- Email: [email protected]
Detection of Apoptotic Cartilage Cells in Symptomatic Central Tears of the Triangular Fibrocartilage
Detection of Apoptotic Cartilage Cells in Symptomatic Central Tears of the Triangular Fibrocartilage Frank Unglaub, MD, Jorg Fellenberg, PhD, Guenter Germann, MD, PhD, Berthold Bickert, MD, Michael Sauerbier, MD, PhD, Wiltrud Richter, PhD From the Department of Hand, Plastic and Reconstructive Surgery, Burn Center, BG Trauma Center Ludwigshafen, Plastic and Hand Surgery of the University of Heidelberg, Ludwigshafen, Germany; and Division of Experimental Orthopedics, Orthopedic University Hospital, Heidelberg, Germany.
Purpose: Central traumatic injuries of the triangular fibrocartilage are frequent sources of ulnar wrist pain. It is unknown whether the fibrocartilage cells survive this trauma in the central part of the triangular fibrocartilage. The goal of this study was to determine the viability of cells in traumatic triangular fibrocartilage complex (TFCC) Palmer 1A lesions to estimate the role of apoptosis for the fate of fibrocartilage cells. Methods: Twenty-two patients with a symptomatic central traumatic tear in the triangular fibrocartilage were included in this study. The cartilage was debrided arthroscopically, and histologic sections were used for the analysis of cell viability and apoptosis. Cell viability was quantified by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and apoptotic cells were visualized by poly(ADP-ribose) polymerase (PARP) p85 immunohistochemistry. The number of apoptotic cells was correlated with the total number of cells. Results: In contrast with the control patients with only minimal amounts of TUNEL-positive cells, cell viability was markedly reduced in all analyzed patients with TFCC Palmer 1A lesion. Likewise, the number of PARP p85–positive cells was significantly increased in all patients. This indicates that the observed cell death is attributed to apoptosis. Concerning the distribution of apoptotic cells, the inner zone of the biospy showed much higher numbers of apoptotic cells than the outer zone in both PARP and TUNEL staining. In contrast to the analyzed patients, no apoptosis could be detected in the controls. Conclusions: High amounts of apoptotic cartilage cells could be detected within the inner part of the triangular fibrocartilage suggesting that fibrocartilage cells died in response to trauma and were not quickly replaced by new viable cells. PARP p85–positive cells indicate that the apoptotic cascade plays a crucial role in the TFCC Palmer 1A disorder. (J Hand Surg 2007;32A:618 – 622. Copyright © 2007 by the American Society for Surgery of the Hand.) Key words: Apoptosis, degeneration, immunohistochemistry, Palmer 1A, TFCC.
entral traumatic injuries of the triangular fibrocartilage complex (TFCC) are frequent sources of ulnar wrist pain.1 Although the partial excision of the triangular fibrocartilage tears appears to be a clinically effective technique,2,3 little is known about the healing process and the cell viability of this area after trauma. The articular disk consists of a fibrocartilaginous tissue.4 –9 The disk is the central, thinnest portion of the horizontal part of the TFCC. The peripheral
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The Journal of Hand Surgery
10%– 40% of the articular disk is well vascularized, whereas the inner portion is avascular. Most tears affect this avascular part.10 Bednar et al suggested that tears in the periphery of the articular disk may have sufficient blood supply to enable a reparative response and, in theory, can heal. Tears that occur in the center do not have immediate access to blood supply and are not likely to heal,1 but Bednar et al could not show data to support their hypothesis. Apoptosis, or programmed cell death, is a physi-
Unglaub et al / Apoptosis in TFCC Palmer 1A
ologic process involved in various aspects of mammalian development, including embryogenesis, dependent tissue atrophy, and homeostasis of the immune system. Apoptosis can be triggered in a cell through either the extrinsic pathway or the intrinsic pathway. The extrinsic pathway is initiated through the stimulation of transmembrane death receptors, such as the CD95/Fas receptors, located on the cell membrane. In contrast, the intrinsic pathway is initiated through the release of signal factors by mitochondria within the cell. In both cases, the subsequent autocatalytic activation of a downstream cascade of caspases leads to the cleavage of specific substrates, including poly(ADP-ribose) polymerase (PARP), and thus activates the apoptotic executioners. Apoptosis research has expanded to a variety of different organs, tissues, and cell types.11–16 However, no studies investigated the role of apoptotic mechanisms in the TFCC lesion. Therefore, the goal of the current study was to investigate the extent of apoptotic cell death by regional quantification of apoptotic cells in punch biopsies to estimate the involvement of apoptosis in symptomatic traumatic lesions. Furthermore, the numbers of apoptotic cells were correlated with the time interval after trauma.
