*2.1. PLGA*

PLGA 85% L-lactide and 15% glycolide (*w*/*v*), (Purac: PLG 8531-lot No.1504000089, Amsterdam, The Netherlands) were produced using the randomly oriented electrospinning process. Polymeric solutions were prepared dissolving 5% PLGA (weight/volume) in the solvent-binary of chloroform (CHCl3-Vetec, Rio de Janeiro, Brazil) and dimethylformamide (DMF-Vetec, Rio de Janeiro, Brazil) systems (80/20 *v*/*v*), and magnetically stirred at room temperature.

### *2.2. Electrospinning Process*

The polymeric solution was introduced into a syringe with a needle attached to an ejector pump under the voltage of 15 kV using the Glassman PS/FC 60p02.0-11 (KDS 100 Infusion Syringe Pump) high voltage source. The solution was ejected induced by the potential difference between the needle tip and the screen, with a working distance of 15 cm and a feeding rating of 0.6 mL/h for 6 h. PLGA filaments were deposited onto an aluminum foil and collected as a membrane. The process was standardized for all frameworks produced (dimensions: 10 <sup>×</sup> 10 cm<sup>2</sup> and thickness of 6.6 <sup>µ</sup>m, analyzed by scanning electron microscopy). Next, PLGA membranes were sterilized using gamma radiation (Cobalt 60 source—MDS Nordion, model GC 220 E, Ottawa, ON, Canada), with a total dose of 15 KGy [28].

### *2.3. In Vitro Experiments*

### 2.3.1. MDCK Cells Cultured onto PLGA Membranes

Madin-Darby canine kidney cells (MDCK, ATCC, cat. No. PTA-6503) were plated in 25 mm<sup>2</sup> culture bottles, in complete Cascade Medium 200 (M200-500) culture medium, incubated at 37 ◦C, in an atmosphere of 5% CO<sup>2</sup> and 95% humidity. After confluence, cells were trypsinized (2 mL TrypLE ™ Express Enzyme (1×), phenol red, cat. No.12605036- Gibco®) for 5 min. Complete culture medium (2 mL) was used to inactivate the process. Next, cells were centrifuged at 700 g for 3 min. PLGA samples (1 <sup>×</sup> 1 cm<sup>2</sup> ) were glued on the bottom of the wells of a 6 wells cell culture dish, covered or not with 20µg/mL fibronectin (human plasma fibronectin, Sigma-Aldrich, St. Louis, MI, USA, cat. No. F0895) in culture medium. After 1 h, PLGA samples were washed twice with sterile phosphate saline buffer (PBS) pH 7.4. Next, 1 mL culture medium containing MDCK cells (10<sup>4</sup> ) was applied only onto the surface of each membrane, and incubated at 37 ◦C, in an atmosphere of 5% CO<sup>2</sup> and 95% humidity for 1 h. After 1 h, complete culture medium (2 mL) was added in the well to cover each sample, and changed every 48 h. The experiments were carried out in quadruplicate and analyzed after 3, 6 and 12 days. PLGA membranes were fixed using 4% buffered paraformaldehyde solution for 15 min, washed in PBS, pH 7.4, twice; permeabilized with triton X-100 (0,1%) in PBS for 15 min; washed again in PBS, and incubated in PBS containing 0.5% serum albumin (BSA) for 1 h. Samples were treated with Alexa Fluor® 555 phalloidin 1:100 (Invitrogen, cat. No. A34055-Thermo-Fisher Scientific, Waltham, MA, USA), in PBS-0.3% BSA, and stored protected from light for 1.5 h. Next, samples were washed with PBS and treated with Hoechst solution (Bisbenzimide H33342 trihydrochloride solution in PBS, 1:1000, Sigma-Aldrich, cat. No. 14533) for 30 min, washed with PBS (3 times). Confocal microscopy analysis (LSM 700, Zeiss) was performed using the objective lens (W Plan-Apochromatic 63×/1.0 M27). Ten fields per sample were obtained over time, using the X-Z orthogonal axis and captured in the Image J Fiji software [29], to count the cell nuclei. Mean ± sd was used for analysis over time. The difference in cellularity was verified using the GraphPad Prism 5 program. For scanning electron microscopy (SEM) analysis, each sample was fixed in a solution of 1 mL glutaraldehyde 2.5% (Reagen—Quimibras Industrias Químicas S.A., Rio de Janeiro, Brasil); 1 mL of 4% formaldehyde (Merck) and 2 mL of Sorensen phosphate buffer (disodium hydrogen phosphate-Na2HPO4—11.876/L; potassium di-hydrogen phosphate (KH2PO4) at 0.2 M, pH 7.2, for 1 h. Then, the samples were washed in the same buffer 3× for 10 min, dehydrated in serial ethanol (Merck) solutions (30, 50, 70 and 90%) for 10 min each, ending with absolute ethanol, twice for 20 min and dried in ambient air. Then, the samples were fixed onto a metallic support with carbon tape, metallized by gold sputtering (20 nm) and observed in a FEI Quanta 400 scanning electron microscope.

