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Effects of cell adhesion molecules on adhesion of chondrocytes, ligament cells and mesenchymal stem cells
Materials Science and Engineering C 17 Ž2001. 79–82 www.elsevier.comrlocatermsec
Effects of cell adhesion molecules on adhesion of chondrocytes, ligament cells and mesenchymal stem cells Kohei Tsuchiya a,b, Guoping Chen b, Takashi Ushida b,c,) , Takeo Matsuno a , Tetsuya Tateishi b,c a
b
Department of Orthopedic surgery, Asahikawa Medical College, Midorigaoka, 2-1 Higashi, Asahikawa 078-8510, Japan Tissue Engineering, Research Center, National Institute of AdÕanced Industrial Science and Technology, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan c Biomedical Engineering Laboratory, Graduate School of Engineering, The UniÕersity of Tokyo, Tokyo 113-8656, Japan
Abstract Smooth cell seeding and cell adhesion on temporary porous scaffolds are critical for tissue engineering. We investigated that the effects of type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine, laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid on adhesion of articular chondrocytes, ligament cells and mesenchymal stem cells ŽMSCs., for well understanding the interaction between adhesion molecules and diverse kinds of cells. The cell adhesion molecules were coated on the surfaces of 96-well culture plates. The cells were inoculated and cultured in the wells for 30 min, and subsequently, the number of adhered cells was determined. Type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine promoted significantly more cell adhesion than laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid. MSCs showed a similar adhesion tendency as ligament cells. These two kinds of cells adhered significantly more on fibronectin-coated surface than on type I collagen-, type II collagen-, vitronectin- and poly-L-lysine-coated surfaces, while the effect was decreased for chondrocytes. Fibronectin showed to be most potential to promote cell adhesion, and thus would be effective to be incorporated into porous scaffolds for tissue engineering of cartilage and ligament. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Cell adhesion; Chondrocyte; Ligament cell; Mesenchymal cell; Tissue engineering
1. Introduction Tissue engineering has been shown to be a promising approach to regenerate lost or degenerated tissues or organs for orthopedic applications. In this approach, isolated and expanded cells adhere to a temporary porous scaffold, proliferate, and secrete their own extracellular matrices w1x. The proliferated cells and secreted extracellular matrices will organize into tissues or organs that retain the original shapes of the temporary material scaffold and exhibit cellular, biochemical and biomechanical properties similar to healthy endogenous tissues or organs. The temporary porous scaffolds for tissue engineering can be prepared from various natural or synthetic biomaterials such as collagen, chitosan, hyaluronic acid, polylactic acid, polyglycolic acid and etc. Cell adhesion on the scaffolds is the initial and critical step in the tissue engi) Corresponding author. Tissue Engineering, Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan. Tel.: q81-3-5841-6374; fax: q81-35841-6374. E-mail address: [email protected] ŽT. Ushida..
neering approach w2,3x. The function of seeded cells is strongly dependent on the interaction of cell and scaffold surface. Cell adhesion to scaffolds is mediated by cell surface receptors, an important class of which is the integrin family. Integrins are heterodimeric transmembrane glycoproteins consisting of large globular extracellular domains, which bind to specific extracellular matrix proteins and short cytoplasmic domains that interact with cytoskeletal proteins inside the cells w4–6x. The integrin receptors bind to relatively short amino acid sequences such as Arg–Gly–Asp ŽRGD. on extracellular matrices w7x. The cell adhesion molecules containing RGD and many other cell adhesion sequences include fibronectin, vitronectin, laminin, collagens, and a number of other components of the extracellular matrices. RGD containing sequences and cell adhesion molecules have been incorporated into scaffolds to improve cell adhesion and control cell function. In the present study, the effects of various cell adhesion molecules and extracellular matrices on the initial cellular adhesion properties of articular chondrocytes, ligament cells and mesenchymal stem cells were investigated to target the design of optimal scaffolds for tissue engineering of articular cartilage and ligament.
