Intrinsic Fibroblast-Mediated Remodeling of Damaged Collagenous Matrices in Vivo

Matrix Biology 23 (2005) 543–555 www.elsevier.com/locate/matbio

Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo

Paolo P. Provenzanoa,b,*, Adriana L. Alejandro-Osoriob,c, Wilmot B. Valhmub, Kristina T. Jensena,b, Ray Vanderby Jr.a,b

aDepartment of Biomedical Engineering, University of Wisconsin, Madison, WI, United States bDepartment of Orthopedics and Rehabilitation, University of Wisconsin, Madison, WI, United States cDepartment of Biomolecular Chemistry, University of Wisconsin, Madison, WI, United States

Received 1 July 2004; received in revised form 22 September 2004; accepted 23 September 2004

Abstract

Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-a and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior. D 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Keywords: Subfailure damage; Sprain; Scar tissue formation; Inflammation; Medial collateral ligament; Real-time quantitative polymerase chain reaction (real- time QPCR)

1. Introduction progressive joint degeneration. Although numerous studies have examined Grade III1 ligament injuries, further study is Ligament sprains occur more often than complete tissue rupture and can be chronically debilitating, frequently 1 Grades I, II, and III tissue injuries are described as follows (Andriachi causing persistent joint instability, prolonged pain, and et al., 1987; Scuderi and Scott, 2001): Grade I sprains are mild stretches with no discontinuity of the ligament and no clinically detectable increase in joint laxity. Grade II sprains are moderate stretches in which some * Corresponding author. Present address: Department of Pharmaco- collagen fibers are torn. Enough fibers remain intact so that the ligament has logy, 3619 MSC, 1300 University Avenue, Madison, WI 53706, United not completely failed, but detectable abnormal laxity is present at the joint. States. Tel.: +1 608 265 5094. Grade III sprains are severe and consist of a complete or nearly complete E-mail address: [email protected] (P.P. Provenzano). ligament disruption and result in significant joint laxity.

0945-053X/$ - see front matter D 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2004.09.008 544 P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555

glycosaminoglycan content within the scar tissue out to 40 weeks post-injury (Frank et al., 1983). More specific analysis of collagen reveals a strong increase in type III collagen (Frank et al., 1983; Williams et al., 1984), which constitutes the majority of newly synthesized collagen in scar tissue (Williams et al., 1984). These results are in agreement with gene expression analysis of Grade III injured ligaments showing large increases in type III collagen mRNA, with increases in type III collagen expression exceeding type I expression increases 3 weeks post-injury (Boykiw et al., 1998). Hence, Grade III injuries of collagenous extracellular matrices and subsequent scar Fig. 1. Average stress–strain curves for sham control and subfailure formation have received considerable study. However, less stretched tissue (meanFS.D.). The averaged data were fit with a micro- is known about the biology of healing after a Grade II injury structural model (Hurschler et al., 1997) to illustrate the shape of the stress– and the effects of subfailure tissue deformations. It is known strain curve. Note that matrix damage due to subfailure stretching increases that after a Grade II ligament injury, extracellular matrix the strain-stiffening region of the stress–strain curve and results in decreased elastic modulus, indicating a significant change in the material damage appears to be distributed and that a localized scar properties of the collagenous matrix. formation is not detected macro- or microscopically (Laws and Walton, 1988; Provenzano et al., 2002a,b). This information, combined with reports of a potential intrinsic, required to describe the mechanics and biology of the non-inflammatory, fibroblastic-mediated healing response in collagenous extracellular matrix after a subfailure damage subfailure damaged tissue (Laws and Walton, 1988), (sprain) injury. indicate further study of subfailure tissue deformation and A prospective study of patients with partial tears of the anterior cruciate ligament (ACL) revealed clinically unsta- ble knees in over 50% of the patients after a mean follow-up of 17 months (Fruensgaard and Johannsen, 1989). Such instabilities are likely caused by increased deformations within the physiologic strain-stiffening region of the load- deformation (Panjabi et al., 1996) and stress–strain curves (Provenzano et al., 2002b), and laxity (Provenzano et al., 2002a) of ligaments after a subfailure injury. In collateral ligaments, the initial increase in tissue laxity is likely caused by collagen fiber damage and/or rupture (Provenzano et al., 2002a; Yahia et al., 1990) to fibers that are first recruited during tissue loading (Viidik, 1972). In addition, the initial injury to the tissue results in substantial cellular damage (Provenzano et al., 2002b) followed by apoptosis (Proven- zano et al., 2002c). Hence, it is reasonable to expect a substantial healing response to remove cell and tissue debris and repair the damaged extracellular matrix. Yet this repair is imperfect. Although tissue strength increases with healing time after a subfailure injury, tissue laxity persists (Proven- zano et al., 2002a). An in vivo study of rat ligament healing showed a significant increase in tissue ultimate stress (to 80% of control) 2 weeks after a subfailure injury; yet there was no significant improvement in the tissue laxity over 2 weeks (Provenzano et al., 2002a). Further studies of the causal mechanisms of tissue damage (and the resulting laxity) as well as the in vivo healing response after subfailure injury are required. After a Grade III injury (complete rupture) to ligament, a region of scar tissue is deposited to provide a provisional Fig. 2. Ultimate stress (A) and elastic modulus (B) values after a subfailure matrix for connecting the two residual ends of the ruptured ligament injury are significantly decreased ( p=0.03 and p=0.026, tissue (Frank et al., 1983; Jack, 1950). Biochemical analysis respectively; meanFS.E.M.) following subfailure damage injury to the of this scar tissue reveals changes in total collagen and tissue indicating significant and consistent tissue damage. P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555 545