### 2.3.2. Primary Hamsters' Fibroblasts Cultured onto PLGA Membranes

Primary fibroblasts from left hamsters' cheek-pouch were isolated after surgery using intraperitoneal ketamine (80 mg/kg) and xylazine (60 mg/kg). The excised cheek pouch was washed with povidine-iodine for 5 min, next rinsed with a saline solution 1 min, treated with nystatin (100.000 UI/Laboratorio Teuto S/A-Brasil) for 5 min, and finally immersed in sterile saline solution for 5 min. Tissues were sectioned into small pieces under a laminar flow hood (Vertical laminar flow—Pachane LTDA, model PA 320, No.03201. Pirasicaba, Brasil); immersed in collagenase type 1 solution (1 mg/mL-Gibco/Life Technologies) in culture medium (100 mL) inside an incubator at 37 ◦C under agitation for 3 h and inactivated using fetal bovine serum (10%—Vitrocell/lot 014/18). Finally, the cells were isolated by filtration (cell strainer 100 µm/Sigma-Aldrich) and centrifugation (700 g) of the supernatant. The pellet was suspended in 1 mL low glucose DMEM culture medium, supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, 100 mg/mL streptomycin and 0.01 mg/mL amphotericin (Sigma Aldrich, St. Louis, MO, USA), plated in a 25 cm<sup>2</sup> culture bottle containing 4 mL culture medium, and kept in an incubator at 37 ◦C in a 5% CO<sup>2</sup> atmosphere. Culture medium was changed every 48 h after 3 washes with PBS, and stored at 37 ◦C in a

5% CO<sup>2</sup> atmosphere for 4 days. In another assay PLGA membranes (1 <sup>×</sup> 1 cm<sup>2</sup> ) were fixed with silicone glue and treated with 100 µL of a mouse collagen solution (Sigma-Aldrich) 10% (*v*/*v*) in serum-free DMEM, incubated for 1 h in the laminar flow and washed using PBS. Next, a suspension of 10<sup>4</sup> or 3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells in 100µL of complete DMEM culture medium was added to each membrane and cultured over 1, 3, 6 and 12 days. Over time, the membranes were fixed in 4% paraformaldehyde in PBS for 30 min, washed in PBS for 5 min 3× and kept in a 20 mL Falcon tube with PBS at 4 ◦C. After 12 days, all the membranes with cells were permeabilized with Triton X-100 (0.1%) in PBS for 10 min, washed 3× in PBS for 5 min, immersed in PBS containing 0.5% BSA for 1 h, washed in PBS for 5 min and incubated with phalloidin-Alexa Fluor 488 (Invitrogen, 1:80) in PBS containing 1% BSA, for 1 h; washed 3× in PBS and finally stained with Hoechst solution (1: 1000 in PBS/Sigma-Aldrich) for 30 min. After washing 3× in PBS for 5 min, membranes were mounted on 3 cm diameter imaging dish with a coverslip. The side of cell seeding was fixed facing the bottom of the coverslips, and covered with a new drop of mounting medium to fix another coverslip. The cell nuclei were counted on the confocal microscope (10 fields per membrane) (LEICA-TCS-SPE, inverted O DMi8, using the ACS-APO 63×/1.30 lens). For SEM analysis, membranes were fixed in a solution of 2.5% glutaraldehyde, 4% paraformaldehyde and 0.1 M sodium cacodylate buffer, for 2 h at room temperature, washed with the same buffer for 10 min, post-fixed in 1% buffered osmium tetroxide solution for 40 min, washed with distilled water for 10 min, dehydrated in serial ethanol solutions (30%, 50%, 70% and 90%) for 10 min each and 100% ethanol for 20 min twice, at the end. Membranes were immersed in hexadimethylsilazane in absolute ethanol (*v*/*v*) solution for 5 min twice and dried. Next, the membranes were mounted on carbon strips attached to a metal plate and metallized with gold (20 nm) (BAL-TEC SCD 050) for analysis (SEM FEI QUANTA 250, operated at 15 kV).

