Objective To construct a new type of self-assembling peptide nanofiber scaffolds—RGDmx, and to study the cell compatibility of the new scaffolds and the proliferation and chondrogenic differentiation of precartilaginous stem cells(PSCs) in scaffolds. Methods PSCs were separated and purified from newborn Sprague Dawley rats by magnetic activated cell sorting and indentified by immunohistochemistry and immunofluorescent staining. The RGDmx were constructed by mixing KLD-12 and KLD-12-PRG at volume ratio of 1 ∶ 1. PSCs at passage 3 were seeded into the KLD-12 scaffold (control group) and RGDmx scaffold (experimental group). The proliferation of PSCs in 2 groups were observed with the method of cell counting kit (CCK) -8 after 1, 3, 7, and 14 days after culture. The RGDmx were constructed by mixing KLD-12-PRG and KLD-12 at different volume ratios of 0, 20%, 40%, 60%, 80%, and 100% and the prol iferation of PSCs was also observed. The complete chondrogenic medium (CCM) was used to induce chondrogenic differentiation of PSCs in different scaffolds. The differentiation of PSCs was observed by toluidine blue staining and RT-PCR assay. Results PSCs were separated and purified successfully, which were identified by immunohistochemistry and immunofluorescent staining methods. The results of CCK-8 showed that the absorbance (A) value in the experimental group increased gradually and reached the highest at 7 days; the A value in the experimental group was significantly higher than that in the control group at 7 days and 14 days (P lt; 0.05). Meanwhile, the A value in the RGDmx scaffold with a volume ratio of 40% was significantly higher than those in others (P lt; 0.05). After 14 days of induction culture with CCM, the toluidine blue staining results were positive in 2 groups; the results of RT-PCR showedthat the expression levels of collagen type II and the aggrecan in the experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion The self-assembling peptide nanofiber scaffold—RGDmx is an ideal scaffold for tissue engineer because it has good cell compatibility and more effective properties of promoting the differentiation of PSCs to chondrocytes.
It is very difficult to repair large articular cartilage defect of the hip. From May 1990 to April 1994, 47 hips in 42 patients of large articuler cartilage defects were repaired by allograft of skull periosteum. Among them, 14 cases, whose femoral heads were grade. IV necrosis, were given deep iliac circumflex artery pedicled iliac bone graft simultaneously. The skull periosteum had been treated by low tempreturel (-40 degrees C) before and kept in Nitrogen (-196 degrees C) till use. During the operation, the skull periosteum was sutured tightly to the femoral head and sticked to the accetabulum by medical ZT glue. Thirty eight hips in 34 patients were followed up for 2-6 years with an average of 3.4 years. According to the hip postoperative criteria of Wu Zhi-kang, 25 cases were excellent, 5 cases very good, 3 cases good and 1 case fair. The mean score increased from 6.4 before operation to 15.8 after operation. The results showed, in compare with autograft of periosteum for biological resurface of large articular defect, this method is free of donor-site morbidity. Skull periosteum allograft was effective for the treatment of large articular cartilage defects in hip.
In order to observe the histological changes of the autogenous perichondrium graft from rib in the repair of injured articular cartilage of the condylar process of mandible, 50 rabbits were used, in which 15 were served as control. The articular cartilage with its subchondral bone were resected and an autogenous graft of costal perichondrium was sutured onto the raw surface of the condylar process, and in the controls, only the articular portion of the condylar process was resected without the application of autogenous costal perichondrium graft. The morphological changes of the newly formed cartilage during the process of its development were investigated by hiostological and autoradiog aphic techniques. The result revealed that 10 days after operation, the graft had increased in thickness and was richly populated form the proliferation of mesenchyme-like cells. Twenty to thirty days later, the chondrocytes were matured and the newly formed cartilage had covered the bony surface of mandibular condyle. At 60 days, the newly formed cartilagenous joint surface became glossy, and the morphology and arrangement of cells tended to be regular simulating the morphology of normal articular cartilage. From the experiment, it could be concluded that (1) The autogenous perichondrium graft placed on the condylar surface of mandible could form new articular cartilage which was similar in tissue morphology to the normal condylar cartilage. (2) The process of development of newly formed cartilage was similar to that of the normal cartilage. (3) The motion and loading on the joint could promote the formation of new cartilage and undergo biological reformation, gradually resulting in normal joint morphology. On this basis, the clinical application of autogenous perichondrium graft to repair injured cartilage of the condylar process of the mandible was feasible.
