1、1Adipose derived stem cells to repair full-thickness cartilage defects in rabbits of kneeAbstract observe the evaluation of the patellofemoral joint surface of rabbit knee joint center of artificial full-thickness articular cartilage defects, the pure fat source of calcium alginate gel stem cells tr
2、ansplanted into articular cartilage defects of the repair of full thickness cartilage defect effect, to explore joint cartilage defect repair simple and effective way. Method 27 New Zealand white rabbits were randomly divided into A, B, C three groups, A group of articular cartilage defects disposal
3、 into calcium alginate gel, B group without any treatment of cartilage defects, C Group disposed of into the defect without the fat derived stem cells induced by calcium alginate gel compound, 4,8,12 weeks after operation, respectively, animals were killed by gross, histological observation and tran
4、smission electron microscopy techniques to observe the effect of repair , using the modified Wakitani score to rate the standard of repair tissue obtained score using statistical software SPSS 11.5 for statistical analysis. Results A, B group of the cartilage defect was filled with fibrous tissue ti
5、lting, histological sections can 2be seen covering the original fiber , C group for the class of cartilage defects of hyaline cartilage tissue filling, cartilage cells are seen in tissue sections and around the lacunae, electron microscope, a large number of cells around collagen fibers, C group in
6、each period and A, B group differences in scores between are statistically significant (P 0.01), group C showed two sets of fixed effects is superior than the other. Conclusion adipose derived stem cells combined with pure calcium alginate gel can be an effective full-thickness defects of articular
7、cartilage repair. Keywords: adipose derived stem cells in cartilage calcium alginate Abstract: Objective To evaluate the effect of not induced adipose derived stem cells (ADSCs) with calcium alginate healing full-thickness cartilage defects of rabbit knee after the model was made in center of femora
8、l facies patellaris artificially, in order to find a simple and working way of articular cartilage defect restoration. Method Twenty-seven New Zealand white rabbits were divided in to A, B, C groups randomly. Calcium alginate was transplanted to cartilage defect position of rabbits of group A, nothi
9、ng for group B and calcium alginate with not induced ADSCs for 3group C. The animals were killed in 4th, 8th and 12th week later.Restored tissue was evaluated using naked eyes, histological section and transmission electron microscopy, score was given according to modified Wakitani grade standard. T
10、he total score was analyzed with statistic software SPSS 11.5. Result Articular cartilage defects of the animal of groups A and B were filled with fibrous tissue which contains protofiber when it was checked using histological section and microscopy.The defect was filed with chondrocyte-like tissue
11、. Chondrocyte-like cells surrounded with lacunes were observed in histological section. Plenty of collagen fibers around cells could be seen under transmission electron microscopy. Group C had a significant statistical difference compared with groups A and B (P 0.01) in each period which showed that
12、 group C got a better indication than other two groups. Conclusion Non-induced ADSCs with calcium alginate could repaire full-thickness cartilage defect of rabbit knee effectively. Keywords: ADSCs; calcium alginate; cartilage Self-repair capacity of articular cartilage is limited, its repair after i
13、njury problems has been one of the orthopedic industry. With the stem cell culture and tissue engineering 4technology advances, a variety of tissue engineering methods have been applied to explore the feasibility of treatment of cartilage defects of .2001, Zuk PA, and Zhu Min 1 and other human adipo
14、se tissue suspension from the first time in a number of isolated stem cells to differentiate. has been confirmed that adipose tissue derived stem cells (adipose derived stem cells, ADSCs can be the fat cells, cartilage cells, bone cells, muscle cells and cardiac cells induced differentiation 2. In t
15、his study, adipose derived stem cells combined with pure calcium alginate gel carrier filled with full-thickness repair of rabbit articular cartilage defects, the experimental results were observed and analyzed in order to of pure fat derived stem cells used in the feasibility of repairing the defec
16、t of articular cartilage for articular cartilage defect repair simple and effective way to explore. 1 Materials and methods 1.1 Materials 1.1.1 The main instrument (1) Clean Benches (JHT-DDC type, Jinan combo purification equipment Co., Ltd.), (2) constant temperature incubator (Heraeus, Germany), (
17、3) inverted microscope (Nikon TE2000, Japan), (4 ) 50 ml culture flask (Corning, USA), 5(5 low-temperature high-speed centrifuge (Heraeus, Germany. 1.1.2 Reagents (1) DMEM medium (Gibco Company, USA), (2) collagenase (Sigma, USA), (3) trypsin (Shanghai Huamei Company, China), (4) fetal bovine serum