Fig. 3. Introduction of a subfailure damage injury does not produce an increased macrophage response within the ligament tissue (400). In both control and damaged ligament tissue, the majority of the tissue area was void of macrophages (a, b). However, both tissues also possess very few regions in which macrophages were present (c, d), with no difference between control and injured tissues. Alternatively, the scar tissue surrounding the ligament (resulting from the surgical insult) is strongly positive for macrophage infiltration in both control and damaged tissue (e, f: boundary between ligament (L) tissue and surrounding scar tissue (S); g, h: macrophages in surgically induced scar tissue surrounding the ligament). Furthermore, macrophages are present near the bone–ligament insertion (i, j). 546 P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555

Fig. 4. Introduction of a subfailure damage injury does not produce an increased neutrophil response within the ligament tissue (400). In both control and damaged ligament tissue, the majority of the tissue area was void of neutrophils (a, b). However, both tissues also possessed very few regions in which neutrophils were present (c, d), with no difference between control and injured tissues. Alternatively, the scar tissue surrounding the ligament (resulting from the surgical insult) is strongly positive for neutrophil infiltration in both control and damaged tissue (e, f: boundary between ligament (L) tissue and surrounding scar tissue (S); g, h: neutrophils in surgically induced scar tissue surrounding the ligament). Furthermore, neutrophils are present near the bone–ligament insertion (i, j). P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555 547 injury is necessary and may provide unique insight into ligament (Fig. 4c,d). Similar to macrophages, neutrophils fibroblast behavior and the fibroblast–matrix interaction. were present in the (surgical insult-induced) scar tissue The purpose of this study, therefore, was to determine surrounding the ligament (Fig. 4e–h), and near the bone– whether there is an up-regulated inflammatory response ligament insertion (Fig. 4i–j). after subfailure injury or whether the remodeling process is Induction of a subfailure (Grade II) ligament injury an intrinsic fibroblast-mediated response. Further, gene resulted in substantial changes in gene expression. Analysis expression patterns for extracellular matrix proteins and of GAPDH mRNA levels did not show any significant matrix-degrading proteinases were examined following a differences between control and injured tissues. Collagen I subfailure injury to elucidate the cellular response to tissue expression (Fig. 5A) showed a trend of initial suppression injury. To address these questions, immunohistochemistry after 1 day, followed by an increasing trend at 3 days. By was utilized to determine whether macrophages and/or day 7, collagen I gene expression was significantly neutrophils were infiltrating the connective tissue after increased over the sham side MCL ( p=0.004) with a 48- injury. Real-time quantitative polymerase chain reaction fold increase in mRNA expression in healing tissues. Type (QPCR) analysis was employed to determine the expression III collagen gene expression (Fig. 5B) also revealed a levels of collagen, proteoglycan, and proteinase mRNAs at suppressed trend at 1 day, which by 3 days was virtually 1, 3, and 7 days post-subfailure damage injury in the rat identical to sham values. After 7 days, type III collagen gene medial collateral ligament. In addition, typical mRNA expression was significantly increased over the sham side markers of inflammation were used to lend additional MCL ( p=0.026) with a 42-fold increase in mRNA insight into the intrinsic remodeling hypothesis. expression in healing tissues. Hence, type I collagen revealed a larger fold increase than type III collagen as well as significantly larger mRNA expression than type III 2. Results ( p=0.014) after 7 days. Furthermore, type I collagen expression in damaged tissue was ~20-fold greater than Mechanical analysis following the stretch-induced sub- type III expression, while in sham tissues this ratio was ~17, failure tissue damage indicated a substantial and consistent indicating that the increases in both collagen I and III may injury. Dramatic changes in the stress–strain behavior of the tissues were detected (Fig. 1) and ultimate stress ( p=0.03) and elastic modulus ( p=0.026) values of the subfailure tissues were significantly decreased to approximately 50% of the control value (Fig. 2). Strain at failure was not significantly different. However, tissue length and laxity values were significantly increased. Initial tissue laxity was calculated from ligament length values to be 12.9%. Moreover, the strain at the transition between the nonlinear strain-stiffening region (dtoe-regionT) and the dlinearT region of the stress–strain curve was significantly ( p=0.049) increased in damaged tissues (eTRANS=0.015F0.008 for control tissues and 0.037F0.007 for damaged tissues). Immunohistochemical evaluation of damaged ligaments indicated that injury did not induce an inflammatory response within the tissue. Macrophage and neutrophil levels and localization were not different between sham and damaged tissues (Figs. 3 and 4). Both tissues possessed a very small number of regions in which macrophages were present (Fig. 3c,d) although the majority of the tissue area was void of macrophages (Fig. 3a,b) with one damaged tissue completely negative for macrophage staining. In contrast, macrophages were primarily localized to tissue surrounding the MCLs where tissue was injured due to the surgical procedure (Fig. 3e–h) and near the bone–ligament insertion (Fig. 3i–j). Furthermore, neutrophil localization in control and damaged tissue had a similar pattern to that seen Fig. 5. Type I (A) and III (B) collagen gene expression following a for macrophages (Fig. 4). In both control and damaged subfailure (Grade II) MCL injury. Both collagen I and III expression in subfailure damaged ligaments show a trend of initial suppression after 1 ligaments, the majority of the tissue area was void of day, followed by an increasing trend at 3 days, and a significant increase by neutrophils (Fig. 4a,b), yet both groups had very few 7 days ( p=0.004 and p=0.026, respectively). Note the difference in scale regions in which neutrophils were present within the between A and B. 548 P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555

