The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 89, Issue 6

Scientific Articles | June 01, 2007

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

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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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Improvement of the treatment of tendon injury is an issue of central concern within the field of orthopaedic surgery. The current management of Achilles tendon ruptures focuses on the mechanical aspects of healing. Clinicians weigh their options for achieving and maintaining apposition of the ruptured ends of the tendon against their concerns about muscular atrophy, rerupture, and complications of treatment^1-6. Surgical management directly repairs the tendon, permits early initiation of rehabilitation, and reduces the rate of rerupture. However, nerve injury, dehiscence, infection, and scarring at the site of the wound may compromise the outcomes of treatment. Immobilization with a cast or functional bracing obviates the risks attendant with surgical repair but increases the chance of a rerupture and may prolong the need for therapy to address muscular atrophy and stiffness of the joints. All of these options rely on, but do not capitalize on, the complex biological milieu that governs tendon-healing. The introduction of new strategies to manipulate these molecular and cellular interactions to enhance healing and function may yield more successful outcomes of the treatment of these injuries.

Several studies have focused on the effects of bone morphogenetic proteins (BMPs) on healing tendons. BMP-12, 13, and 14 have been shown to induce neotendon formation in animal models when injected ectopically^7,8. Direct injection of each of these three proteins increased the tensile strength of the reparative tendon that formed in the defects of rat and rabbit Achilles tendon rupture models^9-13. Similar effects on tensile strength have been observed in flexor-tendon-laceration chicken and rabbit models^14-16. In these experimental models of protein therapy, the BMP is delivered to the local environment through a carrier. The carrier facilitates sustained release of the protein to maintain an optimal concentration at the site of healing. However, the therapeutic effect of protein therapy diminishes over time as the quantity of delivered BMP diffuses into the surrounding tissue. Gene therapy potentially obviates the need for a carrier and addresses the problem of diffusion. This system delivers the gene for the BMP directly to the site of injury. The gene may be transferred into host cells, or exogenous cells containing the gene may be introduced directly, to establish a constant source for the local production of BMP through gene expression.

The favorable effects on tendon-healing observed after the direct injection of BMP encouraged us to evaluate the capacity of BMP gene therapy to enhance healing of an Achilles tendon laceration. Our previous experience with the effects of adenovirus-mediated BMP gene transfer on flexor tendon repair suggested that the use of this viral delivery system would permit the expression of human BMP-14 (AdBMP-14) at the site of an Achilles tendon laceration¹⁷. In the present study, we hypothesized that AdBMP-14 is an effective delivery system for gene therapy in a rat model of an Achilles tendon laceration. We also hypothesized that transgene expression of BMP-14 at the site of injury would improve the histological appearance and biomechanical strength of a surgical repair of an Achilles tendon laceration when compared with controls.

Materials and Methods

Materials and Methods | Results | Discussion | References

Construction and Preparation of Recombinant Adenovirus Vector Expressing BMP-14 and Green Fluorescent Protein Controls

We previously described our technique for generating recombinant adenoviral vectors expressing human BMPs for gene therapy^18-21. Briefly, the coding region of the human BMP-14 gene was amplified with use of a polymerase-chain-reaction process and subcloned into an adenoviral shuttle vector (pAdTrack-CMV). Once the integrity of the amplified human BMP-14 gene was verified by DNA sequencing, a recombinant adenovirus plasmid was constructed and subsequently transfected into an HEK 293 cell line to generate high-titer stocks of adenovirus expressing BMP-14 (AdBMP-14). The AdBMP-14 also encoded the gene expressing a green fluorescent protein (GFP) to serve as a marker for infection. With use of an identical process, an adenovirus expressing only green fluorescent protein (AdGFP) was constructed as a control for the effects of adenoviral infection of the cells at the site of tendon repair.

Assignment of Treatment and Control Groups

The institutional animal care and use committee of the University of Chicago approved the protocol governing the use and care of the animals in this study. Ninety seven-week-old male Sprague-Dawley rats were divided into three groups of thirty animals each. One group of animals received the adenovirus expressing GFP and BMP-14 (AdBMP-14, 10⁸ plaque-forming units [pfu] per injection) at the site of the repaired tendon. The second group received the adenovirus expressing only GFP (AdGFP, 10⁸ pfu per injection). The third (surgical control) group received no virus after repair of the tendon. The doses of the adenoviruses used in this investigation were based on our previous work^17,18,20,21 and the results of a pilot study with our animal model.

