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Letters to the Editor to:

Scientific Articles:
Carl T. Brighton, Wei Wang, and Charles C. Clark
The Effect of Electrical Fields on Gene and Protein Expression in Human Osteoarthritic Cartilage Explants
J Bone Joint Surg Am 2008; 90: 833-848 [Abstract] [Full text] [PDF]
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Electronic letters published:

[Read Letter to the Editor] Gene and Protein Expression in Human Osteoarthritic Cartilage Explants
Benedict A Rogers, MA, MSc, MRCGP, MRCS   (13 May 2008)
[Read Letter to the Editor] Dr. Brighton & Dr. Clark respond to Dr. Rogers
Carl Brighton, M.D., Ph.D., Charles Clark, Ph.D.   (13 May 2008)

Gene and Protein Expression in Human Osteoarthritic Cartilage Explants 13 May 2008
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Benedict A Rogers, MA, MSc, MRCGP, MRCS,
Specialist Rgistrar Trauma & Orthopaedics
St Peter's Hospital, Chertsey, UK

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Re: Gene and Protein Expression in Human Osteoarthritic Cartilage Explants

benedictrogers{at}hotmail.com Benedict A Rogers, MA, MSc, MRCGP, MRCS

To The Editor:

I read with great interest the excellent paper by Brighton et al.(1) entitled “The Effect of Electrical Fields on Gene and Protein Expression in Human Osteoarthritic Cartilage Explants” and would like to make the following points:

1. The study would benefit from biometric data (age, sex, weight and height) regarding the patient cohort undergoing total knee replacement. Such factors, as well as previous treatments such including intra- articular steroid injections, all have potential substantial effects on the composition, histology(2,3) and metabolism(4) of articular cartilage.

2. The Outerbridge classification of degenerative chondral changes(5), classified by direct vision during an arthrotomy or arthroscopy, could have been used and would have provided a superior assessment of degenerative changes within the knee joint compared to the radiographic assessment used in this study(6).

3. The study utilized a hexosamine assay to quantify total proteoglycan in the explants(7). How does this biochemical assay compare in relation to other GAG assays, such as dimethylmethylene blue (DMMB) spectrophotometric assay?(8). Was it used to quantify sulphated GAG in the papain-digested cartilage specimens?

4. Total Knee Replacement (TKR) involves distal femoral and proximal tibial cuts guided by either intramedullary or extramedullary referenced gigs. The osteochondral fragments removed during TKR are thus from both high and low load bearing locations within the human knee. For example, the posterior aspect of the femoral condyle, while maximally loaded during deep knee flexion, general exhibits less degenerative change due to lower loading than cartilage at the apex of the femoral condyles. Further, knee malalignment can have a dramatic effect on the load borne by articular cartilage in any one location(9). Did the authors detail the location upon the articular surface from which the articular explants were obtained?

5. The relevance of the above point to this study is that cartilage is a load-sensitive tissue. In a study that I published using human articular cartilage obtained from subjects undergoing above knee amputations, with minimal or no osteoarthritic changes, explants from high load bearing regions exhibited a higher GAG/DNA ratio than explants obtained from lower load bearing regions(10). Such differences in the biochemical characteristics of articular cartilage from differing loci may be a potential confounding factor in this study.

The author did not receive any outside funding or grants in support of his research for or preparation of this work. Neither he nor a member of his immediate family received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which the author, or a member of his immediate family, is affiliated or associated.

References:

1. Brighton CT, Wang W, Clark CC. The effect of electrical fields on gene and protein expression in human osteoarthritic cartilage explants. J Bone Joint Surg Am. 2008;90:833-848.

2. McIntosh, Hanssen AD, Wenger DE, Osmon DR. Recent intraarticular steroid injection may increase infection rates in primary THA. Clin Orthop Relat Res. 2006;451:50-54.

3. Raynauld. Clinical trials: impact of intraarticular steroid injections on the progression of knee osteoarthritis. Osteoarthritis Cartilage. 1999;7:348-349.

