ObjectiveTo observe the feasibility of acellular cartilage extracellular matrix (ACECM) oriented scaffold combined with chondrocytes to construct tissue engineered cartilage.MethodsChondrocytes from the healthy articular cartilage tissue of pig were isolated, cultured, and passaged. The 3rd passage chondrocytes were labeled by PKH26. After MTT demonstrated that PKH26 had no influence on the biological activity of chondrocytes, labeled and unlabeled chondrocytes were seeded on ACECM oriented scaffold and cultivated. The adhesion, growth, and distribution were evaluated by gross observation, inverted microscope, and fluorescence microscope. Scanning electron microscope was used to observe the cellular morphology after cultivation for 3 days. Type Ⅱ collagen immunofluorescent staining was used to check the secretion of extracellular matrix. In addition, the complex of labeled chondrocytes and ACECM oriented scaffold (cell-scaffold complex) was transplanted into the subcutaneous tissue of nude mouse. After transplantation, general physical conditions of nude mouse were observed, and the growth of cell-scaffold complex was observed by molecular fluorescent living imaging system. After 4 weeks, the neotissue was harvested to analyze the properties of articular cartilage tissue by gross morphology and histological staining (Safranin O staining, toluidine blue staining, and typeⅡcollagen immunohistochemical staining).ResultsAfter chondrocytes that were mainly polygon and cobblestone like shape were seeded and cultured on ACECM oriented scaffold for 7 days, the neotissue was translucency and tenacious and cells grew along the oriented scaffold well by inverted microscope and fluorescence microscope. In the subcutaneous microenvironment, the cell-scaffold complex was cartilage-like tissue and abundant cartilage extracellular matrix (typeⅡcollagen) was observed by histological staining and typeⅡcollagen immunohistochemical staining.ConclusionACECM oriented scaffold is benefit to the cell adhesion, proliferation, and oriented growth and successfully constructes the tissue engineered cartilage in nude mouse model, which demonstrates that the ACECM oriented scaffold is promise to be applied in cartilage tissue engineering.
ObjectiveTo explore the anthropometric changes of the auricle after auricular cartilage unfolding in moderate concha-type microtia patients, so as to provide the basis to help evaluate surgical timing and prognostic.MethodsA total of 33 children with moderate concha-type microtia, who were treated with auricular cartilage unfolding between October 2016 and September 2018 and met the inclusive criteria, were included in the study. There were 24 boys and 9 girls with an average age of 1.4 years (range, 1-3 years). Sixteen cases were left ears and 17 cases were right ears. The follow-up time was 12-23 months (mean, 17.5 months). The affected auricular detailed structures were observed and quantitatively analyzed before operation and at immediate after operation. The width, length, and perimeter of auricle before operation and at immediate after operation and at last follow-up were noted with three dimensional-scanning technology. The normal auricle was noted as control.ResultsThere were (7.5±1.0) and (11.3±0.8) structures of the affected auricle at pre- and post-operation, respectively, showing significant difference between pre- and post-operation (t=23.279, P=0.000). The length, width, and perimeter of the affected auricle constantly increased after operation, and there were significant differences between pre-operation and immediately after operation and between immediately after operation and last follow-up (P<0.05). The differences of length, width, and perimeter of the affected auricle between immediately after operation and last follow-up were (3.13±1.44), (2.44±0.92), and (8.50±3.76) mm, respectively. And the differences of length, width, and perimeter of the normal auricle between pre-operation and last follow-up were (3.16±1.54), (2.35±0.86), and (9.79±4.60) mm, respectively. There was no significant difference in the differences of length, width, and perimeter between the affected auricle and the normal auricle (P>0.05).ConclusionThe auricular cartilage unfolding in treatment of the moderate concha-type microtia can receive more ear structures and increase auricle sizes, which make it possible for free composite tissue transplantation. In addition, the affected and the contralateral normal auricles have a very similar growth rate and it offers the theoretical foundation for the early treatment for moderate concha-type microtia.
