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BACKGROUND: H-type vessels are mainly distributed in the metaphysis, which can promote the proliferation of osteocytes, further accelerating osteogenesis.OBJECTIVE: To investigate the expression of H-type vessels in the subchondral bone during the occurrence and development of osteoarthritis.METHODS: 8-week-old C57 mice were randomly divided into experimental and sham groups, followed by the right medial menisectomy to establish the osteoarthritis models or only articular capsulotomy. The knee samples were removed at 4 weeks postoperatively, and were stained with safranin-O-fast green to evaluate the degree of injury. The expression levels of CD31, Emcn and matrix metalloproteinase 13 were detected by immunofluorescent staining. The changes of the subchondral bone were observed by hematoxylin-eosin staining and the changes of bone mass in the subchondral bone were analyzed by micro-CT.RESULTS AND CONCLUSION: Compared with the sham group, the Osteoarthritis Research Society International scores, expression levels of CD31, Emcn, H-type vessels and matrix metalloproteinase 13, as well as the bone mass in the subchondral bone were significantly increased in the experimental group (P < 0.05). To conclude, the increased H-type vessels in the subchondral bone promote the hyperplasia and remodeling of subchondral bone in the progression of osteoarthritis.
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Objective To evaluate graft isometry after anatomic single bundle reconstruction of anterior cruciate ligament (ACL) using dynamic computed tomography (CT).Methods A retrospective study was conducted of the 14 patients who had undergone single bundle ACL reconstruction from June to August 2015.They were all men,with an average age of 28.6 years (range,from 18 to 39 years).At 6 months after operation,they received dynamic CT scanning during a cycle of knee extension to flexion.3D bone models representing the knee at different flexion positions (0°,30°,60°,90°,and 120°) in each patient were reconstructed from the CT images.The grid method was used to locate the positions of the central footprints of the tibial and femoral tunnels.The lengths between the entries of the femoral and tibial tunnels were measured from each tunnel entry to reflect the graft length change.Furthermore,we measured the isometry at the over-the-top position of the femur and at the anatomic tibial position.Results All the tunnel entries were located at the central area of the ACL anatomic attachment.The reconstructed ACL was the longest when the knee was in full extension.The length was gradually shortened between the femoral and tibial tunnels during flexion of the knee from 0° to 90°.The anatomic position showed an average of 4.82 mm shortening and the over-the-top position an average of 3.28 mm shortening.The length excursion increased in early flexion from 0° to 30° (2.91 ±0.91 mm on average) and reduced in later flexion from 90° to 120° (2.98 ± 1.41 mm on average).Conclusions None of the reconstructed ACL was isometric.A graft length may be the longest when the knee is in full extension and decrease gradually during the flexion from 0° to 90° and increase gradually during the flexion of 90° to 120°.The graft should be fixed when the knee is in the flexion of 30°.
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Objective To report our self-designed guide module for placement of posterior column lag screws in anterior-posterior column acetabular plate using CT reconstruction data.Methods The CT scan data of 50 normal adult pelves were collected from February 2012 to April 2013,involving 30 males and 20 females with an average age of 46.4 years(range,from 25 to 69 years).The data were imported into Mimics 10.01 software for reconstruction of semi-pelvic models.Virtual cylindrical implants were placed intraosseously in both the left and the right posterior columns.The perpendicular distance (OP) from the insertion point O of the virtual cylindrical implant to the arcuate margin (P) and the distance (PI) from the point P to the point I,the crosspoint of the extension line of the ischial ramus and the arcuate margin were measured respectively.The angle (∠φ) between the direction of screws and the plane of guide module and the angle (∠θ) between the direction of screws and the long axis of guide module were also measured respectively.Results The average length of PI was 0.98 ± 0.13 cm,with 1.08 ± 0.22 cm in females and 0.95 ± 0.27 cm in males.The difference between genders was not statistically significant (P ＞ 0.05).The average length of OP was 1.09 ± 0.26 cm,with 1.06 ± 0.29 cm in females and 1.12 ± 0.24 cm in males.The gender difference was not statistically significant either (P ＞ 0.05).The mean value of ∠ φ was 55.43° ± 3.64°,with 55.33° ± 4.00° in females and 55.50° ±3.43° in males.The difference between genders was not statistically significant (P ＞ 0.05).The ∠θ value in females was 39.21 ° ± 2.45° and 35.58° ± 2.31 ° in males.The gender difference was statistically significant (P ＜ 0.05).Conclusions In design of the guide module,the nail holes should be located about 1 cm away from both the posterior edge and the medial edge,the angle between the screw direction and the module plane should be approximately 39° in females and 35° in males,and the angle between the screw direction and the long axis of the module approximately 55°.
