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Preventing chondrocyte programmed cell death caused by iatrogenic injury
The Knee 14 (2007) 107 – 111
Preventing chondrocyte programmed cell death caused by iatrogenic injury John G. Costouros ⁎, Hubert T. Kim Department of Orthopaedic Surgery, University of California Medical Center, San Francisco, CA, United States Veterans Affairs Medical Center, San Francisco, CA, United States Received 21 August 2006; received in revised form 20 October 2006; accepted 30 October 2006
Abstract Cartilage repair technology is advancing at a rapid pace. However, all techniques share a common weakness—unintentional chondrocyte cell death resulting from cartilage injury that occurs during preparation of the defect site. The loss of chondrocytes at the edge of host cartilage is likely to contribute to failed integration of regenerated tissue or grafts to the surrounding cartilage. Recent studies have demonstrated that “apoptosis”, or programmed cell death (PCD), may be responsible for much of the cell death caused by cartilage injury. Theoretically, inhibitors of key pathways responsible for PCD could rescue chondrocytes and improve the results of cartilage repair surgery. The purpose of this study was to test the hypothesis that short-term, intra-articular PCD inhibitor treatment can limit chondrocyte death in vivo following simulated preparation of host cartilage for a repair procedure. A microcurette was used to create full-thickness articular cartilage injuries to the femoral condyles of adult New Zealand White rabbits. Animals received daily intra-articular injections either with a potent PCD inhibitor or with vehicle alone. Treatment with the inhibitor resulted in a significant reduction in the percentage of chondrocytes undergoing PCD compared to controls [treated = 10.1 ± 2.4%; controls = 26.5 ± 3.6%; (p = 0.0013)]. These results provide proof of concept for the use of PCD inhibitors to enhance the results of cartilage repair surgeries. © 2006 Elsevier B.V. All rights reserved. Keywords: Cartilage; Apoptosis; Trauma; Caspase; Arthritis
1. Introduction Despite surgical and technological advancements, the treatment of articular cartilage defects of the knee continues to challenge orthopaedic surgeons. Chondral lesions are common, and have been reported in 63% of over 31,000 arthroscopic procedures in one series [1]. Current operative treatments include debridement and stabilization, stimulation of intrinsic repair mechanisms, and repair or transplantation of chondral tissue [2–4]. A key step in all cartilage repair procedures is debridement of the damage site back to healthy cartilage. This procedure most commonly is performed with a ⁎ Corresponding author. University of California, San Francisco, Department of Orthopaedic Surgery, 260, International Circle, San Jose, CA 95119, United States. Tel.: +1 408 972 6326; fax: +1 408 972 3578. E-mail address: [email protected] (J.G. Costouros). 0968-0160/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.knee.2006.10.013
sharp curette. Among the problems common to all repair techniques is difficulty obtaining consistent integration at the wound edge [5–8]. It has been suggested that cell death at the edges of the prepared injury site may contribute to this lack of integration [9]. Recent studies have shown that chondrocyte apoptosis, or programmed cell death (PCD), is associated with acute cartilage injury and may contribute to the development of posttraumatic arthritis [10–16]. Apoptosis is a highly regulated process intrinsic to normal embryogenesis, immunological competence, tissue homeostasis, and skeletal development [17–19]. In vitro studies have demonstrated increased levels of chondrocyte apoptosis following experimental cartilage injuries including compression, drilling, and trephine wounding [15,16,20]. More recently, analysis of cartilage specimens from patients who have sustained knee injuries also demonstrated increased levels of chondrocyte PCD [14,21].
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Theoretically, apoptosis inhibitors could improve outcomes of cartilage repair and regeneration techniques by limiting chondrocyte death at the edge of the prepared cartilage defect, thus facilitating integration of cartilage repair tissue or grafts. Caspases (cysteinyl aspartate-specific proteases) are key enzymatic mediators of apoptosis [18,19,22,23]. The caspase inhibitor most commonly used in PCD inhibition studies is the non-selective, cell permeable, irreversible caspase inhibitor ZVAD-fmk. Recent experiments have shown that ZVAD-fmk can block chondrocyte apoptosis in vitro and preserve chondrocyte function [12,13,21,24,25]. These studies suggest that ZVAD-fmk could be a useful therapeutic agent in the treatment of articular cartilage injuries. The objective of this study was to determine whether intra-articular administration of ZVAD-fmk could decrease chondrocyte PCD following isolated chondral injury in a rabbit model.
0.1% DMSO, 0.5 cm3; Calbiochem, La Jolla, CA). Animals in the control group (N = 10) received daily intra-articular injections of vehicle alone (0.1% DMSO in normal saline, 0.5 cm3). All animals were euthanized 4 days following surgery and tissues harvested immediately for subsequent analysis. Previous experiments demonstrated that chondrocyte apoptosis reaches a peak approximately 4 days after cartilage injury in this model. The research protocol was reviewed and approved by the Animal Research Subcommittee at the Veterans Affairs Medical Center, San Francisco according to the established NIH guidelines.
