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BMP-14 Gene Therapy Increases Tendon Tensile Strength in a Rat Model of Achilles Tendon Injury
Patrick Bolt, MD1; Avnish Neil Clerk, MD1; Hue H. Luu, MD1; Quan Kang, MD1; Jennifer L. Kummer, MD1; Zhong-Liang Deng, MD, PhD1; Kirstina Olson, MD1; Frank Primus, BS1; Anthony G. Montag, MD1; Tong-Chuan He, MD, PhD1; Rex C. Haydon, MD, PhD1; Brian C. Toolan, MD1
1 Section of Orthopaedic Surgery and Rehabilitation Medicine, Department of Surgery, University of Chicago Medical Center, 5841 South Maryland Avenue, MC 3079, Chicago, IL 60637. E-mail address for B.C. Toolan: btoolan@surgery.bsd.uchicago.edu
View Disclosures and Other Information
Disclosure: In support of their research for or preparation of this work, one or more of the authors received, in any one year, outside funding or grants of less than $10,000 from the Orthopaedic Research and Education Foundation, the American Orthopaedic Foot and Ankle Society, and the National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases. Neither they nor a member of their immediate families 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 authors, or a member of their immediate families, are affiliated or associated.
Note: The authors thank Dezheng Huo of the Department of Health Studies for his expert assistance with the statistical analysis of the samples.
Investigation performed at the Section of Orthopaedic Surgery and Rehabilitation Medicine, Department of Surgery, University of Chicago Medical Center, Chicago, Illinois

The Journal of Bone and Joint Surgery, Incorporated
J Bone Joint Surg Am, 2007 Jun 01;89(6):1315-1320. doi: 10.2106/JBJS.F.00257
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Abstract

Background: Molecular and cellular-based enhancements of healing combined with conventional methods may yield better outcomes after the surgical management of tendon injury. We examined the histological and biomechanical effects of adenovirus-mediated transgene expression of bone morphogenetic protein-14 (BMP-14) on healing in a rat Achilles tendon laceration model. Specifically, we hypothesized that this delivery system for gene therapy would hasten the restoration of the normal histological appearance and tensile strength of a surgically repaired tendon.

Methods: The right Achilles tendon of ninety male Sprague-Dawley rats was transected, repaired, and immediately infected with adenovirus expressing either the gene for green fluorescent protein (AdGFP) or the gene for human BMP-14 and green fluorescent protein (AdBMP-14). A sham control group received no viral-mediated infection after repair. Animals from each of the three groups were killed at one, two, and three weeks after surgery. The retrieved tendons were inspected, examined under light and fluorescent microscopy, and tested to determine their tensile strength.

Results: Tendons transduced with BMP-14 exhibited less visible gapping, a greater number of neotenocytes at the site of healing, and 70% greater tensile strength than did either those transduced with GFP or the sham controls at two weeks after repair. Histological examination revealed no inflammatory response to the adenovirus in tendons transduced with BMP-14 or GFP. No ectopic bone or cartilage formed in the tendons transduced with BMP-14.

Conclusions: Adenovirus-mediated gene therapy with BMP-14 expedites tendon-healing in this animal model. No adverse immunological response to the adenoviral vector was detected in the host tissue, and the local production of BMP-14 did not induce unwelcome bone or cartilage formation within the healing tendon.

Clinical Relevance: The results of this animal study suggest that gene therapy with BMPs may improve the capacity of injured musculoskeletal tissue to heal.

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    These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.
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    Brian C. Toolan, M.D.
    Posted on July 19, 2007
    Dr. Toolan et al. respond to Dr. Rickert.
    University of Chicago Medical Center, Chicago, IL 60637

    We appreciate the insightful comments from Dr. Rickert regarding our recent article on the use of adenovirus-mediated transfer of BMP-14 (GDF- 5) for Achilles tendon lacerations in a rat model, as well as the opportunity to respond to his concerns.

    First, our manuscript was originally submitted for publication in February, 2006, only a couple months after Dr. Rickert’s manuscript was published. We were unaware of his study in Connective Tissue Research(1) during preparation of our manuscript; however, we referenced many other articles from the Dr. Rickert’s laboratory at the University of Heidelberg, recognizing his important contributions to this area of research. As such, it was not our intention to ignore his study, and we welcome the chance to discuss the two articles.

