- Email: [email protected]
The effect of growth factors on both collagen synthesis and tensile strength of engineered human ligaments
Biomaterials 33 (2012) 6355e6361
Contents lists available at SciVerse ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
The effect of growth factors on both collagen synthesis and tensile strength of engineered human ligaments Paul Hagerty a, Ann Lee a, Sarah Calve b, Cassandra A. Lee c, Martin Vidal d, Keith Baar a, * a
Department of Neurobiology, Physiology and Behavior, University of California Davis, One Shields Ave, 181 Briggs Hall, Davis, CA 95616, USA Weldon School of Biomedical Engineering, Purdue University, USA c Department of Orthopaedic Surgery, University of California Davis, School of Medicine, USA d School of Veterinary Medicine, University of California Davis, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2012 Accepted 20 May 2012 Available online 12 June 2012
Growth factors play a central role in the development and remodelling of musculoskeletal tissues. To determine which growth factors optimized in vitro ligament formation and mechanics, a Box-Behnken designed array of varying concentrations of growth factors and ascorbic acid were applied to engineered ligaments and the collagen content and mechanics of the grafts were determined. Increasing the amount of transforming growth factor (TGF) b1 and insulin-like growth factor (IGF)-1 led to an additive effect on ligament collagen and maximal tensile load (MTL). In contrast, epidermal growth factor (EGF) had a negative effect on both collagen content and MTL. The predicted optimal growth media (50 mg/ml TGFb, IGF-1, and GDF-7 and 200 mM ascorbic acid) was then validated in two separate trials: showing a 5.7-fold greater MTL and 5.2-fold more collagen than a minimal media. Notably, the effect of the maximized growth media was scalable such that larger constructs developed the same material properties, but larger MTL. These results show that optimizing the interactions between growth factors and engineered ligament volume results in an engineered ligament of clinically relevant function. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: IGF-1 TGF-beta Ascorbic acid EGF Regenerative medicine Design of experiments
1. Introduction The ligaments of synovial joints develop from cartilaginous precursor cells following cavitation of the developing anlagen [1,2]. In order for these joints and ligaments to form normally, cells rely upon both mechanical and soluble cues. Developmentally, the mechanical force is initially the result of the synthesis of hyaluronic acid (HA) within the developing joint space [3,4] and this is further supported by dynamic strain across the developing ligaments [4]. Immobilizing the developing joint decreases HA formation and results in joint fusion [4], whereas mechanical loading results in improved collagen synthesis [5]. The soluble growth factor cues necessary for ligament development have been more difficult to determine. Growth and differentiation factor (GDF) 5 is initially expressed in the interzone of the developing joint [6] and GDF5/6/7 can induce ectopic ligament formation when injected subcutaneously [7], suggesting that these factors are important in ligament development. Transforming growth factor (TGF) b is also important in ligament formation since in the absence of TGFb2 and 3 no cruciate ligaments develop [8]. * Corresponding author. E-mail address: [email protected] (K. Baar). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2012.05.045
Insulin-like growth factor (IGF)-1 is thought to be important in the anabolic response to mechanical loading [9] and can increase the synthesis of extracellular matrix proteins including type I and type III collagens, elastin, tenascin-C, and vimentin [10]. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) are thought to be important for the proliferative capacity of ligament cells [11,12]. However, at present the combination of growth factors needed for ligament development has yet to be determined. We have recently developed a three-dimensional (3D) engineered ligament in which the cells produce a native 3D collagen matrix [5,13e15]. This model has a number of advantages over traditional culture models. First, the cells are placed within a matrix that allows them to form in a manner similar to tendons in vivo [14]. Second, using molded brushite (calcium phosphate cement) anchors, the sinews can be stretched in a uniaxial manner using reverse molded grips [15,16]. This means that mechanical, as well as biochemical, data can be generated from the cultures. Third, since a fibrin matrix is used to engineer the sinews, the cells produce all of the collagen in the grafts. Therefore, collagen content can be measured and used as an indicator of the anabolic response of the sinew to various external interventions. The objective of this study was to determine the optimum growth factor milieu for in vitro engineering ligaments. We focused
6356
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
on growth factors that have been suggested to play a role in ligament development including IGF-1, EGF, PDGF, and GDF-7, and TGFb1. To achieve this objective, we used a design of experiments (DOE) multivariable approach [17]. The strength of the DOE multivariable approach is that it identifies not only the individual factors that play a significant role in ligament development, but also interactions between different factors, where standard onevariable-at-a-time approaches cannot. Using this approach, we determined the concentration of each growth factor that was optimal for both collagen production and tensile strength. This growth factor mix was then validated and combined with a greater volume of fibrin matrix to produce the strongest biologically engineered ligaments to date.
mechanics and collagen content. Immediately following graft formation, the media in which the constructs were cultured was changed to one of the three formulations described above. Ligaments were then cultured for another 14 days, with the appropriate growth factor mix added every two days. 2.5. Mechanical properties of ligament constructs The mechanical properties of the sinews were analyzed as described previously [5,15]. Briefly, prior to testing, the width of each construct was measured with digital calipers. Sinews were placed in a custom-built tensile tester at room temperature and submerged in PBS to maintain tissue hydration. Samples were mechanically tested to failure without preconditioning at a constant elongation rate of 0.4 mm/s. All samples used for data analysis demonstrated a linear portion in the loadeextension curve. The samples failed at the interface in all cases. The load and extension data together with the dimensions of the grafts were used to calculate the maximal tensile load (MTL), tangent modulus, and ultimate tensile stress.
2. Methods 2.6. Collagen content 2.1. Cell isolation Human anterior cruciate ligaments were collected under anesthesia during reconstruction surgery. Prior to collection, all subjects (3 different donors were used for the various experiments) provided informed consent and all surgical procedures were approved by the University of California Davis Institutional Review Board. The ligament remnants were digested as described previously for rat and chick tissues [5]. Briefly, ligaments were digested overnight at 37 C in a 1% collagenase Type II solution (Dulbecco’s Modified Eagles Medium (DMEM) containing 20% fetal bovine serum (FBS) and 1% penicillin and streptomycin). The next morning the resulting fibroblasts (hACL) were collected by centrifugation (1.5 g for 5 min), washed twice with PBS (phosphate buffered saline, pH 7.3), and then plated and cultured in DMEM containing 10% FBS and 1% penicillin and streptomycin. Cells were either passaged or cryopreserved (DMEM, 20% FBS, 10% DMSO). All experiments were performed on cells prior to passage 5.
The collagen content of the ligament constructs was determined using a hydroxyproline assay [18]. Briefly, after mechanical testing, sinews were removed from their cement anchors and dried in an oven for 30 min at 110 C. Each sample was then weighed and hydrolyzed in 200 ml of 6N HCl at 130 C for 3 h. The liquid was removed by allowing the HCl to evaporate for 30 min in a fume hood at 130 C. The resulting pellet was resuspended in 200 ml of hydroxyproline buffer. Samples were further diluted 1:8 in hydroxyproline buffer. 150 ml of Chloramine T Solution was added to each sample, vortexed and left at room temperature for 20 min. 150 ml aldehyde-perchloric acid Solution was then added to each tube before the tubes were vortexed and incubated in a heat block at 60 C for 15 min. Following incubation, tubes were left to cool for 10 min and then samples/standards were read at 550 nm on an Epoch Microplate Spectrophotometer (BioTek Instruments Limited, Winooski, VT). Hydroxproline was converted to collagen mass using a factor of 13.8% as reported previously [19].
