Insulin-like growth factor-I gene expression patterns during spontaneous repair of acute articular cartilage injury

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Journal of Orthopaedic Research Journal of Orthopaedic Research 19 (7001) 720-718 www.elsevier.nl/locate/orthres

Insulin-like growth factor-I gene expression patterns during spontaneous repair of acute articular cartilage injury Lisa A. Fortier a, Cheryl E. Balkman a, Linda J. Sandell b, Anthony Ratcliffe ', Alan J. Nixon a,* '' Comnpuruiire Orthopueriics Laborntor}: College i f I 'eterinury Medicine, Cornell Uniiiersity, Iihacii, New York, USA Department of' Orthopuedic Surgery, W'ushingion Unitwsitj~School of Medicine, St Louis, MO. US.4 Adiwced Tissue Scirnces, Inc., La Jollu, USA

Received 21 December 1999; accepted 13 March 1000

Abstract

This study evaluated the constitutive insulin-like growth factor-I (IGF-I) gene expression pattern in spontaneously healing cartilage defects over the course of 16 weeks, and correlated the tissue morphology and matrix gene expression with IGF-I m R N A levels. Full-thickness 15 mm cartilage defects were debrided in the femoral trochlea of both femoropatellar joints of 8 horses and the healing defects examined 2, 4, 8, or 16 weeks after surgery. Samples were harvested for histologic assessment of tissue healing using H&E staining, toluidine blue histochemical reaction for proteoglycan deposition, and in situ hybridization and immunohistochemistry procedures to demonstrate collagen type I1 mRNA and protein expression. Total R N A was isolated for Northern analysis to measure cartilage matrix molecule expression, and for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) to determine IGF-I gene expression patterns in healing cartilage defects. Full-thickness cartilage defects in horses were slow to heal compared to smaller lesions in similar locations in other animals. However, a progressive decline in tissue cellularity and vascularity, and increased tissue organization were observed on H&E stained specimens over the 16-week experiment. Evidence of early chondrogenic repair was detected through collagen type 11 in situ hybridization and immunohistochemistry. However, levels of collagen type I1 and aggrecan mRNA in lesions were not abundant on Northern analysis indicating incomplete chondrogenesis. IGF-I message expression followed a cyclic pattern with low levels at 2 weeks, followed by an increase at 4 and 8 weeks, and a subsequent decline at 16 weeks. There was no direct correlation between the stage of healing and cartilage matrix message expression, and the abundance of IGF-I mRNA in the healing lesions. In conclusion, this study demonstrated that the spontaneous healing of articular defects was accompanied by a temporal fluctuation in IGF-I gene expression which was discoordinate to the steady rise in expression of cartilage matrix molecules such as procollagen type 11. 0 1001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Introduction

Many studies show that articular cartilage defects are repaired with fibrocartilage and that extensive cartilage loss can precipitate the development of osteoarthritis [4,12,13,30,24]. Maintaining or restoring normal cartilage homeostasis depends on an intricate balance between anabolic and catabolic peptides influencing cartilage metabolism [18]. Injury to a cartilage surface can destabilize this normal homeostatic balance in favor of catabolism resulting in cartilage destruction and joint * Corresponding author. Tel.: + 1-607-253-3050; fax: + 1-607-333271. E-miif uddrt..,&.ajn l(@ornell.edu (A.J. Nixon).

dysfunction. Insulin-like growth factor-I (IGF-I) is one of the more important of these anabolic peptides. It has been shown in vitro to enhance cartilage matrix metabolism, both through increased synthesis of aggrecan, hyaluronan, and link protein, and through the inhibition of proteoglycan degradation, culminating in maintenance of a net positive proteoglycan balance [6,17,33, 36,371. In addition, IGF-I aids in maintenance of the mechanical properties of cartilage, including the equilibrium modulus and electrokinetic coefficient [32], and it protects cartilage from the matrix degradative events following exposure to interleukin- 1 and/or tumor necrosis factor-ct [11,37]. These effects suggest a role for IGF-I in restoring cartilage homeostasis following insult or injury.

