Objective To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle. Methods Acellular cartilage matrix was made from human auricle cartilage. The MSCs were isolated from the nucleated cells fraction of mice marrow by centrifuge.The MSCs were embedded in acellular cartilage matrix. After 10 day’s combined culture, the specimens were observed with optical and electrical microscope.Results The MSCs could well proliferate in the acellular cartilage matrix. The cells were not well-distributed in acellular cartilage matrix. There were more cells in the peripheral part of the matrix than in the central part of the matrix. Most of the cells were in cartilaginous lacunae. There were 1 or 2 cells in every cartilaginous lacunae.Conclusion The MSCs can be used as seed cells of tissue engineering and can well proliferate in the acellular cartilage matrix and become tissue engineering cartilage.
ObjectiveTo investigate the relationships between the expression of integrin β1 and activated cells in a partial-thickness articular cartilage injury model of adult rats. MethodForty-five male Sprague Dawley rats (aged 10 weeks and weighing 300-400 g) were randomly divided into operated group (n=15) , sham-operated group (n=15) , and control group (n=15) . Partial-thickness articular cartilage injury model was made by scarification in operated group, direct suture after opening of the knee joint was performed in sham-operated group, and no operation was done in control group. Five rats were sacrificed at 1, 7, and 14 days after operation respectively for macroscopic evaluation, HE staining, Safranin O staining, CD105, BrdU, CD105/integrin β1 immunofluorescence and double labeling staining. The histological score of HE staining, gray value of Safranin O staining and CD105-positive cells count were compared among groups at each time point. ResultsMacroscopic evaluation showed chondromalacia and cartilage fibrosis around the linear injury with aggravating tendency with time in operated group, but no chondromalacia and cartilage fibrosis in sham-operated and control groups. HE staining demonstrated a number of activated cells accumulating around the linear injury with nonuniform distribution in operated group, and uniform size and distribution in sham-operated and control groups. The histological scores at each time point in operated group were significantly higher than those in sham-operated group and control group (P<0.05) , but no significant difference was found between different time points in 3 groups (P>0.05) . Safranin O staining was nonuniform with hypochromasia around linear injury in operated group, but the staining was uniform in sham-operated group and control group. Gray value of Safranin O staining had no significant difference among groups and among different time points in the same group (P>0.05) . BrdU-positive and CD105-positive cells distributed unevenly around the linear injury in operated group, uniform distribution was observed in sham-operated group and control group. CD105-positive cells count in operated group was significantly higher than those in sham-operated group and control group at each time point (P<0.05) ; CD105-positive cells increased significantly with time in operated group (P<0.05) . CD105/integrinβ1-positive cells were observed around the linear injury in operated group, but was not observed in sham-operated group and control group. ConclusionsThe partial-thickness articular cartilage injury model is successfully established in rats, and cartilage injury could not be repaired completely in the model. The activated cells aggregation around the linear injury can be observed, but there is no obvious relationships between activated cells and cartilage matrix. These activated cells are in proliferation and could express both CD105 and integrin β1.
Objective To evaluate the operative methods and therapeutic effects of nasal septum cartilage-sil ica gel complex for two-stage repair of nasal deformities of unilateral cleft l ip. Methods From June 2001 to June 2007, 38 cases of secondary nasal deformity and septum deviation of cleft l ip were treated with transplanting nasal septum cartilage-sil ica gelcomplex. Among of them, there were 21 males and 17 females, aging 14-23 years with an average of 17.6 years. All cases were with nasal deformities of unilateral cleft l ip, including 21 cases of complete cleft l ip and 17 cases of incomplete cleft l ip. The locations were left side in 26 cases and right side in 12 cases. Nasal deformities were columella nasi deflexion, flattened nasal tip, pteleorrhine and alanasi collapse. The patients received 1-4 times operations, and the interval of two operations was 3-10 years (mean 5.5 years). According to nasal deformity, the nasal septum cartilage of 1.8 cm × 1.2 cm was cut, and transplanted into the nose point phantom surface forming “the shield” to extend nose column and to raise the tip of the nose. At the same time the nasal tip fat-connective tissue flap graft with fat knot was given. After fixation, the nasal alar cartilage and soft tissues were reduced to normal position. Results Primary heal ing of the incisions was achieved in all cases. The nasal deformity was corrected. The postoperative follow-up period was 12-18 months with an average of 15.6 months. All the patients of regional cartilage scars had no compl ication. The figure of nose was sl inky, the height of apex of nose and the shape of nose was natural,the apex of nose, nasal ala, nostrils and nasal columella were satisfactory [(the results were satisfactory in 30 cases (78.9%), general in 8 cases (21.1%)]. The nose department overall esthetics shape was improved in all the patients, no compl ications of the phantom sl iding, shifting and exposure, hemorrhage and infection occurred. Conclusion The nasal septum cartilagesil ica gel complex to repair the nasal deformities of unilateral cleft l ip is an ideal operation style.
Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes (P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers (P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B (P<0.05), and the level of TIMP-1 in group C was significantly higher (P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B (P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B (P<0.05).Conclusion CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
【Abstract】 Objective To review the recent progress of BMSCs acting as seeding cell for tissue engineeredcartilage. Methods The recent ten years l iterature about BMSCs acting as seeding cell for tissue engineered cartilage was extensively reviewed. Results Scaffold provided an optimal environment for the growth of BMSCs. Cytokine and gene del ivery could promote BMSCs to differentiate toward chondrocytes. All of them played important roles in the field of cartilage tissue engineering. Conclusion The improvement of three-dimensional scaffolds, the rational use of cytokine, and the enhancement of gene del ivery will promote the development of cl inical cartilage reconstruction.
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.
【Abstract】 Objective To investigate the protective effect of early motion on articular cartilage after joint allograft by performing a controlled trial between different post-operation strategies after joint allograft in an animal model. Methods Twenty hemi-knee joints were harvested from 10 6-month-old New Zealand white rabbits (male or female, weighing 2.5-3.0 kg); 10 hemi-knee joints by deep frozen treatment (donors) were transplanted to unilateral knee joints (recipients) of 10 6-month-old Chinchilla rabbits (male or female, weighing 2.5-3.0 kg), which were divided into early motion group (n=5) and sustained fixation group (n=5); and 10 hemi-knee joints were used as blank control (n=5) and frozen control (n=5). The articular cartilage of allogenic joints was detected by X-ray film, gross, and histology at 6 weeks after operation. Results Gross observation: no obvious limitation of joint movements was observed in early motion group, but obvious limitation in sustained fixation group. X-ray films: the bone ends between donor and recipient healed well with good paraposition and alignment on the operation day and 2 weeks after operation; at 6 weeks, angulation deformity was observed in early motion group of 3 rabbits, and paraposition and alignment were satisfactory in sustained fixation group. Histological observation: HE staining showed that the chondrocytes had normal quantity and morphology with few nuclear fragmentation and karyolysis in early motion group, but the quantity of chondrocytes sharply decreased with dissolved nuclei and numerous fibrous tissues in the cartilage matrix in sustained fixation group. The cell survival rate of the early motion group (49.66% ± 2.15%) was significantly higher than that of the sustained fixation group (20.68% ± 1.24%) (P lt; 0.05). Scanning electron microscopy observation: nuclear membrane was intact with chromatin condensation and edema of mitochondria and rough surfaced endoplasmic reticulum in early motion group, and that the membrane of chondrocyte vanished with blurring border between chondrocyte and matrix, rupture of nuclear membrane and the disappearance of chromatin and organelles could be found in sustained fixation group. Conclusion Early motion has protective effect on articular cartilage after joint allograft, but cannot completely prevent degeneration of the allogenic articular cartilage.
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.
Objective To review the recent research progress on relationshi p between subchondral bone and cartilage degeneration in osteoarthritis (OA), and to predict future research directions. Methods Recent l iteratures about the pathological changes of subchondral bone in OA were reviewed and analyzed in terms of biomechanics, bone remodel ingand biological factors. Results Subchondral bone sclerosis or softening was the result of osteoarthritis and also closely related to the occurrence and development of OA. Inhibiting the bone metabol ism of subchondral bone could slow the degeneration of articular cartilage. Conclusion For the treatment of OA, it is necessary to pay close attention to cartilage changes and the prevention of subchondral bone degeneration.
ObjectiveTo explore the relationship between subchondral bone reconstruction and articular cartilage regeneration in a rabbit model of spontaneous osteochondral repair. MethodsTwenty-four 6-month-old New Zealand white rabbits were included. The osteochondral defects (4 mm in diameter and 3 mm in depth) were created in the trochlear groove of the unilateral femur, which penetrated the subchondral bone without any treatment. The rabbits were sacrificed at 1, 4, 12, and 24 weeks after operation, respectively. The specimens were obtained for macroscopic, histological, and immunohistochemical observations. According to the International Cartilage Repair Society (ICRS) histological scoring, the effect of cartilage repair was assessed. The histomorphometrical parameters of subchondral bone were analyzed by micro-CT scan and reconstruction, and the relationship between cartilage repair and the histomorphometrical parameters of the subchondral bone were also analyzed. ResultsOsteochondral defects could be repaired spontaneously in rabbit model. With time, defect was gradually filled with repaired tissue, subchondral bone plate under the defect region gradually migrated upward. Bone mineral density, bone volume fraction, tissue mineralized density, trabecula number, and trabecula thickness were increased, while trabecula spacing was decreased. Significant difference was found in the other parameters between different time points (P<0.05) except for trabecula thickness between at 4 and 12 weeks after operation (P>0.05). Histological examination showed that fibrous repair was predominant with rare hyaline cartilage. With time, ICRS scores increased gradually, showing significant differences between other time points (P<0.05) except for between at 4 and 12 weeks after operation (P>0.05). Among the histomorphometrical parameters of subchondral bone, the trabecula spacing was negatively correlated with ICRS score (r=-0.584, P=0.039), and the other histomorphometrical parameters were positively correlated with ICRS score (r=0.680-0.891). ConclusionThere is relevant correlation as well as independent process between cartilage regeneration and subchondral bone reconstruction in the rabbit model of spontaneous osteochondral repair, and fast subchondral bone remodeling may adversely affect articular cartilage repair.