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Objective:To study the in vitro construction of functional and self-renewing cartilage organoids based on cartilage acellular extracellular matrix (ECM) microcarriers.Methods:Fresh porcine articular cartilage was taken. The merely crushed cartilage particles were set as natural cartilage group and ECM microcarriers of appropriate particle size, which were prepared by the acellular method of combining physical centrifugation and chemical extraction, were set as microcarrier group. Cartilage organoids were constructed by loading human umbilical cord mesenchymal stem cells (hUCMSCs) and human chondrocytes (hCho) with a ratio of 3∶1 with microcarriers through a rotating bioreactor. The organoids with different induction times were divided into 0-, 7-, 14-, and 21-day induction groups. The cell residues of the microcarrier group and natural cartilage group were evaluated by 4′, 6-diaminidine 2-phenylindole (DAPI) fluorescence staining and DNA quantitative analysis. The retention of microcarrier components was observed by Safranin O and toluidine blue stainnings, and the collagen and glycosaminoglycan (GAGs) levels in the microcarrier group and the natural cartilage group were determined by colorimetric method and dimethyl-methylene blue (DMMB) method. The microcarriers were further characterized by scanning electron microscopy and energy dispersive spectroscopy. The hUCMSCs cultured with Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with fetal bovine serum (FBS) in a volume fraction of 10% was used as the control group and the hUCMSCs cultured with the microcarrier extract was used as the experimental group. Subgroups of hUCMSCs cultured at 3 time points: 1, 3 and 5 days were set up in the two groups separately. Cell Counting Kit 8 (CCK-8) was used to detect the biocompatibility of the two groups. The cellular activity of the organoids of the 0-, 7-, 14-, and 21-day induction groups was detected by live/dead staining and the self-renewal ability of the cartilage organoids of the 14-day induced group was identified by Ki67 fluorescence staining. The organoids of the 7-, 14-, and 21-day induction groups were detected by RT-PCR in terms of the expression levels of chondrogenesis-related marker aggrecan (ACAN), type II collagen (COL2A1), SRY-related high mobility group-box gene-9 (SOX9), cartilage hypertrophy-and mineralization-related marker type I collagen (COL1A1), Runt-related transcription factor-2 (RUNX2), and osteocalcin (OCN). Colorimetric and DMMB assays were performed to determine the ability of organoids in the 0-, 7-, 14-, and 21-day induction groups to secrete collagen and GAGs.Results:The results of DAPI fluorescent staining showed that the natural cartilage group had a large number of nuclei while the microcarrier group hardly had any nuclei. The DNA content of the microcarrier group was (7.8±1.8)ng/mg, which was significantly lower than that of the natural cartilage group [(526.7±14.7)ng/mg] ( P<0.01). Saffranin O and toluidine blue staining showed that the microcarrier was dark- and uniform-colored and it kept a lot of cartilage ECM components. The collagen and GAGs contents of the microcarrier group were (252.9±1.4)μg/mg and (173.4±0.8)μg/mg, which were significantly lower than those of the natural cartilage group [(311.9±2.2)μg/mg and (241.3±0.7)μg/mg] ( P<0.01). Scanning electron microscopy showed that the surface of the microcarriers had uneven and interleaved collagen fiber network. The results of energy spectrum analysis showed that elements C, O and N were evenly distributed in the microcarriers, indicating that the composition of the microcarrier was uniform. The microcarrier had good biocompatibility and there was no statistical significance in the results of CCK-8 test between the control group and the experimental group after 1 and 3 days of culture ( P>0.05). After 5 days of culture, the A value of the experimental group was 0.53±0.02, which was better than that of the control group (0.44±0.03) ( P<0.05). In the 0-, 7-, 14-, and 21-day induction groups, hUCMSCs and hCho were attached to the surface of the microcarriers, with good cellular activity, and the live/death rates were (70.6±1.1)%, (80.5±0.6)%, (94.5±0.9)%, and (90.8±0.5)% respectively ( P<0.01). There were a large number of Ki67 positive cells in cartilage organoids. RT-PCR showed that the expression levels of ACAN, COL2A1, SOX9, COL1A1, RUNX2 and OCN were 1.00±0.09, 1.00±0.24, 1.00±0.18, 1.00±0.03, 1.00±0.06 and 1.00±0.13 respectively in the 7-day