Superficial cartilage defect is an important factor that causes osteoarthritis. Therefore, it is very important to investigate the influence of superficial cartilage defects on its surface morphology and mechanical properties. In this study, the knee joint cartilage samples of adult pig were prepared, which were treated by enzymolysis with chymotrypsin and physical removal with electric friction pen, respectively. Normal cartilage and surface treated cartilage were divided into five groups: control group (normal cartilage group), chymotrypsin immersion group, chymotrypsin wiping group, removal 10% group with electric friction pen, and removal 20% group with electric friction pen. The surface morphology and structure of five groups of samples were characterized by laser spectrum confocal microscopy and environmental field scanning electron microscopy, and the mechanical properties of each group of samples were evaluated by tensile tests. The results show that the surface arithmetic mean height and fracture strength of the control group were the smallest, and the fracture strain was the largest. The surface arithmetic mean height and fracture strength of the removal 20% group with electric friction pen were the largest, and the fracture strain was the smallest. The surface arithmetic mean height, fracture strength and fracture strain values of the other three groups were all between the above two groups, but the surface arithmetic mean height and fracture strength of the removal 10% group with electric friction pen, the chymotrypsin wiping group and the chymotrypsin soaking group decreased successively, and the fracture strain increased successively. In addition, we carried out a study on the elastic modulus of different groups, and the results showed that the elastic modulus of the control group was the smallest, and the elastic modulus of the removal 20% group with electric friction pen was the largest. The above study revealed that the defect of the superficial area of cartilage changed its surface morphology and structure, and reduced its mechanical properties. The research results are of great significance for the prevention and repair of cartilage injury.
Objective To investigate the feasibility of repairing thyroid cartilage defects by implantation of chondrocyte-allogenous acellular cartilaginousmatrix(chondrocyte-ACM) composite in rabbits. Methods The thyroid chondrocyteswere isolated and co-cultured in vitro with allogenous acellular cartilaginousmatrix(ACM) to form the chondrocyte-ACM composite. The composite was analyzed histologically and was used to repair defects of thyroid cartilage. Eighteen New Zealand adult rabbits were made the defect models of thyroid cartilage at the two sides and divided into three groups. The defects were repaired with chondrocyte-ACM composite in the experimental group(n=6), with simple ACM in the ACM group (n=6)and without any material in the control group(n=6). The animals were sacrificed at 8 weeks after operation. The specimens were evaluated histologically. Results In vitro, the growth of chondrocytes was observed on the surface of allogenous acellular cartilaginous matrix and no chondrocytes grew inside the matrix. The defect filled with muscle and connective tissues in control group; the lymphocyte infiltration was observed in the matrix and no new cartilage formationoccurred at 8 weeks after operation in simple ACM group and experimental group.So the defect repair of rabbits thyroid cartilage failed. Conclusion The allogenous acellular cartilaginous matrixfailed to serve as a scaffold for chondrocytes both in vitro and in vivo. The allogenous acellular cartilaginous matrixshould be improved.
Objective Platelet-rich plasma (PRP) can enhance the chondrocyte prol iferation and repair of cartilage defects. To explore the safety and efficacy of intra-knee-articular injection of PRP to treat knee articular cartilage degeneration by comparing with injecting sodium hyaluronate (SH). Methods Thirty consecutive patients (30 knees) with knee articular cartilage degeneration were selected between January 2010 and June 2010. According to different injections, 30 patients wererandomly divided into PRP group (test group, n=15) and SH group (control group, n=15). There was no significant difference in gender, age, body mass index, and Kellgren-Lawrence grade between 2 groups (P gt; 0.05). Test group received 3.5 mL of PRP intra-knee-articular injections while control group received 2 mL of SH during the same time period. Both treatments were administered in series of 3 intra-knee-articular injections at 3-week intervals. Then, adverse reactions were recorded. International Knee Documentation Committee (IKDC) score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, and Lequesne index were used for evaluation of treatment results. Results The patients of 2 groups were followed up 6 months. There were significant differences in IKDC score, WOMAC score, and Lequesne index between pre- and post-injection in 2 groups (P lt; 0.05); no significant difference was found between different time points (3, 4, and 6 months) in test group (P gt; 0.05), while significant differences were found between the postoperative 6th month and the postoperative 3rd and 4th months in control group (P lt; 0.05). There was no significant difference in IKDC score, WOMAC score, and Lequesne index between 2 groups within 4 months (P gt; 0.05), but the effectiveness of test group was significantly better than that of control group at 6 months after injection (P lt; 0.05). Adverse reactions occurred in 12 patients (31 injections) of test group and in 12 patients (30 injections) of control group. No significant difference in onset time, termination time, and duration of adverse reactions were found between 2 groups (P gt; 0.05). Conclusion Intra-knee-articular injection of PRP to treat knee articular cartilage degeneration is safe, which can alleviate symptoms of pain and swell ing and improve the qual ity of l ife of patients; however, further data of large samples and long-term follow-up are needed to confirm the safety and effectiveness.
