell clone is cultivated and gone down to posterity.
Describedly be stored in the microcapsule in the culture fluid or be stored in microcapsule particle size range in the normal saline between 100～1000 μ m.The particle size range of described microcapsule can be made different magnitude range as required.(as 100～200 μ m; 200～300 μ m; 300～550 μ m; 500～750 μ m; Or 700～950 μ m etc.).Gained cartilage cells of epiphyseal plate compound microcapsule preparation is used for the epiphyseal plate damage and early closes treatment.
Three of purpose of the present invention provides the application that contains cartilage cells of epiphyseal plate or other chondrocyte compound microcapsule preparations, is used for the treatment of the epiphyseal plate damage, is used for filling and effectively secretion after epiphyseal plate excises.
It is the core of microcapsule technology that immunity is isolated, adopt nontoxic high molecular polymer to make little sacculus with semipermeable membrane function, to treat that transplanted cells is wrapped in this sacculus, the capsule inner cell can see through this microcapsule membrane and obtain nutrition, secreted bioactive substance also can be discharged by microcapsule, have immunocompetent macromolecular substances such as immunocyte, immunoglobulin etc. simultaneously and can not pass through this film, thereby play the immunologic barrier effect.This artificial cell is transplanted in the host, can be prevented the host immune system attack and long-term surviving.
Because collagen protein has and prevents the ability of ossify and for the regeneration of chondrocyte provides substrate, thereby further synthetic II Collagen Type VI recovers epiphyseal plate and keeps open.Cartilage cells of epiphyseal plate is behind alginate-collagen protein-polylysine one sodium citrate and calcium chloride parcel, without the implantable immediately epiphyseal plate defective region of In vitro culture, should be more suitable for filling of epiphyseal plate defective region and reparation, and more effectively stop blood vessel to be grown into and prevent that the bone bridge from forming.Reconstruction is for normal epiphyseal plate organizational structure under local physiological environment, and can recover the growth function of epiphyseal plate preferably, provides the required active graft of good function that has for the epiphyseal plate disease undermines the treatment of early closing.
Because it is more that the epiphyseal plate damaged portion is early closed the origin cause of formation, the treatment of epiphyseal plate damage is the clinical a difficult problem of orthopaedics always, the Clinical Processing method is various, after the damaged generation of bone, the physiology of bone is seriously damaged continuously, when utilizing exogenous cell therapy, suitable carrier must be arranged, make growth factor release that cell produces to the bony defect position by carrier, induce new bone formation to reach therapeutic purposes.Can set up frozen reserve cell storehouse by the method for obtaining cell source and immortalized cells from body or allogeneic, obtain ideal cell source, increase application prospect clinically.
After having been arranged, good cell select alginate and collagen protein as main carrier, adopt the high-pressure electrostatic generator for microcapsules that cartilage cells of epiphyseal plate is wrapped in the microcapsule, cell can obtain permanently effective existence after being implanted to bone bridge defective region, the secretion somatomedin, promote cartilage-derived growth, difficult problems such as solution is aging easily to the histiocyte after transplanting both at home and abroad at present, and effect is bad.Utilize the implant of transplanting after back microencapsulation cartilage cells of epiphyseal plate excises as the bone bridge, and can overcome immunological rejection effectively, bone bridge excision back prolongs the epiphyseal plate open hour by the microencapsulation cartilage cells of epiphyseal plate that implantation has physiological property, solve bone bridge excision future trouble limb energy for growth still be lower than normally then reach tender just less than the difficult problem of 20 degree angulation deformities.According to the size of epiphyseal plate defective region decision microencapsulated cell amount what, the microencapsulated cell amount is implanted in control, reaches to grasp the epiphyseal plate speed of growth.
Syringeability microencapsulation artificial cell is a kind of less invasive techniques, have easy and simple to handle, advantage such as plastotype is random, and wound is little.Because it is very similar that microencapsulation cartilage cells of epiphyseal plate itself and natural growth plate cartilage are organized, therefore can replace original growth plate cartilage after the transplanting, become the reparation epiphyseal plate and damage comparatively ideal graft, great clinical application potential is arranged.
The invention will be further described below by embodiment, but do not mean that limiting the scope of the invention.