Ten rats from each group were killed at one, two, and three weeks after surgical repair. The entire Achilles tendon with the attached gastrocnemius muscle body and the proximal half of the calcaneus were harvested en bloc. Eight tendons from each of the nine subgroups underwent biomechanical testing immediately after the animals were killed. Two tendons from each subgroup were designated for histological examination.

Achilles Tendon Laceration and Surgical Repair

Each rat was anesthetized with isoflurane delivered through a mask and a chambered gas-anesthesia machine. A posterior midline incision was made over the right hindpaw of each rat to the level of the peritenon. The peritenon was identified and was incised in line with the skin. The Achilles and plantaris tendons were isolated, and the Achilles tendon was sharply transected at a point halfway between the musculotendinous junction and the insertion onto the calcaneus. The plantaris muscle and tendon were preserved throughout the procedure.

Each end of the transected tendon was injected with an aliquot of its designated viral suspension (10⁸ pfu in 10 µL). The animals in the surgical control group received no injection. A single, 6-0 horizontal mattress Ticron suture (Syneture, Norwalk, Connecticut) was used to appose the tendon ends. Skin was apposed with veterinary surgical clips. Each rat recovered from the anesthesia alone in a cage under a warming lamp and received a single subcutaneous injection of buprenorphine (50 µg/kg) for pain control after the surgery. The rats were then housed in pairs, allowed to eat, drink, and move ad libitum, and monitored on a daily basis until they were killed.

Histological Analysis

Immediately after harvest, one tendon from each subgroup was processed and was examined with fluorescent microscopy to evaluate the extent of adenovirus-mediated gene transfer to host cells. Another tendon from each subgroup was decalcified, thin-sectioned in the coronal plane, and stained with hematoxylin and eosin. Microscopic slides were evaluated for the presence of an inflammatory host response, ectopic bone or cartilage formation, and the appearance of the reparative tissue formed at the site of the tendon repair independently by at least three investigators blinded to the group assignments.

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Fig. 1: Construction and characterization of an adenoviral vector expressing BMP-14. A: Construction of AdBMP with use of the AdEasy system. B: AdBMP-14 and AdGFP (the control virus) were shown to effectively transduce mouse myoblastic line C2C12. GFP signals were recorded at twenty-four hours after infection. C: AdBMP-14 and AdGFP vectors were shown to efficiently transduce rat Achilles tendons through direct injection. GFP signals were detected on frozen sections of the retrieved tendons at six days after injection. BF = bright field, and FF = GFP fluorescent field (magnification, ×100).

Assessment of Gapping of the Retrieved Samples

The remaining eight tendons from each subgroup were designated for mechanical testing, and the site of repair was inspected for gapping. The location of tendon apposition was defined as the region between the cephalad and caudad passage of the horizontal mattress suture through the tendon. Two observers blinded to group assignment independently measured the gap once with a metric ruler. Gapping was defined as a distance of >1 mm between the healed ends of the proximal and distal tendon stumps. Each observer recorded the presence or absence of gapping for each repair in a separate table for subsequent comparison.

Biomechanical Analysis

The ultimate tensile strength of each repair was determined with use of a published methodology established for the biomechanical testing of healing lacerations of Achilles tendons in rats^12,13,22-26. Briefly, each specimen was mounted on a tensiometer. At the distal end of the specimen, the calcaneus was fixed with a clamp positioned at a 30° angle to the direction of the applied load, and the proximal end of the specimen was clamped at the musculotendinous junction. Before the initiation of testing, the plantaris tendon was transected to isolate loading to the repaired Achilles tendon. The tensile load was applied at a constant rate of 1 mm/sec until failure. The ultimate load to failure was recorded for each specimen.

Statistical Analysis

We used analysis of variance to test whether there was a significant difference across the three treatment groups for the three time-points separately. If the F test in the analysis of variance showed a significant difference across the groups, additional pairwise comparisons were conducted. We were interested in two comparisons: between the AdBMP and sham groups and between the AdBMP and AdGFP groups. The stepdown Bonferroni procedure was used to control the family-wide type-I error rate.

Results

Materials and Methods | Results | Discussion | References

Recombinant adenoviruses expressing human BMP-14 (i.e., AdBMP-14) or GFP only (i.e., AdGFP) were constructed with the AdEasy system (Stratagene, La Jolla, California)^17,18,20,21 (Fig. 1, A). The AdBMP-14 also expresses GFP as a convenient marker to monitor gene transduction efficiency (Fig. 1, B). As shown in Figure 1, C, the AdBMP-14 infected the rat Achilles tendons, as an expression of the GFP was observed in the AdBMP-14 and AdGFP groups.