2. Methods
2.3. Apoptosis analysis
2.1. Animal surgery: experimental chondral injury Adult female New Zealand White Rabbits (Western Oregon Rabbit Company, Philomath, OR) were anaesthetized with an intramuscular injection of ketamine (100 mg/kg) and xylazine (8 mg/kg). Antibiotic prophylaxis was administered (Trimethoprim–Sulfadiazine, 30 mg/kg), and the right knees were shaved and disinfected with Betadine solution in the standard sterile fashion. A standard medial parapatellar arthrotomy was performed to expose the underlying distal femoral condyles. A twomillimeter microcurette was then used to create a pair of linear full-thickness chondral defects in the weight-bearing portions of the medial and lateral femoral condyles. Close attention was paid to avoiding injury to the subchondral bone. An indwelling intra-articular epidural catheter (Arrow, Redding, PA) was then introduced subcutaneously from the upper thigh into the knee joint to enable consistent intra-articular drug delivery in controlled fashion. Animals received a second dose of antibiotic 24 h following surgery. Postoperatively, the animals were permitted cage activity without immobilization and monitored for signs of infection and distress. Animals in the treatment group (N = 10) received daily intra-articular injections of the broad-spectrum caspase inhibitor ZVAD-fmk (100 μM in
2.2. Tissue preparation The harvested femoral condyles were split in the sagittal plane within the patellar groove. Excess subchondral bone was removed, and the specimens were fixed in 10% buffered formalin at 4 °C overnight. Following fixation, the specimens were decalcified for 3 weeks (0.45 M EDTA/0.1 M Tris, pH 8.0). Tissues were then dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. 5 μm sections were cut on a rotary microtome and placed on Superfrost Plus slides.
Chondrocyte apoptosis was quantified by TUNEL analysis using the ApopTag® Direct in situ apoptosis detection kit (Intergen, Purchase, NY). Enzyme and substrate concentrations, as well as washing stringencies, were modified from the manufacturer's protocol in order to optimize the sensitivity and specificity of the assay [26]. Slides were mounted using Vectashield mounting media (Vector, Peterborough, UK) containing DAPI (1 μg/ml). Fluorescence images were captured using an Axiocam digital camera (Zeiss, NJ) at 1 megapixel resolution. The captured fields included the full thickness of articular cartilage extending 1.5 mm from either edge of curette injury. The captured fields were divided into three “Zones” with Zone 1 encompassing the full thickness of articular cartilage extending from 0–0.5 mm away from the injury; Zone 2 extending from 0.5–1 mm away from the injury; and Zone 3 extending from 1–1.5 mm away from the injury (Fig. 1). Semiautomated data collection and analysis were performed using digital image acquisition software (Scion, MD). An apoptotic index was calculated and defined as follows: [apoptotic index= (# of TUNEL positive / # of DAPI positive) × 100]. Each injury site was treated as an independent specimen, and a value for each “Zone” from each specimen was recorded as the mean of measurements from three separate near-adjacent sections. In order to validate the data obtained from TUNEL analysis, alternative methods of PCD analysis were employed including examination of nuclear
Fig. 1. Schematic representation of an osteochondral sample cut in the sagittal plane. Arrow represents the 2-millimeter microcurette used to create a fullthickness chondral lesion. Each zone, represented by a grey bar, corresponds to 0.5 mm in length. [Zone 1 = 0–0.5 mm from area of injury. Zone 2 = 0.5–1.0 mm from area of injury. Zone 3 = 1.0–1.5 mm from area of injury. C = cartilage. B = bone.]
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formed using Mann–Whitney test for non-parametric data. Statistical significance was defined as p b 0.05.
3. Results 3.1. Intra-articular caspase inhibition blocks chondrocyte PCD in vivo
Fig. 2. Inhibition of chondrocyte apoptosis in vivo with ZVAD-fmk following isolated chondral injury. Y-axis: depicts percentage of total chondrocytes undergoing apoptosis (# TUNEL-positive cells / # DAPI positive cells × 100). X-axis: depicts contiguous 0.5 mm full-thickness cartilage segments starting at the edge of the curette injury site (Zone 1 = 0– 0.5 mm; Zone 2 = 0.5–1.0 mm; Zone 3 = 1.0–1.5 mm). Asterisk indicates statistical significance between groups (Zone 2: p = 0.004; Zone 3: p = 0.0001; Total: p = 0.001).
morphology, and anti-ssDNA antibody (Chemicon, Temecula, CA) staining of formamide and heat-treated specimens. The anti-ssDNA staining technique has been demonstrated to be highly specific for apoptosis with an excellent ability to distinguish between apoptotic and necrotic cell death [27,28].