In order to observe the effects of different facing directions of the germinal layer of periosteum on the cartilage regeneration, the human fibrin adhesive agent was used to adhere autogenous periosteum to repair the articular cartilage defect of rabbits. Twentyfour rabbits with 48 knee joints were divided randomly into two groups. A 0.6cm×1.2cm articular cartilage defect was created on the femoral trochlea until there was bleeding from the subchondral bone. A piece of periosteum, sized 0.75cm×1.5cm, was removed from the medial aspect of upper tibia. The periosteum was adhered to the defect by human fibrin adhesive agent. In Group 1 the germinal layer faced the subchondral bone and in Group 2 the germinal layer faced the joint cavity. The cartilage regeneration in both groups was observed by naked eyes and light microscope in 2nd and 6th weeks and by electron microscope after Safronin Ostained in 12th and 20th weeks. The results showed that before the 6th week, the cartilage regeneration was faster in Group 2 than that in Group 1. After that there was no significant difference in regeneration between the two groups. This suggested that the facing direction of the germinal layer was not a critical factor on cartilage regeneration. It was also found that the strength of the adhesive agent was not enough. The regenerated cartilage was proved to be hyaline cartilage.
ObjectiveTo review the research progress of different cell seeding densities and cell ratios in cartilage tissue engineering. MethodsThe literature about tissue engineered cartilage constructed with three-dimensional scaffold was extensively reviewed, and the seeding densities and ratios of most commonly used seed cells were summarized. ResultsArticular chondrocytes (ACHs) and bone marrow mesenchymal stem cells (BMSCs) are the most commonly used seed cells, and they can induce hyaline cartilage formation in vitro and in vivo. Cell seeding density and cell ratio both play important roles in cartilage formation. Tissue engineered cartilage with good quality can be produced when the cell seeding density of ACHs or BMSCs reaches or exceeds that in normal articular cartilage. Under the same culture conditions, the ability of pure BMSCs to build hyaline cartilage is weeker than that of pure ACHs or co-culture of both. ConclusionDue to the effect of scaffold materials, growth factors, and cell passages, optimal cell seeding density and cell ratio need further study.
ObjectiveTo investigate the effects of micro-fracture and insul in-l ike growth factor 1 (IGF-1) in treatment of articular cartilage defect in rabbits. MethodsTwenty-four New Zealand white rabbits (aged, 4-6 months; weighing, 2.5-3.5 kg) were randomly divided into 4 groups (n=6):micro-fractures and recombinant human IGF-1 (rhIGF-1) treatment group (group A), micro-fracture control group (group B), rhIGF-1 treatment control group (group C), and blank control group (group D). Full thickness articular cartilage defects of 8 mm×6 mm in size were created in the bilateral femoral condyles of all rabbits. The micro-fracture surgery was performed in groups A and B. The 0.1 mL rhIGF-1 (0.01 μg/μL) was injected into the knee cavity in groups A and C at 3 times a week for 4 weeks after operation, while 0.1 mL sal ine was injected in groups B and D at the same time points. At 4, 12, and 24 weeks, the gross, histological, and immunohistochemical observations were performed, and histological score also was processed according to Wakitani's score criteria. The collagen contents in the repair tissues and normal patellofemoral cartilage were detected by the improved hydroxyproline (HPR) method at 24 weeks. Electron microscope was used to observe repair tissues of groups A and B at 24 weeks. Results All animals were survival at the end of experiment. At 24 weeks after operation, defect was repaired with time, and the repair tissue was similar to normal cartilage in group A; the repair tissue was even without boundary with normal cartilage in group B; and the repair tissue was uneven with clear boundary with normal cartilage in groups C and D. Histological staining showed that the repair tissues had no difference with normal cartilage in group A; many oval chondrocytes-l ike cells and l ight-colored matrix were seen in the repair tissues of group B; only a few small spindle-shaped fibroblasts were seen in groups C and D. Moreover, histological scores of group A were significantly better than those of groups B, C, and D (P<0.05) at 4, 12, and 24 weeks. Electron microscope observation showed that a large number of lacuna were seen on the surface of repair tissue in group A, and chondrocytes contained glycogen granules were located in lacunae, and were surrounded with the collagen fibers, which was better than that in group B. Collagen content of the repair tissue in group A was significantly higher than that in groups B, C, and D (P<0.05), but it was significantly lower than that of normal cartilage (P<0.05). Conclusion Combination of micro-fracture and rhIGF-1 for the treatment of full thickness articular cartilage defects could promote the repair of defects by hyaline cartilage.
