ObjectiveTo observe the effect of using tungsten drills to prepare mouse knee osteochondral injury model by comparing with the needle modeling method, in order to provide an appropriate animal modeling method for osteochondral injury research.MethodsA total of 75 two-month-old male C57BL/6 mice were randomly divided into 3 groups (n=25). Mice in groups A and B were used to prepare the right knee osteochondral injury models by using needles and tungsten drills, respectively; group C was sham-operation group. The general condition of the mice was observed after operation. The samples were taken at 1 day and 1, 2, 4, and 8 weeks after modeling, and HE staining was performed. The depth, width, and cross-sectional area of the injury site at 1 day in groups A and B were measured, and the percentage of the injury depth to the thickness of the articular cartilage (depth/thickness) was calculated. Toluidine blue staining and immunohistochemical staining for collagen type Ⅱ were performed at 8 weeks, and the International Cartilage Research Society (ICRS) score was used to evaluate the osteochondral healing in groups A and B.ResultsAll mice survived to the completion of the experiment. HE staining showed that group C had normal cartilage morphology. At 1 day after modeling, the injury in group A only broke through the cartilage layer and reached the subchondral bone without entering the bone marrow cavity; the injury in group B reached the bone marrow cavity. The depth, width, cross-sectional area, and depth/thickness of the injury in group A were significantly lower than those in group B (P<0.05). At 1, 2, 4, and 8 weeks after modeling, there was no obvious tissue filling in the injured part of group A, and no toluidine blue staining and expression of collagen type Ⅱ were observed at 8 weeks; while the injured part of group B was gradually filled with tissue, the toluidine blue staining and the expression of collagen type Ⅱ were seen at 8 weeks. At 8 weeks, the ICRS score of group A was 8.2±1.3, which was lower than that of group B (13.6±0.9), showing significant difference (t=−7.637, P=0.000).ConclusionThe tungsten drills can break through the subchondral bone layer and enter the bone marrow cavity, and the injury can heal spontaneously. Compared with the needle modeling method, it is a better method for modeling knee osteochondral injury in mice.
Objective To explore heterotopic chondrogenesis of canine myoblasts induced by cartilage-derived morphogenetic protein 2 (CDMP-2) and transforming growth factor β1 (TGF-β1) which were seeded on poly (lactide-co-glycolide) (PLGA) scaffolds after implantation in a subcutaneous pocket of nude mice. Methods Myoblasts from rectus femoris of 1-year-old Beagle were seeded on PLGA scaffolds and cultured in medium containing CDMP-2 and TGF-β1 for 2 weeks in vitro. Then induced myoblasts-PLGA scaffold, uninduced myoblasts-PLGA scaffold, CDMP-2 and TGF-β1-PLGA scaffold, and simple PLGA scaffold were implanted into 4 zygomorphic back subcutaneous pockets of 24 nude mice in groups A, B, C, and D, respectively. At 8 and 12 weeks, the samples were harvested for general observation, HE staining and toluidine blue staining, immunohistochemical staining for collagen type I and collagen type II; the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were determined by RT-PCR, the glycosaminoglycans (GAG) content by Alician blue staining, and the compressive elastic modulus by biomechanics. Results In group A, cartilaginoid tissue was milky white with smooth surface and slight elasticity at 8 weeks, and had similar appearance and elasticity to normal cartilage tissue at 12 weeks. In group B, few residual tissue remained at 8 weeks, and was completely degraded at 12 weeks. In groups C and D, the implants disappeared at 8 weeks. HE staining showed that mature cartilage lacuna formed of group A at 8 and 12 weeks; no cartilage lacuna formed in group B at 8 weeks. Toluidine blue staining confirmed that new cartilage cells were oval and arranged in line, with lacuna and blue-staining positive cytoplasm and extracellular matrix in group A at 8 and 12 weeks; no blue metachromatic extracellular matrix was seen in group B at 8 weeks. Collagen type I and collagen type II expressed positively in group A, did not expressed in group B by immunohistochemical staining. At 8 weeks, the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were detected by RT-PCR in group A at 8 and 12 weeks, but negative results were shown in group B. The compressive elastic modulus and GAG content of group A were (90.79 ± 1.78) MPa and (10.20 ± 1.07) μg/mL respectively at 12 weeks, showing significant differences when compared with normal meniscus (P lt; 0.05). Conclusion Induced myoblasts-PLGA scaffolds can stably express chondrogenic phenotype in a heterotopic model of cartilage transplantation and represent a suitable tool for tissue engineering of menisci.