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10.3969/j.issn.2095-4344.2013.25.001
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Objective To explore the method and effect of transinfection of rabbit early knee osteoarthritis models via chitosan microsphere with gene of recombined human IL-1Ra gene and TGF-β1 gene. Methods Chitosan microspheres with plasmids of IL-1Ra gene and TGF-β1 gene, and rabbit early knee osteoarthritis models were prepared. Rabbits in different groups had intra-articular injections of chitosan microsphere containing IL-1Ra gene and / or TGF-β1 gene, and chitosan solution as control group before being executed regularly and randomly. The joint specimens were evaluated by HE staining, lycopene red O staining and immunohistochemical analysis and Mankin's score. ELISA was used for detection of IL-IRa and TGF-β1 concentration of articular cavity fluid in each group. Results The control group was consistent with the pathological changes of early OA. In co-transinfection group, judging from the appearance and staining of cartilage,the OA damage of the specimens was less serious than other groups'. Its Mankin's score was significantly lower than single-gene transinfection group (P ＜ 0.05), and the latters Mankin's score were significantly lower than control group (P ＜ 0.05). Conclusion Intra-articular injection of chitosan microspheres containing both IL-1Ra gene and TGF-β1 gene could inhibit the degeneration of cartilage and promote cartilage repair.
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Objective To introduce experience of using the new AO anterolateral distal tibia locking com-pression plate (LCP) for treatment of Pilon fractures. Methods Between February and August of 2009,8 closed Pi-lon fractures were treated by open reduction and internal fixation. The distal fibula was fixed with a one-third tubular plate or an recontruction plate via a straight incision posterior to the fibular crest. The distal tibia was approched by a straight incision over the ankle joint, and the fracture was stabilized using an anterolateral distal tibia LCP. Regular follow up was made to observe and evaluate the preliminary clinical outcomes. Results Seven of the 8 patients were availabe for follow up for 3 ～ 6 months (average 4.5 months). All incisions obtained primary healing, though one ex-perienced mild superficial inflammation,and none developed deep infections. Based on the Burwell and Charnley radi-ographic criteria,anatomical reduction was obtained in 5 cases,good in 1 ,and fair in 1. Among the 5 cases exceeding 5 months postsurgery,4 were evaluated as excellent and 1 us good according to Tometta' s clinically based criteria for Pilon fractures. Conclusion With good surgical timing,internal fixation with anterolateral LCP for Pilon fractures is reliable and warrants less complications.