Results herein point toward an intrinsic process of collagenous ECM remodeling that relies more heavily on replacing type I collagen directly than producing a predomi- nantly type III collagen provisional matrix (scar) to be later replaced by type I collagen. Furthermore, proteoglycan mRNA levels indicate significant increases in conjunction with increased collagen expression. Since proteoglycans such as decorin, lumican, and fibromodulin are associated with collagen fibrillogenesis (Birk et al., 1995; Chakravarti et al., 1998; Ezura et al., 2000; Jepsen et al., 2002; Svensson et al., 1999; Vogel and Trotter, 1987), as well as cell behavior (see Kresse and Schonherr, 2001), it appears likely that they are, at least in part, up-regulated to help modulate the fibrillogenesis of the newly synthesized collagen. Fig. 6. Proteoglycan expression following a subfailure ligament stretch During embryonic tendon development in vivo, type I reveals significantly (*) increased levels of decorin, fibromodulin, lumican, and versican, but not biglycan, mRNA in stretched ligaments 7 days post- collagen fibrillogenesis is a multi-step fibroblast-mediated injury. Sham=gray bars; damaged=black bars; F=fibromodulin; D=decorin; process (Birk and Trelstad, 1984, 1986; Birk et al., 1989) L=lumican; V=versican; B=biglycan. during which fibril segments polymerize within deep channels in the fibroblast surface and form fibril bundles be the result of an acceleration of the normal remodeling within a peripheral extracytoplasmic compartment of the response that produces much more collagen I. Analysis of fibroblast (Birk and Trelstad, 1986). These collagen fibril proteoglycan mRNA levels (Fig. 6) indicated significant segments then form intermediate structures that assemble increases in fibromodulin ( p=0.017), decorin ( p=0.013), into collagen fibrils and undergo post-depositional growth lumican ( p=0.006), and versican ( p=0.030) 7 days post- during embryonic development (Birk et al., 1989, 1995, injury, corresponding to increases in collagen expression. 1997), a process that is, in part, mediated by small leucine- Biglycan mRNA levels were low with no significant rich proteoglycans. For instance, studies have shown that changes at any time point. In addition, proteinase expression decorin plays a role in regulating collagen fibril organization (Fig. 7) displayed a trend of initial suppression at 1 day, with and maturation by delaying fibril assembly (Vogel and significantly increased levels of cathepsin K ( p=0.048) and Trotter, 1987), inhibiting fibril maturation (Birk et al., 1995; L(p=0.037), but not MMP-13 ( p=0.19) or MMP-2 Scott et al., 1981), and regulating fibril tip-to-tip fusion ( p=0.88), at 7 days. Matrix metalloproteinase-2 and -13 (Graham et al., 2000). Furthermore, both fibromodulin and expression were not significantly different at any time point, lumican have also been implicated in regulating collagen while TIMP-1 mRNA levels were significantly increased fibrillogenesis. When collagen fibrils from mice deficient in after 3 ( p=0.046) and 7 ( p=0.024) days. Tartrate-resistant fibromodulin and/or lumican are examined, they exhibit acid phosphatase levels were low with no significant abnormal development in terms of diameter and shape, changes at any time point. TNF-a mRNA levels were lower resulting in abnormal tissue function (Chakravarti et al., in damaged tissue when compared to sham tissues: 1998; Ezura et al., 2000; Jepsen et al., 2002; Svensson et al., 57.7F15.0%, 57.4F22.1%, and 49.8F21.0% of control 1999). Therefore, proteoglycans, such as decorin, fibromo- values for 1, 3, and 7 days, respectively, with no statistically dulin, and lumican, strongly regulate multiple aspects of significant between any time point.

