Special extraction device for main ingredient collagen II of natural cartilage matrix
Technical field
The present invention relates to a kind of professional production device of typeⅡ Collagen, be specifically related to a kind of Special extraction device for main ingredient collagen II of natural cartilage matrix, the typeⅡ Collagen of extraction is mainly used in the preparation of Injectable tissue engineering cartilage gel matrix.
Background technology
By wound or osteopathy joint caused position cartilage defect clinical common, have a strong impact on patients ' life quality, become the one of the main reasons of current deformity of limbs.U.S.'s sickness rate is 1.5 ‰ ~ 3 ‰, and China is about 5 ~ 6 times of the U.S., and in ascendant trend year by year.Joint cartilage belongs to hyaline cartilage, lacks neurovascular dystrophy, self is difficult to repair after damage.All there is open defect in clinical existing remedy measures, its expectant treatment and joint debridement art can only respite pain, can not stop the development of the course of disease; Bone and cartilage autotransplantation art can cause for district's damage, and limited source, be difficult to the defect of repairing larger area; There is immunological rejection and pathophorous possibility in allograph bone cartilage grafting; Artificial joint replacement then somewhat expensive, complication is more, revision rate is higher, particularly, economical load weight large on the impact of the body and mind of young patient.
Birth and the fast-developing Regeneration and Repair for joint cartilage thereof of organizational engineering provide new technology.The three-dimensional rack that tissue engineering bracket material provides for the cell building tissue, is conducive to sticking, breed and even breaking up of cell, for Growth of Cells provides suitable external environment.In organizational project, timbering material plays the effect of extracellular matrix, is the bionical of the structure and function of extracellular matrix.It not only plays a supportive role, and keeps former organized shape, but also plays template action, for cell provides the place of rely boarding, growth, Differentiation and proliferation, thus guides the regeneration of damaged tissue and controls the structure of regenerating tissues.
But, because tissue engineering bracket material generally chooses the material identical with human normal articular chondrocytes epimatrix main component as the bionical starting material preparing bionic cartilage extracellular matrix for tissue engineering, need through multiple step and process for a long time in preparation process, and in preparation process, need long stirring, centrifugal treating, prepare the time that a tissue engineering bracket material will spend 20 days usually, if all adopt manual operation, then can cause the defects such as the efficiency reduction of human cost increase and manufacture.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of Special extraction device for main ingredient collagen II of natural cartilage matrix, this typeⅡ Collagen extracts the preparation that special purpose device can complete tissue engineering bone/cartilage collagen protein automatically, effectively can save manpower, reduce cost of manufacture and enhance productivity.
For achieving the above object, the invention provides following technical scheme:
A kind of Special extraction device for main ingredient collagen II of natural cartilage matrix, comprising thermostat container, being arranged on the centrifugal device in described thermostat container and the Controlling System for controlling constant temperature the temperature inside the box and centrifugal device action, described centrifugal device comprises whizzer, the vessel assembly be arranged on whizzer, for add in vessel assembly reagent liquid feeding system, for the post-filter drainage system of waste liquid in amount discharge container device and the stirring system for stirring reagent;
Described whizzer comprises base and is arranged on the rotating disk on base, is provided with the motor for driving turntable rotation in described base;
Described vessel assembly comprises the container holder be fixedly mounted on described rotating disk, described container holder is vertically provided with at least two-stage for holding the container of reagent, and in adjacent two-stage container, superposed container bottom is provided with the passage communicated with the container being positioned at bottom;
Described post-filter drainage system comprises waste liquid tank and is positioned at the described container bottom of bottom and the apocenosis passage for discharging waste liquid with being arranged on, described passage and apocenosis passage are equipped with semi-permeable membranes and control valve, correspondingly with described apocenosis passage on described base be provided with sump pit, between described sump pit and described waste liquid tank, be provided with waste collection passage;
Described stirring system comprises the seat of honour be arranged on above described vessel assembly, and the bottom surface at the described seat of honour is provided with the agitator arm group arranged with described container one_to_one corresponding, is provided with the drive unit for driving the described seat of honour to move up and down between the described seat of honour and base;
Described liquid feeding system comprises and is arranged on described seat of honour end face for holding reagent or installing the reagent trough of reagent container, and between described reagent trough and described container, one_to_one corresponding is provided with liquid feeding passage, and described liquid feeding passage is provided with control valve;
Described Controlling System comprises controller, all be connected with described controller and for measure described constant temperature the temperature inside the box temperature sensor, for the timing register of timing, for controlling the motor-drive circuit of described motor action and the control module for controlling described drive unit lifting action, be provided with temperature in described thermostat container and control output system, described controller controls output system with described control valve and temperature respectively and is connected.
