ELSEVIER Journal of Orthopaedic Research 23 (2005) 949-957

Journal of Ort hopa edic

Research www.elsevier.com/locate/orthres

Differential MMP-2 activity of ligament cells under mechanical stretch injury: An in vitro study on

human ACL and MCL fibroblasts

David Zhou a, Hwa Sung Lee a, Francisco Villarreal ', Annabelle Teng a,

Erick Lu a, Shirley Reynolds ', Chuan Qin a, Joel Smith b, K.L. Paul Sung a,b9*

a Department of Bioengineering, University of California, San Diego, 9500 Gilman Drioe, La Jolla, CA 92093-0412. USA Department of Orthopaedics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412, USA

Departmenl of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412, USA

Accepted 28 January 2005

Abstract

Recent studies have revealed that following injuries, ligament tissues such as anterior cruciate ligaments (ACL), release large amounts of matrix metalloproteinases (MMPs). These enzymes have a devastating effect on the healing process of the injured lig- aments. Although these enzymes are produced following ligament injuries, because of different healing capacities seen between the medial collateral ligament (MCL) and ACL, we were curious to find if the MMP activity was expressed and modulated differently in these tissues. For this purpose ACL and MCL fibroblasts were seeded on equi-biaxial stretch chambers and were stretched in dif- ferent levels. The stretched cells were assayed using Zymography, Western Blot and global MMP activity assays. The results showed that within 72 h after injurious stretch, production of 72 kD pro-MMP-2 increased in both ACL and MCL. However, the ACL fibroblasts generated significantly more pro-MMP-2 than the MCL fibroblasts. Furthermore we found in ACL pro-MMP-2 was converted more into active form. With 4-aminophenyl mercuric acetate (APMA) treatment, large amounts of pro-MMP-2 were con- verted into active form in both. This indicates that there is no significant difference between ACL and MCL fibroblasts in post-trans- lational modification of MMP-2. The fluorescent MMP activity assays revealed that the MMP family activities were higher in the injured ACL fibroblasts than the MCL. Since the MMPs are critically involved in extracellular matrix (ECM) turnover, these find- ings may explain one of the reasons why the injured ACL hardly repairs. The higher levels of active MMP-2 seen in the ACL injuries may disrupt the delicate balance of ECM remodeling process. These results suggest that the generation and modulation of MMP-2 may be directly involved in the different responses seen in ACL and MCL injuries. 0 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: ACL; MCL; Matrix metalloproteinase-2 (MMP-2); Ligament injury; Tissue remodeling; Zymography

Introduction

The ACL of the human knee plays a pivotal role in controlling and stabilizing the knee joint. Injuries to the ACL result in knee instability, pain and even pro- gressive degeneration of other joint structures. Ligament injuries result in significant disability in over 100,000 patients annually in the US alone [35]. In addition,

* Corresponding author. Address: Department of Orthopaedics and Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412, USA. Tel,: + I 858 534 5252; fax: + I 858 534 6896.

E-mail address: klsung@ucsd.edu (K.L. Paul Sung).

0736-0266/$ - see front matter 0 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. doi: 10.1016/j.orthres.2005.01.022

950 D. Zhou rt nl. I Journal of Orthopaedic Research 23 (200.5) 949-957

approximately 50,000 ACL reconstructions are carried out annually and 45-50'% of these patients will later de- velop osteoarthritis (OA) [13]. An injured ACL does not heal satisfactorily, whereas an injured MCL can heal rel- atively well and restore full functionality. Currently the preferred choice of treatment for ACL injuries is recon- struction, but it remains difficult to satisfactorily repro- duce ACL's biomechanics. Another option of treatment is surgical repair of torn ACL, which has low success rate and can be associated with long-term joint stiffness [8]. This is opposed to MCL injuries, which are generally amenable to non-operative treatment [43].