0928-4931r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 1 . 0 0 3 4 1 - 1
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K. Tsuchiya et al.r Materials Science and Engineering C 17 (2001) 79–82
2. Materials and methods 2.1. Isolation of chondrocytes and ligament cells Bovine articular chondrocytes were isolated from the shoulder articular cartilage of a 4-week-old calf, obtained from a local abattoir within 4 h of slaughter. The cartilage was sliced and minced into 1–2-mm3 pieces. After being rinsed twice in cold Dulbecco’s phosphate buffered saline ŽPBS., the minced cartilage was digested by 0.2% type I collagenase ŽWorthington Biochemical, Lakewood, NJ. in Dulbecco’s modified Eagle’s minimal essential medium ŽDMEM. supplemented with antibiotics Žpenicillin, 100 Urml and streptomycin, 100 mgrml. under shaking overnight at 37 8C. The digestion solution was filtered through a sterile 70-mm nylon mesh to remove any undigested fragments, and the chondrocytes were subsequently collected by centrifugation and washed twice with PBS. The cell number and viability were determined by using a hemocytometer and the trypan blue exclusion dye test. The isolated chondrocytes were suspended in DMEM medium containing 10% FBS Žculture medium., seeded in a 75-cm2 flask at a high cell density, and cultured under an atmosphere of 5% CO 2 at 37 8C for 1 week. Ligament cells were isolated from the anterior cruciate ligament of a knee joint from a 2-week-old Japanese white rabbit. The specimens were sliced after removing of superficial synovium. The manipulation of cell isolation and collection was the same as that for bovine articular chondrocytes. The isolated ligament cells were suspended in culture medium, seeded in 75-cm2 flask at a high cell density, and subcultured under an atmosphere of 5% CO 2 at 37 8C. The second passaged ligament cells were used for adhesion experiment. 2.2. Coating of cell adhesion molecules The cell adhesion molecules and extracellular matrices used in the present study were bovine type I collagen ŽICN Biomedicals, Costa Mesa, CA., bovine type II collagen ŽSigma, St. Louis, MO., human laminin ŽFunakoshi, Japan., bovine fibronectin ŽFunakoshi., bovine vitronectin ŽFunakoshi., bovine chondroitin-4-sulfate ŽSigma., bovine chondroitin-6-sulfate ŽSigma., bovine aggrecan ŽSigma., chicken hyaluronic acid ŽARTZ, Seikagaku, Japan. and poly-L-lysine ŽSigma.. They were diluted with PBS to the final concentration of 500 mgrml, which is higher than the concentration that allows the monolayer absorption of these molecules, and added to 96-well culture plates Ž200 mlrwell.. The plates were kept overnight at 4 8C and then rinsed once with PBS. Subsequently, the plates were added with 1% bovine serum albumin Ž200 mlrwell. and incubated at room temperature for 1 h to block the non-coated sites. The wells were rinsed twice with PBS and used for cell culture.