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Objective To summarize the experience in the perioperative nursing of one-stage bilateral total hip arthroplaaty (THA) for the systemic lupus erythematosus (SLE) patients with avascular necrosis of the femoral head (ANFH).Methods From June 1998 to April 2007,17 cases of patients who were diagnosed as SLE with bilateral ANFH were treated by one-stage bilateral THA.Perioperative nursing included psychological support,diet control,observation and nursing of various complications,correct functional exercise and health education.Harris score and SF-36 score were evaluated before and after operation.Results There were 2 cases with delayed incision healing,1 with early prosthesis dislocation,1 with thigh pain for 10 months and 1 with acute renal failure postoperatively,which were improved and recovered after proper treatments.Asymptomatic deep vein thrombosis (DVT) in low extremities were detected by color Doppler ultrasonography in 6 cases.There were no pulmonary embolism and no deep infection around prosthesis.There was no Addisonian crisis postoperatively.The pain was relieved and the motion of joint was improved during follow-up.There was no radiological evidence of implant loosening.The Harris hip score and SF-36 score greatly alleviated after operation.All patients were followed up and the mean follow-up time was 28 months.Conclusions One-stage bilateral THA has good results for the patients with SLE and AVNH.Strengthening of perioperative nursing contributed to improvement of success rate of surgery and reduction of complication.
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Objective To investigate and compare the clinical effects of intraarticular injection of sodium hyaluronate and diprospan on knee osteoarthritis. Methods 94 patients with knee osteoarthritis were divided into two groups, the HA group and Cortieosteroid group. Each patient in the HA group was treated with intra-articular injection of sodium hyaluronate at 2.5 ml every week for 5 weeks, and each patient in the Corticosteroid group was treated with intra-articular injection of diprospan at 1ml on the first and fourth week. The clinical assessments included pain,joint effusion,and Lequesne Index. Assessments were done at baseline, at week 4, and week 12. Results 88 cases were followed up for 3 months. A significant decrease in VAS scores for pain and in Lequesne Index was found in both groups at week 4 when compared to baseline and there were no significant differences between the two groups. However,at 12 week improvement in pain score and Lequesne Index was found in favour of hyaluronic acid. In addition,diprospan seemed to have preferable short-term effect on patient with joint effusion. Conclusion Both intra-articular injection of sodium hyaluronate and diprospan provided clinically significant improvement in short-term and demonstrated that hyaluronic acid had a more long-term beneficial effect in patients with knee osteoarthritis.
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Objective To investigate the effect of treatment for frozen shoulder by manipulation release combined with hydraulic pressure distension.Methods In this randomized trial,78 patients with frozen shoulder syndrome randomly divided into three groups were enrolled in the study.28 patients were treated by manipulation release under interscalene brachial plexus anesthesia.23 patients were treated by sodium hyaluronate injection into glenohumeral joint combined with manipulation release.27 patients were treated by hydraulic pressure distension combined with manipulation release.All patients were followed for six months.Effective rate evaluation combined with pain evaluation and functional evaluation were adopted for final evaluation.Results There were significant difference between pre-treatment and post-treatment in each group.Effective rate of hydraulic pressure distension combined with manipulation release was 92.6%,which was superior to manipulation release (78.6% ) and sodium hyahronate injection combined with manipulation release ( 82.6% ).There were no significant difference in functional evaluation of the three groups.Conclusion Hydraulic pressure distension combined with manipulation release under interscalene brachial plexus anesthesia is a successful treatment for idiopathic frozen shoulder.