Materials and Methods
619
years (19 and 29 years). In both, the TFCC had to be removed because of a tumor with hemiresection of the ulna. No wrist disorder was known in these patients. Operative Technique Wrist distraction was provided by a wrist traction tower that maintained approximately 4 kg of distraction throughout the procedure via finger traps placed on the index, middle, and ring fingers. The wrist was distended with 5–10 mL of sterile saline solution, and the arthroscope (2.7 mm) was introduced in accordance with standard technique through the 3-4 portal. Sufficient material of the articular disk could be obtained in all cases; the palmar or dorsal radioulnar ligaments or the TFCC insertion at the base of the styloid were not violated in any procedure. Knowing ahead of time that the diameter of the biopsy punch was 3 mm (Arthrex, Munich, Germany), we routinely resected so far into the disk until we found all degenerative tissue. Most of the resection could be performed with the scope in the 3-4 portal and the biopsy punch in the 4-5 portal. The articular disk finally was debrided with a shaver. After completion of the procedure, the skin portals were closed with a 4-0 nonabsorbable suture. A compressive dressing and wrist splint were applied for 2 weeks.
Patients The study design was approved by the institutional review board. Twenty-two patients (15 men, 7 women) with a symptomatic Palmer 1A lesion were included. All patients had a history of trauma and complained of ulnar wrist pain and showed both a positive sign during the ulnocarpal stress test and tenderness at the distal end of the ulna. The fovea sign was positive in all patients. Fifteen patients reported clicking when moving the wrist. Before arthroscopy, all patients were treated conservatively with a brace for 3– 4 weeks and anti-inflammatory medication without showing signs of improvement of symptoms. Exclusion criteria were other Palmer lesions than 1A, patient age over 40 years, positive ulnar variance, distal radioulnar joint instability, carpal instability, acute carpal bone, radius or ulna fracture. The average age at the time of operation was 29 years ⫾ 6. Duration of symptoms before surgical intervention averaged 11 months ⫾ 138.
Histology The tissue was fixed in 4% formalin for 3 days and embedded in paraffin. Specimens were sectioned axially into slides of 5 m.
Control Group The TFCC complex of 2 patients (1 man, 1 woman) was used as the control. The average age was 24
In Situ Cell Death Detection Detection of cell death was done by terminal deoxyribonucleotidyl transferase-mediated dUTP nick
Immunohistochemistry The cleavage of the caspase substrate PARP was analyzed by immunohistochemistry using an antiPARP p85 antibody (Promega Corp., Madison, WI) specific for the 85-kD cleavage fragment that is produced by caspases exclusively in apoptotic cells. Paraffin sections were dewaxed in Xylol replacement medium (XEM)-200, rehydrated, and pretreated with Triton X-100 (0.2% in phosphate-buffered saline [PBS]) for 5 minutes at ambient temperature. For detection, the ImmunoCruz Staining System for use with rabbit primary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used according to the manufacturer’s protocol. The primary antibody was diluted 1:100.
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Figure 1. Immunohistochemical analysis of paraffin sections from patients with symptomatic Palmer 1A lesion (inner zone) and controls. (A and D) Hemalum (HE) staining. (B and E) TUNEL-staining using FastRed as substrate (arrows) and HE counterstaining. (C and F) PARP p85 staining using diaminobenzidine (DAB) as substrate (arrows) without counterstaining. Representative sections from each staining are shown.
