ObjectiveTo study the ectopic osteogenesis and biocompatibility of bone morphogenetic protein 2 (BMP-2)-derived peptide P24 loaded chitosan-4-thio-butylamidine (CS-TBA) hydrogel.MethodsFirst, the CS-TBA/hydroxyapatite (HA) solution was prepared by using chitosan, 2-iminothiolane hydrochloride, and HA. Then, the different amount of P24 peptides were added to the CS-TBA/HA to prepare the CS-TBA/5%P24/HA and CS-TBA/10%P24/HA solutions. Finally, β-glycerophosphate disodium (β-GP) was added to the CS-TBA/HA, CS-TBA/5%P24/HA, and CS-TBA/10%P24/HA to prepare the CS-TBA/HA/β-GP, CS-TBA/5%P24/HA/β-GP, and CS-TBA/10%P24/HA/β-GP hydrogels, respectively. Eighteen Sprague Dawley female rats were randomly divided into 3 groups (n=6), which were injected into the back muscle pouches with equal volume CS-TBA/HA/β-GP hydrogel (group A), CS-TBA/5%P24/HA/β-GP hydrogel (group B), and CS-TBA/10%P24/HA/β-GP hydrogel (group C). The animals were sacrificed at 4 and 8 weeks and conducted micro-CT. The ability of biodegradation and osteogenesis of hydrogl was detected by trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and histological staining (HE and Masson).ResultsAll the rats survived to the time point of the harvest. Micro-CT results showed that the new bones gradually increased in each group after operation. At the same time, the new bone formation was more obvious in groups B and C than in group A, and with the increase of P24 concentration, new bone formation in group C was much more than that in group B. The Tb.Th, Tb.N, and BMD increased gradually in 3 groups, and the differences between 4 and 8 weeks were significant (P<0.05) except the Tb.Th in group A. At different time points, the Tb.Th, Tb.N, and BMD were significantly higher in groups B and C than in group A (P<0.05), and in group C was higher than in group B (P<0.05), showing significant differences between groups. Histological staining showed that the materials of groups B and C were biodegradable, and the osteogenic effect was increased with the increase of P24 concentration.ConclusionP24 peptide can improve the ectopic osteogenesis of CS-TBA hydrogel, and the 10% concentration is more effective.
Objective To study the effect of sodium hyaluronate hydrogel in treating residual cavity on body surface after abscess drainage so as to provide new method to speed up the heal ing of residual cavity after body surface abscess drainageand reduce the frequency of dressing change and cl inic nursing workload. Methods From June 2007 to March 2008, 60 outpatients with body surface abscess drainage were randomly divided into hydrogel group (group A, 30 cases) and the control group (group B, 30 cases). In group A, there were 16 males and 14 females aged (49.5 ± 6.1) years, the disease course was (3.8 ± 0.6) days, and the volume of residual cavity was (4.19 ± 1.31) mL. In group B, there were 18 males and 12 females aged (50.2 ± 7.6) years, the disease course was (4.3 ± 0.5) days, and the volume of residual cavity was (4.04 ± 1.22) mL. There was no significant difference between two groups in gender, age, disease course and volume of residual cavity (P gt; 0.05). Residual cavity was smeared with 1 mL/cm2 sodium hyaluronate hydrogel in group A and drained by sal ine gauze in group B, the dressing was changed every two to three days. Residual cavity volume was recorded every four days, and the residual cavity volume, the frequency of out-patient dressing and the heal ing time residual of cavity were compared. Results The volume of residual cavity was (3.11 ± 1.12), (1.75 ± 0.95) and (0.55 ± 0.56) mL in group A, and was (3.39 ± 1.12), (2.64 ± 0.99) and (1.81 ± 0.81) mL in group B at 4, 8 and 12 days after treatment respectively, showing no significant differences at 4 days (P gt; 0. 05), but significant difference at 8 and 12 days (P lt; 0.01). Residual cavity heal ing time was (12.70 ± 2.78) days in group A and (20.27 ± 3.89) days in group B, and the frequency of dressing change was 5.53 ± 1.33 in group A and 9.13 ± 1.81 in group B, indicating significant differences between two groups (P lt; 0.01). Conclusion Sodium hyaluronate hydrogel can promote residual cavity heal ing, reduce the frequency of dressing change of out-patient and decrease the cl inic nursing care workload.
