| Objective: To explore radial regional variations in the cellular, biochemical, and biomechanicalcharacteristics of rabbit annulus fibrosus (AF). Methods: The AF of a six-month-old New Zealandwhite rabbit was harvested. With the nucleus pulposus (NP) removed, the inner (iAF), middle (mAF),and outer AF (oAF) were carefully separated into three equal-thickness sections along its radialdirection. After tissue being digested, primary cells from iAF (iAFCs), mAF (mAFCs) and oAF (oAFCs)were isolated and culutred. Cellular morphology was observed. Cell Counting Kit-8was used to detectcell proliferation. The gene expression of collagen-I, collagen-II and aggrecan was tested by RT-qPCR.Histological staining of H&E and Safranin Orange-Fast Green and immunohistochemistry staining ofcollagen-I and collagen-II were used to assay the distribution of collagen and proteoglycan (PG) intissues. The contents of DNA, glycosaminoglycan (GAG), hydroxyproline (HYP), collagen-I andcollagen-II in tissues were quantified using enzyme-linked immunosorbant assay (ELISA) kits orcommercially available kit. Cell traction force microscopy (CTFM) technique was employed to measurethe cell traction forces (CTFs) of individual AF cells. Nanoindentation technique was used to measurethe elastic modulus of AF tissue at micro/nano-level while tensile tests for AF tissues were performedat macro-scale. Result: We found iAFCs had rounded, chondrocyte-like morphology, while oAFCspredominantly displayed spindle-shaped, fibroblastic morphology. The mAFCs represented a mixture ofiAFCs and oAFCs. These region-specific AFCs appeared to proliferate at similar rate during culture.According to RT-qPCR analysis, iAFCs exhibited the least expression of collagen-I gene, while oAFCsexhibited the greatest. In contrast, iAFCs exhibited the greatest expression of collagen-II and aggrecan genes, while oAFCs had the least expression of them. Expression of the above genes in mAFCs wasbetween those in iAFCs and oAFCs. According to H&E staining, the AF tissue was mainly composedof collagen. In addition, upon Safranin Orange and Fast Green staining iAF was intensively stainedorange, indicating the existence of high PG content. In contrast, oAF was markedly stained with FastGreen, evidencing the presence of massive collagen fibers. From the immunohistochemical staining ofAF it was clear that there was less collagen-I in iAF compared to oAF, on the contrary, more collagen-IIin iAF but less in oAF. Biochemical analysis found that the DNA, HYP and collagen-I contentgradually increased from iAF, mAF to oAF, while the GAG and collagen-II content gradually decreased.From CTFM result, the CTF gradually decreased from iAFCs, mAFCs to oAFCs. According to thenanoindentation results measured at different indentation frequencies, oAF and iAF consistently showedthe highest and lowest storage moduli (equivalent to Young’s moduli), respectively. Such regionalvariations in the modulus of AF tissue were further confirmed by the results of tensile tests.Conclusion: Radial regional variations in the cellular, biochemical, and biomechanical characteristicsexist in rabbit AF. Objective: To isolate and identify rabbit annulus fibrosus stem cell (AFSC) in vitro. Methods: TheAF of a six-week-old New Zealand white rabbit was harvested. After tissue being digested, AF-derivedcells were isolated and culutred. A colony forming unit-fibroblast (CFU-F) assay was performed to testthe colony forming capability of AF-derived cells. MTT kit was used to detect the rabbit AF-derivedcolony-forming cells (AFDCFCs) proliferation. The gene expression of typical MSC-associated surfaceantigens in cells was tested using RT-PCR. Further, the expression of MSC markers including Oct-4,Nucleostemin (NS) and SSEA-4was examined at protein level using immunofluorescence. The rabbitAFDCFCs were subjected to induced differentiation processes including osteogenesis, chondrogenesis,and adipogenesis to examine the multi-differentiation potential. The cellular morphology, biochemical,gene expression and cellular mechanical characteristics were investigated after rabbit AFDCFCsculturing on the different elastic moduli polyacrylamide hydrogels (PAGs). Result: When the rabbit AF-derived cells were cultured in the growth medium supplemented with20%FBS, they remainedquiescent for3to4days and then started to form colonies. The colony formation capacity was largelydependent on the initial cell seeding density. An initial seeding density of200cells/cm2was found toresult in the highest efficiency of colony formation, in which about3.4%of plated cells formed colonies.MTT assay shows that the rabbit AFDCFCs have the strong self-renewing capacity. These cells hadstrong expression of CD29, CD44and CD166, while they had little expression of CD4, CD8and CD14.Meanwhile, Oct-4, NS and SSEA-4were extensively expressed in the AF-derived cells. After beinginduced, these cells succeeded