MAPs/bFGF-PLGA Microspheres Composite Coated Titantium Surface Increaseed Humam Fibroblasts Functionality

BackgroundThe use of transcutaneous implants which need to penetrate the skin first before it is fixed to the bone can provide a reliable, stable and aesthetic option for craniofacial prostheses. Nevertheless, transcutaneous implants are still facing clinical challenges such as bacterial infections, epithelial downgrowth and mechanical avulsion, which destabilize the soft tissue-implant interface and lead to implant loosening over time. Thus, it is very important to think over the formation of a stable soft tissue closure at the transcutaneous site, which can act as a defense to prevent epithelial down growth and also bacterial invasion on sub epithelial connective tissues and the implant interface.Current approaches to improve the formation of a stable implant-skin interface are aiming at avoiding bacterial infection rather than stimulating skin fibroblasts growth around the transcutaneous site. These approaches include altering the design of implant, altering surgical surgery methods, and coating implant surface with antibacterial agents. Although these coated surfaces can reduce the accumulation of bacteria compared to uncoated control groups, the bacteria infection rates were still high and there were few reported effects on stable biologic closure formation. The dermal fibroblasts play an important role for the formation of a soft tissue seal by producing glycosaminoglycan and type I collagen et al, which are essential components of connective tissues. Previous research have indicated that the quality of the fibrous tissue at the transcutaneous area act as an important factor for the long-term stability of maxillofacial implants.Mussel adhesive proteins (MAPs) have been used as a surface coating for cell adhesion due to their unique features of remarkable adhesion on multitudinous inorganic and organic surfaces in a wet environment and good biocompatibility. MAPs are mainly composed of the post-transnationally modified amino acid3,4-dihydroxy-phenylalanine (DOPA), flexible glycine residues, and positively charged lysine. All of above factors have contributed to a strong adhesion.in-vitro studies of adhesion and tissue biocompatibility have been widely reported,in vivo evaluation as candidate medical adhesives has also been investigated in an extra hepatic syngeneic islet transplantation murine model. BD Cell-Tak adhesive is a mixture of MAPs, which has been used as a coating on a substrate to immobilize cells or tissue. It can simplify a number of common in vitro techniques, including:establishment of primary cultures, immunohistochemistry and In situ hybridization. The first purpose of this work was to coat the smooth Titanium surface with MAPs to investigate whether MAPs-coated Ti surface could promote the attachment and growth of fibroblasts.Also,micro-particles can be easily attracted to and combined onto MAPs-coated Ti surfaces due to the remarkable wet adhesion ability of MAPs and also the negative charge surface of Poly(lactide-co-glycolide)(PLGA) which is opposite to Maps’positive charge.The basic fibroblast growth factor (bFGF) is an effective stimulator of fibroblast proliferation and plays an important role in skin-healing processes,the factor binds into and activates of fibroblast growth factor receptors(FGFRs) to exert its function.PLGA has been approved by the US FDA, and widely used in drug delivery system, basic and clinical science research and diagnostics. It is used for controlled drug-delivery systems for many advantageous characteristics such as favorable biocompatibility, their safety profile for human use and easily degrading into natural metabolites.so we fabricate PLGA microsphere to secure bFGF biological activity and control its release in a sustained and controllable manner in a relatively long time.ObjectivesTo investigate the proliferation and adhesion effects of human skin-derived fibroblasts on titanium surfaces coated with Usun-afix and BD CELL-TAK foot proteins extracted from Mytilus edulis. To fabricate bFGF-PLGA microsphere and fabricate MAPs-coated and MAPs/bFGF-PLGA microspheres composite coated Ti surface. Then effects of MAPs and MAPs-microsphere composite coated Ti surface on biological responses of dermal fibroblasts were evaluated, including cell adhesion and viability, ECM secretion and actin cytoskeleton; in vivo, to verify the effect of three kinds of titanium samples under the skin of SD rats after4weeks.Materials and methodsPure Ti foils were used as the substrate. MAPs was dissolved in5%acetic acid at the concentration of5μg/μl. Using a handy tool, such as a glass rod or micropipette tip, MAPs in5%acetic acid is spread in a thin liquid film. After the acetic acid evaporates, a coating of MAPs is left behind.The bFGF-loaded PLGA microspheres were prepared using the double-emulsion solvent evaporation method (W/O/W).microspheres were dissolved in sterilized distilled water at a concentration of5mg/ml. With200μl of the solution was pipetted onto MAPs-coated Ti surface, then air dried in laminar flow cabinet, as such we fabricated the MAPs/bFGF-PLGA microspheres composite of polished Ti surface.The morphology of three kinds of titanium samples was