3. Discussion

Mechanical evaluation of ligaments after subfailure injury revealed a substantial strength deficit and increased deformations within the strain-stiffening toe-region of the load-deformation, consistent with previous reports (Proven- zano et al., 2002a,b) and indicating a significant tissue injury. This early portion of the stress–strain curve is physiologically relevant. Changes in mechanical behavior in this region indicate a longer functional length prior to load bearing, which in turn can result in joint laxity and abnormal Fig. 7. Proteinase expression following a subfailure ligament stretch reveals significantly (*) increased cathepsin K and L, but not MMP-13, -2, or motion leading to degenerative joint disease. As such, tissue TRAP in stretched ligaments 7 days post-injury. Sham=gray bars; remodeling following this type of injury and the biologic damaged=black bars; 2=MMP-2; 13=MMP-13; CK=cathepsin K; CL=ca- attempts to resolve this abnormal behavior require study. thepsin L; T=TRAP. P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555 549 collagen fibrillogenesis during embryonic development, in injured tissues. Hence, these data support the conclusions while versican can promote fibroblast proliferation (Zhang of Laws and Walton (1988) that Grade II injuries in et al., 1998). Consequently, the up-regulation of these ligaments may remodel intrinsically and differ from proteoglycans in concurrence with type I collagen after complete tissue tears that involve a coagulation/inflamma- subfailure injury implies fibrillogenesis is occurring to tion/provisional-matrix response. remodel the tissue and restore functional mechanical After subfailure tissue damage, one would intuitively properties. expect an up-regulation of debris-clearing proteinases to In addition to changes in expression levels of extrac- precede collagen up-regulation. The lack of inflammatory ellular matrix molecules, cysteine proteinase mRNA levels markers, however, suggests anabolic and catabolic events were also up-regulated following subfailure injury. While are more intrinsically regulated and occur simultaneously. these mRNA levels may only be reflective of protease As such, it is possible that matrix secretion and resorption activity, this level of regulation may indicate increases in a are taking place in different locations within the tissue or non-MMP protease family to clear damaged tissue and help they are acting in synergy to remove damaged tissue and facilitate tissue remodeling For instance, frequently three replace it. Although further analyses, such as in situ main classes of enzymes are strongly implicated in matrix hybridization and histological protein localization, are (particularly collagen) degradation: the MMP family (e.g. required to lend insight into the relationship between the MMP-2 and -13), the cysteine proteinase family (e.g. anabolic and catabolic processes, current information does cathepsin K and L), and the acid phosphatase family (e.g. raise interesting questions about the mechanisms of tissue TRAP). Traditionally, the matrix metalloproteinase family laxity in light of previously reported microstructural data has received the most study and MMP-2 and -13 have been (Provenzano et al., 2002a,b). For instance, examination of implicated in matrix remodeling and increased expression collagen microstructure with scanning electron microscopy during wound healing (Creemers et al., 1998; Hellio Le after subfailure damage revealed ruptured collagen fibers Graverand et al., 2000). However, the cysteine proteinase and fibrils, and fiber and fibril dretractionT appeared to have family is emerging as an equally important class of enzymes taken place (Fig. 8). If proteinases are to clear debris and (Chapman et al., 1997). Cathepsin K is a member of the remove damaged tissue after injury, it is unlikely that the cysteine proteinase family that possesses strong collageno- initial gap between ruptured fibers and/or fibrils would, at lytic activity, at pH range of 4.0–7.0, which exceeds the that point, decrease. As such, if the tissue gap is present activity of MMP-1, -9, and -13 (Bromme et al., 1996; when new tissue is dfilled inT, the repaired fiber or fibril Garnero et al., 1998; Hou et al., 2001; Li et al., 2000). would be longer than its pre-injury length and as such more Moreover, in addition to known intracellular lysosomal roles lax. It is unclear whether long-term remodeling would for cathepsin K and L (Kirschke et al., 1998), cathepsin L is resolve this issue or not, although examination of remodel- known to act as a potent extracellular protease (see Kirschke ing ligaments following subfailure injury revealed substan- et al., 1998) while there is increasing evidence of an tial laxity remained 2 weeks post-injury (Provenzano et al., extracellular role for cathepsin K (Parak et al., 2000; Tepel 2002a). Hence, further study is required of long-term and Bromme, 2000) in cell types other than osteoclasts remodeling, the presence of myofibroblasts (which may (Saftig et al., 1998; Xia et al., 1999). Another collagenase pull the fibril/fiber ends together), the location of newly examined in this study, TRAP, has been implicated in synthesized collagen, and the anabolic/catabolic processes collagen degradation in bone (Henthorn et al., 1999; Oddie and their interaction. and Schenk, 2000), identified in fibroblasts (Dwyer et al., Lastly, numerous studies have examined healing after a 2004, Muir et al., 2002), and is associated with macro- complete tissue rupture or transection. However, few studies phages and inflammation (Bune et al., 2001; Lord et al., have examined the biologic response after a subfailure 1990; Schindelmeiser et al., 1989). Interestingly in this injury in which a localized concentration of scar tissue does study, expression levels for the MMPs did not change at any not appear to form. In contrast to Grade III injuries in which time point while cysteine proteinases cathepsin K and L scar tissue forms at the rupture site (Frank et al., 1983), via were both significantly increased 7 days post-injury, mediation from the coagulation system and a largely suggesting a role for these proteinases in ligament tissue extrinsic inflammatory response (Frank et al., 1983; Riches, remodeling after injury. Their specific roles, however, 1996; Williams et al., 1984), Grade II injury appears to be a remain to be elucidated, particularly their potential intra- largely intrinsically mediated response. Therefore, since cellular versus extracellular functions. Furthermore, TRAP inflammation (as a source of growth factors and cytokines) levels did not increase, implying that a significant macro- plays a large role in wound healing by inducing cell phage infiltration did not take place over the time period migration and proliferation and controlling extracellular examined, consistent with immunohistochemical data. This matrix scar formation (Eckes et al., 1996; Riches, 1996), its conclusion is also supported by the constant MMP levels, absence leaves fibroblasts to direct tissue healing. This which typically show rapid increases with inflammation (see difference in extrinsically versus intrinsically dominated Everts et al., 1996) and the relatively constant, but healing response may account for the relatively low ratio of decreased, TNF-a levels (a typical marker for inflammation) type III to type I collagen expression after subfailure injury 550 P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555

