3D Printed PLGA Scaffold/Hydrogel Loaded With Nucleus Pulposus-Like Urine-Derived Stem Cells To Promote Intervertebral Disc Degeneration

ObjectiveThe PLGA scaffold/hydrogel composite scaffold precursor was prepared,and the nucleus pulposus-like urine-derived stem cells(NP-USCs)were obtained by co-culture induced differentiation,and combined with urine-derived stem cell exosomes(USC-EXOs)to construct a composite scaffold.Combining with the rabbit model of intervertebral disc degeneration,the in vivo and in vitro experiments were conducted to study whether the composite scaffold could improve intervertebral disc degeneration.Methods3D printing to obtain high-precision PLGA scaffolds,using scanning electron microscopy(SEM)to observe the microstructure of the scaffolds,using infrared spectroscopy and Raman spectroscopy to characterise the composition of PLGA scaffold materials.Using simulated body fluid(SBF)to measure the swelling rate of scaffolds,absolute ethanol was used to measure the porosity of the scaffold.A variety of biological enzymes and SBF were used to conduct simulated degradation experiments in vitro.Trilineal differentiation experiments and flow cytometry identified the stem cell characteristics of urine-derived stem cells(USCs).NP-USCs were obtained by inducing USCs to differentiate into nucleus pulposus-like co-culture;CCK8 was used to detect the proliferation-promoting ability of USC-EXOs,and the cytocompatibility of the composite scaffold was detected by live-dead staining.L5-6 rabbits were CT-guided punctured to obtain the lumbar degeneration model,and an MRI examination was performed before the puncture.The lumbar degeneration was observed one month after the puncture and one month after composite stent implantation.After each anesthesia,the blood was drawn and the liver and kidney function of the rabbit was checked to observe the biocompatibility of the composite scaffold.Rabbits were executed after the MRI examination,and then intervertebral disc tissue was removed.The protein samples were obtained by grinding,and the changes of COL-Ⅱ and ACAN protein contents were measured by Western blot.ResultsThe shape of the PLGA scaffold is a multi-cavity structure with a large specific surface area,which is beneficial to the adhesion and growth of cells.Furthermore,the depressions on the scaffold facilitate the migration of stem cells.The PLGA scaffold has a low swelling rate and will not undergo large deformation in vivo.The high porosity provides a good proliferation environment for cells,and the high anti-degradation properties indicate that the scaffold can provide sufficient compression resistance for a long time to maintain structural stability.USCs osteogenic differentiation,adipogenic differentiation,and chondrogenic differentiation were all successfully induced.Flow cytometry identification of stem cell marker proteins CD29(99.105),CD44(98.85%),and CD73(94.27%)indicated that USCs had similar characteristics to mesenchymal stem cells MSCs characteristics.USC-EXOs has the ability to promote cell proliferation,and the composite scaffold has good cytocompatibility.The composite scaffold significantly improved the degeneration of the rabbit lumbar intervertebral disc,and its biocompatibility was good.Compared with the puncture group,the protein content of COL-Ⅱ and ACAN in the transplantation group was significantly increased,but there was still a significant gap compared with the normal group.ConclusionThe composite scaffold system can improve the effect of intervertebral disc degeneration through the secretion of COL-Ⅱ and ACAN by NP-USCs.NP-USCs not only have a large number of proliferation ability of stem cells but also can play a similar function of nucleus pulposus cells.USC-EXOs can promote NP-USCs proliferation in vivo and in vitro.