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 beta 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 viabilities 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 liquid at room temperature and solidified into gel at 37 degrees C (after 15 minutes) due to crosslinking reaction. Acridine orange/propidium iodide staining showed that the viability 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 < 0.05).
Conclusion: The thermo-sensitive chitosan hydrogel has good cellular compatibility. BMSCs culture with chitosan hydrogel maintains better cell shape, proliferation, and extracellular matrix biosynthesis than NPCs.