Objective To evaluate the influence of PKH26 labeling on the biological function of the goat nucleus pulposus cells and the biological function of seeded cells in nude mice by in vivo imaging techonology. Methods Primary nucleus pulposus cells were isolated by enzymatic digestion from the nucleus pulposus tissue of the 1-year-old goat disc. The nucleus pulposus cells at passage 1 were labeled with PKH26 and the fluorescent intensity was observed under the fluorescence microscopy. The labeled cells were stained with toluidine blue and collagen type II immunocytochemistry. The cells viability and proliferation characteristics were assessed by trypan blue staining and MTT assay, respectively. Real-time fluorescent quantitative PCR was used to detect the gene expressions of collagen types I and II, and aggrecan. The fluorescent intensity and scope of the nucleus pulposus cells-scaffold composite in vivo for 6 weeks after implanting into 5 6-week-old male nude mice were measured by in vivo imaging technology. Results Primary nucleus pulposus cells were ovoid in cell shape, showing cluster growth, and the cells at passage 1 showed chondrocyte-like morphology under the inverted phase contrast microscope. The results of toluidine blue and collagen type II immunocytochemistry staining for nucleus pulposus cells at passage 1 were positive. The fluorescent intensity was even after labeling, and the cell viability was more than 95% before and after PKH26 labeling. There was no significant difference in cell growth curve between before and after labeling (P gt; 0.05). The real-time fluorescent quantitative PCR showed that there was no significant difference in gene expressions of collagen types I and II, and aggrecan between before and after labeling (P gt; 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology. Conclusion The PKH26 labeling has no effect on the activity, proliferation, and cell phenotype gene expression of the nucleus pulposus cells. A combination of PKH26 labeling and in vivo imaging technology can track the biological behavior of the cells in vivo.
ObjectiveTo evaluate the most efficient method for transfection of human umbilical cord mesenchymal stem cells (HUMCSs) in vivo. MethodsHUCMSCs were isolated from human umbilical cord and cultured, which were labelled by PKH26 and lentivirus-GFP, then were observed by using a fluorescence microscope. Sixty SD rats were randomly divided into PKH26 transfection group and lentivirus-GFP transfection group. The right hepatic lobe of rat was resected, then the transfected stem cells were injected into portal vein. The rats were sacrificed on day 3, 8, and 13 after transfection. The liver specimens were observed by using a fluorescence microscope. Flow cytometry was used to evaluate the percentage of transfected stem cells and the apoptotic stem cells. ResultsThe third generation of HUCMSCs labelled by PKH26 and lentivirus-GFP were spindle shaped. PKH26 red dye was evenly distributed in the cell membrane of HUCMSCs and could be clearly labelled. The HUCMSCs labelled by lentivirus-GFP were green fluorescence under the fluorescence microscope, and it was clear and stable. The HUCMSCs were clear and could be clearly distinguished on day 3 after transfection by two methods in vivo. As the time went by, red was faded and blurred, then was gradually disappeared on day 13 after transfection in the HUCMSCs stansfected by PKH26; but the color in the HUCMSCs stansfected by lentivirus-GFP were clear at all the time points. The transfection rate of the lentivirus-GFP was significantly higher that that of the PKH26 (P < 0.05), the rate of apoptotic stem cells had no significant differences at all the time points between these two groups (P > 0.05). ConclusionLentivirus-GFP transfection is a higher efficient method for stem cell labelling in vivo, it could be used to observe transplantation cells for a long time in future.
