The Effect of Cytokines on the Proliferation and ... - SAGE Journals

0363-5465/99/2727-0636$02.00/0 THE AMERICAN JOURNAL OF SPORTS MEDICINE, Vol. 27, No. 5 © 1999 American Orthopaedic Society for Sports Medicine

The Effect of Cytokines on the Proliferation and Migration of Bovine Meniscal Cells* Madhu M. Bhargava, PhD, Erik T. Attia, George A. C. Murrell, MD, PhD, Martin M. Dolan, Russell F. Warren, MD, and Jo A. Hannafin,† MD, PhD

From the Laboratory for Soft Tissue Research, Hospital for Special Surgery, affiliated with New York Hospital, Cornell University Medical Center, New York, New York absorption,2, 21, 32 stability,18 and lubrication. Complete loss of the meniscus has been shown to result in accelerated degeneration of the knee joint.9, 13, 14, 15, 19, 36 Even partial meniscectomy has been demonstrated to be detrimental, and methods of reconstruction and repair continue to be explored.30 The variable healing response of meniscal injuries, particularly in the avascular zones, has led to the search for factors that will enhance healing.3, 4, 12, 25, 32 The ability of the meniscus to function in the normal knee and to heal after injury depends on the ability of the meniscal fibrochondrocytes present within the meniscus and of nearby undifferentiated cells to respond to changes in their environment. Several studies have suggested that cytokines may play a role in meniscal healing.3, 4, 12, 15, 16, 27, 34 Injuries to the peripheral vascular region of the meniscus have a more favorable prognosis than do injuries in the avascular region.15, 16 Cytokines present in blood may be responsible for the more-favorable prognosis for healing of the more-vascular peripheral meniscal lesions. Both in vitro and animal in vivo studies have demonstrated the potential value of cytokines in the healing of meniscal injuries.3, 4, 12, 15, 16, 29, 33, 34 Placement of a fibrin clot3 or endothelial growth factor12 in the injured avascular region of the meniscus has been demonstrated to improve meniscal healing. Pituitary-derived fibroblast growth factor and platelet lysate have been shown to stimulate the growth of fibrochondrocytes isolated from the meniscus.34 Spindler et al.29 have demonstrated that a regional mitotic response to plateletderived growth factor-AB will occur. Since cytokines stimulate healing by effecting cell proliferation and cell migration in a variety of tissues,7, 8 we compared the relative effect of several cytokines on cell proliferation by measuring DNA synthesis, and we compared the relative effect of several cytokines on cell migration by measuring chemotaxis in a Boyden chamber using cells isolated from different regions of the meniscus.

ABSTRACT We determined the effect of cytokines on the proliferation and migration of cells isolated from the inner-third (white-white), middle-third (red-white), and outer-third (red-red) regions of bovine meniscus. Cells from the outer, or peripheral, region of the meniscus exhibited higher DNA synthesis in the presence of 10% serum compared with cells from the inner or central regions. Recombinant human platelet-derived growth factorAB, hepatocyte growth factor/scatter factor, and bone morphogenic protein-2 stimulated DNA synthesis of all meniscal cells in a dose-dependent manner, with a two- to threefold maximal stimulation at 10 ng/ml. Cell migration was also stimulated by addition of cytokines. Platelet-derived growth factor and hepatocyte growth factor caused an increase in the migration of cells derived from all three zones, while interleukin-1 selectively stimulated the migration of outer-zone meniscal cells. Epidermal growth factor was much less effective and stimulated the migration of cells in the inner and outer zones by 40% to 50%, while bone morphogenic protein-2 and insulin-like growth factor-1 stimulated the migration of meniscal cells from the middle zone by 40% to 50%. The identification of cytokines that stimulate both the growth and migration of meniscal cells may provide new tools for modulation of meniscal healing.