The specific embodiment
Embodiment 1
The preparation of the cartilage cells of epiphyseal plate of fresh separated (or other chondrocytes) compound microcapsule
1, the preparation before the parcel:
1. the processing of glass drying oven and rustless steel apparatus:
With glass drying oven and the rustless steel apparatus airing that cleans up, be placed in the high temperature baking box in 180～260 degrees centigrade of bakings 3 hours down (degerming reduce phlegm and internal heat source).
2. the preparation of cartilage cells of epiphyseal plate separation solution:
A. the preparation of one's mother's sister's protein enzyme solution:
Take by weighing the commercially available one's mother's sister's protease of 5 grams, place in the above-mentioned glass drying oven, add sterile water for injection till whole dissolvings, get one's mother's sister's protein enzyme solution.
B. the preparation of hyaluronic acid enzymatic solution:
Take by weighing the commercially available hyaluronidase of 2 grams, place in the above-mentioned glass drying oven, add sterile water for injection till whole dissolvings, get the hyaluronic acid enzymatic solution.
The preparation of C.I Collagen Type VI enzyme:
Take by weighing the commercially available type i collagen enzyme of 1 gram, place in the above-mentioned glass drying oven, add sterile water for injection till whole dissolvings; Get the type i collagen enzymatic solution.
D.DMEM/Ham F12 culture fluid, hyclone is all purchased the Gibico company in the U.S..
The purification of the new fresh cell of described growth plate cartilage (or other chondrocytes): take from aspiration birth control person 4 monthly age induced labor foetus (or childhood rabbit), under aseptic condition, get distal femur growth plate cartilage piece, be cut into the piece of 1mm * 1mm * 1mm size, after one's mother's sister's enzymic digestion, centrifugal, washing, the hyaluronic acid enzymic digestion, centrifugal, washing, type i collagen enzymic digestion, centrifugal, washing, extract cartilage cells of epiphyseal plate, carry out primitive cell culture in the DMEM culture medium and go down to posterity.
3. the preparation of sodium alginate soln:
Take by weighing 2 kilograms of commercially available sodium alginates, place in the glass drying oven, stir on one side, add normal saline on one side, all dissolve until sodium alginate, get sodium alginate soln.
4. the preparation of collagen solution:
Take by weighing 0.5 and restrain commercially available collagen protein, place in the glass drying oven, add normal saline, get collagen solution to all dissolvings.
5. the preparation of calcium chloride solution:
Take by weighing 1 kilogram of commercially available calcium chloride, place in the glass drying oven, add sterile water for injection, get calcium chloride solution to all dissolvings;
6. the preparation of polylysine solution:
Take by weighing 1 and restrain commercially available import polylysine, place in the glass drying oven, add sterile water for injection, get polylysine solution to all dissolvings;
7. the preparation of sodium citrate solution:
Take by weighing 2 and restrain commercially available sodium citrate, place in the glass drying oven, add sterile water for injection, get sodium citrate solution to all dissolvings;
8. the cartilage cells of epiphyseal plate (or other chondrocytes) with above-mentioned collagen solution, sodium alginate soln and fresh separated mixes, and gets mixed solution; Draw above-mentioned mixed liquor with disposable sterilized injector, splash in the above-mentioned calcium chloride solution by high-pressure electrostatic bull microcapsule drop generating device, with Ca2+Be cross-linked to form cancellated calcium alginate collagen type microcapsule rapidly, Na in the consolidation liquid+Can with Ca in the calcium alginate2+Crosslinked, make calcium alginate collagen surface of microcapsule have a considerable amount of negative charges all the time, add positively charged polylysine solution, intermolecular cross-linking takes place again, the formed network structure of this intermolecular cross-linking is more close than the network structure of calcium alginate intramolecular crosslinking.The gained microcapsule sinks to below the container, sucts to add polylysine solution after the clear liquid and make reinforcement, and then through sodium citrate liquefaction, must need the microcapsule of scope.Microcapsule is added culture fluid or normal saline is stand-by.
With the upper solution decant of said vesse, following microcapsule is given a baby a bath on the third day after its birth time with normal saline, inhale microcapsule with the large size needle applicator with a small amount of normal saline, change suitable syringe needle and be injected directly into the epiphyseal plate damage location.
