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NANOFIBER ALIGNMENT ENHANCES THE DEVELOPMENT OF ENGINEERED MENISCUS CONSTRUCTS
Baker, BM; Tan, AR; Metter, RB; Nathan, AS; +Mauck, RL McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA
INTRODUCTION The meniscus is a load-bearing, fiber-reinforced, fibrocartilaginous wedge vital to proper knee function [1]. Damage to the meniscus through injury or degeneration inhibits efficient load transfer. Since much of the meniscus is avascular, healing in these regions is limited. Current treatment of meniscus tears involves removal of the torn segment, however resection increases stress on adjacent articular surfaces and predisposes patients to osteoarthritis (OA) [2]. Filling a meniscal defect with a functional tissue may promote healing, return function to the knee, and avert the onset of OA. While there have been numerous studies whose goals have been to deliver meniscal fibrochondrocytes (FC) or mesenchymal stem cells (MSC) to meniscus defects, few groups have focused on engineering scaffolds that mimic both the structural organization of the native tissue and its associated anisotropy [3,4]. To this end, we use electrospinning to create scaffolds comprised of nanometer scale fibers that are conducive to cell attachment and proliferation. These scaffolds can be formed with nonaligned (NA) or aligned (AL) fibers. In the AL formulation, scaffold architecture mimics the mechanical and structural anisotropy of the native tissue [5]. In this study, we explored the effect of fiber organization on long-term construct maturation, hypothesizing that alignment would promote and direct neo-tissue formation. Furthermore, as MSCs are commonly used in musculoskeletal tissue engineering, we assessed their ability to form functional constructs with culture in chondrogenic media.
METHODS Scaffold Fabrication: A 14.3% w/v solution of poly-ε-caprolactone (Sigma, 80 kD) dissolved in 1:1 tetrahydrofuran:N,N-dimethylformamide was electrospun at 13kV over 20cm and collected for 12h on a stationary plate (NA) or rotating mandrel (AL) at ~10m/s. Cell Culture: For degradation studies, unseeded scaffolds were incubated in PBS at 37oC for up to 10 weeks. For cell-seeding studies, scaffolds were prepared and seeded as described previously [6] using p2 bovine meniscal FCs, isolated from calf menisci, and MSCs, harvested from the tibial bone marrow of the same donors [7]. Constructs were cultured in chondrogenic medium as in [7] changed twice weekly over a 10-week culture period. Mechanical Testing: At defined intervals, samples were preconditioned with 10 cycles of 0.5% strain at 0.1Hz, and tested to failure at 0.1% ε/sec. Tensile modulus was calculated from the linear region of the stress-strain curve (0-5%) and the initial geometry. Imaging: A JEOL 6400 SEM was used to image acellular scaffolds. Images of calcein AM-stained MSCs on AL and NA scaffolds after 1 day of culture were acquired (Molecular Probes, Eugene, OR). Biochemistry: Samples were papain digested [8] and DNA, sulfated glycosaminoglycan (sGAG), and collagen content were determined using the Picogreen dsDNA assay (Molecular Probes, Eugene, OR), DMMB dye-binding assay [9], and the orthohydroxyproline assay [10], respectively. Histology: Constructs were fixed in 4% paraformaldehyde and 8μm thick cross-sections were stained with Hematoxylin and Eosin (H&E), Alcian Blue (AB, pH 1.0), or Picrosirius Red (PSR) for cells, proteoglycans, or collagens, respectively. Statistics: ANOVA with Fisher’s LSD post-hoc tests was used compare groups. RESULTS
The results of this study show that nanofibrous scaffolds can be produced with preferential alignment, directing cell-polarity within the scaffold (Fig 1). AL scaffolds exhibited higher initial tensile moduli (~12MPa) than their NA counterparts (~4MPa, p<0.05). After 10 weeks of degradation, neither AL nor NA acellular scaffolds changed in modulus (data not shown, p>0.23). When seeded with MSCs or FCs, both AL and NA scaffolds showed an increase in mechanical properties over time (Fig 2,
p<0.05). Notably, AL constructs increased by ~10MPa while NA constructs increased by only ~1MPa with both cell types. In culture, cells proliferated on both NA and AL scaffolds and deposited fibrocartilaginous matrix components (Fig 2), with MSCs producing more sGAG and collagen than FCs by day 70 (p<0.001). Histology of transverse sections shows successful cellular infiltration into the entirety of the scaffold and increasing matrix deposition with time (Fig 3).
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Figure 2 - Tensile modulus, DNA content, sGAG/DNA, and collagen/DNA for
MSC and FC constructs with time in culture (n=5/time point), *p<0.05. DISCUSSION Engineered meniscus tissue for implantation should recapitulate the mechanical and structural anisotropy of the native tissue. To address this goal, we have developed fiber-aligned anisotropic biodegradable scaffolds that support both FC and MSC attachment, proliferation, and matrix deposition. In this study, both cell types colonized scaffolds and deposited fibrocartilaginous matrix. Interestingly, similar amounts of matrix (sGAG and collagen) were produced for each cell type on NA and AL scaffolds. With the instructive environment of AL scaffolds directing cell and matrix orientation, marked improvements (8-10 MPa) were observed in AL scaffolds. NA constructs made similar amounts of matrix, yet exhibited much lower increases in properties (~1MPa), suggesting that without a blueprint, tissue formation is disorganized. We also observed higher levels of ECM production by MSCs compared to FCs. These findings, along with comparable increases in mechanical properties, support the use of MSCs in meniscus tissue engineering. By day 70 of this study, AL scaffolds approached 1/3 the properties of the native tissue [6]. Future work will enhance growth by improving cellular homogeneity in the construct, while accelerating matrix deposition via mechanical preconditioning to produce constructs that match the properties of the native meniscus.
ACKNOWLEDGEMENTS This work was supported by the National Institutes of Health (AR050950). REFERENCES 1) Fithian+, Clin Ortho, 1990(252):19-31. 2) Rath and Richmond, Br J Sports Med, 2000. 34(4):252-7. 3) Murphy+, Trans ORS, 2001. 26:193. 4) Izuta+, Knee, 2005. 12(3):217-23.5) Li+, J. Biomech, 2006, in press. 6) Baker+, Proc. of ASME-BED 2006, #157580. 7) Mauck+, OA Cart, 2006. 14(2):179-89. 8) Mauck+, Trans. ORS, 2005. 30:1722. 9) Farndale+, Biochim Biophys Acta, 1986. 883(2):173-7. 10) Stegemann+, Clin Chim Acta, 1967. 18(2):267-73.
Figure 1 - SEM images of acellular NA (top left) and AL (top right) scaffolds. MSCs on NA (bottom left) and AL (bottom right) scaffolds. Scale bars: 50um.
Figure 3 - H&E (left), AB (middle), and PSR (right) staining of FC- (top) and
MSC- seeded (bottom) aligned constructs on Day 70. Scale bar: 1mm.
53rd Annual Meeting of the Orthopaedic Research Society
Poster No: 0779
