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Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films
Materials Science and Engineering C 20 (2002) 63 – 69 www.elsevier.com/locate/msec
Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films H.W. Ouyang a, J.C.H. Goh a,*, X.M. Mo b, S.H. Teoh b, E.H. Lee a a
Tissue Engineering Laboratory, Department of Orthopaedic Surgery, Faculty of Medicine, National University of Singapore, 10 Lower Kent Ridge Road, 119260 Singapore, Singapore b BIOMAT Centre, Department of Mechanical Engineering, National University of Singapore, Singapore, Singapore
Abstract In this study, the adhesion, proliferation and morphology of rabbit anterior cruciate ligament (ACL) cells and bone marrow stromal cells (bMSCs) on synthetic biodegradable polymeric films were investigated. Tissue culture polystyrene (TCP) was used as control. Seven biodegradable polymers were used; they are as follows: poly(q-caprolactone) (PCL), poly(DL-lactide) (D-PLA), poly(L-lactide) (L-PLA), PLA/PCL (50:50), PLA/PCL (75:25), high molecular weight (HMW) poly(DL-lactide – co-glycolide (PLGA50:50) and HMW PLGA75:25. Polymeric film substrates were manufactured using solvent spin-casting technique. After 8 h of cell culture, a high percentage of ACL cells was found attached to PLGA50:50 (38.6 F 8.4%) and TCP (39.3 F 6.1%) as compared to the other six polymeric films ( p V 0.001). As for bMSCs, 76.4 F 10%, 76.3 F 16% and 76.1 F 19% of seeded bMSCs were adhered to TCP, PLGA50:50 and PLGA75:25, respectively. These were significantly more than those of the other five polymeric films ( p < 0.001). At Day 5, bMSCs were found to proliferate faster on TCP (by 7 F 0.8-fold of initial cell seeding number), D-PLA (by 5.6 F 1.6-fold), PLGA50:50 (by 9.3 F 1.3-fold) and PLGA75:25 (by 5.8 F 1.3fold) than on PCL, PLLA and PCL/PLA (50:50, 25:75) ( p < 0.001). ACL cells had a greater fold expansion on TCP (by 3.5 F 0.2-fold), PLGA50:50 (by 3.1 F 0.4-fold) and PLGA75:25 (by 3.9 F 0.4-fold) than on the other five polymer substrates ( p < 0.001). From these results, HMW PLGA (50:50, 75:25) was shown more likely to allow bMSCs and ACL cells to attach and proliferate, and bMSCs attached and proliferated faster than ACL cells. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Biodegradable polymers; ACL; bMSCs; Cell adhesion; Cell proliferation; Cell morphology
1. Introduction The anterior cruciate ligament (ACL) is the most frequently disrupted ligament in the knee joint [1]. Left untreated, a torn ACL can cause knee instability, meniscal tears and osteoarthritis [2]. However, the injured ACL has poor ability to heal itself. It has been reported that primary repair of a torn ACL often resulted in an unsatisfactory outcome [3]. Therefore, surgical reconstruction of ACL is recommended to restore knee joint function. Currently, autografts and allografts are frequently used to reconstruct ACL, but the disadvantages of autografts and the risk associated with allografts have encouraged research for alternative solutions [4]. The emerging field of tissue engineering offers the potential to find an ideal substrate for ACL regeneration. The fundamental approach in tissue engineering involves
*
Corresponding author. Tel.: +65-772-4423; fax: +65-774-4082. E-mail address: [email protected] (J.C.H. Goh).
the regeneration of tissue through expanded cultured cells on scaffolds to produce a three-dimensional tissue for implantation. Generally, bone marrow and specific organs are the optimal cell sources for tissue engineering. Bone marrow stromal cells (bMSCs) have been extensively studied for bone, cartilage and tendon regeneration [5– 10], while fibroblasts from ACL have been investigated by several groups [11– 13] working on in vitro ACL analog engineering. On the other hand, an appropriate scaffold for ACL regeneration must be able to provide mechanical strength to initially withstand the in vivo force and degrade safely at an appropriate rate in vivo. Most synthetic biodegradable polymers are biocompatible and can be fabricated into three-dimensional scaffolds with various structures. They are reproducible and offer a wide range of mechanical and degradation properties. They are therefore a promising group of materials for use as scaffold in ACL tissue engineering. However, limited data have been reported in using such biodegradable polymers for ACL repair [14,15].
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 ( 0 2 ) 0 0 0 1 4 - 0
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H.W. Ouyang et al. / Materials Science and Engineering C 20 (2002) 63–69
Not only must the appropriate choice of cells and the matrices be considered carefully, their interactions are also important for normal cell activity. Laurencin et al. [16] reported that adhesion of fibroblast from different sources was cell-type sensitive on poly(DL-lactide – co-glycolide) (PLGA50:50), thus reflecting the particular surface requirements of the respective soft-tissue cells. This observation warrants further investigation so as to provide more information on the cells’ interaction with chemical surface. In order to develop an appropriate substrate for ACL reconstruction using cell-seeded synthetic biodegradable polymer scaffold, it was imperative to undertake this study. The objective was to characterize the adhesion, proliferation and phenotype of anterior cruciate ligament cells and mesenchymal stem cells on several synthetic biodegradable polymeric materials.
