ObjectiveTo observe the effect of using tungsten drills to prepare mouse knee osteochondral injury model by comparing with the needle modeling method, in order to provide an appropriate animal modeling method for osteochondral injury research.MethodsA total of 75 two-month-old male C57BL/6 mice were randomly divided into 3 groups (n=25). Mice in groups A and B were used to prepare the right knee osteochondral injury models by using needles and tungsten drills, respectively; group C was sham-operation group. The general condition of the mice was observed after operation. The samples were taken at 1 day and 1, 2, 4, and 8 weeks after modeling, and HE staining was performed. The depth, width, and cross-sectional area of the injury site at 1 day in groups A and B were measured, and the percentage of the injury depth to the thickness of the articular cartilage (depth/thickness) was calculated. Toluidine blue staining and immunohistochemical staining for collagen type Ⅱ were performed at 8 weeks, and the International Cartilage Research Society (ICRS) score was used to evaluate the osteochondral healing in groups A and B.ResultsAll mice survived to the completion of the experiment. HE staining showed that group C had normal cartilage morphology. At 1 day after modeling, the injury in group A only broke through the cartilage layer and reached the subchondral bone without entering the bone marrow cavity; the injury in group B reached the bone marrow cavity. The depth, width, cross-sectional area, and depth/thickness of the injury in group A were significantly lower than those in group B (P<0.05). At 1, 2, 4, and 8 weeks after modeling, there was no obvious tissue filling in the injured part of group A, and no toluidine blue staining and expression of collagen type Ⅱ were observed at 8 weeks; while the injured part of group B was gradually filled with tissue, the toluidine blue staining and the expression of collagen type Ⅱ were seen at 8 weeks. At 8 weeks, the ICRS score of group A was 8.2±1.3, which was lower than that of group B (13.6±0.9), showing significant difference (t=−7.637, P=0.000).ConclusionThe tungsten drills can break through the subchondral bone layer and enter the bone marrow cavity, and the injury can heal spontaneously. Compared with the needle modeling method, it is a better method for modeling knee osteochondral injury in mice.
OBJECTIVE To review the recent research progress of bone-marrow stromal stem cells (BMSCs) in the conditions of culture in vitro, chondrogenic differentiation, and the application in cartilage tissue engineering. METHODS: Recent original articles related to such aspects of BMSCs were reviewed extensively. RESULTS: BMSCs are easy to be isolated and cultivated. In the process of chondrogenesis of BMSCs, the special factors and interaction between cells are investigated extensively. BMSCs have been identified to form cartilage in vivo. One theory is the committed chondrocyte from BMSCs is only a transient stage. CONCLUSION: BMSCs are the alternative seeding cells for cartilage tissue engineering. The conditions promoting mature chondrocyte should be further investigated.
Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
Objective To study the biological characteristic and potential of chondrocytes grafting cultured on fascia in repairing large defect of articular cartilage in rabbits. Methods Chondrocytes of young rabbits were isolated and subcultured on fascia. The large defect of articular cartilage was repaired by grafts of freeze-preserved and fresh chondrocytes cultured on fascia, and free chondrocytes respectively; the biological characteristic and metabolism were evaluated bymacroscopic, histological and immunohistochemical observations, autoradiography method and the measurement of nitric oxide content 6, 12, 24 weeks after grafting. Results The chondrocytes cultured on fascia maintained normal growth feature and metabolism, and there was no damage to chondrocytes after cryopreservation; the repaired cartilage was similar to the normal cartilage in cellular morphology and biological characteristics. Conclusion Chondrocytes could be cultured normally on fascia, which could be used as an ideal carrier of chondrocytes.
An experimental study, repairing of articular cartilage by free periosteal graft in rabbit, was designed. Two 4.5mm wide circular full-thikness cartilage defects were drilled on the medial femoral condyle, in 24 adult rabbits. A graft of periosteum from the proximal tibia was fitted into the defect (right side), by using fibrinogen glue. On the control side (left side), the defect was fulled with fibrinogen glue, or repaired by periehondrial grafts which were taken from their own ribs. This experiment indicated that both periosteum and perichnodrium have the same potential of cartilagious regeneration. And the processes of regeneration are also the same. So we suggest to use free autologous periosteal grafts to replace free autologous perichondrial grafts to cure the articular cartilage defects.
Objective To review the latest progress of seeding cells for articular cartilage tissue engineering. Methods The recent original l iteratures on seeding cells for articular cartilage tissue engineering were extensively reviewed. Results The chondrocytes derived from BMSCs’ differentiation would be a main source of seeding cells articular cartilage for tissue engineering. Three-dimensional scaffolds and cultivation surroundings played important roles in the field of articular cartilage tissue engineering. Conclusion The util ization of cytokine and transgenic technology as well as improvements of three-dimensional scaffolds and cultivation surroundings will promote the development of articular cartilage tissue engineering.