2.4. Histological assessment Sections were stained with hematoxylin and eosin (H and E) and Safranin-O/Fast Green under the following conditions: dye concentration 0.1%, staining time 6 min, and pH 5.7 at a temperature of 37 °C. Histological grading was performed based on cellularity and Safranin-O stain distribution as described by Mankin and modified by van der Sluijs [29].
2.5. Statistical analysis Statistical analysis between groups was performed using the two-tailed Students t-test for TUNEL data. Analysis of histological scores was per-
In order to determine whether or not intra-articular administration of ZVAD-fmk could decrease chondrocyte apoptosis after chondral damage, rabbits were subjected to full-thickness cartilage injuries using a two-millimeter microcurette. One group of animals received daily injections of ZVAD-fmk while a second group of animals received injections of vehicle alone. Previously published experiments using this model have demonstrated that chondrocyte PCD is at a maximum 4 days following injury [15,16,21]. Cartilage specimens from animals treated with ZVAD-fmk for 4 days following chondral injury showed a statistically significant decrease in chondrocyte PCD as quantified by TUNEL analysis (Fig. 2). Within 1.5 mm from the edge of the injury site, the percentage of TUNEL-positive chondrocytes in control specimens was 26.5 ± 3.6%. In the treated specimens, the amount of chondrocyte PCD was 10.1 ± 2.4%. This difference was highly statistically significant (p = 0.0013). This reduction in PCD was most pronounced in areas greater than 1 mm from the edge of the injury, where 21.8 ± 1.3% of control specimens displayed PCD compared to 3.1 ± 0.7% in specimens treated with the caspase inhibitor (p = 0.0001). In control specimens, a relatively diffuse distribution of apoptotic chondrocytes was noted, primarily confined to the superficial and middle zone of articular cartilage, and extending up to 1.5 mm away from either edge of the experimental injury site (Fig. 3). In treated specimens, apoptotic chondrocytes were primarily located in the superficial and middle zones within a 0.5 mm segment of cartilage immediately adjacent to the wound edge (Fig. 3). Chondrocyte apoptosis was confirmed by examination of nuclear morphology and immunohistochemistry analysis of formamide/heat-treated sections using an antibody specific for ssDNA as described. The amount and distribution of positively staining cells
Fig. 3. Representative cartilage sections which have undergone TUNEL analysis (A, B) and DAPI (C, D) staining. Apoptotic cells are TUNEL-positive and are identified by bright nuclear staining. DAPI identifies all cells (both apoptotic and non-apoptotic) by a non-specific nuclear staining. Control specimens to the left (A, C) and specimens treated with ZVAD-fmk to the right (B, D). Location of experimental injury is indicated by an arrow. TUNEL analysis reveals an overall decrease of apoptosis in treated specimens (B) in comparison to controls (A). The treatment effect is most pronounced moving away from the area of injury.
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Fig. 4. Left panel: anti-ssDNA staining of an untreated cartilage specimen from an area adjacent to a curette injury. Numerous true apoptotic cells are stained brown. Right panel: anti-ssDNA staining of an uninjured specimen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
were very similar to the results of the TUNEL analysis, with markedly decreased staining in treated specimens (Fig. 4). 3.2. Histological grading In order to determine whether or not treatment with ZVAD-fmk would have other beneficial, or possibly detrimental effects on the articular cartilage, all specimens were also processed for routine histology and scored using a modified Mankin scale. We observed some chondrocyte loss as defined by empty lacunae in both treated and control specimens. Mild proteoglycan loss as measured by Safranin-O staining was also noted in both treated and control specimens. However, using the modified Mankin grading scale, no statistically significant difference in histological scores was observed between treated and untreated specimens [treated = 2.6 ± 0.5; controls = 2.9 ± 0.5; (p = 0.60)].