Some researchers argued that the different healing ability could result from several factors. The ACL is an extracellular structure surrounded by a thin layer of synovial tissue within an intra-articular environment such that when this synovial tissue is ruptured, ACL is exposed to synovial fluids, hemorrhagic breakdown products and proteolytic enzymes [4]. Due to its extra- articular location and very limited vascular bed environ- ment, the ACL is incapable of forming intermediate scar tissue and lacks an initial inflammatory response, result- ing in poor tissue healing [29,7]. The ACL also has a poor vascular response after injury that make its repair more difficult [3]. The intrinsic differences between ACL and MCL can help to explain their differential healing abilities. However, ACLs and MCLs injury and repair processes are very complex. Previous studies on ACL injuries were mostly focused on ACL reconstruction [30,41,24].

It was reported that mRNA expression of the type I and type I11 collagen was increased in ACL after stretch injury. The transforming growth factor beta-1 (TGF-PI) released by ACL was also increased by the stretch [23]. Wiig et al. [42] reported that the normal MCL had a higher level of procollagen mRNA than normal ACL. At the injury sites of the MCL and ACL, the levels of type I procollagen mRNA increased at all post-lacera- tion periods, reaching its highest level at 14 days post- surgery. The MCL healing site had a considerably higher level of procollagen mRNA than the ACL healing site (i.e., injury site) at all post-operative intervals. This dem- onstrates that procollagen mRNA levels are higher in MCL tissue than in ACL tissue for both normal and injury states [l].

It was reported that expression of platelet-derived growth factor (PDGF), TGF-Dl and basic fibroblast growth factor (bFGF) was increased in and around the wound site in the rabbit MCL seven days following surgical injury [27]. In the ACL, however, these protein expressions were limited to the injury site periphery and were lower in intensity than in MCL injuries [27]. Although this is from the rabbit model, however, be- cause of the high genomic homology and the fact that it have been shown that in the knee joint fluids of human injured knee, the TGF-P1 level was significantly elevated

and the administration of TGF-PI, bFGF and PDGF can improve the ACL healing [31], we can reasonably postulate that the same could also happen in human tis- sue. Levels of IL-la, IL-6 and IL-8 were increased in synovial fluids of the human ACL-deficient knees and levels of those cytokines were very similar in patients 4 weeks after ACL injury and in chronic patients [9]. Blu- teau et al. [6] reported that early after ACL surgery, MMP- 13 (collagenase-3) gene expression increased dra- matically and remained high thereafter. An increase in MMP-1 (collagenase-1) and MMP-3 expression was also noted with an absence of variation for TIMP-1 expres- sion. In addition, the global MMPs activities paralleled the MMP gene expression, which implies the involve- ment of MMPs in ACL injury response and remodeling

The modulation of MMP activities in response to stretch injury has been addressed in other biological sys- tems. He [17] applied 10% cyclic equi-biaxial tensional and compressive forces in vitro to human periodontal ligament fibroblasts and found that under compression the MMP-2 was increased but the Col-I was decreased. But the tensional forces increased both the MMP-2 and Col-I. Archambault [2] showed that rabbit tendon fibro- blasts responded to fluid-induced shear stress by increas- ing the release of pro-MMP-3 in a dose-response manner with the level of fluid shear stress. Adam's result indicated that prolonged spinal loading induced MMP-2 activation in intervertebral disc [22]. Mechanical strain to MH7A rheumatoid synovial cells caused reduction of cytokine-induced expression and activity of MMP-I and MMP-13 [36].

MMP-2 is a member of the matrix metalloproteinases family and has been found to be involved in many cellu- lar processes such as tissue remodeling, repair and base- ment membrane degradation, the healing of the acute tears, tumor invasion, neovascularization and metastasis [40,11]. Our studies have shown that MMP-2 is directly involved in ACL and MCL injuryhemodeling processes and could be an important factor responsible for their differential healing ability. Recent studies have revealed that ligament tissues including the ACL produce various MMPs including MMP-2 [14]. In this study we have fur- ther investigated how ACL and MCL cells modulate their MMP-2 expression levels and activities in stretch induced injuries.

[61.