2.3. Cell culture and adhesion assay Chondrocytes were used 1 week after subculture. Ligament cells were used after one passage. Human mesenchymal stem cells ŽMSCs. were purchased from Biowhittaker, in the second passage. Thawed cells were cultured at 5 = 10 5rcm 2 with defined growth medium under an atmosphere of 5% CO 2 at 37 8C, and the medium was changed twice a week. The third passaged cells were used for adhesion assay. The chondrocytes, ligament cells and MSCs were harvested with PBS containing 0.15% Žwrv. trypsin Ž2000 unitsrg. and 0.02% EDTA. The cells were washed once with medium containing FBS, twice with serum-free medium, and then suspended in serum-free DMEM at the concentration of 2.5 = 10 5 cellsrml Žchondrocytes. and 5 = 10 4 cellsrml Žligament and MSCs.. The cell suspensions were added to the coated culture plates Ž200 mlrwell. and incubated under an atmosphere of 5% CO 2 at 37 8C for 30 min. Unattached cells were removed by washing twice with PBS, while attached cells were quantified with a cell counting kit F assay according to manufacture’s protocol. Briefly, 100 ml PBS and 100 ml of the CalceinAMrDMSO solution diluted to 50 times were added to each well of the plates. The plates were incubated at 37 8C for 1 h. Calcein-AM fluorescence dye was hydrolyzed by endocellular elastase and generated fluorescence. This signal reflects the endocellular elastase activity, which directly correlates with the number of attached cells w8x. The fluorescence intensity was measured at 485 nm of excitation and 530 nm of emission by a fluorescence plate reader ŽCyteFluor2350, MILLIPORE.. Eight wells per adhesion molecule were measured and averaged. By taking the data of non-coated well in each measurement as the controls, statistical analysis was performed using Student’s t-test. 3. Results The standard curves for the relation between the fluorescence intensity and the amount of cells using limiting dilution method were shown to be linear for all the three types of cells Ždata were not shown.. After washing twice with PBS, almost no cells were detected on the surfaces blocked with albumin without coating any adhesion molecules or any extracellular matrices. Fig. 1 shows the calculated number of cells adhered on the surfaces coated with the cell adhesion molecules and extracellular matrices. Lots of cell adhered on the surfaces coated with type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine, whereas few cells adhered on the surfaces coated with laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid for all the three types of cells. Chondrocytes showed the preferable attachment to the coated surfaces in rank of fibronectin, vitronectin, type I collagen, type II collagen and poly-L-lysine ŽFig. 1A.. The
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number of chondrocytes adhered on the surface coated with fibronectin was about 50% of the seeded chondrocytes. The number of cell adhered on poly-L-lysine-coated surface was about half of that on fibronectin-coated surface. Almost 80% of ligament cells adhered on the fibronectin-coated surface. They demonstrated a preference
Fig. 2. Comparison of relative adhesion percent of chondrocyte, ligament cell and mesenchymal cell. The data were derived from the fluorescence intensities by standardizing with those on poly-L-lysine-coated surfaces. ) Statistically significant differences between the cell types Ž P - 0.03..
in attachment to fibronectin) type I collagen) type II collagen) vitronectin) poly-L-lysine. The ligament cells adhered on poly-L-lysine-coated surface was only 20% of that on fibronectin-coated surface ŽFig. 1B.. The difference among type I collagen, type II collagen, vitronectin and poly-L-lysine was not remarkable. MSC cells showed a similar adhesion property as did the ligament cells. Almost all the seeded MSCs adhered on fibronectin-coated surface. Type I collagen-, type II collagen-, vitronectin- and poly-L-lysine-coated surfaces possessed almost the same potential of cell adhesion, which was about 40% of that on fibronectin-coated surface ŽFig. 1C.. The difference of cell adhesion among various cell types was summarized in Fig. 2 by standardizing the adhesion data of chondrocyte, ligament cells and MSCs with their respective adhesion on poly-L-lysine-coated surface. Although fibronectin promoted most adhesion of these cells, the effects seemed to be strongly dependent on cell type. It stimulated the adhesion of ligament cells more than chondrocytes and MSCs. The other adhesion molecules and extracellular matrices also showed some difference between cell types, but not so evident as fibronectin.
4. Discussion Fig. 1. Cell number of chondrocyte ŽA., ligament cell ŽB. and mesenchymal stem cell ŽC. attached to the surfaces coated with different adhesion molecules. Cell number was calculated from their fluorescence intensity using standard curves. COL1: type I collagen, COL2: type II collagen, FN: fibronectin, VN: vitronectin, LN: laminin, AG: aggrecan, HA: hyaluronic acid, C4S: chondroitin-4-sulfate, C6S: chondroitin-6-sulfate, PLL: poly-L-lysin. Data are given as mean"standard error Ž ns8..
