Cloxacillin

ABSTRACT

Purpose. To investigate the harmful effects of a single episode of intra-articular bleeding on articular cartilage of rabbit knees using scanning electron microscopy.

Methods. Autologous blood was injected into the right knee joints of 18 New Zealand white rabbits. Surface and cellular damages were examined by the scanning electron microscope (n=9) and light microscope (n=34), respectively. The injected right knees were then compared with the corresponding control left knees at one, 3, and 6 weeks after the blood injection.

Results. The articular surface of the injected knees turned uniformly rough with multiple pits after one week. Maximal changes with elevations and depressions were observed at 3 weeks. These changes reversed at 6 weeks with the irregularities smoothing out. A similar pattern of transient cartilage damage was noted histologically.

Conclusion. Both scanning electron microscopic and light microscopic findings suggest that a single episode of intra-articular bleeding leads to articular cartilage damage but this appears to be reversible. Our findings of transient damage to the articular cartilage suggest that there is no need for intra-articular evacuation and washout following an acute episode of haemarthrosis.

Key words: blood; cartilage, articular; hemarthrosis; microscopy, electron, scanning

INTRODUCTION

Blood in a joint cavity is unphysiological and potentially harmful. Much of our knowledge of irreversible articular damage is derived from haemophiliacs with repeated episodes of intra-articular bleeding.1,2 However, it is not certain if a single bleeding episode after a trauma would lead to permanent articular damage. This is difficult to prove because the harmful effects may take many years to manifest or the haemarthrosis may be associated with other injuries that actually lead to the long-term damage. Opinions differ regarding the long-term effects of a single bleeding episode. Some authors reported lasting damage to the articular cartilage,3-6 whereas others reported no harmful effects or transient changes only.7-10 So far, most of these studies have relied on biochemical analyses to determine cartilage damage. We decided to use a different technique-scanning electron microscopy (SEM)-to study the effects of a single bleeding episode on articular cartilage. SEM has a great depth of focus and a high degree of resolution and has been shown to be most adaptable to the surface study of articular cartilage.11 It has been proven to be very useful in the study of articular changes associated with osteoarthritis,12-16 rheumatoid arthritis,11 and chondromalacia patellae.16,17 In blood-induced cartilage damage, the cartilage surface is expected to be most affected as blood comes into direct contact with it. Any changes in the cartilage surface would be easily observed by SEM. Light microscopy (LM) was used to study cellular damage and we correlated our LM findings with SEM findings.

MATERIALS AND METHODS

18 mature New Zealand white rabbits (17 males, one female) with a mean age of 6 months and mean weight of 2.8 kg were used in our study. They were randomly divided into 3 groups with 6 rabbits each for assessment of cartilage damage at one, 3, and 6 weeks after an intra-articular blood injection. The rabbits were sedated with intramuscular injection of diazepam (0.5 mg/kg) and ketamine (50 mg/kg), then 2 ml of blood was drawn from the most prominent dorsal vein of the ear using a 21-gauge heparinised needle. This whole blood was injected immediately into the right knee joint. A single dose of antibiotics (cloxacillin and ampicillin, each 40 mg/kg) was given intramuscularly after the blood injection. The left knee joints served as controls. 17 rabbits were euthanised by giving intracardiac phenobarbitone. The only female rabbit was spared because it had delivered 4 babies a day prior to the scheduled euthanasia. Within 30 minutes of euthanasia, both knee joints were exposed and the weight-bearing portions of both medial femoral condyles were harvested. The harvested samples were halved (3-4 mm each) and preserved in 10% phosphate-buffered formalin. All procedures were carried out aseptically in the animal operating theatre. Adequate precautions were taken to prevent damage to the articular surface during harvesting and handling of samples. Because of cost constraints, only 9 samples (2 tests and one control from each of the 3 study groups i.e. one, 3, and 6 weeks after the blood injection) were processed for the SEM study. Each sample was washed and dehydrated through an ascending ethanol series at room temperature and dried in a critical point dryer using paint. The specimen was then mounted to the SEM stub and the articular surface of the specimen was sputter-coated with gold. All prepared specimens were then examined using the JEOL JSM-5600LV scanning electron microscope (JEOL, Peabody [MA], US). All 34 samples (17 tests, 17 controls) were then processed for the LM study. The dehydrated specimens were impregnated with wax and mounted as histology blocks, which were then cut into 5 µm-thin sections using a Leica 2135 microtome (Leica, Wetzlar, Germany). Sections were dewaxed by heating and then stained with haemotoxylin and eosin and finally mounted on glass slides.