inspected using a field emission scanning electron microscope (S-4800; Hitachi High Technologies, Tokyo, Japan). Images with a range of scan sizes were acquired from the top and cross-sections. The changes in the chemical composition of the surface layer was determined by X-ray photoelectron spectroscopy (XPS, ESCALAB MK-II).Contact angle measurements were carried out by the sessile drop method on an Easy Drop Standard instrument (KRUSS GmbH, Hamburg, Germany) at room temperature. Distilled water was used in the contact angle measurements. The surface free energy and its components were calculated using the van Oss acid-base method. The contact angle was measured by analyzing the drop shape surface and the calculations of free energy using DSA1software (KRUSS GmbH, Hamburg, Germany).Human newborn foreskin tissues from surgical resection were obtained with written informed consent. Primary human skin fibroblasts were isolated from newborn foreskin as previously described. Cells were used at passages three. The cells were seeded on the samples placed in24well plates, after culturing for0.25,0.5,1and2h, unattached cells were removed, the adherent cells were fixed and stained with4’,6-diamidino-2-phenylindole (DAPI, Sigma, USA) and then the cell numbers were counted in five random5x fields on each sample under a fluorescence microscope. Fibroblasts were seeded on the samples placed in24well plates,after culturing for1,3, and5days, cell proliferation was assessed using the CCK-8(Dojindo Molecular Technologies Inc, Kumamoto, Japan) assay. Fluorescence microscopy was used to compare the morphological alterations both in regarding to the organization of the actin cytoskeleton and cellular shape. The distribution of actin was observed at2hours,4hours, and1day of culture. Expressions of the ECM-related genes were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR).Three kinds of titanium samples were transplanted under the skin of SD rats for4weeks,to evaluate the soft tissue reaction. To assess the bonding strength between the three different Ti samples and the soft tissue. Slice the soft tissue, and stain the soft tissue by HE staining, then observe the morphology and the organization of the soft tissue.Results1. The influence of two kinds of MAPs-coated Ti surface on fibroblast functionality(1)We coated Ti surface with MAPs with a density of50ug/cm2according to the instructions of the product. bFGF-PLGA microspheres were dissolved in sterilized distilled water with a concentration of5mg/ml. Scanning electron micrographs showed MAPs were highly ordered and aligned. The unmodified Ti surface showed relatively smooth.(2)The results of EDS (Energy Dispersive Spectrometer)showed that Compared to unmodified titanium surface, MAPs-coated Ti surface demonstrated reduced Ti,O concentrations and increased N, C concentrations.(3)Cell proliferation results indicated that there was no significant difference among the samples at day1,3,5between unmodified titanium surface, Usun-afix-coated Ti surface and BD CELL-TAK-coated Ti surface.(p>0.05)(4)Cell-adhesion analyses revealed that cell attachment rates of groups2and3were higher than control group (p<0.05) at15min、30min、1h、2h time points, however there was no significant difference at4h time point (p>0.05).There was no significant difference between groups2and3at any time point (p>0.05). DiI-AcLDL staining results showed the morphologies of cells grown on protein-coated surfaces were better at1h、2h、4h time points compared with control group.2. Fabricated of MAPs/bFGF-PLGA microspheres composite coated Ti surface and its physical characters.(1)The MAPs/bFGF-PLGA microspheres composite-coated Ti surface were loaded with uniform and sleek microspheres, the average diameter of the microspheres was1.848±0.304μm.(2)Adopting the BCA kit to determinate absorbance of the (A) value of the bFGF standard substance and sample solution, the bFGF concentration and (A) value were proportional to the standard curve. The encapsulation rate and drug loading rate were calculated using the standard curve, and the encapsulation rate was82.6%, while drug loading rate was0.1237%.(3) The early release kinetics of the microspheres was fast, with release rates of18.5%,32.4%and41.5%on days1,2and3, respectively. Approximately66.7%of the bFGF was released from the PLGA microspheres during the first10days; after20days, the cumulative release rate reached87%. (4)MAPs were highly ordered and aligned on the smooth Ti surface, and loaded with uniform and sleek bFGF-PLGA microspheres.