Polydopamine-modified Poly(L-lactic Acid) Nanofiber Scaffolds Immobilized With An Osteogenic Growth Peptide For Bone Tissue Regeneration

Part I:preparation and physicochemical property of the PLLA-PDA-OGP scaffoldObjective:To prepare the polydopamine(PDA)and osteogenic growth peptide(OGP)functionalized PLLA scaffold and evaluate its physicochemical properties.Methods:PLLA solution with concentrations at 1%,2%,3%and 4%was used to prepare fibrous scaffold by electrospinning technology,and the appropriate concentration was determined through SEM and fiber diameter analysis.And then the polydopamine(PDA)coating on the PLLA fiber was formed via self-polymerization capability of PDA under alkaline conditions.The osteogenic growth peptide(OGP)was then immobilized onto PLLA fibers assisted by PDA-OGP interaction,and finally the PLLA-PDA-OGP scaffold was obtained.The scaffolds were divided into three groups:the PLLA,the PLLA-PDA and the PLLA-PDA-OGP scaffold.The microstructure of the scaffold was characterized by scanning electron microscopy(SEM).The diameter of the scaffold fiber was analyzed by NanoMeasurer software.The chemical bonds of the scaffold were analyzed by infrared spectrometer(FTIR).The surface composition of the scaffold was analyzed by X-ray photoelectron spectroscopy(XPS).The hydrophilicity of the scaffold was measured by water contact angle(WCA).The release of OGP from the scaffold in vitro was detected via ELISA at 0.5d?1d?2d?3d?5d?7d?14d?21d and 28d.Results:The SEM and fiber diameter analysis showed that the PLLA concentration at 3%was most suitable for preparing nanofiber scaffold.The SEM images showed that the fibers of each group are uniform and distribute networks.The fiber diameters of PLLA,PLLA-PDA and PLLA-PDA-OGP scaffolds were731.40±85.89 nm,747.01± 105.90 nm,751.31±126.34 nm(p>0.05),respectively.The WCA results showed that the hydrophilicity of the PLLA scaffolds gradually increased after PDA coating and OGP immobilization.The contact angles of three groups were 122.50±7.25°,32.73±3.95° and 15.57±4.30°(p>0.05),respectively.XPS results showed that new nitrogen(N1s)peaks were found in the spectra of both PLLA-PDA scaffold and PLLA-PDA-OGP scaffold,whereas no nitrogen(N1s)peak was observed in PLLA scaffold.N component percentages found in PLLA,PLLA-PDA,and PLLA-PDA-OGP groups were 0%,3.9%,and 8.7%,respectively.FTIR results indicated that new absorption peaks at 1501 cm-1 and 1615 cm-1 appeared after functionalized by PDA and OGP.Tensile strength of the PLLA,PLLA-PDA and PLLA-PDA-OGP scaffolds was 4.64±0.24 MPa?4.76±0.22 MPa and 4.73±0.19 MPa(p>0.05),respectively.The release of OGP from PLLA-PDA-OGP scaffolds was 4.97± 1.28%at 1 day,and the cumulative release was 25.91 ±4.23%after 28 days.Conclusions:The PLLA-PDA-OGP scaffold has three-dimensional porous nanostructure,good hydrophilicity,certain mechanical property and achieves a sustained release of OGP.Part II:Effects of promoting BMSCs osteogenic differentiation by the PLLA-PDA-OGP scaffold in vitroObjective:To evaluate the capability of PLLA-PDA-OGP scaffold in promoting in vitro cell adhesion,proliferation and osteogenic differentiation.Methods:BMSCs of rat were extracted via whole bone marrow adherence method.The phenotype of BMSCs was detected by flow cytometry.Osteogenesis,fat formation and cartilage induction were performed to evaluate the multi-directional differentiation capability of BMSCs.To investigate the capability of PLLA-PDA-OGP scaffold in promoting in vitro cell adhesion,proliferation and osteogenic differentiation.Samples were divided into four groups:Control,PLLA,PLLA-PDA,and PLLA-PDA-OGP group,and BMSCs were seeded on the scaffolds.Viability and proliferation of cells in different groups was measured by dead/live staining and CCK-8 technique,respectively.The morphological changes of cytoskeleton and nucleus of BMSCs were evaluated by FTIC-phalloidin and DAPI fluorescence staining,respectively.Cell adhesion and spreading on the materials was evaluated by SEM imaging,integrin?1 and vinculin immunofluorescent staining.ALP staining,alizarin red staining,Runx2?Col-1?OPN and OCN cell immunofluorescence staining were used to evaluate the capability of the scaffold in promoting BMSCs differentiation.The expression of osteogenic genes such as Runx2.Col-1?OPN and OCN were detected