To evaluate the effect of deacetylation degree (DDA) on the gelation behavior of thermosensitive chitosan-β glycerol phosphate disodium salt pentahydrate (CH-GP) system and to compare their rheological behaviors before and after gelation. Methods A series of thermosensitive CH-GP samples with different DDAs (70%, 85%, 90%, 97%)were prepared by dissolving CH with 0.1 mol/L HCl solution, 5 samples for every single DDA, and then all these CH-GP solution samples processed the frequency sweep test and temperature sweep test (10-70℃ , 1℃ /min) on AR 2000ex rheometer, with pH value of 7.02. Also, all the results of hydrogel samples were processed a frequency sweep test. Results With CH concentration of 2% (w/v) and pH value of 7.02 , the gelating temperature of CH-GP systems with different DDAs (85%, 90%, 97%) were (59.90 ± 0.08), (48.10 ± 0.08), (37.10 ± 0.11) ℃ , respectively. While the gelating temperature of CH-GP system with 70% DDA was over 70℃ . There were statistically significant differences in temperature and time of gelation among groups with different DDAs (P lt; 0.05). Furthermore, storage modulus of such system raised from dozens Pa to a magnitude of several kPa during gelation , while loss modulus kept almost steady. Conclusion Gelating temperature and mechanical property of the system could be measured objectively by rheological characterization. Thus during designing tissue engineered scaffolds for various purposes, it is helpful applying selected CH with optimal DDA to different target tissues.
Objective
To investigate the application potential of alginate-strontium (Sr) hydrogel as an injectable scaffold material in bone tissue engineering.
Methods
The alginate-Sr/-calcium (Ca) hydrogel beads were fabricated by adding 2.0wt% alginate sodium to 0.2 mol/L SrCl2/CaCl2 solution dropwise. Microstructure, modulus of compression, swelling rate, and degradability of alginate-Sr/-Ca hydrogels were tested. Bone marrow mesenchymal stem cells (BMSCs) were isolated from femoral bones of rabbits by flushing of marrow cavity. BMSCs at passage 5 were seeded onto the alginate-Sr hydrogel (experimental group) and alginate-Ca hydrogel (control group), and the viability and proliferation of BMSCs in 2 alginate hydrogels were assessed. The osteogenic differentiation of cells embeded in 2 alginate hydrogels was evaluated by alkaline phosphate (ALP) activity, osteoblast specific gene [Osterix (OSX), collagen type I, and Runx2] expression level and calcium deposition by fluorescent quantitative RT-PCR and alizarin red staining, Von Kossa staining. The BMSCs which were embeded in alginate-Ca hydrogel and cultured with common growth medium were harvested as blank control group.
Results
The micromorphology of alginate-Sr hydrogel was similar to that of the alginate-Ca hydrogel, with homogeneous pore structure; the modulus of compression of alginate-Sr hydrogel and alginate-Ca hydrogel was (186.53 ± 8.37) and (152.14 ± 7.45) kPa respectively, showing significant difference (t=6.853, P=0.002); there was no significant difference (t=0.737, P=0.502) in swelling rate between alginate-Sr hydrogel (14.32% ± 1.53%) and alginate-Ca hydrogel (15.25% ± 1.64%). The degradabilities of 2 alginate hydrogels were good; the degradation rate of alginate-Sr hydrogel was significantly lower than that of alginate-Ca hydrogel on the 20th, 25th, and 30th days (P lt; 0.05). At 1-4 days, the morphology of cells on 2 alginate hydrogels was spherical and then the shape was spindle or stellate. When three-dimensional cultured for 21 days, the DNA content of BMSCs in experimental group [(4.38 ± 0.24) g] was significantly higher than that in control group [(3.25 ± 0.21) g ] (t=8.108, P=0.001). On the 12th day after osteogenic differentiation, the ALP activity in experimental group was (15.28 ± 1.26) U/L, which was significantly higher than that in control group [(12.07 ± 1.12) U/L] (P lt; 0.05). Likewise, the mRNA expressions of OSX, collagen type I, and Runx2 in experimental group were significantly higher than those in control group (P lt; 0.05). On the 21th day after osteogenic differentiation, alizarin red staining and Von Kossa staining showed calcium deposition in 2 groups; the calcium nodules and phosphate deposition in experimental group were significantly higher than those in control group (P lt; 0.05).
Conclusion
Alginate-Sr hydrogel has good physicochemical properties and can promote the proliferation and osteogenic differentiation of BMSCs, so it is an excellent injectable scaffold material for bone tissue engineering.