4. Discussion Recent studies indicate that commonly performed arthroscopic procedures such as chondral shaving, debridement, and laser abrasion lead to death of chondrocytes bordering the edge of the treatment site and may have a long-term deleterious effect on articular cartilage [9,30]. In a recent study, Hunziker reported a significant chondrocyte loss at the wound edge of partial-thickness chondral defects in pigs repaired with an autologous fascial flap sutured over a fibrinous matrix [31]. Even seemingly benign procedures such as the suturing of a periosteal flap during autologous chondrocyte transplantation (ACI) may result in chondrocyte death along the path of the suture needle. Thus, the iatrogenic creation of an acellular or hypocellular zone of cartilage may be a major factor affecting edge healing and incorporation of transplanted repair tissue. Until recently, the specific mediators responsible for stimulating chondrocyte death in response to traumatic injury were largely unknown. Some chondrocytes clearly die as a result of catastrophic cellular injury in an unregulated fashion (i.e. via necrosis). However, an increasing body of evidence
supports the hypothesis that matrix injury initiates an apoptotic response in chondrocytes, and that it is possible to intervene in this process by blocking key mediators of apoptosis [13,24,25]. The experimental data in the present study demonstrate the in vivo effectiveness of intra-articular caspase inhibition in limiting chondrocyte PCD following experimental full-thickness cartilage injury. We noted a 38% overall reduction in chondrocyte PCD as measured by TUNEL analysis. These observations are consistent with previous in vitro studies. For instance, D'Lima and colleagues demonstrated a 50% reduction in PCD with ZVAD-fmk treatment following mechanical compression of cartilage explants [21]. Hopefully, other drugs or combinations of drugs will be able to block PCD even more effectively. However, it is likely that some of the TUNELpositive cells either cannot be rescued from cell death or are “false positives”. Only long-term in vivo studies can determine whether short-term PCD inhibition will actually improve chondrocyte viability and cartilage preservation. Recent studies have, in fact, demonstrated that PCD inhibition does result in cartilage preservation in rabbit models of osteoarthritis and osteochondral injury [32,33,34]. Consequently, we hypothesize that PCD inhibition will also have beneficial long-term effects on chondrocyte survival in the setting of cartilage repair surgeries. The most striking effect of ZVAD-fmk treatment was observed in areas of cartilage greater than 1 mm away from the injury site. In this region, PCD was decreased to essentially background levels. A recent study using a cartilage explant model of compression injury provided convincing evidence that a diffusible mediator or mediators are responsible for propagation of chondrocyte PCD beyond the original site of injury [34]. We hypothesize that therapeutic approaches based upon apoptosis inhibition may be particularly effective in limiting the “zone of injury” following articular cartilage injury by interfering with the effects of these diffusible secondary injury mediators.
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In summary, the intra-articular administration of a broadspectrum caspase inhibitor significantly reduces the amount of chondrocyte apoptosis in vivo following experimental chondral injury. Therefore, intra-articular caspase inhibitor treatment may be a useful adjunct to existing cartilage repair and regeneration techniques that continue to be plagued by inconsistent graft incorporation and “edge-healing”. These findings also have broader significance in that they support PCD inhibition as a potential therapeutic strategy for cartilage injury in general. Further studies on the effects of apoptosis inhibitors in preventing cartilage degeneration and the progression of arthritis should be explored aggressively. Acknowledgements This work was supported by grants from the Arthroscopy Association of North America (AANA) and the Musculoskeletal Transplant Foundation. Conflict of interest statement: None of the authors have any financial or personal relationships with other people or organizations that could inappropriately influence the work presented herein, all within 3 years of beginning the work submitted. References [1] Curl WW, Krome J, Gordon ES, Rushing J, Smith BP, Poehling GG. Cartilage injuries: a review of 31,516 knee arthroscopies. Arthroscopy 1997;13(4):456–60. [2] O'Driscoll SW. The healing and regeneration of articular cartilage. J Bone Jt Surg Am Vol 1998;80(12):1795–812. [3] Brittberg M, Winalski CS. Evaluation of cartilage injuries and repair. J Bone Jt Surg Am Vol 2003;85-A(Suppl 2):58–69. [4] Browne JE, Branch TP. Surgical alternatives for treatment of articular cartilage lesions. J Am Acad Orthop Surg 2000;8(3):180–9. [5] Newman AP. Articular cartilage repair. Am J Sports Med 1998;26 (2):309–24. [6] Shapiro F, Koide S, Glimcher MJ. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J Bone Jt Surg Am Vol 1993;75(4):532–53. [7] Brittberg M, Nilsson A, Lindahl A, Ohlsson C, Peterson L. Rabbit articular cartilage defects treated with autologous cultured chondrocytes. Clin Orthop Relat Res 1996(326):270–83. [8] Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI, et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Jt Surg Am Vol 1994;76(4):579–92. [9] Hunziker EB, Quinn TM. Surgical removal of articular cartilage leads to loss of chondrocytes from cartilage bordering the wound edge. J Bone Jt Surg Am Vol 2003;85-A(Suppl 2):85–92. [10] Blanco FJ, Guitian R, Vazquez-Martul E, de Toro FJ, Galdo F. Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology. Arthritis Rheum 1998;41(2):284–9. [11] Hashimoto S, Ochs RL, Komiya S, Lotz M. Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Arthritis Rheum 1998;41(9):1632–8. [12] D'Lima DD, Hashimoto S, Chen PC, Colwell Jr CW, Lotz MK. Impact of mechanical trauma on matrix and cells. Clin Orthop 2001(391 Suppl): S90–9. [13] D'Lima DD, Hashimoto S, Chen PC, Lotz MK, Colwell Jr CW. Prevention of chondrocyte apoptosis. J Bone Jt Surg Am Vol 2001;83A(Suppl 2(Pt 1)):25–6.