2.2. Ligament formation
2.7. Statistics
Tissue engineered ligament constructs were engineered as described previously [14] with modification [5]. Briefly, two circular 4 mm diameter brushite anchors were pinned 12 mm apart in a Sylgard coated 35 mm dish. Human ACL cells (2.5 105) were suspended in 1 ml of growth media containing 10U thrombin (Calbiochem), 400 mM aminohexanoic acid, 20 mg aprotinin, and 40 mg of fibrinogen. The solution was rapidly spread over the surface of the dish and the gel was allowed to set for 10 min at 37 C. Once set, the constructs were given 2 ml of growth media every other day until day 14 when all of the constructs were tested. For the volume experiments, all constituents of the gel were increased proportionally. Constructs made with an initial fibrin volume of 1 mL (containing 2.5 105 cells) formed in an average of 7 days, while constructs made with an initial fibrin volume of 1.5 mL (containing 3.75 105 cells) formed in an average of 8 days, and constructs made with an initial fibrin volume of 2 mL (containing 5 105 cells) formed in 10 days.
Data are presented as means SEM. Differences in mean values were compared within groups and significant differences were determined by ANOVA with post hoc TukeyeKramer HSD test using BrightStat (www.brightstat.com). The significance level was set at p < 0.05.
2.3. Design of experiments The concentrations of the growth factors were determined using a Box-Behnken design [17] using Design-ExpertÒ software (Stat-Ease, Inc., Minneapolis, MN). BoxBehnken describes a subset of DOE techniques that uses an incomplete factorial design where three levels of any given factor are tested and the resulting data is analyzed using quadratic response surface plots [17]. In our case, each construct was fed a different combination of ascorbic acid, TGFb1, IGF-1, GDF-7, EGF, and PDGF across a 10-fold range for 2 weeks. The range for ascorbic acid (AA) was 25e250 mM, EGF ranged from 0.5 to 5 ng/ml, and GDF-7, IGF-1, PDGF, and TGFb1 were added at a range from 5 to 50 ng/ml. To determine the variability from sample to sample and provide the statistical basis for selection, 5 grafts received the identical treatment. A total of 54 samples were treated with the various growth factor combinations. Maximal tensile load and collagen content for each of the conditions were measured following 14 days in culture and the significance of each growth factor and predicted optimum media was determined using the statistical and point prediction tools of the Design-ExpertÒ software. The optimal media was then validated in two independent experiments with 6 grafts per group. 2.4. Validation experiments The predicted optimal growth factor combination from the DOE as well as a growth factor mix lacking EGF and PDGF were validated in two independent experiments using different donors and 6 grafts per group. Briefly, the predicted worst (minimal: 5 ng/ml TGFb1, IGF-1, GDF-7, and EGF and 50 ng/ml PDGF), and best DOE media (DOE: 50 ng/ml TGFb1, IGF-1, and GDF-7, 5 ng/ml PDGF, and 0.5 ng/ml EGF) were tested against a media that lacked EGF and PDGF (maximal: 50 ng/ml TGFb1, IGF-1, and GDF-7) since these growth factors have a negative effect on graft
3. Results 3.1. Growth factors and maximal tensile load Treating constructs with differing levels and combinations of ascorbic acid and growth factors resulted in grafts that demonstrated an 11.6-fold range of maximal tensile load from 67 to 770 mN. The variability of the 5 control ligaments was low (17 mN) showing the repeatability of the model. Both TGFb1 and IGF-1 showed a significant positive relationship with MTL (Fig. 1A). The combination of GDF-7 and TGFb1 showed a curvilinear relationship. At low concentrations of TGFb1, GDF-7 had a negative effect on MTL, whereas at high TGFb1 levels GDF-7 had a positive effect on MTL (Fig. 1B). Lastly, both EGF and PDGF showed a negative relationship with MTL, with the effects of EGF being significant (Fig. 1C; p ¼ 0.0001). 3.1.1. Growth factors and collagen content Treating the ligaments with differing levels and combinations of ascorbic acid (AA) and growth factors resulted in grafts that demonstrated a 2.9-fold range of collagen content from 180 to 516 mg. Further analysis showed that ascorbic acid, TGFb1, and IGF-1 all significantly increased collagen content (p < 0.0001; Fig. 2). EGF on the other hand showed a significant negative relationship with collagen (p ¼ 0.0027; data not shown). The combination of GDF-7 and TGFb1 once again showed a curvilinear relationship where at low concentrations of TGFb1 GDF-7 had a negative effect on collagen content, whereas at high TGFb1 levels GDF-7 had a positive effect on collagen content (Fig. 2B). Interestingly, at concentrations of AA higher than 200 mM, there was a progressive decline
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
Fig. 1. 3D response surface plots of growth factor effects on maximal tensile load. The plots show the effect on maximal tensile load of combinations of: A) IGF-1 and TGFb1; B) GDF-7 and TGFb1; and C) EGF and TGFb1. Note that the near linear response to both IGF-1 and TGFb1 indicates that the effects of the growth factors are additive while the curvilinear response to GDF-7 indicates a much more complex relationship between these two growth factors. Additionally the linear negative relationship between EGF and TGFb1 indicates that EGF competitively inhibits TGFb1 function. All growth factor values are ng/ml. All of these relationships are statistically significant.
Fig. 2. 3D response surface plots of growth factor effects on the collagen content of grafts. The plots show the effect on collagen content of: A) IGF-1 and TGFb1; B) GDF-7 and TGFb1; and C) IGF-1 and ascorbic acid (AA). Note once again that the linear response to IGF-1 and TGFb1 showing that both agents have independent positive effects on collagen synthesis, whereas the relationship between GDF-7 and TGFb1 and IGF-1 and AA are more complex. Growth factors were used at the ng/ml level. All of these relationships are statistically significant. 6357
6358
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
in collagen content indicating that too much ascorbic acid can be detrimental for collagen deposition (Fig. 2C). 4. Validation of the growth factor DOE In order to validate the optimized growth factor mix, six grafts were engineered using the predicted minimal growth factor mix (minimal: 5 ng/ml TGFb1, IGF-1, GDF-7, and EGF, 50 ng/ml PDGF, and 200 mM ascorbic acidAA), the DOE predicted best mix (DOE: 50 ng/ml TGFb1, IGF-1, and GDF-7, 5 ng/ml PDGF, 0.5 ng/ml EGF, and 200 mM AA), and a mix lacking EGF and PDGF (maximal: 50 ng/ml TGFb1, IGF-1, and GDF-7, and 200 mM AA). As predicted from the DOE, ligaments grown in the minimal growth factor mix showed a 4.5- lower MTL and a 4.1-fold lower collagen content then ligaments grown in the optimized DOE media (Fig. 3). Further, removing the EGF and PDGF from the mix (maximal media) resulted in a significantly greater increase in MTL (5.7-fold) and collagen content (5.2-fold). 4.1. Construct volume and ligament strength Having improved graft mechanics 5.7-fold by altering the growth factor environment, we next sought to determine the effect of increasing the construct volume while maintaining the fibrin:cell ratio. Increasing the volume of fibrin from 1 ml (2.5 105 cells) to 1.5 ml (3.75 105 cells) or 2 ml (5 105 cells) resulted in a stepwise increase in both MTL and collagen content (Fig. 4). Further, combining the optimized growth factor mix with the greater volume increased MTL 77% (Fig. 5) and tended to increase the collagen content of the grafts. 5. Discussion Using a design of experiments multivariable approach, we have determined a combination of growth factors that increases the mechanical strength (5.7-fold) and collagen content (5.2fold) of fibrin-based 3D engineered ligaments. The multivariable approach identified significant positive effects of IGF-1 and TGFb1, negative effects of EGF, and a significant interaction between GDF-7 and TGFb1. This interaction effect meant that the relationship between GDF-7 levels and MTL and collagen content of the grafts was completely reversed depending on the levels of TGFb1 in the media. Combining the optimized growth factor mix with a greater volume of fibrin gel resulted in a further 77%
increase in MTL, indicating that the technique is scalable and will continue to increase in strength as the size of the construct is increased. When designing the DOE, we used MTL and collagen content as the selection parameters since we felt that the absolute tensile strength (MTL) and amount of collagen of the grafts is more important in designing a functional ligament than modulus, ultimate tensile strength, or the percent collagen within the graft. This decision was made with the understanding that interventions that decreased the cross-sectional area of a graft could dramatically increase the modulus, ultimate tensile strength, and percent collagen without improving the absolute strength of the grafts. This is best seen by the fact that increasing the volume of the constructs from 1 ml to 2 ml resulted in a 77% increase in MTL without altering either modulus or ultimate tensile strength due to the increase in the cross-sectional area of the graft. However, it is important to note that while there was a 79% difference in the cross-sectional area of the grafts in the original DOE, the MTL showed a range of 11.6-fold. Regardless, we feel that if the ultimate goal is to use the engineered ligaments as a clinical source of graft material, it is the absolute strength of the graft that will be most important to withstand initial implantation. With that in mind, it is important to note that the largest MTL recorded for these grafts was 2.35N corresponding to an ultimate tensile stress of 1.7 MPa and a tangent modulus of 5.16 MPa. These values are significantly less than the native human ACL (UTS ¼ 55 MPa; tangent modulus ¼ 200 MPa [20];). However, it is important to note that all of the constructs failed at the boneeligament interface during the tensile tests so the measured MTL is likely an underestimation of the true tissue mechanics. This suggests that improving the interface between the soft tissue and the brushite bone cement will be key to further development of this technology. TGFb signaling is known to play a critical role in the development of ligaments [8], fibrosis [21], and collagen synthesis [5,22]. TGFb is thought to mediate these effects in tendons and ligaments by activating the early growth response factors (EGR)1 and 2 [23]. EGR1 and EGR2 are transcription factors that bind to the tendon specific elements within the Col1a1 promoter [24] and together with scleraxis and nuclear factor of activated T-cells (NFAT) increase collagen expression [25]. In the absence of EGR1/2 there is a significant decrease in the collagen content of tendons and ligaments [24], suggesting that the increase in collagen synthesis in our engineered ligaments is due to the regulation of ERG1/2 through the addition of TGFb.