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Articular chondrocytes synthesize IGF-I, and this synthesis is increased both in animal models of early osteoarthritis and in naturally occurring osteoarthritis [26,29,40]. In the osteoarthritic state, chondrocytes are less responsive to the anabolic effects of IGF-I, even though there is increased ligand available in the synovial fluid and cartilage matrix [2,8,22,35]. By contrast, in a study investigating a rabbit model of full-thickness cartilage defects, repair cells were significantly more responsive to IGF-I supplementation than normal articular cartilage retrieved from the same animal [28]. Repair cells were cultured ex vivo after 3, 6, and 12 weeks of intrinsic healing and treated with exogenous IGF-I. The repair cells responded to IGF-I supplementation through increased synthesis of type I1 collagen, glycosaminoglycans, and increased [35S]-incorporation into newly synthesized proteoglycans. Similarly, bone marrow-derived mesenchymal stem cells, which likely repopulate a full thickness cartilage defect, have enhanced chondrogenic potential when cultured in vitro and supplemented with exogenous IGF-I [9]. In a rabbit model of fullthickness cartilage injury, type I procollagen mRNA was detected in the repair tissue by in situ hybridization just 3 days after creating the defect [25]. Type I1 procollagen mRNA was identified after 14 days, and continued to increase throughout the month study period; however, type I collagen was still expressed at high levels. The study also reported Northern blot analysis data, however, the tissue from which the RNA was derived not only contained tissue from within the defect, but also intentionally included 1 mm of cartilage from the surrounding area and therefore is not solely representative of intrinsic repair tissue. Combined, these studies indicate that the cells which initially populate an articular defect following full-thickness cartilage loss are capable of synthesizing collagen type I1 and proteoglycans. Further, that repair cells are receptive to stimulation by IGF-I, and respond by enhanced synthesis of the major matrix molecules, suggesting a possible role for IGF-I in enhanced cartilage repair. However, there are no data concerning the intrinsic expression of IGF-I in acute or chronic healing cartilage defects to provide insight into appropriate timing for exogenous IGF-I supplementation as an aid in cartilage repair. The objective of this study was to examine IGF-I gene expression over time in healing cartilage lesions and to correlate the expression of IGF-I with expression of major matrix molecules such as aggrecan and type I1 collagen. Our hypothesis was that the expression of IGF-I in healing articular cartilage defects would increase over time, and that the increase in expression would be correlated with expression of the chondrocyte phenotype.

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Materials and methods This project was approved and performed according to guidelines of the Institutional Animal Care and Use Committee of Cornell University. Surgical prucedurr

Eight adult horses ranging in age from 3-5 years were studied. Horses were treated pre-operatively and post-operatively, with antibiotics and a non-steroidal antiinflammatory agent. Anesthesia was administered and each femoropatellar joint was arthroscopically explored to rule out any pre-existing cartilage lesions. A 15-mm diameter spade bit cutter, pre-measured to drill 3 mm deep, was introduced into the femoropatellar joints and used to create two full-thickness cartilage defects, 25 mm apart, in the lateral trochlear ridge of each femur. To mimic current clinical recommendations following surgery for cartilage injury, horses were confined to box stall rest for 4 weeks, then allowed light exercise. Two horses were euthanized at 2, 4. 8, and 16 weeks post-operative by IV administration of an overdose of sodium pentobarbital. Tissue harivst Following euthanasia, the joints were opened and photographed, and cartilage was removed from the lesion, 1 cm peripheral to the lesion (perilesional), and >2 cm peripheral to the lesion (remote). Tissue harvested from the two defect sites within each joint were pooled, but tissues from left and right joints were kept separate. Tissue from each of the three locations (lesion, perilesion, remote) was snap frozen in liquid nitrogen for later isolation of RNA, and samples for histology were harvested and fixed in 4Yo paraformaldehyde. Insufficient sample was available for protein analysis. Histulugy

Fixed cartilage specimens were processed for H&E and toluidine blue staining, in situ hybridization to collagen types I and 11, and immunohistochemistry to collagen type I1 as previously described [lo]. RNA Isolation