9. the damaged Preparation of model of young rabbit tibia upper end epiphyseal plate inside part:
Select for use 5 the week age health large ear rabbit, the capable intraperitoneal anesthesia of 1% pentobarbital sodium, conventional sterile working, bilateral tibial upper end inner incision is got in operation, appear tibia upper end growth plate cartilage, excision epiphyseal plate inside part is about 1/3～1/2, layer-by-layer suture periosteum, subcutaneous and skin.Cartilage cells of epiphyseal plate compound microcapsule with preparation injects left side epiphyseal plate injury region then, and right side proximal tibia epiphyseal plate injury region is not transplanted group in contrast, raises routine observation in the postoperative cage.
Embodiment 2
The preparation of cryopreservation resuscitation cartilage cells of epiphyseal plate (or other chondrocytes) compound microcapsule
1, the preparation before the parcel:
1. the processing of glass drying oven:
With the glass drying oven airing that cleans up, be placed in the high temperature baking box in 180～260 degrees centigrade of bakings 3 hours down (degerming reduce phlegm and internal heat source).
2. the processing of recovery cartilage cells of epiphyseal plate:
From liquid nitrogen container, take out frozen pipe, drop into rapidly in 37 ℃ of warm water, and shake frequently, make it melt as early as possible.Take out frozen pipe from water-bath, sucking-off cell suspension injects centrifuge tube and drips culture fluid more than 10 times, mixes the back low-speed centrifugal, wash 3 times, detects cell survival rate with trypan blue dyeing, and the cartilage cells of epiphyseal plate of purification is pressed 2～8 * 106Cell number is wrapped up, and it is stand-by to make recovery back cartilage cells of epiphyseal plate compound microcapsule.
3. the preparation of sodium alginate soln:
Take by weighing 2 kilograms of commercially available sodium alginates, place in the glass drying oven, stir on one side, add normal saline on one side, all dissolve until sodium alginate, get sodium alginate soln.
4. the preparation of collagen solution:
Take by weighing 0.5 and restrain commercially available collagen protein, place in the glass drying oven, add normal saline, get collagen solution to all dissolvings.
5. the preparation of calcium chloride solution:
Take by weighing 1 kilogram of commercially available calcium chloride, place in the glass drying oven, add sterile water for injection, get calcium chloride solution to all dissolvings;
6. the preparation of polylysine solution:
Take by weighing 1 and restrain commercially available import polylysine, place in the glass drying oven, add sterile water for injection, get polylysine solution to all dissolvings;
7. the preparation of sodium citrate solution:
Take by weighing 2 and restrain commercially available sodium citrate, place in the glass drying oven, add sterile water for injection, get sodium citrate solution to all dissolvings;
8. above-mentioned collagen solution, sodium alginate soln and cartilage cells of epiphyseal plate are mixed, get mixed solution; Draw above-mentioned mixed liquor with disposable sterilized injector, splash in the above-mentioned calcium chloride solution by high-pressure electrostatic bull microcapsule drop generating device, the gained microcapsule sinks to below the container, adding polylysine solution makes reinforcement after sucting clear liquid, and then, must need the microcapsule of scope through sodium citrate liquefaction.Microcapsule is added culture fluid or normal saline is stand-by.
With the upper solution decant of said vesse, following microcapsule is given a baby a bath on the third day after its birth time with normal saline, inhale microcapsule with the large size needle applicator with a small amount of normal saline, change suitable syringe needle and be injected directly into the epiphyseal plate damage location.
9. the damaged Preparation of model of young rabbit tibia upper end epiphyseal plate inside part:
Select for use 5 the week age health large ear rabbit, the capable intraperitoneal anesthesia of 1% pentobarbital sodium, conventional sterile working, bilateral tibial upper end inner incision is got in operation, appear tibia upper end growth plate cartilage, excision epiphyseal plate inside part is about 1/3～1/2, layer-by-layer suture periosteum, subcutaneous and skin.Cartilage cells of epiphyseal plate compound microcapsule with preparation injects left side epiphyseal plate injury region then, and right side proximal tibia epiphyseal plate injury region is not transplanted group in contrast, raises routine observation in the postoperative cage.
