Experimental Study on Effect of Drynaria Combining with Tissue Engineered Cartilage to Promote Cartilage Regeneration / 现代生物医学进展

Objective:To evaluate the effect of rhizome drynaria combined with tissue engineering cartilage on cartilage regeneration in experimental rabbits with cartilage defects.Methods:The hIGF-1 gene was transfected into MSCs by using the method of isola tion,purification and recombination of transgenic stem cells.The MSCs were transplanted into rabbit bone marrow mesenchymal stem cells (MSCs) in vitro.The cells were further amplified and mixed with acellular dermal matrix (ADM) to construct tissue engineered cartilage.Twenty-four New Zealand white rabbits,aged 6 months,were randomly divided into 4 groups (A,B,C and D).six rabbits in each group.Group A and C were transplanted with autologous cartilage.Group B and D were transplanted with modified cells.Group C and D group were fed with 40％ Drynaria Decoction,150ml/d for 4 weeks.Animals were sacrificed at 12 weeks postoperatively,and articular cartilage defects were isolated.Cartilage defect samples were embedded in paraffin blocks and stained with hematoxylin and eosin (H&E).Cartilage regeneration was evaluated by gross morphology,including sclerotic shape,color,contour and homogeneity.The quality of regenerated cartilage was assessed by histological scoring.Toluidine blue staining was used to evaluate the occurrence of chondrogenic glycosaminoglycans (GAG).Results:Compared with group B,the cartilage coverage,the color of new bone marrow,the edge of defect and the surface roughness of group C and D were significantly improved (P＜0.05);the cartilage surface score of regenerated cartilage was significantly improved P＜0.05).Groups C and D had better matrix,cell distribution and surface index than the other groups.And had a thick like hyaline cartilage tissue,with the normal glycosaminoglycan production.It is indicated that drynaria combined with tissue engineering cartilage can reduce cartilage defects by regenerating hyaline cartilage.Conclusion:Cartilage combined with drynariae can significantly improve the quality of cartilage defect repair in rabbit knee joint,and provide an important theoretical basis for clinical treatment of cartilage lesions.

Influence of Recombinant Human Bone Morphogenetic Protein-2 on the Remodeling of Subchondral Bone and Cartilage Healing in the Articular Cartilage Defect of the Rabbit / 대한정형외과학회잡지

PURPOSE: The purpose of this study is to evaluate the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) after microfracture on the remodeling of subchondral bone and cartilage healing in a model of full-thickness articular cartilage injury in a rabbit. MATERIALS AND METHODS: A full thickness articular cartilage defect of 6x3-mm-size was created in the trochlear groove of the right femur in 24 rabbits. The defect was left empty in six rabbits, and microfracture was done in 18 rabbits. After microfracture, no treatment was done in six rabbits, defect was filled with fibrin glue in six rabbits, and with fibrin glue and rhBMP-2 in six rabbits. The effect of rhBMP-2 after microfracture was evaluated based on histological analysis and real-time polymerase chain reaction (PCR) for analysis of collagen type at 8 weeks after surgery. RESULTS: The score of histological grade scale of six rabbits in which the defect was filled with fibrin glue and rhBMP-2 was better than that of others and real-time PCR also showed a higher amount of collage type 1 and collage type 2 in these six rabbits. CONCLUSION: We consider that fibrin glue and rhBMP-2 after microfracture may accelerate cartilage healing in an articular cartilage defect and maybe helpful in healing the articular cartilage defect into more closely native hyaline cartilage.

Experiment study of rabbit joint articular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells / 中华显微外科杂志

Objective To investigate the experiment effects of rabbit joint articular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells. Methods Bone marrow mesenchymal stem cells were isolated and cultured in vitro. The positive rate of transfection was defected by cell immunohistochemistry methods after Ad-hTGF-β1 transfected for 1 week. Twenty-four adult New Zealand white rabbits with full articular cartilage defects were randomly divided into 4 groups, each group had 6 animals, both hind limbs were used in the experiment. Group A: hydrogel combined with transfected cells; Group B: hydrogel combined with untransfected cells; Group C: hydrogel group; Group D: blank control group. Specimens and histological observation were used to evaluate the repair effect after 16 weeks according to Pineda's score. Results The positive rate of hTGF-β1 expression in BMSCs was about 85.4% after transfection. After 16 weeks the defects of group A were repaired by cartilage-like tissue, the cell arrangement and densities of regenerated cartilage were similar to normal cartilage, type Ⅱ collagen immunohistochemistry were positive. There was a significant difference in Pineda's score compaired with other groups (P ＜ 0.05). Conclusion Rabbit articiular cartilage defects could be repaired by CS/PVA hydrogel engineered hTGF-β1-transfected bone marrow mesenchymal stem cells.

