CN101829362A - Experimental method for establishing tissue engineering cartilage in vitro by taking knee-joint cartilage unit of rabbit as seed cell - Google Patents

Abstract

The invention belongs to the technical field of in-vitro establishment of tissue engineering cartilages, more particularly to an experimental method for establishing tissue engineering cartilage in vitro by taking a knee-joint cartilage unit of a rabbit as a seed cell. The experimental method comprises the following steps of: taking a keen joint of a rabbit, repeatedly cutting the joint cartilage into fragmentized tissues, washing, removing supernatant away and putting in a sterile conical flask for later use; adding dispase and II-type collagenase into the conical flask according to 12mL DMEM-F12 culture solution/g cartilage, stirring and digesting to form a digested suspension solution; filtering and centrifuging the digested suspension solution, adding a primary chondrocyte culture solution to prepare a sterile chondrocyte suspension solution, and carrying out sodium alginate gel three-dimensional culture on the obtained cartilage unit to construct the tissue engineering cartilage. The invention has the beneficial effects that: the cartilage unit has stable form and multiplication situation in the in-vitro long-term culturing process, the physiological function of substrate ingredients, such as secreted II-type collagen, and the like is improved, and the effect of repairing cartilage injury by adopting a tissue engineering method is expectedly accelerated.

Description

With the rabbit knee cartilage unit is seed cell, the experimental technique of external structure tissue engineering bone/cartilage

Technical field

The invention belongs to the organization engineered cartilage technical field of in-vitro, being specifically related to a kind of is seed cell with the rabbit knee cartilage unit, the experimental technique of external structure tissue engineering bone/cartilage.

Background technology

In organization engineered cartilage external structure process, the selection of desirable seed cell is the most key, also is the difficult point of studying at present.At present, in the domestic and foreign literature report, the seed cell of Chang Yong articular cartilage tissue engineering is a chondrocyte the most.Its external conventional enzymolysis, digestion method is: the 0.4%Pronase enzyme is pressed 12mLDMEM-F12 culture fluid/g cartilage and was digested 90 minutes for 37 ℃, and behind the replacing culture fluid, 0.025%II Collagen Type VI enzyme digested overnight is obtained the knee cartilage cell.But in the chondrocyte In vitro culture and the process that goes down to posterity, viscoelasticity reduces gradually, and fragility increases, and the regression of chondrocyte function is than very fast, repair in the cartilage defect process in the body and can not form the hyaline cartilage tissue that meets the normal cartilage characteristic, become one of biggest obstacle of restriction tissue engineering technique development.

Chondron (Chondron) is as the articular cartilage functional structure of just paying attention to gradually in recent years, and by cell Zhou Jizhi and be wrapped in one or several chondrocyte and constitute jointly, it has brought into play important function at chondrocyte metabolism and mechanical environment aspect keeping.We attempt to obtain on the basis of rabbit knee cartilage unit at external successful enzymolysis, digestion, are seed cell with it, set up the experimental technique of the engineered articular cartilage of external external structure.

Summary of the invention

The object of the invention be at existing be the deficiency of organization engineered cartilage seed cell with the chondrocyte, a kind of seed cell---the external acquisition methods of chondron (Chondron) and constructing technology of vitro tissue through engineering approaches cartilage thereof of new articular cartilage tissue engineering proposed.Specifically being on the basis of the external enzymolysis, digestion of a kind of rabbit knee cartilage unit, separation method, is seed cell with the chondron, is carrier with the sodium alginate gel, sets up the experimental technique of the engineered articular cartilage of external structure.

The present invention adopts following technical scheme to realize: a kind of is seed cell with the rabbit knee cartilage unit, and the experimental technique of external structure tissue engineering bone/cartilage is characterized in that step is as follows:

(1), leave and take rabbit knee under the aseptic condition, in superclean bench, knee joint is opened, scraped and get knee joint femur and tibial plateau holostrome cartilage, articular cartilage is cut into the fragment tissue repeatedly, and supernatant is abandoned in aseptic D-Hanks liquid flushing, weigh, be positioned over sterile conical flask;

(2), adopt dispase enzyme and II Collagen Type VI enzyme to add in the above-mentioned conical flask by 12mL DMEM-F12 culture fluid/g cartilage, be that every gram cartilage adds DMEM-F12 culture fluid 12mL, contain mass/volume concentration in the DMEM-F12 culture fluid and be 0.3% dispase enzyme and mass/volume concentration and be 0.2% II Collagen Type VI enzyme

Above-mentioned two kinds of enzymes all adopt aseptic DMEM-F12 culture fluid dissolving, through the filtration of 0.22 μ m disposable filter, degerming,