    There are three important differences between the model of Achilles tendon injury that we used and that of Dr. Rickert. Most notably, we repaired the Achilles tendon with a single stitch to appose the ends of the lacerated tendon, thereby permitting tensile loading across the repair site. We did not lacerate the adjacent plantaris tendon, to ensure that functional ambulation would not be altered after the procedure. In Dr. Rickert’s model, the Achilles and plantaris tendons were lacerated and not repaired. Lastly, we used a significantly lower titer of virus for transduction of the ends of the lacerated tendon (108 PFU as opposed to 1- 3 x 1010).

    These differences may account for the differences in results between the two studies. Dr. Rickert asserts that previous reports from the University of Manchester(2,3) suggest that “cartilage and bone formation is essential during rat Achilles tendon healing.” This hypothesis is debatable, and worthy of further study; however, it should be emphasized that cartilage and bone formation occurred mainly in models in which no repair was performed. Forslund & Aspenberg(4) found that heterotopic ossification was infrequent and extremely limited in tendons that were exposed to tensile loads; whereas, robust heterotopic bone formation was found in nearly all unloaded tendons. This was one reason why we chose to repair the tendon after infection with the adenovirus. Although it would be of interest to look at later time points, the main point of this study was to examine tensile properties at early time points that would be of clinical interest, not to examine the impact of mechanical loading on lineage commitment in cells that mediate tendon repair. Nonetheless, based on our previous analysis of the 14 types of BMPs for their osteogenic activity, we believe that BMP14 is among the least osteogenic BMPs both in vitro and in vivo(5,6).

    Dr Rickert also questions how tendon healing and mechanical strength were assessed in our study. As clinicians, we are most concerned about re- rupture of the Achilles tendon early after repair. Our study was more narrowly focused in this regard compared to his study, and we did not include cross-sectional area of the repair tissue and Young’s modulus measurements. Assessment of cross-sectional area can be highly subjective, since values can vary depending on which part of the repair tissue is selected for analysis. For that reason, it was not included. Instead, we used tensile load to failure and gap formation as, perhaps, more clinically relevant methods to determine the feasibility of this approach in patients. Dr. Rickert mentions that measurements of gap formation are “imprecise and unconventional;” however, in a study by Gelberman et al.(7), gaps in the repair tendons were found to correlate with “the ultimate force, repair- site rigidity, and repair-site strain” in a canine model of tendon healing. For this reason, we felt that gap formation was a more clinically meaningful measurement to determine the risk of re-rupture after repair.

    Lastly, Dr. Rickert questioned the justification behind our conclusion that there was “no evidence of an acute inflammatory reaction to the adenoviral vectors.” We have previously published work on the dose -response relationship between different titers of adenoviral vectors and inflammation in a rabbit flexor-tendon model(8). Using standard HE stains, we could identify histologic evidence of acute inflammation at titers over 109. These parameters were used to determine the doses which were used in this study, and the histologic data presented in this study suggest that this titer does not cause excessive inflammation in the rat Achilles tendon model. There is no mention made of inflammation in Dr. Rickert’s publication, and it would be interesting to compare results, as his doses were considerably higher. He also mentions that standard HE stains may not be sufficient to rule out the presence of inflammatory cells in healing tendons, suggesting that we consider approaches that specifically identify CD4+ or CD8+ T-cells. Although this is a valid point, histologic evaluation with HE stains only have been used by others(9) to assess inflammation in tendons after adenoviral gene transfer, without the need for additional tests. Given that inflammation is an important part of the healing process in tendons, some inflammatory cells would be expected at the repair site, regardless of whether an adenovirus was present or not. HE stains, however, represent a reasonable method to determine the presence of excessive inflammation that might impact on clinical outcomes.

    We believe that the differences cited above between the two studies highlight some of the important factors that affect tendon healing, and the utility of adenoviral delivery of BMP14 for Achilles tendon repair. Instead of being contradictory, we believe the findings of our two studies should be viewed as complementary because both contribute meaningful data to the debate over the role of gene therapy in the future treatment of Achilles tendon ruptures.

    References:

    1. Rickert, M, Wang, H, Wieloch, P, Lorenz, H, Steck, E, Sabo, D, and Richter, W. Adenovirus-mediated gene transfer of growth and differentiation factor-5 into tenocytes and the healing rat Achilles tendon. Connect Tissue Res. 46(4-5):175-83,2005.

    2. Rooney, P, Grant, M. E, and McClure, J. Endochondral ossification and de novo collagen synthesis during repair of the rat Achilles tendon. Matrix. 12(4): 274-81,1992.