Materials and methods

Cell culture

Human ACL and MCL cells were harvested from four donor tis- sues with age from 23 to 56. The donor ligament tissue was isolated within 24 h after patient death and immediately washed with lx PBS with 3x PSF and then cut into small pieces of dimension 1 x 2 x 2 mm'. The small pieces of ligament tissue were suspended in

D. Zhou et al. I Journal of Orthopaedic Research 23 (2005) 949-957 95 1

10% FBS-DMEM and incubated at 37 "C in a humidified atmosphere of 5% C02 and 95% air. After the ligament cells migrate out of the small tissues and attached to the bottom, the tissues were transferred to another flask and let the remaining cells grow to confluency. The cells were then frozen into arrest with liquid nitrogen until use. Cells were then cultured in 10% FBS media (low glucose DMEM, 0.1 mM non-essential amino acids, 4 mM L-glutamine and antibiotics) at 37°C in a humidified atmosphere of 5% C 0 2 and 95% air. In our experiments, only cells from passage 1 to passage 5 were used and ACL fibroblast strains from four different donor patients were used. All experiments were repeated at least three times.

In uitro injury

Cells were trypsinized and seeded onto a silicone membrane within an equi-biaxial stretch chamber [27,28] at the concentration of 500,000 cellslchamber. Cells were allowed 48 h to seed and equilibrate [19]. The culture media was removed and replaced by 2% FBS media for 16 h of starvation (under 12% injurious conditions, we have found that the cells are more vulnerable to death in 0.5% FBS media). Right before stretching the culture media was replaced with fresh 1% FBS media. ACL and MCL cells were then subjected to physiologic (6%), injurious (12% and 14%), and control (0%) stretch conditions. Two hundred mircolitres culture media samples were collected at 0, 4, 8, 12 h and every 24 h after stretching up to 9 days.

Zymography

MMP-2 activity from collected samples was assayed using 0.05% gelatin zymography. Briefly, 10 pI of each sample was mixed with equal amount of BioRad Laemmli sample buffer (62.5mM Tris- HCI, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, no p-mercaptoethanol) and separated in 10% SDS-PAGE gel copolymer- ized with 0.05% gelatin. To regain enzyme activity by removing SDS, gels were washed in 2.5% Triton-X-100 three times for 1.5 h at room temperature (RT) after electrophoresis. Washed gels were then bathed in proteolysis buffer (50 mM CaC12, 0.5 M NaCI, 50 mM Tris, pH 7.8) and incubated at 37 "C for 15 h. Following incubation, gels were rinsed in 2.5% Triton-X-100 solution and stained at room temperature with coomassie blue (45% methanol, 44.75% HzO, 1Ooh acetic acid, 0.25% coomassie blue R250) for 1 h on a rotator. Gels were destained (40% methanol, 7.5% acetic acid, 52.5% H20) until white bands appeared clearly from the coomassie blue background.

Western blot

Protein samples were prepared by mixing 1 part of sample with 1 part of BioRad Laemmli Sample Buffer Sample Buffer (62.5mM Tris-HCI, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 5% P-mercaptoethanol) and then boiled at 100 "C for 5 min. Proteins were separated in 10% SDS-PAGE and transferred to a NC membrane at 250 mA for 1 h at room temperature (RT). The blot was blocked with 5% non-fat dry milk suspended in l x TBS (25 mM Tris, 137 mM NaC1, 2.7 mM KC1) for 1 h at room temperature. The resulting blot was incu- bated with 1:500 rabbit MMP-2 polyclonal antibody from Santa Cruz Biotechnology (H-76, sc-10736) for 1 h at RT (or 4 "C overnight), fol- lowed by incubation with 1500 goat anti-rabbit IgG-HRP from Santa Cruz Biotechnology for 1 h at RT (or 4 "C overnight). Between the first and second incubation, the blot was washed three to four times with Ix TTBS (25 mM Tris, 137 mM NaC1, 2.7 mM KCI, 0.05% Tween-20) for 10 min each time. Signals from blots were obtained using Santa Cruz Western Blotting Luminol Reagent Kit (sc-2048). Proteins were visual- ized via chemiluminescence with hydrogen peroxide using Kodak X-AR (Eastman Kodak Co. Scientific Imaging Systems, Rochester, New York, USA) and luminol as substrate.