Total cellular RNA was isolated according to the method of Chomczynski and Sacchi [ 3 ] , combined with silica-based spin columns for total RNA isolation (RNeasy, Qiagen, Chatsworth, CA). The approximate yield was 10-15 pg of RNAlsample. RNA was used for quantitative polymerase chain reaction and Northern blot analysis. Reoer.w trunscripiionlquantitatiiiepol.~wwrasechain reuciion Methods for establishing a non-competitive quantitative polymerase chain reaction (qPCR) assay have been described [7,30,3 1,381. Reverse transcription (RT) was performed with 1 pg total RNA, oligodT primers (Gibco, Gaithersburg, MD) and Moloney murine leukemia virus reverse transcriptase (Gibco). A 1.5 dilution of the R/T mixture was used for subsequent polymerase chain reaction (PCR) utilizing equine IGF-I gene-specific intron-spanning primers to exclude product from any contaminating genomic DNA. Tuq DNA polymerase (Promega) was used to amplify the cDNA samples for quantitation. Standards for quantitation of PCR products were generated by RTI PCR of equine liver RNA, using the same primers as described above. The RTlPCR product was ligated into the pCRII TA cloning vector using the TA cloning kit (Invitrogen, San Diego, CA. The ligation mix was transformed, cultured, and plasmids were isolated. Plasmid DNA was analyzed by sequence analysis to confirm presence of the IGF-I insert, and the DNA concentration was determined by ultraviolet spectroscopy at A260.Plasmid DNA was used to determine the cycle number necessary to give product at approximately the midpoint of the linear amplification range. PCR was then simultaneously performed on 10 p1 of diluted (1:5) RIT samples and on seventeen-10 p1 aliquots of standard plasmid DNA containing IGF-I template at concentrations covering a range of template copy numbers known to produce a lop/ log linear standard curve of pixel intensity vs. copy number when separated on an ethidium-stained agarose gel. PCR products were

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electrophoresed on an agarose gel, the gel was stained with ethidium bromide and photographed. The photograph was scanned, and the pixel intensity of each PCR product-band was determined (Adobe PhotoShop 5.0, NIH ImagePCcl9 software). Using the standard curve data, pixel intensity of the RT-PCR product was transformed into copy number of IGF-I, and the data were normalized to 1.18total RNA. Northern blot analysis

Ten pg of total RNA was resolved on a 1.5% agarose-formaldehyde gel and transferred by capillary blotting to a nylon membrane (Micron Separations, Westborough, MA) in 1OX SSPE, overnight. The RNA was cross-linked to the membrane by ultraviolet light exposure. A 201 bp cDNA probe for total type I1 collagen (IIB + IIA) was made from the same construct used for in situ hybridization. A 416 bp cDNA probe for equine COLl A1 procollagen mRNA, 65 nucleotides coding for the signal peptide and 351 bp coding for the NH? propeptide, corresponding to positions 135-551 of the available equine coding sequence (Genbank accession number-AF034691), was cloned into pGEM-3zf(+) expression vector. The aggrecan probe coded the entire GI--G? interglobular domain (447 nt) plus 35 upstream and 65 downstream nucleotides. The cDNA for elongation factor l a (EF-la; courtesy Dr. Roy Levine, Department of Molecular Medicine, Cornell University) was used to standardize variable RNA loading [16]. Each of the cDNA probes was restriction digested, purified, and [32P]-CTP labeled according to the manufacturers directions (Random Primers DNA Labeling System, Gibco). Hybridization was performed overnight at 54"C, with 1 t lo6 cpmlml hybridization solution. Kodak BioMax MS film was exposed with BioMax screens for various lengths of time at -70°C. After each hybridization, the blot was washed with ? i SSC/O.l% SDS for 1 h with 2 changes followed by 0.1 Y SSC/O.l% SDS for 1 h with 2 changes. The radiographs were scanned and the pixel intensity of each hybridization product was determined (Adobe PhotoShop 5.0, NIH ImagePCa9 software). The pixel intensity of each hybridization band was normalized to the pixel intensity of EF-lr from the same sample. Stiitistical analysis

To limit the number of animals euthanized for this study, the results of biochemical and molecular analyses from two horses at each time point were evaluated by regression analysis with Y = u a(effect of joint) b(effect of time), to determine if the left and right joints in a single animal could be considered independent variables to arrive at an n = 4 for further statistical evaluation. Regression analysis results indicated that there was no effect of joint operated on any outcome measured, confirming that each joint could be considered independently. Following regression analysis, a one-way analysis of variance (ANOVA)was used to compare copy # of IGF-I in lesional, perilesional, and remote samples at 2, 4, 8, and 16 weeks, and to assess differences in Northern blot message hybridization between the same 3 sites and 4 time periods. A Tukey's post-hoc test was utilized for ANOVA's with significant F-tests. A P 0.05 was considered significant.