The conical flask that adds culture fluid is positioned over 37 ℃ of 5%CO ₂On the magnetic stirring apparatus, stir at a slow speed in the incubator, unite enzymolysis and stir digestion 3h, form the digestion suspension;

(3), with above-mentioned digestion suspension with 100 μ m cell screen filtrations in centrifuge tube, the centrifugal 5min of 1200rpm, abandon supernatant, then the aseptic DMEM-F12 culture fluid of 10mL wash repeatedly, centrifugal 3 times, add former generation cultured chondrocytes liquid 10mL, in former generation,, cultured chondrocytes liquid comprised DMEM-F12 culture medium 9mL and peptide Ox blood serum 1mL, made aseptic chondron suspension, promptly obtained seed cell---chondron;

(4), the chondron of above-mentioned acquisition carries out the sodium alginate gel stereoscopic culture, the external structure tissue engineering bone/cartilage, step is as follows:

1., 120mg sodium alginate powder is dissolved in 10mL0.9%NaCl solution, vibration, mixing, 0.45 μ m disposable filter filters, degerming is standby,

2., with chondron suspension counting, centrifugal, abandon supernatant, by 4 * 10 ⁶Individual unit/mL sodium alginate gel is mixed, blows and beats repeatedly evenly with aseptic straw,

3., draw the sodium alginate gel of 40 μ L mix homogeneously, slowly splash into aseptic 102mMCaCl with micropipettor ₂Solution left standstill 5～10 minutes, made the sodium alginate gel ball, and 0.9%NaCl solution dips in to be washed, and was inoculated in 6 orifice plates and cultivated, and 4 gel balls of every hole inoculation add former generation culture medium 3mL, former generation culture medium comprise DMEM-F12 culture medium 2.7mL and peptide Ox blood serum 0.3mL.

4., culture plate is positioned over 37 ℃ of 5%CO ₂Incubator is cultivated, and changes 1 culture fluid, and makes articular cartilage unit at the sodium alginate gel stable growth in 2nd～3.

The present invention utilizes chondron as the organization engineered cartilage seed cell, and prior art has following beneficial effect relatively:

(1), utilize dispase enzyme and II Collagen Type VI enzyme associating enzymolysis to stir digestion can successfully to obtain rabbit knee cartilage unit in 3 hours, acquisition methods is simple, good reproducibility.

(2), the chondron that obtains of enzymolysis, digestion comprise complete cell Zhou Jizhi and be wrapped in one or several chondrocyte, make chondrocyte at In vitro culture and make up in the organization engineered cartilage process and meet chondrocyte growth in vivo environment and biological characteristics more.

(3), find, by the micropipette aspiration biomechanics experiment acute digestion chondron equilibrium modulus (1.573 ± 0.151kPa), instantaneous modulus (6.850 ± 0.607kPa) and the viscoelasticity index (13.423 ± 1.027kPas) apparently higher than simple chondrocyte equilibrium modulus (0.370 ± 0.013kPa), instantaneous modulus (0.672 ± 0.015kPa) and viscoelasticity index (6.293 ± 0.134kPas).Illustrate that articular cartilage unit compares biomechanics characteristic than simple cell and obviously improves, improved chondrocyte as the relatively poor shortcoming of seed cell biomechanics characteristic.

(4), compare, form in the external long-term cultivation process of chondron, propagation situation are stable, and matrix components physiologic functions such as secretion II Collagen Type VI improve, and are expected to promote organizational project method reparation cartilage injury's effect with chondrocyte.

The specific embodiment

Concrete steps of the present invention are as follows:

(1), leaves and takes rabbit knee under the aseptic condition.In superclean bench knee joint is opened, sharp tackle is scraped and is got knee joint femur and tibial plateau holostrome cartilage.With little shears articular cartilage is cut into the fragment tissue repeatedly, supernatant is abandoned in aseptic D-Hanks liquid flushing, weighs, and is positioned over sterile conical flask.

(2), adopt 0.3%dispase enzyme (a kind of neutral lyases, U.S. Sigma company) and 0.2%II Collagen Type VI enzyme (U.S. Sigma company) to add in the above-mentioned conical flask by 12mL DMEM-F12 culture fluid/g cartilage.Two kinds of enzymes all adopt aseptic DMEM-F12 culture fluid (U.S. HyClone company) dissolving, through the filtration of 0.22 μ m disposable filter, degerming.To add the culture fluid conical flask and be positioned over 37 ℃ of 5%CO ₂In the incubator on the magnetic stirring apparatus, stir at a slow speed, unite enzymolysis stir digestion 3h (observe discovery continuously by us, when stirring digestion 3h naked eyes cannot see residue cartilaginous tissue piece).