by RT-qPCR.Results:BMSCs phenotype test indicated that the expression of surface antigens of CD29 and CD90 were 97.9%and 99.9%,respectively.While the expression of both CD34 and CD45 molecules were negative.After corresponding induction,BMSCs were observed to differentiate into osteogenesis,adipogenic and chondrogenic.Dead/live cell staining results showed that cells on the PLLA-PDA-OGP scaffolds grew well,and no obvious dead cells with red staining was observed.and viability of cells adhered on this type of samples was significantly higher than the other two groups(p<0.05).The CCK-8 results showed that BMSCs proliferated on all types of scaffolds.Particularly,cells on PLLA-PDA-OGP scaffold had the highest cell proliferation rate(p<0.05).The cytoskeleton and nuclear staining and SEM images showed that cells adhered and exhibited normal morphology on the three types of scaffolds.The integrin?1 and vinculin immunofluorescent staining showed that BMSCs on PLA-PDA-OGP scaffold had a higher intensity than the other two types scaffolds(p<0.05).The ALP staining revealed that BMSCs on the PLA-PDA-OGP scaffold showed a large range deeper staining,while the other two groups had only a small amount of light staining.The ALP activity of cells on PLLA-PDA-OGP scaffold was significantly stronger than the other two types of scaffolds(p<0.05).The alizarin red staining revealed that BMSCs on PLLA-PDA-OGP scaffold showed a large range of red staining,while the other two groups had only a small amount of light red.The calcium content on PLLA-PDA-OGP scaffold was significantly greater than the other two types of scaffolds(p<0.01).Immunofluorescence staining showed that Runx2?Col-1?OPN and OCN proteins were significantly expressed on PLA-PDA-OGP scaffolds,while only a small amount was expressed on the other two types scaffolds.Gene expression results showed Runx2 and Col-1 had an increased expression after a 3-day?7-day and 14-day culture(p<0.05),and OPN and OCN had an increased expression after a 7-day and 14-day culture(p<0.05).Conclusion:The PLLA-PDA-OGP scaffold has good biocompatibility and promoted the adhesion,proliferation,and osteogenic differentiation of BMSCs.Part ?:The Study of the ability of PLLA-PDA-OGP scaffold to promote the repair of rat skull defectObjective:To evaluate the capacity of the PLLA-PDA-OGP scaffold in vivo bone formation,and feasibility on repairing skull defects in ratsMethods:Skull defects with a diameter of 4 mm in rat model was created.The scaffold was implanted into the skull defect area.Control group,PLLA group,PLLA-PDA group,and PLLA-PDA-OGP group were included in the in vivo study.The new bone formation in the skull defect area was characterized by Micro-CT 4 weeks and 8 weeks after surgery.The image analysis software was used to calculate the new bone volume/total tissue volume(BV/TV)and bone mineral density(BMD).New bone formation and scaffold degradation were evaluated by H&E and Masson staining of the tissue sections.Results:After 4 and 8 weeks,Micro-CT results showed a few new bone formation occurred along the edge of bone defect in Control group and a small amount of sheet-shaped new bone formation in PLLA group and PLLA-PDA group.PLLA-PDA-OGP group showed more new bone formation at 4 W,and most of the bone defect area was covered with new formed bone at 8 W.BV/TV value was used to evaluate the new bone in the defective area.BV/TV values of PLLA-PDA-OGP group at 4 weeks and 8 weeks were 35.74±4.61%and 61.57±8.75%,respectively,and BMD of PLLA-PDA-OGP group at 4 weeks and 8 weeks were 198.77±58.49(mg/cm3)and 366.08±65.16(mg/cm3),respectively.Both BV/TV and BMD values of PLLA-PDA-OGP group were significantly higher than those in the other groups(p<0.05).The results of H&E and Masson staining showed that Control group only had loose connective tissue,and PLLA group as well as PLLA-PDA group showed scaffolds partial degraded as along with a small amount of new bone formation,whereas PLLA-PDA-OGP group exhibited scaffolds mostly degrade as along with a large amount of new bone formation and vascular tissue ingrowth.Conclusions:PLLA-PDA-OGP scaffold can significantly promote new bone formation and improve bone mineral density in vivo,and the ability to repair the skull defect in rats is obviously superior to PLLA scaffold and PLLA-PDA scaffold.