Fig. 3. Optimized media improves maximal tensile load and collagen content of engineered ligaments. A) The maximal tensile load (MTL) and B) collagen content of engineered ligament grafts was determined following 14 days of treatment with either the minimally effective media or best media (DOE) as determined by the DOE. The maximal group was fed the DOE media without any EGF and PDGF. * indicates significantly higher than the minimal group and y indicates significantly greater than the DOE group (p < 0.05). Results are representative of 2 independent trials and presented as mean S.E.M. of n ¼ 6 for all groups.
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
6359
Fig. 4. Increasing fibrin volume and cell number increases maximal tensile load and collagen content. A) The maximal tensile load (MTL) and B) collagen content of engineered ligament grafts was determined 10 days after formation for fibrin gels of either 1 mL (2.5 105 cells), 1.5 mL (3.75 105 cells), or 2 mL (5 105 cells). * indicates significantly higher than 1 ml group and y indicates significantly higher than the 1.5 ml group (p < 0.05). Results are representative of 2 independent trials and presented as mean S.E.M. of n ¼ 6 for all groups.
The molecular mechanism underlying the effects of IGF-1 is less well established. Doessing et al. [26] have demonstrated that supplementing healthy volunteers with recombinant human growth hormone results in an increase in IGF-1 mRNA within the patellar tendon and this local increase in IGF-1 correlated with the resulting rise in Col1a1 expression. Furthermore, this same group has shown that exercise resulted in a greater than 5-fold increase in IGF-1 protein within the peritendinous space [27]. This increase in IGF-1 was associated with a 5.7-fold increase in markers of collagen synthesis (COOH-terminal telopeptide of type 1 collagen), indicating that IGF-1 may be involved in the anabolic response of tendons to loading [27]. In agreement with a role of IGF-1 in collagen turnover within ligaments, Sciore et al. [28] showed that IGF-1 mRNA levels within a scarred ligament are 5times higher following three weeks of repair than in an uninjured ligament. Lastly, in a recent study Hansen et al. [29] showed that injections of IGF-1 into the patellar tendon resulted in a 24% increase in the fractional synthetic response of collagen. In the current study, IGF-1 had a positive independent effect on MTL and collagen content and was additive with TGFb1, suggesting that the two growth factors act independently to control collagen synthesis. The negative effects of EGF and PDGF on engineered ligament MTL and collagen content was surprising given that a previous in vivo study had shown that PDGF improved collagen synthesis
and repair in skin wounds [30] and EGF has been shown to increase fibroblast proliferation and collagen synthesis [11]. However, the data from the current study are quite clear. EGF had a significant negative effect on both MTL (p < 0.0001) and collagen content (p ¼ 0.0027). Furthermore, completely removing both EGF and PDGF had a significant positive effect on both MTL and collagen, suggesting that even small amounts of these growth factors impair engineered ligament function. Lastly, the Box-Behnken analysis demonstrated that in human ACL cells AA had a positive effect on collagen content up to approximately 200 mM. Concentrations greater than 200 mM AA tended to decrease the collagen content of the grafts. The positive effect of AA is consistent with its role as an essential co-factor for prolyl-4-hydroxylase [31]. The absence of AA leads to inactivation of prolyl-4-hydroxylase and retention of procollagen within the endoplasmic reticulum [32]. In primary tendon cells, AA increases collagen synthesis [33,34], and this has been seen in many other cell types as well [35e38]. There are reports of a plateau in collagen synthesis with doses of AA over 20 mM [39]; we have not found other reports of a negative effect of high concentrations of ascorbic acid on collagen synthesis and have no ready explanation for this observation. High levels of AA cause a significant decrease in construct size with chick tendon fibroblasts [40], however in the current work the effect was only seen in collagen content and not construct diameter.
Fig. 5. Combining greater volume and optimal media results in greater engineered ligament MTL. A) The maximal tensile load (MTL) and B) collagen content of engineered ligament grafts was determined 10 days after formation for fibrin gels of either 1 ml (2.5 105 cells) or 2 ml (5 105 cells) grown in maximal media determined in Fig. 3. * indicates significantly higher than 1 ml group (p < 0.05). Results are representative of 2 independent trials and presented as mean S.E.M. of n ¼ 6 for all groups.
6360
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
Combining the optimized growth factor mix with a greater volume of fibrin and cells resulted in a significant increase in MTL and a trend towards increased collagen content. This is significant since it shows that the ligaments are not currently at their maximal viable radius. For engineered tissues, the maximal viable radius is the size at which metabolic failure (necrosis) begins to occur at the core of the tissue and function is reduced [41]. For metabolically active tissues such as cardiac and skeletal muscle this radius is w50 mm [42] and w150 mm [43], respectively. For ligaments this value appears to be significantly higher, which is not surprising given the avascular nature of ligaments in vivo. The fact that human ligaments can be engineered with greater cross-sectional areas may help make the transition to the clinic more realistic than it is for heart or skeletal muscle. 6. Conclusions Using a multivariable approach designed to identify interactions, we have determined that increasing levels of TGFb1 and IGF-1 are beneficial for engineered ligament function. Further, at high levels of TGFb1, GDF-7 shows a positive effect on both ligament function and collagen content. This, together with removal of EGF and PDGF and an increase in fibrin and cell volume, has resulted in human ligament grafts with an MTL of up to 2.35N and this corresponds to an UTS of 1.7 MPa and a tangent modulus of 5.17 MPa; approximately 1/40 that of the native ACL. Acknowledgments This work was supported in part by a University of California Davis Health System, Vision Grant (54885) and by the E. Baar Memorial Research Trust (KB). Further support came from a project grant from the Grayson-Jockey Club Research Foundation, Inc. (MV). References [1] Archer CW, Dowthwaite GP, Francis-West P. Development of synovial joints. Birth Defects Res C Embryo Today 2003;69:144e55. [2] Soeda T, Deng JM, de Crombrugghe B, Behringer RR, Nakamura T, Akiyama H. Sox9-expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons. Genesis 2010;48: 635e44. [3] Edwards JC, Wilkinson LS, Jones HM, Soothill P, Henderson KJ, Worrall JG, et al. The formation of human synovial joint cavities: a possible role for hyaluronan and CD44 in altered interzone cohesion. J Anatomy 1994;185(Pt 2):355e67. [4] Bastow ER, Lamb KJ, Lewthwaite JC, Osborne AC, Kavanagh E, WheelerJones CP, et al. Selective activation of the MEK-ERK pathway is regulated by mechanical stimuli in forming joints and promotes pericellular matrix formation. J Biological Chemistry 2005;280:11749e58. [5] Paxton JZ, Grover LM, Baar K. Engineering an in vitro model of a functional ligament from bone to bone. Tissue Eng Part A 2010;16:3515e25. [6] Francis-West PH, Abdelfattah A, Chen P, Allen C, Parish J, Ladher R, et al. Mechanisms of GDF-5 action during skeletal development. Development 1999;126:1305e15. [7] Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, et al. Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family. J Clin Invest 1997;100: 321e30. [8] Pryce BA, Watson SS, Murchison ND, Staverosky JA, Dunker N, Schweitzer R. Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation. Development 2009;136:1351e61. [9] Dahlgren LA, Nixon AJ, Brower-Toland BD. Effects of beta-aminopropionitrile on equine tendon metabolism in vitro and on effects of insulin-like growth factor-I on matrix production by equine tenocytes. Am J Vet Res 2001;62: 1557e62. [10] Steinert AF, Weber M, Kunz M, Palmer GD, Noth U, Evans CH, et al. In situ IGF1 gene delivery to cells emerging from the injured anterior cruciate ligament. Biomaterials 2008;29:904e16. [11] Throm AM, Liu WC, Lock CH, Billiar KL. Development of a cell-derived matrix: effects of epidermal growth factor in chemically defined culture. J Biomed Mater Res Part A 2010;92:533e41.