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Results Gross

At euthanasia, the cartilage defects were healed with areas of white fibrous-appearing tissue, occupying 2040% of the lesion circumference and 10-200/0 of the lesion depth (Fig. I ). Perilesional and remote cartilages were grossly normal. Within each joint, the amount of repair tissue in each of the two defects appeared different, but there was no consistent pattern as to improved or less healing in the proximal vs. distal lesion.

Fig. 1. Macroscopic photograph of repair tissue formed 16 weeks after surgery. There is incomplete filling in both surgically created defects in the lateral trochlear ridge of the femur. 850091 =case number, L = left limb.

Histologji

Repair tissue evaluated through H&E staining displayed signs of limited chondrogenesis, similar to previously described studies in smaller animal species [25]. There was an increase in overall tissue and cellular organization and a decrease in repair tissue cellularity over time (Fig. 2). Two weeks post-operative, tissue samples consisted of disorganized granulation tissue with stellate-shaped cells and small blood vessels. Repair tissue in 4- and 8-week samples was fibrous in appearance, and more organized, particularly in the superficial layers. By 16 weeks, there was increased organization and decreased cellularity of the repair tissue. Although no semi-quantitative assessments of histologic parameters were made, there were some obvious trends in matrix molecule expression and synthesis. There was no evidence of collagen type I1 on immunohistochemistry or in situ hybridization in the 2 week lesion-tissue samples, and similarly, no evidence of matrix metachromasia indicative of proteoglycan synthesis. Collagen type I1

L.A. Fortier et al. I Journal of Orthopaedic Research 19 (20011 720-728

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pattern of IGF-I gene expression. Expression of IGF-I was significantly greater in the healing cartilage defects than in the perilesional (1 cm from lesion) and remote (>2 cm from lesion) cartilages during all time points examined (Fig. 4). IGF-I expression increased in the healing cartilage defects during the later phases of cartilage repair, peaking in the 8-week samples and decreasing at 16 weeks. Expression of IGF-I peaked at 8 weeks in perilesional cartilage, and at 4 weeks in remote cartilage. Matrix gene expression

Fig. 2. Repair cell morphology in healing articular cartilage defects at 2 (A), 4 (B), 8 ( C ) , and 16 (D) weeks after full-thickness cartilage debridement. Cellularity declined and structural organization of the new tissue increased over time. Bar = 40 pm.

was detected at 4 weeks, and was increased at 8 and 16 weeks (Fig. 3). Hybridization to type I collagen was apparent in repair tissue at all time periods. Comparison of defect tissue to normal (remote) cartilage at 16 weeks indicated reduced type I1 collagen message and protein expression on in situ hybridization and immunoreaction studies, respectively, and reduced matrix metachromasia. Conversely, increased collagen type I persisted at 16 weeks. In all perilesional cartilage samples, matrix metachromasia was decreased and type I collagen gene expression was focally increased immediately adjacent to the lesion edge. However, there were no changes in intensity of collagen type I1 immunoreaction or in type I1 gene expression, relative to the adjacent normal cartilage. No histologic abnormalities were detected in remote cartilage samples.

IGF-I mRNA expression Analysis of mRNA derived from the various articular sites demonstrated a time- and location-dependent

There was no type I1 procollagen expression on Northern blots from 2 week lesional tissue samples and low levels of expression in the 4-, 8-, and 16-week lesional tissue samples. Expression of procollagen type I1 in remote and perilesional sites was similar when compared at each time period (Fig. 5(A)). However, assessment of collagen type I1 expression over time within the perilesional cartilage samples, revealed reduced type I1 mRNA levels at 2 weeks, which returned to levels equal to that of remote cartilage by 4 weeks, and were maintained at normal levels in the 8- and 16-week samples. There was no aggrecan expression in 2 week lesional samples, and low levels of expression in the 4-, 8-, and 16-week samples. Expression of aggrecan mRNA in perilesional and remote cartilages increased with time, and was increased in remote cartilage compared to lesional and perilesional areas (Fig. 5(B)). Expression of aggrecan in remote cartilage was significantly greater than in perilesional cartilage during all time points. The expression pattern of procollagen type I followed an inverse pattern compared to procollagen type I1 and aggrecan (Fig. 5(C)). There was significantly greater expression of type I procollagen mRNA in lesional tissue compared to perilesion and remote during all time points examined. In perilesion tissue, expression of type I procollagen increased from week 2 to week 4 where it plateaued for the remainder of the study. There was no detectable expression of type I collagen in remote cartilage during any time point.

