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.
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 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 observe the long-term clinical results of repairing large articular cartilage defects of the hip and the knee with free autogeneous periosteum. Methods Based on the results of experimental studies, the authors used free autogeneous periosteum transplantation and postoperative continuous passive motion (CPM) to repair large articular cartilaginous defects in 52 patientsfrom February 1987 to August 1995. Of 37 patients with complete follow-up data, 16 had congenital dislocation of the hip, 6traumatic arthritis of hip, 1 femoral head destruction following mild infection, 2 ankylosing spondylitis, 6 intra-articular fracture of the knee, 4 arthritisof the knee and 2 stiff knee following joint infection. The patients with dislocation of hip were given relieving traction before operation. The cartilages of pathological changes were excised to bleeding bone. The defects were repairedwith periosteum removing from tibia. CPM were immediately applied for 4-6 weeksand no bearing was allowed 6 months after discharge. The silicon membrane was taken out in the 6th month. Results Thirty-seven patients (17 males, 20 females) were followed up 7-15 years with an average of 10.5 years. The functional evaluation referred to joint pain degree，joint mobile range，daily activity and X-ray findings. The results were excellence in 11 patients , good in 18 patients , poor in 8 patients. Conclusion The method to repair articular cartilage defect with free autogeneous -periosteum is effective and may be applied clinically.
Objective To establish an effective way to cryopreserveprecartilaginous stem cells(PSCs) of neonate rat. Methods PSCs ［fibroblast growth factor-3(FGFR-3) positive cells］ were isolated and purified by magnetic cell sorting method. PSCs were cultured and amplified to the third generation. PSCs were preserved in liquid nitrogen. The biological properties of cryopreserved PSCs were investigated by reverse transcriptase polymerase chain reaction(RT-PCR), immunohistochemistry, and immunofluorescence. Results Immunohistochemical and immunofluorescent analysis showed widespread expression of FGFR-3 in cryopreserved PSCs. FGFR-3 could be dectected by RT-PCR in cryopreserved PSCs.Cryopreserved PSCs kept high cell viability, and phenotypic and proliferation characteristics of PSCs in vivo.Conclusion Cryopreservation of PSCs can supply adequate qualified cells for repairing the defects of epiphyseal growth plate by tissue engineering technique.
Objective To review the recent progress of the researches in the field of cartilage tissue engineering, and to discuss the challenges in construction of tissue engineered cartilage. Methods Literature related with cartilage tissue engineering was reviewed and analyzed. Results Some techniques have been appl ied in cl inical. As far as the seeding cells, induced pluripotent stem cells have attracted much more attention. Current strategies of scaffold designing are trying to imitate both component and structure of natural extracellular matrix. Cartilage regeneration through the autologous cell homing technique el iminate the transplantation of exotic cells and has become the hot topic. Conclusion Successful treatment of the damaged cartilage using tissue engineering method will depend on the advances of stem cell technology development, biomimetic scaffolds fabrication and proper appl ication of growth factors.
ObjectiveTo study the effect of transforming growth factor β3 (TGF-β3), bone morphogenetic protein 2 (BMP-2), and dexamethasone (DEX) on the chondrogenic differentiation of rabbit synovial mesenchymal stem cells (SMSCs). MethodsSMSCs were isolated from the knee joints of 5 rabbits (weighing, 1.8-2.5 kg), and were identified by morphogenetic observation, flow cytometry detection for cell surface antigen, and adipogenic and osteogenic differentiations. The SMSCs were cultured in the PELLET system for chondrogenic differentiation. The cell pellets were divided into 8 groups: TGF-β3 was added in group A, BMP-2 in group B, DEX in group C, TGF-β3+BMP-2 in group C, TGF-β3+DEX in group E, BMP-2+DEX in group F, and TGF-β3+BMP-2+DEX in group G; group H served as control group. The diameter, weight, collagen type II (immuohistochemistry staining), proteoglycan (toluidine blue staining), and expression of cartilage related genes [real time quantitative PCR (RT-qPCR) technique] were compared to evaluate the effect of cytokines on the chondrogenic differentiation of SMSCs. Meanwhile, the DNA content of cell pellets was tested to assess the relationship between the increase weight of cell pellets and the cell proliferation. ResultsSMSCs were isolated from the knee joints of rabbits successfully and the findings indicated that the rabbit synovium-derived cells had characteristics of mesenchymal stem cells. The diameter, weight, collagen type II, proteoglycan, and expression of cartilage related genes of pellets in groups A-F were significantly lower than those of group G (P<0.05). RT-qPCR detection results showed that the relative expressions of cartilage related genes (SOX-9, Aggrecan, collagen type II, collagen type X, and BMP receptor II) in group G were significantly higher than those in the other groups (P<0.01). Meanwhile, with the increase of the volume of pellet, the DNA content reduced about 70% at 7 days, about 80% at 14 days, and about 88% at 21 days. ConclusionThe combination of TGF-β3, BMP-2, and DEX can make the capacity of chondrogenesis of SMSCs maximized. The increase of the pellet volume is caused by the extracellular matrix rather than by cell proliferation.