[12] Wang XT, Liu PY, Tang JB. Tendon healing in vitro: genetic modification of tenocytes with exogenous PDGF gene and promotion of collagen gene expression. J Hand Surg Am 2004;29:884e90. [13] Bayer ML, Yeung CY, Kadler KE, Qvortrup K, Baar K, Svensson RB, et al. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension. Biomaterials 2010;31: 4889e97. [14] Kapacee Z, Richardson SH, Lu Y, Starborg T, Holmes DF, Baar K, et al. Tension is required for fibripositor formation. Matrix Biol 2008;27:371e5. [15] Paxton JZ, Donnelly K, Keatch RP, Baar K, Grover LM. Factors affecting the longevity and strength in an in vitro model of the bone-ligament interface. Ann Biomed Eng 2010;38:2155e66. [16] Paxton JZ, Donnelly K, Keatch RP, Baar K. Engineering the bone-ligament interface using polyethylene glycol diacrylate incorporated with hydroxyapatite. Tissue Eng Part A 2009;15:1201e9. [17] Box GEP, Behnken DW. Some new three level designs for the study of quantitative variables. Technometrics 1960;2:455e75. [18] Woessner Jr JF. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961;93:440e7. [19] Arruda EM, Calve S, Dennis RG, Mundy K, Baar K. Regional variation of tibialis anterior tendon mechanics is lost following denervation. J Appl Physiol 2006; 101:1113e7. [20] Woo SL, Hollis JM, Adams DJ, Lyon RM, Takai S. Tensile properties of the human femur-anterior cruciate ligament-tibia complex. The effects of specimen age and orientation. Am J Sports Med 1991;19:217e25. [21] Sarrazy V, Billet F, Micallef L, Coulomb B, Desmouliere A. Mechanisms of pathological scarring: role of myofibroblasts and current developments. Wound Repair Regen 2011;19(Suppl. 1):s10e5. [22] Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M, Varga J. Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad 3. J Invest Dermatol 1999;112:49e57. [23] Chen SJ, Ning H, Ishida W, Sodin-Semrl S, Takagawa S, Mori Y, et al. The earlyimmediate gene EGR-1 is induced by transforming growth factor-beta and mediates stimulation of collagen gene expression. J Biol Chem 2006;281: 21183e97. [24] Lejard V, Blais F, Guerquin MJ, Bonnet A, Bonnin MA, Havis E, et al. EGR1 and EGR2 involvement in vertebrate tendon differentiation. J Biol Chem 2011;286: 5855e67. [25] Lejard V, Brideau G, Blais F, Salingcarnboriboon R, Wagner G, Roehrl MH, et al. Scleraxis and NFATc regulate the expression of the pro-alpha1(I) collagen gene in tendon fibroblasts. J Biol Chem 2007;282:17665e75. [26] Doessing S, Heinemeier KM, Holm L, Mackey AL, Schjerling P, Rennie M, et al. Growth hormone stimulates the collagen synthesis in human tendon and skeletal muscle without affecting myofibrillar protein synthesis. J Physiol 2010;588:341e51. [27] Olesen JL, Heinemeier KM, Gemmer C, Kjaer M, Flyvbjerg A, Langberg H. Exercise-dependent IGF-I, IGFBPs, and type I collagen changes in human peritendinous connective tissue determined by microdialysis. J Appl Physiol 2007;102:214e20. [28] Sciore P, Boykiw R, Hart DA. Semiquantitative reverse transcriptionpolymerase chain reaction analysis of mRNA for growth factors and growth factor receptors from normal and healing rabbit medial collateral ligament tissue. J Orthop Res 1998;16:429e37. [29] Hansen M, Boesen A, Holm L, Flyvbjerg A, Langberg H, Kjaer M. Local administration of insulin-like growth factor-I (IGF-I) stimulates tendon collagen synthesis in humans. Scand J Med Sci Sports; 2012. [30] Lynch SE, Colvin RB, Antoniades HN. Growth factors in wound healing. Single and synergistic effects on partial thickness porcine skin wounds. J Clin Invest 1989;84:640e6. [31] Kadler KE, Baldock C, Bella J, Boot-Handford RP. Collagens at a glance. J Cell Sci 2007;120:1955e8. [32] Peterkofsky B. Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy. Am J Clin Nutr 1991;54:1135Se40S. [33] Schwarz RI, Farson DA, Soo WJ, Bissell MJ. Primary avian tendon cells in culture. An improved system for understanding malignant transformation. J Cell Biol 1978;79:672e9. [34] Schwarz RI, Bissell MJ. Dependence of the differentiated state on the cellular environment: modulation of collagen synthesis in tendon cells. Proc Natl Acad Sci USA 1977;74:4453e7. [35] Chu AJ, Prasad JK. Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling. J Am Coll Surg 1999; 188:271e80. [36] Ganta DR, McCarthy MB, Gronowicz GA. Ascorbic acid alters collagen integrins in bone culture. Endocrinology 1997;138:3606e12. [37] Graham MF, Willey A, Adams J, Yager D, Diegelmann RF. Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells. J Cell Physiol 1995;162:225e33. [38] Sawaguchi S, Yue BY, Chang IL, Wong F, Higginbotham EJ. Ascorbic acid modulates collagen type I gene expression by cells from an eye tissueetrabecular meshwork. Cell Mol Biol 1992;38:587e604. [39] Pinnel SR, Murad S, Darr D. Induction of collagen synthesis by ascorbic acid. A possible mechanism. Arch Dermatol 1987;123:1684e6.
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361 [40] Paxton JZ, Wudebwe U, Wang A, Woods D, Grover LM. Monitoring sinew contraction during formation of tissue-engineered fibrin-based ligament constructs. Tissue Eng Part A; 2012. [41] Dennis RG, Smith B, Philp A, Donnelly K, Baar K. Bioreactors for guiding muscle tissue growth and development. Adv Biochem Eng Biotechnol 2009; 112:39e79.
6361
[42] Baar K, Birla R, Boluyt MO, Borschel GH, Arruda EM, Dennis RG. Self-organization of rat cardiac cells into contractile 3-D cardiac tissue. Faseb J 2005;19: 275e7. [43] Dennis RG, Kosnik 2nd PE, Gilbert ME, Faulkner JA. Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines. Am J Physiol Cell Physiol 2001;280:C288e95.