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BACKGROUND:Bone marrow stromal stem cells(BMSCs)do not allow for single differentiation of chondrocytes due to their multi-directional differentiation,bone morphogenetic protein secreted from osteoblasts affect the non-differentiated precursor cells and promote their osteoblast differentiations,while those differentiated cells are bound to form tissues.OBJECTIVE:To in vitro induce canine BMSCs differentiate into chondrocytes,and to investigate the method and conditions of chondrocyte differentiation in vitro.DESIGN,TIME AND SETTING:Single sample observation was performed in the Laboratory of Tissue Engineering,Sun Yat-sen University between March 2005 and January 2006.MATERIALS:One male dog,aged 4 months,was involved to harvest BMSCs from the rib.METHODS:Rib BMSCs extracted from bone marrow of 2.0-3.0 mL were cultured in vitro. When cells reached a confluence at 8-11 days,trypsinization was conducted and then halted with L-DMEM synthesis culture solution containing 10% fetal bovine serum. Cellular suspension was collected and centrifuged,cells were rssuspended and incubated at a ratio of 1:3. The third generation of cells were cultured and amplified,10 μg/L basic fibroblast growth factor 2 mL was added to replenish culture medium twice,then 1 mg/L transforming growth factor β1 of 2 mL was applied to induce BMSCs differentiation into chondrocytes.MAIN OUTCOME MEASURES:Toluidine blue and alcian blue stains were applied to determine cartilage matdx secretion,immunohistochemistTy was used for the detection of cartilage specific Ⅱ collagen expression.RESULTS:After BMSCs were primarily cultured and subcultured in vitro,they were shown to grow well at the fourth generation,those induced by basic fibroblast growth factor and transforming growth factor β1 were positive for toluidine blue and aician blue staining;immunohistochemistry showed a positive outcome for type Ⅱ collagen,indicating the induced BMSCs exhibited chondrocyte's characteristics.CONCLUSION:Utilizing basic fibroblast growth factor and transforming growth factor β1,the induced canine BMSCs could differentiate into chondrocytes,which is considered as an ideal seed cells for cartilage tissue engineering.
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BACKGROUND: The meniscal allograft transplantation has been proved good outcome in clinical, however, the source and matching restrict its application. By means of a patent technique of separating antigen, a new biological meniscus can be obtained.OBJECTIVE: To observe the histological changes of transplanted biological meniscus in goat's knee joint, in addition, to investigate the feasibility of reconstituting meniscus by using biological meniscus. DESIGN, TIME AND SETTING: The animal experiment of goats was performed at the Central Laboratory in the Third Affiliated Hospital of Sun Yat-sen University between June 2005 and June 2006.MATEAIALS: A total of 15 healthy goats were used in the experiment. Fresh porcine meniscus was harvested for the preparing of biological meniscus.METHODS: The medial meniscus of goat was excised, and then the absent part was repaired using biological meniscus. MAIN OUTCOME MEASURES: The animals were sacrificed at months 1, 3, 6 and 12 after operation. And the samples were detected by histology, scanning electron microscopy (SEM) and Indian ink perfusion. RESULTS: The incision of all animals healed well, without infection or death. The transplanted meniscus healed well with the surrounding tissue, and the connection was tight. With time prolonged, the transplanted meniscus slowly soaked, and live cells existed inside the meniscus, but there were no live cells in about 1/4 district at 1 year after the surgery. Indian ink perfusion showed that the surrounding tissue grew inside and set up microcirculation, but only limited to 10% periphery of the meniscus (normal goat approximately 15%). The transplanted meniscus began the necrosis at 6 months after surgery. Under SEM, the surface of the meniscus became crude and cracked in some places, but still maintains the gross shape at 1 year after the surgery.CONCLUSION: There is no obvious rejection after the biological meniscus was transplanted into knee joint of goats. During the process of absorption, autologous tissue of goats can grow into and set up microcirculation in the transplanted meniscus. The transplanted meniscus protects the'knee cartilage notably, only slightly damage in cartilage can be observed.
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Objective To evaluate one-stage arthroscopic reconstruction of anterior crueiate ligament (ACL)and posterior cruciate ligament(PCL)using Achilles tendon-bone allografts. Methods From July 2000 to February 2005.we treated 15 patients(11 males and 4 females)whose ACL and PCL were ruptured at one knee but the eontralateral knee was intact.Their associated meniscus injuries were treated arthroscopically according to established procedures prior to ligament reconstruction.Thirty Achilles tendon-bone allografts were used to reconstruct torn ACL and PCL in 15 knees at one stage.Reconstruction of both ligaments was performed at subacute or chronic phase(>3 to 8 weeks)in 12 casses,and at acute phase in 3 cases(<3 weeks).All knees were graded pre-and postoperatively using the International Knee Documentation Committee(IKDC)and Lysholm scoring systems.At follow-up,functions were evaluated for all patients and compared with those of the contralateral healthy knee. Results All patients were followed up for a minimum of 3 years(mean,38 months).Preoperatively,the IKDC ratings showed all the injured knees were severely abnormal.At final postoperative f0Uow-up,9 knees received a normal rating,5 a nearly normal one and 1 an abnormal one.The differences in Lysholm score were statistically significant (t=15.660,P<0.05)between pre-and postoperative analyses.The most noticeable postoperative complication was a short localized fever coupled with arthroedema in 1 case. Conclusions Achilles tendon-bone allograft offers an alternative for simultaneous arthroseopic reconstruction of ACL and PCL.However,problems inherent in allograft tissues entail further investigation to ensure future application.