2. Materials and methods 2.1. Polymeric film preparation Seven poly(hydroxyesters) were used in this study. They are listed in Table 1 along with their reported inherent viscosity and source. Spin-casting technique was used to manufacture the polymeric films. For the spin-casting process, 0.1 g/ml of each polymer was dissolved in 0.2 ml solution of chloroform and drip onto the center of a circular glass coverslip (diameter 15 mm) (Bellco Glass, Vineland, NJ), which was spun at 100 rpm. The polymeric films produced were left under the fume hood for 48 h to allow the chloroform to evaporate and subsequently placed under vacuum for 24 h to remove the remaining solvent. The polymeric films were placed on the well bottom of a 24-well plate (Iwaki, Japan), and tissue culture polystyrene (TCP) on the well bottom of the 24-well plate served as control. These substrates were sterilized under UV for 1 h and pre-wetted with 100% ethanol for 1 h, then rinsed three times
Table 1 Source of the polymers used in this study and water contact angles of the substrates (n = 4, mean F S.D.) Polymer
Source
Substrate contact angle (deg)
Poly(q-caprolactone) Poly(DL-lactide) Poly(L-lactide) 50:50 Poly (DL-lactide – co-caprolactone) 75:25 Poly (DL-lactide – co-caprolactone) HMW 50:50 Poly (DL-lactide – co-glycolide) HMW 75:25 Poly (DL-lactide – co-glycolide)
Aldrich chemical Polysciences Polysciences Gunze Kyoto
95 F 1 78 F 1 70 F 2 74 F 2
Gunze Kyoto
77 F 1
Alkermes
79 F 2
Alkermes
72 F 2
Fig. 1. Adhesion kinetics of anterior ligament cells to tissue culture polystyrene (TCP), poly(q-carprolactone) (PCL), 50:50 poly(lactide – cocaprolactone) (PCL/PLA) and 50:50 poly(DL -lactide – co-glycolide) (PLGA) expressed as percent of seeded cells that remain adhered to the substrates after 4, 6, 8 and 10 h of cell culture (n = 3 per substrate).
(at 1-h intervals) with phosphate-buffered saline (PBS). After that, the substrates were soaked in culture medium [RPMI1640, 15% FBS]. The water contact angles were measured on dry substrates using a contact angle measurement system described previously [17] (Model VCA-2500 XE, Advanced Surface Technology). 2.2. Bone marrow stromal cell Bone marrow stromal cells were isolated by short-term adherence to plastic as described by Beresford and Owen [18], Kuznetsov and Gehron Robey [19] and Wakitani et al. [9] with several modifications. Bone marrow was aspirated from iliac crest of 4-month female NZW rabbits. Nucleated cells were isolated by density gradient centrifugation over Ficoll/Paque (Pharmacia). Following this, the nucleated cell layers were carefully removed and resuspended in culture medium containing DMEM (Gibco), 15% (wt/vol) fetal bovine serum (FBS; Hyclone) 100 U/ml penicillin, 100 Ag/ml streptocycin (Gibco). The nucleated cells were plated at the density of 5 million nucleated cells per 100-mm dish and incubated at 37 jC with 5% humidified CO2. After 24 h, non-adherent cells were discarded and adherent cells were cultured. Medium was changed every 3 days. When culture dishes became nearly confluent, the cells were detached and serially subcultured. 2.3. Anterior cruciate ligament cell As Kobayashi et al. [20] and Nagineni et al. [21] described, ACL were harvested from adult NZW rabbits with No15 blade under sterile conditions. The ligaments were washed three times with 0.9% (wt/vol) sodium chloride containing 200 U/ml penicillin and 200 Ag/ml streptomycin. After the femoral and tibia insertions were removed, each ligament was dissected from the synovial sheath and periligamentous tissue. For cell culture, the ligaments were cut into small pieces and digested for 2.5 h with 0.25% (wt/vol)
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Fig. 2. Number of anterior ligament cells (ACL) and bone marrow stromal cells (bMSCs) adhered to the various polymeric films after 8 h of cell culture expressed as percent of seeded cells that remain adhered to the substrates (seeding density was 20,000 cells/cm2) (n = 4 per substrate).
collagenase supplement with 0.2% (wt/vol) albumin (Sigma, St. Louis, MO, USA). Cells from ligaments were isolated by centrifugation at 1500 rpm for 10 min. The cell pellet was washed three times with culture medium containing RPMI1640 (Gibco), 15% (wt/vol) fetal bovine serum (Hyclone), 100 U/ml penicillin, 100 Ag/ml streptocycin (Gibco). The ACL cells were counted (5 106 cells) and then suspended in 15 ml culture medium in a 75-cm2 flask (Corning). The culture medium was changed every 3 days. When they became confluent usually 7 –10 days after isolation of cells, the fibroblasts were detached by treatment with 1 ml 0.25% trypsin –EDTA (Gibco) and subcultured.
2.4. Cell adhesion studies For the assessment of the kinetics of cell adhesion, ligament cells were seeded onto TCP, poly(q-caprolactone) (PCL), PCL/PLA (50:50) and PLGA (50:50) at the density of 20,000 cells/cm2. The cells were allowed to attach to the substrates undisturbed in a humidified incubator (at 37 jC, 5% CO2) for 4, 6, 8 and 10 h. At each time point, parallel samples of n = 3 were used. The number of attached cells was qualified by MTS assay. Since the difference of ligament cells attachment at 10 h has no significance, the effects of polymer substrate on li-
Fig. 3. Bone marrow stromal cell (bMSCs) proliferation on various substrates. The numbers of cells were normalized to initial density of seeded cells (10,000 cells/cm2) (n = 4 per substrate).
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gament cells and bMSCs were studied using 8 h attachment time. The cells were seeded on each substrate at the same density and incubated for 8 h. The number of attached cells was assessed by MTS assay (n = 4). 2.5. Cell proliferation studies
2.7. Phase contrast microscopy Phase contrast micrographs were taken of cells attached to the substrates after 12 h of attachment (seeding density: 20,000 cells/cm2). The cells attached to the substrates following the PBS rinse were visualized on an inverted microscopy.
Cells were seeded onto each polymeric film and TCP at the density of 10,000 cells/cm2. The cells were allowed to attach and proliferate for 3 and 5 days, at which time point, the number of attached cells was determined by MTS assay. bMSCs usually get confluence in 1 week after passage, so Days 3 and 5 were chosen as the observation time points which ensure enough room for cell proliferation.