Damaged articular cartilage and intraarticular ligament such as anterior cruciate ligament have limited capacity for self-repair. Cell-based therapy using tissue engineering technology has a great future as a next generation therapeutic approach for these tissues. The initial event for seeded cells to function in temporary porous scaffolds is
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cell adhesion on the surfaces of the scaffolds. To improve cell adhesion on scaffolds, various cell adhesion molecules such as collagen w9x, fibronectin w10x, vitronectin w11x, laminin w12x, and poly-L-lysine w13x have been coated or incorporated into the scaffolds. Collagen-coated scaffolds facilitate cell adhesion of hepatocytes and promote liver regeneration w14x. Laminin-coated scaffolds facilitate nerve regeneration. The mature chondrocytes, ligament cells and mesenchymal stem cells have been the main cell sources for tissue engineering of cartilage and ligament. In the present study, we compared the effects of various cell adhesion molecules and extracellular matrices on the adhesion behavior of these cells to search for the preferable cell adhesion molecules for the tissue engineering of cartilage and ligament. Type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine promoted significantly more cell adhesion than laminin, chondroitin4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid. MSCs showed a similar adhesion tendency as did the ligament cells. These two kinds of cells adhered significantly more on fibronectin-coated surface than on type I collagen-, type II collagen-, vitronectin- and poly-Llysine-coated surfaces, while the effect was decreased for chondrocytes. The cell adhesion ability interacts with cell surface receptors such as integrins. Integrins function as a physical link between the cell surface and extracellular matrices, and also connect the extracellular matrices to the intracellular cytoskeleton. In an articular lesion, chondrocytes express a1, a 3, a 5, a6, a10, a v, and b1 integrin family w15x. Anterior cruciate ligament cells express a1, a 5, a6 and b1 integrin family w16,17x. Although the whole integrins family of mesenchymal stem cells has not been still clarified, they express a1, a 2, a 3, a a, a v and b1 w18x. The three kinds of cells possess some common integrin family, which might explain their similar adsorption behaviors. The variant adhesion behaviors of these cells to different adhesion molecules may be probably due to the different expression and expression level of integrins family. Fibronectin showed to be most potential to promote cell adhesion, and thus would be effective to be incorporated into porous scaffolds for tissue engineering of cartilage and ligament.
Acknowledgements The authors acknowledge support from the New Energy and Industrial Technology Development Organization of Japan.
References w1x C.R. Chu, R.D. Coutts, M. Yoshioka, F.L. Harwood, A.Z. Monosov, D. Amiel, Articular cartilage repair using allogeneic perichondrocyte-seeded biodegradable porous polylactic acid ŽPLA.: a tissue-engineering study, J. Biomed. Mater. Res. 29 Ž1995. 1147–1154. w2x C.E. Holy, M.S. Shoichet, J.E. Davies, Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell-seeding density and culture period, J. Biomed. Mater. Res. 51 Ž1999. 76–382. w3x H.A. Award, D.L. Butler, M.T. Harris, R.E. Ibrahim, Y. Wu, R.G. Young, S. Kadiyara, G.P. Boivin, In vitro characterization of mesenchymal stem cell-seeded collagen scaffolds for tendon repair: effects of initial seeding density on contraction kinetics, J. Biomed. Mater. Res. 51 Ž1999. 233–240. w4x B.M. Gumbinar, Cell adhesion: the molecular basis of tissue architecture and morphogenesis, Cell 84 Ž1993. 249–263. w5x R.O. Hynes, Integrins: versatility, modulation, and signaling in cell adhesion, Cell 69 Ž1992. 11–25. w6x K.M. Yamada, Adhesive recognition sequences, J. Biol. Chem. 266 Ž1991. 12809–12812. w7x E. Ruoslahti, M.D. Pierschbacher, New perspectives in cell adhesion: RGD and integrins, Science 23 Ž1987. 491–497. w8x A.L. Akeson, C.W. Woods, A fluorometric assay for the quantitation of cell adherence to endothelial cells, J. Immunol. Methods 9 Ž1993. 181–185. w9x G. Chen, T. Ushida, T. Tateishi, A biodegradable hybrid sponge nested with collagen microsponges, J. Biomed. Mater. Res. 51 Ž2000. 273–279. w10x C.F. Chu, A. Lu, M. Liszkowski, R. Sipehia, Enhanced growth of animal and human endothelial cells on biodegradable polymers, Biochim. Biophys. Acta 1472 Ž1999. 