A P M A treatment

After the injury, ACL and MCL fibroblast media samples were incubated in 0.05 M borate (pH 9.0), 0.01 mM ZnCI2, 5 mM CaC12 and 0.5 mM APMA at 37 "C for I . 2 and 3 h. APMA treatment trun-

cates the pro-peptide in 72 kD MMP-2, thereby converting it into 62 kD MMP-2 whose activities can be measured other methods such as ELISA. The samples are then analyzed using zymography assay.

Global M M P ussay

The quenched fluorescent peptide Mca-Pro-Leu-Gly-Leu-Dpa- Ala-Arg-NH2 (Biomol, Plymouth Meeting, PA; Calbiochem, La Jol- la, CA) acts as a substrate for cleavage by multiple MMPs (including MMP-2). Reaction: 50 pl of [SAMPLE: cell lysate, culture medium, or whatever it is] in 149 pl of reaction buffer (50mM Tris, 150 mM NaCI, 5 mM CaCI2, 0.2 mM NaN3, pH 7.6) with 1 pI of 2 mM Omni-MMP Fluorescent Substrate (final concentration 0.1 mM). We performed kinetic analyses for global MMP activity in a BioTek FLx800 plate reader at 37"C, reading once a minute for 1 h. The reaction rate is determined from the linear portion of the kinetic curve, and is expressed as relative fluorescence units per minute (RFUlmin). Negative controls were run using buffer rather than sample. Phenanthroline, a global MMP inhibitor, was used ( 1 mM final concentration) as a control for cleavage specifically by MMPs.

Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) to determine whether differences existed among groups. Post hoc analysis utilized Fisher's protected least significant differences (PLSD). In each analysis, critical significance levels will be set to D! = 0.05.

Results

MMP-2 levels are increased in a stretch-level-dependent manner in ACL jibroblast

Compared with the non-stretch control (O%), the physiological stretch (6%) increased the MMP-2 expres- sion in ACL fibroblast by 35%, 10% stretch by 105%,, 12% stretch by 423% and 14% stretch by 670%. There is a clear doseedependence response of MMP-2 expres- sions to stretch levels (Fig. 1).

72 kD MMP-2 and 62 kD MMP-2 are both increased in injured A CLIMCL jibroblast

Both ACL and MCL fibroblasts increased their pro- duction of 72 kD MMP-2 according to the extent of injurious stretching. Pro-MMP-2 is visible at approxi- mately 72 kD and active MMP-2 is visible at 62 kD (Fig. 2a). The ACL fibroblast showed two distinct differ- ences from the MCL fibroblast. First, only the ACL fibroblast showed a noticeable stepwise, time-dependent conversion of 72 kD MMP-2 to 62 kD MMP-2 that started approximately 8 h after stretch induced injury. Increased stretch is correlated with an increasing conver- sion to the active form within the first 24 h. Second, the ACL fibroblast also produced more 72 kD MMP-2. MCL fibroblast produces a small increase in the 72 kD MMP-2 with increasing levels of stretch. There was no detectable 62 kD MMP-2 in MCL fibroblast at 24 h after stretching. The ACL fibroblast had a relatively large increase in the 72 kD MMP-2 with increasing

952

Time (hr) 4

0%

6%

10 %

12%

D. Zhou et al. I Journal of Orthopaedic Research 23 (2005) 949-957

8 12

Fig. 2. ACL fibroblasts produced more MMP-2 than MCL fibroblasts after incremented stretch-induced injury. (a) Samples were taken from stretch chambers with stretch levels from 6% to 12% and applied to zymography (b) MMP-2 levels increased over time after 14% injurious stretching of ACL and MCL fibroblasts. Note that because of high stretch level, the samples were collected early from 8 h after stretch.

r * more 72 kD MMP-2 than the MCL fibroblast in all the

*

1

0 0% 6% 10% 12% 14%

(b) Stretch Percentage

Fig. I . MMP-2 levels increased in an amplitude-dependent manner in ACL fibroblasts. (a) Samples were taken from stretch chambers and applied to zymography. O%, 6%, lo'%, 12% and 14% indicate the stretch percentages. (b) MMP-2 quantification of stretch samples at 12 h with NIH ImageJ software. Statistic analysis was done by ANOVA method. *Significant difference with respect to control (p < 0.05).

levels of stretch. Strong bands of 62 kD MMP-2 can be seen in the ACL fibroblast even at 24 h after injury (Fig. 2a).