Objective To explore the mechanisms for repairing spinal cord injury (SCI) with tetramethylpyrazine-loaded electroconductive hydrogel (hereinafter referred to as “TGTP”). Mehtods A total of 72 female Sprague-Dawley rats were randomly divided into 4 groups: sham operation group (group A), SCI group (group B), SCI+electroconductive hydrogel group (group C), and SCI+TGTP group (group D). Only the vertebral plate was removed in group A, while the remaining groups were subjected to a whole transection model of spinal cord with a 2 mm gap in the lesions. The recovery of hindlimb motor function was evaluated by Basso, Beattie, Bresnahan (BBB) score and modified Rivlin-Tator inclined plate test before operation and at 1, 3, 7, 14, and 28 days after operation, respectively. Animals were sacrificed at 7 days and 28 days after modeling. Neovascularisation was observed by immunofluorescence staining of CD31 and the expression levels of angiopoietin 1 (Ang-1) and Tie-2 were assessed by Western blot assay. At 28 days postoperatively, the expression levels of pro-angiogenic related proteins, including platelet-derived growth factor B (PDGF-B), PDGF receptor β (PDGFR-β), vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2), were also assessed by Western blot. The fibrous scar in the injured area was assessed using Masson staining, while neuronal survival was observed through Nissl staining. Furthermore, LFB staining was utilized to detect myelin distribution and regeneration. Immunofluorescence and Western blot assay were employed to evaluate the expression of neurofilament 200 (NF200). Results The hindlimb motor function of rats in each group gradually recovered from the 3rd day after operation. The BBB score and climbing angle in group D were significantly higher than those in group B from 3 to 28 days after operation, and significantly higher than those in group C at 14 days and 28 days after operation (P<0.05). Masson staining showed that the collagen volume fraction in groups B-D were significantly higher than that in group A, and that in group D was significantly lower than that in groups B and C (P<0.05); a small amount of black conductive particles were scattered at the broken end in group D, and the surrounding collagen fibers were less than those in group C. Nissl and LFB staining showed that the structure of neurons and myelin sheath in the injured area of spinal cord in group D was relatively complete and continuous, and the number of Nissl bodies and the positive area of myelin sheath in group D were significantly better than those in groups B and C (P<0.05). NF200 immunofluorescence staining and Western blot assay results showed that the relative expression of NF200 protein in group D was significantly higher than that in groups B and C (P<0.05). CD31 immunofluorescence staining showed that the fluorescence intensity of group D was better than that of groups B and C at 28 days after operation, and tubular or linear neovascularization could be seen. The relative expressions of Ang-1 and Tie-2 proteins in group D were significantly higher than those in groups B and C at 7 and 28 days after operation (P<0.05). The relative expressions of PDGF-B and PDGFR-β proteins in group D were significantly higher than those in groups B and C, and group B was significantly higher than group C at 28 days after operation (P<0.05). The relative expressions of VEGF-A and VEGFR2 proteins in group D were higher than those in groups B and C, showing significant difference when compared with group B (P<0.05), but only the expression of VEGF-A protein was significantly higher than that in group C (P<0.05). There was significant difference only in VEGFR-2 protein between groups B and C (P<0.05). Conclusion TGTP may enhance the revascularization of the injured area and protect the neurons, thus alleviating the injury of spinal cord tissue structure and promoting the recovery of neurological function after SCI in rats.