479–485. w11x B.S. Kim, J. Nikolovski, J. Bonadio, E. Smiley, D.J. Mooney, Engineered smooth muscle tissues: regulating cell phenotype with the scaffold, Exp. Cell Res. 251 Ž1999. 318–328. w12x T. Hadlock, C. Sundback, D. Hunter, M. Cheney, J.P. Vacanti, A polymer foam conduit seeded with Schwann cells promotes guided peripheral nerve regeneration, Tissue Eng. 6 Ž2000. 119–127. w13x G.X. Chen, Y. Peng, P.L. Lou, J.P. Liu, Bioartificial thyroid. The in vitro culture of microencapsulated rabbit thyroid tissue, ASAIO Trans. 37 Ž1991. M439–M440. w14x P.M. Kaufman, S. Heinrath, B.S. Kim, D.J. Moony, Highly porous polymer matrices as a three-dimensional culture system for hepotocyte, Cell Transplant 6 Ž1997. 463–468. w15x R.F. Leoser, Chondrocyte integrin expression and function, Biorheology 37 Ž2000. 109–116. w16x S.S. AbiEzzi, D.S. Gesink, P.J. Schreck, D. Amiel, W.H. Akeson, V.L. Woods, Increased expression of the beta 1, alpha 5, and alpha v integrin adhesion receptor subunits occurs coincident with remodeling of stress-deprived rabbit anterior cruciate and medial collateral ligaments, J. Orthop. Res. 13 Ž1995. 594–601. w17x D.S. Gesink, H.O. Pachec, S.D. Kuiper, P.J. Schreck, D. Amiel, W.H. Akeson, V.L. Woods, Immunohistochemical localization of b1-integrins in anterior cruciate and medial collateral ligaments of human and rabbit, J. Orthop. Res. 10 Ž1992. 596–599. w18x M.K. Pittenger, A.M. Mackey, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Creig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 Ž1999. 143–147.
Effects of cell adhesion molecules on adhesion of chondrocytes, ligament cells and mesenchymal stem cells Kohei Tsuchiya a,b, Guoping Chen b, Takashi Ushida b,c,) , Takeo Matsuno a , Tetsuya Tateishi b,c a
b
Department of Orthopedic surgery, Asahikawa Medical College, Midorigaoka, 2-1 Higashi, Asahikawa 078-8510, Japan Tissue Engineering, Research Center, National Institute of AdÕanced Industrial Science and Technology, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan c Biomedical Engineering Laboratory, Graduate School of Engineering, The UniÕersity of Tokyo, Tokyo 113-8656, Japan
Abstract Smooth cell seeding and cell adhesion on temporary porous scaffolds are critical for tissue engineering. We investigated that the effects of type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine, laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid on adhesion of articular chondrocytes, ligament cells and mesenchymal stem cells ŽMSCs., for well understanding the interaction between adhesion molecules and diverse kinds of cells. The cell adhesion molecules were coated on the surfaces of 96-well culture plates. The cells were inoculated and cultured in the wells for 30 min, and subsequently, the number of adhered cells was determined. Type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine promoted significantly more cell adhesion than laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid. MSCs showed a similar adhesion tendency as ligament cells. These two kinds of cells adhered significantly more on fibronectin-coated surface than on type I collagen-, type II collagen-, vitronectin- and poly-L-lysine-coated surfaces, while the effect was decreased for chondrocytes. Fibronectin showed to be most potential to promote cell adhesion, and thus would be effective to be incorporated into porous scaffolds for tissue engineering of cartilage and ligament. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Cell adhesion; Chondrocyte; Ligament cell; Mesenchymal cell; Tissue engineering
1. Introduction Tissue engineering has been shown to be a promising approach to regenerate lost or degenerated tissues or organs for orthopedic applications. In this approach, isolated and expanded cells adhere to a temporary porous scaffold, proliferate, and secrete their own extracellular matrices w1x. The proliferated cells and secreted extracellular matrices will organize into tissues or organs that retain the original shapes of the temporary material scaffold and exhibit cellular, biochemical and biomechanical properties similar to healthy endogenous tissues or organs. The temporary porous scaffolds for tissue engineering can be prepared from various natural or synthetic biomaterials such as collagen, chitosan, hyaluronic acid, polylactic acid, polyglycolic acid and etc. Cell adhesion on the scaffolds is the initial and critical step in the tissue engi) Corresponding author. Tissue Engineering, Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan. Tel.: q81-3-5841-6374; fax: q81-35841-6374. E-mail address: [email protected] ŽT. Ushida..