RESULTS

All rabbits remained healthy throughout the study. There was an initial effusion of blood-injected right knees and a limp was noted which subsided within 2 to 3 days after the injection. No gross articular or other bony changes were detected in the joints while harvesting the samples.

SEM examination

Test samples in each group showed similar changes. The articular surface of the control samples appeared smooth throughout the field (Fig. 1). The articular surface of the test samples at one week was no longer smooth and revealed multiple small pits (Fig. 2). Maximal surface changes were seen at 3 weeks. Multiple elevations and depressions were noted (Fig. 3a) that, at higher magnification, mimic the gross appearance of sulci and gyrii of the human brain (Fig. 3b). Interestingly, repair had already taken place in the test samples. At 6 weeks, the irregularities disappeared and the surface became smooth again (Fig. 4).

LM examination

Similar changes within each LM study group were also noted. The control samples showed chondrocytes arranged tangentially in 2 to 3 layers in the superficial zone and in vertical clusters in the deeper zones (Fig. 5). At one week, the chondrocytes of test samples were noted to be more sparse and smaller. The tangential arrangement was maintained but the deeper vertical cluster pattern was disturbed (Fig. 6). Like the SEM findings, the most significant finding was noted at 3 weeks. Empty lacunae arranged tangentially in the superficial zone were observed at 3 weeks (Fig. 7), representing dead chondrocytes. In the deeper zones, the chondrocytes were spared. Interestingly, at 6 weeks, the empty lacunae were repopulated by chondrocytes and an attempt to re-organise into the vertical cluster pattern was noted (Fig. 8).

DISCUSSION

The surfaces of the normal and abnormal articular cartilage have been studied frequently by SEM alone or in combination with other methods. The articular cartilage surface of a normal joint has been described as smooth by Bloebaum and Radley18 using SEM. Our study also showed the normal articular surface (control) to be smooth. In SEM studies of articular cartilage in various conditions (e.g. chondromalacia patellae, osteoarthritis, and rheumatoid arthritis), features such as clefts, scaly irregularities, craters, fissures were observed in the diseased cartilage.11-22 In our study, the surface changed from a smooth surface to one scattered with pits after one week, and subsequently at 3 weeks, to one scattered with elevations and depressions indicative of increasing surface damage. These changes were similar to those described by other SEM studies of diseased cartilage mentioned above. These SEM findings also correlated well with the LM findings. An interesting finding of the SEM studies was that repair of the surface was observed at 6 weeks, which correlated with the LM findings. Both findings suggest that a single bleeding episode is sufficient to cause cartilage damage but this is reversible. Several studies have suggested that a single episode of haemarthrosis causes lasting damage to the articular cartilage.3-6 Findings include permanent changes to the proteoglycan and collagen content. Roosendaal et al.4 proposed reconsidering the treatment of acute haemarthrosis such as joint aspiration to remove the intra-articular blood. On the other hand, other authors have shown that a single episode of bleeding only resulted in transient changes.7-10 Safran et al.7 demonstrated in a rabbit model that a single injection of blood into the ankle joints resulted in only transient changes in joint stiffness and synovial and cartilage histomorphology. It was suggested that therapeutic arthrocentesis is not necessary in the treatment of acute post-traumatic haemathrosis.

CONCLUSION

We believe our study to be the first to look into the harmful effects of a single episode of intra-articular bleeding using SEM. Our SEM findings correlated well with the LM findings and demonstrated only transient changes to the articular cartilage. These findings suggest that there is no need for evacuation of intra-articular blood and washout following acute haemarthrosis.

Although known for its limited regenerative potential, the articular cartilage in our study showed an ability to regenerate following an insult of a single bleeding episode. Early mobilisation following traumatic haemarthrosis whenever possible would be an appropriate measure to enhance this regeneration. We conclude that a single episode of intra-articular bleeding results in transient damage of articular cartilage. Therefore, there is no need for evacuation of intra-articular blood and washout following acute haemarthrosis.

ACKNOWLEDGEMENTS

The authors thank Dr Balazs Borsizsky, Mr Robert Ng, Ms Song Im Chin, and the staff of the Department of Experimental Surgery, Singapore General Hospital for their advice and assistance in this project.

REFERENCES

1. Arnold WD, Hilgartner MW. Hemophilic arthropathy. Current concepts of pathogenesis and management. J Bone Joint and Surg Am 1977;59:287-305.

2. Stein H, Duthie RB. The pathogenesis of chronic haemophilic arthropathy. J Bone Joint Surg Br 1981;63:601-9.

3. Roosendaal G, Vianen ME, Marx JJ, van den Berg HM, Lafeber FP, Bijlsma JW. Blood-induced joint damage: a human in vitro study. Arthritis Rheum 1999;42:1025-32.