Autologous Chondrocyte Implantation for Articular Cartilage Defect of Femoral Condyle / 대한정형외과학회잡지

PURPOSE: To evaluate the midterm clinical and histological results after autologous chondrocyte implantation (ACI) for an articular cartilage defect of the distal femoral condyle. MATERIALS AND METHODS: Twenty four cases with an articular cartilage defect (Outerbridge grade IV) of the femoral condyle that was confirmed by MRI and the arthroscopic findings underwent ACI. Their mean age at the time of surgery was 42.8 years and the mean follow-up period was 53.2 months (range, 20-82 months). At the last follow up, the articular cartilage view (SPGR) of MRI was examined and the clinical results were evaluated using the HSS and Lysholm scores. In 8 cases, second-look arthroscopy and biopsy were performed and evaluated using histological and histochemical methods. RESULTS: All cases except for one showed well-regenerated articular cartilage on MRI. All cases showed significant clinical improvement in the HSS and Lysholm scores (p<0.0001), with the exception of the Lysholm score of an articular cartilage fracture. Histologically, the regenerated tissue appeared to be a hyaline-like cartilage in all specimens. CONCLUSION: ACI for the treatment of articular cartilage defects of the distal femoral condyle showed a good clinical and MRI results. In OA, the clinical results were relatively acceptable after an associated high tibial valgus osteotomy. However, a longer term follow-up study will be needed to reach a final conclusion.

Cartilage Repair Using Mesenchymal Stem Cells

Articular cartilage defect rarely heals spontaneously due to its avascularity and low cellularity. Even small articular cartilage defects can develop into osteoarthritis, and subsequently, its management has been a major clinical concern. Although there are several treatment options for cartilage defect, no treatment has been established as a gold standard procedure. Bone marrow stimulation techniques which is equivalent to microfracture these days has been adapted as first line treatment, attributed to their technical easiness and minimal invasiveness to patients. However, this procedure has limitation in reproducing hyaline cartilage, so recent cell-based therapies using autologous chondrocytes or mesenchymal stem cells have drawn particular attention. MSCs regardless of its origin have shown significant potential for chondrogenesis. Novel approaches using MSCs as an alternative cell source for patient derived chondrocytes are currently on trial. In this review, stem cells from various origins considered as cell sources and potential application of mesenchymal stem cells to promote cartilage repair will be discussed. While differentiation of stem cell can be well controlled in vitro, it is not easy to predict the course of differentiation when the stem cell is transplanted. Some novel methods using physical stimulation and material based techniques for differentiation control are introduced in this context. Such differentiation control will be beneficial when it is adapted before transplantation. We call it preconditioning.

Repair of rabbit cartilage defects by composite of cocultures of autogenic bone marrow-derived mesenchymal stem cells and chondrocytes with allogenicfully deproteinized bone / 中国矫形外科杂志

[Objective]To investigate the rlpairing effect on articular cartilage defects by composite of cocultures of autogenic bone marrow-derived mesenchymal stem cells(BMSCs) and chondrocytes with allogenic fully deproteinized bone(FDB),in order to provide basis for optimizing seeding cells resources.[Method]Seeding cells were collected from two-passaged BMSCsand chondroeytes and then cocultured at the rate of 2 to 1.Full thickness articular cartilage defects in the knee joints of rabbits repaired by cocultured cells seeded into allogenic FDB were served as experimental group A,by simple FDB as control group B and by nothing as blank control group C.Repaired tissues were evaluated with macroscopic views,histological scores and immunohistochemistrical stains at 8 and 16 weeks postoperatively.[Result]Chondrocytes cocultured riched in extracellular matrix and proliferated promptly.In A regenerated tissues represented hyaline-like,smoothness and flat.In group B and C,repaired tissues were fiberous and no repaire in group C.Histological scores of experimental group A excelled group B and C with statistically significant differences(P0.05).Immunohistochemistrical stains showed that cells in the zones of repaired tissues were larger in size,arranged columnnedly,riched in type-Ⅱ collagen matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in the experimental group A at 16 weeks postoperatively.[Conclusion]Cocultures of autogenic BMSCS with chondrocytes can promote proliferation of chondrocytes and production of chondral matrix.Cocultures as seeding cells can save a number of chondrocytes,shorten culturing periods and reduce subcultured times.Cocultures embedded into FDB can repair articular cartilage defects effectively.