Contents lists available at SciVerse ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
The effect of growth factors on both collagen synthesis and tensile strength of engineered human ligaments Paul Hagerty a, Ann Lee a, Sarah Calve b, Cassandra A. Lee c, Martin Vidal d, Keith Baar a, * a
Department of Neurobiology, Physiology and Behavior, University of California Davis, One Shields Ave, 181 Briggs Hall, Davis, CA 95616, USA Weldon School of Biomedical Engineering, Purdue University, USA c Department of Orthopaedic Surgery, University of California Davis, School of Medicine, USA d School of Veterinary Medicine, University of California Davis, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2012 Accepted 20 May 2012 Available online 12 June 2012
Growth factors play a central role in the development and remodelling of musculoskeletal tissues. To determine which growth factors optimized in vitro ligament formation and mechanics, a Box-Behnken designed array of varying concentrations of growth factors and ascorbic acid were applied to engineered ligaments and the collagen content and mechanics of the grafts were determined. Increasing the amount of transforming growth factor (TGF) b1 and insulin-like growth factor (IGF)-1 led to an additive effect on ligament collagen and maximal tensile load (MTL). In contrast, epidermal growth factor (EGF) had a negative effect on both collagen content and MTL. The predicted optimal growth media (50 mg/ml TGFb, IGF-1, and GDF-7 and 200 mM ascorbic acid) was then validated in two separate trials: showing a 5.7-fold greater MTL and 5.2-fold more collagen than a minimal media. Notably, the effect of the maximized growth media was scalable such that larger constructs developed the same material properties, but larger MTL. These results show that optimizing the interactions between growth factors and engineered ligament volume results in an engineered ligament of clinically relevant function. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: IGF-1 TGF-beta Ascorbic acid EGF Regenerative medicine Design of experiments
1. Introduction The ligaments of synovial joints develop from cartilaginous precursor cells following cavitation of the developing anlagen [1,2]. In order for these joints and ligaments to form normally, cells rely upon both mechanical and soluble cues. Developmentally, the mechanical force is initially the result of the synthesis of hyaluronic acid (HA) within the developing joint space [3,4] and this is further supported by dynamic strain across the developing ligaments [4]. Immobilizing the developing joint decreases HA formation and results in joint fusion [4], whereas mechanical loading results in improved collagen synthesis [5]. The soluble growth factor cues necessary for ligament development have been more difficult to determine. Growth and differentiation factor (GDF) 5 is initially expressed in the interzone of the developing joint [6] and GDF5/6/7 can induce ectopic ligament formation when injected subcutaneously [7], suggesting that these factors are important in ligament development. Transforming growth factor (TGF) b is also important in ligament formation since in the absence of TGFb2 and 3 no cruciate ligaments develop [8]. * Corresponding author. E-mail address: [email protected] (K. Baar). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2012.05.045
Insulin-like growth factor (IGF)-1 is thought to be important in the anabolic response to mechanical loading [9] and can increase the synthesis of extracellular matrix proteins including type I and type III collagens, elastin, tenascin-C, and vimentin [10]. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) are thought to be important for the proliferative capacity of ligament cells [11,12]. However, at present the combination of growth factors needed for ligament development has yet to be determined. We have recently developed a three-dimensional (3D) engineered ligament in which the cells produce a native 3D collagen matrix [5,13e15]. This model has a number of advantages over traditional culture models. First, the cells are placed within a matrix that allows them to form in a manner similar to tendons in vivo [14]. Second, using molded brushite (calcium phosphate cement) anchors, the sinews can be stretched in a uniaxial manner using reverse molded grips [15,16]. This means that mechanical, as well as biochemical, data can be generated from the cultures. Third, since a fibrin matrix is used to engineer the sinews, the cells produce all of the collagen in the grafts. Therefore, collagen content can be measured and used as an indicator of the anabolic response of the sinew to various external interventions. The objective of this study was to determine the optimum growth factor milieu for in vitro engineering ligaments. We focused
6356
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
on growth factors that have been suggested to play a role in ligament development including IGF-1, EGF, PDGF, and GDF-7, and TGFb1. To achieve this objective, we used a design of experiments (DOE) multivariable approach [17]. The strength of the DOE multivariable approach is that it identifies not only the individual factors that play a significant role in ligament development, but also interactions between different factors, where standard onevariable-at-a-time approaches cannot. Using this approach, we determined the concentration of each growth factor that was optimal for both collagen production and tensile strength. This growth factor mix was then validated and combined with a greater volume of fibrin matrix to produce the strongest biologically engineered ligaments to date.
mechanics and collagen content. Immediately following graft formation, the media in which the constructs were cultured was changed to one of the three formulations described above. Ligaments were then cultured for another 14 days, with the appropriate growth factor mix added every two days. 2.5. Mechanical properties of ligament constructs The mechanical properties of the sinews were analyzed as described previously [5,15]. Briefly, prior to testing, the width of each construct was measured with digital calipers. Sinews were placed in a custom-built tensile tester at room temperature and submerged in PBS to maintain tissue hydration. Samples were mechanically tested to failure without preconditioning at a constant elongation rate of 0.4 mm/s. All samples used for data analysis demonstrated a linear portion in the loadeextension curve. The samples failed at the interface in all cases. The load and extension data together with the dimensions of the grafts were used to calculate the maximal tensile load (MTL), tangent modulus, and ultimate tensile stress.
2. Methods 2.6. Collagen content 2.1. Cell isolation Human anterior cruciate ligaments were collected under anesthesia during reconstruction surgery. Prior to collection, all subjects (3 different donors were used for the various experiments) provided informed consent and all surgical procedures were approved by the University of California Davis Institutional Review Board. The ligament remnants were digested as described previously for rat and chick tissues [5]. Briefly, ligaments were digested overnight at 37 C in a 1% collagenase Type II solution (Dulbecco’s Modified Eagles Medium (DMEM) containing 20% fetal bovine serum (FBS) and 1% penicillin and streptomycin). The next morning the resulting fibroblasts (hACL) were collected by centrifugation (1.5 g for 5 min), washed twice with PBS (phosphate buffered saline, pH 7.3), and then plated and cultured in DMEM containing 10% FBS and 1% penicillin and streptomycin. Cells were either passaged or cryopreserved (DMEM, 20% FBS, 10% DMSO). All experiments were performed on cells prior to passage 5.
The collagen content of the ligament constructs was determined using a hydroxyproline assay [18]. Briefly, after mechanical testing, sinews were removed from their cement anchors and dried in an oven for 30 min at 110 C. Each sample was then weighed and hydrolyzed in 200 ml of 6N HCl at 130 C for 3 h. The liquid was removed by allowing the HCl to evaporate for 30 min in a fume hood at 130 C. The resulting pellet was resuspended in 200 ml of hydroxyproline buffer. Samples were further diluted 1:8 in hydroxyproline buffer. 150 ml of Chloramine T Solution was added to each sample, vortexed and left at room temperature for 20 min. 150 ml aldehyde-perchloric acid Solution was then added to each tube before the tubes were vortexed and incubated in a heat block at 60 C for 15 min. Following incubation, tubes were left to cool for 10 min and then samples/standards were read at 550 nm on an Epoch Microplate Spectrophotometer (BioTek Instruments Limited, Winooski, VT). Hydroxproline was converted to collagen mass using a factor of 13.8% as reported previously [19].
2.2. Ligament formation
2.7. Statistics
Tissue engineered ligament constructs were engineered as described previously [14] with modification [5]. Briefly, two circular 4 mm diameter brushite anchors were pinned 12 mm apart in a Sylgard coated 35 mm dish. Human ACL cells (2.5 105) were suspended in 1 ml of growth media containing 10U thrombin (Calbiochem), 400 mM aminohexanoic acid, 20 mg aprotinin, and 40 mg of fibrinogen. The solution was rapidly spread over the surface of the dish and the gel was allowed to set for 10 min at 37 C. Once set, the constructs were given 2 ml of growth media every other day until day 14 when all of the constructs were tested. For the volume experiments, all constituents of the gel were increased proportionally. Constructs made with an initial fibrin volume of 1 mL (containing 2.5 105 cells) formed in an average of 7 days, while constructs made with an initial fibrin volume of 1.5 mL (containing 3.75 105 cells) formed in an average of 8 days, and constructs made with an initial fibrin volume of 2 mL (containing 5 105 cells) formed in 10 days.