Objective To review the mechanism of extracellular vesicles (EVs) in treating intervertebral disc degeneration (IVDD). Methods The literature about EVs was reviewed and the biological characteristics and mechanism of EVs in the treatment of IVDD were summarized. Results EVs are a kind of nano-sized vesicles with a double-layered lipid membrane structure secreted by many types of cells. EVs contain many bioactive molecules and participate in the exchange of information between cells, thus they play important roles in inflammation, oxidative stress, senescence, apoptosis, and autophagy. Moreover, EVs are found to slow down the process of IVDD by delaying the pathological progression of the nucleus pulposus, cartilage endplates, and annulus fibrosus. Conclusion EVs is expected to become a new strategy for the treatment of IVDD, but the specific mechanism remains to be further studied.
Objective To investigate the effect of resveratrol (RES) on inflammation-induced cartilage endplate (CEP) degeneration, and its regulatory mechanism on high mobility group box-1 protein (HMGB1) signaling pathway. Methods The intervertebral CEP cells of Sprague Dawley (SD) rats aged 3 weeks were extracted and identified by toluidine blue staining and immunofluorescence staining of rabbit anti-rat collagen type Ⅱ. The cell counting kit 8 (CCK-8) method was used to screen the optimal concentration of RES on intervertebral CEP cells. Gene chip analysis was used to determine the target of RES on intervertebral CEP cells. Interleukin 1β (IL-1β) was used to construct the intervertebral CEP cell degeneration model caused by inflammation and the 7-8-week-old SD rat intervertebral disc degeneration model, and pcDNA3.1-HMGB1 (pcDNA3.1) was used as the control of RES effect. Flow cytometry and TUNEL staining were used to detect the apoptotic rate of intervertebral CEP cells and rat intervertebral disc tissue cells, respectively. ELISA kit was used to detect the content of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in the cell supernatant and rat serum. Western blot was used to detect the expressions of HMGB1, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (p-ERK), B cell lymphoma/leukemia 2 gene (Bcl-2), and Bcl-2-associated X protein (Bax). ResultsThe extracted cells were identified as rat intervertebral CEP cells. CCK-8 method screened out the highest activity of intervertebral CEP cells treated with 30 μmol/L RES. The gene chip analysis confirmed that the HMGB1-ERK signal was the target of RES. Both cell experiments and animal experiments showed that RES treatment can significantly down-regulate the apoptosis rate of intervertebral CEP cells, inhibit the release of TNF-α, and increase the content of IL-10; and down-regulate the expressions of HMGB1, p-ERK, and Bax, and increase Bcl-2; and pcDNA3.1 could partially reverse these effects of RES, and the differences were all significant (P<0.05). ConclusionRES can significantly inhibit the apoptosis of intervertebral CEP cells induced by inflammation, which is related to inhibiting the expression of HMGB1.
Objective To establ ish a porcine model of articular full-thickness cartilage defect characterized byremaining cartilage calcified zone on femoral trochlea, so as to provide a considerable and comparative control group forinvestigating repair effects of tissue engineered scaffolds in articular cartilage defects with cartilage calcified zone remaining.Methods The full-thickness cartilage column defects (6 mm in diameter, 0.2-0.5 mm in depth) without damage on calcifiedcartilage zone were made on the femoral trochlea in 9 clean-grade 6-month-old Guizhou mini pigs by standard cartilage-defectmakingsuites. Microscopical observation was performed after modeling. Scanning were made by 3.0T MRI at 4 weeks. Thengeneral observation, stereomicroscope, and histological staining were used to observe cartilage repair. Results All animals wereal ive. No infection of incisions or patellar dislocations occurred; they were able to walk with partial weight-bearing immediatelyafter surgery and could move freely without limp at 1 week. Obvious signal discontinuity in trochlea and subchondral bone couldbe observed in MRI, without deep signal change in defects surrounding. Microscopical observation showed a few repair tissueand petechia at base of the defect with clear boundary. Nearly intact calcified zone of cartilage and zonal collapse of subchondralbone in defects could be observed with stereomicroscope. Under common microscope, no chondrocytes was found in defects,as well as negative staining of fast green-safranin O and alcian blue. Under polarized microscope, the bottom of defects werefilled with a l ittle of fibrous tissue presenting continuous and b l ight-refraction by sirius red staining. Conclusion Theanimal model of articular full-thickness cartilage defect on femoral trochlea by standard cartilage-defect-making suites can beapplied for the research of cartilage disease in early human osteoarthritis and function of calcified cartilage zone in pig.