Fig. 8. Collagen fiber and fibril damage above the damage threshold. After axial tissue strains were induced in medial collateral ligaments, control tissues (0% strain; A, C, D) possess normal microstructure, while tissues stretched above the defined damaged threshold for rat MCL (~5% or 40–60% of the failure strain) show collagen fiber and fibril damage, disorganized fibril organization, and ruptured fibrils (B, E–H). (A) Control tissue. (B) Tissue stretched to 7.7% strain showing collagen fiber damage. (C, D) Control ligaments displaying normal fibril organization and morphology. (E) Tissue stretched to 7.7% strain revealing a region in which collagen fibril morphology and organization appear normal (the collagen fiber that contains this region appeared intact). (F) Tissue stretched to 6.7% strain showing substantial fibril disorganization (the fiber that contains this region was ruptured indicating a possible retraction of fibrils). (G–H) Tissue stretched to 5.8% strain displaying collagen fibril rupture (the fiber containing this region appeared intact). Hence, shortly after injury some fibers are intact and appear normal, some fibers are completely ruptured and a dgapT exists between ruptured fibers, and additionally some fibers are intact but contain ruptured collagen fibrils that contain a dgapT between ruptured fibril ends. As such, if the tissue gap remains and is present when new tissue dfills inT without substantial contracture, the repaired fiber or fibril would be longer than its pre-injury length and as such it will be more lax. as compared to healing from a complete tear. In a more 1996; Frank et al., 1983; Riches, 1996; Williams et al., typical wound model, type III expression is substantial and 1984). Herein type I collagen, and supporting proteoglycans, precedes type I expression (Boykiw et al., 1998; Eckes et al., appears to be up-regulated at an earlier stage with a more P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555 551 substantial response. Therefore this model could lend unique effects of tissue handling during harvest and to ensure that insight into wound healing, tissue remodeling, and fibroblast potential mechanical differences between control and behavior, and hence additional work is required to differ- stretched tissues were due to matrix damage and not a entiate intrinsic behavior from the more extensively studied viscoelastic effect (Provenzano et al., 2001, 2002b). For extrinsic healing response. mechanical testing, the tissue was pulled to failure as In conclusion, this study demonstrates that subfailure previously described (Provenzano et al., 2002a). Briefly, damage to connective tissue induces intrinsically mediated MCL area was computed from optical measurements and remodeling with increased collagen, proteoglycan, and then the femur–MCL–tibia complex, with optical markers proteinase gene expression 7 days after a significant near the tissue insertion sites for measuring displacement, subfailure stretch was applied to collateral ligament. The was placed into a custom designed tissue bath system apparent harmonization of these events provides compel- inserted into our testing machine. A small preload of 0.1 N ling evidence that between 3 and 7 days, or at 7 days, the was applied to the tissue in order to obtain a uniform zero tissue is undergoing a dramatic remodeling response. In (start) point. In displacement control, the ligaments were addition, the strong increase in type I collagen expression, preconditioned (~1% strain for 10 cycles), allowed to which is significantly greater than type III expression, recover, and pulled to failure at ~10%/s. Strains were supports the concept of remodeling without significant scar calculated from videotape (described below) after displace- deposition. These findings distinguish this injury from ment-controlled tests. Tissue displacement was obtained pathologies such as tendonitis where inflammation is using video dimensional analysis to measure the change known to play a role. Furthermore, this model raises the in distance between markers. The change in distance likely possibility that fibroblasts within additional tissues between optical markers was calculated by analyzing in the body respond in a similar fashion and deposit type I stored digital frames with N.I.H. Image using a custom collagen to repair moderately (i.e. non-clotting and non- macro to calculate the change in x–y coordinate center scarring) damaged tissue instead of depositing a type III (centroid coordinates) of each marker. Force was displayed fibrotic matrix. As such this model can provide unique on the video screen and synchronized with displace- insight into acute tissue damage and offer a potentially ment. From displacement data, strain was calculated as powerful model to examine fibroblast behavior and the change between stretched length and gage length collagen fibrillogenesis. divided by gage length: DL/L0. Stress was calculated as the force divided by the initial area. Stress–strain