Objective To provide experimental evidence for the clinical application of ischemia therapy to treating pancreatic cancer. Methods After the model of pancreatic transplanted cancer was established in nude mice with orthotransplantation of human pancreatic cancer cell line into the pancreas, the ischemia of the right lobe of the pancreas was induced with ligation of the gastroduodenal, inferior pancreaticoduodenal and dorsal pancreatic arteries. Effects of regional ischemia on the growth of transplanted cancer and the pathomorphology of the transplanted cancer and pericancerous tissue were investigated. Results The transplanted cancer grew slower and its doubling time was longer in the ischemic group than in the control. On the 3rd, 7th and 14th day after operation, the size of transplanted cancer, the proliferative index and protein content of the cancer cells were significantly lower in the ischemic group than in the control (Ｐ＜0.01). Optical microscopy revealed large areas of coagulation necrosis, necrobiotic cells and the infiltration of inflammatory cells. The atrophy of acini, fibrosis and the infiltration of lymphocyte cells were found in pericancerous tissue. Conclusion Regional ischemia can destroy and inhibit the pancreatic transplanted cancer in nude mice effectively. The ischemia changes of pericancerous tissue may be unfavourable for the growth of the pancreatic transplanted cancer.
In this study, the role of newcastle disease virus (NDV) combined thermic solidified tumor vaccine in inhibiting growth of tumor and immune control was investigated, and rate of inhibiting tumor and cellular immunity were measured. The results showed that rate of inhibiting tumor in experimental group Ⅰ and Ⅱ were 24.8% and 41.1% respectively; average weight of tumor was significantly lower in both experimental groups than in control group, and activity of natural killing (NK) cells in experimental groups was higher than that in control group (Ｐ＜0.01). This suggests that NDV combined thermic solidified tumor vaccine can inhibit growth of tumor and improve activity of NK cells, and their effects are better than that of NDV.
In the development of radio frequency (RF) coils for better quality of the mini-type permanent magnetic resonance imager for using in the small animal imaging, the solenoid RF coil has a special advantage for permanent magnetic system based on analyses of various types of RF coils. However, it is not satisfied for imaging if the RF coils are directly used. By theoretical analyses of the magnetic field properties produced from the solenoid coil, the research direction was determined by careful studies to raise further the uniformity of the magnetic field coil, receiving coil sensitivity for signals and signal-to-noise ratio (SNR). The method had certain advantages and avoided some shortcomings of the other different coil types, such as, birdcage coil, saddle shaped coil and phased array coil by using the alloy materials (from our own patent). The RF coils were designed, developed and made for keeled applicable to permanent magnet-type magnetic resonance imager, multi-coil combination-type, single-channel overall RF receiving coil, and applied for a patent. Mounted on three instruments (25 mm aperture, with main magnetic field strength of 0.5 T or 1.5 T, and 50 mm aperture, with main magnetic field strength of 0.48 T), we performed experiments with mice, rats, and nude mice bearing tumors. The experimental results indicated that the RF receiving coil was fully applicable to the permanent magnet-type imaging system.
Objective To review the in vivo imaging research progress of two-photon microscopy (TPM) in spinal cord. Methods The recent literature concerning in vivo two-photon imaging of axon, microglia, and calcium in transgenic mice spinal cord was extensively consulted and reviewed. Results In vivo two-photon imaging of spinal cord provide dynamic information about axonal degeneration and regeneration, microglial accumulation, and calcium influx after spinal cord injury. Conclusion TPM in vivo imaging study on spinal cord will provide theoretical foundation for pathophysiologic process of spinal cord injury.
ObjectiveTo investigate the effect of circulating estrogen level on the outcome of free fat grafting in nude mice.MethodsEighteen female nude mice aged 6-8 weeks (weighing, 20-25 g) were randomly divided into 3 groups (n=6). The nude mice in the ovariectomized group were treated with ovariectomy. The nude mice in the high estrogen group and the normal estrogen group only made the same incision to enter the peritoneum without ovariectomy. The nude mice in the high estrogen group were given the estradiol (0.2 mg/g) every 3 days for 30 days. The other two groups were given the same amount of PBS every 3 days. At 30 days after operation, the tail vein blood of nude mice in 3 groups were detected by estradiol ELISA kit, and the free fat (0.3 mL) donated by the females was injected into the sub-scalp of nude mice. After 8 weeks of fat grafting, the samples were taken for gross observation and weighing, and the prepared slices were stained with HE staining, CD31-perilipin fluorescence staining, immunohistochemical staining of uncoupling protein 1 (UCP1), and immunofluorescence staining of estrogen receptor α. The diameter of adipocytes and vascular density of adipose tissue were measured. The mRNA expressions of UCP1 and estrogen receptor α were detected by realtime fluorescence quantitative PCR (qRT-PCR).ResultsAll nude mice survived during experiment. ELISA test showed that the concentration of estradiol significantly decreased in the ovariectomized group and increased in the high estrogen group compared with the normal estrogen group (P<0.05). At 8 weeks after fat grafting, the graft volume from large to small was ovariectomized group, normal estrogen group, and high estrogen group. There was significant difference in wet weight between the ovariectomized group and high estrogen group (P<0.05). Section staining showed that compared with the normal estrogen group, the adipocytes in the ovariectomized group were larger, the expression of peri-lipoprotein was weaker, the vascular density decreased, and the expressions of UCP1 was negative, and the estrogen receptor α positive cells reduced. The above observation results in the high estrogen group were contrary to those in the ovariectomized group. There were significant differences in the diameter of adipocytes, the vascular density of adipose tissue, the number of the estrogen receptor α positive cells between groups (P<0.05). The results of qRT-PCR showed that the mRNA expressions of UCP1 and estrogen receptor α significantly increased in the high estrogen group and decreased in the ovariectomized group compared with the normal estrogen group, and the differences were significant (P<0.05).ConclusionThe level of circulating estrogen has a significant effect on the outcome of free fat grafting in nude mice. Low estrogen level leads to hypertrophy of graft adipocytes, while high estrogen level leads to the production of a large amount of beige fat and high vascular density in fat grafts, which may be related to the activation of estrogen receptor α on adipocytes.
