JBJS | Immunolocalization of Matrix Metalloproteinases in Partial-Thickness Defects in Pig Articular Cartilage : A Preliminary Report

The Journal of Bone & Joint Surgery, Volume 83, Issue 6

Articles | June 01, 2001

Immunolocalization of Matrix Metalloproteinases in Partial-Thickness Defects in Pig Articular Cartilage : A Preliminary Report

Rosalind M. Hembry, BSc, PhD; Jonathan Dyce, MA, VetMB, DSAO, MRCVS; Iris Driesang, DVM; Ernst B. Hunziker, MD, ME; Amanda J. Fosang, BSc, PhD; Jenny A. Tyler, BSc, PhD; Gillian Murphy, BSc, PhD

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Investigation performed at the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
Rosalind M. Hembry, BSc, PhD
Gillian Murphy, BSc, PhD
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. E-mail address for R.M. Hembry: r.hembry@uea.ac.uk

Jonathan Dyce, MA, VetMB, DSAO, MRCVS
College of Veterinary Medicine, Ohio State University, 601 Vernon L. Tharp Street, Columbus, OH 43210-1089

Iris Driesang, DVM
Ernst B. Hunziker, MD, ME
M.E. Müller Institute for Biomechanics, University of Bern, Murtenstrasse 35, P.O. Box 30, CH-3010 Bern, Switzerland

Amanda J. Fosang, BSc, PhD
University of Melbourne, Department of Paediatrics, Cell and Matrix Biology Research Unit and Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia

Jenny A. Tyler, BSc, PhD
Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 8RN, United Kingdom

Although none of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article, benefits have been or will be received but are directed solely to a research fund, foundation, educational institution, or other nonprofit organization with which one or more of the authors is associated. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were the Medical Research Council (United Kingdom) and Orthogene (Sausalito, California).

The Journal of Bone & Joint Surgery. 2001; 83:826-838

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Abstract

Background: Partial-thickness defects in mature articular cartilage do not heal spontaneously. Attempts at repair often result in limited integration between the repair tissue and the surrounding cartilage, with formation of chondrocyte clusters adjacent to a zone of cartilage necrosis. In wound repair, spatially and temporally controlled expression of matrix metalloproteinases and their inhibitors have been implicated in proteolytic degradation of damaged extracellular matrix components, but the sequence of events following damage to cartilage is unknown. To determine this sequence, we studied the distribution of matrix metalloproteinases and their inhibitors during early in vivo repair of partial-thickness defects in pig articular cartilage.

Methods: With use of a model that elicits the ingrowth of mesenchymal cells into partial-thickness defects, partial-thickness defects were created in knee joint cartilage. The distributions of matrix metalloproteinase-1, 2, 3, 9, 13, and 14; tissue inhibitors of metalloproteinase-1 and 2; and the neoepitope DIPEN341 specifically generated following matrix metalloproteinase cleavage of aggrecan were determined by immunolocalization of repair tissue and surrounding cartilage excised from immature pigs during the first eight weeks of repair and from adult minipigs at eight days and three weeks.

Results: Synthesis of matrix metalloproteinase-13 was usually confined to hypertrophic chondrocytes in immature cartilage and to the radial zone in adult cartilage. Following injury, strong induction of matrix metalloproteinase-13 synthesis was observed in chondrocyte clusters surrounding lesions in all of the animals. The migration of macrophages into defects was prominent at two and eight days, with synthesis and deposition of matrix metalloproteinase-9 onto damaged cartilage matrix and newly synthesized matrix in the defect. The DIPEN341 neoepitope was localized to damaged cartilage matrix at eight days and six weeks, indicating partial degradation of aggrecan. Focal synthesis of matrix metalloproteinase-1, 3, and 14 and of tissue inhibitor of metalloproteinase-1 occurred at later times, suggesting continuous remodeling of the increasingly compact repair tissue.

Conclusions: The expression of matrix metalloproteinase-13 by normal hypertrophic chondrocytes and the induction of synthesis in chondrocyte clusters adjacent to the zone of cartilage necrosis suggest that this enzyme participates in the pericellular proteolysis required for lacunar expansion. The localization of matrix metalloproteinase-9 to damaged cartilage matrix suggested matrix proteolysis, which was confirmed with DIPEN341 localization. Reduced matrix metachromasia persisted and was colocalized with DIPEN341 at six weeks. However, under the conditions investigated, there was only limited proteolytic degradation in the zone of cartilage necrosis. This may render the zone mechanically weakened, thereby contributing to subsequent instability of the region, and may form a barrier to integration of repair tissue with viable cartilage.

Clinical Relevance: Osteoarthritis initially involves the superficial layers of cartilage. The development of procedures to promote the healing or repair of early defects will have major advantages in terms of disease alleviation as well as economic importance. Identification of the enzymes involved in the early repair of partial-thickness defects in articular cartilage is clinically relevant because proteolysis of damaged matrix has to take place in order for repair tissue to integrate with surrounding healthy cartilage.

Figures in this Article

Introduction

Introduction | Materials and Methods | Results | Discussion | References

Matrix metalloproteinases (MMPs) are a family of proteinases that together can degrade all extracellular matrix components. Their activity is controlled by their inhibitors, known as tissue inhibitors of metalloproteinases (TIMPs), and both MMPs and TIMPs have been shown to have a role in developmental processes, normal cartilage turnover, and wound repair¹. In particular, MMP-13 (collagenase 3) may be important in the remodeling of articular cartilage. MMP-13 mRNA is expressed by hypertrophic chondrocytes during human fetal bone development^2,3, increased expression of MMP-13 has been reported to occur in both osteoarthritic cartilage^4-7 and in a guinea-pig model of osteoarthritis⁸, and the purified enzyme preferentially cleaves type-II collagen⁹. The principal proteoglycan in cartilage, aggrecan, is cleaved by MMPs, including MMP-9 and 13, at the N341ØF342 bond in the interglobulin domain and possibly at other C-terminal sites as well^10,11. MMP cleavage-site-specific antibodies have been developed and have been used to immunolocalize the C-terminal neoepitope DIPEN341 in human articular and intervertebral disc cartilage^12,13, indicating that MMPs degrade aggrecan in vivo.