data were fit with the probabilistic microstructural model of Hursch- 4. Experimental procedures ler et al. (1997) to determine elastic modulus and strain at the transition between the nonlinear strain-stiffening re- 4.1. Animal procedures and experimental design gion and the approximately linear portion of the stress– strain curve (Hurschler et al., 1997; Hurschler and This study was approved by the Institutional Animal Provenzano, 2003), which has functional implications in Use and Care Committee. Thirty-two male Sprague– physiologic loading range (see Appendix A). Parameters Dawley rats were used as an animal model. Under for comparison are tissue length, tissue laxity, strain at the general anesthesia, one randomly selected MCL of each transition between the strain-stiffening and linear regions animal was approached via skin incision without damage of the stress–strain curve, ultimate stress, strain at failure, to other joint structures. The MCL received a subfailure and elastic modulus. Tissue laxity was taken to be the (Grade II) injury in the same manner as previously difference between control ligament length (LC)and described in lateral collateral ligaments (Provenzano et al., stretched ligament length (LS), normalized by the control 2002a). This injury method pulls laterally on the ligament length: [i.e. %Laxity=100(LSLC)/LC]aspreviously with a controlled displacement while restraining the described (Provenzano et al., 2002a). insertion sites. The contralateral MCL received a sham surgery. The incision was closed in a routine manner. 4.3. Immunohistochemistry Animals were euthanized at time zero for mechanical analysis (n=4 rats) to characterize the injury, after 24 h to To test for the possible occurrence of infiltrating test for the presence of infiltrating macrophages and macrophages and neutrophils into the ligaments following neutrophils (n=4 rats), and at 1, 3, or 7 days for gene injury, immunohistochemistry was employed. Tissues were expression analysis (n=8 rats/group). frozen in OCT embedding media and cut into 5-Am frozen sections. Following tissue sectioning, the tissues were 4.2. Mechanical testing thawed to room temperature, fixed for 30 min in formalin and then washed in PBST (1% Tween-20 in PBS). To For mechanical testing, four pairs of contralateral quench endogenous peroxidase activity, tissue sections ligaments were harvested and allowed to recover for 15 were blocked in 3% H2O2 and washed again in PBST. min while remaining hydrated in order to reduce the Tissue sections were then subjected to nonspecific protein 552 P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555 blocking in 5% goat serum in PBS, washed in PBST, and DNase-, RNase-, and protease-free phosphate-buffered incubated with the primary antibody. To detect infiltrating saline to remove blood or other contaminants from the macrophages the mouse anti-rat ED1 (rat CD68) antibody tissues, placed in RNAlater solution (Ambion, Austin, (Serotec, Oxford, UK) was utilized at a 1:50 dilution for 2 TX), and stored at 20 8C. Utilizing methods similar to h at room temperature. To detect neurtrophils, a mouse Reno et al. (1997), which combines the TRIzol method anti-rat antibody (Serotec) to the CD43 antigen was with the column fractionation steps of the RNeasy Total utilized at a 1:100 dilution for 2 h at room temperature. RNA kit (Qiagen, Valencia, CA), total RNA was later After extensive washing in PBST, immunochemical labels isolated. Tissues were pooled in groups of two to ensure were applied using the STAT-Q-DAB kit (Innovex quantifiable RNA. Tissues were placed in a liquid nitrogen Biosciences, San Ramon, CA) with mouse-linked (rat- cooled Braun Mikro-Dismembrator Vessel (B. Braun adsorbed) secondary antibody. Tissue sections were incu- Biotech International, Allentown, PA) and reduced to bated with the secondary linking antibody, labeled with powder. One milliliter of Trizol Reagent (Life Technolo- peroxidase (HRP)-labeled strepavidin, followed by appli- gies, Grand Island, NY) was then added to the homogenate cation of 3,3-diaminobenzidine substrate. The tissue and incubated at room temperature for 5 min. The sections were then washed, dehydrated, cover-slipped, homogenate was then centrifuged to remove insoluble and imaged with light microscopy. Spleen was stained as tissue debris, followed by the addition of 0.2 mL of a positive control. chloroform to the supernate. After the addition of chloroform, the samples were mixed vigorously, incubated for 3 4.4. RNA isolation and reverse transcription min at room temperature, and transferred to a pre-spun 2.0 mL heavy phase-lock gel tube (Brinkman-Eppendorf, For gene expression analysis, eight contralateral pairs of Westbury, NY). The organic and aqueous phases were tissues were harvested per group. Under anesthesia, tissues then separated by centrifugation at 14,000g for 5 min, were harvested without surrounding scar tissue, rinsed with after which an equal volume of 70% EtOH was added to