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[14] Kim HT, Lo MY, Pillarisetty R. Chondrocyte apoptosis following intraarticular fracture in humans. Osteoarthr Cartil 2002;10(9):747–9. [15] Costouros JG, Dang AC, Kim HT. Inhibition of chondrocyte apoptosis in vivo following acute osteochondral injury. Osteoarthr Cartil 2003;11 (10):756–9. [16] Costouros JG, Dang AC, Kim HT. Comparison of chondrocyte apoptosis in vivo and in vitro following acute osteochondral injury. J Orthop Res 2004;22(3):678–83. [17 Aizawa T, Kon T, Einhorn TA, Gerstenfeld LC. Induction of apoptosis in chondrocytes by tumor necrosis factor-alpha. J Orthop Res 2001;19 (5):785–96. [18] Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995;146(1):3–15. [19] Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cyt 1980;68:251–306. [20] Tew SR, Kwan AP, Hann A, Thomson BM, Archer CW. The reactions of articular cartilage to experimental wounding: role of apoptosis. Arthritis Rheum 2000;43(1):215–25. [21] D'Lima DD, Hashimoto S, Chen PC, Colwell Jr CW, Lotz MK. Human chondrocyte apoptosis in response to mechanical injury. Osteoarthr Cartil 2001;9(8):712–9. [22] Lockshin RA, Zakeri Z, Tilly JL. When cells die. New York: John Wiley and Sons; 1998. [23] Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends Biochem Sci 1997;22(8):299–306. [24] Lee D, Long SA, Adams JL, Chan G, Vaidya KS, Francis TA, et al. Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality. J Biol Chem 2000;275 (21):16007–14. [25] Nuttall ME, Nadeau DP, Fisher PW, Wang F, Keller PM, DeWolf Jr WE, et al. Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro. J Orthop Res 2000;18(3):356–63. [26] Aigner T, Hemmel M, Neureiter D, Gebhard PM, Zeiler G, Kirchner T, et al. Apoptotic cell death is not a widespread phenomenon in normal aging and osteoarthritis human articular knee cartilage: a study of proliferation, programmed cell death (apoptosis), and viability of chondrocytes in normal and osteoarthritic human knee cartilage. Arthritis Rheum 2001;44(6):1304–12. [27] Frankfurt OS, Robb JA, Sugarbaker EV, Villa L. Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp Cell Res 1996;226(2):387–97. [28] Zunino SJ, Singh MK, Bass J, Picker LJ. Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes. Am J Pathol 1996;149(2):653–63. [29] van der Sluijs JA, Geesink RGT, van der Linden AJ, Bulstra SK, Kuyer R, Drukker J. The reliability of the Mankin score for osteoarthritis. J Orthop Res 1992;10:58–61. [30] Hunziker EB. Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable? Osteoarthr Cartil 1999;7(1):15–28. [31] Hunziker EaD, IMK. Surgical suturing of adult articular cartilage is associated with a loss of chondrocytes and an absence of wound healing. Transactions of the 49th annual meeting of the Orthopaedic Research Society; 2003. [32] D'Lima D, Hermida J, Hashimoto S, Colwell C, Lotz M. Caspase inhibitors reduce severity of cartilage lesions in experimental osteoarthritis. Arthritis Rheum Jun 2006;54(6):1814–21. [33] Dang AC, Warren AP, Kim HT. Beneficial effects of intra-articular caspase inhibition therapy following osteochondral injury. Osteoarthr Cartil Jun 2006;14(6):526–32. [34] Levin A, Burton-Wurster N, Chen CT, Lust G. Intercellular signaling as a cause of cell death in cyclically impacted cartilage explants. Osteoarthr Cartil 2001;9(8):702–11.