The Effect of Chitosan-TGF-beta1 Conjugate on Full Thickness Articular Cartilage Defect in the Rabbit Knee Model (Preliminary Report) / 대한정형외과연구학회지

PURPOSE: To evaluate the effect of the chitosan-TGF-beta1 conjugate on articular cartilage defects of rabbits' knees MATERIALS AND METHODS: Full thickness cartilage defect(6mm, round shape) was made at both knees of 20 rabbits and, after the lapse of 3 days, chitosan-TGF-beta1(15ng/ml, 1ml) was injected into one side(experimental group) of knees and PBS(1ml) was injected into the other side(control group). 5 rabbits were sacrificed 6 weeks after the injection and the rest of 15 rabbits 12 weeks later. Then, gross morphology and histological evaluation(Mendelson scoring) was conducted. RESULTS: No arthritic findings was observed and histological results in the experimental group at 6(Ave. 11.3+/-1.5) and 12(Ave. 4.5+/-1.9) weeks postoperatively were superior to those in the control group at 6(Ave. 14.6+/-1.7) and 12(Ave. 9.8+/-2.2) weeks. Especially at 12 weeks, the experimental group was superior to the control group statistically in results of 5 subgroups except for filling of defects. The difference of two groups at 12 weeks was more remarkable than those at 6 weeks. CONCLUSION: Regeneration of something very close to normal cartilage was observed in the experimental group. It shows that the biological activity of TGF-beta1 is sustained by the action of conjugate with chitosan, through prolonged half life of TGF-beta1.

Relationship between meniscal injury & articular cartilage defect in arthroscopy

PURPOSE: We studied to know the relationship between meniscal injury & defect of articular cartilage and its clinical importance. MATERIALS AND METHODS: 252 patients were selected, who undergone arthroscopic surgery for meniscal injury from May 1997 to December 2001. We excluded patients with severely progressed osteoarthritis or degenerative changes in simple X-ray. Physical examinations, simple X-rays, MRI & arthroscopic photographs were taken for all patients. And the results were analyzed according to age, duration from accident to operation and type of meniscal injury RESULTS: 40 cases (15.9%) showed defects of articular cartilage with meniscal injury. Among them, 18 cases (45%) in femur, 10 cases (25%) in tibia, and 12 cases (30%) in both. Incidence of articular cartilage defect was significantly higher in patients over the age of forty (p<0.01) and in those with a history longer than six months (p<0.05). Posterior horn tears were associated with the highest incidence of articular cartilage defect, but were also associated with a longer history and with older patients. CONCLUSION: In patients with meniscal injury, older age, long duration of symptom and tear at posterior horn have higher incidence of articular cartilage defect, especially femoral condyle. The reason for this is not entirely clear but these patients need close attention and early treatment.

Gene Transfer Into Human Chondrocyte Derived Cells Using A Liposome Mediated Transfection System / 대한정형외과학회잡지

PURPOSE: To introduce the CMV promoter driven luciferase and -galactosidase marker gene into previously permeabilized human chondrial cells. MATERIALS AND METHODS: The cultured chondrocyte cells were transfected with a liposome/DNA mixture (pCMV-Luc or pSV40-lacZ). Cultured cells not transfected by liposome/DNA were used as a control. After forty-eight hours of incubation, the cells were used for reporter gene assays and the polymerase chain reaction (PCR). RESULTS: Fifteen percent of the chondrocyte cells treated with liposome/ pSV40-lacZ DNA were positive for -gal staining. Chondrocyte cells transfected with pCMV-Luc yielded a 70-fold increase in luciferase activity over that of the control cells. A PCR product corresponding to the luciferase gene appeared only in the transfected chondrocyte cells. These results indicate that the human chondrocyte cells can be transfected with pCMV-Luc and pSV40-lacZ. CONCLUSION: This system is particularly suitable for gene therapy, as well as for the use of genetically modified cartilage cells for resurfacing full thickness articular cartilage defects.

Clinical Application of Free Periosteal Autografting in Repairing Articular Cartilage / 中华创伤杂志

We repaired articular cartilage defect in 13 cases using free periosteal autografting. and followed up for 15～29 months with average of 22.4 months. Good results were obtained in 76.9%. The operation method and factors of influencing free periosteal autografting are discussed in the paper.