Embodiment 3
The preparation of immortalization cartilage cells of epiphyseal plate (or other chondrocytes) compound microcapsule
1, the preparation before the parcel:
1. the processing of glass drying oven:
With the glass drying oven airing that cleans up, be placed in the high temperature baking box in 180～260 degrees centigrade of bakings 3 hours down (degerming reduce phlegm and internal heat source).
2. the amplification and the frozen processing of immortalization cartilage cells of epiphyseal plate (or other chondrocytes):
The amplification of described immortalization cartilage cells of epiphyseal plate: take from aspiration birth control person 4 monthly age induced labor foetus (or childhood rabbit), under aseptic condition, get piece → one's mother's sister's enzymic digestion of distal femur growth plate cartilage piece → be cut into 1mm * 1mm * 1mm size after → centrifugal, washing → hyaluronic acid enzymic digestion → centrifugal, washing → type i collagen enzymic digestion → centrifugal, carrying out primitive cell culture → with one's mother's sister's enzyme in washing → extraction cartilage cells of epiphyseal plate → DMEM culture medium digests primary cell again → cultivates and make cell confluency about 70% → with SV40LTAg gene transfecting cell → positive cell clone → cultivate and go down to posterity (or frozen).
From liquid nitrogen container, take out frozen pipe, drop into rapidly in 37 ℃ of warm water, and shake frequently, make it melt as early as possible.Take out frozen pipe from water-bath, sucking-off cell suspension injects centrifuge tube and drips culture fluid more than 10 times, mixes the back low-speed centrifugal, wash 3 times, detects cell survival rate with trypan blue dyeing, and the cartilage cells of epiphyseal plate of purification is pressed 2～8 * 106Wrap up, it is stand-by to make immortalization cartilage cells of epiphyseal plate (or other chondrocytes) compound microcapsule.
3. the preparation of sodium alginate soln:
Take by weighing 2 kilograms of commercially available sodium alginates, place in the glass drying oven, stir on one side, add normal saline on one side, all dissolve until sodium alginate, get sodium alginate soln.
4. the preparation of collagen solution:
Take by weighing 0.5 and restrain commercially available collagen protein, place in the glass drying oven, add sterile water for injection, get collagen solution to all dissolvings.
5. the preparation of calcium chloride solution:
Take by weighing 1 kilogram of commercially available calcium chloride, place in the glass drying oven, add sterile water for injection, get calcium chloride solution to all dissolvings;
6. the preparation of polylysine solution:
Take by weighing 1 and restrain commercially available import polylysine, place in the glass drying oven, add sterile water for injection, get polylysine solution to all dissolvings;
7. the preparation of sodium citrate solution:
Take by weighing 2 and restrain commercially available sodium citrate, place in the glass drying oven, add sterile water for injection, get sodium citrate solution to all dissolvings;
8. above-mentioned collagen solution, sodium alginate soln and immortalization cartilage cells of epiphyseal plate (or other chondrocytes) are mixed, get mixed solution; Draw above-mentioned mixed liquor with disposable sterilized injector, splash in the above-mentioned calcium chloride solution by high-pressure electrostatic bull microcapsule drop generating device, the gained microcapsule sinks to below the container, adding polylysine solution makes reinforcement after sucting clear liquid, and then, must need the microcapsule of scope through sodium citrate liquefaction.Microcapsule is added culture fluid or normal saline is stand-by.
With the upper solution decant of said vesse, following microcapsule is given a baby a bath on the third day after its birth time with normal saline, inhale microcapsule with the large size needle applicator with a small amount of normal saline, change suitable syringe needle and be injected directly into the epiphyseal plate damage location.
9. the damaged Preparation of model of young rabbit tibia upper end epiphyseal plate inside part:
Select for use 5 the week age health large ear rabbit, the capable intraperitoneal anesthesia of 1% pentobarbital sodium, conventional sterile working, bilateral tibial upper end inner incision is got in operation, appear tibia upper end growth plate cartilage, excision epiphyseal plate inside part is about 1/3～1/2, layer-by-layer suture periosteum, subcutaneous and skin.Immortalization cartilage cells of epiphyseal plate compound microcapsule with preparation injects left side epiphyseal plate injury region then, and right side proximal tibia epiphyseal plate injury region is not transplanted group in contrast, raises routine observation in the postoperative cage.