Repair Of Articular Cartilage Defect With Homogeneous Chondrocytes Combined With Hyaluronic Acid Benzylester Gel

PREFACEAfter damaged through injury, articular cartilage does not heal rapidly or effectively by itself. Until recently methods for treatment have not produced good results in clinical work. Therefore, it is a difficult spot in the field of orthopaedics. For conducting medical research, we adopted the method of tissue engineering, and applied hyaluronic acid benzylester gel combined with chondrocyte to repair the defect of articular cartilage of rabbit to observe its effects. Now we discuss the feasibility of the medication used as carrier for transplantation.MATERIALS AND METHODS1. Acquisition and culture of chondrocytes Two pieces of Newzealand rabbit at two weeks of age were killed by intravenous injection of overdose pento-barbital. Articular cartilage of the rabbits were cut in sterilized condition and sheared into pieces of 1mm~3 , paying attention to keep the articular cartilage in moistened condition. Cells were digested at 37°C according to the procedures below: ①digesting with 2 mg/ml hyaluronidase for 45 minutes; (2)digesting with 2 mg/ml trypsin for 45 minutes; ③ after digesting with 4 mg/ml of type II colla-genase for 3 hours, the cells were rinsed and centrifuged for 5 minutes. The precipitate was culture with DMEM medium containing 15% fetal bovine serum. Taking a plate smeared with a few primary chondrocyte, then detecting immunity with collagen antibody SABC. The chondrocyte was dyed to brown -yellow color, which demonstrated that type II collagen had been secreted. Chondrocytewas confirmed by this method. The cells were cultured at a constant temperature of 37 C in a 5% C02incubator. After the proliferated cells have become mono-layer cells, 80% chondrocytes fusion had been seen under microscope. The cells were genetically cultured. The form, adherence, and survival conditions of the cells were observed..2. Culturing of chondrocytes in transparent gel o f hyaluronic acid benzylester. Chondrocytes within 4 passages were made into chondrocytes suspension (1 x 10~6/ml). Then they were transformed into compound by infusing with hyaluronic acid benzylester gel (a final concentration of 500 μg/ml) , and cultured with DMEM medium at a constant temperature of 37oC in a 5% CO2incubator. After 72 hours, the medium was changed for the first time, and the change was repeated every other day. The form, adherence, survival condition, and reproductive activity of the cells were observed. .3. Building and reparation of defect models of articular cartilage. Thirty pieces of Newzealand white rabbit at two months of age (totally 60 knees) were anesthetized by muscular injecting of 5% ketamine( 15 mg/kg).. The external condyles of femur of double posterior limb of the rabbits were exposed by aseptic operation, and drilled with crank brace along the top of lateral condyle. The drilled diameters were 4 mm, depth 4 mm. The wound was sutured after rinsing, but the knee joints were not fixed. The rabbits were randomly divided into 3 groups. Group A was the experimental group ( passage 2 chondrocytes cells cultured in vitro were mixed with hyaluronic acid benzylester gel. The final cellular concentration was 2.5 107/ml. It was injected into the joint capsule of the built animal model). Group B was the group of chondrocytes suspension (at a final cellular concentration of 2. 5 107/ml). Group C was a blank control group. The rabbits were allowed to move freely, and then were killed respectively after 4,8, and 12 weeks. The condition of their recovery was examined by observing the lateral condyle of their knee joint. After fixed, the specimens were treated with HE dying, observed under light microscope and electron microscope, and detected for II collagen antibody mediated immunity. The results were elaborately observed.1. Culturing of chondrocytes in vitro. The inoculated chondrocytes initially showed round form, suspended in the medium of culture. After 24 hours, they adhered to the wall of the test tube. The cells proliferated quickly after three generations of culturing. After seven generations, 80% chondrocytes fusion had been seen under microscope. It shows that there is hereditary passage. The shape of cells shifted from round or polygon into short spindles, then into long spindles. Almost all of the cells were transformed into long spindle cells by the sixth generation.2. Culturing of chondrocytes in the gel of hyaluronic acid benzylester. After the suspension of chondrocytes was added into transparent hyaluronic acid benzylester gel, the chondrocytes were seen to diffuse uniformly into the gel. The cells showed round or polygon form. In the early period of culture, prominence occurred on a few chondrocyte cells similar to the condition of monolayer adherent culture. Then, the percentage of chondrocytes shown in adherent -like growing condition increased gradually. The amount of cells also increased. Meanwhile, the transparency of the gel gradually decreased. The compound became turbid after 6 weeks. The chondrocytes adhered to each other and became a whole piece. A great quantity of chondrocytes was released after digested with typsin. Inoculated into a cell - culture bottle, the chondrocyte cells still adhered to the bottle wall. The collagen antibody mediated immunity dying displayed positive, which demonstrated that the characteristic of articular chondrocytes still existed.3. The repairing effect of chondrocytes-----hyaluronic acid benzylester gelcompound on defect articular cartilage3. 