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.
To investigate the feasibility of using the pedicled patella for repaire of the superior articular surface of the medial tibial condyle, 37 lower limbs were studied by perfusion. In this series, there were 34 obsolete specimens and 3 fresh specimens of lower legs. Firstly, the vessels which supply to patella were observed by the methods of anatomy, section and casting mould. Then, the form and area of the patellar and tibial medial conylar articular surface were measured in 30 cases. The results showed: (1) the arteries supplied to patella formed a prepatellar arterial ring around patella, and the ring gave branches to patella; (2) medial inferior genicular artery and inferior patellar branches of the descending genicular arterial articular branch merge and acceed++ to prepatellar ring at inferior medial part of patella; (3) the articular surface of patella is similar to the superior articular surface of the tibial medial condyle on shape and area. It was concluded that the pedicled patella can be transposed to medial tibial condyle for repaire of the defect of the superior articular surface. The function of the knee can be reserved by this method.
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 develop a novel porous three-dimensional scaffold and to investigate its physico-chemical properties for tissue engineering cartilage.Methods Refined 88% deacetylation degree chitosan was prepared and dissolved in 0.2 mol/L acetate acid and fully mixed with highly purified porcine type Ⅱcollagen in 0.5 mol/L acetate acid solution in a ratio of 4 to 1 (wt/wt). Freeze-drying process was employed to fabricate the composite scaffold. The construct wascross-linked by use of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS). A mechanical tester was utilized to determine the tensilestrength change before and after cross-linking. The microstructure was observed via scanning electron microscopy (SEM). The lysozyme degradation was performedto evaluate the degradability of the scaffold in vitro. Results A bulk scaffold with desired configuration was obtained. The mechanical test showed that the crosslinking treatment could enhance the mechanical strength of the scaffold. The SEM results revealed that the two constituents evenly distributed in the scaffold and that the matrix was porous, sponge-like with interconnected pore sizing 100250 μm. In vitro lysozyme degradation indicated that crosslinked or uncross-linked composite scaffolds had faster degradation rate than the chitosan matrix. Conclusion Chitosan and typeⅡcollagen can be developed into a porous three-dimensional scaffold. The related physico-chemical tests suggest that the composite socaffold meets requirements for tissue engineered scaffold and may serve as an alternative cellcarrier for tissue engineering cartilage.
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.
ObjectiveTo explore the clinical application and effectiveness of a personalized tissue engineered cartilage with seed cells derived from ear or nasal septal cartilage and poly-glycolic acid (PGA)/poly-lactic acid (PLA) as scaffold in patients with nasal reconstruction. MethodsBetween March 2014 and October 2015, 4 cases of acquired nasal defects and 1 case of congenital nasal deformity were admitted. The patient with congenital nasal deformity was a 4-year-old boy, and the source of seed cells was nasal septal cartilage. The other 4 patients were 3 males and 1 female, aged 24-33 years, with an average of 28.5 years. They all had multiple nasal subunit defects caused by trauma and the source of seed cells was auricular cartilage. The tissue engineered cartilage framework was constructed in the shape of normal human nasal alar cartilage and L-shaped silicone prosthesis with seed cells from cartilage and PGA-PLA compound biodegradable scaffold. The boy underwent nasal deformity correction and silicone prosthesis implantation in the first stage, and the prosthesis was removed and implanted with tissue engineered cartilage in the second stage; the remaining 4 adult patients all used expanded forehead flaps for nasal reconstruction. All 5 patients underwent 1-4 nasal revisions. The implanted tissue engineered cartilage was observed during the operation and taken from 2 patients for histological examination.ResultsAll the incisions healed by first intention after the tissue engineered cartilage implantation, and the expanded forehead flaps survived. Postoperative low fever occurred in 3 patients. No complications such as infection, obvious immune rejection response, and tissue engineered cartilage protrusion were found in all patients. All patients were followed up 9-74 months (mean, 54.8 months). During follow-up, the patients had no obvious discomfort in the nose and the ventilation function were good. All patients were satisfied with the nasal contour. Early-stage histological examination showed the typical cartilage characteristics in 1 patient after the implantation of tissue engineered cartilage. Late-stage histological examination in 1 patient of tissue engineered cartilage showed the characteristics of fibrous connective tissue; and the other showed there was remaining cartilage.ConclusionThe safety of tissue engineered cartilage constructed in vitro for reconstruction is preliminarily confirmed, but the effectiveness still needs further verification.