MMP-2 levels increased variably with time under 14% injurious stretch in ACL versus MCL fibroblast

Under 14% injurious stretch 72 kD MMP-2 further increased for both ACL and MCL fibroblast from 8 to 24 h (Fig. 2b). However, the conversion from the 72 kD MMP-2 to 62 kD MMP-2 was increased in inju- rious stretch conditions only in the ACL fibroblast. It should be noted that this effect is visible beginning at 8 h after initiating the injury in ACL fibroblast. Also, as we saw in Fig. la, the ACL fibroblast produced much

time intervals tested (Fig. 2b).

A CL cells produce more 72 kD MMP-2 in a time- dependent manner during continued injurious stretch

Western analysis of ACL samples from 12% stretch injury revealed that pro-MMP-2 was present before the stretch injury (Fig. 3). There is a general trend of

r 3 11 $ T

0 12 24 48 (b) Time (hr)

Fig. 3. Increased MMP-2 production by ACL fibroblasts after 12% injurious stretch. (a) Western Blot of stretched ACL fibroblast samples at indicated times. (b) Quantification with NIH ImageJ software. Statistic analysis was done by ANOVA method. Note in zymography, both pro- and active MMP-2 were detectable but in Western Blot, only the pro-form were detected. *Significant difference with respect to control (p < 0.05).

D. Zhou et al. I Journal of Orthopuedic Reseurch 23 (2005) 949-957 953

increased 72 kD MMP-2 levels with time after the stretch injury. This is consistent with our Zymography results mentioned above (Fig. 2a and b). As previously reported, the Western Blot was difficult to detect 62 kD MMP-2 because it is present in trace amounts compared to 72 kD MMP-2 [lo].

Both ACL and ,MCL$broblast’s 72 kD MMP-2 can be eflciently converted into 62 kD MMP-2 by APMA

Injured ACL and MCL fibroblast conditioned media samples were treated with APMA, which can truncate the N-terminal of 72 kD MMP-2 and convert it into 62 kD MMP-2. If there were any post-translational modifications in MMP-2 that can block the APMA acti- vation of pro-MMP-2, then from the amount of active 62kD MMP-2 converted from the pro-MMP-2 we could know whether there is such modification in the ACL and MCL fibroblast MMP-2. The 72 kD MMP-2 in ACL and MCL fibroblast can both be converted into 62 kD MMP-2 efficiently (Fig. 4). We found that the ACL fibroblast sample initially had a higher amount of 62 kD MMP-2 than the MCL fibroblast sample. No other extra bands appeared after APMA treatment of ACL and MCL fibroblast samples. This means that there may be no significant post-translational modifica- tion differences between ACL and MCL fibroblast MMP-2 that may affect MMP-2’s activity modulation (Fig. 4). NIH image analysis shows that after APMA treatment, 62 kD MMP-2 activity is amplified 19 (19.23 k2.63) and 9 (9.34k 1.17) fold in ACL and MCL fibroblast, respectively. On average, more than

twice the amount of pro-MMP-2 (72 kD) is converted to active MMP-2 (62 kD) in ACL than in MCL fibroblast.