neering approach w2,3x. The function of seeded cells is strongly dependent on the interaction of cell and scaffold surface. Cell adhesion to scaffolds is mediated by cell surface receptors, an important class of which is the integrin family. Integrins are heterodimeric transmembrane glycoproteins consisting of large globular extracellular domains, which bind to specific extracellular matrix proteins and short cytoplasmic domains that interact with cytoskeletal proteins inside the cells w4–6x. The integrin receptors bind to relatively short amino acid sequences such as Arg–Gly–Asp ŽRGD. on extracellular matrices w7x. The cell adhesion molecules containing RGD and many other cell adhesion sequences include fibronectin, vitronectin, laminin, collagens, and a number of other components of the extracellular matrices. RGD containing sequences and cell adhesion molecules have been incorporated into scaffolds to improve cell adhesion and control cell function. In the present study, the effects of various cell adhesion molecules and extracellular matrices on the initial cellular adhesion properties of articular chondrocytes, ligament cells and mesenchymal stem cells were investigated to target the design of optimal scaffolds for tissue engineering of articular cartilage and ligament.
0928-4931r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 1 . 0 0 3 4 1 - 1
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2. Materials and methods 2.1. Isolation of chondrocytes and ligament cells Bovine articular chondrocytes were isolated from the shoulder articular cartilage of a 4-week-old calf, obtained from a local abattoir within 4 h of slaughter. The cartilage was sliced and minced into 1–2-mm3 pieces. After being rinsed twice in cold Dulbecco’s phosphate buffered saline ŽPBS., the minced cartilage was digested by 0.2% type I collagenase ŽWorthington Biochemical, Lakewood, NJ. in Dulbecco’s modified Eagle’s minimal essential medium ŽDMEM. supplemented with antibiotics Žpenicillin, 100 Urml and streptomycin, 100 mgrml. under shaking overnight at 37 8C. The digestion solution was filtered through a sterile 70-mm nylon mesh to remove any undigested fragments, and the chondrocytes were subsequently collected by centrifugation and washed twice with PBS. The cell number and viability were determined by using a hemocytometer and the trypan blue exclusion dye test. The isolated chondrocytes were suspended in DMEM medium containing 10% FBS Žculture medium., seeded in a 75-cm2 flask at a high cell density, and cultured under an atmosphere of 5% CO 2 at 37 8C for 1 week. Ligament cells were isolated from the anterior cruciate ligament of a knee joint from a 2-week-old Japanese white rabbit. The specimens were sliced after removing of superficial synovium. The manipulation of cell isolation and collection was the same as that for bovine articular chondrocytes. The isolated ligament cells were suspended in culture medium, seeded in 75-cm2 flask at a high cell density, and subcultured under an atmosphere of 5% CO 2 at 37 8C. The second passaged ligament cells were used for adhesion experiment. 2.2. Coating of cell adhesion molecules The cell adhesion molecules and extracellular matrices used in the present study were bovine type I collagen ŽICN Biomedicals, Costa Mesa, CA., bovine type II collagen ŽSigma, St. Louis, MO., human laminin ŽFunakoshi, Japan., bovine fibronectin ŽFunakoshi., bovine vitronectin ŽFunakoshi., bovine chondroitin-4-sulfate ŽSigma., bovine chondroitin-6-sulfate ŽSigma., bovine aggrecan ŽSigma., chicken hyaluronic acid ŽARTZ, Seikagaku, Japan. and poly-L-lysine ŽSigma.. They were diluted with PBS to the final concentration of 500 mgrml, which is higher than the concentration that allows the monolayer absorption of these molecules, and added to 96-well culture plates Ž200 mlrwell.. The plates were kept overnight at 4 8C and then rinsed once with PBS. Subsequently, the plates were added with 1% bovine serum albumin Ž200 mlrwell. and incubated at room temperature for 1 h to block the non-coated sites. The wells were rinsed twice with PBS and used for cell culture.