### *2.4. In Vivo Experiment*

Golden Syrian hamsters were purchased at Oswaldo Cruz Foundation-Rio de Janeiro, Brazil, at six weeks of age and maintained at the Health Science Center bioterium of Federal University of Rio de Janeiro-UFRJ-Brazil, in environmental, nutritional, and healthcontrolled conditions, according to "The Guide for Care and Use of Laboratory Animals" (DHHS Publication No (NIH) 85-23, Office of Science and Health Reports, Bethesda, MD 20892—available at: http://www.nih.gov). The Ethics Committee on the Use of Animals in Scientific Experimentation at the Health Sciences Center of the Federal University of Rio de Janeiro, registered in the National Council for Animal Experimentation Control (CONCEA-Brazil), certified the use of hamsters in the present study (protocol No. 003/15, approved on 15 April 2015). Animals between 120 and 205 g were included in the present study. The animals were kept in appropriate cages containing 3 animals/cage, lined with sterile shavings, under constant temperature (23 ± 2 ◦C), in the standard light/dark cycle (12/12 h), with unrestricted access to water and feed. After the experiments animals were euthanized according to the "American Veterinary Medical Association Guidelines on Euthanasia", 2007 (available at http://www.nih.gov).

### *2.5. PLGA Implantation and Material Collection*

PLGA membranes (1 <sup>×</sup> 1 cm<sup>2</sup> ) were implanted in the dorsal region of the hamsters over time (7, 15, 30 and 90 days). The animals were randomly separated in 4 groups and were anesthetized with Ketamine (80 mg/Kg) and Xilasina (60 mg/Kg), in the intraperitoneal region (i.p.) for surgical procedures of PLGA implantation. PLGA was implanted in the upper dorsal region, after asepsis using 70% alcohol and shaving. Two PLGA samples (1 <sup>×</sup> 1 cm<sup>2</sup> ) were implanted in the subcutaneous upper dorsal region, one in each side. Next, the skin edges were sutured (5.0 nylon-Technofio). The animals were kept under observation for 24 h to control post-surgical behavior. Over time (7, 15, 30 and 90 days), PLGA samples were surgically removed with the adjacent skin, and divided in two pieces

for morphological analysis. Next, the animals were euthanized, by inhaling CO<sup>2</sup> followed by decapitation through a guillotine.

### *2.6. Sample Preparation for Morphological Analyzes by Light and Electron Microscopies*

The implanted materials were fixed in 4% paraformaldehyde in PBS, pH 7.4 at 4 ◦C for 2 h. Next, washed in running water, dehydrated in serial solutions of ethanol from 30% to 100% twice for 20 min, clarified in xylene (2 baths of 30 min) and embedded in paraffin. Sections of 5 µm-thick were performed on a rotary microtome (Leica Microsystem RM 2125®, Wetzlar, Germany) and collected on glass slides. Dried histological sections were stained with hematoxylin-eosin (HE) and Masson's trichrome stains. The other samples were immersed in Karnovsky's fixative solution (4% paraformaldehyde solution, 2.5% glutaraldehyde and 0.1 M sodium cacodylate) for 2 h, washed 3 times in the same buffer, cleaved in cubes of about 1 mm<sup>2</sup> and post-fixed with 0.1 M osmium tetroxide solution for 30 min. Next, they were washed with cacodylate buffer 3 times for 10 min, dehydrated in serial ethanol solutions to absolute 2 times for 20 min and embedded in Spurr resin. The selected region was cut into 90 nm thickness and contrasted with 1% uranyl acetate and 2% lead citrate for transmission electron microscopy analysis in a Morgagni 268 (FEI company, Hillsborough, OR, USA) operated at 80 KV.