The importance of the meniscus in knee function is well established. It provides load transmission,1, 5, 17, 27 shock

*Presented at the annual meeting of the Orthopaedic Research Society, San Francisco, California, February 1997. † Address correspondence and reprint requests to Jo A. Hannafin, MD, PhD, Hospital for Special Surgery, 525 East 70th Street, New York, NY 10021. No author or related institution has received any financial benefit from research in this study. See “Acknowledgments” for funding information.

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Effect of Cytokines on Meniscal Cell Proliferation and Migration

MATERIALS AND METHODS Isolation and Culture of Cells from the Different Regions of the Bovine Knee Meniscus Bovine stifle joints from skeletally mature animals were obtained from a local abattoir. The joints were transported to the laboratory on ice, cleaned thoroughly with povidone-iodine, and aseptically dissected. The menisci were harvested and were sharply dissected free of adherent synovium and capsule. The specimens were immersed in medium M199 containing 10% antibiotic-antimycotic solution (antibiotic-antimycotic solution: 10,000 mg/ml penicillin G sodium, 10,000 mg/ml streptomycin sulfate, 25 mg/ml amphotericin B in 0.85% saline) (Gibco BRL, Grand Island, New York). The intact menisci were sectioned into outer one-third (peripheral, red-red), middle one-third (red-white), and inner one-third (avascular, white-white) zones as shown schematically in Figure 1. The sections were washed three times for 30 minutes with M199 containing 10% antibiotic-antimycotic solution. Each of the three sections was finely diced into small pieces, placed into M199 containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution (Gibco BRL) in T-75 flasks, and incubated at 37°C in a humidified atmosphere of 5% carbon dioxide and 95% air. The cells migrated out of the explants and reached confluence in 2 to 3 weeks. Microscopic examination revealed that cells had a fibrochondrocytic appearance, with plump cell bodies. Approximately 20%, 30%, and 40% of the cells from the inner, middle, and outer regions of the meniscus, respectively, had fibroblastic phenotype. This proportion of fibroblastic cells did not vary significantly from isolation to isolation. Determination of Tritium Incorporation into DNA The incorporation of 3H-thymidine as a measure of DNA synthesis was determined using the Multiscreen method

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(Millipore Corp., Bedford, Massachusetts).22, 23 Multiscreen-HV (low protein binding, 0.45-m, hydrophilic Durapore membrane, 96-well filtration plates) were coated with type-I rat tail collagen (3 mg/ml) (Collaborative Research, Inc., Lexington, Massachusetts). Collagen was diluted 1:4 in 60% ethanol; 50 ml was added to each well of the plate, the plates were gently shaken for an even coating, then allowed to air-dry overnight in a laminar flow hood with the plate cover slightly open. Equal amounts (5 3 104) of cells from the three regions of the meniscus were plated in the individual wells. After 24 hours, the cells were washed twice with M199, incubated for 2 hours in M199, and then cytokines were added. All cytokines were obtained from R&D systems (Minneapolis, Minnesota). The cytokine solutions contained 0.1, 1, or 10 ng of the cytokine per milliliter of M199. This range of concentration of cytokines was chosen because of previously demonstrated effects on cell proliferation in knee-ligament fibroblasts and in periodontal fibroblasts and chondrocytes.6, 19, 26 The cytokines tested were human recombinant platelet-derived growth factor-AB, insulinlike growth factor-I, insulin-like growth factor-II, interleukin-1, hepatocyte growth factor, bone morphogenic protein-2, fibroblast growth factor, transforming growth factor, or epidermal growth factor. Incubations with purified cytokines were performed in absence of serum. After 24 hours, 200 ml of 10-mCi/ml 3H-thymidine in M199 medium was added to each well, and the plates were incubated at 37°C for 2 hours. The controls were prepared by adding the equivalent 3H-thymidine to wells containing media in the absence of cells to determine nonspecific binding of thymidine to the Durapore filter. An equal volume of water was added to the unused wells of the plate. After incubation, the plates were vacuum-filtered, washed three times with cold phosphate-buffered saline and three times with 5% trichloroacetic acid. The underdrain support was removed from the multiscreen vacuum filtration unit, the bottom of the plate was blotted and dried under a heat lamp for 10 minutes (membrane side up). Disks from the multiscreen plates were then punched into 5.0-ml scintillation vials containing 0.5 ml of sodium hypochlorite (0.42%). Vials were agitated for 30 minutes on a rotary table and 3.5 ml scintillation cocktail was added. The specimens were mixed and radioactivity was measured in a Beckman Model LS 2800 scintillation counter (Beckman Instruments, Inc., Fullerton, California). A blank consisting of liquid scintillation cocktail and hypochlorite solution was included. The DNA synthesis (3H-thymidine incorporation) was expressed as counts per minute per well. Three independent experiments were performed and each cytokine concentration was tested in triplicate. Measurement of Chemotactic Response to Cytokines