Data are presented as means SEM. Differences in mean values were compared within groups and significant differences were determined by ANOVA with post hoc TukeyeKramer HSD test using BrightStat (www.brightstat.com). The significance level was set at p < 0.05.
2.3. Design of experiments The concentrations of the growth factors were determined using a Box-Behnken design [17] using Design-ExpertÒ software (Stat-Ease, Inc., Minneapolis, MN). BoxBehnken describes a subset of DOE techniques that uses an incomplete factorial design where three levels of any given factor are tested and the resulting data is analyzed using quadratic response surface plots [17]. In our case, each construct was fed a different combination of ascorbic acid, TGFb1, IGF-1, GDF-7, EGF, and PDGF across a 10-fold range for 2 weeks. The range for ascorbic acid (AA) was 25e250 mM, EGF ranged from 0.5 to 5 ng/ml, and GDF-7, IGF-1, PDGF, and TGFb1 were added at a range from 5 to 50 ng/ml. To determine the variability from sample to sample and provide the statistical basis for selection, 5 grafts received the identical treatment. A total of 54 samples were treated with the various growth factor combinations. Maximal tensile load and collagen content for each of the conditions were measured following 14 days in culture and the significance of each growth factor and predicted optimum media was determined using the statistical and point prediction tools of the Design-ExpertÒ software. The optimal media was then validated in two independent experiments with 6 grafts per group. 2.4. Validation experiments The predicted optimal growth factor combination from the DOE as well as a growth factor mix lacking EGF and PDGF were validated in two independent experiments using different donors and 6 grafts per group. Briefly, the predicted worst (minimal: 5 ng/ml TGFb1, IGF-1, GDF-7, and EGF and 50 ng/ml PDGF), and best DOE media (DOE: 50 ng/ml TGFb1, IGF-1, and GDF-7, 5 ng/ml PDGF, and 0.5 ng/ml EGF) were tested against a media that lacked EGF and PDGF (maximal: 50 ng/ml TGFb1, IGF-1, and GDF-7) since these growth factors have a negative effect on graft
3. Results 3.1. Growth factors and maximal tensile load Treating constructs with differing levels and combinations of ascorbic acid and growth factors resulted in grafts that demonstrated an 11.6-fold range of maximal tensile load from 67 to 770 mN. The variability of the 5 control ligaments was low (17 mN) showing the repeatability of the model. Both TGFb1 and IGF-1 showed a significant positive relationship with MTL (Fig. 1A). The combination of GDF-7 and TGFb1 showed a curvilinear relationship. At low concentrations of TGFb1, GDF-7 had a negative effect on MTL, whereas at high TGFb1 levels GDF-7 had a positive effect on MTL (Fig. 1B). Lastly, both EGF and PDGF showed a negative relationship with MTL, with the effects of EGF being significant (Fig. 1C; p ¼ 0.0001). 3.1.1. Growth factors and collagen content Treating the ligaments with differing levels and combinations of ascorbic acid (AA) and growth factors resulted in grafts that demonstrated a 2.9-fold range of collagen content from 180 to 516 mg. Further analysis showed that ascorbic acid, TGFb1, and IGF-1 all significantly increased collagen content (p < 0.0001; Fig. 2). EGF on the other hand showed a significant negative relationship with collagen (p ¼ 0.0027; data not shown). The combination of GDF-7 and TGFb1 once again showed a curvilinear relationship where at low concentrations of TGFb1 GDF-7 had a negative effect on collagen content, whereas at high TGFb1 levels GDF-7 had a positive effect on collagen content (Fig. 2B). Interestingly, at concentrations of AA higher than 200 mM, there was a progressive decline
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
Fig. 1. 3D response surface plots of growth factor effects on maximal tensile load. The plots show the effect on maximal tensile load of combinations of: A) IGF-1 and TGFb1; B) GDF-7 and TGFb1; and C) EGF and TGFb1. Note that the near linear response to both IGF-1 and TGFb1 indicates that the effects of the growth factors are additive while the curvilinear response to GDF-7 indicates a much more complex relationship between these two growth factors. Additionally the linear negative relationship between EGF and TGFb1 indicates that EGF competitively inhibits TGFb1 function. All growth factor values are ng/ml. All of these relationships are statistically significant.
Fig. 2. 3D response surface plots of growth factor effects on the collagen content of grafts. The plots show the effect on collagen content of: A) IGF-1 and TGFb1; B) GDF-7 and TGFb1; and C) IGF-1 and ascorbic acid (AA). Note once again that the linear response to IGF-1 and TGFb1 showing that both agents have independent positive effects on collagen synthesis, whereas the relationship between GDF-7 and TGFb1 and IGF-1 and AA are more complex. Growth factors were used at the ng/ml level. All of these relationships are statistically significant. 6357
6358
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
in collagen content indicating that too much ascorbic acid can be detrimental for collagen deposition (Fig. 2C). 4. Validation of the growth factor DOE In order to validate the optimized growth factor mix, six grafts were engineered using the predicted minimal growth factor mix (minimal: 5 ng/ml TGFb1, IGF-1, GDF-7, and EGF, 50 ng/ml PDGF, and 200 mM ascorbic acidAA), the DOE predicted best mix (DOE: 50 ng/ml TGFb1, IGF-1, and GDF-7, 5 ng/ml PDGF, 0.5 ng/ml EGF, and 200 mM AA), and a mix lacking EGF and PDGF (maximal: 50 ng/ml TGFb1, IGF-1, and GDF-7, and 200 mM AA). As predicted from the DOE, ligaments grown in the minimal growth factor mix showed a 4.5- lower MTL and a 4.1-fold lower collagen content then ligaments grown in the optimized DOE media (Fig. 3). Further, removing the EGF and PDGF from the mix (maximal media) resulted in a significantly greater increase in MTL (5.7-fold) and collagen content (5.2-fold). 4.1. Construct volume and ligament strength Having improved graft mechanics 5.7-fold by altering the growth factor environment, we next sought to determine the effect of increasing the construct volume while maintaining the fibrin:cell ratio. Increasing the volume of fibrin from 1 ml (2.5 105 cells) to 1.5 ml (3.75 105 cells) or 2 ml (5 105 cells) resulted in a stepwise increase in both MTL and collagen content (Fig. 4). Further, combining the optimized growth factor mix with the greater volume increased MTL 77% (Fig. 5) and tended to increase the collagen content of the grafts. 5. Discussion Using a design of experiments multivariable approach, we have determined a combination of growth factors that increases the mechanical strength (5.7-fold) and collagen content (5.2fold) of fibrin-based 3D engineered ligaments. The multivariable approach identified significant positive effects of IGF-1 and TGFb1, negative effects of EGF, and a significant interaction between GDF-7 and TGFb1. This interaction effect meant that the relationship between GDF-7 levels and MTL and collagen content of the grafts was completely reversed depending on the levels of TGFb1 in the media. Combining the optimized growth factor mix with a greater volume of fibrin gel resulted in a further 77%
increase in MTL, indicating that the technique is scalable and will continue to increase in strength as the size of the construct is increased. When designing the DOE, we used MTL and collagen content as the selection parameters since we felt that the absolute tensile strength (MTL) and amount of collagen of the grafts is more important in designing a functional ligament than modulus, ultimate tensile strength, or the percent collagen within the graft. This decision was made with the understanding that interventions that decreased the cross-sectional area of a graft could dramatically increase the modulus, ultimate tensile strength, and percent collagen without improving the absolute strength of the grafts. This is best seen by the fact that increasing the volume of the constructs from 1 ml to 2 ml resulted in a 77% increase in MTL without altering either modulus or ultimate tensile strength due to the increase in the cross-sectional area of the graft. However, it is important to note that while there was a 79% difference in the cross-sectional area of the grafts in the original DOE, the MTL showed a range of 11.6-fold. Regardless, we feel that if the ultimate goal is to use the engineered ligaments as a clinical source of graft material, it is the absolute strength of the graft that will be most important to withstand initial implantation. With that in mind, it is important to note that the largest MTL recorded for these grafts was 2.35N corresponding to an ultimate tensile stress of 1.7 MPa and a tangent modulus of 5.16 MPa. These values are significantly less than the native human ACL (UTS ¼ 55 MPa; tangent modulus ¼ 200 MPa [20];). However, it is important to note that all of the constructs failed at the boneeligament interface during the tensile tests so the measured MTL is likely an underestimation of the true tissue mechanics. This suggests that improving the interface between the soft tissue and the brushite bone cement will be key to further development of this technology. TGFb signaling is known to play a critical role in the development of ligaments [8], fibrosis [21], and collagen synthesis [5,22]. TGFb is thought to mediate these effects in tendons and ligaments by activating the early growth response factors (EGR)1 and 2 [23]. EGR1 and EGR2 are transcription factors that bind to the tendon specific elements within the Col1a1 promoter [24] and together with scleraxis and nuclear factor of activated T-cells (NFAT) increase collagen expression [25]. In the absence of EGR1/2 there is a significant decrease in the collagen content of tendons and ligaments [24], suggesting that the increase in collagen synthesis in our engineered ligaments is due to the regulation of ERG1/2 through the addition of TGFb.