### *2.7. Immunohistochemistry*

Paraffin sections were collected on silanized histological slides (Sakura Finetek, Staufen, Germany) for immunohistochemistry. After adhesion, histological sections were dewaxed in xylene and hydrated. The samples were washed with 50 mM ammonium chloride solution, in phosphate buffered saline (PBS) pH 8.0, for 15 min, to block free aldehyde residues and washed in PBS; permeabilized with Triton X100 (0.5%) in PBS for 15 min, followed by a bath containing 0.3% hydrogen-peroxide in methanol to inhibit endogenous peroxidase for 15 min, in the dark. After washing with PBS, pH 7.4, sections were submitted to heat-mediated antigen retrieval in either the microwave (potency 800 W) or steamer, according to the antibody used (Table 1). After cooling, the histological sections were incubated with PBS containing 5% bovine serum albumin (BSA), normal 5% goat serum (1 h) in a humid chamber, at room temperature, and then, primary antibodies (Table 1) were incubated. Sections were maintained in a humid chamber, for about 20 h, in the refrigerator. Afterwards, the sections were washed in a PBS solution containing 0.25% Tween-20 (PBS-Tween), followed by incubation with the secondary antibody conjugated to peroxidase (Envision TM Dual link system-HRP—cat. No. K4601, Dako, CA, USA), for 1 h, followed by washes in PBS-Tween. Peroxidase was developed with the chromogenic substrate diaminobenzidine (Liquid DAB, Dako, cat. No. K3468), followed by washes in PBS-Tween and distilled water and assembly of sections between slide and coverglass with Entellan®. Negative controls were performed by incubating the histological sections with non-immune rabbit or mouse serum or with the antibody diluent in place of the primary antibody.


**Table 1.** Characteristics of antibodies used and antigenic recovery in the immunohistochemistry assays.

### *2.8. Morphometrical and Statistical Analysis*

Semi-quantitative analyses of histological sections were performed using an Eclipse E800 light microscope (Nikon, Japan) coupled to a digital camera (Evolution VR Cooled Color 13 bits (Media Cybernetics, Bethesda, Rockville, MD, USA) using the 2×, 4×, 10×, 20× and 40× objectives lens. The interface capture used was Q-Capture 2.95.0, version 2.0.5 (Silicon GraphicsInc, Milpitas, CA, USA), and the images were transmitted to a color LCD monitor, captured (2048 × 1536 pixel buffer) in TIFF format and digitized. The images were captured after calibration of the appropriate color and contrast parameters and remained constant for each type of staining or immunohistochemistry. The quantification of CD3<sup>+</sup> cells (T lymphocytes) or myeloperoxidase + (neutrophils), were performed on the captured images (10 microscopic fields of the slides stained with the respective antibodies, using the 40× objective lens). The results were expressed as number of reactive cells/histological field ± standard error of the mean. The amount of multinucleated giant cells and capillaries (transversal sections) were calculated on 20 photomicrographs randomly obtained from HE stained histological sections (objective lens 20×). Results were expressed as number of multinucleated giant cells or number of capillaries, /histological field ± standard error of the mean.

For statistics, the in vitro analysis of cells was performed by one investigator (AC). Cells nuclei were counted on Image J Fiji software or in Image ProPlus 5.0 (Media Cybernetics, MD, USA). For the in vivo quantification, one investigator (pathologist, CMT) acquired all histological images and performed the quantification, in a blinded manner. The data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The differences between the groups were analyzed using one-way ANOVA on ranks (Kruskal–Wallis test), followed by a post hoc test (Dunn's multiple comparison test or Neuman–Keuls). Differences were considered significant at *p* < 0.05, with asterisks indicating the level of significance: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001; ns indicates no significant differences.

