Abstract
Background
Tissue engineering techniques using biodegradable three-dimensional (3D) scaffolds with cultured cells offer more potential alternatives for the treatment of severe ligament and tendon injuries. In tissue engineering, one of the crucial roles of 3D scaffolds is to provide a temporary template with the biomechanical characteristics of the native extracellular matrix (ECM) until the regenerated tissue matures. The purpose of the present study was to assess the effect of various cyclic mechanical stresses on cell proliferation and ECM production in a 3D scaffold made from chitosan and hyaluronan for ligament and tendon tissue engineering.
Methods
Three-dimensional scaffolds seeded with rabbit patella tendon fibroblasts were attached to a bioreactor under various conditions: static group, no strain; stretch group, tensile strain; rotational group, rotational strain; combined group, rotational and tensile strain. In the Static group, 3 weeks of stationary culture was performed. In the remaining three groups, a loading regimen of 0.5 Hz for 18 h and then 6 h rest was carried out for 2 weeks after 1 week of static culture. The DNA content was determined to quantify cell proliferation. Real-time reverse transcription polymerase chain reaction analysis was performed to assess the mRNA levels of the ECM products.
Results
DNA content of the combined group was significantly higher than that of the static and stretch groups, and that of the rotational group was significant higher than that of the static and stretch groups at 21 days after cultivation. The mRNA level of types I and III collagen and fibromodulin in the combined group was significantly higher than that in the other three groups. The amount of collagen synthesis in the combined group was higher than that in the static group, but the difference was not significant.
Conclusions
Multidimensional cyclic mechanical strain to mimic the physiological condition in vivo has the potential to improve or accelerate tissue regeneration in ligament and tendon tissue engineering using 3D scaffolds in vitro.
Similar content being viewed by others
References
Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–966.
Hynes RO. Cell adhesion: old and new questions. Trends Cell Biol 1999;9:M33–M37.
Funakoshi T, Majima T, Iwasaki N, Suenaga N, Sawaguchi N, Shimode K, et al. Application of tissue engineering techniques for rotator cuff regeneration using a chitosan-based hyaluronan hybrid fiber scaffold. Am J Sports Med 2005;33:1193–1201.
Funakoshi T, Majima T, Iwasaki N, Yamane S, Masuko T, Minami A, et al. Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering. J Biomed Mater Res A 2005;74:338–346.
Kim SG, Akaike T, Sasagaw T, Atomi Y, Kurosawa H. Gene expression of type I and type III collagen by mechanical stretch in anterior cruciate ligament cells. Cell Struct Funct 2002;27:139–144.
Altman GH, Horan RL, Martin I, Farhadi J, Stark PR, Volloch V, et al. Cell differentiation by mechanical stress. FASEB J 2002;16:270–272.
Toyoda T, Matsumoto H, Fujikawa K, Saito S, Inoue K. Tensile load and the metabolism of anterior cruciate ligament cells. Clin Orthop 1998;(353):247–255.
Beynnon BD, Fleming BC. Anterior cruciate ligament strain in-vivo: a review of previous work. J Biomech 1998;31:519–525.
Kim YJ, Sah RL, Doong JY, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168–166.
Miller EJ, Rhodes RK. Preparation and characterization of the different types of collagen. Methods Enzymol 1982;82(Pt A):33–64.
Lin VS, Lee MC, O’Neal S, McKean J, Sung KL. Ligament tissue engineering using synthetic biodegradable fiber scaffolds. Tissue Eng 1999;5:443–452.
Park SA, Kim IA, Lee YJ, Shin JW, Kim CR, Kim JK, et al. Biological responses of ligament fibroblasts and gene expression profiling on micropatterned silicone substrates subjected to mechanical stimuli. J Biosci Bioeng 2006;102:402–412.
Yang G, Crawford RC, Wang JH. Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions. J Biomech 2004;37:1543–1550.