18、(Tianjin Hao Yang Biological Products Technology Co., Ltd.), (5) D-Hanks balanced salt (Sigma Corporation, USA, (6 alginate (Shanghai Chemical Reagent Station-packing plant, (7 calcium chloride (Shanghai Health Bioengineering Co., Ltd 2 Experimental Methods 2.1 fat source for stem cells, culture and
19、 identification New Zealand white rabbits were killed, sterile taken iliac fossa adipose tissue of about 10 ml, 2 times the volume with D-Hanks balanced salt solution rinsing 3 times, cut into 1 mm 1 mm 1 mm size, add 0.2% 5 ml collagenase at 37 in temperature inside the digestive 0.5 h, low-tempera
20、ture high-speed centrifuges 1 000 r / min 10 min centrifugation supernatant was removed by adding 160 mmol / L NH4Cl about 5 ml red cell lysis 10 min, filter mesh by 8 000 / cm2 culture flasks were grown in 50 ml. flasks at 37 in 5% volume fraction of CO2 constant temperature inside the .24 h 6incub
21、ation medium was changed after the first time, to remove residual red blood cells and no adherent cells every 2 3 d after the medium was changed once, 80% integration of the passage above, animal experiments with cells spread to the 4th generation. 2 cells using a portion containing 10% FBS, 0.1 mol
22、 / L dexamethasone, 50 mol / L vitamin C, 10 mmol / L -glycerophosphate, 100 U / ml penicillin, 100 U / ml streptomycin in DMEM medium induced by the direction of the induction of osteoblasts. 2.2 Animal experiments Select 27 healthy adult New Zealand white rabbits (purchased from Experimental Anima
23、l Center of Shandong Lukang, weight 2.5 3.5 kg, male or female. No were randomly divided into A, B, C 3 groups, each nine, and the knees are used Experimental .10% chloral hydrate anesthesia ear vein, take the bilateral medial incision, the patella toward the outside, into the joint cavity. the righ
24、t and left, respectively 3.5 mm diameter drill in the center of the patellofemoral joint surface caused by Full-thickness articular cartilage defects, the depth to spotting far, about 2.5 mm.A Group: (18 knees implanted defect in calcium alginate gel alone. B group: (18 knees, control, defect withou
25、t any 7treatment. C: (18 knees, into cultured adipose derived stem cell / alginate gel complexes, cell final concentration of not less than 2.0 106 / ml. of surgery in rabbits regained consciousness, place the cage free, intramuscular injection of penicillin Continuous 5 d. 2.3 Detection 2.3.1 cytol
26、ogy Biological characteristics: To observe the cell growth, a portion 2 cells crawl into cells to do with oil red O staining film, and the other to take some direction to the osteoblast cells induced by 2-piece climbing into cells to do mine Von Kossa staining of nodules, to confirm whether the stem
27、 cell characteristics of cultured cells. 2.3.2 Detection of repair tissue 4,8,12 weeks after surgery, respectively, animals were killed, drawing the line in general, histological examination by light microscopy and electron microscopy. (1) general observation: whether the congestion and edema of kne
28、e synovial membrane, joint capsule with or without adhesions and repair tissue formation, gloss, (2) histology: The decalcified specimens, graded ethanol dehydration, paraffin embedded, sliced, HE staining and toluidine blue staining, for 8histological observation under light microscopy, (3 transmis
29、sion Observation: Take 12 weeks, repair tissue, embedding ultra-thin sections, cells and matrix observed situation. 2.4 Statistical analysis According to the modified Wakitani score standards (see Table 1) using blinded samples of each histological score. Each specimen had three people score, taking
30、 both the number of scoring results from application of SPSS 11.5 statistical software for multivariate analysis of variance, compared with the control group by Dunnett test. Table 1, the organization of the cartilage defect score of histological scores of standard indicators 3 Results 3.1 adipose d
31、erived stem cell culture and induction Cells 24 h after inoculation around adherent fusiform cell shape, triangular or polygonal (Figure 1. Cell growth strong, 3 4 d to passage. Take some climbing 2 cells into cells to do the oil film red O staining, hematoxylin nucleus, cytoplasm is not colored wit
32、h oil red O (Figure 2, showing that the cytoplasm does not contain lipids, and no correlation between fat cell lineage. cell culture proliferative even after 7 9generations. to the direction of osteoblast cells induced into cell 2 did Von Kossa staining climbing film can be observed in the formation
33、 of mineralized nodules Ag + deposition was black (see Figure 3, indicating that the adipose derived stem cells cultured with osteogenic the ability of cell differentiation, the differentiation of the potential, with stem cell characteristics. Links to free paper download 3.2 General observation 3 4
