Establishment And Osteogenesis Evaluation Of Novel Hydrogel-Based Aspirin Sustained Release Scaffolds

BackgroundBone defect caused by inflammation,trauma,neoplasm,etc.has become a major health concern worldwide.Effcient and stable neo-bone formation is a demanding clinical issue which remains unsolved.With synergistic effects of scaffolds,seeded cells and bioactive factors,bone tissue engineering provides new opportunities to obtain ideal bone tissue regeneration.Hydrogels are a category of highly hydrated,extracellular matrix-like,three dimentional network biomimetic materials,which are widely used as tissue engineering scaffolds or drug release reservoirs.Among them,easy-synthesized,highly homogeneous,and high-strength tetra-polyethylene glycol(PEG)hydrogel has promising biomedical application prospects.Chitosan(CS)is a widely-distributed natural basic polysaccharide with advantages of superb biocompatibilities,excellent cell adhesion and anti-inflammatory effects.Harvested from periodontal ligaments,periodontal ligament stem cells(PDLSCs)are a kind of adult mesenchymal stem cells(MSCs)possessing strong proliferation,osteogenic differentiation and immunomodulation abilities,which make them preferential seeded cells in periodontal tissue regeneration and bone tissue regeneration.It has been substaintially reported that aspirin is capable to enhance MSCs osteogenic differentiation and MSCs-mediated bone regeneration.However,to date,few publicated articles have reported PDLSCs-laden,aspirin sustained release scaffolds for bone tissue engineering.ObjectivesPresent study aims to synthesize tetra-PEG hydrogel based bone tissue engineering scaffolds with outstanding biocompatibilities in a economical and handy method.To achieve gentle drug release curves,drug loading strategies including physical entrapment and electrostatic interaction were applied.The novel-designed scaffolds are capable to load and promote seeded cells' function,as well as modulate host MSCs' behavior and local microenvironment,so as to reach a better bone regeneration prognosis and provide references for henceforth scaffolds designing and clinical practice.Materials and Methods1.The osteogenic effect of tetra-PEG hydrogel aspirin sustained release scaffold ex vivo and in vivoPDLSCs were isolated from healthy premolars due to orthodontic extraction then identified and cultured for further experiments.A series of aspirin gradient concentration were set to induce PDLSCs osteogenic differentiation.At Day 7 and Day 14,RT-qPCR,alkaline phosphatase staining and alizarin red staining were used to examine the optimal concentration to promote PDLSCs in vitro osteogenesis.Adopting the mechanisms of active ester-amine condensation and drug physical entrapment,tetra-PEG hydrogel aspirin sustained release scaffolds were synthesized.Morphology of hydrogel scaffolds and the laden PDLSCs was observed and aspirin release curve was aquired.A series of cytotoxicity tests were applied to examine in vitro biocompatibility of the scaffolds.In vitro osteogenic effects exerted by drug loaded tetra-PEG hydrogel were assessed by RT-qPCR,alkaline phosphatase staining and alizarin red staining.The hydrogel scaffolds were implanted subcutaneously in nude mice.The degradation of the scaffolds and the gross state of the implant sites were observed.The tissue sections were stained to observe the inflammatory status of the local tissues.After loading PDLSCs,tetra-PEG hydrogel scaffolds were implanted into critical defect of calvarial bone in mice.After 8 weeks,the samples were taken for micro-CT scanning to calculate the proportion of new bone area.After decalcification,tissue sections were prepared and stained to observe the morphological structure of new bone and fibrous connective tissue in bone defect areas.IFN-y immunofluorescence staining was used to evaluate the microenvironment immunomodulation effect of aspirin loaded tetra-PEG hydrogel.2.Construction of tetra-PEG-CS aspirin sustained release scaffold and evaluation its effects on PDLSCs-mediated bone regenerationWith the mechanisms of active ester-amine condensation,hydrogen bond and molecular entanglement between tetra-PEG and CS,drug physical entrapment and CS-aspirin electrostatic interaction,tetra-PEG-CS composite gel aspirin sustained release scaffolds were constructed.Flourier tranfromation infrared spectroscopy was applied to analyze chemical bonds and secondary interaction between three gel ingredients.Morphology of material surface and attached cells,pore sizes were also measured.The porosity of tetra-PEG-CS composite gels were tested by ethanol substitution method.Swelling curves,degradation curves and aspirin release curve were obtained by immersing gels in PBS.Scaffolds'biocompatibility and influences on PDLSCs osteogenic differentiation were evaluate via in vitro assays.RT-qPCR,alkaline phosphatase staining and alizarin red staining were used to evaluate in vitro osteogenic effects exerted by drug loaded tetra-PEG-CS composite gel.Eventually,after loading PDLSCs,tetra-PEG-CS composite gel scaffolds were implanted into calvarial bone defect in mice.After 8 weeks,the samples were taken for micro-CT scanning to calculate the bone volume/total volume and proportion of new bone area.After decalcification,tissue sections were prepared and stained to observe the morphological structure of new bone and fibrous connective tissue in bone defect areas.Results1.The osteogenic effect of tetra-PEG hydrogel aspirin sustained release scaffold ex vivo and in vivo(1)Aspirin has a concentration-dependent effect on the osteogenic differentiation of PDLSCs.According to present study,aspirin has the strongest osteogenic effect towards PDLSCs at 100?g/ml;(2)Aspirin loaded tetra-PEG hydrogel can release aspirin continuously for 14 days;(3)Cytotoxicity experiments showed that the tetra-PEG hydrogel aspirin sustained release scaffolds exert no negative effect on cell viability and proliferation,and effectively promoted PDLSCs mediated osteogenesis in vitro;(4)Compared with aspirin the hydrogel scaffolds loaded with PDLSCs,the new PDLSCs loaded hydrogel scaffolds showed better bone repair results;(5)Tetra-PEG hydrogel aspirin sustained-release scaffolds are able to lower the number of local inflammatory cells and reduce the level of IFN-? in bone defects.2.Construction of tetra-PEG-CS aspirin sustained release scaffold and evaluation its effects on PDLSCs-mediated bone regeneration(1)Compared with the tetra-PEG hydrogel aspirin sustained release scaffold,the PEG-CS hydrogel aspirin sustained release scaffolds possess gentler drug release curve;(2)Tetra-PEG-CS composite aspirin sustained release carrier had good biocompatibility.Compared with the tetra-PEG hydrogel,the tetra-PEG-CS composite hydrogel promoted PDLSCs osteogenic differentiation in vitro;(3)8 weeks later,compared with empty carrier or PDLSCs,aspirin singly-loaded carriers,tetra-PEG-CS composite aspirin sustained release carrier exhibited much greater osteogenic capability.Compared with PDLSCs-loaded,tetra-PEG hydrogel sustained release carrier,tetra-PEG-CS composite gel aspirin sustained release scaffold loaded with PDLSCs is more advantangeous in both ex vivo and in vivo osteogenesis.Conclusion1.Tetra-PEG hydrogel aspirin sustained release scaffolds are non-toxic,fully degradable and biocompatible.It has certain cell adhesion ability and aspirin sustained release ability.After loading PDLSCs,the scaffolds could achieve good osteogenic effect in vivo,which may be promoted by improving bone immune microenvironment;2.CS was introduced to modify tetra-PEG hydrogel.The tetra-PEG-CS composite gel aspirin sustained release scaffolds have approperiate physical properties,excellent biocompatibility and good cell adherence.Compared with the tetra-PEG hydrogel aspirin sustained-release scaffolds,it achieved a more gentle drug release curve and a better augmentation effect on PDLSCs-mediated osteogenesis both in vitro and in vivo.