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BACKGROUND: Poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) is a novel scaffold made by solvent casting/particulate leaching procedure, composed of polyhydroxybutyrate and polyhyclroxyvalerate at certain ratio, which has good biocompatibility as well as high intensity and modulus. It has three-dimensional porous net structure and good biodegradation. OBJECTIVE: To evaluate the biocompatibility between copolymers of PHBV and canine bone marrow mesenchymal stem cells(BMSCs).DESIGN, TIME AND SETTING: In vitro comparative observation. The study was performed at the Laboratory of Histology and Embryology, Sun Yat-sen University between June 2003 and March 2004.MATERIALS: PHBV scaffold, film porosity > 85% and 100-350 μ m aperture size.METHODS: Canine BMSCs were isolated and cultured. The 3-4 passage cells were seeded onto the PHBV films and three-dimensional foam scaffold. Cells cultured alone served as control.MAIN OUTCOME MEASURES: The seeded cells were observed under inverted microscope; at 1, 2, 3 weeks after seeding, the BMSCs were treated with 4% paraformaldehyde and stained with hematoxylin-eosin (HE); The protein content in seeded cells was determined by bicinchoninic acid assay (BCA), and the content of DNA was quantified using Hoechst33258 assay at 5, 10, 14 days after culture.RESULTS: Inverted microscopic observation showed that the PHBV fibers were fairly thick with weak lucency, and the fibers were hardly detectable under contrast phase microscope. Majority of cells attached onto the PHBV films 2 hours after seeding, and extended well in a spindle shape at 3 days. One week after culture, 2 PHBV were fixed, and BMSCs proliferation was observed after HE staining. At two weeks, cells continued to proliferate and densely covered the PHBV film. The cells grew in the three-dimensional pores, connected at 1 week, extended at 3 weeks, secreting a large amount of material around cells. Cell proliferation did not change much at 3 weeks compared with 2 weeks, and there was no significant difference in DNA and protein contents between control and PHBV groups (P > 0.05).CONCLUSION: As a kind of tissue-engineered scaffold material for BMSCs, PHBV displays good biocompatibility.
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@#Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.
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Objective To explore transinfection of rabbit chondrocytes via chitosan microsphere with human IL-1Ra and TGF-β1 gene. Methods Chitosan-DNA microspheres carrying plasmids with IL-1 Ra and TGF-β1 genes were prepared.The encapsulation efficiency,DNA-released kinetics and lysozyme degradation in vitro were performed.Articular rabbit chondrocytes were co-transinfected with the plasmids with IL-1Ra and TGF-β1 genes via chitosan-DNA mierosphere,evaluated by fluorescence microscope,TaqMan real-time PCR assay and MTF test. Results The chitosan microspheres with IL-1Ra and TGF-β1 genes were(2.8±0.2)μm and(2.6±0.1)μm in diameters respectively.The encapsulation efficiency were(88.3±4.1)%and(87.2±2.6)%.During the degradation,significant morphological changes were noticed.The plasmids could be released in a multiphasic fashion.Enhanced green fluorescent protein and Real-Time PCR analysis showed that genes were expressed in chondrocytes.lasting near 30 days.MTT indicated that the cotransinfection promoted the chondrocytes'proliferation. Conclusion IL-1Ra and TGF-β1 genes cotransfected into chondrocytes via chitosan-DNA microsphere could be expressed in a long term and cotransinfection promoted the chondroeytes'proliferation,which is the base of inhibiting the degeneration of cartilage and promote cartilage repair.