ObjectiveTo investigate the mechanism of muscle-derived cells (MDCs) in repairing sciatic nerve defects in mice by observing the early growth of damaged peripheral nerves.MethodsThe hind limb skeletal muscles of mice carrying enhanced green fluorescent protein (EGFP) was collected to extract and culture EGFP-MDCs to P1 generation for later experiments. Five-mm-long nerve defects were created in the right sciatic nerves of C57BL/6 mice to establish a peripheral nerve defect model. The two stumps of sciatic nerve were bridged with 7-mm-long polyurethane (PUR) conduit. For the MDC group, EGFP-MDCs were injected into the PUR conduit. The PUR group without EGFP-MDCs was used as the negative control group. At 1 and 2 weeks after operation, the proximal and distal nerve stumps of the surgical side were collected to generally observe the early growth of nerve. Immunofluorescence staining of S100β, the marker of Schwann cells, was performed on longitudinal frozen sections of nerve tissues to calculate the maximum migration distance of Schwann cells, and observe the source of the Schwann cells expressing S100β. Immunofluorescence staining of phosphorylated erb-b2 receptor tyrosine kinase 2 (p-ErbB2) and phosphorylated focal adhesion kinase (p-FAK) in transverse frozen sections of nerve tissue was performed to calculate the positive rates of both proteins.ResultsThe general observation showed that the proximal and distal stumps of the surgical side in PUR group were not connected at 1 and 2 weeks after operation, while the bilateral nerve stumps in the MDC group were connected at 2 weeks after operation. Immunofluorescence staining showed that the Schwann cells expressing S100β in proximal and distal nerve stumps of PUR group and MDC group was not connected at 1 week after operation. At 2 weeks after operation, the Schwann cells expressing S100β in the two nerve stumps of the MDC group were connected, but not in the PUR group. At 2 weeks after operation, the sum of the maximum migration distance of Schwann cells in the regenerated nerve in both two groups was significantly increased when compared with that in each group at 1 week after operation, and that of MDC group was significantly higher than that in the PUR group at both 1 and 2 weeks after operation, the differences were all significant (P<0.05). At 1 week after operation, the positive rates of p-ErbB2 and p-FAK in the proximal nerve stump of MDC group were significantly higher than those in PUR group (P<0.05). There was no significant difference in the positive rate of p-ErbB2 of proximal stump between the two groups at 2 weeks after operation (t=0.327, P=0.747), while the positive rate of p-FAK of MDC group was significantly higher than that of PUR group (t=4.470, P=0.000). At 1 and 2 weeks after operation, the positive rates of p-ErbB2 and p-FAK in the distal stump of MDC group were significantly higher than those in PUR group (P<0.05). At 1 and 2 weeks after operation, part of Schwann cells expressing S100β, which were derived from EGFP-MDCs, could be observed in the regenerated nerves of MDC group.ConclusionMDCs can promote the phosphorylation of ErbB2 and FAK in the nerve stumps of mice, and promote the migration of Schwann cells. MDCs can be differentiated into cells expressing the Schwann cell marker S100β, or as other cellular components, to involve in the early repair of peripheral nerves.