After soft-tissue wounding, the spatially and temporally controlled expression of several MMPs by inflammatory cells and mesenchymal cells is associated with proteolytic degradation of damaged extracellular matrix components, and newly synthesized matrix is remodeled until repair tissue is completely integrated with the surrounding tissues^14-17. Although considerable progress has been made to develop procedures to elicit cartilage formation in full-thickness^18-21 and partial-thickness^22-25 cartilage defects, there is often poor integration between the repair tissue and the surrounding cartilage^20,26,27. In an early study of the histological response of immature dog cartilage to damage, Calandruccio and Gilmer²⁸ observed a zone of cell death (zone of cartilage necrosis) and loss of metachromasia along the marginal wall of each defect, with formation of new, multinucleated chondrocyte clusters in the surrounding cartilage. In a study of full-thickness articular-cartilage defects in rabbits, this zone of chondrocyte necrosis appeared not to be replaced over a forty-eight-week period²⁷. The pattern of MMP and TIMP expression by chondrocytes and repair cells immediately following cartilage damage by defect formation is not known, and failure of the repair tissue to integrate with surrounding cartilage could indicate that their expression is inappropriate.

To determine the sequence of events following cartilage damage, we created partial-thickness defects in pig articular cartilage^24,25. We determined by immunolocalization which MMPs (MMP-1 [collagenase 1], MMP-2 [gelatinase A], MMP-3 [stromelysin 1], MMP-9 [gelatinase B], MMP-13 [collagenase 3], and MMP-14 [membrane type-1 MMP]) and TIMPs (TIMP-1 and TIMP-2) were present at two and eight days and at three, six, and eight weeks following the creation of partial-thickness defects in immature pigs. Since our antibody to MMP-9 does not distinguish between pro and active enzyme forms, we also used specific antibodies to the C-terminal MMP cleavage neoepitope in the aggrecan interglobulin domain (DIPEN341) to determine whether degradation of aggrecan had occurred. For comparison, we also analyzed partial-thickness defects from two adult minipigs (at eight days and three weeks), since immature cartilage has been shown to have greater repair potential than adult cartilage^29,30. Our data show that, under the conditions tested, there was limited proteolytic degradation in the zone of cartilage necrosis, which may render it mechanically weakened and restrict integration of repair tissue with surrounding healthy cartilage.

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Fig. 1:: Anteroposterior diagram of the distal aspect of the left femur, showing the location of the partial-thickness articular-cartilage defects on the trochlear sulcus and the medial femoral condyle.

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Fig. 2:: Matrix metalloproteinase (MMP) synthesis by pig cartilage remote from the defects. The cartilage remote from the defects in four-month-old (a through e) or adult (f) pigs was excised either two days (a, b, c, and e) or eight days (d and f) after the formation of the defects, cultured with monensin for twenty-four hours, frozen, sectioned, and stained by indirect immunofluorescence for MMP-3 (a), MMP-9 (b and d), MMP-1 (c), or MMP-13 (e and f). The nuclei were counterstained and appear red (a through d). The bars indicate 30 mm (a through d), 250 mm (e),or 50 mm (f). a and b: Green juxtanuclear immunofluorescence indicates the synthesis of MMP-3 (a) and MMP-9 (b) in random transitional-zone chondrocytes remote from the defects. c and d: Green fluorescence indicates the synthesis of MMP-1 (c) and MMP-9 (d) in terminal hypertrophic chondrocytes. e: Most of the hypertrophic chondrocytes are strongly positive for MMP-13. No counterstain was used, and the section was exposed to show the weak autofluorescence of the cell nuclei in the superficial and transitional zones. The arrow indicates the articular surface. f: The lower radial-zone chondrocytes in the adult minipig cartilage adjacent to bone (B) are positive for MMP-13 (no counterstain).

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Fig. 3:: The response of chondrocytes to the creation of defects. Partial-thickness defects were created in immature pigs (a through i) and adult minipigs (j through m). The joint was closed without further treatment (a through d), or the defects were treated with chondroitinase AC and closed (e and f), or the defects were treated with chondroitinase AC, filled with transforming growth factor-b/matrix as described in the Materials and Methods section, and closed (g through m). The defects and the surrounding cartilage were excised either two days (a through i) or eight days (j through m) later, cultured with monensin, frozen, sectioned, and stained by indirect immunofluorescence for MMP-13 (b,d, f, i, and k), MMP-3 (l), or TIMP-1 (m), followed by either hematoxylin and eosin (a, c, g, h, and j) or by toluidine blue (e). The bars indicate 100 mm (a, b, c, e, f, g, j, and k), 50 mm (d and h), and 30 mm (i, l, and m). a: A hematoxylin-and-eosin-stained section of an untreated (control) defect excised on day 2, showing no defect contents (average number of cells per section, 2.0) as well as an unusually wide necrotic zone on the lateral margin, where chondrocyte nuclei are absent. The arrows in a and b indicate identical areas. b: A section (same as in a) stained with anti-MMP-13 but not with a counterstain. The chondrocytes adjacent to the zone of necrosis stain strongly. c: A hematoxylin-and-eosin-stained section of an untreated (control) defect, excised on day 2, showing the defect to be filled with fibrinous material and cells (a higher-power view is shown in Fig. 4, a). d: A section (same as in c) stained with anti-MMP-13 but not with a counterstain. The chondrocytes in the cartilage beneath the middle of the defect have strong MMP-13 immunofluorescence. e: A toluidine-blue-stained section of a chondroitinase-AC-treated defect in immature cartilage, excised on day 2, showing almost complete loss of metachromasia on the basal and lateral margins of the defect and along the articular surface. f: A section (same as in e) stained with anti-MMP-13 (green) and propidium iodide (red). The chondrocytes in the lateral margin immediately adjacent to the zone of necrosis are strongly positive for MMP-13. g: A hematoxylin-and-eosin-stained section of a defect filled with transforming growth factor-b/matrix, showing Gelfoam, fibrin, and cells (a higher-power view is shown in Fig. 4, d). h: A higher-power view of the basal defect margin (same as in g), showing an absence of nuclei in the zone of necrosis and adjacent chondrocyte lacunae containing two nuclei (arrows). i: A section similar to that in h, stained with anti-MMP-13. The nuclei are counterstained red. Green immunofluorescence indicates chondrocyte synthesis of MMP-13. j and k: A section of adult minipig cartilage, excised on day 8, stained with hematoxylin and eosin (j) and MMP-13 immunofluorescence (with no nuclear counterstain) (k). The chondrocyte clusters adjacent to the zone of cartilage necrosis have multiple nuclei and strong staining for MMP-13. The lower radial-zone chondrocytes adjacent to the bone also stain strongly, and they appear to be unaffected by the presence of the defect. l: A section (adjacent to j and k) stained with anti-MMP-3 and nuclear counterstain. Chondrocyte synthesis of MMP-3 is visible in the expanding chondrocyte clusters. m: A section (adjacent to j and k) stained with anti-TIMP-1 and nuclear counterstain. TIMP-1 synthesis is present in the newly forming chondrocyte clusters.