induction group; 4.16±0.28, 5.09±1.25, 5.65±1.05, 0.47±0.01, 1.68±0.02 and 0.21±0.06 respectively in the 14-day induction group; 13.42±0.92, 3.07±0.21, 1.84±1.08, 2.72±0.17, 2.91±0.18 and 3.32±1.20 respectively in the 21-day induction group. Compared with the 7-day induction group, the expression levels of ACAN, COL2A1, SOX9 and RUNX2 in the 14-day group were increased ( P<0.05), but COL1A1 expression level was decreased ( P<0.05), with no significant difference in OCN expression level ( P>0.05). Compared with the 7-day induction group, the expression levels of ACAN, COL1A1 and RUNX2 in the 21-day induction group were significantly increased ( P<0.01), with no significant differences in the expression levels of COL2A1, SOX9 and OCN ( P>0.05). Compared with the 14-day induction group, the expression levels of ACAN, COL1A1, RUNX2 and OCN in the 21-day group were increased ( P<0.05 or 0.01), with no significant difference in the expression level of COL2A1 ( P>0.05), but the expression level of SOX9 was decreased ( P<0.05). The contents of collagen in 0-, 7-, 14-and 21-day induction groups were (219.15±0.48)μg/mg, (264.07±1.58)μg/mg, (270.83±0.84)μg/mg and (280.01±0.48)μg/mg respectively. The GAGs contents were (171.18±1.09)μg/mg, (184.06±1.37)μg/mg, (241.08±0.84)μg/mg and (201.14±0.17)μg/mg respectively. Compared with the 0-day induction group, the contents of collagen and GAGs in 7-, 14-, and 21-day induction groups were significantly increased ( P<0.01), among which the content of collagen was the lowest in 7-day induction group ( P<0.01) but the highest in the 21-day induced group ( P<0.01); the content of GAGs was the lowest in the 7-day induced group ( P<0.01) but the highest in the 14-day induction group ( P<0.01). Conclusions:The microcarriers prepared by combining physical and chemical methods are decellularized successfully, with more matrix retention, uniform composition and on cytotoxicity. By loading microcarriers with hUCMSCs and hCho, cartilage organoids are successfully constructed in vitro, which are characterized by good cell activity, self-renewal ability, strong expression of genes related to chondrogenesis and secretion of collagen and GAGs. The cartilage organoids constructed at 14 days of induction have the best chondrogenic activity.
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Objective To observe biomechanical characteristics and advantages of the self-developed anatomical locking plate of sternoclavicular joint by comparison with the radial distal oblique T-shaped locking plate and sternoclavicular hook plate. Methods Nine embalming and moistening adult corpses were selected, including 6 males and 3 females. Model of complete dislocation of the sternoclavicular joint caused by complete removal of the sternoclavicular joint specimen. The bilateral sternoclavicular joints of 9 specimen models were randomly numbered, matched and divided into Group A(experimental group, sternoclavicular joint anatomical locking plate), Group B(control group 1, the radial distal oblique T-shaped locking plate) and Group C(control group 2, sternoclavicular hook plate), with 6 sternoclavicular joints in each group. All specimens were placed with steel plates on both sides and fixed on the universal mechanical test machine. Three biomechanical experiments were carried out, including loading of distal clavicle, torsion of distal clavicle and anti-pull out of sternum handle screw. Results Distal clavicle loading test: the load-displacement of specimens in three groups showed a linear relationship. The compressive deformation resistance in experimental group was stronger than that in two control groups. Distal clavicle torsion test: the relationship between torque and torsion angle was linear. The torsional deformation resistance in experimental group was stronger than that in two control groups. Anti-pullout test of sternum handle screw: there was a significant difference in the maximum anti-pullout force of sternum handle screw among the three groups (P＜0.05). The anti-pullout performance of the sternum handle screw in the experimental group was better than that in the two control groups. Conclusions The self-developed sternoclavicular joint anatomical locking plate is superior to the oblique T-shaped locking plate of distal radius and the plate of sternoclavicular hook in terms of anti-compression, anti-torsion and anti-pullout of sternoclavicular screw, so as to provide an ideal internal fixation device for the treatment of fracture and dislocation of sternoclavicular joint.
