Fabrication Of Phage Films On Titanium Surface And Their Biological Properties

Ideal bone implants should have the ability to modulate the immune response and induce bone formation.Although titanium implants have been widely used in the field of hard tissue implantation,their surface lack some of the biological activity and can cause the inflammatory response around the implants.Specific binding phages to the inorganic materials based on the phage display technology can not only form stable matrix on the material surfaces to regulate the biological function of adherent cell and tissue,but also provide peptides and drug molecules for cell growth by means of chemical modification.Transforming growth factor-?1(TGF-?1)plays an important role in inflammatory response and wound healing.Tailoring the TGF-?1 regulation on the immune and tissue cells can promote the bone healing and reduce the incidence of fibrosis formation around the implants.This study used the phages displayed the titanium-binding peptide(TBP-phages)to modify the titanium surface.These TBP-phages were identified from the M13 phage display library,and had the specific binding to the acid-alkali treated titanium surface.To give more biofunctions to the titanium surface,the phage films made of TBP54-phage were grafted with the TGF-?1 affinity peptides.Through culturing with mesenchymal stem cells(MSCs),macrophages in vitro,and animal bone implantation in vivo,a comprehensive evaluation of the regulation of titanium surface with different phage films on the inflammatory response and bone remodeling was made.Titanium implant with the best osteoimmunomodulatory property was obtained.First of all,TBP-phages,which can specifically bind to the acid-alkali treated titanium,were identified from the phage display library.The binding peptide TBP54 on the TBP54-phage bound with the hydroxyl groups on the titanium surface,especially the basic hydroxyl groups,through coordination and electrostatic interactions.The affinity between the phage and the titanium surface was controlled by the surface structure and chemical groups.Prolonging the alkali treatment time of the titanium surface will enhance the binding of the phages to the titanium surface.By a simple drop-coating method,uniform nano-porous phage films made of TBP54-phage were prepared on the titanium surface.The phage films had good stability and substrate selectivity.The addition of PVP into the phage suspension can result in a more uniform film(named as APP).The composite film,composed of TBP54-phage and chitosan,had the better hydrophilicity,degradation resistance and film/substrate adhesion,compared to the pure chitosan film.Thirdly,the osteogenic differentiation,mineralization and macrophage inflammatory response of the pretreated titanium surface,acid-alkali treated titanium surface,titanium surface with phage film(APP)and phage film cross-linked by glutaraldehyde were investigated.Titanium treated by acid-alkali improved the osteoblast adhesion,differentiation,and mineralization in simulated body fluid,while reduced the inflammatory response.After modified with phage films,the osteogenic differentiation and mineralization were more significantly enhanced.At the later stage of macrophage culture APP also showed a more pronounced inflammatory decline.When cross-linked by glutaraldehyde,the biological properties of the phage films was reduced.Then,phage display technology was used to identify the TGF-?1 affinity peptide(T?ms).The T?m16 had a relatively higher affinity to TGF-?1,but a poor blockage of the immunoreactivity of TGF-?1,while T?m4 showed the opposite properties.T?ms alone promoted the early osteoblast proliferation and had a negligible effect on the alkaline phosphatase activity(ALP).In the presence of exogenous TGF-?1,T?ms improved the ALP activity,Ca deposition and expression of osteocalcin,along with impaired phosphorylation of Smad2/3.After that,T?m4,T?m14 and T?m16 were grafted onto the APP surface to investigate the regulation of T?ms on the osteogenic differentiation of mesenchymal stem cells(MSCs)and the inflammatory response of macrophages.With TGF-?1 binding to the phage films,phage films grafted with T?m14 could significantly promote the osteogenic differentiation and mineralization of MSCs.TGF-?1 could effectively promote the early osteogenesis of MSCs.In the absence of TGF-?1,APP was more favorable for mineralization of MSCs.Phage films grafted with T?ms were incubated with macrophages further.T?ms had an effect on the protein adsorption,integrin express and the early inflammatory response.After the cell apoptosis,inflammatory enhancement,persistence and polarization transformation,macrophages entered the stage of response decline.The characterization of macrophage phenotype at different stages indicated that phage films upregulated the early inflammatory expression of macrophages.This related to the T?ms and was regulated by pro-inflammatory pathways of TGF-?1 and NF-?B.With the gradual enhancement of TGF-?1 anti-inflammatory effect,macrophage phenotype changed into anti-inflammatory,and the inflammatory decline was accelerate.Among the phage films,the inflammatory response of the films with T?m4 lasted longer.Finally,the early inflammatory response and later osseointegration of titanium implants with different phage films were investigated in rats.Compared with acid-alkali treated titanium implants,phage films significantly improved the osseointegration of titanium implants,and the APP had the best effect in inducing bone formation.In the inflammatory response of the first 7 days,there were more infiltration of macrophages above the implants with T?m4,while more multinucleated macrophages were distributed around the surfaces with T?m16.Due to the influence of the early inflammatory response,the formation of fibrous capsule was observed on the implants with T?m4 at 2 weeks,which hindered the later bone integration.For the implants with T?m16,the bone remodeling was slower at the first 4 weeks because of the matrix degradation by macrophage.Among the implants modified by the three phage films with T?ms,the implants with T?m14 had the better osseointegration.In general,the phage films prepared by the phages displayed the titanium-binding peptide and PVP had better osteoimmunomodulatory property.Although the phage films with TGF-?1 affinity peptides can regulate osteogenic differentiation and inflammatory response,they had no advantage in the normal bone healing.