(3), with above-mentioned digestion suspension with 100 μ m cell screen filtrations in the 15mL centrifuge tube, the centrifugal 5min of 1200rpm abandons supernatant, the aseptic DMEM-F12 culture fluid of 10mL washes repeatedly, centrifugal 3 times.Add former generation cultured chondrocytes liquid 10mL (DMEM-F12 culture medium 9mL and peptide Ox blood serum 1mL), make aseptic chondron suspension.

(4), the chondron that obtains of above-mentioned digestion carries out the sodium alginate gel stereoscopic culture, step is as follows:

1., 120mg sodium alginate (U.S. Sigma company) powder is dissolved in 10mL0.9%NaCl solution, vibration, mixing, 0.45 μ m disposable filter filters, degerming is standby.

2., with chondron suspension counting, centrifugal, abandon supernatant.By 4 * 10 ⁶Individual unit/mL sodium alginate gel is mixed, and blows and beats repeatedly evenly with aseptic straw.

3., draw the sodium alginate gel of 40 μ L mix homogeneously, slowly splash into aseptic 102mMCaCl with micropipettor ₂Solution left standstill 5～10 minutes, made the sodium alginate gel ball.0.9%NaCl solution dips in to be washed, and is inoculated in 6 orifice plates and cultivates, and 4 gel balls of every hole inoculation add former generation culture medium 3mL (DMEM-F 12 culture medium 2.7mL and peptide Ox blood serum 0.3mL).

4., culture plate is positioned over 37 ℃ of 5%CO ₂Incubator is cultivated, and changes 1 culture fluid, and makes articular cartilage unit at the sodium alginate gel stable growth in 2nd～3.

Claims (1)

1. one kind is seed cell with the rabbit knee cartilage unit, and the experimental technique of external structure tissue engineering bone/cartilage is characterized in that step is as follows:

(1), leave and take rabbit knee under the aseptic condition, in superclean bench, knee joint is opened, scraped and get knee joint femur and tibial plateau holostrome cartilage, articular cartilage is cut into the fragment tissue repeatedly, and supernatant is abandoned in aseptic D-Hanks liquid flushing, weigh, be positioned over sterile conical flask;

(2), adopt dispase enzyme and II Collagen Type VI enzyme to add in the above-mentioned conical flask by 12mL DMEM-F12 culture fluid/g cartilage, be that every gram cartilage adds DMEM-F12 culture fluid 12mL, contain mass/volume concentration in the DMEM-F12 culture fluid and be 0.3% dispase enzyme and mass/volume concentration and be 0.2% II Collagen Type VI enzyme

Above-mentioned two kinds of enzymes all adopt aseptic DMEM-F12 culture fluid dissolving, through the filtration of 0.22 μ m disposable filter, degerming,

The conical flask that adds culture fluid is positioned over 37 ℃ of 5%CO ₂On the magnetic stirring apparatus, stir at a slow speed in the incubator, unite enzymolysis and stir digestion 3h, form the digestion suspension;

(3), with above-mentioned digestion suspension with 100 μ m cell screen filtrations in centrifuge tube, the centrifugal 5min of 1200rpm, abandon supernatant, then the aseptic DMEM-F12 culture fluid of 10mL wash repeatedly, centrifugal 3 times, add former generation cultured chondrocytes liquid 10mL, in former generation,, cultured chondrocytes liquid comprised DMEM-F12 culture medium 9mL and peptide Ox blood serum 1mL, made aseptic chondron suspension, promptly obtained seed cell---chondron;

(4), the chondron of above-mentioned acquisition carries out the sodium alginate gel stereoscopic culture, the external structure tissue engineering bone/cartilage, step is as follows:

1., 120mg sodium alginate powder is dissolved in 10mL0.9%NaCl solution, vibration, mixing, 0.45 μ m disposable filter filters, degerming is standby,

2., with chondron suspension counting, centrifugal, abandon supernatant, by 4 * 10 ⁶Individual unit/mL sodium alginate gel is mixed, blows and beats repeatedly evenly with aseptic straw,

3., draw the sodium alginate gel of 40 μ L mix homogeneously, slowly splash into aseptic 102mMCaCl with micropipettor ₂Solution left standstill 5～10 minutes, made the sodium alginate gel ball, and 0.9%NaCl solution dips in to be washed, and was inoculated in 6 orifice plates and cultivated, and 4 gel balls of every hole inoculation add former generation culture medium 3mL, former generation culture medium comprise DMEM-F 12 culture medium 2.7mL and peptide Ox blood serum 0.3mL,

4., culture plate is positioned over 37 ℃ of 5%CO ₂Incubator is cultivated, and changes 1 culture fluid, and makes articular cartilage unit at the sodium alginate gel stable growth in 2nd～3.