| Background: Disc degeneration is a serious progressive disease with no approved pharmacological interventions or innovative therapies to prevent exacerbation.Stem cell,biologic factor and genetic based disc regeneration therapies are not yet mature.Patients with moderate to severe disc degeneration eventually opt for surgical treatment,particularly spinal fusion.However,this procedure has many drawbacks,such as fusion device subsidence,long fusion period,and fusion failure,so there is an urgent need to find a new intervertebral bone fusion method.Tissue engineering strategies based on bio-scaffolds,stem cells and bio-factors offer new hope for spinal fusion.Objective:(1)To construct GelMA/GO nanocomposite hydrogels with different concentrations using graphene oxide(GO)and Gelatin Methacryloyl(GelMA),and to investigate their physicochemical properties,bone forming peptide-1(BFP-1)release characteristics and biocompatibility.(2)To explore the migration promotion and osteogenesis induction of bone marrow mesenchymal stem cells(BMSCs)by GelMA/GO composite hydrogels containing different concentrations of BFP-1.(3)To further investigate the in vitro osteogenic properties of GelMA/GO@BFP-1 complex on BMSCs.Methods:(1)GelMA/GO composite hydrogel precursor solutions with different GO ratios were prepared and irradiated by ultraviolet(UV)light to form gels.The microscopic morphology and chemical structure of the composite hydrogels were characterized by scanning electron microscope(SEM)and Fourier transform infrared(FTIR)spectroscopy.The swelling,degradation and mechanical properties of the composite hydrogels were examined by dissolution,degradation and mechanical tests,respectively.The release of BFP-1 from the composite hydrogels was detected by enzymatic standardization.The biocompatibility of GelMA/GO composite hydrogels was assessed by Cell counting kit-8(CCK-8)and Calcein-AM/PI live/dead cell staining kit.(2)GelMA/GO composite hydrogels containing different concentrations of BFP-1 were prepared and co-cultured with BMSCs for a certain period of time to observe cell migration and the production of alkaline phosphatase(ALP)and mineralized substrate.(3)The most comprehensive BFP-1-containing hydrogel in(2)was defined as GelMA/GO@BFP-1 complex and co-cultured with BMSCs for a period of time to detect the production of ALP and mineralized nodules as well as the expression of osteogenic genes such as ALP,Run-related transcription factor 2(Runx-2),Osteocalcin(OCN),Osteopontin(OPN)and Osteogenic protein(Runx-2,OCN).Results:(1)The GelMA/GO composite hydrogel had a porous structure inside and no secondary chemical structure was formed between GO and GelMA chains.Overall,the doping of GO enhanced the mechanical properties,decreased the swelling rate,and slowed down the in vitro degradation of the composite hydrogels.In the BFP-1 release test,GO attenuated the sudden release of the hydrogel and effectively slowed down the BFP-1 release during the subsequent process.The results of CCK-8assay and live/dead cell staining assay showed that the composite hydrogel containing high GO concentration(GelMA/GO-3)inhibited the proliferation and viability of BMSCs,while the remaining group showed better cytocompatibility.(2)The cell migration results showed that the GelMA/GO composite hydrogel containing 0.4mg/mL BFP-1 induced the highest number of cell migration.the ALP staining results showed that the GelMA/GO+0.4 mg/mL BFP-1 group had the highest relative ALP activity.Alizarin red staining results showed the highest percentage of mineralized area between GelMA/GO groups containing 0.4 mg/mL and 0.8 mg/mL BFP-1.(3)The results of GelMA/GO@BFP-1 complex experiments were as follows:in the results of ALP staining and alizarin red staining,the ALP activity and mineralized area ratio were significantly higher in the GelMA/GO@BFP-1 complex group.In the results of qRT-PCR experiments,the expression levels of ALP,Runx-2,OCN and OPN genes were significantly higher in the GelMA/GO@BFP-1 complex group than in the control and GelMA/GO groups on day 7;similarly,the expression of the above four genes remained higher than the other two groups on day 14,but the expression of ALP and Runx-2 in them decreased relative to the control group compared to day 7,while the opposite was true for OCN and OPN.The immunofluorescence staining results showed that the fluorescence intensity of Runx-2 and OCN in the GelMA/GO@BFP-1 complex group was significantly stronger than the rest of the groups,suggesting more protein expression.Conclusion:(1)Based on the physicochemical properties,BFP-1 release characteristics and biocompatibility,the GelMA/GO nanocomposite hydrogel containing 1.2 mg/mL GO had the best overall performance.(2)The GelMA/GO composite hydrogel containing 0.4 mg/mL BFP-1 showed the most significant pro-migration and osteogenic induction effects on BMSCs.(3)GelMA/GO@BFP-1composite significantly promoted osteogenic differentiation of BMSCs and has the potential to be used as a bone implant for improving intervertebral bone fusion. |
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