ObjectiveTo investigate the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on wound healing in diabetic mice.MethodsThe ADSCs were isolated from the adipose tissue donated by the patients and cultured by enzymatic digestion. The supernatant of the 3rd generation ADSCs was used to extract Exos (ADSC-Exos). The morphology of ADSC-Exos was observed by transmission electron microscopy. The membrane-labeled proteins (Alix and CD63) were detected by Western blot, and the particle size distribution was detected by nanoparticle tracking analyzer. The fibroblasts were isolated from the skin tissue donated by the patients and cultured by enzymatic digestion. The 5th generation fibroblasts were cultured with PKH26-labeled ADSC-Exos, and observed by confocal fluorescence microscopy. The effects of ADSC-Exos on proliferation and migration of fibroblasts were observed with cell counting kit 8 (CCK-8) and scratch method. Twenty-four 8-week-old Balb/c male mice were used to prepare a diabetic model. A full-thickness skin defect of 8 mm in diameter was prepared on the back. And 0.2 mL of ADSC-Exos and PBS were injected into the dermis of the experimental group (n=12) and the control group (n=12), respectively. On the 1st, 4th, 7th, 11th, 16th, and 21st days, the wound healing was observed and the wound healing rate was calculated. On the 7th, 14th, and 21st days, the histology (HE and Masson) and CD31 immunohistochemical staining were performed to observe the wound structure, collagen fibers, and neovascularization.ResultsADSC-Exos were the membranous vesicles with clear edges and uniform size; the particle size was 40-200 nm with an average of 102.1 nm; the membrane-labeled proteins (Alix and CD63) were positive. The composite culture observation showed that ADSC-Exos could enter the fibroblasts and promote the proliferation and migration of fibroblasts. Animal experiments showed that the wound healing of the experimental group was significantly faster than that of the control group, and the wound healing rate was significantly different at each time point (P<0.05). Compared with the control group, the wound healing of the experimental group was better. There were more microvessels in the early healing stage, and more deposited collagen fibers in the late healing stage. There were significant differences in the length of wound on the 7th, 14th, and 21st days, the number of microvessels on the 7th and 14th days, and the rate of deposited collagen fibers on the 14th and 21st days between the two groups (P<0.05).ConclusionADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.
ObjectiveTo investigate the effects of icariin (ICA) on serum bone turnover markers expressions and histological changes of cartilage and subchondral bone in mouse osteoarthritis (OA) model.MethodsEighty 8-week-old male C57BL/6J mouse were randomly divided into 8 groups (n=10). The OA model was established by anterior cruciate ligament transaction (ACLT). Group A: sham operation/early-stage normal saline administration; group B: sham operation/early-stage ICA administration; group C: ACLT/early-stage normal saline administration; group D: ACLT/early-stage ICA administration; group E: sham operation/late-stage normal saline administration; group F: sham operation/late-stage ICA administration; group G: ACLT/late-stage normal saline administration; group H: ACLT/late-stage ICA administration. Each animal received either ACLT or simply opening joint capsule, respectively. For groups B and D, ICA was given by gavage [10 mg/(kg·day)] on the first day after ACLT. For groups F and H, ICA was given with the same volume at 4 weeks after operation. The blood serum of the mouse was collected and prepared at 8 weeks after operation. Serum bone turnover markers and cytokines, including C-telopeptide of type I collagen (CTX), osteocalcin (OC), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1β, were measured by ELISA. Tissue samples from the knee were stained by alcian blue/hematoxylin & orange G (AB/H&OG). Histological changes of cartilage and subchondral bone were observed and evaluated by Osteoarthritis Research Society International (OARSI) scoring system.ResultsComparison between each group with early-stage administration (groups A, B, C, and D): Compared with groups A and B, the levels of CTX and OC in group C were significantly reduced (P<0.05); the levels of IL-6, TNF-α, and IL-1β and OARSI score was significantly increased (P<0.05). Compared with group C, the levels of CTX and OC in group D were significantly increased (P<0.05); the level of IL-6 was significantly reduced (P<0.05); the levels of TNF-α and IL-1β were not changed (P>0.05), and OARSI score was significantly reduced (P<0.05). Histological observation showed that the tibial cartilage loss was significantly improved. Comparison between each group with late-stage administration (groups E, F, G, and H): Compared with groups E and F, the levels of CTX and OC in group G were significantly reduced (P<0.05); the levels of IL-6, TNF-α, and IL-1β and OARSI score were significantly increased (P<0.05). Compared with group G, the level of CTX in group H were increased (P<0.05); the levels of OC, IL-6, TNF-α, and IL-1β and OARSI score were not changed (P>0.05). Histological observation showed that the tibial cartilage loss had no changes after late-stage ICA administration.ConclusionICA plays protective effects on subchondral bone, hyaline, and calcified cartilage. Meanwhile, ICA can improve bone remodeling in subchondral bone of OA to some extent. The consistent changes of serum bone markers and pathological morphology suggest that early intervention of ICA on OA is more effective.