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 examine the biological characteristic changes in thededifferenciated human articular chondrocytes by the bioreactor culturing in vitvo.Methods The cartilage tissue was obtained from the joints of the adult human. The chondrocytes were isolated from the cartilage tissue with the type Ⅱ collagenase digestion（0.2%, 37℃, 3 h）and were cultured in DMEMF12 supplemented with 20% fetal bovine serum (FBS) with 1 ng/ml of TGF-β1and 5 ng/mlof FGF-2. After about 20 passages by the monolayer culture，the cells were then transferred to the bioreactor culturing of the rotational cell culture system (RCCS) for a 3-week sequence culture. The cell counting was performed with the platelet counter, and the doubling time for each passage of thecells was determined. The frozen section was stained with HE. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry. Results When the monolayer culture was performed without any growth factors, the chondrocytes were rapidly proliferated within 3 passages (average doubling time, 59 h),but at the same time, dedifferentiation was also progressing rapidly. After the4th passage, most of the cells were dedifferenciated and the proliferation was decreased. With the growth factors (TGF-β1/FGF-2), the speed of the expansion was accelerated (average doubling time, 47 h), but the speed of the dedifferentiation was slowed down. After 20 passages were performed with the monolayer culture, the dedifferentiated chondrocytes could be redifferentiated when they were cultured for 3 weeks with RCCS. Then, the Safranine-O staining was bly positive for the cells, positive for aggrecan and collagen Ⅱ, but negative for collagen Ⅰ, with a wellregained phenotype. Conclusion The bioreactor culturing of the dedifferenciated human articular condrocytes can regain the differentiated phenotype and it is a useful method of obtaining the human articular chondrocytes in large amounts and in a differentiated phenotype in vitro.
ObjectiveTo evaluate the effect of bone cement filling on articular cartilage injury after curettage of giant cell tumor around the knee. MethodsFifty-three patients with giant cell tumor who accorded with the inclusion criteria were treated between January 2000 and December 2011, and the cl inical data were retrospectively analyzed. There were 30 males and 23 females, aged 16-69 years (mean, 34.2 years). The lesion located at the distal femur in 28 cases and at the proximal tibia in 25 cases. According to Campanacci grade, there were 6 patients at grade I, 38 at grade Ⅱ, and 9 at grade Ⅲ. Of 53 patients, 42 underwent curettage followed by bone cement fill ing, and 11 received curettage followed by bone grafts in the subchondral bony area and bone cement fill ing. Two groups were divided according to whether secondary osteoarthritis occurred or not during postoperative follow-up. The gender, age, lesion site, the subchondral residual bone thickness, tumor cross section, preoperative Campanacci grade, subchondral bone graft, and Enneking function score were compared between 2 groups, and multivariate logistic regression analysis was done. ResultsAll incisions healed by first intention. The average follow-up time was 65 months (range, 23-158 months). Of 53 cases, 37 (69.8%) had no osteoarthritis, and 16 (30.2%) had secondary osteoarthritis. Three cases (5.7%) recurred during the follow-up period. Univariate logistic regression analysis showed no significant difference in gender, age, lesion site, and Campanacci grade between 2 groups (P>0.1); difference was significant in the subchondral residual bone thickness, tumor cross section, Enneking function score, and subchondral bone graft (P<0.1). The multivariate logistic regression analysis showed that the decreased subchondral residual bone thickness, the increased tumor cross section, and no subchondral bone graft are the risk factors of postoperative secondary osteoarthritis (P<0.05). ConclusionCurettage of giant cell tumor around the knee followed by bone cement filling can increase the damage of cartilage, and subchondral bone graft can delay or reduce cartilage injury.
Objective To compare biological characteristics between articular chondrocyte and meniscal fibrochondrocyte cultured in vitro andto investigate the possibility of using cultured cartilage as a substitute for meniscus.Methods Chondrocytes isolated from articular cartilage and meniscus of rabbits aged 3 weeks were respectively passaged in monolayer and cultured in centrifuge tube. Cartilages cultured in centrifuge tube and meniscus of rabbit aged 6 weeks were detected by histological examination and transmission electron microscopy. Growth curves of articular chondrocytes and meniscalfibrochondrocytes were compared; meanwhile, cell cycles of articular chondrocytes and meniscal fibrochondrocytes in passage 2and 4 were separately measured by flow cytometry.Results Articular chondrocytes in passage 4 were dedifferentiated. Articular chondrocytes formed cartilage 2 weeks after cultivation in centrifuge tube, but meniscal fibrochondrocytes could not generate cartilage. The differences in ultrastructure and histology obviously existed between cultured cartilage and meniscus; moreover, apoptosis of chondrocytes appeared in cultured cartilage. Proportion of subdiploid cells in articular chondrocytes passage 2 and 4 was markedly higher than that in passage 2 and 4 fibrochondrocytes(Plt;0.05). Conclusion Meniscal fibrochondrocytes can not form cartilage after cultivationin centrifuge tube, while cartilage cultured in centrifuge tube from articular chondrocytes can not be used as graft material for meniscus. Articular cartilage ismarkedly different from meniscus.