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Fig. 4:: MMPs and TIMP-1 in repair tissue cells and matrix. Partial-thickness defects were created in immature pigs. The joint was closed without treatment (a and b), or the defects were treated with chondroitinase AC and closed (c), or the defects were treated with chondroitinase AC, filled with transforming growth factor-b/matrix as described in the Materials and Methods section, and closed (d through l). The defects and the surrounding cartilage were excised either two days (a through j) or eight days (k and l) later, cultured with monensin, frozen, sectioned, and stained with either hematoxylin and eosin (a, d, and l) or by indirect immunofluorescence for MMP-9 (b, c, e, g, and k), macrophage marker (f, h, and j), MMP-14 (j), or TIMP-1 (i). The bars indicate 30 mm (a and c through i), 100 mm (b and l), 250 mm (k), and 25 mm (j). a: Higher-power view of the hematoxylin-and-eosin-stained untreated (control) defect (seen in Fig. 3, c) showing fibrin and cells (arrows) (average number of cells per section, 31.7). b: The section in a, shown at a lower power (as in Fig. 3, c), stained with anti-MMP-9 without counterstain. Cells within the defect stain strongly, and there is staining on the zone of cartilage necrosis (arrow). c: A chondroitinase-AC-treated, unfilled defect stained with anti-MMP-9 (green) and nuclear counterstain. The cells within the defect are strongly immunofluorescent, and there is staining on the adjacent damaged matrix (arrow). d: Higher-power view of the hematoxylin-and-eosin-stained section (seen in Fig. 3, g), showing the Gelfoam, fibrin, and cells (average number of cells per section, 48.6). e and f: Dual localization of MMP-9 (e, green) and macrophage marker (f, red) on the articular-cartilage surface adjacent to a defect excised on day 2. The cells on the articular surface stain with both antibodies, but there is no staining of the cartilage matrix below them. g and h: A section (same as in e and f) showing the lateral margin of the defect. The cells adherent to the damaged cartilage stain with both antibodies, and there is additional MMP-9 staining of the damaged cartilage matrix (arrows). i: A section (adjacent to e through h) stained with anti-TIMP-1 and nuclear counterstain. The green juxtanuclear immunofluorescence of cells within the defect indicates TIMP-1 synthesis. j: A section (as in e through i) stained with both anti-MMP-14 and macrophage marker. The cells in the defect have macrophage marker immunofluorescence (red) as well as MMP-14. k: A section of a defect, excised on day 8, stained with anti-MMP-9 without nuclear counterstain. The lateral and basal margins of the defect stain strongly for MMP-9, as do the cells and matrix within the defect. The insert is a higher-magnification view of the cells marked with the arrow, and it shows a group of macrophage-like cells with juxtanuclear immunofluorescence, probably in the Golgi apparatus. l: A hematoxylin-and-eosin-stained section (same as in k).

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Fig. 5:: Immunolocalization of DIPEN341 and ITEGE373 G1 domains in the zone of cartilage necrosis. Partial-thickness defects were created in immature pigs. The defects were treated with chondroitinase AC, filled with transforming growth factor-b/matrix as described in the Materials and Methods section, and closed (a, b, h, i, and j),or they were filled with fibrinogen alone (c through g). The defects and the surrounding cartilage were excised either eight days (a and b) or six weeks (c through j) later, frozen, sectioned, and stained with either toluidine blue (c, e, and h) or by indirect immunofluorescence with anti-DIPEN341 (a, d, f, and i) or anti-ITEGE373 (b, g, and j). The bars indicate 30 mm (a and b), 100 mm (c, e, and h), and 20 mm (d, f, g, i, and j). a and b: A defect excised on day 8 has strong DIPEN341 immunofluorescence on the frayed lateral wall but no ITEGE373 staining. c and d: A defect originally filled with fibrinogen has no contents when excised at six weeks. The matrix in the left-hand corner has reduced metachromasia (c) and bright DIPEN341 immunofluorescence (d, rotated 90Â°). e, f, and g: A defect originally filled with fibrinogen is completely filled with repair tissue at six weeks, and the zone of necrosis lateral and basal to the defect has little matrix metachromasia and a chondrocyte cluster in the upper-right corner (e). This cluster is surrounded by matrix with DIPEN341 immunofluorescence (f). A similar section stained with anti-ITEGE373 has no matrix staining but has intracellular immunofluorescence in both chondrocytes and repair cells (g). h, i, and j: A defect originally filled with transforming growth factor-b/matrix, excised at six weeks, is completely filled with repair tissue. The zone of necrosis lateral and basal to the defect has little matrix metachromasia (h), and the cartilage matrix beneath the defect has DIPEN341 staining (i) but not ITEGE373 staining (j). The chondrocytes and repair cells have intracellular staining for both antigens (probably internalized material).

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Fig. 6:: A summary of the major features following formation of partial-thickness defects in pig articular cartilage. Macrophages infiltrating the defects at days 2 and 8 synthesize and deposit MMP-9 onto the damaged cartilage matrix, the zone of cartilage necrosis. The MMP-derived DIPEN341 neoepitope and the loss of matrix metachromasia are colocalized to the damaged cartilage matrix at eight days and at six weeks, indicating aggrecan degradation. Strong induction of MMP-13 synthesis occurs in chondrocyte clusters that form at the interface between the zone of cartilage necrosis and the normal cartilage.

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TABLE I: Summary of Matrix Metalloproteinase (MMP) and Tissue Inhibitor of Metalloproteinase (TIMP)-Staining of Cartilage Remote from Defects*

*The sections were stained as described in the Materials and Methods section. MMP-1, 3, and 9 and TIMP-1 and 2 were scored according to both the number of positive cells and the brightness of intracellular staining, with + indicating one to ten positive cells with faint staining, ++ indicating eleven to 100 positive cells generally with faint staining and occasional bright cells, +++ indicating 101 to 200 positive cells and many bright cells, and ++++ indicating more than 200 positive cells with mostly bright staining.

