Assembly of laser structured electrospun scaffolds for bone tissue engineering Æ1McCullen, SD; Æ1Miller PR; Æ1Narayan, RJ; Æ1Loboa, EG

Æ1Joint Department of Biomedical Engineering at UNC-Chapel Hill and North Carolina State University, Raleigh, NC Senior author egloboa@ncsu.edu

INTRODUCTION: Electrospun nanofibrous scaffolds have been investigated extensively for bone tissue engineering due to their three dimensional structure, large surface area, and similar size-scale to the natural extracellular matrix. Though electrospun scaffolds provide many advantages, they are limited by their inherently small pore size and thickness. One method to overcome these limitations is to integrate multiple electrospun nanofibrous scaffolds in an appropriate gel binding system. The integration of a gelatinous system could assist in laminating multiple nanofibrous scaffolds together. Type I collagen is an ideal gel system due to its natural presence in bone and ability to undergo rapid contraction to assist with layer integration. To assist with collagen gel permeation between multiple layers, we assessed the benefits of structured pore arrays fabricated by laser ablation against non-ablated electrospun scaffolds. The objective of this research was to compare assembled electrospun scaffolds with and without laser structured features to assist with collagen gel impregnation and evaluate their behavior in vitro with human adipose-derived stem cells (hASCs) in both expansion and osteogenic differentiating medium conditions. METHODS: Scaffold Fabrication Electrospun scaffolds were prepared using the electrospinning technique. Commerical PLA with an Mw of 70,000 Da was solubilized in chloroform and dimethyl formamide at a ratio of 4:1 to yield a 12 wt% solution. The solubilized polymer was electrospun at a flow rate of 50 μl/min, working distance of 15 cm, applied voltage of 15 kV, and a deposition time of 1.5 hrs. After electrospinning, scaffolds were laser ablated into 10x10 mm squares. A 6x6 array of 300 μm pores were ablated in one half of the scaffolds. Cell Culture and Construct Fabrication Human ASCs (49 y.o. African American female) were cultured on electrospun scaffolds at an initial seeding density of 20,000 cells/cm2 for 48 hrs in complete growth medium (CGM). Scaffolds were layered (3 layers/construct) and polymerized with 400 μl type I collagen gel and cultured for 3 weeks. Constructs were evaluated on days 7, 14, and 21 for mass, cell number, and cell deposited calcium. On day 21, the constructs were fixed and evaluated by histological staining (Hematoxylin and Eosin (H&E)) and electron microscopy. Data is presented as average ± standard error mean. Statistical analysis was performed using ANOVA and Tukey-Kramer HSD tests (significance at p-value < 0.05). RESULTS: Scaffolds with nanofiber dimensions were produced using the electrospinning technique (Figure 1a). Using the method of laser ablation, 300 μm diameter pores were ablated into the electrospun PLA scaffold with minimal thermal degradation around the edge of the pore (Figure 1b). Large arrays were constructed in order to compare the ability of the collagen gel to penetrate through either non-ablated scaffolds (control) or ablated scaffolds (Figure 1c).

Figure 1:SEM images of electrospun nanofibers (a) representative ablated pore (b) and pore array (c) in electrospun scaffold and picture of assembled construct (d). The addition of pore arrays assisted the assembled scaffolds with collagen contraction and subsequent mass loss (data not shown). Histological images indicated that ablated constructs were able to maintain a tight intact structure while non-ablated constructs demonstrated delamination (Figure 2a-d). Scanning electron microscopy also indicated a compact structure for the ablated constructs, while the non-ablated scaffolds were loosely organized (Figure 2 e-h). Aerial view of the ablated scaffolds indicated that hASCs were able to adhere and display a well spread morphology around and within the ablated features (Figure 3a-c).

Figure 2a-h: Histological images (a-d) of H&E stained constructs and electron micrographs of scaffold cross-section after 3 weeks in vitro. Non-ablated constructs (a,b,e,f) displayed delamination (indicated by arrows) of the layers in both CGM (a,e) and ODM (b,f), while the addition of ablated pores assisted with collagen gel impregnation and maintained an intact construct for both CGM (c,g) and ODM (d,h).

Figure 3a-c: SEM images of assembled ablated construct showing pore array (indicated by white arrows) (a) and magnified view of pores (b-c). Human ASCs were able to adhere and proliferate around and within the ablated features (indicated by yellow arrows). To determine the potential of the assembled constructs for bone tissue engineering, hASCs were osteogenically differentiated for 21 days and mineral content was assessed (Figure 4). By day 21 it was noted that there was a significant increase in the amount of calcium deposited by hASCs on the ablated scaffolds under osteogenic stimulation.

Figure 4: Human ASC mineralization data within assembled constructs at days 7, 14, 21. Calcium deposition increased in a temporal manner for the ODM medium groups and it was noted that by day 21, there was significantly more mineral present on the ablated constructs (p < 0.5). DISCUSSION: These findings indicate that the addition of laser structured features in electrospun scaffolds can assist in collagen gel impregnation and minimize delamination as determined via histological and electron microscopy images. Scanning electron microscopy revealed that hASCs were well spread and attached around and within laser-ablated features. In addition, mineralization data indicated a significant difference in mineral content for the ablated construct by day 21. This is the first study to demonstrate the use of ablated assembled electrospun scaffolds, and that the addition of structured pore arrays in stacked electrospun scaffolds facilitates collagen gel impregnation, preserves layer integration, and promotes the mineralization of hASCs under osteogenic stimulation. ACKNOWLEDGEMENTS: We would like to thank Dr. Susan Bernacki, for assistance with histological staining.

Paper No. 288 • 56th Annual Meeting of the Orthopaedic Research Society