### **3. Results**

### *3.1. In Vitro Experiments*

### 3.1.1. MDCK-Cell Line

MDCK cell line is a widely used epithelial cell line to test the monolayer formation. They were used in the present work to test the electrospun PLGA (85:15) membranes produced herein. The cells adhered and proliferated on both fibronectin-coated (FN) and uncoated (no-FN) PLGA membranes, independently, over time. These results showed that electrospun membranes with random fibers, with or without FN promoted cell proliferation, such as shown in Figure 1A–C, with cells forming a monolayer onto the membranes. MDCK cells preferentially adhered and proliferated on the fibronectin (FN) coated PLGA membranes as seen in (Figure 1C). After 3 days in culture, the number of epithelial MDCK cells on FN-coated or no-FN membranes showed no difference (Figure 1C: 3 days FN: <sup>27</sup> <sup>±</sup> 2 cells/ mm<sup>2</sup> to specify; 3 days no-FN: 24 <sup>±</sup> 1 cells/ mm<sup>2</sup> , *n* = 4). An increase in the number of cells was observed after 6 and 12 days (Figure 1C), regardless of treatment with fibronectin. However, after 12 days, there was a greater number of cells on FN-coated membranes in comparison to no-FN membranes (Figure 1C: 6 days FN: 59 <sup>±</sup> 8 cells/mm<sup>2</sup> ; no-FN: 50 <sup>±</sup> 8 cells/mm<sup>2</sup> ; 12 days FN: 131 <sup>±</sup> 16 cells/mm<sup>2</sup> ; no-FN: 102 <sup>±</sup> 16 cells/cm<sup>2</sup> ).

To illustrate, a confocal image is showing MDCK cells through the fibers of no-FN membrane fibers (Figure 1(Aa)) after 3 days in culture. For comparison, image of FNcoated membrane (Figure 1(Af)) is showing cells proliferating through the fibers after 3 days in culture (thick arrow). In an equivalent view of Figure 1(Aa,Af), the cell nuclei (Figure 1(Ab,Ag)) are exhibiting different distribution, showing that cells are following the random design of the fibers. In another equivalent view of A-a (Figure 1(Ac)), confocal image of no-FN membrane (Figure 1(Ac), arrows) after phalloidin staining after 3 days reveals that cells are distributed among the fibers but are not connected since no circumferential actin belt is visible at that time (Figure 1(Ac), circle). For comparison, confocal image of FN-coated membrane (Figure 1(Ah)) exhibits regions where cells appeared to be connected since circumferential actin belt is sometimes visible (Figure 1(Ah), thin arrows) among cells without adhesions (Figure 1(Ah), thick arrows), still not taking the classic cuboidal

morphology after 3 days. Image of orthogonal projections (Figure 1(Ad,Ai)) showing cells after 3 days reveals the monolayer formation onto no-FN (Figure 1(Ad)) or FN-coated membranes (Figure 1(Ai)), both with few in-depth cell migration after 3 days.

Image of cells onto no-FN membrane after 12 days (Figure 1(Ba,Bf)) reveals more localized MDCK cells among PLGA fibers (Figure 1(Ba), arrow; double arrow) in comparison to FN-coated membrane showing a dense monolayer of cells among the fibers (Figure 1(Bf), double arrow). An equivalent image of Figure 1(Ba), f reveals the cells' nuclei on no-FN and FN coated membranes (Figure 1(Bb,Bg)), respectively in close contact to each other and exhibiting different directions according to fibers' organization. Moreover, images after phalloidin staining of no-FN or FN-coated membranes (Figure 1(Bc,Bh)), depicts increased cell connections with circumferential actin belt; both taking their classic cuboidal morphology after 12 days (Figure 1(Bc,Bh), arrows). Note that orthogonal projections images after 12 days (Figure 1(Bd,Bi)) show MDCK cells taking decreased size in comparison to cells after 3 days in culture. This can be explained by the proliferation of the cells, causing them to thicken and take the classic cuboidal morphology of epithelial cells at confluence, visible on orthogonal projections. SEM images of MDCK cells reveals the presence of isolated cells after 3 days (Figure 1(Ae,Aj), thin arrows and asterisks), exhibiting less amount of MDCK cells onto no-FN and FN membranes, in comparison with the cluster of cells with extracellular matrix visible after 12 days (Figure 1(Be,Bj), white arrows and asterisks). In addition, SEM image of FN-coated membrane after 12 days (Figure 1(Bj), white arrows) shows cells spreading in close contact with the membrane fibers', by forming cell extensions towards areas without cells following PLGA fibers direction.