Discussion This study confirmed the slow but progressive healing of full-thickness cartilage defects over time, corresponding to previous reports of healing in full-thickness cartilage defects [ 12,331. While the patterns of histologic repair observed in this study were no different to previous studies in smaller animal species, the rate of chondrogenic repair in this large animal model was apparently decreased compared to the rabbit [12]. Despite this, the larger experimental animal allowed a more complete spatial and quantitative analysis of spontane-

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Fig. 3. Expression of collagen type I1 in healing cartilage defects from 2 weeks to 16 weeks. In situ hybridization data, shown as bright field (A-D) and corresponding dark field photomicrographs (E-H), indicate increasing collagen type I1 expression. Formation of collagen type IT is confirmed by inimunohistochemistry (ILL), where coordinate increases in collagen with time are evident. Photomicrographs represent surface one-half to twothirds of healing cross-sections. Bar = 40 pm.

ous articular cartilage repair through the increased quantity of retrieved tissue. After sampling cartilage for histologic examination, the remaining repair tissue from the two 15 mm defects within each joint was pooled for RNA isolation, providing sufficient RNA for Northern blot analysis and RT-PCR. The lateral trochlear ridge of the distal femur was chosen as the site for cartilage injury because it is a large flat articular surface, a common site for clinical disease, and a site that is not loaded as aggressively in the immediate postoperative period compared to the femoral condyle. Uncontrolled

weight bearing on the condyle within hours of surgery is a poor model for most rehabilitative programs in use today. Expression of IGF-I increased with time after injury, to reach a peak at 8 weeks. The impact of high levels of IGF-I on the homeostasis of normal cartilage is well known, however, its role in healing defects is less well understood. Numerous studies document the ability of IGF-I to stimulate the synthesis of cartilage matrix proteins [6,17,23], protect cartilage proteoglycan from the catabolic effects of interleukin-1 and tumor

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Fig. 4. Scanned, ethidium bromide-stained agarose gel containing IGF-1 RT-PCR products from lesion, perilesion, and remote sample site harvested at 2, 4, 8, and 16 weeks after surgically creating the cartilage defects. There is increased IGF-I RT-PCR product in lesional tissues compared to perilesional and remote sites at each time period, with a peak in IGF-I expression at 8 weeks post-operative (A). Graphic representation of IGF-I RT-PCR products (B). Each bar represents n = 4 i SEM. Tukey's classification letters indicate significant differences within a site (lesion, perilesion, or remote) due to time (2,4, 8, or 16 weeks).

necrosis factor-a [11,37], and to maintain the normal biomechanical properties of cartilage tissue [32]. Of particular relevance to the current study, repair cells populating full-thickness cartilage lesions are more responsive to IGF-I supplementation [28]. Ex vivo exposure of repair tissue to IGF-I resulted in enhanced matrix synthesis, compared to the response in normal articular cartilage [28]. Additionally, other studies of bone marrow-derived mesenchymal stem cells, which gain access to full-thicknesscartilage defects through the perforated subchondral spaces, show enhanced chondrogenic potential when cultured in vitro and supplemented with exogenous IGF-I [9]. These anabolic effects of IGF-I on cartilage and repair tissue metabolism suggest a role for IGF-I supplementation in cartilage repair procedures. However, there has been little information available to guide when exogenous IGF-I might be most appropriately administered, especially if the aim is to supplement low constitutive levels. The spontaneous healing of articular defects was accompanied by a temporal fluctuation in IGF-I gene expression, but not in the expression of mRNA for the major cartilage matrix molecules procollagen type I1 and aggrecan, which tended to steadily increase with time. There was an increase in IGF-I mRNA expres-