After injury the A CL fibroblast continuously rrlrusrd MMP-2

After ACL fibroblast injury the medium samples were collected up to nine days and subjected to zymo- graphic analysis. For the first two days samples were collected without any media change. After two days media was changed immediately after daily sample col- lection. We found that on day 4, MMP-2 level de- creased to previous level of 4 h stretch condition and remained at that level. For days 7-9, however, 1% FBS media was replaced with 10% FBS media and 72 kD MMP-2 level dramatically increased. Sixty-two kD MMP-2 level did not show any significant increase in that time frame (Fig. 5). These data suggest that in the injured state, ACL fibroblast MMP-2 expression level is initially increased for about two days and then decreased to the level same as at 4 h after stretch. In 10% FBS, the MMP-2 level generated is much higher than the amount generated in 1% FBS by 12% injuri- ous stretch.

Time

1% FBS

Incubation Time 0 1 2 3 (W

ProMMP-2 + ActiveMMP-I + ACL 1% FBS

MCL 10% FBS

Fig. 5 . ACL fibroblasts continuously released MMP-2 into the media after injury. ACL-conditioned media samples were collected at 4, 8, 12. 24 and 48 h after stretch injury with no media change. After the 48-h sample collection, media was changed daily following each sample collection. After day 7 (D7), the media was switched from 1x1 FBS media to 10% FBS. All samples were subjected to zymographic analysis.

Fig. 4. Seventy-two kD MMP-2 in ACL and MCL can both be converted into 62 kD MMP-2 after APMA treatment. Media samples from 12% stretched ACL and MCL fibroblasts were incubated with APMA for up to 3 h. Samples were subjected to zymographic analysis. Note both before and after APMA treatments, MMP-2 levels were higher in ACL samples than MCL samples.

954 D. Zhou et al. I Journal of Orthopaedic Research 23 (2005) 949-957

Table 1 Omni fluorescent assay of total MMP activity

Control 12% Stretch

ACL -4 86.7 MCL -6.2 -4

Higher MMP activity in the injured ACL than MCL jibroblast

A fluorescence-based global MMP activity assay was used to evaluate the total MMP activity in the samples. The peptide substrate is specific for most MMP enzymes but with different affinity, including MMP-1-3, 7-17,25, 26, and ADAMl7/TACE. MMPs cleave the Gly-Leu bond in substrates with sequence Mca-Pro-Leu-Gly- Leu-Dpa-Ala-Arg-NH2-AcOH. Among these MMPs, MMP-8 has the highest activity ( 5 x 10’) and ADAM17/TACE (1 x lo3) has the lowest activity against this substrate. It was found that the ACL fibro- blast released about 20 times more MMP than the MCL fibroblast after injury (Table 1).

Discussion

A number of intrinsic differences exist between fibro- blasts from ACL and MCL fibroblast [30]. For example, delivery of exogenous tropomodilin (Tmod) into MCL fibroblasts significantly increased their adhesion to fibronectin whereas a monoclonal antibody (mAb) against Tmod significantly decreased cell adhesiveness [37]. Both ACL and MCL fibroblast adhesion rely on cytoskeletal assembly, but the dependence differs in many ways between ACL and MCL fibroblast [37]. There are obviously intrinsic differences between ACL and MCL fibroblast in their signal transduction path- ways upon binding to fibronectin [38]. These results indicated the importance of the cyclic adenosine mono- phosphate and Ca2+/phospholipid signaling in integrin- mediated adhesion of MCL fibroblasts to fibronectin, but only a partial role of Ca2+/phospholipid signaling in integrin-mediated adhesion of ACL fibroblasts to fibronectin [38]. Depending on the laminin concentra- tion, adhesion strength of human ACL fibroblasts to laminin was greater and significantly different from that of human MCL fibroblasts [39]. The ACL fibroblasts exhibited greater adhesion forces to poly-D-lysine bound Collagen type 1 and Collagen type I11 (Coll and ColIII) [MI. Whereas ACL fibroblasts responded to cyclic strains by expressions of higher levels of type-1 collagen message with almost no significant increases in type-I11 collagen, MCL fibroblasts exhibited statistically signifi- cant increases in type-111 collagen mRNA at all times after initiation of strain with almost no significant in- creases in type-I collagen [19]. In addition, human

ACL fibroblasts generally have lower proliferation rates than MCL fibroblasts and ACLs responses to growth factors also vary from that of MCL [33,45].