end labeling (TUNEL) assay (Roche, Mannheim, Germany) according to the manufacturer’s protocol. In brief, 10 mol/L tissue sections from paraffinembedded disks were dewaxed in XEM-200, rehydrated, and pretreated with proteinase K (20 g/mL in 100 mmol/L Tris pH 8.0/50 mmol/L edetic acid [EDTA]) for 10 minutes at 37°C. Slides were rinsed twice in PBS and incubated with TUNEL reaction mixture for 1 hour at 37°C in a humidified chamber. After washing with PBS 50 L, Converter-AP solution was applied, and the slides were incubated for an additional 30 minutes at 37°C. The slides were washed again 3 times in PBS and incubated for 10 minutes at ambient temperature after adding the chromogenic substrate FastRed (Roche, Basel, Switzerland). Slides were counterstained with hemalum and mounted under glass coverslips using Eukitt (Kindler, Freiburg, Germany). Nucleus Staining After deparaffinization, the sections were stained with hemalum (Merck, Darmstadt, Germany). The staining was performed according to standard protocols. Briefly, after deparaffinization, specimens were incubated with hemalum for 6 minutes and eosin
(Chroma, Munster, Germany) for 10 minutes. The slides were rinsed in tap water for 20 minutes. Next, a dehydration was performed with 70%, 96%, and 100% propanol then washed with XEM and embedded in Eukitt (Kindler, Freiburg, Germany). Analysis Total number of cells (hemalum) and the number of positive cells (PARP, TUNEL, respectively) were counted separately in 3 high-power fields (HPF) (⫻100) for statistical analysis. Counting was done separately in the inner, middle, and outer zones, respectively. Relative numbers of apoptotic cells were calculated in relation to the total number of cells. Statistical Analysis The Mann-Whitney U test and Wilcoxon test were used for statistical analysis. Furthermore, the Spearman rank correlation test was used for correlating time of trauma and frequency of apoptosis. For all tests, a p value ⱕ .05 was considered to be significant. Data analysis was performed with SPSS for Windows 10.0 (SPSS inc. Chicago, IL).
Unglaub et al / Apoptosis in TFCC Palmer 1A
621
Table 1. Cell Viability and Occurence of Apoptosis in Fibrocartilaginous Cells of the Discus Articularis TUNEL
Controls Patients
PARP p85
Inner Zone
Middle Zone
Outer Zone
Inner Zone
Middle Zone
Outer Zone
4.00 34.90*
3.15 32.80*
3.80 27.00*
0.00 36.70**
0.00 32.60**
0.00 21.86**
p ⬎ .05
p ⬎ .05
p ⬎ .05 p ⬎ .05
p ⬍ .05
p ⬎ .05
*p ⬎ .05, **p ⬎ .01 compared with controls.
Results
Discussion
Nucleus Staining High numbers of total cells in the control group could be observed by hemalum staining. Patients with TFCC lesion showed significantly decreased numbers of total cells (p ⬎ .01) (Fig. 1A, D).
Apoptosis was detected and quantified using 2 independent methods of biopsies for Palmer 1A lesions. The widely used TUNEL assay was applied to detect DNA fragmentation in the cartilage disk cells. As DNA fragmentation is not restricted to apoptotic cells, but also occurs in necrotic cells, the apoptosisspecific cleavage product PARP p85 was also analyzed. Poly(ADP-ribose) polymerase is a substrate of caspases, the key enzymes of the apoptotic signaling pathway, and therefore not cleaved in vital or necrotic cells. With both assays, high amounts of fibrocartilaginous cell death could be detected within all symptomatic traumatic TFCC lesions. Up to 74% of the fibrocartilaginous cells were apoptotic in the central part of the disk. In comparison with other disorders of fibrocartilagenous tissue (ie, intervertebral disk degeneration12), there was a greatly higher proportion of apoptotic cells in the TFCC lesion. The highest amount of cell death was observed in the inner part of the biopsies. We suppose that the avascularity of the central TFCC is responsible for the high number of cell death. As mentioned above, the peripheral 10%– 40% of the articular disk is well vascularized, whereas the inner portion is avascular.1 However, no correlation was found between number of apoptotic cells and the interval before surgical intervention. Thus, we suppose that the apoptotic cascade is initiated rather quickly in response to the trauma and, once initiated, is not reversible and timeindependent. Mikic evaluated the TFCC of 180 cadaver wrists ranging in age from premature infants to 97 years.5 There was no evidence of degenerative change of any part in the third decade of life. He found no perforations before the age of 30. As a consequence, to exclude patients with degenerative TFCC lesions, inclusion criteria was limited to age 40 years or less in our study.