Age of animal	MMP-1	MMP-3	MMP-9	TIMP-1	TIMP-2
Immature, 4 months	++++	++++	+++	++	+
Immature, 5.5 months	++	++	+	+	+
Adult	+	+	+	+	+

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TABLE I: Summary of Matrix Metalloproteinase (MMP) and Tissue Inhibitor of Metalloproteinase (TIMP)-Staining of Cartilage Remote from Defects*

Age of animal	MMP-1	MMP-3	MMP-9	TIMP-1	TIMP-2
Immature, 4 months	++++	++++	+++	++	+
Immature, 5.5 months	++	++	+	+	+
Adult	+	+	+	+	+

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TABLE II: Matrix Metalloproteinase (MMP)-13 Staining Surrounding Defects*

*The sections were stained as described in the Materials and Methods section. MMP-13 was scored according to both the number of positive cells and the brightness of intracellular staining, with + indicating one to ten positive cells with faint staining, ++ indicating eleven to 100 positive cells generally with faint staining and occasional bright cells, and +++ indicating 101 to 200 positive cells and many bright cells. †ND = not determined.

	Immature	Adult†
2 days	+++	ND
8 days	+++	+++
3 weeks	+	+

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TABLE II: Matrix Metalloproteinase (MMP)-13 Staining Surrounding Defects*

	Immature	Adult†
2 days	+++	ND
8 days	+++	+++
3 weeks	+	+

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Materials

Asolectin, cholesterol, triolein, chloroform, and ether were obtained from Fluka Chemicals (Buchs, Switzerland), and recombinant human transforming growth factor-b2 was obtained from Celtrix Pharmaceuticals (Santa Clara, California). All other chemicals were obtained from Sigma-Aldrich (Poole, Dorset, United Kingdom).

Surgical Procedures

Three-to-four-month-old female large white cross pigs weighing 23 to 28 kg were obtained from the breeding unit at Cambridge University Farm and were maintained on proprietary pig chow ad libitum. The animals received humane care in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. Prior to surgery, the animals were sedated and anesthesia was induced with intravenous injection of ketamine (Vetalar; Parke-Davis Veterinary, Crawley, Sussex, United Kingdom). Surgical anesthesia was maintained with halothane/oxygen/nitrous oxide delivered through an endotracheal tube. Intravenous saline solution (2.5 mL/kg/hr) was administered throughout the procedure. Both stifle joints were exposed through lateral arthrotomy, and longitudinal defects (0.5 mm wide, 0.5 mm deep, and 15 mm long) were cut into cartilage with use of a specially designed gouge²⁵ at five sites: the proximal, distal, medial, and lateral portions of the trochlear sulcus and the medial femoral condyle (Fig. 1). The defects were treated with chondroitinase AC (one unit per milliliter for four minutes²⁵) (Sigma-Aldrich) and were blotted dry, and then the joint was flushed with sterile saline solution and was swabbed dry. A sterile, absorbable gelatin powder (50 mg) (Gelfoam; Upjohn, Kalamazoo, Michigan) was mixed to a paste with fibrinogen solution (20 mg/mL in phosphate-buffered saline solution with transforming growth factor-b2 at a concentration of 5 ng/mL) with liposomes containing transforming growth factor-b2 (500 ng/mL; prepared as described previously³¹). This paste (transforming growth factor-b/matrix) was applied to the defects to fill the groove, and the excess was removed. Thrombin (fifty units per milliliter) was applied sparingly to the filled defects to produce a cross-linked fibrin matrix. Soft tissues were closed routinely in three layers; skin sutures were removed after ten days. Postoperative analgesia (buprenorphine, 0.03 mg/kg) (Vetergesic; Animalcare, York, Yorkshire, United Kingdom) was routinely given, with further opioid analgesia administered as required. At the conclusion of the experiment, the animals were killed with a lethal injection of pentobarbitone (Pentoject; Animalcare) and the defects were excised with the surrounding intact cartilage for immunohistochemical analysis.

Animals with partial-thickness defects containing transforming growth factor-b/matrix were killed at two days (ten defects, two pigs), eight days (ten defects, two pigs), three weeks (ten defects, two pigs), six weeks (twenty defects, four pigs), or eight weeks (ten defects, one pig). In addition, five defects were made in one joint, the joint was closed without further treatment, and the pig was killed at two days. Two other joints received chondroitinase AC treatment only and the animals were killed at two days (five defects, one pig) or eight days (five defects, one pig), and ten defects (two joints, two pigs) were filled with Gelfoam/fibrinogen without growth factor and the animals were killed at six weeks.

Two adult Goettingen minipigs were obtained from the Department of Reproduction Science, University of Zurich, and were housed in the large-animal facility at the University of Bern Veterinary School. They were maintained on proprietary pig chow and received humane care in compliance with the Swiss State Veterinary Office. Bilateral arthrotomy of the stifle joints was performed, and partial-thickness defects were created and were filled with transforming growth factor-b/matrix as described above. The first animal (ten defects), a twenty-eight-month-old, 30-kg male, was killed at eight days, and the second animal (ten defects), a twenty-one-month-old, 30-kg female, was killed at three weeks.