4. Roosendaal C, TeKoppele JM, Vianen ME, van den Berg HM, Lafeber FP, Bijlsma JW. Blood-induced joint damage: a canine in vivo study. Arthritis Rheum 1999;42:1033-9.

5. Niibayashi H, Shimizu K, Suzuki K, Yamamoto S, Yasuda T, Yamamuro T. Proteoglycan degradation in hemarthrosis. Intraarticular, autologous blood injection in rat knees. Acta Orthop Scand 1995;66:73-9.

6. Fabry C. Early changes in the ground substance of articular cartilage in experimental hemarthrosis in dogs, measured by the fixed-charge density method. Arch Orthop Trauma Surg 1989;108:76-91.

7. Safran MR, Johnston-Jones K, Kabo JM, Meals RA. The effect of experimental hemarthrosis on joint stiffness and synovial histology in a rabbit model. Clin Orthop 1994;303:280-8.

8. Hoaglund FT. Experimental hemarthrosis. The response of canine knees to injections of autologous blood. J Bone Joint Surg Am 1967;49:285-98.

9. Roy S, Ghadially FN. Pathology of experimental haemarthrosis. Ann Rheum Dis 1966;25:402-15.

10. Wolf CR, Mankin HJ. The effect of experimental hemarthrosis on articular cartilage of rabbit knee joints. J Bone Joint Surg Am 1965;47:1203-10.

11. Redler I, Zimny ML. Scanning electron microscopy of normal and abnormal articular cartilage and synovium. J Bone Joint Surg Am 1970;52:1395-404.

12. Gulisano M, Delrio AN, Fadda M, Marceddu S. Human articular cartilage during osteoarthrosis: a study under the scanning electron microscope. Ital J Anat Embryol 1993;98:175-85.

13. O'Sullivan FX, Koopman WJ, Gay S. Scanning electron microscopic evaluation of the arthritis in MRL/lpr mice. Rheumatol Int 1992;12:115-20.

14. Bendele AM, White SL, Hulman JF. Osteoarthrosis in guinea pigs: histopathologic and scanning electron microscopic features. Lab Anim Sci 1989;39:115-21.

15. van Lent PE, Wilms FH, van den Berg WB. Interaction of polymorphonuclear leucocytes with patellar cartilage of immobilised arthritic joints: a scanning electron microscopic study. Ann Rheum Dis 1989;48:832-7.

16. Candolin T, Videman T. Surface changes in the articular cartilage of rabbit knee during immobilization. A scanning electron microscopic study of experimental osteoarthritis. Acta Pathol Microbiol Scand 1980;88:291-7.

17. Mori Y, Kubo M, Okumo H, Kuroki Y. A scanning electron microscopic study of the degenerative cartilage in patellar chondropathy. Arthroscopy 1993;9:247-64.

18. Bloebaum RD, Radley KM. Three-dimensional surface analysis of young adult human articular cartilage. J Anat 1995;187: 293-301.

19. Szilagyi I, Demel Z, SalaczT. Patellar chondropathy. II. Ultrastructure, scanning electron microscopic studies. Acta Chir Hung 1986;27:115-25.

20. Clark JM, Simonian PT. Scanning electron microscopy of "fibrillated" and "malacic" human articular cartilage: technical considerations. Microsc Res Tech 1997;37:299-313.

21. Thompson RC Jr, Vener MJ, Griffiths HJ, Lewis JL, Oegema TR Jr, Wallace L. Scanning electron-microscopic and magnetic resonance-imaging studies of injuries to the patellofemoral joint after acute transarticular loading. J Bone Joint Surg Am 1993;75:704-13.

22. Wampler HW, Tebo HG, Pinero GJ. Scanning electron microscopic and radiographie correlation of articular surface and supporting bone of the mandibular condyle. J Dent Res 1980;59:754-61.

AHC Tan, AK Mitra, PCC Chang, BK Tay

Department of Orthopaedic Surgery, Singapore General Hospital, Singapore

HL Nag

Department of Orthopaedic Surgery, All India Institute of Medical Sciences, India

CS Sim

Department of Pathology, Gleneagles Hospital, Singapore

Address correspondence and reprint requests to: Dr Andrew HC Tan, Consultant, Department of Orthopaedic Surgery, Singapore General Hospital, Singapore. E-mail: goothc@sgh.com.sg

Copyright Western Pacific Orthopaedic Association Dec 2004
Provided by ProQuest Information and Learning Company. All rights Reserved