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.
Survivors from myocardial infarction (MI) eventually develop heart failure due to the post-infarct ventricular remodeling which could not be suppressed by existing treatments. Currently, coronary heart disease has become the major cause of heart failure instead of rheumatic heart disease in China. For this reason, seeking effective treatment to prevent post-infarct ventricular remodeling is urgent. Intramyocardial injection of hydrogels as a new strategy for MI treatment has made great progress recently. This review discusses the principle, present status, mechanisms and prospects of injectable hydrogel therapies for MI.
Objective To introduce an injectable andin situ gelling gelatin hydrogel, and to explore the possibility as a carrier for demineralized bone matrix (DBM) powder delivery. Methods First, thiolated gelatin was prepared and the thiol content was determined by Ellman method, and then the injectable andin situ gelling gelatin hydrogel (Gel) was formed by crosslinking of the thiolated gelatin and poly (ethylene oxide) diacrylate and the gelation time was determined by inverted method. Finally, the DBM-Gel composite was prepared by mixing Gel and DBM powder. The cytotoxicity was tested by live/dead staining and Alamar blue assay of the encapsulated cells in the DBM-Gel. Forin vitro cell induction, C2C12 cells were firstly incubated onto the surface of the DBM and then the composite was prepared. The experiment included two groups: DBM-Gel and DBM. The alkaline phosphatase (ALP) activity was determined at 1, 3, 5,and 7 days after culture.In vivo osteoinductivity was evaluated using ectopic bone formation model of nude rats. Histological observation and the ALP activity was measured in DBM-Gel and DBM groups at 4 weeks after implantation. Results The thiol content in the thiolated gelatin was (0.51±0.03) mmol/g determined by Ellman method. The gelation time of the hydrogel was (6±1) minutes. DBM powder can be mixed with the hydrogel and injected into the implantation site within the gelation time. The cells in the DBM-Gel exhibited spreading morphology and connected each other in part with increasing culture time. The viability of the cells was 95.4%±1.9%, 97.3%±1.3%, and 96.1%±1.6% at 1, 3, and 7 days after culture, respectively. The relative proliferation was 1.0±0.0, 1.1±0.1, 1.5±0.1, and 1.6±0.1 at 1, 3, 5, and 7 days after culture respectively.In vitro induction showed that the ALP activity of the DBM-Gel group was similar to that of the DBM group, showing no significant difference (P>0.05). With increasing culture time, the ALP activities in both groups increased gradually and the activity at 5 and 7 days was significantly higher than that at 1 and 3 days (P<0.05), while there was no significant difference between at 1 and 3 days, and between 5 and 7 days (P>0.05). At 4 weeks after implantationin vivo, new bone and cartilage were observed, but no bone marrow formation in DBM-Gel group; in DBM group, new bone, new cartilage, and bone marrow formation were observed. The histological osteoinduction scores of DBM-Gel and DBM groups were 4.0 and 4.5, respectively. The ALP activities of DBM-Gel and DBM groups were respectively (119.4±22.7) and (146.7±13.0) μmol/mg protein/min, showing no significant difference (t=–2.085,P=0.082). Conclusion The injectable andin situ gelling gelatin hydrogel for delivery of DBM is feasible.
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 therapeutic effect of BMSCs- chitosan hydrogel complex transplantation on intervertebral disc degeneration and to provide experimental basis for its cl inical appl ication. Methods Two mill il iter of bone marrow from 6 healthy one-month-old New Zealand rabbits were selected to isolate and culture BMSCs. Then, BMSCs at passage 3 were labeled by 5-BrdU and mixed with chitosan hydrogel to prepare BMSCs- chitosan hydrogel complex. Six rabbitswere selected to establ ish the model of intervertebral disc degeneration and randomized into 3 groups (n=2 per group): control group in which intervertebral disc was separated and exposed but without further processing; transplantation group in which 30 μL of autogenous BMSCs- chitosan hydrogel complex was injected into the center of defected intervertebral disc; degeneration group in which only 30 μL of 0.01 mol/L PBS solution was injected. Animals were killed 4 weeks later and the repaired discs were obtained. Then cell 5-BrdU label ing detection, HE staining, aggrecan safranin O staining, Col II immunohistochemical staining and gray value detection were conducted. Results Cell label ing detection showed that autogenous BMSCs survived and prol iferated after transplantation, forming cell clone. HE staining showed that in the control and transplantation groups, the intervertebral disc had a clear structure, a distinct boundary between the central nucleus pulposus and the outer anulus fibrosus, and the obviously stained cell nuclear and cytochylema; while the intervertebral disc in the degeneration group had a deranged structure and an indistinct division between the nucleus pulposus and the outer anulus fibrosus. Aggrecan safarine O stainning notified that intervertebral disc in the control and transplantation groups were stained obviously, with a clear structure; while the intervertebral disc in the degeneration group demonstrated a deranged structure with an indistinct division between the nucleus pulposus and the anulus fibrosus. Col II immunohistochemical staining showed that the tawny-stained region in the control group was located primarily in the central nucleus pulposus with a clear structure of intervertebral disc, the central nucleus pulposus in the transplantation group was positive with obvious tawny-stained intercellular substances and a complete gross structure, while the stained color in the degeneration group was l ighter than that of other two groups, with a indistinct structure.Gray value assay of Col II immunohistochemical staining section showed that the gray value of the control, the ransplantation and the degeneration group was 223.84 ± 3.93, 221.03 ± 3.53 and 172.50 ± 3.13, respectively, indicating there was no significant difference between the control and the transplantation group (P gt; 0.05), but a significant difference between the control and transplantation groups and the degeneration group (P lt; 0.05). Conclusion The rabbit BMSCs-chitosan hydrogel complex can repair intervertebral disc degeneration, providing an experimental foundation for the cl inical appl ication of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.