Figure 1. Schematic representation of the sectioning of meniscus into zones for obtaining outer, middle, and inner meniscal cells.

Cell-migration studies were performed with purified cytokines in the absence of serum as previously described6, 20 with minor modifications. The confluent monolayers of

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meniscal fibrochondrocytes were washed with M199 and incubated in serum-free M199 for 1 to 2 hours and detached by treatment with trypsin (0.25%) EDTA (1 mM) in calcium- and magnesium-free Hanks Balanced Salt Solution (Gibco BRL) for 2 minutes at 37°C. Once the cells were released, the trypsin was neutralized with medium M199 containing 10% fetal bovine serum. Cells were collected by centrifugation, washed, and suspended in medium M199 containing 2 mg/ml bovine serum albumin (Sigma, St. Louis, Missouri). Solutions containing 1, 10, or 100 ng/ml of cytokines were pipetted separately into the bottom wells of a chemotaxis chamber (Neuro Probe, Cabin John, Maryland). Eight-micron-pore polycarbonate filters coated with type I collagen were placed above the solution, the chamber was assembled, and 50 ml of cell suspension containing 1 to 5 3 104 cells was placed in the top wells of the chamber. The cells were incubated for 4 hours at 37°C in a humidified atmosphere of air and 5% carbon dioxide. At the end of this period, the filters were removed, and the adherent cells were fixed and stained with a nuclear stain (Diff Quick, Fisher Scientific, Springfield, New Jersey). The filters were washed with water and placed on a microscope slide. Cells that did not migrate through the filter but settled on the filter because of gravity were wiped off with a cotton swab, and the cells that migrated through were counted using a microscope at 340 magnification. Three independent experiments were performed, each cytokine concentration was tested in triplicate, and three representative areas were counted from each well.

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Figure 2. The effect of serum on DNA synthesis by outer- , middle- , and inner-zone cells. Results are mean 6 SEM (N 5 12). The effect of 10% serum on DNA synthesis was significantly higher (P , 0.001) than the effect of 0.1% serum in cells from all three zones. The difference between outer and middle zone cells was significant (P , 0.05), but differences between middle and inner zone cells or between inner and outer zone cells were not significant.

Statistical Analysis The mean 6 SD was determined by the Sigma Plot statistical analysis program (SPSS, Chicago, Illinois). The statistical significance of the differences between groups of observations was evaluated using the Student’s t-test. The threshold for statistical significance was set at P , 0.05.

