Biodegradable Electrospun Scaffolds for Annulus Fibrosus Tissue Engineering: Effect of Scaffold Structure and Composition on Annulus Fibrosus Cells in Vitro
Authors: Grad S. , Wismer N. , Fortunato G. , Ferguson S. J. , Alini M. , Eglin D. 
Published: June 2012

Introduction

Lumbar disk herniation is the pathological condition for which spinal surgery is most often performed. One significant shortcoming is the high recurrence rate after conventional discectomy. This is partly due to the fact that existing methods for annulus fibrosus repair are not able to restore normal tissue structure and biomechanical function. A biological annular repair may improve the surgical outcome by providing an environment which mimics the AF tissue mechanics and structure. A cell-seeded membrane patch is likely to be a promising approach for AF repair, though the adequate properties of a suitable membrane patch have not been described yet. In this study, the effect of structural and material properties of poly(ester-urethane) (PU) and poly(?-caprolactone) (PCL) based membranes, processed by electrospinning technology, on AF cells cultured onto their surface was investigated. The aim was to assess the potential of these membranes to support the regeneration of damaged AF.

Materials and Methods

PU (Mn = 500,000 g/mol), produced as previously reported,1 and PCL (Mn = 80,000 g/mol) were processed into nonoriented and oriented electrospun scaffolds to mimic collagen fiber alignment of AF tissue. The scaffolds were evaluated using scanning electron microscopy; image analysis software was used to quantitatively analyze fiber diameter and orientation. The mechanical behavior was characterized by uniaxial tensile testing and compared to reported values for human outer AF lamella.2 AF cells were isolated from bovine tail disks, seeded onto the scaffolds (7.8 × 104 cells/cm2) and cultured in DMEM containing 10% FCS and 50 µg/mL ascorbic acid for up to 21 days. Spin-coated flat membranes were used as controls. Analyses included DNA, glycosaminoglycan (GAG), collagen content, histology, and collagen type I immunohistochemistry. Gene expression levels of collagen type I, collagen type II, aggrecan, elastin, biglycan, versican, and decorin were determined.

Results

Mean fiber diameters were 3.10 ± 1.49 µm and 2.17 ± 1.53 µm for non-oriented and oriented PCL, whereas 0.92 ± 0.36µm and 0.84 ± 0.32µm were measured for nonoriented and oriented PU. The failure stress of an AF lamella is 5.6 to 10.3 MPa.2 The oriented PU scaffold was able to withstand similar forces (4.65 ± 0.42MPa) before reaching the yield point and was the only material reaching the range of the failure strain (26.81 ± 1.16%) of a native AF lamella (9.2 to 12.7%). Despite this, the Young's modulus of oriented PU (17.96 ± 0.66MPa) remained below that of a single lamella of the AF (82–136MPa). Only the oriented PCL (46.00 ± 1.09MPa) came close to the values of the native AF. An increase in DNA was observed after 14 days relative to day 1 for all groups. At day 7, the DNA content was higher in electrospun PU than PCL scaffolds. After 14 and 21 days, there was an overall higher DNA amount in electrospun compared to spin-coated surfaces. In all groups, the amounts of GAG and collagen increased between day 7 and 21. GAG retention in the scaffolds was higher in electrospun scaffolds compared to flat control surfaces. GAG contents were increased in nonoriented compared to oriented scaffolds (Fig. 1). Moreover, by day 21 the collagen content was enhanced in the electrospun scaffolds compared to the spin-coated surfaces. Relative gene expression of elastin was upregulated, while collagen type II and aggrecan expression was downregulated by day 21 compared to day 0. The remaining genes were unchanged over the culture time. There was no difference in gene expression between the different membranes. In all scaffolds, deposition of extracellular matrix (ECM) could be observed by Toluidine blue staining and immunohistochemistry. Cell and matrix ingrowth was increased in PCL compared to PU scaffolds, which may be due to the higher porosity of PCL materials. Fig. 1. GAG content after 7, 14, and 21 days of AF cell culture on spin-coated PCL (PCL sc), spin-coated PU (PU sc), nonoriented PCL (PCL no es), nonoriented PU (PU no es), oriented PCL (PCL o es), and oriented PU (PU o es). GAG retained in scaffolds and released into medium is shown as * p < 0.05 for total GAG. Mean ± SD. N = 5 experiments (triplicates each)

Conclusion

Both PU and PCL membranes supported AF cell growth and ECM production and accumulation. Electrospinning improved the retention of collagen and GAG in the scaffolds. Based on their mechanical characteristics oriented electrospun PU scaffolds may be appropriate materials for AF tissue engineering. Further studies with an improved 3D environment to increase cell ingrowth and to prevent cell de-differentiation will be required. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Boissard CI, et al. Acta Biomater 2009;5:3316–3327 Skaggs DL, et al. Spine (Phila Pa 1976.) 1994;19:1310–1319