Preventing chondrocyte programmed cell death caused by iatrogenic injury John G. Costouros ⁎, Hubert T. Kim Department of Orthopaedic Surgery, University of California Medical Center, San Francisco, CA, United States Veterans Affairs Medical Center, San Francisco, CA, United States Received 21 August 2006; received in revised form 20 October 2006; accepted 30 October 2006
Abstract Cartilage repair technology is advancing at a rapid pace. However, all techniques share a common weakness—unintentional chondrocyte cell death resulting from cartilage injury that occurs during preparation of the defect site. The loss of chondrocytes at the edge of host cartilage is likely to contribute to failed integration of regenerated tissue or grafts to the surrounding cartilage. Recent studies have demonstrated that “apoptosis”, or programmed cell death (PCD), may be responsible for much of the cell death caused by cartilage injury. Theoretically, inhibitors of key pathways responsible for PCD could rescue chondrocytes and improve the results of cartilage repair surgery. The purpose of this study was to test the hypothesis that short-term, intra-articular PCD inhibitor treatment can limit chondrocyte death in vivo following simulated preparation of host cartilage for a repair procedure. A microcurette was used to create full-thickness articular cartilage injuries to the femoral condyles of adult New Zealand White rabbits. Animals received daily intra-articular injections either with a potent PCD inhibitor or with vehicle alone. Treatment with the inhibitor resulted in a significant reduction in the percentage of chondrocytes undergoing PCD compared to controls [treated = 10.1 ± 2.4%; controls = 26.5 ± 3.6%; (p = 0.0013)]. These results provide proof of concept for the use of PCD inhibitors to enhance the results of cartilage repair surgeries. © 2006 Elsevier B.V. All rights reserved. Keywords: Cartilage; Apoptosis; Trauma; Caspase; Arthritis
1. Introduction Despite surgical and technological advancements, the treatment of articular cartilage defects of the knee continues to challenge orthopaedic surgeons. Chondral lesions are common, and have been reported in 63% of over 31,000 arthroscopic procedures in one series [1]. Current operative treatments include debridement and stabilization, stimulation of intrinsic repair mechanisms, and repair or transplantation of chondral tissue [2–4]. A key step in all cartilage repair procedures is debridement of the damage site back to healthy cartilage. This procedure most commonly is performed with a ⁎ Corresponding author. University of California, San Francisco, Department of Orthopaedic Surgery, 260, International Circle, San Jose, CA 95119, United States. Tel.: +1 408 972 6326; fax: +1 408 972 3578. E-mail address: [email protected] (J.G. Costouros). 0968-0160/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.knee.2006.10.013
sharp curette. Among the problems common to all repair techniques is difficulty obtaining consistent integration at the wound edge [5–8]. It has been suggested that cell death at the edges of the prepared injury site may contribute to this lack of integration [9]. Recent studies have shown that chondrocyte apoptosis, or programmed cell death (PCD), is associated with acute cartilage injury and may contribute to the development of posttraumatic arthritis [10–16]. Apoptosis is a highly regulated process intrinsic to normal embryogenesis, immunological competence, tissue homeostasis, and skeletal development [17–19]. In vitro studies have demonstrated increased levels of chondrocyte apoptosis following experimental cartilage injuries including compression, drilling, and trephine wounding [15,16,20]. More recently, analysis of cartilage specimens from patients who have sustained knee injuries also demonstrated increased levels of chondrocyte PCD [14,21].
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Theoretically, apoptosis inhibitors could improve outcomes of cartilage repair and regeneration techniques by limiting chondrocyte death at the edge of the prepared cartilage defect, thus facilitating integration of cartilage repair tissue or grafts. Caspases (cysteinyl aspartate-specific proteases) are key enzymatic mediators of apoptosis [18,19,22,23]. The caspase inhibitor most commonly used in PCD inhibition studies is the non-selective, cell permeable, irreversible caspase inhibitor ZVAD-fmk. Recent experiments have shown that ZVAD-fmk can block chondrocyte apoptosis in vitro and preserve chondrocyte function [12,13,21,24,25]. These studies suggest that ZVAD-fmk could be a useful therapeutic agent in the treatment of articular cartilage injuries. The objective of this study was to determine whether intra-articular administration of ZVAD-fmk could decrease chondrocyte PCD following isolated chondral injury in a rabbit model.
0.1% DMSO, 0.5 cm3; Calbiochem, La Jolla, CA). Animals in the control group (N = 10) received daily intra-articular injections of vehicle alone (0.1% DMSO in normal saline, 0.5 cm3). All animals were euthanized 4 days following surgery and tissues harvested immediately for subsequent analysis. Previous experiments demonstrated that chondrocyte apoptosis reaches a peak approximately 4 days after cartilage injury in this model. The research protocol was reviewed and approved by the Animal Research Subcommittee at the Veterans Affairs Medical Center, San Francisco according to the established NIH guidelines.
2. Methods
2.3. Apoptosis analysis
2.1. Animal surgery: experimental chondral injury Adult female New Zealand White Rabbits (Western Oregon Rabbit Company, Philomath, OR) were anaesthetized with an intramuscular injection of ketamine (100 mg/kg) and xylazine (8 mg/kg). Antibiotic prophylaxis was administered (Trimethoprim–Sulfadiazine, 30 mg/kg), and the right knees were shaved and disinfected with Betadine solution in the standard sterile fashion. A standard medial parapatellar arthrotomy was performed to expose the underlying distal femoral condyles. A twomillimeter microcurette was then used to create a pair of linear full-thickness chondral defects in the weight-bearing portions of the medial and lateral femoral condyles. Close attention was paid to avoiding injury to the subchondral bone. An indwelling intra-articular epidural catheter (Arrow, Redding, PA) was then introduced subcutaneously from the upper thigh into the knee joint to enable consistent intra-articular drug delivery in controlled fashion. Animals received a second dose of antibiotic 24 h following surgery. Postoperatively, the animals were permitted cage activity without immobilization and monitored for signs of infection and distress. Animals in the treatment group (N = 10) received daily intra-articular injections of the broad-spectrum caspase inhibitor ZVAD-fmk (100 μM in
2.2. Tissue preparation The harvested femoral condyles were split in the sagittal plane within the patellar groove. Excess subchondral bone was removed, and the specimens were fixed in 10% buffered formalin at 4 °C overnight. Following fixation, the specimens were decalcified for 3 weeks (0.45 M EDTA/0.1 M Tris, pH 8.0). Tissues were then dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. 5 μm sections were cut on a rotary microtome and placed on Superfrost Plus slides.