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sion in the repair tissue, with a definitive peak in expression occurring at 8 weeks post-injury, and a decrease in expression at 16 weeks. At all time points, expression of IGF-I was greater in the lesional tissue than in the perilesion and remote cartilages. Although the pattern of IGF-I message expression was clear, the coordinate translation into IGF-I ligand was not established by IGF-I protein analyses in this study due to limited sample quantity. A peak IGF-I expression level at 8 weeks correlated well with histologic evidence of increased cellular organization. However, there was no observed direct relationship between IGF-I expression and expression of aggrecan or procollagen type 11. There was, however, collagen type I1 in situ hybridization data to indicate a temporal increase in expression up to 8 weeks following injury, which then intensified at 16 weeks despite declining IGF-I message levels. The molecular and histochemical responses of the repair tissue in this study were slightly delayed compared to previous studies investigating repair tissue in rabbit models of full-thickness cartilage defects [12,25,28,33]. In this study, matrix metachromasia and procollagen type I1 in situ hybridization reactions, both cellular markers of intrinsic healing attempts, were not detected until week 4, compared to evidence of early chondrogenesis at 2 weeks in rabbits. The apparent discrepancy in intrinsic repair is likely due to the species differences in animal models used in the studies. All previous studies employed rabbits 4-18 months of age, in which 1.5-3 mm full-thickness cartilage defects were made. Rabbits become skeletally mature when physes close at the age of 6 to 8 months [21]. This study employed 3-5-year old horses, in which 15 mm cartilage defects were created. Horses typically reach skeletal maturity by the age of 18-22 months, when longitudinal bone growth ceases [ls]. The type and age of species used, as well as the size of the cartilage lesions, may account for the differences between the studies. An agerelated decline in the response of cartilage to IGF-I has been reported [5,14], as have studies demonstrating the impact of larger sized lesions on diminished cartilage repair [34,39]. Both factors were important in selecting this model for study. The removal of cartilage had a detrimental effect on the perilesional cartilage immediately adjacent to the lesion. There was significantly less expression of aggrecan and type I1 procollagen in perilesion tissue compared to remote cartilage in the 2-week samples. During the course of the study, the expression of type I1 collagen increased to the level of expression in remote cartilage, but expression of aggrecan in perilesional cartilage did not reach expression levels evident in remote cartilage. Expression of type I procollagen in perilesional cartilage peaked at 4 weeks, and remained elevated compared to remote cartilage throughout the

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Fig. 5 . Matrix collagen and aggrecan gene expression at 2, 4, 8 and 16 weeks after full-thickness cartilage debridement. Northern blot analysis was done on total RNAs isolated from within the healing cartilage defects (lesion), adjacent to the defects (perilesion), and remote to the defects (remote). Total RNAs were isolated 2, 4, 8, and 16 weeks after the defects were surgically created. The blots were hybridized to procollagen type IIB (A), aggrecan (B), and procollagen type I (C). Each bar represents n = 4 iSEM. Tukey’s classification letters indicate significant (P<0.05) differences within a tissue site (lesion, perilesion, or remote) due to time (2,4,8, 16 weeks). Different numbers of asterisks indicate significant differences within a time period, due to tissue location.

study, indicating sustained cartilage matrix gene expression abnormalities. The cause of these persistent aggrecan and type I collagen effects were unclear, but may have been the result of post-operative synovial inflammation, loss of contiguous structural support at the defect edge, or indirect mechanical trauma such as sheer forces as a result of drilling the defect. There were no significant variations in matrix gene expression in remote cartilages, suggesting local responses were primarily driving these aberrant perilesional cartilage matrix changes. After 4 months of spontaneous healing the final repair tissue was histologically and grossly fibrocartilaginous, and attained neither morphologic nor molecular expression characteristics of normal articular cartilage. Such a mediocre response has functional consequences and it is well documented that intrinsic repair of fullthickness cartilage defects with fibrocartilage results in inferior biomechanical tissue properties, breakdown of the repair tissue, and frequently osteoarthritis [1,19,27]. Longer term studies may have revealed better hyaline cartilage formation, however, the focus of this study was

the characterization of early healing and the attendant changes in growth factor gene expression. The limited intrinsic repair of articular cartilage following injury has stimulated extensive interest in methods to enhance articular cartilage repair. Further controlled studies will help define whether exogenous administration of growth factors such as IGF-I may have a role in improved cartilage healing. Certainly it is apparent, even at 2 weeks, that the healing cartilage tissue mounts a constitutive IGF-I mRNA response compared to normal cartilage, and previous experiments indicate that this IGF-I flux should contribute to chondrogenesis and ,differentiated chondrocyte function. Further studies are required to determine if the endogenously expressed IGF-I message is translated into functional protein, secreted into the extracellular matrix, and binds with the IGF-I cell surface receptor or becomes bound to IGF-I binding proteins. If the IGF-I that is constitutively expressed is not available for cell surface binding, it is plausible that exogenous supplementation may enhance the intrinsic repair response.

L.A. Fortier et al. I Journal of Orthopaedic Research 19 (2001) 720-728

Acknowledgements This research was supported by National Institutes of Health Individual National Research Service Award AR08360 (LAF) and a grant from the Harry M Zweig Memorial Fund for Equine Research (AJN, LAF).