Table 1 PCR primer sequences Target gene Primer sequence 5V–3V Amplicon length Collagen I (col1a1) FP: CGT GAC CAA AAA CCA AAA GTG C 186 RP: GGG GTG GAG AAA GGA ACA GAA A Collagen III (col3a1) FP: CGA GGT AAC AGA GGT GAA AGA 349 RP: AAC CCA GTA TTC TCC GCT CTT Fibromodulin FP: CTG CTG TAT GTC CGG CTG TCT T 267 RP: GCT GCG CTT GAT CTC GTT CC Decorin FP: GAG GGC TCC GGT GGC AAA TA 218 RP: TGG CAT CGG TAG GGG CAC ATA Lumican FP: GTC CGC TCC CAA AGT CCC TAC A 296 RP: AAG CCC GGT GAA GCG ATT GA Versican FP: TTC CAA GGG CAA TGC TAC AAG T 140 RP: TCA TGG CCC ACA CGA TTC ACA A Biglycan FP: CAG GGG AAG GGG AAC AAA GAA G 264 RP: ATG CCC GGA TGG AAC AGT AAC MMP-13 FP: AAA GAA CAT GGT GAC TTC TAC C 283 RP: ACT GGA TTC CTT GAA CGT C MMP-2 FP: GGT CGC AGT GAT GGC TTC CTC T 376 RP: CAC ACC ACA CCT TGC CAT CGT T Cathepsin K FP: TGC GAC CGT GAT AAT GTG AAC C 205 RP: ATG GGC TGG CTG GCT TGA ATC Cathepsin L FP: GAC GGT GGG GCC TAT TTC TG 237 RP: TTC CGG TCT TTG GCT ATT TTG A TRAP FP: CGC CAG AAC CGT GCA GAT TAT G 297 RP: AAG ATG GCC ACG GTG ATG TTC G TIMP-1 FP: ACA GCT TTC TGC AAC TCG 335 RP: CTA TAG GTC TTT ACG AAG GCC TNF-a FP: TCA GCC GAT TTG CCA TTT CAT A 251 RP: AAC ACG CCA GTC GCT TCA CA GAPDH FP: GAC TGT GGA TGG CCC CTC TG 239 RP: CGC CTG CTT CAC CAC CTT CT GenBank accession numbers for col1a1, col3a1, decorin, lumican, fibromodulin, versican, biglycan, MMP-13, MMP-2, cathepsin K, cathepsin L, TRAP, TIMP-1, TNF-a, and GAPDH are Z78279, XM_343563, Z12298, X84039, X82152, AF072892, U17834, M60616, NM_031054, AF010306, NM_013156, M76110, L31883, AF329982, and AF106860, respectively. P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555 553 the RNA-containing aqueous phase. Total RNA was then (3) by examining PCR melt-curves following QPCR to isolated from the ethanol mixture using the column ensure specificity. All reactions were specific. The cDNA fractionation steps of the RNeasy Total RNA kit. Yield copy numbers of the target gene were analyzed after being and purity of RNA were quantified by spectrophotometric normalized to the copy number of GAPDH. measurement at 260, 280, and 320 nm (UltraSpec 3000 Spectrophotometer, Pharmacia Biotech, Cambridge, UK). 4.6. Scanning electron microscopy After RNA quantification, reverse transcription into cDNA was performed at 42 8Cina20ALtotalvolume Scanning electron microscopy was performed as pre- containing 5 AL total RNA, 1 AL oligo(dT)15 (500 ng/ viously described (Provenzano et al., 2002a) on additional AL; Promega, Madison, WI), RNase-free water, and 9 AL (to those listed above) ligaments stretched to measured of First Strand Synthesis buffer containing 40 units of levels of strain. RNase inhibitor (Ambion), 500 AM dNTP mix, 10 mM DTT, and 200 units of Superscript II reverse transcriptase 4.7. Statistical analysis (Invitrogen, Life Technologies). For statistical analysis, paired t-tests were applied to 4.5. Real-time quantitative PCR analyze differences in mechanical properties at time zero and gene expression within a time group. Gene expression For real-time quantitative PCR, primers for collagen I data were log-transformed to conform to the t-test (col1a1), collagen III (col3a1), decorin, lumican, fibro- assumptions. All groups that were significantly different modulin, biglycan, versican, rat collagenase (matrix metal- had at least a 10-fold change in gene expression, except two loproteinase (MMP)-13), gelatinase-A (MMP-2), cathepsin samples that had 3- and 4-fold changes, respectively. K, cathepsin L, tartrate-resistant acid phosphatase (TRAP), tissue inhibitor of metalloproteinase 1 (TIMP-1), tumor