Antibodies

To determine which antibodies to MMPs and TIMPs most efficiently cross-reacted with their respective pig antigens, cross-reaction was initially assessed with Western blotting and immunolocalization on monolayers of pig chondrocytes stimulated with either interleukin-1³² or interleukin-1 and oncostatin M³³. Sheep antisera to the following MMPs were chosen (with the characterizations given in the references): MMP-1³⁴, MMP-2³⁵, MMP-3³⁶, MMP-9³⁷, MMP-13^33,38, and MMP-14³⁹. Immunoglobulins were prepared from each serum by triple ammonium sulfate precipitation⁴⁰ and were used at 50 mg/mL in phosphate-buffered saline solution. The sheep antiserum to pig MMP-9 was further purified to F(ab)2⁴¹ and was used at 10 mg/mL. Normal sheep serum immunoglobulins were used as the controls. For double-labeling of MMP-1 and MMP-13, a mouse monoclonal antibody to MMP-1⁴² (Mac 064; Celltech, Slough, United Kingdom) was used at 5 mg/mL. Mouse monoclonal antibodies to human TIMP-1⁴² (Mac 015; Celltech, used at 5 mg/mL) and human TIMP-2³⁶ (Mac 093; Celltech, used at 0.5 mg/mL), were also shown to cross-react with the respective pig antigens. A mouse monoclonal antibody to pig monocyte-macrophages (MCA 1218) was obtained from Serotec (Oxford, United Kingdom)⁴³, and normal mouse immunoglobulins were obtained from Sigma-Aldrich. Rabbit anti-DIPEN341 (0.5 mg/mL) and rabbit anti-ITEGE373 (5 mg/mL) immunoglobulins⁴⁴ were purified with use of protein A-Sepharose followed by fractionation on ovalbumin-coupled Sepharose to remove reactivity against the ovalbumin carrier conjugated to the peptide immunogen⁴⁵. Normal rabbit immunoglobulins (5 mg/mL), obtained from Sigma-Aldrich, were used as the controls. These immunoglobulins were diluted in phosphate-buffered saline solution with 1% bovine serum albumin. Secondary antibodies were either fluorescein-isothiocyanate-labeled pig anti-sheep Fab¢⁴⁰ (1:200 in phosphate-buffered saline solution), fluorescein isothiocyanate-labeled sheep anti-mouse immunoglobulins (1:200) (Sigma-Aldrich), Texas Red-labeled donkey anti-mouse F(ab)2, or fluorescein-isothiocyanate-labeled donkey anti-rabbit F(ab)2 (1:200) (Jackson Immunoresearch Labs, West Grove, Pennsylvania).

Immunohistochemical Methods

Partial-thickness defects with surrounding cartilage (cut approximately 3 mm on either side and to the bone interface below) were removed from the joints and were cut in half. The proximal portion of each specimen was incubated in Dulbecco modified Eagle medium (Gibco BRL, Paisley, United Kingdom) with 10% fetal bovine serum (Gibco BRL) and monensin (5 mmol) (Sigma-Aldrich) for twenty-four hours at 37Â°C with 5% carbon dioxide in air and then was frozen in liquid nitrogen. The ionophore monensin prevents translocation of secreted proteins, facilitating identification of cells actively synthesizing the proteinase of interest³⁴. The distal portion of each specimen was frozen without culture. Frozen sections were cut to a thickness of 7 mm, placed onto glass slides precoated with poly-l-lysine (Sigma-Aldrich), air-dried briefly, fixed, and stained by indirect immunofluorescence as described previously⁴⁶. Sections for neoepitope antibody staining were pretreated with chondroitinase ABC (Proteus vulgaris; Sigma-Aldrich) for one hour at 37Â°C, as previously described⁴⁵; to remove chondroitin sulfate chains. After antibody treatments, some sections were counterstained with either methyl green or propidium iodide to locate nuclei by red fluorescence. For double-label experiments, the primary antibodies were applied together (one-hour incubation) followed by the sequential addition of fluorescein-isothiocyanate-labeled pig anti-sheep Fab¢ and then Texas Red-labeled donkey anti-mouse F(ab)2 (thirty-minute incubations). Controls were included in which each primary antibody was replaced with the respective normal species’ immunoglobulins and counterstain was omitted. Sections were mounted in either Citifluor (University of Kent at Canterbury, United Kingdom) or Vectashield (Vector Laboratories, Burlingame, California) and were viewed on either a Zeiss photomicroscope III (Carl Zeiss, Welwyn Garden City, Hertfordshire, United Kingdom) or a Bio-Rad MRC 600 confocal microscope with a krypton/argon laser (Bio-Rad Microscience, Hemel Hempstead, Hertfordshire, United Kingdom). Blocks were cut at a minimum of three positions, with at least three sections per site. To provide quantitation, the sections were scored according to the number of positive cells and the brightness of intracellular staining, with + indicating one to ten positive cells with faint staining, ++ indicating eleven to 100 positive cells with generally faint staining and occasional bright cells, +++ indicating 101 to 200 positive cells and many bright cells, and ++++ indicating more than 200 positive cells with mostly bright staining.

After they were photographed with Kodak EPH 1600 film (Hemel Hempstead, Hertfordshire, United Kingdom) processed at 1600 ASA, the sections were stained either with Harris hematoxylin and eosin to determine the morphology or with toluidine blue to determine proteoglycan metachromasia, observed with bright-field microscopy, and rephotographed with Kodak Ektachrome 64 film. The number of cells within the matrix/defect in each of the ten sections stained with hematoxylin and eosin was counted and averaged. Some unfixed air-dried tissue sections were stained for neutral esterase as a macrophage marker with use of a-naphthyl acetate and hexazotised pararosanilin⁴⁷, counterstained with Harris hematoxylin, and observed with bright-field microscopy. Photographs were scanned at 3200 dots per inch on a Nikon LS2000 scanner (Kingston upon Thames, Surrey, United Kingdom), and plates were compiled with PowerPoint and a Fujifilm Pictrography 4000 printer (Bedford, Bedfordshire, United Kingdom).

Results

Introduction | Materials and Methods | Results | Discussion | References

Cartilage explants containing the partial-thickness defects and repair tissue, their surrounding cartilage, and cartilage remote (>5 mm) from the defect site were excised from the joint up to eight weeks after surgery. Cartilage frozen directly without monensin treatment was consistently negative for all MMPs and TIMPs, except for MMP-9, as described in detail below. Cartilage explants that had been cultured in monensin for twenty-four hours before they were frozen had chondrocytes and cells within the defects that stained with green intracellular immunofluorescence, indicating synthesis of MMPs and TIMPs. Sections that had been stained with control normal sheep or mouse immunoglobulins had no green immunofluorescence and had only the red nuclear counterstain, confirming specificity of the immunofluorescence. Data collected from sequential sectioning of these explants showed that the distribution of MMPs and TIMPs in the three areas changed during the eight-week experimental period. These data are described below.

