Objectives To observe the changes of axonal transport in the rabbit optic nerve under acute ocular hypertensions. Methods 24 adult healthy New Zealand white rabbits were divided into 4 groups according to the intraocular pressure (IOP), with 6 rabbits in each group. There are 3 experimental groups with an IOP of 20, 30, 40 mm Hg (1 mm Hg=0.133 kPa). respectively, and 1 control group with an IOP of 10 -15 mm Hg.Two 25-gauge cannulas were inserted into each rabbitprime;s anterior chamber to create the model of acute ocular hypertension. At the beginning of the experiment, rhodamine-beta;-isothiocyanate (RITC) was injected into the vitreous of each eye to label axonal transport. After 3 hours of high intraocular pressure, rabbits were sacrificed with anesthetic overdose. The retina and the optic nerve were then carefully exposed. Fluorescent microscopy was used for quantitative measurements of the changes of optic nerve axonal transport. Statistically analyze the average grey level in different groups and sites by Leica RITC Q500IW image analysis software. Results RITC, a fluorescent tracer, was transported in the anterograde direction by axonal transport. With the increasing of the intraocular pressure, the distance of the axonal transport was declined (F=159.3, P＜0.05). The difference of the grey level in the pre-laminar region of 20, 30, 40 mm Hg group was not statistically significant (F=0.2545,P＞0.05 ). Compared the grey level of 40 mm Hg group with control group, the differences in lamina cribrosa（t=5.684）and the proximal 350 mu;m of the post-laminar (t=5.124) were statistically significant (P＜0.05). Compared the grey level of 20, 30 mm Hg group with control group, the difference was not statistically significant(t=1.747, P＞0.05 ).Conclusion 40 mmHg intraocular pressure lasts for 3 hours can reduce axonal transport in the lamina cribrosa and post laminar of optic nerve.
ObjectiveTo study the inducting differentiation effect of the sciatic nerve extracts on rabbit adipose-derived stem cells (ADSCs) in vitro. MethodsThe ADSCs were isolated from 2 healthy 4-month-old New Zealand rabbits (weighing, 2.0-2.5 kg) and cultured to passage 3, which were pretreated with 10 ng/mL basic fibroblast growth factor (bFGF) for 24 hours before induction. Then the induction media containing the extracts of normal sciatic nerve (group B) and injured sciatic nerve at 3, 7, and 14 days (group C, group D, and group E) were used, and D-Hank was used in group A as blank control group. The morphological changes of the cells were observed. At 7 days of induction, the gene expressions of neuron-specific enolase (NSE), nestin (NES), and S-100 were detected by real-time fluorescent quantitative PCR. The S-100 protein expression was tested by immunocytochemical staining. ResultsAt 4 days after induction, some ADSCs of groups C, D, and E showed the morphology of Schwann-like cells or neuron-like cells, the change of group D was more obvious; and the ADSCs of group A and B had no obvious change, which were still spindle. The S-100 immunocytochemical staining showed positive expression in groups C, D, and E (more obvious in group D) and negative expression in groups A and B. The gene expression of S-100 displayed time-dependent increases in groups C and D, which was significantly higher than that of groups A, B, and E (P<0.05), but no significant difference was found between groups C and D (P>0.05). The gene expression of NSE showed the same tendency to S-100, which reached the peak in group D; the gene expression of NSE in groups D and E was significantly higher than that of groups A, B, and C (P<0.05), and groups D and E showed significant difference (P<0.05). However, the gene expression of Nestin showed no significant difference among different groups (P>0.05). ConclusionThe ADSCs can be induced to differentiate into Schwann-like cells or neuron-like cells with sciatic nerve extracts; and the early stage (3-7 days) after injury is the best time for stem cell transplantation.