Fig. 3. Optimized media improves maximal tensile load and collagen content of engineered ligaments. A) The maximal tensile load (MTL) and B) collagen content of engineered ligament grafts was determined following 14 days of treatment with either the minimally effective media or best media (DOE) as determined by the DOE. The maximal group was fed the DOE media without any EGF and PDGF. * indicates significantly higher than the minimal group and y indicates significantly greater than the DOE group (p < 0.05). Results are representative of 2 independent trials and presented as mean S.E.M. of n ¼ 6 for all groups.
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
6359
Fig. 4. Increasing fibrin volume and cell number increases maximal tensile load and collagen content. A) The maximal tensile load (MTL) and B) collagen content of engineered ligament grafts was determined 10 days after formation for fibrin gels of either 1 mL (2.5 105 cells), 1.5 mL (3.75 105 cells), or 2 mL (5 105 cells). * indicates significantly higher than 1 ml group and y indicates significantly higher than the 1.5 ml group (p < 0.05). Results are representative of 2 independent trials and presented as mean S.E.M. of n ¼ 6 for all groups.
The molecular mechanism underlying the effects of IGF-1 is less well established. Doessing et al. [26] have demonstrated that supplementing healthy volunteers with recombinant human growth hormone results in an increase in IGF-1 mRNA within the patellar tendon and this local increase in IGF-1 correlated with the resulting rise in Col1a1 expression. Furthermore, this same group has shown that exercise resulted in a greater than 5-fold increase in IGF-1 protein within the peritendinous space [27]. This increase in IGF-1 was associated with a 5.7-fold increase in markers of collagen synthesis (COOH-terminal telopeptide of type 1 collagen), indicating that IGF-1 may be involved in the anabolic response of tendons to loading [27]. In agreement with a role of IGF-1 in collagen turnover within ligaments, Sciore et al. [28] showed that IGF-1 mRNA levels within a scarred ligament are 5times higher following three weeks of repair than in an uninjured ligament. Lastly, in a recent study Hansen et al. [29] showed that injections of IGF-1 into the patellar tendon resulted in a 24% increase in the fractional synthetic response of collagen. In the current study, IGF-1 had a positive independent effect on MTL and collagen content and was additive with TGFb1, suggesting that the two growth factors act independently to control collagen synthesis. The negative effects of EGF and PDGF on engineered ligament MTL and collagen content was surprising given that a previous in vivo study had shown that PDGF improved collagen synthesis
and repair in skin wounds [30] and EGF has been shown to increase fibroblast proliferation and collagen synthesis [11]. However, the data from the current study are quite clear. EGF had a significant negative effect on both MTL (p < 0.0001) and collagen content (p ¼ 0.0027). Furthermore, completely removing both EGF and PDGF had a significant positive effect on both MTL and collagen, suggesting that even small amounts of these growth factors impair engineered ligament function. Lastly, the Box-Behnken analysis demonstrated that in human ACL cells AA had a positive effect on collagen content up to approximately 200 mM. Concentrations greater than 200 mM AA tended to decrease the collagen content of the grafts. The positive effect of AA is consistent with its role as an essential co-factor for prolyl-4-hydroxylase [31]. The absence of AA leads to inactivation of prolyl-4-hydroxylase and retention of procollagen within the endoplasmic reticulum [32]. In primary tendon cells, AA increases collagen synthesis [33,34], and this has been seen in many other cell types as well [35e38]. There are reports of a plateau in collagen synthesis with doses of AA over 20 mM [39]; we have not found other reports of a negative effect of high concentrations of ascorbic acid on collagen synthesis and have no ready explanation for this observation. High levels of AA cause a significant decrease in construct size with chick tendon fibroblasts [40], however in the current work the effect was only seen in collagen content and not construct diameter.
Fig. 5. Combining greater volume and optimal media results in greater engineered ligament MTL. A) The maximal tensile load (MTL) and B) collagen content of engineered ligament grafts was determined 10 days after formation for fibrin gels of either 1 ml (2.5 105 cells) or 2 ml (5 105 cells) grown in maximal media determined in Fig. 3. * indicates significantly higher than 1 ml group (p < 0.05). Results are representative of 2 independent trials and presented as mean S.E.M. of n ¼ 6 for all groups.
6360
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361
Combining the optimized growth factor mix with a greater volume of fibrin and cells resulted in a significant increase in MTL and a trend towards increased collagen content. This is significant since it shows that the ligaments are not currently at their maximal viable radius. For engineered tissues, the maximal viable radius is the size at which metabolic failure (necrosis) begins to occur at the core of the tissue and function is reduced [41]. For metabolically active tissues such as cardiac and skeletal muscle this radius is w50 mm [42] and w150 mm [43], respectively. For ligaments this value appears to be significantly higher, which is not surprising given the avascular nature of ligaments in vivo. The fact that human ligaments can be engineered with greater cross-sectional areas may help make the transition to the clinic more realistic than it is for heart or skeletal muscle. 6. Conclusions Using a multivariable approach designed to identify interactions, we have determined that increasing levels of TGFb1 and IGF-1 are beneficial for engineered ligament function. Further, at high levels of TGFb1, GDF-7 shows a positive effect on both ligament function and collagen content. This, together with removal of EGF and PDGF and an increase in fibrin and cell volume, has resulted in human ligament grafts with an MTL of up to 2.35N and this corresponds to an UTS of 1.7 MPa and a tangent modulus of 5.17 MPa; approximately 1/40 that of the native ACL. Acknowledgments This work was supported in part by a University of California Davis Health System, Vision Grant (54885) and by the E. Baar Memorial Research Trust (KB). Further support came from a project grant from the Grayson-Jockey Club Research Foundation, Inc. (MV). References [1] Archer CW, Dowthwaite GP, Francis-West P. Development of synovial joints. Birth Defects Res C Embryo Today 2003;69:144e55. [2] Soeda T, Deng JM, de Crombrugghe B, Behringer RR, Nakamura T, Akiyama H. Sox9-expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons. Genesis 2010;48: 635e44. [3] Edwards JC, Wilkinson LS, Jones HM, Soothill P, Henderson KJ, Worrall JG, et al. The formation of human synovial joint cavities: a possible role for hyaluronan and CD44 in altered interzone cohesion. J Anatomy 1994;185(Pt 2):355e67. [4] Bastow ER, Lamb KJ, Lewthwaite JC, Osborne AC, Kavanagh E, WheelerJones CP, et al. Selective activation of the MEK-ERK pathway is regulated by mechanical stimuli in forming joints and promotes pericellular matrix formation. J Biological Chemistry 2005;280:11749e58. [5] Paxton JZ, Grover LM, Baar K. Engineering an in vitro model of a functional ligament from bone to bone. Tissue Eng Part A 2010;16:3515e25. [6] Francis-West PH, Abdelfattah A, Chen P, Allen C, Parish J, Ladher R, et al. Mechanisms of GDF-5 action during skeletal development. Development 1999;126:1305e15. [7] Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, et al. Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family. J Clin Invest 1997;100: 321e30. [8] Pryce BA, Watson SS, Murchison ND, Staverosky JA, Dunker N, Schweitzer R. Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation. Development 2009;136:1351e61. [9] Dahlgren LA, Nixon AJ, Brower-Toland BD. Effects of beta-aminopropionitrile on equine tendon metabolism in vitro and on effects of insulin-like growth factor-I on matrix production by equine tenocytes. Am J Vet Res 2001;62: 1557e62. [10] Steinert AF, Weber M, Kunz M, Palmer GD, Noth U, Evans CH, et al. In situ IGF1 gene delivery to cells emerging from the injured anterior cruciate ligament. Biomaterials 2008;29:904e16. [11] Throm AM, Liu WC, Lock CH, Billiar KL. Development of a cell-derived matrix: effects of epidermal growth factor in chemically defined culture. J Biomed Mater Res Part A 2010;92:533e41.
