Effects of Mechanical Stimulation on Tenocyte Morphology and Gene Expression in Electrospun Collagen Scaffolds 1Chou, A; 1Juneja, SC; 1Chen, T; 2Bradica, G; 1Wu Y; 1Clark R; 1Benoit, D; +1Awad, HA

1University of Rochester, Rochester, NY; 2Kensey Nash Corporation, Exton, PA + hani_awad@urmc.rochester.edu

INTRODUCTION: A variety of fabrication methods can be used to produce polymeric multi-scale fibers including drawing, template synthesis, phase separation, self-assembly, and electrospinning [1]. Recent reports from multiple laboratories have demonstrated that electrospinning can be adapted to fabricate micro- or nano-fibers from a variety of biopolymers. Electrospun nanofibrous scaffolds have been used in a large number of experimental applications in tissue engineering, including tendon [2]. To engineer functional tendon constructs from electrospun scaffolds, significant barriers, including optimizing cell seeding and differentiation on these scaffolds, must still be overcome. In this study, we evaluated the effects of mechanical tensile stimulation on tenocyte-seeded electrospun collagen scaffolds. METHODS: Scaffold fabrication: Randomly oriented and parallel aligned collagen type I scaffolds were electrospun as previously described [3] using 80 mg/ml collagen (Kensey Nash, Exton, PA) in HFP (Figure 1). Scaffolds with dimensions of 4 mm wide by 45 mm in length were crosslinked with 200 mM EDC in ethanol. After crosslinking, the scaffolds were washed with 1X PBS and sterilized under the UV lamp in the cell culture hood for 45 minutes prior to cell seeding. A B

Cell isolation and seeding: Tenocytes were isolated from flexor tendons of 5 four-month old C57BL/6 WT mice. The cells were allowed to form colonies on Petri dishes in 20% FBS, 1% Pen/Strep and 6.5 µL/L of 2-mercaptoethanol. Cells were then passaged four to six times. The scaffolds were seeded at a 96,000 cells per scaffold for cell morphology studies (n=4 scaffolds per group) and 500,000 cells per scaffold for RT-PCR studies (n=4 scaffolds per group) in DMEM supplemented with 10% FBS and 1% Pen/Strep. Mechanical stimulation: 24 hours post-seed, scaffolds were loaded into the LigaGen® bioreactor (Tissue Growth Technologies, Minnetonka, MN). Constructs were mechanically stretched at 4 or 8% tensile strain for 1 second every 5 minutes for a total duration of 6 hours. Four scaffolds were cultured in Petri dishes without stimulation, which served as controls. Cell Morphology: All samples were washed with sterile 1X PBS and fixed in 3.7% paraformaldehyde in 1X PBS for 10 minutes. The scaffolds were then washed with 1X PBS (3x) and permeabilized in 0.1% Triton in 1X PBS and washed 3 times. 10µL of the methanolic stock solution of Phalloidin 488 F-actin stain was diluted in 200 µL for each scaffold. The staining solution was placed on the scaffolds for 20 minutes at room temperature and washed 3 times with PBS. 300 nM of DAPI in PBS was placed on the scaffolds for 5 minutes and washed and mounted on slides using ProLong Gold anti-fade mounting media. The samples were viewed using the FV1000 Olympus confocal microscope. Images were post-processed to adjust brightness and contrast. Gene Expression: The samples were then placed in Trizol and snap frozen in liquid nitrogen and stored in -140°C until RNA extraction. RNA was extracted from samples using the Qiagen RNEasy extraction kit. RT-PCR studies were conducted using primers for mouse β-actin, collagen I, collagen 3, tenascin-c, decorin, scleraxis, and the mechanogrowth factors IGF-Iea, and IGF-Ieb. All gene expression values were normalized to the housekeeping gene (β-actin) and then normalized by the unstimulated controls from each group to obtain the fold-increase in gene expression. Statistics: Two-way ANOVA and Bonferroni’s post-hoc test were used to compare the effects of scaffold alignment and mechanical stimulation on cell morphology and gene expression. Significant differences were determined if p<0.05.

RESULTS: Scaffold fiber orientation influenced the alignment and the aspect ratio of the tenocytes (Figure 2). Mechanical stimulation further

enhanced the alignment and elongation of tenocytes on the parallel-aligned scaffolds. Cells cultured on the randomly oriented scaffold did not appear to have a preferred direction of alignment with mechanical stimulation. Aspect ratio measurements of the cells in both groups revealed an increase with mechanical stimulation with statistical differences found in the parallel-aligned scaffolds (Figure 3). There did not seem to be fiber alignment-induced changes in gene expression. However gene expression of col1a1, col3a1, decorin and scleraxis were enhanced at 8% tensile stretch (Figure 4). Tensile stretch (8%) also resulted in increased expression of Igf-Iea and tenascin-c.

 Figure   2:  Confocal   images  of   tenocytes  stained  with  DAPI  and  Phalloidin  488  on  parallel  aligned  and  randomly  oriented  scaffolds  under  mechanical  simulation.      

 Figure 4: Effects of scaffold alignment and mechanical stimulation on gene expression normalized by the housekeeping gene (β-actin), and expressed as multiples of unstimulated controls from each group. Data represented as mean values and error bars represent SEM.

DISCUSSION:

Electrospinning offers the ability to control both the macro and micro scaffold architecture to recreate the extracellular matrix of the tendon. Our results indicate that both fiber alignment and mechanical stimulation had effects on tenocyte cell morphology and gene expression responses. Cell alignment and elongation were enhanced with mechanical stimulation in cells seeded on parallel scaffolds, mimicking the cell morphology in native tendon. Mechanical stimulation caused increased expression of various tendon genes and mechanoresponsive growth factors, which might have implications for functional tendon tissue engineering. Long-term studies to evaluate the effects of mechanical stimulation parameters on these tenocyte-seeded scaffolds will allow us to improve their biomechanical properties for in vivo implantation. Significance: The repair of tendon/ligament injuries remains a challenge in orthopaedic surgery. The ability to fabricate collagen scaffolds using electrospinning may be a scalable means of creating biomimitic scaffolds for tendon tissue engineering. Mechanical conditioning of these scaffold might be a prerequisite prior to in vivo implantation. REFERENCES: 1. Jayaraman K, et al. (2004) J Nanosci Nanotechnol. 4(1-2):52-65. 2. Sahoo S, et al. (2006) Tissue Eng. 12(1):91-9. 3. Wu YQ et al. (2007) Polymer. 48(19 ):5653-61.

Figure 1: SEM images of fabricated electrospun collagen type I scaffolds with two scaffold orientations: (A) Random alignment. (B) Parallel alignment.

Figure 3: Aspect ratio measurements for actin projections of tenocytes seeded on aligned and randomly oriented fiber scaffolds. Data represented as mean values and error bars represent standard error of mean (SEM).

40 µm Scale bar:

Poster No. 0655 • ORS 2012 Annual Meeting