    3. Rooney, P, Walker, D, Grant, M. E, and McClure, J. Cartilage and bone formation in repairing Achilles tendons within diffusion chambers: evidence for tendon-cartilage and cartilage-bone conversion in vivo. J Pathol. 169(3): 375-81,1993.

    4. Forslund, C, and Aspenberg P. CDMP-2 induces bone or tendon-like tissue depending on mechanical stimulation. J Orthop Res. 20(6):1170-4,2002.

    5. Cheng, H. et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am. 85-A(8):1544-52,2003.

    6. Kang, Q. et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther. 11(17):1312-20,2004.

    7. Gelberman, RH, Boyer, MI, Brodt MD, Winters SC, and Silva MJ. The effect of gap formation at the repair site on the strength and excursion of intrasynovial flexor tendons. An experimental study on the early stages of tendon-healing in dogs. J Bone Joint Surg Am. 81(7):975-82,1999.

    8. Mehta V, Kang Q, Luo J, He TC, Haydon, RC, and Mass DP. Characterization of adenovirus-mediated gene transfer in rabbit flexor tendons. J Hand Surg Am. 30(1):136-41, 2005.

    9. Zhu B, Cao Y, Xin KQ, Wang XT, Summerhayes IC, Liu PY, and Tang JB. Tissue reactions of adenoviral, adeno-associated viral, and liposome-plasmid vectors in tendons and comparison with early-stage healing responses of injured flexor tendons. J Hand Surg Am. 31(10):1652-60, 2006.

    Markus Rickert, M.D., Ph.D
    Posted on June 13, 2007
    BMP-14 gene transfer into rat Achilles tendon
    University of Heidelberg, Orthopedic Department, GERMANY

    To The Editor:

    We read with interest the article, “BMP-14 Gene Therapy Increases Tendon Tensile Strength in a Rat Model of Achilles Tendon Injury”(1). The article appears to be important in demonstrating the feasibility of this new technique but we would like to question some aspects of the study. We published a very similar experiment two years ago (2), which surprisingly was not cited in the article. In our study, we found some different results:

    As published in 1992 and 1993 by Rooney (3,4), cartilage and bone formation is essential during rat Achilles tendon healing. The important question is, whether the use of BMPs facilitates this this process or not. It should be noted that the observation period in this study (3 weeks) is probably too short to find any differences between the groups. We found an increase of cartilage and bone formation in the BMP14- (GDF5) group 8 weeks after virus administration (2).

    Furthermore, the authors describe that no adverse immunological response to the adenoviral vector was detected, but they do not describe specific methods, eg. CD4+ or CD8+ T-cells, that might be used to confirm this conclusion. HE staining is neither sensitive nor specific enough to clarify this question within the healing tendon callus.

    The assessment of “gapping” of the healing tendon seems to be an imprecise and unconventional method to determine structural and biomechanical data in this model. Why didn´t the authors measure the cross -sectional area ( e.g. using a non-contact laser method) and why didn´t they calculate Young`s modulus? To which variable did the authors normalize their results, e.g. left side, tendon thickness?

    Finally, the authors should try to provide an explanation for the increase in tendon tensile strength. Is it because of the thicker tendon callus or did they find further changes within the quality of the callus? A thicker tendon does not necessarily mean a stronger tendon.

    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. Bolt P, Clerk AN, Luu HH, Kang Q, Kummer JL, Deng ZL, Olson K, Primus F, Montag AF, He TC, Haydon RC, and Toolan BC. BMP-14 gene therapy increases tendon tensile strength in a rat model of achilles tendon injury. J Bone Joint Surg Am. 2007:89:1315-1320.

    2. Rickert M, Wang H, Wieloch P, Lorenz H, Steck E, Sabo D, Richter W Adenovirus-mediated gene transfer of growth and differentiation factor-5 into tenocytes and the healing rat Achilles tendon (2005). Connective Tissue Research, 46: 175-183.

    3. Rooney P, Grant ME, McClure J (1992), Enchondral ossification and de novo collagen synthesis during repair of the rat Achilles tendon. Matrix, 12: 274-281.

    4. Rooney P, Walker D, Grant ME, McClue J (1993), Cartilage and bone formation in repairing Achilles tendons within diffusion chambers: Evidence for tendon-cartilage and cartilage-bone conversion in vivo. J Pathol, 169: 375-381.

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