Number of cells/mm<sup>2</sup> over time is illustrated in Figure 1C. The results are expressed as mean ± SEM (standard error of the mean). Images of cell nuclei (*n* = 10) stained with Hoechst on each sample (*n* = 4) were captured by CLSM (63× objective lens) and counted. Data were submitted to one-way ANOVA followed by Bonferroni's multiple comparison test (ns: not significant, \*\* *p* < 0.001, \*\*\* *p* < 0.0001).

### 3.1.2. In Vitro Experiment Using Primary Fibroblast-like Cells

Primary fibroblasts isolated from hamsters' cheek paunch were seeded onto collagencoated electrospun PLGA (85:15) membranes. To select the best construct, two cell concentrations (10<sup>4</sup> or 3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells) were tested and compared over time (1, 3, 6 and 12 days) using confocal microscopy (CLSM). Both concentrations of cells adhered and proliferated over time onto PLGA-coated membranes. The construct with 3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells showed increased cell proliferation after 6 and 12 days in culture; such cells took an elongated morphology and migrated towards the interior of PLGA membrane, posing an ideal dressing for tissue engineering and future tests as cell delivery therapy.

The primary culture of fibroblast-like cells is represented on a phase-contrast image (Figure 2A) (fourth passage) after 4 days, and the random electrospun PLGA fibers covered with collagen is represented on a CLSM image (Figure 2B). After 1 day in culture, cells adhered on the surface of the fibers (Figure 2C, white arrows depict the PLGA fibers), and took an elongated morphology, taking different directions following the fibers (Figure 2D, white arrows). The maximum projection confocal images show cell proliferation (3 <sup>×</sup> <sup>10</sup><sup>5</sup> ) over time and their respective in depth migration (Figure 2(Ee,Ff,Gg,Hh)). After one day, cells were only on the surface of the membrane (Figure 2(Ee)). After 3 days (Figure 2(Ff), white arrows) confocal image shows cells initiating the in-depth migration. After 6 days (Figure 2(Gg), white arrows) more cells were migrating to the interior of the membrane, while a deeper in-depth migration was observed after 12 days (Figure 2(Hh), white arrows), in three different areas of the same membrane. Results of cell proliferation in the assay with <sup>3</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> cells after 6 and 12 days, reveal significantly higher proliferation of cells compared to other times (Figure 2I). However, it was possible to observe an increase in the number of cells in each assay with 10<sup>4</sup> cells (1 day: 9 ± 2; 3 days: 30 ± 2; 6 days: 43 ± 2; 12 days: <sup>47</sup> <sup>±</sup> 4; or 3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells) or 3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells (1 day: 35 ± 4; 3 days: 45 ± 10; 6 days: 64 ± 3; 12 days: 76 ± 2) (Figure 2J).