Further, described drive unit comprises and is arranged on tail rod between the described seat of honour and base and for driving the cylinder of tail rod stretching motion, the gas circuit of described cylinder is provided with magnetic valve, and described control module is connected with described magnetic valve.
Further, be arranged on container on described container holder all in the coaxial bulge arranged, and in adjacent two-stage container, the cross-sectional area being positioned at the container of bottom is greater than the cross-sectional area of superposed container.
Further, described agitator arm group correspondence is arranged on described in adjacent two-stage between container, and the agitator arm group that arrange corresponding to the described container being positioned at the top is arranged on above the vessel port of this container.
Further, described agitator arm group comprises at least one agitator arm.
Further, described liquid feeding passage comprises and to be arranged on liquid feeding passage I in the described seat of honour with described reagent trough one_to_one corresponding and to be arranged on the liquid feeding passage II be connected with described liquid feeding passage I respectively in described agitator arm.
Further, described control valve is arranged on described liquid feeding passage I.
Further, described container holder is provided with for being placed in described container and PH sensor for measuring pH value, and described PH sensor is connected with described controller.
Further, described agitator arm is provided with agitating vane.
Beneficial effect of the present invention is:
Special extraction device for main ingredient collagen II of natural cartilage matrix of the present invention, by arranging centrifugal device, under the effect of whizzer, can drive vessel assembly high speed rotating, carrying out centrifugal treating to the reagent be contained in vessel assembly; By arranging multi-stage vessel in vessel assembly, and passage is set between adjacent container, in conjunction with filtrate drainage system, under the effect of semi-permeable membranes, can separation of supernatant and throw out, and select to retain supernatant liquor or throw out continues preparation; By arranging stirring system, when needs stir time, agitator arm group being extend in corresponding container, driving vessel assembly relative to agitator arm low speed rotation by whizzer, the agitating function of solution can be realized; By arranging liquid feeding system, in preparation process, reagent can be added in the container of correspondence;
By arranging Controlling System, controller controls output system by the temperature signal of temperature sensor to the temperature of thermostat container and sends steering order, enables the temperature in thermostat container meet preparation requirement all the time; Motor startup-shutdown, rotating speed and rotational time etc. can be controlled, and by the up-and-down movement of control module accessory drive by motor-drive circuit, can stirred solution or centrifugal solution when needed; By the control to control valve, required reagent can be injected when needed in container;
Concrete, adopt Special extraction device for main ingredient collagen II of natural cartilage matrix of the present invention to prepare the method for typeⅡ Collagen as follows:
1) under room temperature, aseptic condition, from fresh pig femur, cut knee cartilage with scalpel, be placed in physiological saline and keep in;
2) the cartilage thin slice of washes clean is placed in low-temperature freeze dryer carries out freeze-drying, pulverize with Lowtemperaturepulverizer after removing moisture completely, and take chondroconia weight;
3) cartilage after pulverizing is placed in the superiors' container, and be in 75% medical alcohol of cartilage volume 5 times to injected slurry volume in container, sterilize 24h in 4 DEG C and gnotobasis, after having sterilized, utilize whizzer with the rotating speed centrifugal solution 20min of 8000rpm, open control valve after centrifugal, utilize semi-permeable membranes to filter out supernatant liquor, the throw out distilled water of reservation fully washs;
