Objective To compare the growth and extracellular matrix biosynthesis of nucleus pulposus cells (NPCs)and bone marrow mesenchymal stem cells (BMSCs) in thermo-sensitive chitosan hydrogel and to choose seed cells for injectable tissue engineered nucleus pulposus. Methods NPCs were isolated and cultured from 3-week-old New Zealand rabbits (male or female, weighing 150-200 g). BMSCs were isolated and cultured from bone marrow of 1-month-old New Zealand rabbits (male or female, weighing 1.0-1.5 kg). The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium β glycerophosphate, and hydroxyethyl cellulose. Then, NPCs at the 2nd passage or BMSCs at the 3rd passage were mixed with chitosan hydrogel to prepare NPCs or BMSCs-chitosan hydrogel complex as injectable tissue engineered nucleus pulposus. The viabil ities of NPCs and BMSCs in the chitosan hydrogel were observed 2 days after compound culture. The shapes and distributions of NPCs and BMSCs on the scaffold were observed by scanning electron microscope (SEM) 1 week after compound culture. The histology and immunohistochemistry examination were performed. The expressions of aggrecan and collagen type II mRNA were analyzed by RT-PCR 3 weeks after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at37 (after 15 minutes) due to crossl inking reaction. Acridine orange/propidium iodide staining showed that the viabil ity rates of NPCs and BMSCs in chitosan hydrogel were above 90%. The SEM observation demonstrated that the NPCs and BMSCs distributed in the reticulate scaffold, with extracellular matrix on their surfaces. The results of HE, safranin O histology and immunohistochemistry staining confirmed that the NPCs and BMSCs in chitosan hydrogel were capable of producing extracellular matrix. RT-PCR results showed that the expressions of collagen type II and aggrecan mRNA were 0.564 ± 0.071 and 0.725 ± 0.046 in NPCs culture with chitosan hydrogel, and 0.713 ± 0.058 and 0.852 ± 0.076 in BMSCs culture with chitosan hydrogel; showing significant difference (P lt; 0.05). Conclusion The thermo-sensitive chitosan hydrogel has good cellular compatibil ity. BMSCs culture with chitosan hydrogel maintains better cell shape, prol iferation, and extracellular matrix biosynthesis than NPCs.
Objective To investigate the feasibil ity of using thermo-sensitive chitosan hydrogen as a scaffold to construct tissue engineered injectable nucleus pulposus (NP). Methods Three-month-old neonatal New Zealand rabbits (male or female) weighing 150-200 g were selected to isolate and culture NP cells. The thermo-sensitive chitosan hydrogel scaffold wasmade of chitosan, disodium β-glycerophosphate and hydroxyethyl cellulose. Its physical properties and gross condition were observed. The tissue engineered NP was constructed by compounding the scaffold and rabbit NP cells. Then, the viabil ity of NP cells in the chitosan hydrogel was observed 2 days after compound culture and the growth condition of NP cells on the scaffold was observed by SEM 7 days after compound culture. NP cells went through histology and immunohistochemistry detection and their secretion of aggrecan and expression of Col II mRNA were analyzed by RT-PCR 21 days after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at 37 (15 minutes) due to crossl inking reaction. Acridine orange-propidiumiodide staining showed that the viabil ity rate of NP cells in chitosan hydrogel was above 90%. Scanning electron microscope observation demonstrated that the NP cells were distributed in the reticulate scaffold, with ECM on their surfaces. The results of HE, toluidine blue, safranin O and histology and immunohistochemistry staining confirmed that the NP cells in chitosan hydrogel were capable of producing ECM. RT-PCR results showed that the secretion of Col II and aggrecan mRNA in NP cells cultured three-dimensionally by chitosan hydrogen scaffold were 0.631 ± 0.064 and 0.832 ± 0.052, respectively,showing more strengths of producing matrix than that of monolayer culture (0.528 ± 0.039, 0.773 ± 0.046) with a significant difference (P lt; 0.05). Conclusion With good cellular compatibilities, the thermo-sensitive chitosan hydrogel makes it possible for NP cells to maintain their normal morphology and secretion after compound culture, and may be a potential NP cells carrier for tissue engineered NP.
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.