Objective To construct a ultraviolet-cross-linkable chitosan-carbon dots-morin (NMCM) hydrogel, observe whether it can repair cartilage injury by in vivo and in vitro experiments, and explore the related mechanism. Methods The chitosan was taken to prepare the ultraviolet (UV)-cross-linkable chitosan by combining methacrylic anhydride, and the carbon dots by combining acrylamide. The two solutions were mixed and added morin solution. After UV irradiation, the NMCM hydrogel was obtained, and its sustained release performance was tested. Chondrocytes were separated from normal and knee osteoarticular (KOA) cartilage tissue donated by patients with joint replacement and identified by toluidine blue staining. The 3rd generation KOA chondrocytes were co-cultured with the morin solutions with concentrations of 12.5, 25.0, 50.0 μmol/L and NMCM hydrogel loaded with morin of the same concentrations, respectively. The effects of morin and NMCM hydrogel on the proliferation of chondrocytes were detected by cell counting kit 8 (CCK-8). After co-cultured with NMCM hydrogel loaded with 50 μmol/L morin, the level of collagen type Ⅱ (COL-Ⅱ) of KOA chondrocytes was detected by immunofluorescence staining, and the level of reactive oxygen species (ROS) was detected by 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Twenty 4-week old Sprague Dawley rats were selected to construct a articular cartilage injury of right hind limb model, and were randomly divided into two groups (n=10). The cartilage injury of the experimental group was repaired with NMCM hydrogel loaded with 25 μmol/L morin, and the control group was not treated. At 4 weeks after operation, the repair of cartilage injury was observed by micro-CT and gross observation and scored by the International Cartilage Repair Association (ICRS) general scoring. The cartilage tissue and subchondral bone tissue were observed by Safranine-O-fast green staining and COL-Ⅱ immunohistochemistry staining and scored by ICRS histological scoring. The expressions of tumor necrosis factor α (TNF-α), nuclear factor κB (NK-κB), matrix metalloproteinase 13 (MMP-13), and COL-Ⅱ were detected by Western blot and real-time fluorescence quantitative PCR. Results NMCM hydrogels loaded with different concentrations of morin were successfully constructed. The drug release rate was fast in a short period of time, gradually slowed down after 24 hours, and the amount of drug release was close to 0 at 96 hours. At this time, the cumulative drug release rate reached 88%. Morin with a concentration ≤50 μmol/L had no toxic effect on chondrocytes, and the proliferation of chondrocytes improved under the intervention of NMCM hydrogel (P<0.05). NMCM hydrogel loaded with morin could increase the level of COL-Ⅱ in KOA chondrocytes (P<0.05) and reduce the level of ROS (P<0.05), but it did not reach the normal level (P<0.05). Animal experiments showed that in the experimental group, the articular surface was rough and the defects were visible at 4 weeks after operation, but the surrounding tissues were repaired and the joint space remained normal; in the control group, the articular surface was rougher, and no repair tissue was found for cartilage defects. Compared with the control group, the experimental group had more chondrocytes, increased COL-Ⅱ expression, and higher ICRS gross and histological scores (P<0.05); the relative expressions of MMP-13, NF-κB, and TNF-α protein and mRNA significantly decreased (P<0.05), and the relative expressions of COL-Ⅱ protein/COL-2a1 mRNA significantly increased (P<0.05). Conclusion NMCM hydrogel can promote chondrocytes proliferation, down regulate chondrocyte catabolism, resist oxidative stress, protect chondrocytes from cartilage injury, and promote cartilage repair.
Polyvinyl alcohol (PVA) hydrogel was made for simulating human's soft tissue in our experiment. The image acquisition device is composed of an optical platform, a camera and its bracket and a light source. In order to study the law of soft tissue deformation under flexible needle insertion, markers were embedded into the soft tissue and their displacements were recorded. Based on the analysis of displacements of markers in X direction and Y direction, back propagation (BP) neural network was employed to model the displacement of Y direction for the markers. Compared to the experimental data, fitting degree of the neural network model was above 95%, the maximum relative error for valid data was limited to 30%, and the maximum absolute error was 0.8 mm. The BP neural network model was beneficial for predicting soft tissue deformation quantitatively. The results showed that the model could effectively improve the accuracy of flexible needle insertion into soft tissue.
Objective
To investigate the effect of porcine small intestinal submucosa extracellular matrix (PSISM) on the vitality and gene regulation of hepatocyte so as to lay the experimental foundation for the application of PSISM in liver tissue engineering.
Methods
The experiment was divided into two parts: ① BRL cells were cultured with 50, 100, and 200 μg/mL PSISM-medium which were prepared by adding PSISM into the H-DMEM-medium containing 10%FBS in groups A1, B1, and C1, and simple H-DMEM-medium served as a control (group D1); ② BRL cells were seeded on 1%, 2%, and 3% PSISM hydrogel which were prepared by dissolving PSISM in sterile PBS solution containing 0.1 mol/L NaOH in groups A2, B2, and C2, and collagen type I gel served as a control (group D2). At 1, 3, and 5 days after culture, the morphology and survival of liver cells were detected by the Live/Dead fluorescent staining. The cell vitality was tested by cell counting kit-8 (CCK-8) assay. And the relative expressions of albumin (ALB), cytokeratin 18 (CK18), and alpha-fetoprotein (AFP) in hepatocytes were determined by real-time fluorescent quantitative PCR (RT-qPCR).