RESULTS Effect of Cytokines on DNA Synthesis There was 700%, 300%, and 500% more DNA synthesis in cells from the outer, middle, and inner zones of the meniscus, respectively, in presence of 10% serum than in the presence of 0.1% serum (Fig. 2). In media containing 10% serum, cells from the outer zone of the meniscus exhibited 40% more DNA synthesis than cells from the inner zone, and 130% more DNA synthesis than cells from the middle zones (Fig. 2). Platelet-derived growth factor-AB (Fig. 3), hepatocyte growth factor (Fig. 4), and bone morphogenic protein-2 (Fig. 5) stimulated the DNA synthesis of cells from all three regions of the meniscus in a dose-dependent manner, with the maximum stimulation of 200% at 10 ng/ml. Insulin-like growth factor-I had no significant effect on DNA synthesis at any of the concentrations tested. The stimulatory effect of platelet-derived growth factor-AB on DNA synthesis was similar on cells isolated

Figure 3. Effect of platelet-derived growth factor-AB on DNA synthesis in cells from the three zones of the meniscus. Results are mean 6 SEM (N 5 12). The effect of plateletderived growth factor at 1 and 10 ng/ml on DNA synthesis (in all three cell types) compared with control was significant (P , 0.05).

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Figure 4. Effect of hepatocyte growth factor on DNA synthesis in cells from the three zones. Results are mean 6 SEM (N 5 12). The effect of hepatocyte growth factor at 1 and 10 ng/ml on DNA synthesis (in all three cell types) compared with control was significant (P , 0.05).

Figure 5. Effect of bone morphogenic protein-2 on DNA synthesis in cells from the three zones. Results are mean 6 SEM (N 5 12). The effect of bone morphogenic protein-2 at 1 and 10 ng/ml on DNA synthesis (in all three cell types) compared with control was significant (P , 0.05). from outer, middle, and inner zones of the meniscus at all concentrations tested. At 10 ng/ml, the effect of hepatocyte growth factor on DNA synthesis in cells from the outer zone was slightly more pronounced than its effect on DNA

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Figure 6. Effect of platelet-derived growth factor on the migration of outer- , middle- , and inner-zone cells. Results are mean 6 SEM (N 5 12). The effect of platelet-derived growth factor at 1 and 10 ng/ml on cell migration (in all three cell types) compared with control was significant (P , 0.05). At higher concentration of platelet-derived growth factor (100 ng/ml) less cell migration was observed.

Figure 7. Effect of hepatocyte growth factor on the migration of meniscal cells. Results are mean 6 SEM (N 5 12). The effect of hepatocyte growth factor at 1, 10, and 100 ng/ml on cell migration (in all three cell types) compared with control was significant (P , 0.05). There was no significant difference in the effects on cell migration in inner- , middle- , and outer-zone cells between 1, 10, and 100 ng/ml of hepatocyte growth factor.

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synthesis in cells from the inner zone. The effect of bone morphogenic protein-2 on DNA synthesis in the cells from middle zone was slightly higher than its effect in cells from inner or outer zones of the meniscus at all three concentrations tested. Effect of Cytokines on Chemotactic Migration of Meniscal Cells Platelet-derived growth factor stimulated the migration of all meniscal cells with the maximum stimulation (200% to 300%) at 10 ng/ml. An inhibition of migration was observed at higher concentrations (Fig. 6). A similar degree of stimulation of migration of meniscal cells isolated from the three different zones was observed with hepatocyte growth factor, but maximum stimulation was observed at 1 and 10 ng/ml (Fig. 7). Since the concentrations of hepatocyte growth factor between 1 and 10 ng/ml were not tested, the precise concentration that caused maximum stimulation of cell migration cannot be determined from these data. Bone morphogenic protein-2 was less stimulatory, with significant stimulation seen only with cells isolated from the middle zone of the meniscus at 100 ng/ml (Fig. 8). With insulin-like growth factor-1, significant stimulation was observed only with cells isolated from the middle and inner zones at 100 ng/ml (Fig. 9). Interleukin-1 was a potent stimulator of cells isolated from the outer third of the meniscus, with 100% stimulation at 1 ng/ml (Fig. 10). At higher concentrations of interleukin-1 the stimulation of meniscal-cell migration was slightly less (Fig. 10). Epidermal growth factor was most effective in stimulating the migration of cells from the inner zone, with its maximum stimulation effect in that zone, unlike in the other two zones, at 10 ng/ml (Fig. 11).