In Situ Cell Death Detection In all samples analyzed, high amounts of TUNELpositive fibrocartilaginous cells were detected in the traumatic lesions. The relative number of TUNELpositive fibrocartilaginous cells was 35% in the inner zone, 33% in the middle zone, and 27% in the outer zone. In contrast, only less than 5% TUNEL-positive cells could be detected in the controls. No significant differences (p ⬎ .05) of cell viability could be detected between the different zones (Table 1; Fig. 1B, E). In addition, there was no correlation between time of trauma, age, or gender in comparison with the number of TUNEL-positive fibrocartilaginous cells. PARP Cleavage Similar to the results of the TUNEL staining, the relative number of PARP p85–positive apoptotic fibrocartilaginous cells was markedly increased in the traumatic lesions of all analyzed patients. The number of apoptotic cells in the inner zone was significantly higher than in the outer zone (p ⬎ .05). However, no significant difference (p ⬎ .05) could be observed between the inner and the middle zones. In the inner zone, 37% of apoptotic fibrocartilaginous cells could be detected, whereas the middle zone showed 33% and the outer zone 22% of apoptotic cells. (Table 1; Fig. 1C, F). In the control group, no PARP p85–positive fibrocartilaginous cells were found. In addition, there was no correlation between time of trauma, age, or gender in comparison with the number of PARP p85–positive cells.
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Uchiyama and Nakatsuchi concluded from their study that ulnar plus variance plays an important role in inducing or causing progression of degenerative changes in the wrist.17 Thus, patients with ulnar plus variance were excluded from our study as well. The high number of apoptotic fibrocartilaginous cells indicates that Palmer 1A lesions have little potential to heal. This supports the theory of Bednar et al,1 who suggest that the avascularity of the central part may be responsible for the poor healing potential. Nitric oxide production increases with the development of osteoarthritis and has been shown to have a catabolic effect on chondrocytes. Kobayashi et al and Hashimoto et al showed that increased synthesis of nitric oxide is associated with an increased apoptosis of chondrocytes.18,19 Although the observed differences between the controls and the TFCC patients are very clear and sound, the fact that only 2 control TFCC disks were available is one limitation of this study. However, only TFCC material can be used for this study; it is fixed in formalin immediately after harvesting, therefore, cadaver tissue is not suitable for these kinds of assays. Received for publication February 6, 2006; accepted in revised form February 27, 2007. The authors thank K. Goetzke for excellent technical assistance. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Supported by the Albert-J.B.-Sturm Stiftung, Villingen-Schwenningen, Germany. Corresponding author: F. Unglaub, MD, Department of Hand, Plastic and Reconstructive Surgery, Burn Center, BG Trauma Center Ludwigshafen, Plastic and Hand Surgery of the University Heidelberg, Ludwig-Guttmannstr. 13, 67071 Ludwigshafen, Germany; e-mail: [email protected] Copyright © 2007 by the American Society for Surgery of the Hand 0363-5023/07/32A05-0005$32.00/0 doi:10.1016/j.jhsa.2007.02.023
References 1. Bednar MS, Arnoczky SA, Weiland A. The microvasculature of the triangular fibrocartilage complex: its clinical significance. J Hand Surg 1991;16A:1101–1105. 2. Osterman AL. Arthroscopic debridement of triangular fibrocartilage complex tears. Arthroscopy 1990;6:120 –124.