Objective To observe the main biological characteristics and chondrogenesis potency of bone marrow -derived stromal cells(MSCs) after cytokinesinduction or gene modification in vitro. Methods MSCs from an adult New Zealand white rabbit were isolated and cultivated, and then MSCs were divided into the common medium group(Group A, 15%FBS in DMEM), the induced group by cytokines (Group B), the transfected group(Group C)with adenovirus-hepatocyte growth factor transgene (adHGF). The medium of group B consisted of transforming growth factor-β1(TGF-β1,10 ng/ml), basic fibroblast growth factor(bFGF,25 ng/ml) addexamethasone (DEX,10-7mol/L) with 15%FBS in DMEM. Cartilage slices wereobtained from femoral condyles and patellar grove in the same rabbit. The minced cartilage was digested in Ⅱ collagenase (3 mg/ml) to obtain chondrocytes(Group D). The change of cell appearance, proliferation capacity, glycosaminoglycans(GAG), immunohistochemical staining for type Ⅰ, Ⅱ collagen were observed during the 5th passage MSCs and MSCs after induction or gene modification. Expression of mRNA for type Ⅰ and Ⅱ collagen was detected by RT-PCR. Results Primary MSCs proliferated as shortspindle shape, while the 5th MSCs showed longspindle shape. Positive stain of type Ⅰ collagen could be found in groups A, B and C, while positivestain of type Ⅱ collagen was shown in groups B and D. The content of GAG in group B was higher than that in group A, but there was no significant difference between them（Pgt;0.05）, and there was significant difference between groups A and D（Plt;0.05）. No significant difference was noted in groups A,B and C on proliferation by MTT（Pgt;0.05）,except that of at the fourth day after transfection between groups A and C（Plt;0.05）. RT-PCR demonstrated that MSCs always had higher levelsof mRNA type Ⅰ collagen in groups A, B and C. The expression of mRNA type Ⅱ collagen was identified in groups B and D, and only low levels of mRNA type Ⅱ collagen in group C. Conclusion The above results indicate MSCs have a natural tendency of osteogenic differentiation in vitro culture, and also demonstrate the chondrogenic potency with the technique of cytokines induction or gene modification after passage. MSCs can be transfected efficiently being seed cells in tissue engineered bone or cartilage to accept target genes such as adHGF, and have a higher levels of expression in vitro, which lasted 4 weeks at least.
Objective To explore the relationship of the limited resource of the autologous bone marrow mesenchymal stem cells (MSCs) in articularcavity to the treatment results of full-thickness articular cartilage defect, and to investigate whether the extrogenous sodium hyaluronate(SH) promotes the migration of MSCs cultured in vitro tothe articular defect in vivo. Methods Sixty-six Japan rabbits were made the model of the full-thickness articular cartilage defect (5 mm width and 4 mm depth).The autologous MSCs were extracted from the rabbit femur, cultured in vitro, labeledby Brdu, and injected into the injured articular cavity with or without SH. Theexperiment was divided into 4 groups; group A (MSCs and SH, n=15); group B (MSCs, n=15); group C (SH, n=18); and group D (non-treatment, n=18). The morphologic observation was made by HE staining, Mallory staining and immunohistochemical staining after 5 weeks, 8 weeks and 12 weeks of operation. Results There were significant differences in the thickness of repairing tissue between group A and group B(Plt;0.01); but there were no significant differences between group A and group C, and between group B and group D(P＞0.05). Thehistological observation showed that the main repairing tissue was fibrocartilage in group A and fiber tissue in group B. Conclusion MSCs cultured in vitro and injected into the articular cavity can not improve the treatment results of the articular cartilage defect. Extrogenous SH has effect on repairing cartilage defect. The extrogenous SH has no effect on the chemotaxis of the MSCs, and on the collection of MSCs into the joint defect.
OBJECTIVE: To study chondrogenesis of calcium alginate-chondrocytes predetermined shapes. METHODS: Chondrocytes isolated from ears of rabbit by type II collagenase digestion, and then were mixed with 1.5% solidium alginate solution. The suspension was gelled to create three spatial shapes as triangle, circle and quadrilateral by immersed into 2.5% CaCl2 for 90 minutes, and then was implanted into the subcutaneous pocket on the dorsum of the rabbit. Samples were harvested at 6 and 12 weeks after implantation. RESULTS: Gross examination of excised specimens at 6 and 12 weeks after implantation revealed the presence of new cartilage of approximately the same dimensions as the original construct. Histologic evaluation using hematoxylin and eosin stains confirmed the presence of cartilage nodules at 6 weeks after implantation. After 12 weeks, mature cartilage was observed and histologic analysis confirmed the presence of well formed cartilaginous matrix. CONCLUSION: Predetermined shapes neocartilage can be regenerated using calcium alginate as a carrier of chondrocytes in the bodies of immune animals.
Objective To investigate the feasibility of the complex of the fibrin sealant (FS) and the bone marrow mesenchymal stem cells（MSCs） to createanew cartilage in the nude mice by the issue engineering technique. Methods T he MSCs were isolated from healthy humans and were expanded in vitro. And then the MSCs were induced by the defined medium containing the transforming growth factor β1 (TGF-β1), dexamethasone, and ascorbic acid. The biomechanical properties of the chondrocytes were investigated at 7 and 14 days. The MSCs induced for 7days were collected and mixed with FS. Then, the FSMSCs mixture was injectedby a needle into the dorsum of the nude mice in the experimental group. In the tw o control groups, only FS or MSCs were injected respectively. The specimens were harvested at 6 and 12 weeks，and the ability of chondrogenesis in vivo was inve stigated by the gross observation, HE, Alcian Blue staining, and type Ⅱ collagen immunohistochemistry. Results The MSCs changed from a spindlel ike fibroblastic appearance to a polygonal shape when transferred to the defined medium, and couldbe induced to express the chondrocyte matrix. After an injection of the mixture , the cartilage-like tissue mass was formed, and the specimens were harvested from the mass at 6 and 12 weeks in the experimental group. The tissue mass at 6 we eks was smaller and relatively firm in texture, which had a distinct lacuna structure. And glycosaminoglycan (GAG) and Type II Collagen expressions were detecte d. The tissue mass at 12 weeks was bigger, firmer and glossier with the mature c hondrocytes lying in the lacuna structure. The positive Alcian blue and Collagen II immunohistochemistry stainings were ber at 12 weeks than at 6 weeks. But there was no cartilage-like tissue mass formed in the two control groups. Conclusion This study demonstrates that the fibrin sealant and the bone marrow mesenchymal stem cells can be successfully used in a constructing technique for the tissue engineered injectable cartilage.