2.8. Statistical analysis
2.6. MTS assay
3. Results
In order to study the cells adhesion and proliferation, the number of viable cells on the polymer sheets was determined with use of Cell Titer 96 (Promega), a colorimetric assay in which metabolically active cells react with a tetrazolium salt to produce a soluble formazan dye that was read at 490 nm and compared with a standard curve to calculate the number of viable cells. As Lin et al. [15] described, before testing, the polymer sheets were rinsed with PBS twice and then 250 Al MTS reagent diluted to 5% concentration in culture medium was added into each well. The culture plates were then incubated for 1.5 h, after which 100-Al aliquots of each well was placed into individual wells of a 96-well plate. The 96-well plates were then placed into a spectrophotometric plate reader to have the absorbance of the content of each well.
3.1. Polymer films
All attachment and proliferation measurements were collected in quadruplicate and expressed as mean F 1 standard deviation (S.D.). Single-factor analysis of variance (ANOVA) was employed to assess statistical significance.
Table 1 shows the various biodegradable polymers and corresponding substrate contact angle measured. Most of the water contact angles were between 70j and 80j with the exception of PCL, which had contact angle of 95j ( p < 0.05). 3.2. Cell adhesion The kinetics of anterior cruciate ligament (ACL) cells adhesion on three polymeric films and TCP are as shown in Fig. 1. ACL cells attached to all the substrates with increasing numbers over the 10-h period. The rate of cell adhesion slowed down dramatically after 8 h. There was statistical difference in cell attachment among the substrates after 4 h
Fig. 4. Anterior ligament cells proliferation on polymeric films and TCP. The cell numbers were normalized to the initial density of seeded cells (10 000 cells/ cm2). Fibroblasts from ACL were found to proliferate better on PLGA (50:50, 75:25) and TCP than on PCL, D-PLA, L-PLA and PCL/PLA (50:50, 75:25) (n = 4 per substrate).
H.W. Ouyang et al. / Materials Science and Engineering C 20 (2002) 63–69
( p V 0.001), 6 h ( p < 0.05) and 8 h ( p V 0.001). By 10 h, the percentage of attached cells to TCP, PCL, PCL/PLA (50:50) and PLGA (50:50) was 49.3 F 13%, 34.5 F 5.4%, 39.9 F 4.7% and 42.7 F 6.1%, respectively, which showed no statistical significance ( p > 0.05). ACL cells and bMSCs adhesion on all polymer films and TCP were studied. As shown in Fig. 2, both kinds of cells adhered to all the substrates after 8 h of cell culture. As compare to ACL cells, more bMSCs attached to TCP and polymeric films. These differences were significant on TCP, PLGA (50:50, 75:25), PLLA and PCL/PLA (50:50). When multiple comparisons were made between the substrates, a greater percentage of ACL cells were found attached to PLGA50:50 (38.6 F 8.4%) and TCP (39.3 F 6.1%) as compared to the other six polymeric films ( p V 0.001). As for bMSCs, 76.4 F 10%, 76.3 F 16% and 76.1 F 19% of seeded cells were adhered to TCP, PLGA50:50 and PLGA75:25, respectively. These were significantly more than those of the other five polymeric films ( p < 0.001).
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3.3. Cell proliferation ACL cells and bMSCs proliferation on the polymeric substrates were studied at Days 3 and 5 and are as shown in Figs. 3 and 4, respectively. The increased number of ACL cells and bMSCs at the two time points demonstrated that proliferation occurred in all substrates. As the initial cell densities were the same (10,000 cells/cm2), at Day 5 the bMSCs were found to have more folds of expansion on all substrates with the exception of PCL when compared to ACL cells ( p < 0.05). Of all the substrates studied, the bMSCs proliferated faster on TCP (7 F 0.8 folds), poly( DL -lactide) ( D -PLA) (5.6 F 1.6 folds), PLGA50:50 (9.3 F 1.3 folds) and PLGA75:25 (5.8 F 1.3 folds) than on PCL, PLLA and PCL/PLA (50:50, 25:75) ( p < 0.001). As for ACL cells, greater folds of expansion were found on TCP (3.5 F 0.2 folds), PLGA50:50 (3.1 F 0.4 folds) and PLGA75:25 (3.9 F 0.4 folds) when compared to the other five polymeric substrates ( p < 0.001).
Fig. 5. (a) – (d) Morphology of the fibroblasts from anterior cruciate ligament on (a) TCP, (b) PLGA, (c) PCL and (d) D-PLA after 12-h cell culture. It was found that the ACL fibroblasts attached and stretched well on these substrates. There is no significant difference between their morphology (magnification: 100 ).