3. Husby T, Haugstvedt JR. Long-term results after arthroscopic resection of lesions of the triangular fibrocartilage complex. Scand J Plast Reconstr Hand Surg 2001;35:79 – 83. 4. Palmer AK, Werner F. The triangular fibrocartilage complex of the wrist – anatomy and function. J Hand Surg [A] 1981;2:153–162. 5. Mikic Z. Age changes in the triangular fibrocartilage of the wrist joint. J Anat 1978;126:367–384. 6. Chidgey LK, Dell PC, Bittar ES, Spanier SS. Histologic anatomy of the triangular fibrocartilage. J Hand Surg 1991; 16A:1084 –1100. 7. Nakamura T, Yabe Y. Histological anatomy of the triangular fibrocartilage complex of the human wrist. Ann Anat 2000; 182:567–572. 8. Gracia-Elias M, Domenech-Mateu JM. The articular disc of the wrist. Limits and relations. Acta Anat 1987;128:51–54. 9. Schmidt HM. The anatomy of the ulnocarpal complex. Orthopade 2004;6:628 – 637. 10. Palmer AK. Triangular fibrocartilage complex lesions: a classification. J Hand Surg 1989;14A:594 – 606. 11. Gu Z, Shibata T, Cao Z, Cao Z, Feng J, Hu J. Chondrocyte apoptosis in temporomandibular joints with disc displacement. J Oral Maxillofac Surg 2002;60:1026 –1031. 12. Chen B, Fellenberg J, Wang H, Carstens C, Richter W. Occurrence and regional distribution of apoptosis in scoliotic discs. Spine 2005;30:519 –524. 13. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239 –257. 14. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998;281:1305–1308. 15. Unglaub F, Guehring T, Lorenz H, Carstens C, Kroeber MW. Effects of unisegmental dynamic disc compression on adjacent segments: an in vivo animal model. Eur Spine J 2005;10:949 –955. 16. Unglaub F, Guehring T, Omlor G, Lorenz H, Carstens C, Kroeber MW. Controlled distraction as a therapeutic option in moderate degeneration of the intervertebral disc – an in vivo study in rabbit-spine model. Z Orthop Ihre Grenzgeb 2006;1:68 –73. 17. Uchiyama S, Nakatsuchi Y. Anatomical and radiological evaluation of the triangular fibrocartilage complex of the wrist. J Hand Surg 1994;19B:319 –324. 18. Kobayashi K, Mishima H, Hashimoto S, Goomer RS, Harwood FL, Lotz M, et al. Chondrocyte apoptosis and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy. J Orthop Res 200;19:802– 808. 19. Hashimoto S, Takahashi K, Ochs RL, Coutts RD, Amiel D, Lotz M. Nitric oxide production and apoptosis in cells of the meniscus during experimental osteoarthritis. Arthritis Rheum 1999;42:2123–2131.
Purpose: Central traumatic injuries of the triangular fibrocartilage are frequent sources of ulnar wrist pain. It is unknown whether the fibrocartilage cells survive this trauma in the central part of the triangular fibrocartilage. The goal of this study was to determine the viability of cells in traumatic triangular fibrocartilage complex (TFCC) Palmer 1A lesions to estimate the role of apoptosis for the fate of fibrocartilage cells. Methods: Twenty-two patients with a symptomatic central traumatic tear in the triangular fibrocartilage were included in this study. The cartilage was debrided arthroscopically, and histologic sections were used for the analysis of cell viability and apoptosis. Cell viability was quantified by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and apoptotic cells were visualized by poly(ADP-ribose) polymerase (PARP) p85 immunohistochemistry. The number of apoptotic cells was correlated with the total number of cells. Results: In contrast with the control patients with only minimal amounts of TUNEL-positive cells, cell viability was markedly reduced in all analyzed patients with TFCC Palmer 1A lesion. Likewise, the number of PARP p85–positive cells was significantly increased in all patients. This indicates that the observed cell death is attributed to apoptosis. Concerning the distribution of apoptotic cells, the inner zone of the biospy showed much higher numbers of apoptotic cells than the outer zone in both PARP and TUNEL staining. In contrast to the analyzed patients, no apoptosis could be detected in the controls. Conclusions: High amounts of apoptotic cartilage cells could be detected within the inner part of the triangular fibrocartilage suggesting that fibrocartilage cells died in response to trauma and were not quickly replaced by new viable cells. PARP p85–positive cells indicate that the apoptotic cascade plays a crucial role in the TFCC Palmer 1A disorder. (J Hand Surg 2007;32A:618 – 622. Copyright © 2007 by the American Society for Surgery of the Hand.) Key words: Apoptosis, degeneration, immunohistochemistry, Palmer 1A, TFCC.
