Construction Of Tissue-engineered Intervertebral Disc And Preliminary Evaluation Of Its Biological Function In Vivo

Objective:To construct a tissue-engineered intervertebral disc(IVD) with biological activity mimicking structure and function of normal intervertebral disc tissue, and preliminarily investigate its biological function in vivo.Methods:(1) Nucleus pulposus(NP) cells and annulus fibrosus(AF) cells were harvested from 3-week-old New Zealand rabbit with type Ⅱcollagenase digestion method. The passage two(P2) cells were identified by safranin-O staining, collagen Ⅱ immunohistochemical staining and aggrecan(proteoglycan) immunofluorescence staining.(2) The scaffold of nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were manufactured by chitosan hydrogel using crosslinking reaction, PBST [poly(butylene succi-nate-co-terephthalate)] spinning fibers through electrospinning and solid PBST ring respectively. The space structure of chitosan hydrogel and PBST spinning films were observed by scanning electron microscopy(SEM),and the porosity and water absorption rate of PBST spinning films were tested through volume method.(3) NP cells and AF cells were seeded on the chitosan hydrogel and PBST spinning films respectively, and cytocompatibility of the scaffolds were tested by HE staining at 3 weeks culture in vitro.(4) PBST/chitosan hydrogel three-phase tissue-engineered IVD scaffold was assembled by solid PBST as the outer annulus fibrosus scaffold, PBST spinning film as the inner annulus fibrosus scaffold, chitosan hydrogel as the nucleus pulposus scaffold respectively. The mechanical properties of the three phase scaffold were evaluated with biomechanical testing machine.(5) AF cells and NP cells were seeded on the AF and NP regions respectively, and the tissue-engineered IVD were implanted into the subcutaneous of nude mice and harvested at 4 weeks. The histomorphology of the specimens were observed through HE staining, safranin-O staining, collagen Ⅱ immunohistochemical staining, and aggrecan immunofluorescence staining.(6) The tissue-engineered IVD fabricated in nude mice body was implanted in the resected deficit of IVD of three-month-old New Zealand rabbits and the adjacent segments were fixed with plate. The position of the plate was tested by X-ray and the hydration of replaced tissue-engineered IVD was tested by MRI at postoperatively 4 weeks. The histomorphology of the specimens, the expression of collagen Ⅱ and aggrecan were observed through HE staining, safranin-O staining, collagen Ⅱ immunohistochemical staining, and aggrecan immunofluorescence staining respectively.(7) The RNA of nude mice subcutaneous tissue-engineered IVD(nude mice group), replaced experimental rabbit tissue-engineered IVD(experimental rabbit group), normal rabbit IVD(control group) were extracted, and the collagen Ⅱ gene and aggrecan gene were amplified through RT-PCR in vitro.Results:(1) The P2 AF cells and NP cells grew fast with the form of a long fusiform or polygon and secreted collagen Ⅱ and proteoglycanand in vitro.(2) Chitosan hydrogel with white spongiform became transparent gel after absorbing water and there were a large number of interconnected pores in scaffolds observed by SEM. The NP cells proliferated rapidly and distributed homogeneously in scaffold after cultured on Chitosan hydrogel for 3 weeks.(3) The thickness of PBST fibers was uniform under optical microscope view and the PBST spinning film was white and loosen with porosity of(61.83%±7.33%) and absorption rate of(297.34%±57.13%). AF cells grew well and distributed homogeneously in scaffold after cultured on PBST spinning film for 3 weeks.(4) The three phase tissue-engineered IVD scaffolds with size of 9×5×2 mm, connected tightly among each part, without obvious boundary. The compression strength of it was(3.10 MPa±0.13 MPa) and compression modulus was(22.22 MPa±0.92 MPa) while the tensile strength was(3.21 MPa±0.18 MPa), tensile modulus was(18.13 MPa±1.30 MPa).(5) The structure of tissue-engineered IVD in the subcutaneous of nude mice was similar to that of the normal IVD. The outer annulus fibrosus with dense structure, inner annulus fibrosus with layered ring appearance and the nucleus pulposus with white gel formation integrated closely among each part. These cells secreted collagen Ⅱ and aggrecan in the tissue-engineered IVD by histomorphology observation.(6) The fixtion of adjacent segments with plate could maintain the normal height of IVD by X-ray test, and the T2 phase of replaced tissue-engineered IVD appeared as high signal through MRI test.(7) The position of replaced tissue-engineered IVD was fixed in situ, without subside and displacement, and the structure of that was complete at postoperatively 4 weeks by anatomy observation. There were a large number of cells and the expression of collagen Ⅱ and aggrecan in the replaced tissue-engineered IVD.(8) The m RNA expression of collagen Ⅱ and aggrecan in normal IVD tissue was highest, followed by the tissue-engineered IVD in nude mice and then replaced tissue-engineered IVD in rabbit, the difference among each group was statistically significant.Conclusions:(1) The PBST/chitosan three phase tissue-engineered IVD scaffold with good cytocompatibility, porosity, mechanical properties and plasticity is consistent with the structure features of the normal IVD.(2) The structure of the tissue-engineered IVD constructed in the subcutaneous of nude mice is similar to that of the normal IVD, and the three part of it integrated well with cell biological activity.(3) The replaced tissue-engineered IVD with reliable fixation, have good cell activity and biological functions in vivo.(4) This study provide a basic theory for construction of tissue-engineered IVD.