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BACKGROUND: Currently, the materials used in clinical practice to repair cruciate ligament of knee joint contain auto-graft bone- mid 1/3 patella tendon-bone (B-T-B), auto-semitendinous muscle, gracilis muscle and allogenic tissue graft. All of them are limited to a certain degree in clinical application. Therefore, people hope to consistently develop artificial ligaments to take the place of auto- and allografts. OBJECTIVE: To investigate the feasibility to construct artificial biological ligament (ABL) by applying a novel biochemical technique using porcine tendon as the raw material. DESIGN: Research of new biological material. SETTING: Department of Orthopedics, Third Affiliated Hospital of Sun Yat-sen University. MATERIALS: Adult pigs of either gender were provided by the Animal Center of Sun Yat-sen University. Scanning electron microscope (SEM, S-520) was provided by Hitachi, Japan, and micro-controlled electron tension-testing device (Model LWK-10B) by Guangzhou Experimental Devices Factory. METHODS: The experiment was performed at the Animal Center of Sun Yat-sen University from January 2004 to June 2005. ABL was established by means of treating porcine tendon with epoxy cross-linking fixation, diversified antigen minimization process, mechanic enhancement modification and surface activating process. Under aseptic condition, a 6-month-old goat's bone marrow was abstracted, and then the bone marrow matrix stem cells were cultured in ABL stent for 3 weeks. Scanning electron microscope was used to observe structure and compatibility of artificial ligament, and mechanics test was used to analyze biomechanics characteristics of ABL. MAIN OUTCOME MEASURES: Structural features, cell compatibility and biomechanics characteristics of ABL.RESULTS: ① Structural features of ABL: The appearance of ABL was similar to that of the normal human ligament. Histological examination showed that the ABL was collagen fibers with no cells. Electron microscope examination revealed that the ABL was composed of hair-looking and fiber-like objects running uniformly in a certain direction and closely parallel-arranged. ② Cell compatibility: Three weeks after xenogenic marrow matrix cells were cultured on the surface of the ABL, it was noted that cells adhered and the matrix secreted by the cells precipitated around the cells. There were no cells found inside the ABL. ③ Mechanical strength of the ligament: The average diameter of ABL was 5 mm and the mechanical test at a speed of 100 mm/min showed that its averaged tensile limit was 927.19 N and the tension-resistant strength was 47.22 N/mm those were close to the corresponding parameters of the normal goat's ACL. The normal goat's ACL was 5 mm. The greatest tensile load was 807.50 N and the tension-resistant strength was 41.13 N/mm.CONCLUSION:As we used the unique biochemical technique and minimized the xenogenic protein immunogenicity of the porcine tendon, ABL has acceptable biomechanical properties and superior biocompatibility. As a substitute of the ligament in the reconstruction of the ACL, ABL has a promising prospect in clinical applications.