Objective To investigate the effectiveness of LARS ligament and three-dimensional (3D) printed prosthesis on the combined reconstruction of radial hemicarpal joint after distal radius tumor resection. Methods The clinical data of 12 patients with combined reconstruction of radial hemicarpal joint with LARS ligament and 3D printed prosthesis after distal radius tumor resection between September 2017 and March 2021 were retrospectively analyzed. There were 7 males and 5 females with an average age of 41.8 years (range, 19-63 years). There were 8 cases on the left side and 4 cases on the right side, and 10 cases of giant cell tumor of bone and 2 cases of osteosarcoma. The disease duration ranged from 1 to 20 months, with an average of 8.1 months. The osteotomy length, operation time, and intraoperative blood loss were recorded, and the wrist function was evaluated by Mayo wrist score and Musculoskeletal Tumor Society (MSTS) score before and after operation. The grip strength of the affected limb was expressed by the percentage of grip strength of the healthy upper limb, and the range of motion (ROM) of the wrist joint was measured, including extension, flexion, radial deviation, and ulnar deviation; the bone ingrowth and osseointegration at the bone-prosthesis interface of the wrist joint were observed by radiographic follow-up; the possible wrist complications were recorded. ResultsAll 12 patients successfully completed the operation. The osteotomy length was 5.0-10.5 cm (mean, 6.8 cm), and the operation time was 180-250 minutes (mean, 213.8 minutes). The intraoperative blood loss was 30-150 mL (mean, 61.7 mL). All patients were followed up 11-52 months (mean, 30.8 months). Radiographic follow-up showed that bone ingrowth and osseointegration at the bone-prosthesis interface were observed in all patients, and biological fixation was gradually achieved. During the follow-up, the stability, motor function, and ROM of the wrist joint were good. There was no complication such as arthritis, subluxation, prosthesis loosening, and infection, and no tumor recurrence and metastasis. At last follow-up, the Mayo score was 82.1±5.4, and MSTS score was 27.5±1.5, which were significantly improved when compared with those before operation (48.8±13.5, 16.4±1.4; t=−10.761, P<0.001; t=−26.600, P<0.001). The grip strength of the affected side was 59%-88% of that of the healthy side, with an average of 70.5%. The ROM of wrist joint were 55°-80° (mean, 65.42°) in extension, 35°-60° (mean, 44.58°) in flexion, 10°-25° (mean, 17.92°) in radial deviation, 10°-25° (mean, 18.33°) in ulnar deviation. Conclusion The combined application of LARS ligament and 3D printed prosthesis is an effective way to reconstruct bone and joint defects after distal radius tumor resection. It can improve the function of wrist joint, reduce the incidence of complications, and improve the stability of wrist joint.
Objective To fabricate a novel composite scaffold with acellular demineralized bone matrix/acellular nucleus pulposus matrix and to verify the feasibility of using it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties. Methods Pig proximal femoral cancellous bone rings (10 mm in external diameter, 5 mm in internal diameter, and 3 mm in thickness) were fabricated, and were dealed with degreasing, decalcification, and decellularization to prepare the annulus fibrosus phase of scaffold. Nucleus pulposus was taken from pig tails, decellularized with Triton X-100 and deoxycholic acid, crushed and centrifugalized to prepare nucleus pulposus extracellular mtrtix which was injected into the center of annulus fibrosus phase. Then the composite scaffold was freeze-dryed, cross-linked with ultraviolet radiation/carbodiimide and disinfected for use. The scaffold was investigated by general observation, HE staining, and scanning electron microscopy, as well as porosity measurement, water absorption rate, and compressive elastic modulus. Adipose-derived stem cells (ADSCs) were cultured with different concentrations of scaffold extract (25%, 50%, and 100%) to assess cytotoxicity of the scaffold. The cell viability of ADSCs seeded on the scaffold was detected by Live/Dead staining. Results The scaffold was white by general observation. The HE staining revealed that there was no cell fragments on the scaffold, and the dye homogeneously distributed. The scanning electron microscopy showed that the pore of the annulus fibrosus phase interconnected and the pore size was uniform; acellular nucleus pulposus matrix microfilament interconnected forming a uniform network structure, and the junction of the scaffold was closely connected. The novel porous scaffold had a good pore interconnectivity with (343.00 ± 88.25) µm pore diameter of the annulus fibrosus phase, 82.98% ± 7.02% porosity and 621.53% ± 53.31% water absorption rate. The biomechanical test showed that the compressive modulus of elasticity was (89.07 ± 8.73) kPa. The MTT test indicated that scaffold extract had no influence on cell proliferation. Live/Dead staining showed that ADSCs had a good proliferation on the scaffold and there was no dead cell. Conclusion Novel composite scaffold made of acellular demineralized bone matrix/acellular nucleus pulposus matrix has good pore diameter and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, and good biocompatibility, so it is a suitable scaffold for intervertebral disc tissue engineering.
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.