in differentiating into osteogenesis, chondrogenesis, and adipogenesis byexamining with Alizarin Red S, and Safranin O, and Oil Red O staining, respectively. In addition, thegene of osteocyte specific genes (collagen-I, Runx2), chondrocyte specific genes (collagen-II, Sox-9)and adipocyte specific genes (PPAR-γ, LPL) had strong expression in the induced cells, respectively. Inaddition, we found cellular morphology on soft PAG showed rounded, chondrocyte-like morphology,while on stiff PAG displayed spindle-shaped and polygon. Cells on PAG appeared to proliferate duringculture. According to RT-qPCR analysis, on soft PAG it exhibited the least expression of collagen-Igene, while on stiff PAG exhibited the greatest. In contrast, on soft PAG it exhibited the greatestexpression of collagen-II and aggrecan genes, while on stiff PAG had the least expression. Biochemicalanalysis found that the content of DNA, HYP and collagen-I gradually increased from on soft to stiffPAG group, while the content of GAG and collagen-II gradually decreased. RT-qPCR analysis foundthat the expression of collagen-I gene gradually increased from on soft to stiff PAG group, while theexpression of collagen-II and aggrecan genes gradually decreased. From the CTFM result, the CTFgradually decreased from soft to stiff PAG group. Conclusion: We have successfully isolated andidentified a population of colony-forming cells from rabbit AF tissue. These cells possess clonogenicity,self-renewing capability, multi-differentiation potential and modulus dependent differentiation, whichare the common characteristics of MSCs. Therefore, we define these cells as AF stem cells (AFSCs). Objective: To fabricate different elastic moduli electrospun polyurethane fibre scaffold. Methods:Four different elastic moduli polyurethane that had synthetized before were chose for electrospun. Theelastic moduli was measured firstly by tensile test and nanoindentation. The polyurethane resin wasdissolved in a solvent of hexafluoroisopropanol. The homogenized polyurethane solution wastransferred to a20ml plastic syringe. The syringe, loaded with2ml polymer solution, was fitted with a0.85mm inner diameter cannula connected to the positive potential of a high-voltage power supply. Around glass collector which diameter was similar to a hole of24culture plate was placed at a distance of22cm from the cannula tip. Experiments were carried out with the cannula tip at a potential of15kV.The surface morphology and structural properties of the electrospun fiber scaffolds were inspected usingscanning electron microscopy (SEM). Result: The elastic moduli was measured by tensile test andnanoindentation. The moduli values of tensile test were2.5±0.4MPa,5.1±0.4MPa,6.8±0.5MPa,13.4±0.7MPa, respectively. Then fibrous scaffolds were manufactured with a horizontalelectrospinning setup. SEM-micrograph revealed a highly porous three dimensional structure composedof randomly oriented straight fibers. Conclusion: Four groups different elastic moduli electrospunpolyurethane fibre scaffold were fabricated. Objective: To investigate the cellular, biochemical, gene expression and cellular mechanicalcharacteristics after AFSCs cultured on the different elastic moduli electrospun polyurethane fibrescaffolds. Methods: After rabbit AFSCs cultured on the different elastic moduli electrospunpolyurethane fibre scaffolds, cell morphology was observed at1st,4th,7th,14th day usingFITC-Phalloidin staining. Cell Counting Kit-8was used to detect cell proliferation. After AFSCscultured on the substrate7days, the contents of DNA, PG, collagen-I and collagen-II in cells were quantified by biochemical assay. The gene expression of collagen-I, collagen-II and aggrecan was testedby RT-qPCR. CTFM technique was employed to measure the CTF of individual AF cells. Result: Wefound the cell morphology kept the same at the four different moduli fibre scaffolds that were indicatingpolygon. Cells on substrate appeared to proliferate during culture. According to RT-qPCR analysis, onsoft fibre scaffold it exhibited the least expression of collagen-I gene, while on stiff fibre scaffoldexhibited the greatest. In contrast, on soft fibre scaffold it exhibited the greatest expression ofcollagen-II and aggrecan genes, while on stiff fibre scaffold had the least expression of them.Biochemical analysis found that the DNA, HYP and collagen-I content gradually increased from soft tostiff fibre scaffold group, while the GAG and collagen-II content gradually decreased. From the CTFMresult, the CTF gradually decreased from soft to o stiff fibre scaffold group. Conclusion: Thesubstrate elastic moduli regulated the differentiation of rabbit AFSCs. After these cells cultured on thedifferent elastic moduli electrospun polyurethane fibre scaffolds, they tend to differed into the cellcharacteristics of radial regional cells in native rabbit annulus fibrosus. |
PDF Full Text Request