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Objective To explore the feasibility of building tissue engineered cartilage by bone marrow stromal cells and pbotografting modified copolymers of 3-hydroxybutymte and 3-hydroxyvalerate.Methods Sheep BMSCs were seeded in three-dimensional photografting modified PHBV scaffoids.Twenty-four hours later.composites were cultured with ehondrogenically inductive medium(DMEM)containing TGF-B(10 ng/m1),IGF-1(150 ng/m1)and 20% fetal bovine serum.Three weeks later,the constructs were evaluated by scanning electron microscopy(SEM)and light microscopy with alcian blue,safrine 0 and type Ⅱ collage immunohistochemical staining.GAG contents of constructs were determined by DMB(1,9-dimethylmethylene blue)binding assay at weekly intervals up to 3 weeks.The composites were implanted subcutaneously in sheep abedoml and were evaluated macroscopically and bistologically at 4 weeks postoperatively.Results SEM photograph showed.after one week culture,cell morphology changed from fibroblast-like elongated spindle to the flat rounded like chondrocytes,and the extra cellular matrix also increased obviousl~.Furthmore,with the culture time extension,this change were more evident.HE staining showed that cells filled all the inter-connected pores in the constructs.And more cells were observed in the outer layer of the constructs.ECM(extraeellular matrix)Was strongly positive by Aleian blue,Safrine O staining and type Ⅱ collage immunohistechemical staining.DMB binding assay revealed that the induced BMSCs GAG secretion(1306.7±192.3)wag significantly higher than BMSCs(205.0±26.2)(P<0.001),but it was significantly lower than passage 2 ehondrocytes(1969.2±235.3)(P<0.001).Saltine O and type Ⅱ collage immunohistochemical staining were positive in constructs implanted subcutaneously.Conclusion Tissue engineered cartilage could be obtained using BMSCs and photografting modified PHBV,but there are still gaps physiologically between the constructs and the nature cartilage.
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BACKGROUND: A new composite scaffold for cartilage tissue engineer ing has been employed to culture chondrocytes and overcome many limits related to traditional scaffolds, such as poor biocompatibility, inferior mechanical property, inappropriate biodegradability, and simplex structure which can not match layered structure of articular cartilage, etc. The new composite scaffolds provided a new approach for the research of cartilage tissue engineering.OBJECTIVE: To evaluate the feasibility and value of layered cylindrical collagen/hydroxyapatite (HA) composite for cartilage tissue engineering by observing how it absorbs chondrocytes and affects its cellular characteristics.DESIGN: Completely randomized design and controlled experimental study.SETTING: Department of Orthopaedics, Third Hospital Affiliated to Sun Yat-sen University, and College of Material Science, South China University of Technology.MATERIALS: The experiment was conducted at the central experimental laboratory of the Third Hospital Affiliated to Sun Yat-sen University from June to November 2004. One two-week-old male healthy New Zealand rabbit,which was bred in 20 ℃ and 40% humidity, was used in this experiment.METHODS: ①Right amount of deionized water was added into HA, collagen I solution was added to disperse HA, then carbodiimide was added in the mixture at a proportion for getting the collagen/HA composite at different ratios. Pour to the certain mould in successive layers. The upper layer was pure collagen and the bottom was pure HA. The prepared layered cylindrical collagen/HA composite was put into the ultra low temperature freezer, lyophilized, and sterilized by ethylene oxide for the following procedures. ② Chondrocytes of juvenal rabbit were isolated and multiplied in vitro, then chondrocytes were seeded onto porous collagen/HA composite scaffold and cultured. The effects of composite scaffold on chondrocytes'morphological changes, proliferation, and function were evaluated through a series of examinations such as inverted phase-contrast microscopy, histological, scanning electron microscopy and immunohistochemistry.MAIN OUTCOME MEASURES: ①Morphology of collagen/HA porousscaffold. ②The characteristics of chondrocytes cultured in vitro. ③ The chondrocytes' compatibility with the collagen/HA porous scaffold.RESULTS: ①Morphology of collagen/HA porous scaffold: Collagen/ HA was a cylindrical three-dimensional porous scaffold in layer structure with certain mechanical strength. The top layer was pure collagen, the bottom was pure HA, and the intermediate layer was collagen and HA composite.Under scanning electron microscope, the scaffold had irregular blowhole structure, the apertures were even and pores were communicated each other. The average pore diameter was about 147 μm. ②The characteristics of chondrocytes cultured in vitro: The chodrocytes that had just been isolated were spherical; rate of living cells was 95%. 24 hours later, the cells were going to attach to the wall, extend to be triangle or polygon. The cytoplasms were abundant with secretary granules inside. The cells kept on growing in monolayer and the multiply rate accelerated after passage, and chondrocytes overspreaded the culture flask in 4 days. After passaged for several times, the multiply rate decreased. In passage Ⅵ, the ability of cell division was decreased obviously, the cytomorphosis was clear. Cellular sizes were larger. Most of cells were shuttle-shape, less refracted with blur edges, which trended to age and dedifferentiate to fibroblast. ③The chondrocytes' compatibility with the collagen/HA porous scaffold: Cylindrical collagen/ HA composite scaffold in layer structure had good hydrophilicity. The chondrocytes adhered to the surface of the scaffold, proliferated and migrated toward the scaffold through the pore. Chondrocytes attached to wall of microholes of the scaffold had largely maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold.CONCLUSION: Cylindrical collagen/ HA composite scaffold in layer structure has good cellular compatibility.It is stronger in mechanical property than pure collagen, is anideal scaffold for cartilage tissue engineering.