Injuries to the ACL have become increasingly preva- lent and account for a large proportion of knee ligament injuries among young and active individuals. Thus, researchers and clinicians have paid more attention to the ACL injury. Biomechanical studies that quantify strains in ACL and MCL have shown that these liga- ments are subjected to 4 5 % stretch during normal activities and can be strained to 7.7% during external application of loads to the knee joint [20,21,26]. The cur- rent standard of treatment for ACL injuries in active adults with functional instability is ligament reconstruc- tion. Reconstruction still does not satisfactorily restore normal ACL biomechanics [34]. In addition, after com- bined ACL and MCL injuries, the absence of a function- ing ACL adversely affects the healing processes in the MCL [43]. This raises the necessity to detail the differ- ences in the injury and repair processes of the ACL and MCL at the molecular level.

During the ligament injury and repair processes, old ECM molecules are removed and new ECM molecules are synthesized and deposited. This protein digestion and synthesis in the ACL and MCL is an intricately mod- ulated process. Regulation of this process will greatly affect the ability of ligaments. The balance between the degradative and biosynthetic arms of tissue remodeling process is controlled by MMPs and their inhibitors (tis- sue inhibitors of metalloproteases, TIMPs). In general, during the degradative process of tissue remodeling, the influence of MMPs is greater than TIMPs and the opposite is true in the reparative process [14]. MMP activity is tightly coordinated at different levels including transcriptional regulation, activation of latent zymogen and interaction with endogenous inhibitors and other proteins such as MMPs. It is possible that different MMPs may be regulated by different factors mentioned above via different signal pathways, which combine to finally contribute to the differential healing ability be- tween ACL and MCL.

The equi-biaxial stretch chamber has been used in our laboratory for numerous years, enabling the application of two-dimensional mechanical strains at 5-1 YO, to ACL and MCL ligament fibroblast. This stretch appara- tus also has been used widely by other labs for many years and many results have been published using this system [20,2 1,26,28]. This apparatus (Country Machines and Plastics, San Diego, CA) consists of a plexiglass frame into which a chamber has been machined. Clamps secure the ends of a 50 mm x 104 mm silicone elastomer sheet (Specialty Manufacturing Inc., Saginaw, MI) and are positioned in notches in the frame. These notches have been designed to allow us to stretch the membrane incrementally by 10% and 20% from a reference notch. The seeded fibroblasts will attach to the membrane, pro-

D. Zhou et al. I Journal of Orthopaedic Research 23 (2005) 949-957 955

liferate and release ECM components into the surround- ing environment in which the cells will interact either with themselves or with the ECM components. It hence mimics the stretching forces applied to the ACL tissue in the knee joint.

In our lab, we found that MMP-2 production is di- rectly related to ACL fibroblast injury. MMP-2, also termed a gelatinase or type IV collagenase, can cleave collagens type IV and V, and degrade collagen I and I11 and denatured collagen of all types [lo]. It appears that two forms (72 kD and 62 kD) can carry out the same enzymatic reaction, but the 72 kD MMP-2 has only about 10% of the activity of the 62 kD MMP-2 [18]. The activation pathway of 72 kD MMP-2 is initi- ated by membrane-type-MMPs (MT-MMPs) including MTl-MMP [12,16]. At the same time, the activation and the activity of MMP-2 are critically dependent on

We found that after injury, the ACL and MCL fibro- blast directly responded by increasing 72 kD MMP-2 production as evidenced by our Western Blot and zymo- graphic analyses. But the ACL fibroblast produces much more 72 kD MMP-2 than the MCL. The ACL fibroblast also converted much more 72 kD MMP-2 into the 62 kD MMP-2 than did MCL. This means that after an injury the ACL fibroblast maintains much higher level of 72kD MMP-2 and 62 kD MMP-2 than the MCL fibroblast. It is conceivable that injured ACL fibroblast will have much higher total MMP-2 activity than MCL. Since MMPs are largely involved in ECM turnover, this may suggest one mechanism by which the ACL repair process differs from that of the MCL. The ACL maintains a higher level of MMP-2 after in- jury. This might disrupt the delicate balance of removing damaged matrix components with the deposition of newly synthesized materials. At the same time, the high level of MMP-2, especially the 62 kD MMP-2, may acti- vate other members of MMP proteins and increases the overall MMP activity in the ACL, thereby hastening it’s degradation [15,25]. We must point out that though injured MCL fibroblast released little active MMP-2, some active MMP-2 is released nevertheless. This can be confirmed by zymography study, using generous amounts of medium sample (data not shown).