ObjectiveTo review the researches of in vivo kinematics in lumbar degenerative spondylol isthesis (DS). MethodsRelated literature concerning the in vivo kinematics in patients with lumbar DS was extensively reviewed and comprehensively analyzed in 4 terms of the instabil ity of lumbar DS, vertebral motion pattern, the morphological changes of spinal canal, and intraoperative biomechanical measurement. ResultsWhether there is lumbar segmental instabil ity in lumbar DS patients is still controversial, which should be based on degenerative stage of lumbar spine and grade of slip. The hypomobility of the lumbar spinous processes and the facet joint is seen in DS. The diameter, cross-sectional area, and volume of spinal canal in lumbar DS patients are significantly smaller than those of the normal control. Because of its invasive procedure and medical ethics, the use of the intraoperative measurement device is limited. These reported researches of in vivo kinematics in DS are almost on the sagittal plane. However, few data have been reported on the 6-degree-of-freedom (6DOF) kinematics of the diseased levels under physiological loading conditions. ConclusionThe 6DOF kinematics data can accurately reflect the segmental motion characteristics in lumbar DS patients, recent studies have been reported, further studies are still needed.
ObjectiveTo review the application and research progress of in vivo bioreactor as vascularization strategies in bone tissue engineering. MethodsThe original articles about in vivo bioreactor that can enhance vascularization of tissue engineered bone were extensively reviewed and analyzed. ResultsThe in vivo bioreactor can be created by periosteum, muscle, muscularis membrane, and fascia flap as well as biomaterials. Using in vivo bioreactor can effectively promote the establishment of a microcirculation in the tissue engineered bones, especially for large bone defects. However, main correlative researches, currently, are focused on animal experiments, more clinical trials will be carried out in the future. ConclusionWith the rapid development of related technologies of bone tissue engineering, the use of in vivo bioreactor will to a large extent solve the bottleneck limitations and has the potential values for clinical application.
This paper investigates the variation of lung tissue dielectric properties with tidal volume under in vivo conditions to provide reliable and valid a priori information for techniques such as microwave imaging. In this study, the dielectric properties of the lung tissue of 30 rabbits were measured in vivo using the open-end coaxial probe method in the frequency band of 100 MHz to 1 GHz, and 6 different sets of tidal volumes (30, 40, 50, 60, 70, 80 mL) were set up to study the trends of the dielectric properties, and the data at 2 specific frequency points (433 and 915 MHz) were analyzed statistically. It was found that the dielectric coefficient and conductivity of lung tissue tended to decrease with increasing tidal volume in the frequency range of 100 MHz to 1 GHz, and the differences in the dielectric properties of lung tissue for the 6 groups of tidal volumes at 2 specific frequency points were statistically significant. This paper showed that the dielectric properties of lung tissue tend to vary non-linearly with increasing tidal volume. Based on this, more accurate biological tissue parameters can be provided for bioelectromagnetic imaging techniques such as microwave imaging, which could provide a scientific basis and experimental data support for the improvement of diagnostic methods and equipment for lung diseases.
Objective To investigate the sensitivity of 5 kinds of chemotherapeutic drugs on human colorectal cancer in vivo. Methods Xenografts in nude mice were set up by tumor tissues from 9 patients with colorectal cancer and nude mice were divided into 6 groups randomly， receiving saline （control group）， 5-fluorouracil （5-FU group）， doxorubicin（ADM group）， mitomycin （MMC group）， oxaliplatin （LOHP group）， and irinotecan （CPT-11 group）， respectively. The inhibitive rates （IR） of xenografts in 5 groups for each patient were calculated. Results The lowest and highest IR of 5 groups were 23.6% and 54.9% in 5-FU group， 23.7% and 69.5% in LOPH group， 23.6% and 82.6% in CPT-11group， 24.1% and 48.1% in MMC group， 5.8% and 20.7% in ADM group， respectively. The IR exceeded 40.0% in 7 patients of LOHP group， 6 patients of CPT-11 group， 4 patients of 5-FU group， and 1 patient of MMC group， respec-tively. Of 9 patients， the IR exceeded 40.0% to 3 kinds of drugs in 3 patients， to 2 kinds of drugs in 4 patients， the IR didn’t exceed 30.0% to 4 kinds of drug （IR was 82.6% to CPT-11） in 1 patient， and the IR didn’t exceed 31.0% to all 5 kinds of drugs in 1 patient. There were statistical differences on the IR of 5 kinds of drugs （H=24.061 2， P=0.000 1）. IR of ADM group was statistical lower than 5-FU group， MMC group， LOHP group， and CPT-11 group （P＜0.05），but there were no statistical differences between 5-FU group， MMC group， LOHP group， and CPT-11 group （P＞0.05）. Conclusions The xenografts from same patient have different sensitivity to different chemotherapy drugs， and the same chemotherapy drug corresponds to different IR in different patients. The IR of LOHP and CPT-11 are the highest， following by 5-FU and MMC.