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4. Discussion In this study, bone marrow stromal cells and anterior cruciate ligament cells were able to adhere and proliferate on the polymeric films used. Phase contrast images showed that ACL cells attached and spread well on all substrates. There was no effect of substrate on cell morphology at 12 h of cell culture (see Fig. 5). These results correlated well with those of previous studies [22 –24], which had shown that water contact angle range from 70j to 90j is suitable for cells to attach and grow. Thus, there are various cells – material systems available for ACL tissue engineering. It was found that less number of ACL cells adhered to the substrates as compared to bMSCs and the difference are significant on PLGA (50:50, 75:25), TCP, PLLA and PLA/ PCL (50:50) ( p < 0.05) but not on D-PLA, PCL, PLA/PCL (75:25). These results showed that bMSCs are more likely to attach to the substrates than ACL cells, and the difference between the adhesion ability of bMSCs and ACL cells are substrate chemical property-sensitive. As for cell proliferation, bMSCs had significantly greater expansion capability than ACL cells on all substrates except for PCL ( p < 0.05). It can be explained by the fact that stem cells are not limited to a fixed number of mitotic divisions, whereas ACL cells are terminally differentiated and have limited lifespan. Hannafin et al. [25] reported that ACL cells possessed some special characteristics and grow more slowly as compared to Medial Collateral Ligament cells. Of all the substrates studied, ACL cells and bMSCs were found more likely to attach to and grow on the high molecular weight (HMW) PLGA (50:50 and 75:25) and TCP control. This result correlated well with previous studies [26] that reported high molecular weight PLGA is more likely to allow epithelium cells to attach and proliferate. It was generally agreed that hydrophilicity of substrates affect biological response, such as cell adhesion and proliferation. Due to the presence of the extra methyl group in lactic acid, PLA is more hydrophobic than PLGA so that a lower percentage of cells attached to PLA as compared to PLGA. Again, PCL has a high olefinic characteristic and was more hydrophobic than PLGA. On the other hand, the amount and type of serum protein that adhered to substrates varied on different chemical surfaces, which also affect cell behavior on the substrates [24]. Although D-PLA and PLLA are composed of the same chemical component, it was shown that bMSCs grew faster on D-PLA than on PLLA, and more bMSCs and ACL cells attached to D-PLA as compared to PLLA. Similar differences have been reported on osteoblasts and chondrocytes [27]. This may be explained by the fact that D-PLA is more crystalline. So far, no general principle has been established to explain and predict the extent of adhesion, proliferation and spreading of cultured cells on different polymer surfaces [28]. Therefore, it is necessary to design and conduct specific experiments similar to this study on the interaction between
specific cells and candidate biomaterials for use as scaffold in tissue engineering research for specific application. 5. Conclusion All polymer substrates studied were able to allow ACL cells and bMSCs to adhere and proliferate. bMSCs are more likely to attach to high molecular weight PLGA (50:50, 75:25) while ACL cells prefer high molecular weight PLGA50:50. MSCs proliferated faster on PLGA (50:50, 75:25) and D-PLA, while ACL cells expanded greatly on PLGA (50:50, 75:25). On all polymer substrates, bMSCs have a higher degree of cell attachment and proliferation than that of ACL cells. The information obtained from this study should be useful for future ACL tissue engineering research. References [1] R.J. Johnson, Int. J. Sports Med. 3 (1982) 71. [2] F.H. Fu, C.D. Harner, D.L. Johnson, M.D. Miller, S.L.Y. Woo, J. Bone Jt. Surg. 75A (1993) 1716. [3] L. Engebretsen, P. Renum, S. Sindalsvoll, Acta Orthop. Scand. 60 (1989) 561. [4] C.T. Laurencin, A.M.A. Ambrosio, M.D. Borden, J.A. Cooper Jr., Annu. Rev. Biomed. Eng. 1 (1999) 19. [5] L.F. Cooper, C.T. Harris, S.P. Bruder, R. Kowalski, S. Kadiyala, J. Dent. Res. 80 (1) (2001) 314. [6] A.I. Caplan, S.P. Bruder, Trends Mol. Med. 7 (6) (2001) 259. [7] H.A. Awad, D.L. Butler, G.P. Boivin, F.N. Smith, P. Malaviya, B. Huibregtse, A.I. Caplan, Tissue Eng. 5 (3) (1999) 267. [8] P. Bianco, M. Riminucci, S. Gronthos, P.G. Robey, Stem Cells 19 (2001) 180. [9] S. Wakitani, T. Goto, S.J. Pineda, R.G. Young, J.M. Mansour, A.I. Caplan, V.M. Goldberg, J. Bone Jt. Surg., Am. Vol. 76 (4) (1994) 579. [10] R.G. Young, D.L. Butler, W. Weber, A.I. Caplan, S.L. Gordon, D.J. Fink, J. Orthop. Res. 16 (4) (1998) 406. [11] L.D. Bellincampi, R.F. Closkey, R. Prasad, J.P. Zawadasky, M.G. Dun, J. Orthop. Res. 16 (1998) 414. [12] M.G. Dune, J.B. Leish, M.L. Tiku, S.H. Maxian, J.P. Zawadsky, Mater. Res. Soc. Symp. Proc., 1994, p. 331. [13] F. Goulet, L. Geramin, D. Rancourt, C. Caron, A. Normand, F.A. Auger, in: R.P Lanza, R. Langer, W.I. Chick (Eds.), Principles of Tissue Engineering, RG Landes Academic, Austin, TX, 1997, p. 639. [14] H.E. Cabaud, J.A. Feagin, W.G. Rodkey, Am. J. Sports Med. 10 (1982) 259. [15] V.S. Lin, M.C. Lee, S. O’Neal, J. McKean, K.L. Sung, Tissue Eng. 5 (5) (1999) 443 – 452. [16] C. Laurencin, M. Attwawia, E. Botchwey, R. Warren, E. Attia, In Vitro Cell. Dev. Biol.: Anim. 34 (1998) 90. [17] S.L. Ishaug-Riley, L.E. Okun, G. Prado, M.A. Applegate, A. Ratcliffe, Biomaterials 20 (23 – 24) (1999) 2245. [18] J.N. Beresford, M.E. Owen, Marrow Stromal Cell Culture, Cambridge Univ. Press, Cambridge UK, 1998. [19] S. Kuznetsov, P. Gehron Robey, Calcif. Tissue Int. 59 (4) (1996) 265. [20] K. Kobayashi, R.M. Healey, R.L. Sah, J.J. Clark, B.P. Tu, R.S. Goomer, W.H. Akeson, H. Moriya, D. Amiel, Tissue Eng. 6 (1) (Feb. 2000) 29. [21] C.N. Nagineni, D. Amiel, M.H. Green, M. Berchuck, W.H. Akeson, J. Orthop. Res. 10 (4) (1992) 465. [22] T. Horbett, M. Schway, J. Colloid Interface Sci. 104 (1985) 28. [23] Y. Ikata, Biomaterials 15 (10) (1994) 725.
H.W. Ouyang et al. / Materials Science and Engineering C 20 (2002) 63–69 [24] Y. Tamada, Y. Ikata, J. Biomed. Mater. Res. 28 (1994) 783. [25] J.A. Hannafin, E.T. Attia, R.F. Warren, M.M. Bhargava, J. Orthop. Res. 17 (1999) 398. [26] G.G. Giordano, R.C. Thomson, S.L. Ishaug, A.G. Mikos, S. Cumber, C.A. Garcia, D. Lahiri-Munir, J. Biomed. Mater. Res. 34 (1) (1997) 87.
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[27] B.D. Boyan, T.W. Hummert, D.D. Dean, Z. Schwartz, Biomaterials 17 (2) (1996) 137. [28] W.M. Saltzman, in: R.P Lanza, R. Langer, J. Vancanti (Eds.), Principles of Tissue Engineering, 2nd edn., Academic Press, California, 2000, p. 221.
Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films H.W. Ouyang a, J.C.H. Goh a,*, X.M. Mo b, S.H. Teoh b, E.H. Lee a a
Tissue Engineering Laboratory, Department of Orthopaedic Surgery, Faculty of Medicine, National University of Singapore, 10 Lower Kent Ridge Road, 119260 Singapore, Singapore b BIOMAT Centre, Department of Mechanical Engineering, National University of Singapore, Singapore, Singapore
Abstract In this study, the adhesion, proliferation and morphology of rabbit anterior cruciate ligament (ACL) cells and bone marrow stromal cells (bMSCs) on synthetic biodegradable polymeric films were investigated. Tissue culture polystyrene (TCP) was used as control. Seven biodegradable polymers were used; they are as follows: poly(q-caprolactone) (PCL), poly(DL-lactide) (D-PLA), poly(L-lactide) (L-PLA), PLA/PCL (50:50), PLA/PCL (75:25), high molecular weight (HMW) poly(DL-lactide – co-glycolide (PLGA50:50) and HMW PLGA75:25. Polymeric film substrates were manufactured using solvent spin-casting technique. After 8 h of cell culture, a high percentage of ACL cells was found attached to PLGA50:50 (38.6 F 8.4%) and TCP (39.3 F 6.1%) as compared to the other six polymeric films ( p V 0.001). As for bMSCs, 76.4 F 10%, 76.3 F 16% and 76.1 F 19% of seeded bMSCs were adhered to TCP, PLGA50:50 and PLGA75:25, respectively. These were significantly more than those of the other five polymeric films ( p < 0.001). At Day 5, bMSCs were found to proliferate faster on TCP (by 7 F 0.8-fold of initial cell seeding number), D-PLA (by 5.6 F 1.6-fold), PLGA50:50 (by 9.3 F 1.3-fold) and PLGA75:25 (by 5.8 F 1.3fold) than on PCL, PLLA and PCL/PLA (50:50, 25:75) ( p < 0.001). ACL cells had a greater fold expansion on TCP (by 3.5 F 0.2-fold), PLGA50:50 (by 3.1 F 0.4-fold) and PLGA75:25 (by 3.9 F 0.4-fold) than on the other five polymer substrates ( p < 0.001). From these results, HMW PLGA (50:50, 75:25) was shown more likely to allow bMSCs and ACL cells to attach and proliferate, and bMSCs attached and proliferated faster than ACL cells. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Biodegradable polymers; ACL; bMSCs; Cell adhesion; Cell proliferation; Cell morphology
1. Introduction The anterior cruciate ligament (ACL) is the most frequently disrupted ligament in the knee joint [1]. Left untreated, a torn ACL can cause knee instability, meniscal tears and osteoarthritis [2]. However, the injured ACL has poor ability to heal itself. It has been reported that primary repair of a torn ACL often resulted in an unsatisfactory outcome [3]. Therefore, surgical reconstruction of ACL is recommended to restore knee joint function. Currently, autografts and allografts are frequently used to reconstruct ACL, but the disadvantages of autografts and the risk associated with allografts have encouraged research for alternative solutions [4]. The emerging field of tissue engineering offers the potential to find an ideal substrate for ACL regeneration. The fundamental approach in tissue engineering involves
*
Corresponding author. Tel.: +65-772-4423; fax: +65-774-4082. E-mail address: [email protected] (J.C.H. Goh).
the regeneration of tissue through expanded cultured cells on scaffolds to produce a three-dimensional tissue for implantation. Generally, bone marrow and specific organs are the optimal cell sources for tissue engineering. Bone marrow stromal cells (bMSCs) have been extensively studied for bone, cartilage and tendon regeneration [5– 10], while fibroblasts from ACL have been investigated by several groups [11– 13] working on in vitro ACL analog engineering. On the other hand, an appropriate scaffold for ACL regeneration must be able to provide mechanical strength to initially withstand the in vivo force and degrade safely at an appropriate rate in vivo. Most synthetic biodegradable polymers are biocompatible and can be fabricated into three-dimensional scaffolds with various structures. They are reproducible and offer a wide range of mechanical and degradation properties. They are therefore a promising group of materials for use as scaffold in ACL tissue engineering. However, limited data have been reported in using such biodegradable polymers for ACL repair [14,15].
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 ( 0 2 ) 0 0 0 1 4 - 0
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H.W. Ouyang et al. / Materials Science and Engineering C 20 (2002) 63–69
Not only must the appropriate choice of cells and the matrices be considered carefully, their interactions are also important for normal cell activity. Laurencin et al. [16] reported that adhesion of fibroblast from different sources was cell-type sensitive on poly(DL-lactide – co-glycolide) (PLGA50:50), thus reflecting the particular surface requirements of the respective soft-tissue cells. This observation warrants further investigation so as to provide more information on the cells’ interaction with chemical surface. In order to develop an appropriate substrate for ACL reconstruction using cell-seeded synthetic biodegradable polymer scaffold, it was imperative to undertake this study. The objective was to characterize the adhesion, proliferation and phenotype of anterior cruciate ligament cells and mesenchymal stem cells on several synthetic biodegradable polymeric materials.