Objective To observe the chondrogenic differentiation of adipose-derived stem cells (ADSCs) by co-culturing chondrocytes and ADSCs. Methods ADSCs and chondrocytes were isolated and cultured from 8 healthy 4-month-old New Zealand rabbits (male or female, weighing 2.2-2.7 kg). ADSCs and chondrocytes at passage 2 were used. The 1 mL chondrocytes at concentration 2 × 104/mL and 1 mL ADSCs at concentration 2 × 104/mL were seeded on the upper layer and lower layer of Transwell 6-well plates separately in the experimental group, while ADSCs were cultured alone in the control group. The morphology changes of the induced ADSCs were observed by inverted phase contrast microscope. The glycosaminoglycan and collagen type II synthesized by the induced ADSCs were detected with toluidine blue staining and immunohistochemistry staining. The mRNA expressions of collagen type II, aggrecan, and SOX9 were detected with real-time fluorescent quantitative PCR. Results ADSCs in the experimental group gradually became chondrocytes-like in morphology and manifested as round; while ADSCs in the control group manifested as long spindle in morphology with whirlool growth pattern. At 14 days after co-culturing, the results of toluidine blue staining and immunohistochemistry staining were positive in the experimental group, while the results were negative in the control group. The results of real-time fluorescent quantitative PCR indicated that the expression levels of collagen type II, aggrecan, and SOX9 mRNA in the experimental group (1.43 ± 0.07, 2.13 ± 0.08, and 1.08 ± 0.08) were significantly higher than those in the control group (0.04 ± 0.03, 0.13 ± 0.04, and 0.10 ± 0.02) (P lt; 0.05). Conclusion ADSCs can differentiate into chondrocytes-like after co-culturing with chondrocytes.
Objective To observe the histological structure and cytocompatibility of novel acellular bone matrix (ACBM) and to investigate the feasibility as a scaffold for bone tissue engineering. Methods Cancellous bone columns were harvested from the density region of 18-24 months old male canine femoral head, then were dealt with high-pressure water washing, degreasing, and decellularization with Trixon X-100 and sodium deoxycholate to prepare the ACBM scaffold. The scaffolds were observed by scanning electron microscope (SEM); HE staining, Hoechst 33258 staining, and sirius red staining were used for histological analysis. Bone marrow mesenchymal stem cells (BMSCs) from canine were isolated and cultured with density gradient centrifugation; the 3rd passage BMSCs were seeded onto the scaffold. MTT test was done to assess the cytotoxicity of the scaffolds. The proliferation and differentiation of the cells on the scaffold were observed by inverted microscope, SEM, and live/dead cell staining method. Results HE staining and Hoechst 33258 staining showed that there was no cell fragments in the scaffolds; sirius red staining showed that the ACBM scaffold was stained crimson or red and yellow alternating. SEM observation revealed a three dimensional interconnected porous structure, which was the microstructure of normal cancellous bone. Cytotoxicity testing with MTT revealed no significant difference in absorbance (A) values between different extracts (25%, 50%, and 100%) and H-DMEM culture media (P gt; 0.05), indicating no cytotoxic effect of the scaffold on BMSCs. Inverted microscope, SEM, and histological analysis showed that three dimensional interconnected porous structure of the scaffold supported the proliferation and attachment of BMSCs, which secreted abundant extracellular matrices. Live/dead cell staining results of cell-scaffold composites revealed that the cells displaying green fluorescence were observed. Conclusion Novel ACBM scaffold can be used as an alternative cell-carrier for bone tissue engineering because of thoroughly decellularization, good mircostructure, non-toxicity, and good cytocompatibility.
【Abstract】 Objective To develop a novel cartilage acellular matrix (CACM) scaffold and to investigate its performance for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and gradient centrifugation and made into 3% suspension after acellularization treatment. After placing the suspension into moulds, 3-D porous CACM scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-l inked by exposure to ultraviolet radiation and immersion in a carbodiimide solution 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysucinimide. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorption rate analysis. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by conditioned medium including TGF-β1, canine BMSCs were seeded into the scaffold. Cell prol iferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed that there are no chondrocytefragments in the scaffolds and that toluidine blue, safranin O and anti-collagen II immunohistochemistry staining werepositive. The novel 3-D porous CACM scaffold had good pore interconnectivity with pore diameter (155 ± 34) μm, 91.3% ± 2.0% porosity and 2 451% ± 155% water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs indicated that cells covered the scaffolds uniformly and majority of the cells showed the round or ell iptic morphology with much matrix secretion. Conclusion The 3-D porous CACM scaffold reserved most of extracellular matrix after thoroughly decellularization, has good pore diameter and porosity, non-toxicity and good biocompatibil ity, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.