Zeichen J, van Griensven M, Bosch U. The proliferative response of isolated human tendon fibroblasts to cyclic biaxial mechanical strain. Am J Sports Med 2000;28:888–892.
Lee CH, Shin HJ, Cho IH, Kang YM, Kim IA, Park KD, et al. Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast. Biomaterials 2005;26:1261–1270.
Gilbert TW, Stewart-Akers AM, Sydeski J, Nguyen TD, Badylak SF, Woo SL. Gene expression by fibroblasts seeded on small intestinal submucosa and subjected to cyclic stretching. Tissue Eng 2007;13:1313–1323.
Hsieh AH, Tsai CM, Ma QJ, Lin T, Banes AJ, Villarreal FJ, et al. Time-dependent increases in type-III collagen gene expression in medical collateral ligament fibroblasts under cyclic strains. J Orthop Res 2000;18:220–227.
Majima T, Yasuda K, Yamamoto N, Kaneda K, Hayashi K. Deterioration of mechanical properties of the autograft in controlled stress-shielded augmentation procedures: an experimental study with rabbit patellar tendon. Am J Sports Med 1994;22:821–829.
Lee IC, Wang JH, Lee YT, Young TH. The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system. Biochem Biophys Res Commun 2007;352:147–152.
Neidlinger-Wilke C, Grood ES, Wang JC, Brand RA, Claes L. Cell alignment is induced by cyclic changes in cell length: studies of cells grown in cyclically stretched substrates. J Orthop Res 2001;19:286–293.
Miyaki S, Ushida T, Nemoto K, Shinjo H, Itabashi A, Ochiai N, et al. Mechanical stretch in anterior cruciate ligament derived cells regulates type I collagen and decorin expression through extracellular signal regulated kinase 1/2 pathway. Master Sci Eng C 2001;17:91.
Henshaw DR, Attia E, Bhargava M, Hannafin JA. Canine ACL fibroblast integrin expression and cell alignment in response to cyclic tensile strain in three-dimensional collagen gels. J Orthop Res 2006;24:481–490.
Bianco P, Fisher LW, Young MF, Termine JD, Robey PG. Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues. J Histochem Cytochem 1990;38:1549–1563.
San Martin S, Zorn TM. The small proteoglycan biglycan is associated with thick collagen fibrils in the mouse decidua. Cell Mol Biol (Noisy-le-grand) 2003;49:673–678.
Lee CY, Liu X, Smith CL, Zhang X, Hsu HC, Wang DY, et al. The combined regulation of estrogen and cyclic tension on fibroblast biosynthesis derived from anterior cruciate ligament. Matrix Biol 2004;23:323–329.
Sung KL, Whittemore DE, Yang L, Amiel D, Akeson WH. Signal pathways and ligament cell adhesiveness. J Orthop Res 1996;14:729–735.
Ezura Y, Chakravarti S, Oldberg A, Chervoneva I, Birk DE. Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons. J Cell Biol 2000;151:779–788.
Noth U, Schupp K, Heymer A, Kall S, Jakob F, Schutze N, et al. Anterior cruciate ligament constructs fabricated from human mesenchymal stem cells in a collagen type I hydrogel. Cytotherapy 2005;7:447–455.
Chen J, Horan RL, Bramono D, Moreau JE, Wang Y, Geuss LR, et al. Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering. Tissue Eng 2006;12:3085–3095.
Vunjak-Novakovic G, Altman G, Horan R, Kaplan DL. Tissue engineering of ligaments. Annu Rev Biomed Eng 2004;6:131–156.
Author information
Authors and Affiliations
About this article
Cite this article
Sawaguchi, N., Majima, T., Funakoshi, T. et al. Effect of cyclic three-dimensional strain on cell proliferation and collagen synthesis of fibroblast-seeded chitosan-hyaluronan hybrid polymer fiber. J Orthop Sci 15, 569–577 (2010). https://doi.org/10.1007/s00776-010-1488-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00776-010-1488-7