entral traumatic injuries of the triangular fibrocartilage complex (TFCC) are frequent sources of ulnar wrist pain.1 Although the partial excision of the triangular fibrocartilage tears appears to be a clinically effective technique,2,3 little is known about the healing process and the cell viability of this area after trauma. The articular disk consists of a fibrocartilaginous tissue.4 –9 The disk is the central, thinnest portion of the horizontal part of the TFCC. The peripheral
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The Journal of Hand Surgery
10%– 40% of the articular disk is well vascularized, whereas the inner portion is avascular. Most tears affect this avascular part.10 Bednar et al suggested that tears in the periphery of the articular disk may have sufficient blood supply to enable a reparative response and, in theory, can heal. Tears that occur in the center do not have immediate access to blood supply and are not likely to heal,1 but Bednar et al could not show data to support their hypothesis. Apoptosis, or programmed cell death, is a physi-
Unglaub et al / Apoptosis in TFCC Palmer 1A
ologic process involved in various aspects of mammalian development, including embryogenesis, dependent tissue atrophy, and homeostasis of the immune system. Apoptosis can be triggered in a cell through either the extrinsic pathway or the intrinsic pathway. The extrinsic pathway is initiated through the stimulation of transmembrane death receptors, such as the CD95/Fas receptors, located on the cell membrane. In contrast, the intrinsic pathway is initiated through the release of signal factors by mitochondria within the cell. In both cases, the subsequent autocatalytic activation of a downstream cascade of caspases leads to the cleavage of specific substrates, including poly(ADP-ribose) polymerase (PARP), and thus activates the apoptotic executioners. Apoptosis research has expanded to a variety of different organs, tissues, and cell types.11–16 However, no studies investigated the role of apoptotic mechanisms in the TFCC lesion. Therefore, the goal of the current study was to investigate the extent of apoptotic cell death by regional quantification of apoptotic cells in punch biopsies to estimate the involvement of apoptosis in symptomatic traumatic lesions. Furthermore, the numbers of apoptotic cells were correlated with the time interval after trauma.
Materials and Methods
619
years (19 and 29 years). In both, the TFCC had to be removed because of a tumor with hemiresection of the ulna. No wrist disorder was known in these patients. Operative Technique Wrist distraction was provided by a wrist traction tower that maintained approximately 4 kg of distraction throughout the procedure via finger traps placed on the index, middle, and ring fingers. The wrist was distended with 5–10 mL of sterile saline solution, and the arthroscope (2.7 mm) was introduced in accordance with standard technique through the 3-4 portal. Sufficient material of the articular disk could be obtained in all cases; the palmar or dorsal radioulnar ligaments or the TFCC insertion at the base of the styloid were not violated in any procedure. Knowing ahead of time that the diameter of the biopsy punch was 3 mm (Arthrex, Munich, Germany), we routinely resected so far into the disk until we found all degenerative tissue. Most of the resection could be performed with the scope in the 3-4 portal and the biopsy punch in the 4-5 portal. The articular disk finally was debrided with a shaver. After completion of the procedure, the skin portals were closed with a 4-0 nonabsorbable suture. A compressive dressing and wrist splint were applied for 2 weeks.
Patients The study design was approved by the institutional review board. Twenty-two patients (15 men, 7 women) with a symptomatic Palmer 1A lesion were included. All patients had a history of trauma and complained of ulnar wrist pain and showed both a positive sign during the ulnocarpal stress test and tenderness at the distal end of the ulna. The fovea sign was positive in all patients. Fifteen patients reported clicking when moving the wrist. Before arthroscopy, all patients were treated conservatively with a brace for 3– 4 weeks and anti-inflammatory medication without showing signs of improvement of symptoms. Exclusion criteria were other Palmer lesions than 1A, patient age over 40 years, positive ulnar variance, distal radioulnar joint instability, carpal instability, acute carpal bone, radius or ulna fracture. The average age at the time of operation was 29 years ⫾ 6. Duration of symptoms before surgical intervention averaged 11 months ⫾ 138.
Histology The tissue was fixed in 4% formalin for 3 days and embedded in paraffin. Specimens were sectioned axially into slides of 5 m.
Control Group The TFCC complex of 2 patients (1 man, 1 woman) was used as the control. The average age was 24
In Situ Cell Death Detection Detection of cell death was done by terminal deoxyribonucleotidyl transferase-mediated dUTP nick
Immunohistochemistry The cleavage of the caspase substrate PARP was analyzed by immunohistochemistry using an antiPARP p85 antibody (Promega Corp., Madison, WI) specific for the 85-kD cleavage fragment that is produced by caspases exclusively in apoptotic cells. Paraffin sections were dewaxed in Xylol replacement medium (XEM)-200, rehydrated, and pretreated with Triton X-100 (0.2% in phosphate-buffered saline [PBS]) for 5 minutes at ambient temperature. For detection, the ImmunoCruz Staining System for use with rabbit primary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used according to the manufacturer’s protocol. The primary antibody was diluted 1:100.