1 Gross observation. Rabbits of all groups moved freely. No significant evidence of inflammation such as red swelling of the skin, and so on was observed , At the fourth week, newly bom shining white tissue surfaces of the defect area in the experiment group were general flat, rough, and soft. It was clearly different from the surrounding cartilages. Observed in the 8th week, thenewly born tissue surfaces of the defect area were flat but not smooth, showing light blue color and tenacious quality, similar to the surrounding cartilage. The limit between its edge and the surrounding cartilage was not very clear, but can still be identified. In the 12th weeks, the defect area in the chondrocyte group had a few light blue membrane -like tissues, soft, rough. It is difficult to identify its contacting surface with the surrounding cartilage. In the 4 week of the cartilage group, there were some white membrane tissues in the defect area. They were soft, rough, but not smooth. There was gap between them and the surrounding tissues. In the 8th week, the newly born tissues displayed white color, soft, with definite limits. In the 12th week, the newly born tissues were uneven, with definite limits. The color was inhomogeneous. In the control group, the defect area displayed inhomogeneous color, with fibrous tissues of definite limits and uneven surface.3. 2 Histological observation. In the experiment group at the4th week, the defect area presented cartilage like tissues. The cells showed round or ellipse form, and had higher density with no directional array. A few isogenous chondrocyte clusters were formed. In the 8' week, the recovered cells displayed round form. Their density decreased, but their matrix composition increased. The chondrocytes showed the tendency of orderly array. The cartilage lacuna was clear. In the 12th week, the surface of chondrocytes was parallel to articular facet. The deeper layer arrayed orderly, and was similar to cortilago hyaline. In the chondrocytes group, at the 4th week, a few fibrous connective tissues appeared in the defect area, occasionally existed among the spherical chondrocytes. Their body is small. At the 8th week, fibrous tissues chiefly spread in the defect area. No definite chondrocytes had been seen. In the 12th week, the defect area was filled with fibrous tissues. The control group was filled with rambling fibrous tissues.3.3 Transmission electron microscope. The chondrocytes of newly born tissues in the experiment group showed round or ellipse form. Their surface had plenty of microvillus. The cell organs in the intracytoplasm included plenty of rough endoplasmic reticulum, ribosome, and colgi complex. In the earlier period, a few chondrocytes existed in the newly born tissues of the chondrocytesgroup. Their body was small, and the number of cell organs was also small. After 8 ~ 12 weeks, the majority of newly born tissues were collagen fibers. The control group had plenty of rambling collagen fibers.DISCUSSIONCurrently, applying the method of tissue engineering to repair the defect of articular cartilage is a hot spot in the research field of orthopaedics, It provides a new path, and reveals a new hope. The selection of biomaterial as a key technique of tissue engineering is highly paid attention to. Ideal biomaterials should have good biocompatibility, controllable degradation rate of materials, fine plasticity , and definite biomechanical property. Meanwhile, it should not only maintain cellular shape and phenotype, but also promote cellular adheranace and proliferation. It should be able to induce tissues regeneration.Transparent hyaluronic acid benzylester is a kind of semi — synthetic ab-sorbable biomaterial. It is formed by esterifying hyaluronic acid solution with benzyl group in free carboxyl group, leading to the increase of water draining composition. Besides maintaining some merits such as biocompatibility of hyaluronic acid and natural biodegradation metabolism mode, etc. , it has increased liposolubility, decreased water solubility, and prolonged half - life of biological action. Meanwhile, the mechanical property of hyaluronic acid benzylester is better than before. It reaches the requirement of an excellent biology vector.Aigner et al. in 1998 studied hyaluronic acid benzylester as a kind of degradation bracket for cartilage cells culture. They found that cartilage cells appeared good survival ability, adhesiveness, re - differentiation, and reproductive activity. After cultured in vitro for a long time, Cartilage cells still remained a regular phenotype.We used hyaluronic acid benzylester gel mixed with homogeneity and variant chondrocyte of rabbit to repair the defect of articular cartilage. The defect depth was 4 mm, not surpassing the down fishbone of the cartilage. Therefore the possibility of the sending down of cartilage fishbone was excluded. The newly produced tissue surface of the defect area was smooth. It showed light blue...