[12] Wang XT, Liu PY, Tang JB. Tendon healing in vitro: genetic modification of tenocytes with exogenous PDGF gene and promotion of collagen gene expression. J Hand Surg Am 2004;29:884e90. [13] Bayer ML, Yeung CY, Kadler KE, Qvortrup K, Baar K, Svensson RB, et al. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension. Biomaterials 2010;31: 4889e97. [14] Kapacee Z, Richardson SH, Lu Y, Starborg T, Holmes DF, Baar K, et al. Tension is required for fibripositor formation. Matrix Biol 2008;27:371e5. [15] Paxton JZ, Donnelly K, Keatch RP, Baar K, Grover LM. Factors affecting the longevity and strength in an in vitro model of the bone-ligament interface. Ann Biomed Eng 2010;38:2155e66. [16] Paxton JZ, Donnelly K, Keatch RP, Baar K. Engineering the bone-ligament interface using polyethylene glycol diacrylate incorporated with hydroxyapatite. Tissue Eng Part A 2009;15:1201e9. [17] Box GEP, Behnken DW. Some new three level designs for the study of quantitative variables. Technometrics 1960;2:455e75. [18] Woessner Jr JF. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961;93:440e7. [19] Arruda EM, Calve S, Dennis RG, Mundy K, Baar K. Regional variation of tibialis anterior tendon mechanics is lost following denervation. J Appl Physiol 2006; 101:1113e7. [20] Woo SL, Hollis JM, Adams DJ, Lyon RM, Takai S. Tensile properties of the human femur-anterior cruciate ligament-tibia complex. The effects of specimen age and orientation. Am J Sports Med 1991;19:217e25. [21] Sarrazy V, Billet F, Micallef L, Coulomb B, Desmouliere A. Mechanisms of pathological scarring: role of myofibroblasts and current developments. Wound Repair Regen 2011;19(Suppl. 1):s10e5. [22] Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M, Varga J. Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad 3. J Invest Dermatol 1999;112:49e57. [23] Chen SJ, Ning H, Ishida W, Sodin-Semrl S, Takagawa S, Mori Y, et al. The earlyimmediate gene EGR-1 is induced by transforming growth factor-beta and mediates stimulation of collagen gene expression. J Biol Chem 2006;281: 21183e97. [24] Lejard V, Blais F, Guerquin MJ, Bonnet A, Bonnin MA, Havis E, et al. EGR1 and EGR2 involvement in vertebrate tendon differentiation. J Biol Chem 2011;286: 5855e67. [25] Lejard V, Brideau G, Blais F, Salingcarnboriboon R, Wagner G, Roehrl MH, et al. Scleraxis and NFATc regulate the expression of the pro-alpha1(I) collagen gene in tendon fibroblasts. J Biol Chem 2007;282:17665e75. [26] Doessing S, Heinemeier KM, Holm L, Mackey AL, Schjerling P, Rennie M, et al. Growth hormone stimulates the collagen synthesis in human tendon and skeletal muscle without affecting myofibrillar protein synthesis. J Physiol 2010;588:341e51. [27] Olesen JL, Heinemeier KM, Gemmer C, Kjaer M, Flyvbjerg A, Langberg H. Exercise-dependent IGF-I, IGFBPs, and type I collagen changes in human peritendinous connective tissue determined by microdialysis. J Appl Physiol 2007;102:214e20. [28] Sciore P, Boykiw R, Hart DA. Semiquantitative reverse transcriptionpolymerase chain reaction analysis of mRNA for growth factors and growth factor receptors from normal and healing rabbit medial collateral ligament tissue. J Orthop Res 1998;16:429e37. [29] Hansen M, Boesen A, Holm L, Flyvbjerg A, Langberg H, Kjaer M. Local administration of insulin-like growth factor-I (IGF-I) stimulates tendon collagen synthesis in humans. Scand J Med Sci Sports; 2012. [30] Lynch SE, Colvin RB, Antoniades HN. Growth factors in wound healing. Single and synergistic effects on partial thickness porcine skin wounds. J Clin Invest 1989;84:640e6. [31] Kadler KE, Baldock C, Bella J, Boot-Handford RP. Collagens at a glance. J Cell Sci 2007;120:1955e8. [32] Peterkofsky B. Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy. Am J Clin Nutr 1991;54:1135Se40S. [33] Schwarz RI, Farson DA, Soo WJ, Bissell MJ. Primary avian tendon cells in culture. An improved system for understanding malignant transformation. J Cell Biol 1978;79:672e9. [34] Schwarz RI, Bissell MJ. Dependence of the differentiated state on the cellular environment: modulation of collagen synthesis in tendon cells. Proc Natl Acad Sci USA 1977;74:4453e7. [35] Chu AJ, Prasad JK. Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling. J Am Coll Surg 1999; 188:271e80. [36] Ganta DR, McCarthy MB, Gronowicz GA. Ascorbic acid alters collagen integrins in bone culture. Endocrinology 1997;138:3606e12. [37] Graham MF, Willey A, Adams J, Yager D, Diegelmann RF. Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells. J Cell Physiol 1995;162:225e33. [38] Sawaguchi S, Yue BY, Chang IL, Wong F, Higginbotham EJ. Ascorbic acid modulates collagen type I gene expression by cells from an eye tissueetrabecular meshwork. Cell Mol Biol 1992;38:587e604. [39] Pinnel SR, Murad S, Darr D. Induction of collagen synthesis by ascorbic acid. A possible mechanism. Arch Dermatol 1987;123:1684e6.
P. Hagerty et al. / Biomaterials 33 (2012) 6355e6361 [40] Paxton JZ, Wudebwe U, Wang A, Woods D, Grover LM. Monitoring sinew contraction during formation of tissue-engineered fibrin-based ligament constructs. Tissue Eng Part A; 2012. [41] Dennis RG, Smith B, Philp A, Donnelly K, Baar K. Bioreactors for guiding muscle tissue growth and development. Adv Biochem Eng Biotechnol 2009; 112:39e79.
6361
[42] Baar K, Birla R, Boluyt MO, Borschel GH, Arruda EM, Dennis RG. Self-organization of rat cardiac cells into contractile 3-D cardiac tissue. Faseb J 2005;19: 275e7. [43] Dennis RG, Kosnik 2nd PE, Gilbert ME, Faulkner JA. Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines. Am J Physiol Cell Physiol 2001;280:C288e95.