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.
Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated frombone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs wereharvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected bylaser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
OBJECTIVE: To provide experimental basis for improving the curative effect of pectus excavatum. METHODS: Twelve rabbits were adopted in this experiment. After the bilateral second and third costal cartilages of the rabbits were resected subperichondrially, their right second and third costal perichondriums were damaged intentionally. Then, the bilateral third costal perichondriums were stitched into a tube-like structure and the second ones were left opened. After 2, 4, 6 of operation, the bilateral second and third neocartilages were measured for their width, and histological character were observed under microscope. RESULTS: 1. After 2, 4, 6 months of operation, the average width of the bilateral second neocartilages were significantly greater than the preoperative ones. 2. 4 and 6 months after operations, there was no significant difference in the average width of the bilateral third neocartilages and the preoperative ones. 3. The amount, distribution of costal neocartilage cells and the arrangement of costal neocartilage matrix within the left second and third costal cartilages were better than the right under the light microscope. 4. The left third costal neocartilage was regenerated and remodeled better than all the others. CONCLUSION: The integrality of costal perichondrium is in favor of the regeneration of costal cartilage, and the sleeve stitch of costal perichondrium facilitates the remodeling of costal neocartilage.
Objective To prepare a novel hyaluronic acid methacrylate (HAMA) hydrogel microspheres loaded polyhedral oligomeric silsesquioxane-diclofenac sodium (POSS-DS) patricles, then investigate its physicochemical characteristics and in vitro and in vivo biological properties. Methods Using sulfhydryl POSS (POSS-SH) as a nano-construction platform, polyethylene glycol and DS were chemically linked through the “click chemistry” method to construct functional nanoparticle POSS-DS. The composition was analyzed by nuclear magnetic resonance spectroscopy and the morphology was characterized by transmission electron microscopy. In order to achieve drug sustained release, POSS-DS was encapsulated in HAMA, and hybrid hydrogel microspheres were prepared by microfluidic technology, namely HAMA@POSS-DS. The morphology of the hybrid hydrogel microspheres was characterized by optical microscope and scanning electron microscope. The in vitro degradation and drug release efficiency were observed. Cell counting kit 8 (CCK-8) and live/dead staining were used to detect the effect on chondrocyte proliferation. Moreover, a chondrocyte inflammation model was constructed and cultured with HAMA@POSS-DS. The relevant inflammatory indicators, including collagen type Ⅱ, aggrecan (AGG), matrix metalloproteinase 13 (MMP-13), recombinant A disintegrin and metalloproteinase with thrombospondin 5 (Adamts5), and recombinant tachykinin precursor 1 (TAC1) were detected by immunofluorescence staining and real-time fluorescence quantitative PCR, with normal cultured chondrocytes and the chondrocyte inflammation model without treatment as control group and blank group respectively to further evaluate their anti-inflammatory activity. Finally, by constructing a rat model of knee osteoarthritis, the effectiveness of HAMA@POSS-DS on osteoarthritis was evaluated by X-ray film and Micro-CT examination. Results The overall particle size of POSS-DS nanoparticles was uniform with a diameter of about 100 nm. HAMA@POSS-DS hydrogel microspheres were opaque spheres with a diameter of about 100 μm and a spherical porous structure. The degradation period was 9 weeks, during which the loaded POSS-DS nanoparticles were slowly released. CCK-8 and live/dead staining showed no obvious cytotoxicity at HAMA@POSS-DS, and POSS-DS released by HAMA@POSS-DS significantly promoted cell proliferation (P<0.05). In the chondrocyte anti-inflammatory experiment, the relative expression of collagen type Ⅱ mRNA in HAMA@POSS-DS group was significantly higher than that in control group and blank group (P<0.05). The relative expression level of AGG mRNA was significantly higher than that of blank group (P<0.05). The relative expressions of MMP-13, Adamts5, and TAC1 mRNA in HAMA@POSS-DS group were significantly lower than those in blank group (P<0.05). In vivo experiments showed that the joint space width decreased after operation in rats with osteoarthritis, but HAMA@POSS-DS delayed the process of joint space narrowing and significantly improved the periarticular osteophytosis (P<0.05). Conclusion HAMA@POSS-DS can effectively regulate the local inflammatory microenvironment and significantly promote chondrocyte proliferation, which is conducive to promoting cartilage regeneration and repair in osteoarthritis.