Figure 8. Effect of bone morphogenic protein-2 on the migration of meniscal cells. Results are mean 6 SEM (N 5 12). The effect of bone morphogenic protein-2 on cell migration compared with controls was significant (P , 0.05) at 100 ng/ml only with middle-zone cells. At other concentrations of bone morphogenic protein-2 and with cells from the other zones, a slight stimulation of migration was observed but was not statistically significant.

DISCUSSION Bovine menisci were used in the present study because of the size and ready availability of these specimens. The adult bovine meniscus is larger than the human meniscus with a more fibrous tissue architecture in the outer 50%.28 However, the cellularity and vascularity of bovine and human meniscus are similar (S. Arnoczky, personal communication, 1998). The larger size of the meniscus allowed reproducible dissection of the menisci into outer, middle, and inner thirds, with adequate tissue for subsequent cell cultures. The enzymatic digestion method for isolating fibrochondrocytes34 gave results similar to that of explant cultures24; thus, explant cultures were used for isolating cells from different zones of the meniscus. Thymidine incorporation into DNA was measured using the Multiscreen assay because it permitted growth of cells, treatment with cytokines, incubation with 3H-thymidine, precipitation, and washing of DNA on a single membrane that could be punched directly into scintillation vials for counting. Very low background binding of 3H-thymidine to membranes (,200 cpm) or incorporation into DNA of confluent cells (,350 cpm) was observed when 1 mCi/ml of 3 H-thymidine was used. This allowed us to test the effect of small amounts of growth factors on DNA synthesis

Figure 9. Effect of insulin-like growth factor-1 on the migration of cells from the three zones. Results are mean 6 SEM (N 5 12). The effect of insulin-like growth factor-1 on cell migration compared with controls was significant (P , 0.05) and dosedependent at 1, 10, and 100 ng/ml with inner- and middle-zone cells but was not significant with outer-zone cells.

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Figure 10. Effect of interleukin-1 on the migration of meniscal cells. Results are mean 6 SEM (N 5 12). The effect of interleukin-1 on cell migration compared with controls was significant (P , 0.05) at 1, 10, and 100 ng/ml with outer- and middle-zone cells but was not significant with inner zone cells. At higher concentration, a less-stimulatory effect was observed with outer-zone cells.

using a limited number of actively dividing cells, in a quantitative and reproducible manner. All measurements dealing with the effect of cytokines on DNA synthesis and cell migration were done in the absence of serum since the effects of added cytokines could potentially be masked by cytokines present in serum. The isolation of two types of meniscal cells has previously been described.10, 35 One type is from the superficial region (closer to the articular surface) and one type is a chondrocyte-like cell from the deeper interior region. In this study, the meniscus was sectioned into outer, middle, and inner zones, and cells were isolated from each of these zones. Cell preparations consisted predominantly of cells derived from deep within the meniscus; however, peripheral fibroblastic cells cannot be completely omitted from these cultures. Furthermore, the fibroblastic cell content varied in the cell preparations from the different zones, with higher levels present in the cells isolated from the outer (peripheral) zones. This may partly account for the different effects of cytokines in DNA synthesis and cell migration on cells isolated from different zones of the meniscus. It is unclear at this time whether both cell types play a role in meniscal repair in vivo; we made no attempt to subtype these cells in the present study. Several cytokines, including transforming growth factor-b and fibroblast growth factor have been observed to stimulate the proliferation of the meniscal fibrochondrocytes in vitro.4, 29, 30 Endothelial cell growth factor has also