2. Materials and methods 2.1. Polymeric film preparation Seven poly(hydroxyesters) were used in this study. They are listed in Table 1 along with their reported inherent viscosity and source. Spin-casting technique was used to manufacture the polymeric films. For the spin-casting process, 0.1 g/ml of each polymer was dissolved in 0.2 ml solution of chloroform and drip onto the center of a circular glass coverslip (diameter 15 mm) (Bellco Glass, Vineland, NJ), which was spun at 100 rpm. The polymeric films produced were left under the fume hood for 48 h to allow the chloroform to evaporate and subsequently placed under vacuum for 24 h to remove the remaining solvent. The polymeric films were placed on the well bottom of a 24-well plate (Iwaki, Japan), and tissue culture polystyrene (TCP) on the well bottom of the 24-well plate served as control. These substrates were sterilized under UV for 1 h and pre-wetted with 100% ethanol for 1 h, then rinsed three times
Table 1 Source of the polymers used in this study and water contact angles of the substrates (n = 4, mean F S.D.) Polymer
Source
Substrate contact angle (deg)
Poly(q-caprolactone) Poly(DL-lactide) Poly(L-lactide) 50:50 Poly (DL-lactide – co-caprolactone) 75:25 Poly (DL-lactide – co-caprolactone) HMW 50:50 Poly (DL-lactide – co-glycolide) HMW 75:25 Poly (DL-lactide – co-glycolide)
Aldrich chemical Polysciences Polysciences Gunze Kyoto
95 F 1 78 F 1 70 F 2 74 F 2
Gunze Kyoto
77 F 1
Alkermes
79 F 2
Alkermes
72 F 2
Fig. 1. Adhesion kinetics of anterior ligament cells to tissue culture polystyrene (TCP), poly(q-carprolactone) (PCL), 50:50 poly(lactide – cocaprolactone) (PCL/PLA) and 50:50 poly(DL -lactide – co-glycolide) (PLGA) expressed as percent of seeded cells that remain adhered to the substrates after 4, 6, 8 and 10 h of cell culture (n = 3 per substrate).
(at 1-h intervals) with phosphate-buffered saline (PBS). After that, the substrates were soaked in culture medium [RPMI1640, 15% FBS]. The water contact angles were measured on dry substrates using a contact angle measurement system described previously [17] (Model VCA-2500 XE, Advanced Surface Technology). 2.2. Bone marrow stromal cell Bone marrow stromal cells were isolated by short-term adherence to plastic as described by Beresford and Owen [18], Kuznetsov and Gehron Robey [19] and Wakitani et al. [9] with several modifications. Bone marrow was aspirated from iliac crest of 4-month female NZW rabbits. Nucleated cells were isolated by density gradient centrifugation over Ficoll/Paque (Pharmacia). Following this, the nucleated cell layers were carefully removed and resuspended in culture medium containing DMEM (Gibco), 15% (wt/vol) fetal bovine serum (FBS; Hyclone) 100 U/ml penicillin, 100 Ag/ml streptocycin (Gibco). The nucleated cells were plated at the density of 5 million nucleated cells per 100-mm dish and incubated at 37 jC with 5% humidified CO2. After 24 h, non-adherent cells were discarded and adherent cells were cultured. Medium was changed every 3 days. When culture dishes became nearly confluent, the cells were detached and serially subcultured. 2.3. Anterior cruciate ligament cell As Kobayashi et al. [20] and Nagineni et al. [21] described, ACL were harvested from adult NZW rabbits with No15 blade under sterile conditions. The ligaments were washed three times with 0.9% (wt/vol) sodium chloride containing 200 U/ml penicillin and 200 Ag/ml streptomycin. After the femoral and tibia insertions were removed, each ligament was dissected from the synovial sheath and periligamentous tissue. For cell culture, the ligaments were cut into small pieces and digested for 2.5 h with 0.25% (wt/vol)
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Fig. 2. Number of anterior ligament cells (ACL) and bone marrow stromal cells (bMSCs) adhered to the various polymeric films after 8 h of cell culture expressed as percent of seeded cells that remain adhered to the substrates (seeding density was 20,000 cells/cm2) (n = 4 per substrate).
collagenase supplement with 0.2% (wt/vol) albumin (Sigma, St. Louis, MO, USA). Cells from ligaments were isolated by centrifugation at 1500 rpm for 10 min. The cell pellet was washed three times with culture medium containing RPMI1640 (Gibco), 15% (wt/vol) fetal bovine serum (Hyclone), 100 U/ml penicillin, 100 Ag/ml streptocycin (Gibco). The ACL cells were counted (5 106 cells) and then suspended in 15 ml culture medium in a 75-cm2 flask (Corning). The culture medium was changed every 3 days. When they became confluent usually 7 –10 days after isolation of cells, the fibroblasts were detached by treatment with 1 ml 0.25% trypsin –EDTA (Gibco) and subcultured.
2.4. Cell adhesion studies For the assessment of the kinetics of cell adhesion, ligament cells were seeded onto TCP, poly(q-caprolactone) (PCL), PCL/PLA (50:50) and PLGA (50:50) at the density of 20,000 cells/cm2. The cells were allowed to attach to the substrates undisturbed in a humidified incubator (at 37 jC, 5% CO2) for 4, 6, 8 and 10 h. At each time point, parallel samples of n = 3 were used. The number of attached cells was qualified by MTS assay. Since the difference of ligament cells attachment at 10 h has no significance, the effects of polymer substrate on li-
Fig. 3. Bone marrow stromal cell (bMSCs) proliferation on various substrates. The numbers of cells were normalized to initial density of seeded cells (10,000 cells/cm2) (n = 4 per substrate).
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gament cells and bMSCs were studied using 8 h attachment time. The cells were seeded on each substrate at the same density and incubated for 8 h. The number of attached cells was assessed by MTS assay (n = 4). 2.5. Cell proliferation studies
2.7. Phase contrast microscopy Phase contrast micrographs were taken of cells attached to the substrates after 12 h of attachment (seeding density: 20,000 cells/cm2). The cells attached to the substrates following the PBS rinse were visualized on an inverted microscopy.
Cells were seeded onto each polymeric film and TCP at the density of 10,000 cells/cm2. The cells were allowed to attach and proliferate for 3 and 5 days, at which time point, the number of attached cells was determined by MTS assay. bMSCs usually get confluence in 1 week after passage, so Days 3 and 5 were chosen as the observation time points which ensure enough room for cell proliferation.
2.8. Statistical analysis
2.6. MTS assay
3. Results
In order to study the cells adhesion and proliferation, the number of viable cells on the polymer sheets was determined with use of Cell Titer 96 (Promega), a colorimetric assay in which metabolically active cells react with a tetrazolium salt to produce a soluble formazan dye that was read at 490 nm and compared with a standard curve to calculate the number of viable cells. As Lin et al. [15] described, before testing, the polymer sheets were rinsed with PBS twice and then 250 Al MTS reagent diluted to 5% concentration in culture medium was added into each well. The culture plates were then incubated for 1.5 h, after which 100-Al aliquots of each well was placed into individual wells of a 96-well plate. The 96-well plates were then placed into a spectrophotometric plate reader to have the absorbance of the content of each well.