(5) The Hybrid resolution XPS spectra results showed that MAPs/bFGF-PLGA microspheres composite-coated Ti surface showed reduced Ti, O concentrations and increased N, C concentrations. In the elemental composition analysis, while N element ratio of the unmodified Ti surfaces was about3%, the elemental N ratios of MAPs-coated Ti surface and MAPs/bFGF-PLGA microspheres composite-coated Ti surface increased to about11%by their successful coating of MAPs, which contains abundant N element (Table), the C ratio of MAPs-coated Ti surface was increased about51.76%,while MAPs/bFGF-PLGA microspheres composite-coated Ti surface was increased about65.23%, because more PLGA microspheres had abundant C element and no nitrogen element. XPS provide unequivocal proof that MAPs and MAPs/bFGF-PLGA microspheres were successfully immobilized onto titanium surfaces(6)Contact angle measurements showed that MAPs-coated Ti surface and MAPs/bFGF-PLGA microspheres composite-coated Ti surface performed an increased surface energy. The water surface contact angles results are indicated in.The water contact angle of unmodified Ti surface is83.2°, The MAPs-coated Ti surface showed higher hydrophily with a contact angle of47.5°,and MAPs/bFGF-PLGA microspheres composite-coated Ti surface displayed similar hydrophily with a contact angle of68.7°.The changes in the surface chemistry led to significantly different water contact angles.3. The influence of MAPs/bFGF-PLGA microspheres composite-coated Ti surface on fibroblast functionality.(1)The fibroblasts proliferation after5days of incubation showed that cell growth on MAPs/bFGF-PLGA microspheres composite-coated Ti surface was significantly higher than the other two surfaces at days,3, and5during incubation (P<0.05).While there was no obvious difference between the MAPs-coated Ti surface and the unmodified surface, although fibroblast proliferated more compared to unmodified Ti surface through the statistical analysis. This finding indicates that MAPs/bFGF-PLGA microspheres composite-coated Ti surface can release bFGF growth factor in a sustaining way which promotes cell proliferation continuously.(2)We used DAPI staining to measure The initial adherent cell number.The results demonstrate evidently increased fibroblasts attachment to MAPs-coated Ti surface (P<0.05) and MAPs/bFGF-PLGA microspheres composite-coated Ti surface (P<0.05) compared to unmodified Ti surface(Figure6).There were both statistically significant differences (P<0.05) in fibroblast adhesion between the two kinds of coating titanium compared to the unmodified Ti surface. The results also indicated that with the time point earlier, the adhesion differences were more significant. And There was not statistically significant difference between MAPs-coated Ti surface (P<0.05) and MAPs/bFGF-PLGA microspheres composite-coated Ti surface at each time point.(3)Results of the cytoskeleton organization indicated a strong preliminary interaction between fibroblasts and MAPs-coated Ti surface and MAPs/bFGF-PLGA microspheres composite-coated Ti surface. Obviously, the morphologies of fibroblasts grown on these three kinds of surfaces were different during the period of culturing. At2and4hours, The cells attach and spread well, compared to unmodified surface, there are more dense filopodia and lamellipodia to stretch out on MAPs-coated Ti surface and MAPs/bFGF-PLGA microspheres composite-coated Ti surface. Whilst at the same time point,the cells grown on the unmodified Ti surface showed lamella formation, and have fewer stress fibers. After1day of incubation, many stress fibers were noticeably observed at the periphery of the fibroblasts on MAPs-coated Ti surface and MAPs/bFGF-PLGA microspheres composite-coated Ti surface, forming good intercellular connection. However, the cells had apparently fewer contractile stress fibers stretching out on the unmodified surfaces.(4) Results of qRT-PCR indicated upregulated Expressions of the ECM-related genes:ICAM1,VEGFA, LAMA1(P<0.05) on3,6days on MAPs-coated Ti surface and MAPs/bFGF-PLGA microspheres composite-coated Ti surface, compared to unmodified Ti surface. (5)Transplanting results showed the similar soft tissue inflammatory reaction between the three different Ti samples, however, an increased bonding strength was found between MAPs coated Ti samples and soft tissue compared to smooth Ti samples.Conclusions(1)Human skin-derived fibroblasts can adhere to titanium surface coated with Usun-afix and BD CELL-TAK foot proteins extracted from Mytilus edulis more easily than normal surface in a short time span especially within4hours after seeding. And these mussel adhesive protein s has no positive effect on cell proliferation.(2)We successfully made bFGF-loaded PLGA microspheres using the double-emulsion solvent evaporation method (W/O/W). The morphology of the bFGF-loaded PLGA microspheres were uniform and sleek. The diameter of the microspheres and the release kinetics of bFGF in vitro showed excellent results.(3) In the present study, we successfully made MAPs-coated and MAPs/bFGF-PLGA microspheres composite-coated Ti surface. Compared to unmodified titanium surface,both MAPs-coated Ti surface and MAPs/bFGF-PLGA microspheres composite-coated Ti surface showed reduced Ti,O concentrations and increased N, C concentrations, and performed an increased surface energy.(4)The present study provides evidence of enhanced fibroblast adhesion, proliferation, actin cytoskeleton organization and Expressions of the ECM-related genes.(5)Transplanted results showed the similar the soft tissue reaction between the three different Ti samples, however, an increased bonding strength was found between MAPs coated Ti samples and soft tissue compared to polished Ti samples. The results suggest that the MAPs/bFGF-PLGA microspheres composite-coated Ti surface may be useful for transcutaneous implants due to the beneficial effects on the formation of a soft tissue biological seal around the transcutaneous site of the implant surface.