Results
The Live/Dead fluorescent staining showed the cells survived well in all groups. CCK-8 results displayed that the absorbance (A) value of group C1 was significantly higher than that of group D1 at 5 days after culture with PSISM-medium, and there was no significant difference between groups at other time points (P>0.05). After cultured with PSISM hydrogels, theA values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days (P<0.05), theA value of group A2 was significantly higher than that of groups B2 and C2 at 5 days (P<0.05), but there was no significant difference between groups at other time points (P>0.05). RT-qPCR showed that the relative expressions of ALB and CK18 mRNA significantly increased and the relative expression of AFP mRNA significantly decreased in groups A1, B1, and C1 when compared with group D1 (P<0.05). The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 (P<0.05). The relative expressions of ALB and CK18 mRNA were significantly higher and the relative expression of AFP mRNA was significantly lower in groups A2, B2, and C2 than group D2 (P<0.05); the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 (P<0.05), and the relative expression of AFP mRNA in group A2 was significantly lower than that in group C2 (P<0.05), but no significant difference was found between other groups (P>0.05).
Conclusion
PSISM has good compatibility with hepatocyte and can promote the vitality and functional gene expression of hepatocyte. PSISM is expected to be used as culture medium supplement or cell carrier for liver tissue engineering.
Spinal cord injury (SCI) is a complex pathological process. Based on the encouraging results of preclinical experiments, some stem cell therapies have been translated into clinical practice. Mesenchymal stem cells (MSCs) have become one of the most important seed cells in the treatment of SCI due to their abundant sources, strong proliferation ability and low immunogenicity. However, the survival rate of MSCs transplanted to spinal cord injury is rather low, which hinders its further clinical application. In recent years, hydrogel materials have been widely used in tissue engineering because of their good biocompatibility and biodegradability. The treatment strategy of hydrogel combined with MSCs has made some progress in SCI repair. This review discusses the significance and the existing problems of MSCs in the repair of SCI. It also describes the research progress of hydrogel combined with MSCs in repairing SCI, and prospects its application in clinical research, aiming at providing reference and new ideas for future SCI treatment.
Objective To investigate the neuroprotective effect of conducting hydrogel loaded with tetramethylpyrazine sustained-release microparticles (hereinafter referred to as “TGTP hydrogel”) on spinal cord injury rats. Methods Forty-eight adult female Sprague Dawley rats were randomly divided into 4 groups: sham operation group (group A), model group (group B), conductive hydrogel group (group C), and TGTP hydrogel group (group D), with 12 rats in each group. Only laminectomy was performed in group A, and complete spinal cord transection was performed in groups B, C, and D. Basso-Bettie-Bresnahan (BBB) score was used to evaluate the recovery of hind limb motor function of each group before modeling and at 1, 3, 7, 14, and 28 days after modeling, respectively. At 28 days after modeling, the rats were sacrificed for luxol fast blue (LFB) staining to detect myelin regeneration. Nissl staining was used to detect the survival of neurons. Immunohistochemical staining was used to evaluate the expression of inflammation-related factors [nuclear factor кB (NF-кB), tumor necrosis factor α (TNF-α), and interleukin 10 (IL-10)]. Immunofluorescence staining and Western blot were used to evaluate the expression of neurofilament 200 (NF200). RseultsBBB scores of group A were significantly better than those of the other three groups at all time points after modeling (P<0.05); at 14 and 28 days after modeling, there was no significant difference in BBB scores between groups C and D (P>0.05), but the BBB score of group D was significantly better than that of group B (P<0.05). LFB staining and Nissl staining showed that the structure of neurons and myelin in group A was intact, and the myelin integrity and survival number of neurons in group D were significantly better than those in groups B and C. Immunohistochemical staining showed that the absorbency (A) value of NF-кB and TNF-α in group A were significantly lower than those in groups B and C (P<0.05), the A value of IL-10 was significantly higher than that in the other three groups (P<0.05); the A value of NF-κB in group D was significantly lower than that in groups B and C, the A value of TNF-α in group D was significantly lower than that in group B, while the A value of IL-10 in group D was significantly higher than that in group B (P<0.05). Immunofluorescence staining showed that the structure of neurons and nerve fibers in group A was clear and the fluorescence intensity was high. The fluorescence intensity of NF200 in group D was higher than that in groups B and C, and some nerve fibers could be seen. Western blot analysis showed that the relative expression of NF200 in group A was the highest, and the relative expression of NF200 in group D was significantly higher than that in groups B and C (P<0.05). Conclusion The TGTP hydrogel can effectively promote the recovery of motor function in rats with spinal cord injury, and its mechanism may be related to the regulation of inflammatory response.