Our global MMP activity assay showed that injured ACL fibroblast released almost 20 times more MMP activity than injured MCL fibroblast (Table 1). After in- jury, there is a substantial increase in total MMP activ- ity in ACL fibroblast compared to an insignificant change in MMP activity in the MCL fibroblast. The ACL fibroblast continuously generated and released MMP-2 into the media even after injury. It has been known that MMPs are involved in tissue remodeling. Taken together, these data suggest that after injury, the ACL will accumulate a very high level of MMP activity that will speed up ligament degeneration. The

TIMP-1.

differing MMP activity, including MMP-2, after liga- ment injury may be one of the main reasons for the dif- ferential healing abilities of the ACL and MCL fibroblast. Since the APMA treatment can convert both ACL and MCL fibroblast 72 kD MMP-2 samples into the 62 kD MMP-2 without generating any extra band, it is possible that after injury, the MMP-2 in ACL and MCL fibroblast does not receive significantly different post-translational modification, although it is possible that ACL and MCL fibroblast may undergo some com- mon post-translational modifications.

However, we have to point out that since our stretch conditions only mimic the real ACL injury, it cannot ex- actly represent the physiological situation. Inside the knee joint fluid surrounding the injured ACL, MMP-2 may come from other sources other than ACL. For in- stance MMP-2 may also come from PCL fibroblast or synovial membrane cells. It is well known that in the knee joint fluids the level of IL-la, TGF-P and TNF-P are all elevated. These factors may further modulate the MMP-2 level from other sources. In our current sys- tem, we only analyzed the ACL and MCL fibroblast injury separately in vitro. In the real physiological situation, the stretching force may exert its effect to- gether with other factors to induce the injurious cascade whereas in our equi-biaxial system, such involvement by other factors cannot be realized. This has to be consid- ered when we compared the ACL or MCL fibroblast data with the clinical data.

It would be very interesting to investigate why the injured ACL fibroblast produces much more pro- MMP-2 and converts much more pro-MMP-2 into ac- tive MMP-2. The mechanism of MMP-2 modulation within ACL and MCL fibroblast is different though they receive the same stretch signal. The promoter re- gion of MMP-2 contains several specific binding sites for other transcriptional factors such as Spl, Sp3, AP-1, AP-2, CAMP response element-binding protein (CREB), PEA3, C/EBP, Est-1, etc. [5,32]. After injuri- ous stretching, these factors within the ACL and MCL fibroblasts may be up-regulated to different extents. Increasing expressions of these factors will lead to the increasing binding to their corresponding binding sites within MMP-2 promotor region resulting in trans-acti- vation of the MMP-2. Thus it would be interesting to investigate the expression levels of these proteins in ACL and MCL fibroblasts under normal and injurious states. Besides, our results indicated that in ACL, the AP-1, JNK and NF-kB pathways can significantly af- fect injured ACL fibroblast MMP-2 expression but this did not happen in injured MCL fibroblast (data not shown). This suggests that after injury, different signal transduction pathway may be activated or suppressed in ACL and MCL fibroblasts that may contribute to the different MMP-2 expression in injured ACL and MCL fibroblast.

956 D. Zhou et al. I Journal of Orthopaedic Research 23 (200s) 949-957

Acknowledgment

Thanks to Dr. Robert Pedowitz and Alexndra Sch- wartz for their detail discussion of ACL injuries and knee trauma. OREF 59121 and NIH AR 45635 (KLPS).

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