Spine 2011, 36:24, 2021-30. 2. Chan et al. PLOS One 2013, 8(8), e72489. PP472. A bioreactor-based 3D culture system for skeletal muscle engineering in fibrin ...
478 Discussion and conclusions: The IVD cells demonstrated cellular stress response in response to loading induced stress with upregulation of the stress response proteins expression. The cells express HSP72 in response to the stress while HSF1 may have a slower and transient expression. The increase in HSP72 and HSF1 expression after two rounds of loading may show that the cells take longer than 2 days to adapt to the stress induced by mechanical loading. Acknowledgments: This project was partially funded by a fellowship exchange award by AO Spine Scientific Research Network (SRN). Disclosure: There is no conflict of interest to be disclosed. References 1. Gantenbein, et al. Spine 31.23 (2006): 2665-2673. 2. Paul C, et al. PloS one 7.3 (2012): e33147.
PP471 Optimizing a complex loading protocol for intervertebral disc tissue engineering
SC Chan1, J Walser2, SJ Ferguson2 and B Gantenbein1 1 Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland; 2Institute for Biomechanics, ETH Z€ urich, Z€ urich, Switzerland Introduction: The intervertebral disc continuously experiences different types of repeated dynamic loading, including compression and torsion. Therefore, when designing a disc tissue-engineering construct, the influence of complex torsion-compression loading should be considered. Our previous study revealed a positive effect on the cell viability when a repetitive short-term (1 h per day) torsion is applied to intervertebral disc organ culture at a physiological magnitude ! 2° [1]. However, after an extended period (8 h per day) of combined torsion-compression loading, substantial cell death was detected in the nucleus pulposus (NP) [2]. In this follow-up study, we aimed to investigate the relationship between the duration of torsion applied to the IVD and the level of IVD cell viability and matrix gene expression. Materials and methods: Bovine caudal discs were harvested and cultured in a custom-built complex loading bioreactor [2]. Complex loading of 0.2 MPa compression and !2° dynamic torsion was applied, at a frequency of 0.2 Hz, to samples for different durations: 0, 1, 4 and 8 hours and repeated for 7 days. After the last day of loading, disc tissue was dissected for analysis of cell viability and gene expression. Results: Disc NP cell viability remained above 85% after torsional loading for 0, 1, or 4 hours per day. Viability was reduced to below 70% when torsion was applied for 8 h per day (P = 0.03; Table 1). The daily duration of torsional loading did not affect the annulus fibrosus (AF) cell viability (> 80% for all loading durations). A trend of collagen 2 gene up-regulation and MMP 13 down-regulation with an increasing duration of torsion was observed in both the NP and AF (figure 1).
Figure 1. Gene expression of the NP and AF after different durations of dynamic torsion.
Poster Presentations Table 1. Cell viability of the disc after different duration of torsion. Mean ! SD. N = 6, *P < 0.05. Time (h)
NP
0
87.17 ! 10.89
1 4 8
AF 89.87 ! 7.34
91.24 ! 6.31*
92.05 ! 12.27
67.19 ! 16.07 *
84.45 ! 13.96
89.31 ! 12.35
81.38 ! 13.89
Discussion and Conclusions: We have shown that dynamic torsion affects disc matrix gene expression and cell viability in organ culture, in a dose-dependent fashion. Torsion may improve matrix synthesis when applied to intervertebral disc tissue engineering constructs for a short duration, however longer loading durations are detrimental. Acknowledgments: Funds from the Orthopedic Department of the Insel University Hospital of Bern and a private donation from Prof. Dr. Paul Heini, Spine Surgeon, Sonnenhof Clinic Bern were received to support this work. Disclosure: The authors have nothing to disclose. References 1. Chan et al. Spine 2011, 36:24, 2021-30. 2. Chan et al. PLOS One 2013, 8(8), e72489.
PP472 A bioreactor-based 3D culture system for skeletal muscle engineering in fibrin scaffolds
P Heher1, C Fuchs2, J Pr€ uller3, B Maleiner3, J Kollmitzer3, 3 2 D R€ unzler , A Teuschl , S Wolbank1 and H Redl1 1 Trauma Care Consult, Ludwig Boltzmann Institute for experimental and clinical Traumatology/AUVA Research Center, Vienna, Austria; 2City of Vienna Competence Team Bioreactors, Department of biochemical Engineering, UAS Technikum Wien, Vienna, Austria; 3Department of biochemical Engineering, UAS Technikum Wien, Vienna, Austria Introduction: Fibrin is a versatile biomaterial that has been used extensively in a variety of tissue engineering applications. We have developed a 3D in vitro culture system using a bioreactor (MagneTissue) which allows mechanical stimulation of myoblasts embedded in a ring shaped fibrin scaffold by application of strain. Using this system we sought to analyze the effects of mechanical strain on cell alignment and distribution, viability and expression of muscle markers. Materials and methods: Fibrin rings were prepared by injection molding (TISSUCOL DUO 500 Fibrin Sealant Kit, Baxter) with following final concentrations: 4 9 106 C2C12 myoblasts embedded per ring, 20 mg/ml fibrinogen and 0.625 U/ml Thrombin. The rings in the strain group were applied to the bioreactor system. At day 3, growth medium was changed to differentiation medium (both supplemented with 100 KIU/ml Aprotinin) for strain and control groups and 10% static strain was applied to the strain group daily for 1 hour for the rest of the culture period (until d9). Results: We demonstrate that over a culture period of at least 9 days the cells remain viable and partially differentiate into myotubes. Additionally, application of mechanical strain leads to parallel cell alignment (Fig. 1A) and also seems to facilitate nutrient diffusion within the scaffold, demonstrated by improved cell distribution (Fig. 1B). Furthermore, myogenic differentiation is confirmed by transcriptional up-regulation of myogenic markers and by histological analysis.
© 2014 The Authors. J Tissue Eng Regen Med 2014; 8 (Suppl. 1): 207–518. Journal of Tissue Engineering and Regenerative Medicine © 2014 John Wiley & Sons, Ltd. DOI: 10.1002/term.1932