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Figure 1. Immunohistochemical analysis of paraffin sections from patients with symptomatic Palmer 1A lesion (inner zone) and controls. (A and D) Hemalum (HE) staining. (B and E) TUNEL-staining using FastRed as substrate (arrows) and HE counterstaining. (C and F) PARP p85 staining using diaminobenzidine (DAB) as substrate (arrows) without counterstaining. Representative sections from each staining are shown.
end labeling (TUNEL) assay (Roche, Mannheim, Germany) according to the manufacturer’s protocol. In brief, 10 mol/L tissue sections from paraffinembedded disks were dewaxed in XEM-200, rehydrated, and pretreated with proteinase K (20 g/mL in 100 mmol/L Tris pH 8.0/50 mmol/L edetic acid [EDTA]) for 10 minutes at 37°C. Slides were rinsed twice in PBS and incubated with TUNEL reaction mixture for 1 hour at 37°C in a humidified chamber. After washing with PBS 50 L, Converter-AP solution was applied, and the slides were incubated for an additional 30 minutes at 37°C. The slides were washed again 3 times in PBS and incubated for 10 minutes at ambient temperature after adding the chromogenic substrate FastRed (Roche, Basel, Switzerland). Slides were counterstained with hemalum and mounted under glass coverslips using Eukitt (Kindler, Freiburg, Germany). Nucleus Staining After deparaffinization, the sections were stained with hemalum (Merck, Darmstadt, Germany). The staining was performed according to standard protocols. Briefly, after deparaffinization, specimens were incubated with hemalum for 6 minutes and eosin
(Chroma, Munster, Germany) for 10 minutes. The slides were rinsed in tap water for 20 minutes. Next, a dehydration was performed with 70%, 96%, and 100% propanol then washed with XEM and embedded in Eukitt (Kindler, Freiburg, Germany). Analysis Total number of cells (hemalum) and the number of positive cells (PARP, TUNEL, respectively) were counted separately in 3 high-power fields (HPF) (⫻100) for statistical analysis. Counting was done separately in the inner, middle, and outer zones, respectively. Relative numbers of apoptotic cells were calculated in relation to the total number of cells. Statistical Analysis The Mann-Whitney U test and Wilcoxon test were used for statistical analysis. Furthermore, the Spearman rank correlation test was used for correlating time of trauma and frequency of apoptosis. For all tests, a p value ⱕ .05 was considered to be significant. Data analysis was performed with SPSS for Windows 10.0 (SPSS inc. Chicago, IL).
Unglaub et al / Apoptosis in TFCC Palmer 1A
621
Table 1. Cell Viability and Occurence of Apoptosis in Fibrocartilaginous Cells of the Discus Articularis TUNEL
Controls Patients
PARP p85
Inner Zone
Middle Zone
Outer Zone
Inner Zone
Middle Zone
Outer Zone
4.00 34.90*
3.15 32.80*
3.80 27.00*
0.00 36.70**
0.00 32.60**
0.00 21.86**
p ⬎ .05
p ⬎ .05
p ⬎ .05 p ⬎ .05
p ⬍ .05
p ⬎ .05
*p ⬎ .05, **p ⬎ .01 compared with controls.
Results
Discussion
Nucleus Staining High numbers of total cells in the control group could be observed by hemalum staining. Patients with TFCC lesion showed significantly decreased numbers of total cells (p ⬎ .01) (Fig. 1A, D).