3.1. Polymer films
All attachment and proliferation measurements were collected in quadruplicate and expressed as mean F 1 standard deviation (S.D.). Single-factor analysis of variance (ANOVA) was employed to assess statistical significance.
Table 1 shows the various biodegradable polymers and corresponding substrate contact angle measured. Most of the water contact angles were between 70j and 80j with the exception of PCL, which had contact angle of 95j ( p < 0.05). 3.2. Cell adhesion The kinetics of anterior cruciate ligament (ACL) cells adhesion on three polymeric films and TCP are as shown in Fig. 1. ACL cells attached to all the substrates with increasing numbers over the 10-h period. The rate of cell adhesion slowed down dramatically after 8 h. There was statistical difference in cell attachment among the substrates after 4 h
Fig. 4. Anterior ligament cells proliferation on polymeric films and TCP. The cell numbers were normalized to the initial density of seeded cells (10 000 cells/ cm2). Fibroblasts from ACL were found to proliferate better on PLGA (50:50, 75:25) and TCP than on PCL, D-PLA, L-PLA and PCL/PLA (50:50, 75:25) (n = 4 per substrate).
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( p V 0.001), 6 h ( p < 0.05) and 8 h ( p V 0.001). By 10 h, the percentage of attached cells to TCP, PCL, PCL/PLA (50:50) and PLGA (50:50) was 49.3 F 13%, 34.5 F 5.4%, 39.9 F 4.7% and 42.7 F 6.1%, respectively, which showed no statistical significance ( p > 0.05). ACL cells and bMSCs adhesion on all polymer films and TCP were studied. As shown in Fig. 2, both kinds of cells adhered to all the substrates after 8 h of cell culture. As compare to ACL cells, more bMSCs attached to TCP and polymeric films. These differences were significant on TCP, PLGA (50:50, 75:25), PLLA and PCL/PLA (50:50). When multiple comparisons were made between the substrates, a greater percentage of ACL cells were found attached to PLGA50:50 (38.6 F 8.4%) and TCP (39.3 F 6.1%) as compared to the other six polymeric films ( p V 0.001). As for bMSCs, 76.4 F 10%, 76.3 F 16% and 76.1 F 19% of seeded cells were adhered to TCP, PLGA50:50 and PLGA75:25, respectively. These were significantly more than those of the other five polymeric films ( p < 0.001).
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3.3. Cell proliferation ACL cells and bMSCs proliferation on the polymeric substrates were studied at Days 3 and 5 and are as shown in Figs. 3 and 4, respectively. The increased number of ACL cells and bMSCs at the two time points demonstrated that proliferation occurred in all substrates. As the initial cell densities were the same (10,000 cells/cm2), at Day 5 the bMSCs were found to have more folds of expansion on all substrates with the exception of PCL when compared to ACL cells ( p < 0.05). Of all the substrates studied, the bMSCs proliferated faster on TCP (7 F 0.8 folds), poly( DL -lactide) ( D -PLA) (5.6 F 1.6 folds), PLGA50:50 (9.3 F 1.3 folds) and PLGA75:25 (5.8 F 1.3 folds) than on PCL, PLLA and PCL/PLA (50:50, 25:75) ( p < 0.001). As for ACL cells, greater folds of expansion were found on TCP (3.5 F 0.2 folds), PLGA50:50 (3.1 F 0.4 folds) and PLGA75:25 (3.9 F 0.4 folds) when compared to the other five polymeric substrates ( p < 0.001).
Fig. 5. (a) – (d) Morphology of the fibroblasts from anterior cruciate ligament on (a) TCP, (b) PLGA, (c) PCL and (d) D-PLA after 12-h cell culture. It was found that the ACL fibroblasts attached and stretched well on these substrates. There is no significant difference between their morphology (magnification: 100 ).
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4. Discussion In this study, bone marrow stromal cells and anterior cruciate ligament cells were able to adhere and proliferate on the polymeric films used. Phase contrast images showed that ACL cells attached and spread well on all substrates. There was no effect of substrate on cell morphology at 12 h of cell culture (see Fig. 5). These results correlated well with those of previous studies [22 –24], which had shown that water contact angle range from 70j to 90j is suitable for cells to attach and grow. Thus, there are various cells – material systems available for ACL tissue engineering. It was found that less number of ACL cells adhered to the substrates as compared to bMSCs and the difference are significant on PLGA (50:50, 75:25), TCP, PLLA and PLA/ PCL (50:50) ( p < 0.05) but not on D-PLA, PCL, PLA/PCL (75:25). These results showed that bMSCs are more likely to attach to the substrates than ACL cells, and the difference between the adhesion ability of bMSCs and ACL cells are substrate chemical property-sensitive. As for cell proliferation, bMSCs had significantly greater expansion capability than ACL cells on all substrates except for PCL ( p < 0.05). It can be explained by the fact that stem cells are not limited to a fixed number of mitotic divisions, whereas ACL cells are terminally differentiated and have limited lifespan. Hannafin et al. [25] reported that ACL cells possessed some special characteristics and grow more slowly as compared to Medial Collateral Ligament cells. Of all the substrates