ObjectiveTo review the research progress of in vivo bioreactor (IVB) for bone tissue engineering in order to provide reference for its future research direction.MethodsThe literature related to IVB used in bone tissue engineering in recent years was reviewed, and the principles of IVB construction, tissue types, sites, and methods of IVB construction, as well as the advantages of IVB used in bone tissue engineering were summarized.ResultsIVB takes advantage of the body’s ability to regenerate itself, using the body as a bioreactor to regenerate new tissues or organs at injured sites or at ectopic sites that can support the regeneration of new tissues. IVB can be constructed by tissue flap (subcutaneous pocket, muscle flap/pocket, fascia flap, periosteum flap, omentum flap/abdominal cavity) and axial vascular pedicle (axial vascular bundle, arteriovenous loop) alone or jointly. IVB is used to prefabricate vascularized tissue engineered bone that matched the shape and size of the defect. The prefabricated vascularized tissue engineered bone can be used as bone graft, pedicled bone flap, or free bone flap to repair bone defect. IVB solves the problem of insufficient vascularization in traditional bone tissue engineering to a certain extent.ConclusionIVB is a promising method for vascularized tissue engineered bone prefabrication and subsequent bone defect reconstruction, with unique advantages in the repair of large complex bone defects. However, the complexity of IVB construction and surgical complications hinder the clinical application of IVB. Researchers should aim to develop a simple, safe, and efficient IVB.
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 design a method for observing pulmonary microcirculation in vivo in a native tissue environment, due to the high incidence of lung diseases and the advantages of animal experiments in vivo.MethodsTen BALB/c male mice were randomly divided into group A and group B, with five mice in each group. A self-made apparatus was used to control the movement towards local lung tissues in order to get a stabilized observation plane, and then a 5-minute video was shot with laser confocal scanning microscope. During the filming, the mice in group A were injected with fluorescein isothiocyanate-dextran via the tail vein, and the mice in group B were injected with green fluorescent protein-platelets (extracted from the blood of tie2-cre&rosa26-tomato-EGFP transgenic black C57 male mice). The data of group A was used to observe the lungs perfusion and the damage to tissue by this method, and the data of group B was used to observe the movement of platelets.ResultsImage of lung structure obtained by this method was clear and stable. Mean areas of alveolus in an instant and at the 30th, 60th, 120th, 180th, and 300th second were (1 603±181), (1 588±183), (1 528±363), (1 506±353), (1 437±369), (1 549±307) μm2, respectively, and there were no significant differences between each time point (P>0.05). The video was smooth, the rapid movement of platelets was recorded and the particles were clear and without tailing; after the observation, hematoxylin-eosin staining showed no obvious damage to the lung tissue.ConclusionThe method can be used for the observation and research of the lung microcirculatory system in the living state of the mouse, and provides a methodological basis for studies of other lung diseases in vivo.
Objective To provide a ventricular assist device for patients with heart failure, Fu Wai （FW） axial blood pump was developed for partly or totally to assist the left ventricular function. Vitro hemolysis and animals tests were also employed to test the hydromechanics and hemocompatibility of the FW left ventricular assist devices developed in Fu Wai hospital. Methods Using vitro test loop, FW axial blood pump has been used to evaluate the performance of hemolysis， the pump has also been tested for hemolysis characteristic through five sheep experiments. Results At 8 400 r/min, the pump generates 5 L/min flow against 100 mm Hg, the normalized index of hemolysis (NIH) was0.17±0.06 mg/L. The plasma free hemoglobin of in vivo tests was around 30 mg/dl. Conclusion The results obtained in vitro and in vivo testing indicate an acceptable design for the blood pump, further in vivo tests will be performed before clinical use.