ObjectiveTo summarize the active changes of Wnt signaling pathway in osteoarthritis (OA) as well as the influence and mechanism of dual-targeted regulation on cartilage and subchondral bone and the role of crosstalk between them on OA process.MethodsThe relevant literature concerning the articular cartilage, subchondral bone, and crosstalk between them in OA and non-OA states by Wnt signaling pathway in vivo and vitro experimental studies and clinical studies in recent years was reviewed, and the mechanism was analyzed and summarized.ResultsWnt signaling can regulate the differentiation and function of chondrocytes and osteoblasts through the classic β-catenin-dependent or non-classical β-catenin-independent Wnt signaling pathway and its cross-linking with other signaling pathways, thereby affecting the cartilage and bone metabolism. Moreover, Wnt signaling pathway can activate the downstream protein Wnt1-inducible-signaling pathway protein 1 to regulate the progress of OA and it also can be established gap junctions between different cells in cartilage and subchondral bone to communicate molecules directly to regulate OA occurrence and development. Intra-articular injection of Wnt signaling inhibitor SM04690 can inhibit the progress of OA, and overexpression of Wnt signaling pathway inhibitor Dickkopf in osteoblasts can antagonize the role of vascular endothelial growth factor work on chondrocytes and inhibit the catabolism of its matrix.ConclusionThe regulation of metabolism and function of cartilage and subchondral bone and crosstalk between them is through interactions among Wnt signaling pathway and molecules of other signaling. Therefore, it plays an vital role in the occurrence and development of OA and is expected to become a new target of OA treatment through intervention and regulation of Wnt signaling pathway.
ObjectiveTo explore the effect of the expanded capsule on the growth of autogenous costal cartilage.
MethodsSixteen New Zealand white rabbits at the age of 3 months (weighing, 2.2-2.5 kg; male or female) were selected and four 15 mL tissue expanders were implanted on the back symmetrically. After 1 month, the expanded capsule formed, the tissue expanders were removed; the capsule of the left side was removed (experimental group), and the capsule of the right side was reserved (control group); meanwhile, the right 7th and 8th costal cartilage without the perichondrium was divided into segments and placed into the capsule of 2 groups symmetrically. At 4 and 8 weeks after transplantation, the cartilage was harvested for the general, weighing, and histological observations.
ResultsOne rabbit died during the experiment, and the other 15 rabbits survived. The differences of cartilage weight between before and after transplantation showed more obvious increase in the experimental group[(0.003 4±0.002 7) g and (0.005 8±0.001 4) g] than those in the control group[(-0.000 3±0.001 9) g and (-0.003 9±0.005 3) g] at 4 and 8 weeks, showing significant differences between 2 gouprs (t=4.331, P=0.029; t=6.688, P=0.008). The change of cartilage weight at 8 weeks was significantly higher than that at 4 weeks in the experimental group (t=-3.098, P=0.001); but the change of cartilage weight at 8 weeks was significantly lower than that at 4 weeks in the control group (t=2.491, P=0.009). The histological observation showed that the activity of the cartilage was enhanced in 2 groups at 4 and 8 weeks when compared with normal cartilage, and more obvious change was observed in the experimental group than in the control group. And the acellular area was seen in the cartilage at 8 weeks in the control group. The Masson staining results showed that the color was deeper in the experimental group than in the control group.
ConclusionThe removal of the expanded capsule during operation is beneficial to the growth of autogenous costal cartilage. The results can provide corresponding experimental guidance for the clinical problems.