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[Objective] To evaluate the cellular biocompatibility of the nano poly(L-lactic acid)-block-poly(?-caprolactone)(Nano-PLLA-b-PCL)with canine articular cells and its feasibility as a scaffold for the cartilage tissue engineering.[Methods]Nano-PLLA-b-PCL was made by liquid-liquid phase separation.Canine articular cells were isolated and multiplied in vitro.The passage 3 cells were seeded onto the PLLA-b-PCL films and cultured in the 2-dimensional environment.The cytotoxicity was measured with MTT assay.Cellular Morphological changes were observed by phase-contrast microscopy and Hoechst33342 fluorometric method.Another passage 3 cells were seeded onto the Nano-PLLA-b-PCL scaffolds(experiment group),PLLA-b-PCL scaffolds(control group)and cultured in the 3-dimensional environment for 3 weeks.The ratio of cell adhesion was detected by cell counting method.The morphological changes of cells were observed by scanning electron microscopy.The protein content in seeded cells were determined by bioinchoninic acid assay(BCA).The content of DNA was quantified using Hoechst33258 assay.[Results]MTT assay showed the PLLA-b-PCL had no cytotoxicity.The seeded cells adhered and proliferated well into the Nano-PLLA-b-PCL scaffolds,and they maintained good cell phenotype.After 21-day cell culture within the Nano-PLLA-b-PCL scaffolds,the chondrocyte DNA and protein contents increased with time.Moreover,the content of DNA and protein was higher in the experiment group than that in the control group,respectively(P
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[Objective]To study the effects of AO double plates in internal fixation of humeral intercondylar fractures and to analyze the causes of the disfunction in elbow.[Method]From March 2001 to March 2006,21 cases (male 14, female 7) of humeral intercondylar fractures were reviewed, with an average age of 35.3 years (ranged,15～64 years).Left elbow:9 cases;right elbow:12 cases.According to AO/ASIF classification,type C1∶6,type C2∶10, and type C3∶5,2 cases of all were open fractures. Posterior operating approach of elbow: trans-olecranon osteotomy or trans-triceps-side approach and internal fixation by standard method of AO double plates were performed in all cases.The patients began the active training of elbow joint as soon as possible postoperatively.[Result]Twenty-one cases were followed up for 8～38 months and the average time was 14.8 months.Bone union was obtained in all cases.One case complicated wound infection,it recovered after 3 weeks of dressing.And 1 case complicated with radial nerve injury,which recovered with neural nutrients.According to the Cassebaum scoring system,the effects were evaluated as excellent 7,good 10,and fair 4,and the excellent-good rate was 80.9%.[Conclusion]The application of AO double plates was proved to be a good method to treat humeral intercondylar fractures at present. But the severity of the bone comminution and soft-tissue lesion, the effective fixation,earlier active exercise were the main causes that influence the functional recovery of elbow joint.