**Figure 1.** Representative images of MDCK cells cultured onto fibronectin-coated (FN) or uncoated (no-FN) PLGA membranes. (**A**,**B**): Confocal Laser Scanning Microscopy images of cells after 3 and 12 days, stained with Hoechst (blue) and phalloidin conjugated to Alexa 568 (red); (**C**): Graphical analysis. (**Aa**): Merge image of cells among no-FN PLGA fibers (arrow, square) after 3 days; (**Af**): Merge image of FN PLGA fibers and cells (white arrow) after 3 days. (**Ab**,**Ag**): Equivalent fields from (**Aa**,**Af**) processed on Image J software to visualize nuclei. Note the different organization of nuclei following the fibers after 3 days. (**Ac**): confocal images of phalloidin staining of no-FN coated fibers after 3 days (**Ac**, arrows). Note few circumferential actin belt staining revealing adhesions between cells (circle) in comparison with FN membranes (**Ah**) after 3 days showing more adhesions between cells (thin arrows), among cells without adhesions (thick arrows). (**Ad**,**Ai**): cells in a X-Z plane after 3 days forming a monolayer with less cells. Scale bars (**Aa**–**Ai**): 20 µm. (**Ae**,**Ai**): SEM images of no-FN or FN membranes exhibiting isolated cells onto the fibers after 3 days (white arrows and asterisks). Scale bars: 100 µm. (**Ba**,**Bf**): Merge image of cells after 12 days. Note fibers and cells among the fibers on no-FN or FN, respectively (white arrows and double arrows). (**Bb**,**Bg**): Equivalent fields from (**Ba**–**Bf**) showing a greater number of nuclei with a differential organization after 12 days. (**Bc**,**Bh**): Equivalent fields from (**Ba**–**Bf**) showing the increase in circumferential actin belts demonstrating formation of adhesions between cells exhibiting a cuboidal morphology onto PLGA fibers. Note a greater number of cells onto FN coated membrane after 12 days. (**Bd**,**Bi**): cells in an X-Z plane after 12 days showing more proliferation. Scale bars (**Ba**–**Bi**): 20 µm. (**Be**,**Bj**): SEM **Figure 1.** Representative images of MDCK cells cultured onto fibronectin-coated (FN) or uncoated (no-FN) PLGA membranes. (**A**,**B**): Confocal Laser Scanning Microscopy images of cells after 3 and 12 days, stained with Hoechst (blue) and phalloidin conjugated to Alexa 568 (red); (**C**): Graphical analysis. (**Aa**): Merge image of cells among no-FN PLGA fibers (arrow, square) after 3 days; (**Af**): Merge image of FN PLGA fibers and cells (white arrow) after 3 days. (**Ab**,**Ag**): Equivalent fields from (**Aa**,**Af**) processed on Image J software to visualize nuclei. Note the different organization of nuclei following the fibers after 3 days. (**Ac**): confocal images of phalloidin staining of no-FN coated fibers after 3 days (**Ac**, arrows). Note few circumferential actin belt staining revealing adhesions between cells (circle) in comparison with FN membranes (**Ah**) after 3 days showing more adhesions between cells (thin arrows), among cells without adhesions (thick arrows). (**Ad**,**Ai**): cells in a X-Z plane after 3 days forming a monolayer with less cells. Scale bars (**Aa**–**Ai**): 20 µm. (**Ae**,**Ai**): SEM images of no-FN or FN membranes exhibiting isolated cells onto the fibers after 3 days (white arrows and asterisks). Scale bars: 100 µm. (**Ba**,**Bf**): Merge image of cells after 12 days. Note fibers and cells among the fibers on no-FN or FN, respectively (white arrows and double arrows). (**Bb**,**Bg**): Equivalent fields from (**Ba**–**Bf**) showing a greater number of nuclei with a differential organization after 12 days. (**Bc**,**Bh**): Equivalent fields from (**Ba**–**Bf**) showing the increase in circumferential actin belts demonstrating formation of adhesions between cells exhibiting a cuboidal morphology onto PLGA fibers. Note a

image showing cells covering the majority of the membrane after 12 days (white arrows and aster-

greater number of cells onto FN coated membrane after 12 days. (**Bd**,**Bi**): cells in an X-Z plane after 12 days showing more proliferation. Scale bars (**Ba**–**Bi**): 20 µm. (**Be**,**Bj**): SEM image showing cells covering the majority of the membrane after 12 days (white arrows and asterisks). Scale bars: 200 µm. (**C**): Graphical representation of number of MDCK cells/mm<sup>2</sup> onto FN or no-FN PLGA membranes. ns: not significant, \*\* *p* < 0.001, \*\*\* *p* < 0.0001.

SEM images of collagen-coated membranes showed a collagen network in the interfibrillar spaces (Figure 2K, fiber: arrow; collagen: asterisk). In another SEM image (Figure 2L), a cluster of cells in extracellular matrix spread on the surface of the PLGA membrane in intimate contact with PLGA fibers (black arrows). Interestingly, sometimes cells showed an elongated morphology following PLGA fibers (Figure 2M, arrow heads), while other cells display more spread morphologies in the interfibrillar space when adhering to multiple fibers (Figure 2M, asterisks).