Chondrocyte apoptosis was quantified by TUNEL analysis using the ApopTag® Direct in situ apoptosis detection kit (Intergen, Purchase, NY). Enzyme and substrate concentrations, as well as washing stringencies, were modified from the manufacturer's protocol in order to optimize the sensitivity and specificity of the assay [26]. Slides were mounted using Vectashield mounting media (Vector, Peterborough, UK) containing DAPI (1 μg/ml). Fluorescence images were captured using an Axiocam digital camera (Zeiss, NJ) at 1 megapixel resolution. The captured fields included the full thickness of articular cartilage extending 1.5 mm from either edge of curette injury. The captured fields were divided into three “Zones” with Zone 1 encompassing the full thickness of articular cartilage extending from 0–0.5 mm away from the injury; Zone 2 extending from 0.5–1 mm away from the injury; and Zone 3 extending from 1–1.5 mm away from the injury (Fig. 1). Semiautomated data collection and analysis were performed using digital image acquisition software (Scion, MD). An apoptotic index was calculated and defined as follows: [apoptotic index= (# of TUNEL positive / # of DAPI positive) × 100]. Each injury site was treated as an independent specimen, and a value for each “Zone” from each specimen was recorded as the mean of measurements from three separate near-adjacent sections. In order to validate the data obtained from TUNEL analysis, alternative methods of PCD analysis were employed including examination of nuclear
Fig. 1. Schematic representation of an osteochondral sample cut in the sagittal plane. Arrow represents the 2-millimeter microcurette used to create a fullthickness chondral lesion. Each zone, represented by a grey bar, corresponds to 0.5 mm in length. [Zone 1 = 0–0.5 mm from area of injury. Zone 2 = 0.5–1.0 mm from area of injury. Zone 3 = 1.0–1.5 mm from area of injury. C = cartilage. B = bone.]
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formed using Mann–Whitney test for non-parametric data. Statistical significance was defined as p b 0.05.
3. Results 3.1. Intra-articular caspase inhibition blocks chondrocyte PCD in vivo
Fig. 2. Inhibition of chondrocyte apoptosis in vivo with ZVAD-fmk following isolated chondral injury. Y-axis: depicts percentage of total chondrocytes undergoing apoptosis (# TUNEL-positive cells / # DAPI positive cells × 100). X-axis: depicts contiguous 0.5 mm full-thickness cartilage segments starting at the edge of the curette injury site (Zone 1 = 0– 0.5 mm; Zone 2 = 0.5–1.0 mm; Zone 3 = 1.0–1.5 mm). Asterisk indicates statistical significance between groups (Zone 2: p = 0.004; Zone 3: p = 0.0001; Total: p = 0.001).
morphology, and anti-ssDNA antibody (Chemicon, Temecula, CA) staining of formamide and heat-treated specimens. The anti-ssDNA staining technique has been demonstrated to be highly specific for apoptosis with an excellent ability to distinguish between apoptotic and necrotic cell death [27,28].
2.4. Histological assessment Sections were stained with hematoxylin and eosin (H and E) and Safranin-O/Fast Green under the following conditions: dye concentration 0.1%, staining time 6 min, and pH 5.7 at a temperature of 37 °C. Histological grading was performed based on cellularity and Safranin-O stain distribution as described by Mankin and modified by van der Sluijs [29].
2.5. Statistical analysis Statistical analysis between groups was performed using the two-tailed Students t-test for TUNEL data. Analysis of histological scores was per-
In order to determine whether or not intra-articular administration of ZVAD-fmk could decrease chondrocyte apoptosis after chondral damage, rabbits were subjected to full-thickness cartilage injuries using a two-millimeter microcurette. One group of animals received daily injections of ZVAD-fmk while a second group of animals received injections of vehicle alone. Previously published experiments using this model have demonstrated that chondrocyte PCD is at a maximum 4 days following injury [15,16,21]. Cartilage specimens from animals treated with ZVAD-fmk for 4 days following chondral injury showed a statistically significant decrease in chondrocyte PCD as quantified by TUNEL analysis (Fig. 2). Within 1.5 mm from the edge of the injury site, the percentage of TUNEL-positive chondrocytes in control specimens was 26.5 ± 3.6%. In the treated specimens, the amount of chondrocyte PCD was 10.1 ± 2.4%. This difference was highly statistically significant (p = 0.0013). This reduction in PCD was most pronounced in areas greater than 1 mm from the edge of the injury, where 21.8 ± 1.3% of control specimens displayed PCD compared to 3.1 ± 0.7% in specimens treated with the caspase inhibitor (p = 0.0001). In control specimens, a relatively diffuse distribution of apoptotic chondrocytes was noted, primarily confined to the superficial and middle zone of articular cartilage, and extending up to 1.5 mm away from either edge of the experimental injury site (Fig. 3). In treated specimens, apoptotic chondrocytes were primarily located in the superficial and middle zones within a 0.5 mm segment of cartilage immediately adjacent to the wound edge (Fig. 3). Chondrocyte apoptosis was confirmed by examination of nuclear morphology and immunohistochemistry analysis of formamide/heat-treated sections using an antibody specific for ssDNA as described. The amount and distribution of positively staining cells
Fig. 3. Representative cartilage sections which have undergone TUNEL analysis (A, B) and DAPI (C, D) staining. Apoptotic cells are TUNEL-positive and are identified by bright nuclear staining. DAPI identifies all cells (both apoptotic and non-apoptotic) by a non-specific nuclear staining. Control specimens to the left (A, C) and specimens treated with ZVAD-fmk to the right (B, D). Location of experimental injury is indicated by an arrow. TUNEL analysis reveals an overall decrease of apoptosis in treated specimens (B) in comparison to controls (A). The treatment effect is most pronounced moving away from the area of injury.