necrosis factor-a (TNF-a), and glyceraldehyde-3-phos- Acknowledgements phate dehydrogenase (GAPDH) were utilized (Table 1). The primer sets were either derived from previously This work was funded through grants from N.A.S.A. published reports for MMP-13 (Knittel et al., 1999)or (NAG9-1152), the N.S.F. (CMS9907977), and a N.I.H. designed from sequences available from GenBank using Predoctoral Training Grant (#T32 GM07215) in Molec- the PrimerSelect module of the Lasergene 5.01 software ular Biosciences (AAO). The authors thank Dr. David (DNAStar, Madison, WI) and BLASTR (Basic Local Hart and Carol Reno for assistance with obtaining sample Alignment Search Tool; http://www.ncbi.nlm.nih.gov:80/ preparation equipment for RNA extraction, Ron McCabe BLAST). Note, versican has four isoforms (Lemire et al., and Anthony Escarcega for technical assistance with 1999), so the versican primer was chosen in the conserved laboratory equipment, the Materials Science Center for region for all four isoforms for mouse, rat, bovine genes. access to the scanning electron microscope, and Phil In order to perform quantitative analysis of each gene, Oshel and the Biological and Biomaterial Preparation, QPCR standards for each of the genes were prepared from Imaging, and Characterization Laboratory (BBPIC) for purified reverse transcription-PCR products of the target technical assistance with electron microscopy specimen sequences. From spectrophotometric quantitation of the preparation. PCR products, the number of copies per AL of each standard was calculated, and 10-fold serial dilutions (ranging from 1109 to 10 copies/AL) were prepared. Appendix A. Determination of the toe-to-linear region For mRNA quantification, real-time quantitative-PCR was transition in fibrous connective tissue performed using a BIO-RAD iCycler iQ Real-time PCR system (BIO-RAD, Hercules, CA). All reactions were The ligament stress–strain curve displays nonlinear carried out in a total volume of 20 AL containing 1 strain-stiffening behavior in the dtoeT region after which Platinum Quantitative PCR Supermix (Life Technologies), a more linear region is encountered. This early portion of 10 nM fluorescein, 200 nM forward primer, 200 nM the stress–strain curve is physiologically relevant. Non- reverse primer, 0.25 Sybr Green, template cDNA, and recoverable stretch changes in the mechanical behavior in RNase-, DNase-, pyrogen-free H2O at an annealing this region can result in joint laxity and abnormal motion temperature of 55–60 8C (depending upon the primers). leading to degenerative joint disease. The model described For each gene, an assessment of quality control was in this appendix quantifies the transition from the dtoeT performed by (1) visualizing RT-PCR products for region to the linear region and has been described in detail expected size and the lack of large molecular weight elsewhere (Hurschler et al., 2003). Stress–stretch data were genomic DNA contamination, (2) by including in each fit with the probabilistic microstructural model of Hursch- QPCR experiment two negative controls (one with no ler et al. (1997) which utilized a Weibull probability template and one with reverse transcriptase excluded), and distribution function (pdf) to represent collagen fiber 554 P.P. Provenzano et al. / Matrix Biology 23 (2005) 543–555 recruitment and hence load bearing capability as the tissue Birk, D.E., Zycband, E.I., Woodruff, S., Winkelmann, D.A., Trelstad, R.L., is stretched and the characteristic crimp pattern of the 1997. 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