4) to injected slurry volume in the superiors' container be the 3rd) the 4mol/L guanidine hydrochloride solution of step gained throw out 10 times of volumes, by throw out and this guanidine hydrochloride solution suspendible, then agitator arm is stretched in container, whizzer is utilized to drive vessel assembly low speed rotation, after stirred solution 24h, agitator arm is exited outside container, utilize whizzer with the rotating speed centrifugal solution 20min of 8000rpm, control valve is opened after centrifugal, utilize semi-permeable membranes to filter out supernatant liquor, the throw out 0.05mol/L Tris-HCl damping fluid of reservation and 0.5mol/L Glacial acetic acid fully wash;
5) to injected slurry volume in first step container be the 4th) the 1g/L stomach en--Glacial acetic acid Digestive system of step gained throw out 5 times of volumes, appropriate scavenging solution is injected in the container of the second stage, then agitator arm is stretched in container, whizzer is utilized to drive vessel assembly low speed rotation, after stirred solution 48h, agitator arm is exited outside container, utilize whizzer with the rotating speed centrifugal solution 20min of 8000rpm, the centrifugal rear scavenging solution first got rid of in the container of the second stage, recycles semi-permeable membranes by supernatant collection in the container of the second stage; Can continue to add after Digestive system carries out digestion 24h in throw out, and by supernatant collection in the container of the second stage;
6) in the container of the second stage, the rapid regulator solution of 2mol/L NaOH is injected to pH7.5, adding NaCl makes its ultimate density be 3mol/L, saltout under 4 DEG C and gnotobasis and spend the night, utilize whizzer with the rotating speed centrifugal solution 20min of 8000rpm, semi-permeable membranes is utilized to filter out supernatant liquor, the throw out of reservation;
7) in the container of the second stage, continue to inject tri-distilled water to dialyse, after having dialysed, with the rotating speed centrifugal solution 20min of 8000rpm, utilize semi-permeable membranes to filter out supernatant liquor, the throw out of reservation, namely obtains collagen protein;
In summary, Special extraction device for main ingredient collagen II of natural cartilage matrix of the present invention can complete the preparation of tissue engineering bone/cartilage collagen protein automatically, effectively can save manpower, reduces cost of manufacture and enhances productivity.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing and being described:
Fig. 1 is the structural representation of Special extraction device for main ingredient collagen II of natural cartilage matrix embodiment of the present invention;
Fig. 2 is the A detail drawing of Fig. 1;
Fig. 3 is control principle block diagram;
Fig. 4 is the sectional view of agitator arm.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
As shown in Figure 1, the structural representation of Special extraction device for main ingredient collagen II of natural cartilage matrix embodiment of the present invention.The Special extraction device for main ingredient collagen II of natural cartilage matrix of the present embodiment, comprising thermostat container 1, being arranged on the centrifugal device in thermostat container 1 and the Controlling System for controlling temperature and centrifugal device action in thermostat container 1, centrifugal device comprises whizzer, the vessel assembly be arranged on whizzer, for add in vessel assembly reagent liquid feeding system, for the post-filter drainage system of waste liquid in amount discharge container device and the stirring system for stirring reagent.
The rotating disk 3 that whizzer comprises base 2 and is arranged on base, is provided with the motor 4 for driving turntable rotation in base 2, rotate or stirring at low speed rotation for driving vessel assembly high speed centrifugation.
Vessel assembly comprises the container holder 5 be fixedly mounted on rotating disk 3, container holder 5 is vertically provided with at least two-stage for holding the container 6 of reagent, and in adjacent two-stage container 6, bottom superposed container 6, be provided with the passage 7 communicated with the container being positioned at bottom.