2.3. Cell culture and adhesion assay Chondrocytes were used 1 week after subculture. Ligament cells were used after one passage. Human mesenchymal stem cells ŽMSCs. were purchased from Biowhittaker, in the second passage. Thawed cells were cultured at 5 = 10 5rcm 2 with defined growth medium under an atmosphere of 5% CO 2 at 37 8C, and the medium was changed twice a week. The third passaged cells were used for adhesion assay. The chondrocytes, ligament cells and MSCs were harvested with PBS containing 0.15% Žwrv. trypsin Ž2000 unitsrg. and 0.02% EDTA. The cells were washed once with medium containing FBS, twice with serum-free medium, and then suspended in serum-free DMEM at the concentration of 2.5 = 10 5 cellsrml Žchondrocytes. and 5 = 10 4 cellsrml Žligament and MSCs.. The cell suspensions were added to the coated culture plates Ž200 mlrwell. and incubated under an atmosphere of 5% CO 2 at 37 8C for 30 min. Unattached cells were removed by washing twice with PBS, while attached cells were quantified with a cell counting kit F assay according to manufacture’s protocol. Briefly, 100 ml PBS and 100 ml of the CalceinAMrDMSO solution diluted to 50 times were added to each well of the plates. The plates were incubated at 37 8C for 1 h. Calcein-AM fluorescence dye was hydrolyzed by endocellular elastase and generated fluorescence. This signal reflects the endocellular elastase activity, which directly correlates with the number of attached cells w8x. The fluorescence intensity was measured at 485 nm of excitation and 530 nm of emission by a fluorescence plate reader ŽCyteFluor2350, MILLIPORE.. Eight wells per adhesion molecule were measured and averaged. By taking the data of non-coated well in each measurement as the controls, statistical analysis was performed using Student’s t-test. 3. Results The standard curves for the relation between the fluorescence intensity and the amount of cells using limiting dilution method were shown to be linear for all the three types of cells Ždata were not shown.. After washing twice with PBS, almost no cells were detected on the surfaces blocked with albumin without coating any adhesion molecules or any extracellular matrices. Fig. 1 shows the calculated number of cells adhered on the surfaces coated with the cell adhesion molecules and extracellular matrices. Lots of cell adhered on the surfaces coated with type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine, whereas few cells adhered on the surfaces coated with laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid for all the three types of cells. Chondrocytes showed the preferable attachment to the coated surfaces in rank of fibronectin, vitronectin, type I collagen, type II collagen and poly-L-lysine ŽFig. 1A.. The
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number of chondrocytes adhered on the surface coated with fibronectin was about 50% of the seeded chondrocytes. The number of cell adhered on poly-L-lysine-coated surface was about half of that on fibronectin-coated surface. Almost 80% of ligament cells adhered on the fibronectin-coated surface. They demonstrated a preference
Fig. 2. Comparison of relative adhesion percent of chondrocyte, ligament cell and mesenchymal cell. The data were derived from the fluorescence intensities by standardizing with those on poly-L-lysine-coated surfaces. ) Statistically significant differences between the cell types Ž P - 0.03..