MMP and TIMP Synthesis in Immature and Adult Pig Cartilage Remote from Defects

The cartilage excised from immature pigs was thick (average, 1.5 mm), with many small chondrocytes not yet organized into distinct zones or columns and with abundant highly metachromatic matrix. Intracellular immunofluorescence for MMP-1, MMP-3, or MMP-9 was present in some chondrocytes remote from the defects, indicating enzyme synthesis. Positive cells were randomly distributed in the upper third of the explants (Fig. 2, a and b), similar to the resting zone of rabbit growth-plate cartilage⁴⁸. The explants removed from all five positions in each joint contained positive cells. However, on serial sectioning of each explant, considerable variation was often observed. Some chondrocytes also contained TIMP-1 or TIMP-2, but the staining was generally weaker and less frequent than that seen for MMPs. The average number of chondrocytes with immunofluorescence in each section decreased over the eight-week period (Table I). Weak staining of MMP-1 and MMP-9 was also observed in terminal hypertrophic chondrocytes adjacent to bone in immature cartilage (Fig. 2, c and d), confirming previous observations in other species^48,49. Strong immunostaining of MMP-13 was seen in all of the hypertrophic chondrocytes, but no staining was observed in the upper zones (Fig. 2, e). No staining of either MMPs or TIMPs was seen in the extracellular matrix.

The cartilage excised from adult minipigs was about 0.7 mm thick, with sparse chondrocytes organized into well-defined zones and highly metachromatic matrix. Immunofluorescence of MMP-1, 3, and 9 and TIMP-1 and 2 was present in a few transitional-zone chondrocytes (Table I). Staining of MMP-13 was present in lower radial-zone chondrocytes adjacent to bone (Fig. 2, f), but these cells did not contain MMP-1 or MMP-9. These data suggest that MMPs and TIMPs play a role in the normal development and growth of pig cartilage, as they do in other species^37,48, and that the rate of synthesis declines with increasing age.

Response of Chondrocytes to Damage

In immature pigs, the defects penetrated approximately one quarter of the cartilage depth. Two days after defect creation, a necrotic area (the zone of cartilage necrosis) was present adjacent to every cut surface, generally extending 100 to 200 mm laterally but less into the articular cartilage beneath the defect (Fig. 3, c). Within this area, chondrocyte nuclei were either absent or pyknotic and the cartilage matrix had slightly reduced metachromasia on toluidine-blue staining. This zone of cartilage necrosis was present in untreated (control) explants (Fig. 3, a and c), chondroitinase-AC-treated defects (Fig. 3, e), and defects treated with transforming growth factor-b/matrix, and it was present in all of the explants taken at later times (eight days to eight weeks) without exception. The defects treated with chondroitinase AC (with or without transforming growth factor-b/matrix) had substantially reduced metachromasia along articular-cartilage surfaces adjacent to the defects and on necrotic cartilage matrix (Fig. 3, e). In adult cartilage, the defects penetrated two-thirds or more of the total depth because of the reduced cartilage thickness, and the zone of cartilage necrosis surrounded all of the defects at eight days (Fig. 3, j) and at three weeks.

Adjacent to the zone of cartilage necrosis, many chondrocytes stained strongly for MMP-13. This induction of chondrocyte MMP-13 synthesis occurred both laterally and in the articular cartilage beneath the middle of the defect. It was present in untreated (control) defects (Fig. 3, b and d), chondroitinase-AC-treated defects (Fig. 3, f), and defects filled with transforming growth factor-b/matrix (Fig. 3, i). Dual-labeling experiments demonstrated that, in some but not all cells, this was coincident with MMP-1 (data not shown). Many chondrocyte lacunae contained two or more nuclei, suggesting induction of cell division together with MMP-13 synthesis (Fig. 3, h).

Eight days after defect creation, chondrocyte clusters bordering the zone of cartilage necrosis contained increased numbers of nuclei, and strong staining of MMP-13 in these chondrocyte clusters was observed both in immature pigs (Table II) and in adult minipigs (Fig. 3, j and k). Staining of MMP-1, MMP-3 (Fig. 3, l), and TIMP-1 (Fig. 3, m) was also observed within the chondrocyte clusters. In immature tissues, this immunofluorescence was more pronounced than in cartilage remote from the defects, suggesting upregulation. In adult minipig explants, where there was lower baseline synthesis in the surrounding tissues, induction of synthesis was seen (Fig. 3, l and m). However, this immunofluorescence was not as strong as the MMP-13 staining.

At three, six (Fig. 5, c), and eight weeks, the expansion of chondrocyte clusters continued and the size of the chondrocyte clusters increased, but only occasional cells were now positive for either MMP-1, 3, 13, or TIMP-1. Chondrocyte clusters containing more than sixteen nuclei per cluster per section were frequently seen lateral to the defects in the immature pigs by eight weeks. Fewer nuclei were observed in the chondrocyte clusters beneath the defects. Close examination of serial histological sections showed that this expansion could lead to blebbing of chondrocyte clusters through the necrotic zone into the defect lumen, a feature also reported to occur in the rabbit⁵⁰.

MMPs and TIMPs in Repair-Tissue Cells and Matrix

Of the five untreated explants excised on day 2, two defects were empty (Fig. 3, a) but the other three contained loose eosinophilic material, probably autologous fibrin, that either lined the defect sides and covered the base or filled the defect (Figs. 3, c and 4, a). Cells were present within this matrix, and the number of cells entrapped was approximately proportional to the amount of fibrin/matrix present. Sections of defects filled with transforming growth factor-b/matrix at the time of the operation contained varying amounts of (heterologous) fibrin and Gelfoam, with increased numbers of cells often surrounding the Gelfoam (Figs. 3, g and 4, d).

When the defects that were excised on day 2 were stained with antibodies to MMPs and TIMPs, intracellular MMP-9 was seen in cells in the untreated (control) defects (Fig. 4, b), chondroitinase-AC-treated defects (Fig. 4, c), and defects treated with transforming growth factor-b/matrix (Fig. 4, e through j). In the third group, the cells were adherent to intact articular-cartilage surfaces (Fig. 4,, e) and to damaged cartilage defect edges (Fig. 4,, g). Dual-labeling with cell-marker antibodies identified these cells as monocyte-macrophages (Fig. 4, f and h); this finding was confirmed by staining consecutive sections for neutral esterase, after which cells within the defects stained with brick-red deposits characteristic of macrophages. MMP-9 staining was also observed on damaged (necrotic) cartilage matrix adjacent to positive cells (Fig. 4, g), suggesting directional secretion, whereas no matrix staining was present on intact articular surfaces (Fig. 4, e). Directional secretion was also seen in chondroitinase-AC-treated defects (Fig. 4, c), and in untreated (control) explants the cartilage matrix in the zone of cartilage necrosis also stained for MMP-9 (Fig. 4, b). A few cells within the defects also stained positively for TIMP-1 (Fig. 4, i), MMP-14 (Fig. 4, j), and MMP-1.