Apoptosis was detected and quantified using 2 independent methods of biopsies for Palmer 1A lesions. The widely used TUNEL assay was applied to detect DNA fragmentation in the cartilage disk cells. As DNA fragmentation is not restricted to apoptotic cells, but also occurs in necrotic cells, the apoptosisspecific cleavage product PARP p85 was also analyzed. Poly(ADP-ribose) polymerase is a substrate of caspases, the key enzymes of the apoptotic signaling pathway, and therefore not cleaved in vital or necrotic cells. With both assays, high amounts of fibrocartilaginous cell death could be detected within all symptomatic traumatic TFCC lesions. Up to 74% of the fibrocartilaginous cells were apoptotic in the central part of the disk. In comparison with other disorders of fibrocartilagenous tissue (ie, intervertebral disk degeneration12), there was a greatly higher proportion of apoptotic cells in the TFCC lesion. The highest amount of cell death was observed in the inner part of the biopsies. We suppose that the avascularity of the central TFCC is responsible for the high number of cell death. As mentioned above, the peripheral 10%– 40% of the articular disk is well vascularized, whereas the inner portion is avascular.1 However, no correlation was found between number of apoptotic cells and the interval before surgical intervention. Thus, we suppose that the apoptotic cascade is initiated rather quickly in response to the trauma and, once initiated, is not reversible and timeindependent. Mikic evaluated the TFCC of 180 cadaver wrists ranging in age from premature infants to 97 years.5 There was no evidence of degenerative change of any part in the third decade of life. He found no perforations before the age of 30. As a consequence, to exclude patients with degenerative TFCC lesions, inclusion criteria was limited to age 40 years or less in our study.
In Situ Cell Death Detection In all samples analyzed, high amounts of TUNELpositive fibrocartilaginous cells were detected in the traumatic lesions. The relative number of TUNELpositive fibrocartilaginous cells was 35% in the inner zone, 33% in the middle zone, and 27% in the outer zone. In contrast, only less than 5% TUNEL-positive cells could be detected in the controls. No significant differences (p ⬎ .05) of cell viability could be detected between the different zones (Table 1; Fig. 1B, E). In addition, there was no correlation between time of trauma, age, or gender in comparison with the number of TUNEL-positive fibrocartilaginous cells. PARP Cleavage Similar to the results of the TUNEL staining, the relative number of PARP p85–positive apoptotic fibrocartilaginous cells was markedly increased in the traumatic lesions of all analyzed patients. The number of apoptotic cells in the inner zone was significantly higher than in the outer zone (p ⬎ .05). However, no significant difference (p ⬎ .05) could be observed between the inner and the middle zones. In the inner zone, 37% of apoptotic fibrocartilaginous cells could be detected, whereas the middle zone showed 33% and the outer zone 22% of apoptotic cells. (Table 1; Fig. 1C, F). In the control group, no PARP p85–positive fibrocartilaginous cells were found. In addition, there was no correlation between time of trauma, age, or gender in comparison with the number of PARP p85–positive cells.
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The Journal of Hand Surgery / Vol. 32A No. 5 May–June 2007
Uchiyama and Nakatsuchi concluded from their study that ulnar plus variance plays an important role in inducing or causing progression of degenerative changes in the wrist.17 Thus, patients with ulnar plus variance were excluded from our study as well. The high number of apoptotic fibrocartilaginous cells indicates that Palmer 1A lesions have little potential to heal. This supports the theory of Bednar et al,1 who suggest that the avascularity of the central part may be responsible for the poor healing potential. Nitric oxide production increases with the development of osteoarthritis and has been shown to have a catabolic effect on chondrocytes. Kobayashi et al and Hashimoto et al showed that increased synthesis of nitric oxide is associated with an increased apoptosis of chondrocytes.18,19 Although the observed differences between the controls and the TFCC patients are very clear and sound, the fact that only 2 control TFCC disks were available is one limitation of this study. However, only TFCC material can be used for this study; it is fixed in formalin immediately after harvesting, therefore, cadaver tissue is not suitable for these kinds of assays. Received for publication February 6, 2006; accepted in revised form February 27, 2007. The authors thank K. Goetzke for excellent technical assistance. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Supported by the Albert-J.B.-Sturm Stiftung, Villingen-Schwenningen, Germany. Corresponding author: F. Unglaub, MD, Department of Hand, Plastic and Reconstructive Surgery, Burn Center, BG Trauma Center Ludwigshafen, Plastic and Hand Surgery of the University Heidelberg, Ludwig-Guttmannstr. 13, 67071 Ludwigshafen, Germany; e-mail: [email protected] Copyright © 2007 by the American Society for Surgery of the Hand 0363-5023/07/32A05-0005$32.00/0 doi:10.1016/j.jhsa.2007.02.023
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