References Buckwalter J, Rosenberg L, Couts R, Hunziker E, Reddi AH, Mow V. Articular cartilage: injury and repair. In: Woo SLY, Buckwalter JA, editors. Injury and Repair of the Musculoskeletal Soft Tissues. Park Ridge: AAOS; 1988. p. 465-82. Chevalier X, Tyler JA. Production of binding proteins and role of the insulin-like growth factor I binding protein 3 in human articular cartilage explants. Br J Rheum 1996;35:515-22. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 1987;162:15&9. Coletti Jr JM, Akeson WH, Woo SL. A comparison of the physical behavior of normal articular cartilage and the arthroplasty surface. J Bone Joint Surg 1972;54A:147-60. Convery FR, Akeson WH, Keown GH. The repair of large osteochondral defects. An experimental study in horses. Clin Orthop 1972;82:253-62. Curtis AJ, Ng CK, Handley CJ, Robinson HC. Effect of insulinlike growth factor-I on the synthesis and distribution of link protein and hyaluronan in explant cultures of articular cartilage, Biochem Biophys Acta 1992;1135:309-17. Djurasovic M, Aldridge JW,Grumbles R, Rosewasser MP, Howell D, Ratcliffe A. Knee joint immobilization decreases aggrecan gene expression in the meniscus. Am J Sports Med 1998;26:46&6. Dore S, Pelletier JP, DiBattista JA, Tardif G, Brazeau P, MartelPelletier J. Human osteoarthritic chondrocytes possess an increased number of insulin-like growth factor I binding sites but are unresponsive to its stimulation. Possible role of IGF-1-binding proteins. Arthritis Rheum 1994;37:25363. Fortier LA, Cable CS, Nixon AJ. Chondrocytic differentiation of mesenchymal cells in long-term three-dimensional fibrin cultures treated with IGF-I. Trans 42nd Ann Mtg ORS 1996;21:110. Fortier LA, Lust G, Mohammed HO, Nixon AJ. Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulin-like growth factor-I. J Orthop Res 1999;17(4):467-74. Fosang AJ, Tyler JA, Hardingham TE. Effect of interleukin-1 and insulin like growth factor-1 on the release of proteoglycan components and hyaluronan from pig articular cartilage in explant culture. Matrix 1991;11:17-24. Furukawa T, Eyre DR, Koide S, Glimcher MJ. Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee. J Bone Joint Surg 1980;62A:79-89. Howell DS. Pathogenesis of osteoarthritis. Am J Med 1986;80: 268. Hurtig MB, Fretz PB, Doige CE, Schnurr DL. Effects of lesion size and location on equine articular cartilage repair. Can J Vet Res 1988;52:13746. Kainer RA. Functional anatomy of equine locomotor organs. In: Stashak TS, editor. Adams’Lameness in Horses, 4th ed. Philadelphia: Lea & Febiger; 1987. p. 38-39. Levine RA, Sendy M, Guo L, Holzschu D. Elongation factor Tu as a control gene for mRNA analysis of lung development and other differentiation and growth-regulated systems. Nucleic Acids Res 1993;21:4426.