The immature pig explants excised on day 8 also had MMP-9-positive cells within the defects, with staining on defect contents and on adjacent damaged cartilage matrix extending throughout the zone of cell necrosis (Fig. 4, k and l). This MMP-9 staining of necrotic matrix occurred in ex vivo tissue (for example, day-8 explants frozen without culture in monensin) and in the zone of cartilage necrosis in adult minipig explants but was absent at three weeks in immature and adult pigs. On day 8 (as on day 2), a few cells within the defects stained positively for TIMP-1, MMP-1, and MMP-14 (Fig. 4, j) in both immature and adult pigs. Dual-labeling of minipig defect cells with antibodies to both MMP-14 and macrophage marker showed that cells staining strongly for MMP-14 were deep in the defect but did not stain with the macrophage marker, whereas surface cells stained strongly with macrophage marker but did not contain MMP-14. However, a few cells stained weakly for both antigens, suggesting that they were undergoing a change in phenotype involving loss of marker antigen with onset of MMP-14 synthesis (Fig. 4, j).

At three, six, and eight weeks, the defect contents became increasingly compact, cellular, and fibrous, with soft tissue often overfilling and overlapping with the adjacent articular cartilage²⁵. At six weeks, seventeen of the twenty defects treated with transforming growth factor-b/matrix and eight of the ten defects filled without growth factor contained repair tissue. Occasional cells staining for MMP-1, 3, or TIMP-1 were seen, suggesting focal remodeling as the repair tissue matured.

The antibody to MMP-9 recognizes both pro and active enzyme forms. Since we observed considerable matrix staining in the zone of cartilage necrosis, we then used specific antibodies to the MMP cleavage C-terminal neoepitope of aggrecan, DIPEN341, to look for evidence of aggrecan degradation. Sections of cartilage that were excised, on day 8, from the region adjacent to those that stained strongly for MMP-9 (Fig. 4, k and l) had bright DIPEN341 immunofluorescence in the zone of cartilage necrosis matrix (Fig. 5, a), suggesting that MMP-9 had been activated, leading to hydrolysis of aggrecan. Parallel sections that were stained with an antibody to the ITEGE373 neoepitope that is formed on hydrolysis of the E373ØA374 bond in the interglobulin domain of aggrecan by aggrecanases had no matrix staining and had only intracellular staining (Fig. 5, b), as reported previously in pig-cartilage cultures stimulated with interleukin-1a and retinoic acid⁴⁵. Since MMP-9 staining in the zone of cartilage necrosis was no longer apparent at three weeks, we then examined tissues excised six weeks after defect creation to determine whether the degraded aggrecan was replaced during repair. Figures 5, c and 5, d show a defect, originally filled with fibrinogen only, in which no contents remained at six weeks (Fig. 5, c); the zone of cartilage necrosis matrix had anti-DIPEN341 immunofluorescence (Fig. 5, d) but no matrix ITEGE373 fluorescence. Defects originally filled with fibrinogen alone (Fig. 5, e, f, and g) or with transforming growth factor-b/matrix (Fig. 5, h, i, and j), both of which were completely filled with repair tissue at six weeks, had a band of anti-DIPEN341 immunofluorescence in the zone of cartilage necrosis matrix surrounding the defects (Fig. 5, f and i), whereas the ITEGE373 epitope was absent in the matrix but appeared to be intracellular (Fig. 5, g and j).

These data, together with the loss of toluidine blue metachromasia, provide evidence for the proteolysis of aggrecan in the zone of cartilage necrosis by MMPs and indicate that this region remains throughout the experimental period examined, without replacement by repair tissue.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

MMP-13 has a broad spectrum of activity against extracellular matrix proteins, including collagenolytic activity⁹, degradation of cartilage aggrecan¹⁰, and an ability to degrade cartilage type-II collagen preferentially^4,9. In this study, we showed specific immunolocalization of MMP-13 in hypertrophic chondrocytes in immature pigs and in the radial zone of adult minipig cartilage. This finding agrees with data obtained from studies of human fetal bone development in which MMP-13 mRNA was found to be specifically expressed by hypertrophic chondrocytes^2,3. No MMP-13 staining occurred in upper cartilage zones; this observation is in agreement with the findings of Moldovan et al.⁵, who showed that only 3% of cells stained for MMP-13 in normal (aged) human cartilage. A study of fracture repair in mouse ribs showed that MMP-13 was induced during fracture repair and was detected primarily in hypertrophic chondrocytes⁵¹. In addition, MMP-13 gene expression in cultured prehypertrophic chick chondrocytes was recently shown to be a hypertrophy-related event⁵². Since the process of hypertrophy involves controlled and efficient digestion of type-II and type-X collagens (and other matrix proteins), the abundant expression of MMP-13 by hypertrophic chondrocytes strongly suggests that this enzyme may participate in this proteolysis.

In an early study of the histological response of immature dog cartilage to damage, formation of new, multinucleated chondrocyte clusters was observed in the surrounding cartilage²⁸. Formation of chondrocyte clusters adjacent to the zone of cartilage necrosis also occurred in immature and adult pigs in our study, and we found that these newly forming chondrocyte clusters stained strongly for MMPs, particularly MMP-13. Chondrocyte clusters are recognized as typical features of osteoarthritic cartilage, and MMP-13 mRNA expression was recently found to codistribute with type-II collagen-degrading activity, assayed by in situ hybridization, only in osteoarthritic cartilage⁵³. An in vitro study has shown that the production of MMP-13 by human osteoarthritic chondrocytes depends on the physiologic state of the cell, with transforming growth factor-b2 able to specifically trigger MMP-13 production over that of MMP-1⁵⁴. However, our data show that newly forming chondrocyte clusters in wounded cartilage stained strongly for MMP-13 irrespective of whether the cartilage had been exposed to exogenous growth factor. This increased expression implicates MMP-13 in the focal matrix remodeling necessary for chondrocyte lacuna expansion (as in the hypertrophic zone), but it may also indicate involvement in chondrocyte proliferation resulting from cartilage damage^28,55. Previous in vitro work has shown that disruption of the cartilage collagen architecture with purified bacterial collagenase stimulates DNA replication⁵⁶. Cleavage of type-II collagen to 1/4 and 3/4 fragments by MMP-13 and proteolysis of pericellular matrix components (such as type-VI collagen, cartilage oligomeric protein, and decorin) either by MMP-13 or MMP-3 synthesized by chondrocyte clusters or MMP-9 (see below) may lead to disruption of chondrocyte-matrix attachment with resultant cell death adjacent to the repair area^57,58.