studied, ACL cells and bMSCs were found more likely to attach to and grow on the high molecular weight (HMW) PLGA (50:50 and 75:25) and TCP control. This result correlated well with previous studies [26] that reported high molecular weight PLGA is more likely to allow epithelium cells to attach and proliferate. It was generally agreed that hydrophilicity of substrates affect biological response, such as cell adhesion and proliferation. Due to the presence of the extra methyl group in lactic acid, PLA is more hydrophobic than PLGA so that a lower percentage of cells attached to PLA as compared to PLGA. Again, PCL has a high olefinic characteristic and was more hydrophobic than PLGA. On the other hand, the amount and type of serum protein that adhered to substrates varied on different chemical surfaces, which also affect cell behavior on the substrates [24]. Although D-PLA and PLLA are composed of the same chemical component, it was shown that bMSCs grew faster on D-PLA than on PLLA, and more bMSCs and ACL cells attached to D-PLA as compared to PLLA. Similar differences have been reported on osteoblasts and chondrocytes [27]. This may be explained by the fact that D-PLA is more crystalline. So far, no general principle has been established to explain and predict the extent of adhesion, proliferation and spreading of cultured cells on different polymer surfaces [28]. Therefore, it is necessary to design and conduct specific experiments similar to this study on the interaction between
specific cells and candidate biomaterials for use as scaffold in tissue engineering research for specific application. 5. Conclusion All polymer substrates studied were able to allow ACL cells and bMSCs to adhere and proliferate. bMSCs are more likely to attach to high molecular weight PLGA (50:50, 75:25) while ACL cells prefer high molecular weight PLGA50:50. MSCs proliferated faster on PLGA (50:50, 75:25) and D-PLA, while ACL cells expanded greatly on PLGA (50:50, 75:25). On all polymer substrates, bMSCs have a higher degree of cell attachment and proliferation than that of ACL cells. The information obtained from this study should be useful for future ACL tissue engineering research. References [1] R.J. Johnson, Int. J. Sports Med. 3 (1982) 71. [2] F.H. Fu, C.D. Harner, D.L. Johnson, M.D. Miller, S.L.Y. Woo, J. Bone Jt. Surg. 75A (1993) 1716. [3] L. Engebretsen, P. Renum, S. Sindalsvoll, Acta Orthop. Scand. 60 (1989) 561. [4] C.T. Laurencin, A.M.A. Ambrosio, M.D. Borden, J.A. Cooper Jr., Annu. Rev. Biomed. Eng. 1 (1999) 19. [5] L.F. Cooper, C.T. Harris, S.P. Bruder, R. Kowalski, S. Kadiyala, J. Dent. Res. 80 (1) (2001) 314. [6] A.I. Caplan, S.P. Bruder, Trends Mol. Med. 7 (6) (2001) 259. [7] H.A. Awad, D.L. Butler, G.P. Boivin, F.N. Smith, P. Malaviya, B. Huibregtse, A.I. Caplan, Tissue Eng. 5 (3) (1999) 267. [8] P. Bianco, M. Riminucci, S. Gronthos, P.G. Robey, Stem Cells 19 (2001) 180. [9] S. Wakitani, T. Goto, S.J. Pineda, R.G. Young, J.M. Mansour, A.I. Caplan, V.M. Goldberg, J. Bone Jt. Surg., Am. Vol. 76 (4) (1994) 579. [10] R.G. Young, D.L. Butler, W. Weber, A.I. Caplan, S.L. Gordon, D.J. Fink, J. Orthop. Res. 16 (4) (1998) 406. [11] L.D. Bellincampi, R.F. Closkey, R. Prasad, J.P. Zawadasky, M.G. Dun, J. Orthop. Res. 16 (1998) 414. [12] M.G. Dune, J.B. Leish, M.L. Tiku, S.H. Maxian, J.P. Zawadsky, Mater. Res. Soc. Symp. Proc., 1994, p. 331. [13] F. Goulet, L. Geramin, D. Rancourt, C. Caron, A. Normand, F.A. Auger, in: R.P Lanza, R. Langer, W.I. Chick (Eds.), Principles of Tissue Engineering, RG Landes Academic, Austin, TX, 1997, p. 639. [14] H.E. Cabaud, J.A. Feagin, W.G. Rodkey, Am. J. Sports Med. 10 (1982) 259. [15] V.S. Lin, M.C. Lee, S. O’Neal, J. McKean, K.L. Sung, Tissue Eng. 5 (5) (1999) 443 – 452. [16] C. Laurencin, M. Attwawia, E. Botchwey, R. Warren, E. Attia, In Vitro Cell. Dev. Biol.: Anim. 34 (1998) 90. [17] S.L. Ishaug-Riley, L.E. Okun, G. Prado, M.A. Applegate, A. Ratcliffe, Biomaterials 20 (23 – 24) (1999) 2245. [18] J.N. Beresford, M.E. Owen, Marrow Stromal Cell Culture, Cambridge Univ. Press, Cambridge UK, 1998. [19] S. Kuznetsov, P. Gehron Robey, Calcif. Tissue Int. 59 (4) (1996) 265. [20] K. Kobayashi, R.M. Healey, R.L. Sah, J.J. Clark, B.P. Tu, R.S. Goomer, W.H. Akeson, H. Moriya, D. Amiel, Tissue Eng. 6 (1) (Feb. 2000) 29. [21] C.N. Nagineni, D. Amiel, M.H. Green, M. Berchuck, W.H. Akeson, J. Orthop. Res. 10 (4) (1992) 465. [22] T. Horbett, M. Schway, J. Colloid Interface Sci. 104 (1985) 28. [23] Y. Ikata, Biomaterials 15 (10) (1994) 725.
H.W. Ouyang et al. / Materials Science and Engineering C 20 (2002) 63–69 [24] Y. Tamada, Y. Ikata, J. Biomed. Mater. Res. 28 (1994) 783. [25] J.A. Hannafin, E.T. Attia, R.F. Warren, M.M. Bhargava, J. Orthop. Res. 17 (1999) 398. [26] G.G. Giordano, R.C. Thomson, S.L. Ishaug, A.G. Mikos, S. Cumber, C.A. Garcia, D. Lahiri-Munir, J. Biomed. Mater. Res. 34 (1) (1997) 87.
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[27] B.D. Boyan, T.W. Hummert, D.D. Dean, Z. Schwartz, Biomaterials 17 (2) (1996) 137. [28] W.M. Saltzman, in: R.P Lanza, R. Langer, J. Vancanti (Eds.), Principles of Tissue Engineering, 2nd edn., Academic Press, California, 2000, p. 221.