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Fig. 4. Left panel: anti-ssDNA staining of an untreated cartilage specimen from an area adjacent to a curette injury. Numerous true apoptotic cells are stained brown. Right panel: anti-ssDNA staining of an uninjured specimen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
were very similar to the results of the TUNEL analysis, with markedly decreased staining in treated specimens (Fig. 4). 3.2. Histological grading In order to determine whether or not treatment with ZVAD-fmk would have other beneficial, or possibly detrimental effects on the articular cartilage, all specimens were also processed for routine histology and scored using a modified Mankin scale. We observed some chondrocyte loss as defined by empty lacunae in both treated and control specimens. Mild proteoglycan loss as measured by Safranin-O staining was also noted in both treated and control specimens. However, using the modified Mankin grading scale, no statistically significant difference in histological scores was observed between treated and untreated specimens [treated = 2.6 ± 0.5; controls = 2.9 ± 0.5; (p = 0.60)].
4. Discussion Recent studies indicate that commonly performed arthroscopic procedures such as chondral shaving, debridement, and laser abrasion lead to death of chondrocytes bordering the edge of the treatment site and may have a long-term deleterious effect on articular cartilage [9,30]. In a recent study, Hunziker reported a significant chondrocyte loss at the wound edge of partial-thickness chondral defects in pigs repaired with an autologous fascial flap sutured over a fibrinous matrix [31]. Even seemingly benign procedures such as the suturing of a periosteal flap during autologous chondrocyte transplantation (ACI) may result in chondrocyte death along the path of the suture needle. Thus, the iatrogenic creation of an acellular or hypocellular zone of cartilage may be a major factor affecting edge healing and incorporation of transplanted repair tissue. Until recently, the specific mediators responsible for stimulating chondrocyte death in response to traumatic injury were largely unknown. Some chondrocytes clearly die as a result of catastrophic cellular injury in an unregulated fashion (i.e. via necrosis). However, an increasing body of evidence
supports the hypothesis that matrix injury initiates an apoptotic response in chondrocytes, and that it is possible to intervene in this process by blocking key mediators of apoptosis [13,24,25]. The experimental data in the present study demonstrate the in vivo effectiveness of intra-articular caspase inhibition in limiting chondrocyte PCD following experimental full-thickness cartilage injury. We noted a 38% overall reduction in chondrocyte PCD as measured by TUNEL analysis. These observations are consistent with previous in vitro studies. For instance, D'Lima and colleagues demonstrated a 50% reduction in PCD with ZVAD-fmk treatment following mechanical compression of cartilage explants [21]. Hopefully, other drugs or combinations of drugs will be able to block PCD even more effectively. However, it is likely that some of the TUNELpositive cells either cannot be rescued from cell death or are “false positives”. Only long-term in vivo studies can determine whether short-term PCD inhibition will actually improve chondrocyte viability and cartilage preservation. Recent studies have, in fact, demonstrated that PCD inhibition does result in cartilage preservation in rabbit models of osteoarthritis and osteochondral injury [32,33,34]. Consequently, we hypothesize that PCD inhibition will also have beneficial long-term effects on chondrocyte survival in the setting of cartilage repair surgeries. The most striking effect of ZVAD-fmk treatment was observed in areas of cartilage greater than 1 mm away from the injury site. In this region, PCD was decreased to essentially background levels. A recent study using a cartilage explant model of compression injury provided convincing evidence that a diffusible mediator or mediators are responsible for propagation of chondrocyte PCD beyond the original site of injury [34]. We hypothesize that therapeutic approaches based upon apoptosis inhibition may be particularly effective in limiting the “zone of injury” following articular cartilage injury by interfering with the effects of these diffusible secondary injury mediators.
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