Repair cells repopulating partial-thickness defects have been shown histologically to include mesenchymal cells and monocyte-macrophages^25,50, and our findings with cell-marker antibodies confirmed this. We found that cells migrate into both unfilled and filled defects, that the number of cells in untreated (control) defects can vary depending on the amount of autologous fibrin deposited, and that the introduction of growth factor, heterologous fibrin, and Gelfoam causes an increase in the number of cells, in agreement with previous studies^22,25,50. However, the full spectrum of differences between defects treated with and without growth factor previously described^22,25,50 was not apparent in our study because of the early times at which the tissues were examined.

We demonstrated that macrophages synthesize and secrete MMP-9 onto both the fibrillar defect contents and the damaged cartilage surfaces depleted of proteoglycan. By day 8, extracellular matrix binding of MMP-9 was present throughout the zone of cartilage necrosis along the marginal walls of each defect. MMP-9 secretion and binding to damaged matrix was observed in both control and growth-factor-treated defects. MMP-9 has wide substrate specificity; it is secreted as a proenzyme and can be activated by MMP-3, plasmin, or cathepsin G, and a further potential activation cascade involving MMP-2, 13, and 14 has been described³³. Because our antibody does not differentiate between pro and active MMP-9, we used neoepitope antibodies to show that proteolysis at the MMP cleavage site of aggrecan along the zone of cartilage necrosis had taken place. The MMP-derived DIPEN341 neoepitope was present at day 8, indicating aggrecan cleavage in the interglobular domain. This was most likely due to macrophage MMP-9, but MMP-13 secreted by adjacent expanding chondrocyte clusters also may have contributed to the cleavage of aggrecan core protein. Loss of metachromasia colocalized with DIPEN341 neoepitope was also noted at six weeks following defect creation regardless of whether the defect had filled with repair tissue; this demonstrated that the aggrecan had been proteolytically cleaved but was neither resorbed nor replaced. Additional long-term studies are now required to determine whether the cleaved aggrecan is ever replaced by newly synthesized intact molecules, and analysis of equivalent human repair tissue would be informative, should the opportunity arise.

Repair cells within defects, probably macrophages, synthesize MMP-14 as well as MMP-1 and TIMP-1, but neither MMP-2 nor TIMP-2 was colocalized with MMP-14 and neither was identified in adjacent cells within the defects. To our knowledge, this is the first report of synthesis of MMP-14 by porcine monocyte-macrophages. However, MMP-14 has been detected immunohistochemically in alveolar macrophages in human pulmonary emphysema⁵⁹, in monocyte-macrophages in periprosthetic tissues from loosened total hip prostheses⁶⁰, and in CD68-positive cells in the lining and sublining layer of rheumatoid synovial tissue⁶¹. MMP-14 degrades many extracellular matrix proteins, including aggrecan^62-64. It may be involved in remodeling the repair tissue as well as in contributing to MMP-mediated aggrecan catabolism in the zone of chondrocyte necrosis.

Recent in vitro studies of the reaction of bovine articular cartilage⁶⁵ and embryonic chick cartilage⁶⁶ to experimental wounding found mechanical damage to type-II collagen fibrils at wound edges on day 0 immediately after wounding, with concomitant loss of metachromasia suggesting that proteoglycan depletion also occurs rapidly after damage. The present study (summarized in Fig. 6) has now shown that, within a few days, aggrecan in the zone of necrosis undergoes proteolytic cleavage brought about by mesenchymal cells and macrophages recruited into defects to begin the repair process and by proteinase secretion by adjacent expanding chondrocyte clusters. Since aggrecan endows cartilage with its capacity to bear load and resist compression, its proteolysis, together with mechanical damage to type-II collagen fibrils, means that this region is mechanically weak and that, over time, compressive stresses will lead to degenerative changes⁶⁷. This weakness may be exacerbated by the perturbation of chondrocyte-extracellular matrix attachment in the adjacent zone of chondrocyte-cluster formation. New strategies are now required to induce complete degradation and removal of the zone of cartilage necrosis and to elicit its replacement by new tissue in which the repair tissue is integrated with the surrounding cartilage

Note: We thank the staff of Cambridge University Farm and the University of Bern Veterinary School large-animal facility for expert care of the pigs, and we thank Mrs. Johanna Burge for technical assistance. Special thanks go to Mr. Chris Green, Department of Biochemistry, Cambridge University, for use of photographic facilities. We are grateful to the Medical Research Council, United Kingdom, and Orthogene, Sausalito, California, for financial support. R.M.H. received a Royal Society United Kingdom study travel award to undertake a University of Melbourne Collaborative Research Program in the Department of Paediatrics.

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TABLE I: Summary of Matrix Metalloproteinase (MMP) and Tissue Inhibitor of Metalloproteinase (TIMP)-Staining of Cartilage Remote from Defects*

Age of animal	MMP-1	MMP-3	MMP-9	TIMP-1	TIMP-2
Immature, 4 months	++++	++++	+++	++	+
Immature, 5.5 months	++	++	+	+	+
Adult	+	+	+	+	+

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TABLE I: Summary of Matrix Metalloproteinase (MMP) and Tissue Inhibitor of Metalloproteinase (TIMP)-Staining of Cartilage Remote from Defects*

Age of animal	MMP-1	MMP-3	MMP-9	TIMP-1	TIMP-2
Immature, 4 months	++++	++++	+++	++	+
Immature, 5.5 months	++	++	+	+	+
Adult	+	+	+	+	+

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TABLE II: Matrix Metalloproteinase (MMP)-13 Staining Surrounding Defects*

	Immature	Adult†
2 days	+++	ND
8 days	+++	+++
3 weeks	+	+

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TABLE II: Matrix Metalloproteinase (MMP)-13 Staining Surrounding Defects*

	Immature	Adult†
2 days	+++	ND
8 days	+++	+++
3 weeks	+	+