Post-filter drainage system comprise waste liquid tank 8 and be arranged on be positioned at bottom container 6 bottom and apocenosis passage 9 for discharging waste liquid, passage 7 and apocenosis passage 9 are equipped with semi-permeable membranes 10 and control valve 11, correspondingly with apocenosis passage 9 on base 2 be provided with sump pit 12, waste collection passage 13 is provided with between sump pit 12 and waste liquid tank 8, by arranging semi-permeable membranes 10 and control valve 11 on passage 7 and apocenosis passage 9, in preparation process, can select to retain throw out or cleaner liquid.
Stirring system comprises the seat of honour 14 be arranged on above vessel assembly, the bottom surface at the seat of honour 14 is provided with the agitator arm group arranged with container 6 one_to_one corresponding, the drive unit for driving the seat of honour 14 to move up and down is provided with between the seat of honour 14 and base 2, by moving up and down of the seat of honour 14, agitator arm group can be stretched into or exit container 6.
Liquid feeding system comprises and is arranged on the seat of honour 14 end face for holding reagent or installing the reagent trough 15 of reagent container, between reagent trough 15 and container 6, one_to_one corresponding is provided with liquid feeding passage 16, liquid feeding passage 16 is provided with control valve 17, by arranging control valve 17 on liquid feeding passage 16, by controlling the opening and closing of control valve 17, kind and the opportunity of in container 6, adding reagent can be controlled.
Controlling System comprises controller 18, all be connected with controller 18 and for measure temperature in thermostat container 1 temperature sensor 19, for the timing register 20 of timing, for controlling the motor-drive circuit 21 of motor 4 action and the control module 22 for accessory drive lifting action, be provided with temperature in thermostat container 1 and control output system 23, controller 18 controls output system 23 with control valve 17 and temperature respectively and is connected.
Preferably, the drive unit of the present embodiment comprises and is arranged on tail rod 24 between the seat of honour 14 and base 2 and for driving the cylinder of tail rod 24 stretching motion, the gas circuit of cylinder is provided with magnetic valve 25, and control module 22 is connected with magnetic valve 25, the seat of honour 14 can be driven to move up and down by cylinder.
Further, be arranged on container 6 on container holder 5 all in the coaxial bulge arranged, and in adjacent two-stage container 6, the cross-sectional area being positioned at the container 6 of bottom is greater than the cross-sectional area of superposed container 6, adopt the vessel assembly of this structure, the stirring of each container 6 can be facilitated.Preferably, agitator arm group correspondence is arranged between adjacent two-stage container 6, and the agitator arm group that arrange corresponding to the container 6 being positioned at the top is arranged on above the vessel port of this container 6, easily agitator arm group can be stretched into or exit container 6.Preferably, agitator arm group comprises at least one agitator arm 26, the container holder 5 of the present embodiment is provided with two-stage container 5, the agitator arm group corresponding with the first step container of the top comprises an agitator arm 26, the agitator arm group corresponding with second stage container comprises the uniform agitator arm 26 of four annulars, agitator arm 26 is provided with agitating vane 26a, can strengthen stirring capacity.
Further, liquid feeding passage 16 comprises and to be arranged on liquid feeding passage I in the seat of honour with reagent trough 15 one_to_one corresponding and to be arranged on the liquid feeding passage II16a be connected with liquid feeding passage I respectively in agitator arm 26, by liquid feeding passage II16a is arranged in agitator arm 26, can simplified construction, and utilize agitator arm 26 to add reagent to container.Preferably, control valve 10 is arranged on liquid feeding passage I.
Further, container holder 5 is provided with for be placed in container 6 and PH sensor 27, PH sensor 27 for measuring pH value is connected with controller 18.