in attachment to fibronectin) type I collagen) type II collagen) vitronectin) poly-L-lysine. The ligament cells adhered on poly-L-lysine-coated surface was only 20% of that on fibronectin-coated surface ŽFig. 1B.. The difference among type I collagen, type II collagen, vitronectin and poly-L-lysine was not remarkable. MSC cells showed a similar adhesion property as did the ligament cells. Almost all the seeded MSCs adhered on fibronectin-coated surface. Type I collagen-, type II collagen-, vitronectin- and poly-L-lysine-coated surfaces possessed almost the same potential of cell adhesion, which was about 40% of that on fibronectin-coated surface ŽFig. 1C.. The difference of cell adhesion among various cell types was summarized in Fig. 2 by standardizing the adhesion data of chondrocyte, ligament cells and MSCs with their respective adhesion on poly-L-lysine-coated surface. Although fibronectin promoted most adhesion of these cells, the effects seemed to be strongly dependent on cell type. It stimulated the adhesion of ligament cells more than chondrocytes and MSCs. The other adhesion molecules and extracellular matrices also showed some difference between cell types, but not so evident as fibronectin.
4. Discussion Fig. 1. Cell number of chondrocyte ŽA., ligament cell ŽB. and mesenchymal stem cell ŽC. attached to the surfaces coated with different adhesion molecules. Cell number was calculated from their fluorescence intensity using standard curves. COL1: type I collagen, COL2: type II collagen, FN: fibronectin, VN: vitronectin, LN: laminin, AG: aggrecan, HA: hyaluronic acid, C4S: chondroitin-4-sulfate, C6S: chondroitin-6-sulfate, PLL: poly-L-lysin. Data are given as mean"standard error Ž ns8..
Damaged articular cartilage and intraarticular ligament such as anterior cruciate ligament have limited capacity for self-repair. Cell-based therapy using tissue engineering technology has a great future as a next generation therapeutic approach for these tissues. The initial event for seeded cells to function in temporary porous scaffolds is
82
K. Tsuchiya et al.r Materials Science and Engineering C 17 (2001) 79–82
cell adhesion on the surfaces of the scaffolds. To improve cell adhesion on scaffolds, various cell adhesion molecules such as collagen w9x, fibronectin w10x, vitronectin w11x, laminin w12x, and poly-L-lysine w13x have been coated or incorporated into the scaffolds. Collagen-coated scaffolds facilitate cell adhesion of hepatocytes and promote liver regeneration w14x. Laminin-coated scaffolds facilitate nerve regeneration. The mature chondrocytes, ligament cells and mesenchymal stem cells have been the main cell sources for tissue engineering of cartilage and ligament. In the present study, we compared the effects of various cell adhesion molecules and extracellular matrices on the adhesion behavior of these cells to search for the preferable cell adhesion molecules for the tissue engineering of cartilage and ligament. Type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine promoted significantly more cell adhesion than laminin, chondroitin4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid. MSCs showed a similar adhesion tendency as did the ligament cells. These two kinds of cells adhered significantly more on fibronectin-coated surface than on type I collagen-, type II collagen-, vitronectin- and poly-Llysine-coated surfaces, while the effect was decreased for chondrocytes. The cell adhesion ability interacts with cell surface receptors such as integrins. Integrins function as a physical link between the cell surface and extracellular matrices, and also connect the extracellular matrices to the intracellular cytoskeleton. In an articular lesion, chondrocytes express a1, a 3, a 5, a6, a10, a v, and b1 integrin family w15x. Anterior cruciate ligament cells express a1, a 5, a6 and b1 integrin family w16,17x. Although the whole integrins family of mesenchymal stem cells has not been still clarified, they express a1, a 2, a 3, a a, a v and b1 w18x. The three kinds of cells possess some common integrin family, which might explain their similar adsorption behaviors. The variant adhesion behaviors of these cells to different adhesion molecules may be probably due to the different expression and expression level of integrins family. Fibronectin showed to be most potential to promote cell adhesion, and thus would be effective to be incorporated into porous scaffolds for tissue engineering of cartilage and ligament.
Acknowledgements The authors acknowledge support from the New Energy and Industrial Technology Development Organization of Japan.
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