Objective To explore the methods of repairing cartilagedefects and to introduce the clinical experience with the autologous osteochondral transplantation. Methods Twenty-five patients with chondral and osteochondral defects of the weight-bearing surfaces were treated by the autologous osteochondral transplantation for the repair of the chondral and osteochondral defects of the unweightbearing surfaces under arthroscope. According to the shape of the defects, the different dimensions of the osteochondral autograft were selected. All the patients began the training of the continuous passive motion after operation. Six weeks after operation, the patients began to walk in the weightbearing habitus. However, in the control group, another 25 patients were retrospectively analyzed, who had chondral and osteochondral defects of the weight-bearing surfaces but were treated only by the cleaning and drilling procedures. The scores evaluated bythe Brittberg-Peterson scoring scale of the 2 group were 98.65±9.87 and 96.98±8.94 respectively. Results The follow-upfor 3-24 months after operation revealed that the treated knee joint had a goodmotion extent. The pain was obviously alleviated. Based on the longitudinal study with the three-dimensional spoiled magnetic resonance imaging (MRI), the signal intensity of the repaired tissues approached to the normal condition. The scores evaluated by the Brittberg-Peterson scoring scale were almost zero 3 monthsafter operation in the experimental group, and the scores were 58.48±6.98 inthe control group. There were significant differences between the experimental group and the control group(P＜0.01). Conclusion Autologous osteochondral transplanation under arthroscope is a good curative method for the cartilage defects, with advantages of minimal invasiveness and avoidanceofrejections resulting from allografts. However, its long-term effect needs to befurther studied. The conventional therapies including cleaning and drilling are useful in alleviating the symptoms.
Objective To investigate the curative effects of homograft of the mesenchymal stem cells(MSCs) compbined with the medical collagen membrane of the guided tissue regeneration(MCMG) on the full thickness defects of the articular cartilage. Methods MSCs derived from New Zealand rabbits aged 3-4 months weighing 2.1-3.4 kg were cultured in vitro with a density of 5.5×108/ml and seeded onto MCMG. The MSC/MCMG complex was cultured for 48 h and transplanted into the fullthickness defects on the inboardcondyle and trochlea. Twenty-seven healthy New Zealand rabbits were randomly divided into 3 groups of 9rabbits in each. The cartilage defects in the inboard condyle and trochlea werefilled with the auto bone marrow MSCs and MCMG complex (MSCs/ MCMG) in Group A (Management A), with only MCMG in Group B (Management B)and with nothing in Group C (Management C). Three rabbits were killed at 4, 8 and 12 weeks after operation in each group, and the reparative tissue samples evaluated grossly,histologically and immunohistochemically were graded according tothe gross and histological scale. Results Four weeks after transplantation, the cartilage and subchondralbone were regenerated in Group A;for 12 weeks, the regenerated cartilage gradually thicked; 12 week after transplantation, the defect was repaired and the structures of the carticular surface and subchondral bone was in integrity.The defects in Group A were repaired by the hylinelike tissue and the defects in Groups B and C were repaired by the fibrous tissues. Glycosaminoglycan and type Ⅱcollagen in Groups A,B and C were reduced gradually.The statistical analysis on the gross at 12 weeks and the histologicalgradings at 4 weeks,8 weeks and 12 weeks showed that the inboardcondylar repairhad no significant difference compared with the rochlearepair(Pgt;0.05).Management A was significantly better than Managements B and C (Plt;0.05), and Management B was better than Management C(Plt;0.05). Conclusion Transplantation of the MSCs combined with MCMG on the full thickness defects of the articular cartilage is a promising approach to the the treatment of cartilage defects. MCMG can satisfy the demands of the scaffold for the tissue-engineered cartilage.
Objective To evaluate the feasibility and the value of the layered cylindric collagenhydroxyapatite composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondrocytes and affe cts the cell behaviors. Methods The chondrocytes were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous collagen／h ydro xyapatite composite scaffold and were cultured in a three-dimensional environme n t for 3 weeks. The effects of the composite scaffold on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by the phase-contrast microscopy, histology, scanning electron micros copy, and immunohistochemistry. Results The pore diameter of the upper layer of the collagen-hydroxyapatite composite scaffold was about 147 μm. and the porosity was 89%; the pore diameter of the bottom layer was about 85 μm and the porosity was 85%. The layered cylindric collagenhydroxyapatite composite scaffold had good hydrophilia. The chondrocytes that adhered to the surface of the scaffold, proliferated and migrated into the scaffold after 24 hours. The chondrocytesattached to the wall of the microholes of the scaffold maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold. Conclusion The layered cylindric collagenhydroxyapatite composite scaffold has a good cellular compatibility, and it is ber in the mechanical property than the pure collagen. It will be an ideal scaffold for the cartilage tissue enginee ring.