The Special extraction device for main ingredient collagen II of natural cartilage matrix of the present embodiment, by arranging centrifugal device, under the effect of whizzer, can drive vessel assembly high speed rotating, carrying out centrifugal treating to the reagent be contained in vessel assembly; By arranging multi-stage vessel 6 in vessel assembly, and passage is set between adjacent container 6, in conjunction with filtrate drainage system, under the effect of semi-permeable membranes 10, can separation of supernatant and throw out, and select to retain supernatant liquor or throw out continues preparation; By arranging stirring system, when needs stir time, agitator arm group being extend in corresponding container, driving vessel assembly relative to agitator arm 26 low speed rotation by whizzer, the agitating function of solution can be realized; By arranging liquid feeding system, in preparation process, reagent can be added in the container of correspondence;
By arranging Controlling System, controller 18 controls output system 23 by the temperature signal of temperature sensor 19 to the temperature of thermostat container 1 and sends steering order, enables the temperature in thermostat container 1 meet preparation requirement all the time; Motor 4 startup-shutdown, rotating speed and rotational time etc. can be controlled, and by the up-and-down movement of control module 22 accessory drive by motor-drive circuit 21, can stirred solution or centrifugal solution when needed; By the control to control valve 17, required reagent can be injected when needed in container 6.
Concrete, the method that employing the present embodiment Special extraction device for main ingredient collagen II of natural cartilage matrix prepares typeⅡ Collagen is as follows:
1) under room temperature, aseptic condition, from fresh pig femur, cut knee cartilage with scalpel, be placed in physiological saline and keep in;
2) the cartilage thin slice of washes clean is placed in low-temperature freeze dryer carries out freeze-drying, pulverize with Lowtemperaturepulverizer after removing moisture completely, and take chondroconia weight;
3) cartilage after pulverizing is placed in the superiors' container, and be in 75% medical alcohol of cartilage volume 5 times to injected slurry volume in container 6, sterilize 24h in 4 DEG C and gnotobasis, after having sterilized, utilize whizzer with the rotating speed centrifugal solution 20min of 8000rpm, open control valve 11 after centrifugal, utilize semi-permeable membranes 10 to filter out supernatant liquor, the throw out distilled water of reservation fully washs;
4) to injected slurry volume in the superiors' container be the 3rd) the 4mol/L guanidine hydrochloride solution of step gained throw out 10 times of volumes, by throw out and this guanidine hydrochloride solution suspendible, then agitator arm 26 is stretched in container 6, whizzer is utilized to drive vessel assembly low speed rotation, after stirred solution 24h, agitator arm is exited outside container, utilize whizzer with the rotating speed centrifugal solution 20min of 8000rpm, control valve is opened after centrifugal, utilize semi-permeable membranes 10 to filter out supernatant liquor, the throw out 0.05mol/L Tris-HCl damping fluid of reservation and 0.5mol/L Glacial acetic acid fully wash;
5) to injected slurry volume in first step container 6 be the 4th) the 1g/L stomach en--Glacial acetic acid Digestive system of step gained throw out 5 times of volumes, appropriate scavenging solution is injected in the container of the second stage, then agitator arm is stretched in container, whizzer is utilized to drive vessel assembly low speed rotation, after stirred solution 48h, agitator arm is exited outside container, utilize whizzer with the rotating speed centrifugal solution 20min of 8000rpm, the centrifugal rear scavenging solution first got rid of in the container of the second stage, recycles semi-permeable membranes by supernatant collection in the container of the second stage; Can continue to add after Digestive system carries out digestion 24h in throw out, and by supernatant collection in the container of the second stage;
6) in the container of the second stage, the rapid regulator solution of 2mol/L NaOH is injected to pH7.5, adding NaCl makes its ultimate density be 3mol/L, saltout under 4 DEG C and gnotobasis and spend the night, utilize whizzer with the rotating speed centrifugal solution 20min of 8000rpm, semi-permeable membranes is utilized to filter out supernatant liquor, the throw out of reservation;
7) in the container of the second stage, continue to inject tri-distilled water to dialyse, after having dialysed, with the rotating speed centrifugal solution 20min of 8000rpm, utilize semi-permeable membranes to filter out supernatant liquor, the throw out of reservation, namely obtains collagen protein;
In summary, Special extraction device for main ingredient collagen II of natural cartilage matrix of the present invention can complete the preparation of tissue engineering bone/cartilage collagen protein automatically, effectively can save manpower, reduces cost of manufacture and enhances productivity.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.