Hierarchically Multi-functionalized Graded Membrane with

Enhanced Bone Regeneration and Self-defensive Antibacterial

Characteristics for Guided Bone Regeneration

Min Hea, Qian Wangb, Li Xiea, Hao Wua, Weifeng Zhao*b, and Weidong Tian*a

a State Key Laboratory of Oral Disease, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan 610041, China

b College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, People’s Republic of China

* Corresponding author. Tel: +86-028-85502122, Fax: 028-85503499; E-mail: drtwd@sina.com; zhaoscukth@163.com

1. The details of the cell test experiment. 1.1 The cell response on the soft tissue interface layer of FGM.

One aim of the specially designed FGM was to improve the biocompatibility of the membrane and regulate the cell behavor via the aligned natural gelatin surface layers. To confirm the superior cell biocompatibility of the aligned natural gelatin outer layer, the random PCL membrane (R-PCL) and random gelatin membrane (R-Gelatin) were electrospun and used as control. Then, a CCK-8 assay was conducted to evaluate the proliferation of the L929 mice fibroblast cell line (State Key Laboratory of Oral Diseases, Sichuan University, China) on the R-PCL, R-Gelatin, and the soft tissue interface layer of the FGM. The samples were sterilized and fixed in the 24-well plate, then 1.5×104 cells were seeded into each well. After culturing for 1, 3, 5, and 7 days, the medium was removed, and 900 μL new medium as well as 100 μL CCK-8 agent were added to each well. After that, the samples were incubated at 37 ºC for 4 hours, then the supernate was distributed into 96-well plates (100 μL for each well), and the absorbance was examined on a microplate reader at a wavelength of 450 nm. To observe the cell morphology, the cell seeded membranes were fixed with 2.5 wt.% glutaraldehyde solution in PBS for 1 h, then dehydrated by immersing in a series of ethanol solutions (30 wt.%, 50 wt.%, 70 wt.%, 90 wt.%, and 100 wt.%). Membranes were dried overnight at room temperature, coated with gold, and observed by SEM. The cell morphology was also observed by using laser scanning confocal microscope. The cell-seeded samples were washed twice with PBS, fixed with 2.5 wt% glutaraldehyde solution, and then stained with FITC-phalloidin solution and flourescein diacetate solution.2.1 The characterization of Pro-MNA

Figure S1. (A) FTIR characteristic of chemical structure of Pro-MNA; (B) the MNA release profiles in CE solution; (C) the MNA release profiles in bacterial solution; (D) plate samples showing colonies of S. aureus incubated with different amounts of Pro-

MNA for 12 h.

Infection associated with GTR implant is mainly caused by anaerobic bacteria. MNA, which has selective activity against anaerobic bacteria, had been incorporated into PCL/gelatin based GTRM by blending electrospinning and coaxial electrospinning.27, 28 Though strong in vitro antibacterial property was exhibited, the issue of uncontrollable continuous elution of MNA, especially the initial burst release limited its in vivo application. To address this issue, we specially synthesized the infection-responsive Pro-MNA, which could release the intact MNA molecular under the bacteria infection microenvironment. The chemical structure of Pro-MNA was characterized by FTIR, as shown in Figure S1(A). The emergence of ester group’ characteristic peak at 1720 cm-1 and the disappearance of the hydroxy group’s characteristic peak at 3300 cm-1 indicated that the MNA molecules were successfully grafted onto the side chains of the polymer via ester groups. The infection induced inflammation microenvironment would lead to the overexpress of esterase, which was able to catalyze the hydrolysis of ester groups.22 Therefore, we studied the enzyme-triggered drug release behaviors by incubating Pro-MNA at 37 ºC in PBS (pH 7.4) with or without esterase. Here, cholesterol esterase was used to simulate the infection-induced inflammation microenvironment. As shown in Figure S1(B), the Pro-MNA displayed negligible hydrolytic release of MNA in a non-enzyme solution, but a remarkably rapid release in the presence of CE was observed, and the release rate increased with the CE concentration, clearly displaying enzyme-triggered responsive release mechanism. It was reported that bacteria could also secrete esterase.16 Thus, we collected bacteria incubation medium after culturing different time intervals by centrifuge to further simulate the bacterial infection microenvironment to investigate the infection-responsive release kinetics. As shown in Figure S1(C), MNA molecular was dramatically released in the bacterial solution, and the longer the culturing time, the faster the drug release for the reason that more esterase was secreted to catalyze the hydrolysis of ester groups. The antibacterial property of the ProMNA was evaluated by spread plate assay, as shown in Figure S1(D). The bacterial colony formation was almost completely inhibited when the bacterial suspension was co-cultured with ProMNA, indicating that intact MNA molecular could be released to kill the bacteria.

Figure S2. The relative survival rates of L929 cells cultured with the leach liquor of the membranes loaded with different Pro-MNA.

Figure S3. The morphology of L929 cells cultured with the leach liquor of the membranes loaded with different Pro-MNA.

Figure S4. SEM observations of the cell morphology after infecting bacteria.

Figure S5. (A) the superficial view of micro-CT images taken at the eighth week after surgery; micromorphometric bone parameters including bone volume fraction (B), trabecular thickness (C), and trabecular separation (D) analyzed after 8

weeks of surgery.

Figure S6. The 3D fluorecent image of the fluoresecent bovine serum albumin adsorbed onto the membrane.