34、 d animals after mild limp, about 1 week back to normal. Knee no redness, swelling, heat and other inflammatory phenomena. After animals were killed, see joint fluid clear, non-joint capsule contracture, adhesions and the formation of new biological, synovial No edema, no erosion of cartilage and jo
35、int edges without osteophyte formation. After 4 weeks, A, B group is basically the same repair tissue defects, the defect is not fully populated, repair tissue was white, the base was dark red, no significant inflammatory granulation tissue, C group has not completely fill the defect However, the ap
36、pearance of repair tissue was yellow-white, rough surface, rolling hills was like, clear boundaries with the surrounding cartilage. 10After 8 weeks, A, B group repair tissue defects is still basically the same, the defect is not fully filled, but more than before, repair tissue is still gray, rough
37、surface, C group did not completely fill the defect, repair tissue than previously and more yellowish white, the surface becomes smooth and elastic, and able to identify the boundaries of the surrounding normal cartilage. After 12 weeks, A, B group Deficit completely filled, tilting base for the fib
38、rous tissue covering the surface flush with the normal cartilage, dark gray, soft, and the surrounding normal cartilage boundaries clear, easy to peel, C group defects District repair tissue color, texture with the normal articular cartilage, smooth surface flush with the surrounding cartilage inter
39、face and is fully integrated, linked closely together, not easy to peel, and the blurring of the surrounding normal cartilage (Figure 4. 3.3 Histological observation After 4 weeks, A, B group, see defect depression, there are blood clots, fibrous tissue covered, stained with toluidine blue staining
40、of the same color of the fibrous tissue, C groups see such a small amount of proliferation of cartilage cells and naive less matrix, cell arrangement is slightly disordered, 11irregular, small cell morphology, no typical lacuna around like structure metachromatic toluidine blue staining was not obvi
41、ous, and no obvious multinucleated inflammatory cells. 8 weeks after surgery, histological similarities with the first 4 weeks, A, B group of fibrous tissue covering the defect, with the original fibers, corrugated courses, metachromatic tissue repair, less and uneven, C group in the repair tissue c
42、an be seen more immature cartilage cells and matrix classes, class, common lacuna cartilage cells are unevenly distributed, were significantly metachromatic toluidine blue staining. After 12 weeks, A, B group repair tissue is thicker and more fiber, a few have not completely filled, corrugated arran
43、gement of fibrils, and no cartilage lacuna formation, toluidine blue staining small and uneven, C group repair The class of normal cartilage and cartilage around the base together into a chondrocyte cell morphology like normal cartilage cells, but the arrangement of non-columnar, irregular than norm
44、al chondrocytes, chondrocyte cartilage cells are seen around the lacunae (Figure 5, no inflammatory cell infiltration, compared with toluidine blue colored uniform. 3.4 TEM 12Transmission electron microscopy shows alginate gel pore size large, cross-linked more closely, the wall thickness. A, B grou
45、p 4 weeks, the defect can be seen to have more red blood cells, 8,12 weeks by the fibrous tissue filling defects, mostly for layered arrangement of fibrous tissue, partially degraded alginate for 4 weeks, 8 weeks, most of the degradation, C group 8,12 week to repair cartilage tissue are seen in imma
46、ture cells, cylindrical or oval, irregular surface processes, cell rich qualitative expansion in the visible rough endoplasmic reticulum and small round mitochondria, there are more around the matrix, surrounding matrix within the thin vertical and horizontal alignment of the collagen fibers (Fig. 6
47、) .4 weeks partially degraded alginate, 8 weeks most of the degradation. 3.5 statistical analysis software Adipose derived stem cell / alginate gel composite score of transplantation group compared with the other two groups were significantly different between periods (P 0.01). Further by Dunnett te
48、st, simply transplanted alginate gel group and blank control group each period between no significant difference (P 0.01). (see Table 2 Table 2 Histological score time 134 Discussion Articular cartilage is a highly differentiated organization, which is generally repair itself after the cartilage fib
49、rous tissue or fibers to form hyaline cartilage tissue is limited. Chondrocytes to form hyaline cartilage repair transplantation 3, but the drawback is that cartilage cells are highly differentiated cells, the limited capacity of in vitro proliferation of autologous transplantation of their derived process will also generate additional damage to the joint damage and the need for secondary surgery, cumbersome and expensive process 4. Tissue engineering cartilage defect of hope. Bone marrow stromal stem cells found in cartilage tissue engineering applications