4. Pelletier, Martel-Pelletier J, Cloutier JM, Woessner JF, Jr. Proteoglycan-degrading acid metalloprotease activity in human osteoarthritic cartilage, and the effect of intraarticular steroid injections. Arthritis Rheum. 1987;30:541-548.

5.Outerbridge. FURTHER STUDIES ON THE ETIOLOGY OF CHONDROMALACIA PATELLAE. J Bone Joint Surg Br. 1964;46:179-190.

6. Cameron, Briggs KK, Steadman JR. Reproducibility and reliability of the outerbridge classification for grading chondral lesions of the knee arthroscopically. Am J Sports Med. 2003;31:83-86.

7. Gatt, Berman ER. A rapid procedure for the estimation of amino sugars on a micro scale. Anal Biochem. 1966;15:167-171.

8. Farndale, Sayers CA, Barrett AJ. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res. 1982;9:247-248.

9. Werner, Ayers DC, Maletsky LP, Rullkoetter PJ. The effect of valgus/varus malalignment on load distribution in total knee replacements. J Biomech. 2005;38:349-355.

10. Rogers, Murphy CL, Cannon SR, Briggs TW. Topographical variation in glycosaminoglycan content in human articular cartilage. J Bone Joint Surg Br. 2006;88:1670-1674.
Dr. Brighton & Dr. Clark respond to Dr. Rogers 13 May 2008
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Carl Brighton, M.D., Ph.D.,
Professor
University of Pennsylvania, Philadelphia, PA,
Charles Clark, Ph.D.

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Re: Dr. Brighton & Dr. Clark respond to Dr. Rogers

ctb{at}mail.med.upenn.edu Carl Brighton, M.D., Ph.D., et al.

Thank you very much for your thought-provoking comments to the editor regarding our recently-published article entitled, “The Effect of Electric Fields on Gene and Protein Expression in Human Osteoarthritic Cartilage Explants”(1). Our responses to your five questions are as follows:

1. The biometric data that you suggested would certainly have been interesting, but correcting such data to the response of the individual explants would be an enormous task and would have required many more patients to achieve statistical significance between the explants. In view of this, we elected to randomize the explants such that any possible effects due to the factors mentioned above would be neutralized.

2. We, of course, had “direct vision” of the articular cartilage and underlying bone removed during the total knee arthroplasties. All eleven patients had a radiographic stage-II osteoarthritis by the Kellgren Scale(2). Full-thickness cartilage plugs measuring either 4 mm in diameter or 8 mm in diameter were taken from all available cartilage removed during the arthroplasty. The plugs were then photographed using reflected light. The amazing thing to us was the great amount of variation of the articular surface topography from one plug to another, but also within the surface of any given plug. That being the case, we elected to randomly assign the plugs to the various experimental groups as in 1 above.

3. Both the hexosamine and DMMB spectrophotometric assays provide an accurate index of the amount of GAG in the tissue. DMMB binds to intact GAG chains whereas the hexosamine assay directly measures one of the constituent GAG chain sugars. Papain-digested cartilage samples were used for our assays.

4. As explained in 1 and 2 above, the explants were randomly assigned to the various experimental groups in order to neutralize any possible effects due to inherent differences due to location, load bearing, etc.

5. Again, your concerns are real and because of such concerns, we elected to randomize the plug explants into the various experimental groups as described above.

We thank you for your interest in our study. The question of “like” being compared to “like” is an important one. However, there are at least two ways of doing so: one, making sure all plugs are the same, and then assigning the plugs to different experimental groups; and two, randomly assigning the plugs to the different experimental groups to compensate for any plug differences.

References:

1. Brighton, CT, Wang W, Clark CC. The effect of electrical fields on gene and protein expression in human osteoarthritic cartilage explants. J Bone Joint Surg Am. 2008;90:833-848.

2. Kellgren JH, Laurence J.S. Radiographic Assessment of Osteo- Arthritis. Ann. Rheum. Dis. 1957;16:494-502.