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[I71 Luyten FP, Hascall VC, Nissley SP, Morales TI, Reddi AH. Insulin-like growth factors maintain steady-state metabolism of proteoglycans in bovine articular cartilage explants. Arch Biochem Biophys 1988;267:416-25. [18] Malemud CJ. The role of growth factors in cartilage metabolism. Rheum Dis Clin North Am 1993;19:569-80. [19] Mankin HJ. The reaction of articular cartilage to injury and osteoarthritis (first of two parts). New Eng J Med 1974;291: 1285-92. [20] Mankin HJ. The response of articular cartilage to mechanical injury. J Bone Joint Surg 1982;64A:460-6. [21] Masoud I, Shapiro F, Kent R, Moses A. A longitudinal study of the growth of the New Zealand white rabbit: cummulative and biweekly incremental growth rates for body length, body weight, femoral length, and tibia1 length. J Orthop Res 1986;4(2):221-31. [22] Matsumoto T, Gargosky SE, Iwasaki K, Rosenfeld RG. Identification and characterization of insulin-likegrowth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases in human synovial fluid. J Clin Endocr Metab 1996;81:150-5. [23] McQuillan DJ, Handley CJ, Campbell MA, Bolis S, Milway VE, Herington AC. Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage, Biochem J 1986;240:423-30. [24] Meachim G, Roberts C. Repair of the joint surface from subarticular tissue in the rabbit knee. J Anat 1971;109:317-27. [2S] Metsaranta M, Kujala UM, Pelliniemi L, Osterman H, Aho H, Vuorio E. Evidence for insufficient chondrocytic differentiation during repair of full-thickness defects of articular cartilage. Matrix Biol 1996;15:3947. [26] Morales TI. The role and content of endogenous insulin-like growth factor-binding proteins in bovine articular cartilage. Arch Biochem Biophys 1997;343:164-72. [27] Mow V, Rosenwasser M. Articular cartilage: Biomechanics. In: Woo SL-Y, Buckwalter JA, editors. Injury and Repair of the Musculoskeletal Soft Tissues. American Academy of Orthopaedic Surgeons: Park Ridge; 1988. p. 427-63. [28] Nakajima H, Goto T, Horikawa 0, Kikuchi T, Shinmei M. Characterization of the cells in the repair tissue of full-thickness articular cartilage defects. Histochem Cell Biol 1998;109:331-8. [29] Neidel J, Blum WF, Schaeffer HJ, Schulze M, Schonau E, Lindschau J, Foll J. Elevated levels of insulin-like growth factor (IGF) binding protein-3 in rheumatoid arthritis synovial fluid inhibit stimulation by IGF-I of articular chondrocyte proteoglycan synthesis. Rheumatol Int 1997;17:29-37. [30] Pane F, Salera A, Mostarda I, Salvatore F, Sacchetti L. Estimation of extremely low amounts of single mRNAs by quantitative non-competitive reverse transcription-polymerase chain reaction assay in biological specimens from normal and neoplastic cells. Anal Biochem 1995;225:362-6. [31] Re P, Valhmu WB, Vostrejs M, Howell DS, Fischer SG, Ratcliffe A. Quantitative polymerase chain reaction assay for aggrecan and link protein gene expression in cartilage. Anal Biochem 1995; 225:356-60. [32] Sah RL, Trippel SB, Grodzinsky AJ. Differential effects of serum, insulin-like growth factor-I, and fibroblast growth factor-2 on the maintenance of cartilage physical properties during long-term culture. J Orthop Res 1996;14(1):4.&52. [33] Shapiro F, Koide S, Glimcher MJ. Cell origin and differentiation in the repair of full thickness defects of articular cartilage. J Bone Joint Surg 1993;75A532-53. [34] Tardif G, Reboul P, Pelletier JP, Geng C, Cloutier JM, MartelPelletier J. Normal expression of type I insulin-like growth factor receptor by human osteoarthritic chondrocytes with increased expression and synthesis of insulin-like growth factor binding proteins. Arthritis Rheum 1996;39:968-78. [35] Tavera C, Abribat T, Reboul P, Dort S, Brazeau P, Pelletier JP, et al. IGF and IGF-binding protein system in the synovial fluid of

728

L.A. Fortier et al. I Journal of Orthopaedic Research 19 (2001) 720-728

osteoarthritic and rheumatoid arthritic patients. Osteoarthritis Cart 1996;4:263-74. [36] Tesch GH, Handley CJ, Cornell HJ, Herington AC. Effects of free and bound insulin-like growth factors on proteoglycan metabolism in articular cartilage explants. J Orthop Res 1992;10(1):1422. [37] Tyler JA. Insulin-like growth factor I can decrease degradation and promote synthesis of proteoglycan in cartilage exposed to cytokines. Biochem J 1989;260:543-8. [38] Valhmu WB, Stazzone EJ, Bachrach NM, Saed-Nejad F, Fischer SG, Mow VC, et al. Load-controlled compression of articular

cartilage induces a transient stimulation of aggrecan gene expression. Arch Biochem Biophys 1998;353:29-36. [39] Venn G, Lauder R, Hardingham TE, Muir H. Effects of catabolic and anabolic cytokines on proteoglycan biosynthesis in young, old and osteoarthritic canine cartilage. Biochem SOCTrans 1990;18: 973-4. [40]Verschure PJ, Van Noorden CJ, Van Marle J, Van den Berg WB. Articular cartilage destruction in experimental inflammatory arthritis: insulin-like growth factor-1 regulation of proteoglycan metabolism in chondrocytes. Histochem J 1996;28:835-57.