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BACKGROUND:Acellular nerve scaffolds have the three-dimensional structure of natural nerves and low immunogenicity,but their effect on long nerve defects is still not ideal.Therefore,it is necessary to construct tissueengineered nerve using acellular nerve and seed cells in order to improve the therapeutic effect.OBJECTIVE:To systemically review the efficacy of combination of acellular nerve grafts (ANGs) and mesenchymal stem cells (MSCs) or Schwann cells (SCs) transplantation in the treatment of sciatic nerve defects in a rat model.METHODS:Randomized controlled trials (RCTs) about the effects of combination of ANGs and MSCs or SCs transplantation for sciatic nerve defects in rats were searched in PubMed,The Cochrane Library,EMbase,CNKI,WanFang and VIP from inception to July 2016.Three reviewers independently screened literature according to the inclusion and exclusion criteria,extracted data,and assessed the risk of bias of included studies.Then,a Meta-analysis was performed using Review Manger5.3 software.RESULTS AND CONCLUSION:A total of 10 RCTs involving 252 rats were included.The results of meta-analysis showed that:compared with the control group (simple acellular nerve scaffold group),the sciatic functional index (SFI) of the combined group (combination of ANGs and MSCs or SCs transplantation) were superior at 2 weeks [SMD=2.73,95％ CI (1.92,3.45),P ＜ 0.000 01],4 weeks [SMD=4.57,95％ CI (3.43,5.70),P ＜ 0.000 01],6 weeks [SMD=1.62,95％CI (0.18,3.06),P=-0.03],8 weeks [SMD=4.90,95％ CI (2.96,6.84),P ＜ 0.000 01] after surgery.The nerve conduction velocity [SMD=1.39,95％ CI (0.99,1.78),P ＜ 0.000 01),latency period (MD=-0.98,95％ CI (-1.19,-0.76),P ＜ 0.000 01],and amplitude [SMD=1.23,95％ CI (0.62,1.85),P ＜ 0.000 1] were superior at 12 weeks after surgery.The myelin sheath thickness was superior at 8 weeks [MD=0.14,95％ CI (0.07,0.21),P ＜ 0.000 1],12 weeks [SMD=1.85,95％ CI (1.63,2.08),P ＜ 0.000 01] and the number of myelinated nerve fibers were superior at 12 weeks [SMD=3.59,95％CI (2.63,4.55),P ＜ 0.000 01] after surgery.The gastrocnemius wet weight was superior at 8 weeks after surgery [SMD=4.22,95％ CI (2.40,6.03),P ＜ 0.000 01].Current evidence indicates that the combination of ANGs and MSCs or SCs can promote the regeneration and functional recovery of the peripheral nerve.Due to the limited quality of the included studies,the above conclusion should be verified by conducting high-quality and large-scale RCTs.
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Objective To evaluate the effect of tissue-engineered nerve graft combined with tetramethylpyrazine in the repair of ischiadic nerve defect in rats.Methods Fifty-five adult SD rats were allocated into groups A (n =15),B (n =15),C (n =15) and D (n =10),according to the random number table.All experimental sciatic nerves were at the right hind side.After the rats were anesthetized with 10 g/L pentobarbital through abdominal injection,a 1.5 cm nerve sciatic nerve was excised at the point 0.5 cm away from the lower margin of the piriformis.Four grafts were used to bridge the nerve defect,including tissue-engineered nerve transplanted neural stem cells (NSCs) that were cultured in medium containing tetramethylpyrazine for 1 week (group A),tissue-engineered nerve transplanted NSCs that were cultured in tetramethylpyrazine-free medium for 1 week (group B),acellular nerve allograft (group C),and nerve autograft (group D).After operation,group A was administered tetramethylpyrazine for 12 weeks in the operative site.For other groups,the same volume of normal saline was used instead of tetramethylpyrazine.Repair results were detected with the sciatic function index (SFI),nerve electrophysiological evaluation,fluorescent microscopy,horseradish peroxidas (HRP) retrograde tracer and nerve fiber regeneration study.Results At postoperative 2 weeks,no differences were found in SFI and electrophysiological evaluation among the groups,there was an intensive fluorescence in groups A and B and no fluorescence in group C,and large nestin-positive cells were observed in groups A and B.At postoperative 12 weeks,SFI was-17.52 ± 2.41 in group A,-25.74 ± 2.85 in group B,-36.12 ± 3.41 in group C and-15.87 ± 2.26 in group D;electrophysiological evaluation showed nerve conduction velocity of (9.43 ± 0.40) m/s in group A,(7.76 ± 0.31) m/s in group B,(5.87 ± 0.67) m/s in group C and (10.16 ± 0.39) m/s in group D;fluorescence microscope and histological staining showed NSCs could survive in vivo,migrate and differentiate into neurons and gliocytes;HRP-positive neurons were 885.40 ± 19.91 in group A,684.57 ± 38.37 in group B,390.33 ± 43.41 in group C and 941.67 ± 32.54 in group D;regenerated nerve fibers,often myelinated were large in group A.All the measures in group A were better compared to groups B and C (P ＜ 0.05),but didn't differ from those in group D (P ＞0.05).Conclusion Tissue-engineered nerve transplanted NSCs cultured in medium including tetramethylpyrazine can effectively repair rat sciatic nerve defect,and promote peripheral nerve regeneration and lower limb motor function recovery.