GelMA/ODex Hydrogel-Loaded KGN For MRI Monitoring Of Situ Cartilage Regeneration

Objective:Osteoarthritis(OA)is a kind of chronic degenerative disease that seriously endangers human health.It mainly affects the articular cartilage,synovium and other intra-articular tissue structures.Articular cartilage has a special tissue structure,which lacks lymphatic,nerve and blood supply.Chondrocytes need to obtain nutrition from subchondral blood vessels and surrounding tissue fluid.The Extracellular matrix(ECM)of chondrocytes limits the migration ability of undifferentiated cells,leading to their poor self-repair ability.In this paper,by analyzing the differences of main clinical treatment methods,the composite hydrogel carrying the inducing factors of cartilage regeneration in situ was prepared,and the magnetic resonance imaging(MRI)was used for continuous monitoring.The main contents of this study were:(1)To compare the advantages and disadvantages of the main clinical treatments for early OA.(2)Synthetic melanin nanoparticles(SMNP)loaded with Kartogenin(KGN)and monitored by MRI were prepared and their physical and chemical properties were evaluated.(3)To evaluate the biological effects and safety of the composite hydrogel.(4)To evaluate the chondrogenic ability of the composite hydrogel.Materials and Methods:(1)A case-control study on the current main treatment methods of early OA,namely arthroscopic microfracture surgery and platelet-rich plasma(PRP)injection in the lesion area,was conducted.Clinical and functional outcomes were assessed by Lysholm score,American knee society knee(KSS)score,the western Ontario and McMaster universities(WOMAC)osteoarthritis index and visual analogue scale(VAS)before operation and at 3,6 and 12 months after operation.Whole-Organ Magnetic Resonance Imaging(WORMS)score was used to evaluate the size of cartilage defects before and 12 months after surgery.(2)A composite hydrogel scaffold modified with Gelatin(Gel),Methacrylic anhydride(MA)and Dextran(Dex)was designed.The SMNP loaded with KGN was prepared and encapsulated in the composite hydrogel scaffold to control the release of KGN.Then the successful modification of GelMA and Dex was verified by infrared spectroscopy.The properties of the mixed hydrogels with different proportions were evaluated in vitro by transmission electron microscopy,dynamic light scattering,rheological properties,and mechanical properties.Then,the in vitro release ability of KGN was measured.Finally,MRI imaging was used to observe the imaging effect of composite hydrogel with different Fe ion content.(3)The cytotoxicity of SMNP nanoparticles with different Fe ion contents was detected and the migration effect of SMNP nanoparticles with different Fe ion contents on bone marrow mesenchymal stem cells(BMSCs)was verified by scratch test.The cytotoxicity of the composite hydrogel was tested by live/dead staining and Cell Counting Kit8(CCK-8),and the osteoinductive and osteogenic ability of the hydrogel was evaluated by PCR.(4)An in vivo cartilage injury model was established,and the composite hydrogel was applied to the rabbit cartilage injury model.The material was implanted into the defect site,and the effect of the composite hydrogel on promoting cartilage regeneration was analyzed by MRI observation and tissue analysis.Results:(1)According to the results of Lysholm score and VAS score,the experimental group and the control group were significantly improved after operation,and the experimental group at 3 months after operation was significantly improved compared with the control group,and the difference was statistically significant(P<0.05).The overall excellent and good rate of KSS was less than 70%in both experimental group and control group.The WOMAC scores at 3,6 and 12 months after surgery were higher than those before surgery,and there was no significant difference(P>0.05)between the two groups.According to the WORMS scores before and 12 months after surgery,there was no significant change in cartilage thickness between the two groups before and after surgery.(2)The GelMA/ODex/SMNP-KGN composite hydrogel was successfully prepared.The physical and chemical properties of the composite hydrogel were tested in vitro,and the imaging effect of SMNP as MRI contrast agent was found to be good.The in vitro release effect of KGN in the hydrogel also confirmed its excellent release performance.(3)In vitro cell experiments verified that SMNP and composite hydrogel materials had almost no cytotoxicity and good migration effect on BMSCs.PCR confirmed that KGN had the effect of inducing BMSCs to differentiate into chondrocytes.(4)MRI observation and histological staining analysis confirmed that the composite hydrogel had the ability to promote cartilage regeneration in situ,and the mechanical properties of the regenerated cartilage were similar to those of normal cartilage by tensile and compressive moduli.Conclusions:This clinical study found that although the current main surgical treatment methods can improve the symptoms of patients,the long-term excellent and good rate is not high,the main reason is that the cartilage structure has not been repaired.Based on the weak ability of microfracture and PRP injection to regenerate hyaline cartilage in situ,we constructed a double-crosslinked polysaccharide hydrogel material with ODex and GelMA loaded with KGN inducing factor to improve the in situ regeneration ability of cartilage.This study shows that the composite hydrogel has good mechanical properties and biocompatibility,and can promote the proliferation of chondrocytes and BMSCs.