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Figure 11. Effect of epidermal growth factor on the migration of meniscal cells. Results are mean 6 SEM (N 5 12). The effect of epidermal growth factor on cell migration compared with controls was significant (P , 0.05) at 1 ng/ml with inner- and outer-zone cells and at 10 ng/ml with inner-zone cells, but was not significant with middle-zone cells at both these concentrations. At 100 ng/ml, a less-stimulatory effect was observed with outer- and inner-zone meniscal cells. For middle-zone cells at higher concentrations (10 and 100 ng/ml), the effect of epidermal growth factor was inhibitory compared with the controls. been shown to stimulate tissue ingrowth in the avascular region of the meniscus in a canine model.12 Webber et al.34 have demonstrated that the central cells of the meniscus can migrate through the matrix toward the fibrin clot. Spindler et al.29 demonstrated that platelet-derived growth factor-AB stimulated the proliferation of cells in the explants from the peripheral region of sheep meniscus but had no significant effect on the cells from the central region. Our studies suggest that at 10 ng/ml, plateletderived growth factor-AB stimulated the proliferation and migration of cells isolated from both inner (white-white) and outer (red-red) regions of the meniscus. In explant studies performed by Spindler et al.,29 much higher concentrations (100 ng/ml) were required for stimulation of DNA synthesis in peripheral (outer) cells in the explants. This could be because many of the cells may not be in direct contact with the growth factor and because the extracellular matrix surrounding the cells in the explant might bind growth factor or impede diffusion of the factor into the depth of the explant. The reasons for the lack of stimulation of proliferation of cells in the explants from the central region of meniscus by platelet-derived growth factor as reported by Spindler et al. are not clear. These cells appear to have receptors for platelet-derived growth factor, since their migration and growth was stimulated by platelet-derived growth factor

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in vitro. Reasons for these differences may be the use of explants versus monolayer cell cultures, species differences, different growth factor preparations, varying times of exposure to the factor, or the percentage of fibroblasts and fibrochondrocytes present in the preparations. Our results indicate that 1) at optimal concentrations, platelet-derived growth factor, hepatocyte growth factor, and bone morphogenic protein-2 are equally effective in stimulating DNA synthesis in the cells isolated from different zones of the meniscus; and 2) platelet-derived growth factor and hepatocyte growth factor are 3 to 4 times stronger than insulin-like growth factor-1, interleukin-1, bone morphogenic protein-2, and epidermal growth factor in stimulating cell migration. The identification of cytokines that stimulated the growth and migration of meniscal cells from the avascular regions should set the stage for in vivo animal studies to evaluate the efficacy and safety of these agents in meniscal healing. This factor also stimulates cell motility, cell proliferation, and proteoglycan synthesis in chondrocytes.31 The use of hepatocyte growth factor, a strong angiogenic factor,11 may be beneficial in initiating neoangiogenesis in the white-white zone during meniscal healing. Appropriate delivery systems and duration of exposure of the cells to these cytokines for generation of an optimal healing response has yet to be determined. Inclusion of these cytokines in collagen scaffold-based repair of meniscal healing30 might lead to better modulation of healing. In certain clinical situations encountered with meniscal injuries, the use of cytokines may be especially beneficial. Failure of meniscal repair is more common in the medial meniscus and is pronounced in the presence of joint instability. A 5% failure rate has been reported when meniscal repair is combined with ACL reconstruction; in stable knees without ACL reconstruction, the failure rate of meniscal repair rises to 15%, and in unstable knees without ACL reconstruction the failure rate is 38% (Ref. 25; S. A. Rodeo et al., personal communication, 1998). The cytokines identified in the present study may be especially useful in optimizing healing in cases in which isolated meniscal injury in the red-white or white-white zone occurs but ACL reconstruction is not performed and in which there is no significant intraarticular clot formation.

ACKNOWLEDGMENTS This work was supported in part by an NIH Development and Feasibility grant (No. 5P60AR383520) (Jo Hannafin), a grant from the Arthroscopy Association of North America, Rosemont, Illinois (George Murrell), a summer student fellowship from the Arthritis Foundation, New York, New York (Martin Dolan), and generous support from the Institute for Sports Medicine Research, New York, New York. The authors are grateful to Dr. J. Wosney, Genetics Institute, Cambridge, Massachusetts, for the gift of bone morphogenic protein-2.


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