Peripheral nerve injury (PNI) is a common neurological dysfunction. In clinical practice, autologous nerve transplantation is used to solve problems related to PNI, such as limited donor resources, neuroma formation and high donor incidence rate. Therefore, searching for new nerve regeneration materials has become a hot research topic. The decellularized extracellular matrix (dECM) hydrogel provides a scaffold for nerve regeneration by removing the cellular components in biological tissues, preserving the extracellular matrix, and is a potential therapeutic material for nerve regeneration. This article reviews the research progress of dECM hydrogel for PNI and looks forward to the clinical prospects of this research direction.
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.
Acute kidney injury is a worldwide public health issue, and its treatment and management strategies continue to advance. In addition to traditional kidney replacement therapy, research in recent years has been focused on whole organ engineering and biofabrication of kidney assistive devices and bioinjections for in-body regeneration. Hydrogel materials show great potential in renal tissue engineering because of their good biocompatibility, thermal stability and controllable biochemical and mechanical properties. This article reviews the application of various hydrogel materials in renal tissue engineering to promote kidney regeneration and discusses the characteristics and applications of natural hydrogels and synthetic hydrogels, which is expected to further promote their clinical applications.
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 investigate the effect of collagen type I concentration on the physical and chemical properties of the collagen hydrogel, and to analyze the effect of different concentrations of collagen type I hydrogel on the phenotype and gene expression of the chondrocytes in vitro. Methods Three kinds of collagen hydrogels with concentrations of 12, 8, and 6 mg/ mL (C12, C8, and C6) were prepared, respectively. The micro-structure, compressive modulus, and swelling ratio of the hydrogels were measured and analyzed. The chondrocytes at 2nd passage were cocultured with three kinds of collagen hydrogels in vitro, respectively. After 1-day culture, the samples were stained with fluorescein diacetate (FDA) / propidium iodide (PI) and the cell activity was observed under confocal laser microscope. After 14-day culture, HE staining and toluidine blue staining were carried out to observe the histological morphology, and mRNA expressions of chondrocytes related genes (collagen type II, Aggrecan, collagen type I, collagen type X, Sox9) were determined by real-time fluorescent quantitative PCR. Results With the increase of collagen type I concentration from 6 to 12 mg/mL, the physical and chemical properties of the collagen hydrogels changed significantly: the fiber network became dense; the swelling ratios of C6, C8, and C12 were 0.260 ± 0.055, 0.358 ± 0.072, and 0.539 ± 0.033 at 192 hours, respectively, showing significant differences among 3 groups (P lt; 0.05); and the compression modulus were (4.86 ± 0.96), (7.09 ± 2.33), and (11.08 ± 3.18) kPa, respectively, showing significant differences among 3 groups (P lt; 0.05). After stained with FDA/PI, most cells were stained green, and few were stained red. The histological observation results showed that the chondrocytes in C12 hydrogels aggregated obviously with b heterochromia, chondrocytes in C8 hydrogels aggregated partly with obvious heterochromia, and chondrcytes in C6 hydrogels uniformly distributed with weak heterochromia. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of collagen type II and Aggrecan were at the same level in C12, C8, and C6; the expressions of collagen type I, Sox9, and collagen type X were up-regulated with the increase of collagen type I hydrogels concentration, and the expressions were the highest at 12 mg/mL and were the lowest at 6 mg/mL, showing significant differences among 3 groups (P lt; 0.05). Conclusion Increasing the concentration of collagen hydrogels leads to better mechanical properties and higher shrink-resistance, but it may induce the up-regulation of cartilage fibrosis and hypertrophy related gene expression.