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Fig. 2: Macrographic examination of the retrieved Achilles tendons from the sham (A), AdGFP (B), and AdBMP-14 (C) groups at two weeks after surgery showed less gapping in those treated with AdBMP-14.

At one week after surgery, the tendons in all three groups demonstrated gapping at the site of repair. At two weeks, most of the tendons in the AdBMP-14 group had robust healing with little or no gapping at the site of repair (six of eight had no gapping compared with two of eight and zero of eight in the AdGFP and sham groups, respectively), whereas the AdGFP and control groups did not demonstrate this quality of healing until three weeks after surgery (Fig. 2).

Histological examination revealed no evidence of an acute inflammatory response to the adenoviral vectors in the tendons that received either AdBMP-14 or AdGFP (Fig. 3). No ectopic bone or cartilage formation was observed in any of the tendons from these two groups (Fig. 3).

The tendons treated with AdBMP-14 exhibited the highest tensile strength at one, two, and three weeks after surgery (Table I). However, the difference was significant only at the two-week time-point, when the repaired tendons in the AdBMP-14 group were approximately 70% stronger than those in the AdGFP and sham groups (p = 0.012 and p = 0.018, respectively) (Table I). No significant difference in the strength of the repaired tendons was detected between the control and AdGFP groups at any of the three time-points.

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TABLE I Ultimate Load to Failure (N) of the Retrieved Rat Achilles Tendons

Group (N = 8 in Each)	Week 1					Week 2				Week 3
	Average	Std. Dev.	P Value	Average	Std. Dev.	P Value	Average	Std. Dev.	P Value
Sham	10.66	4.68	0.591^*	16.69	6.81	0.018^*	30.73	10.75	0.581^*
AdGFP	11.13	4.98	0.591†	15.94	7.96	0.012†	32.89	9.03	0.591†
AdBMP-14	13.77	3.19		27.19	3.62		37.85	8.32
Normal	36.18	8.83

The Bonferroni corrected p value for the AdBMP-14 group compared with the sham groupThe Bonferroni corrected p value for the AdBMP-14 group compared with the AdGFP group

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TABLE I Ultimate Load to Failure (N) of the Retrieved Rat Achilles Tendons

Group (N = 8 in Each)	Week 1					Week 2				Week 3
	Average	Std. Dev.	P Value	Average	Std. Dev.	P Value	Average	Std. Dev.	P Value
Sham	10.66	4.68	0.591^*	16.69	6.81	0.018^*	30.73	10.75	0.581^*
AdGFP	11.13	4.98	0.591†	15.94	7.96	0.012†	32.89	9.03	0.591†
AdBMP-14	13.77	3.19		27.19	3.62		37.85	8.32
Normal	36.18	8.83

The Bonferroni corrected p value for the AdBMP-14 group compared with the sham groupThe Bonferroni corrected p value for the AdBMP-14 group compared with the AdGFP group

Discussion

Materials and Methods | Results | Discussion | References

In this study, adenoviral-mediated gene therapy with BMP-14 enhanced the healing of a lacerated Achilles tendon repaired with surgery in a rat model. The tensile strength of the repaired tendons transduced with the recombinant BMP-14 gene was 70% greater than that in either control group at two weeks after surgery. This increase in strength at two weeks coincided with the observed difference in the gross and histological appearance of the healing tendons. The tendons infected with AdBMP-14 had less gapping and a greater number of neotenocytes at the site of the repair than did the viral and surgical control groups. These results suggest that BMP-14 may stimulate a more active cellular response to tendon injury.

The biomechanical results suggest that the expression of the BMP-14 transgene by the host tenocytes accelerates the cellular processes of tendon-healing. Under our testing conditions, an intact Achilles tendon failed at a mean load of 36 N. The mean tensile strength of the tendons from the two control groups approached the intact condition when their load to failure doubled between the second and third week after repair. The loads to failure of the tendons exposed to BMP-14 doubled, and three-fourths of their normal tensile strength was restored, between the first and second weeks. Their mean load to failure attained the value for the intact tendon by the third week after repair. These results suggest that BMP-14 is an effective adjuvant to the indigenous reparative process of tendon-healing.

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Fig. 3: Histological evaluation of the retrieved tendon tissues, which were fixed in 10% formalin and subjected to paraffin-embedding. Five-micrometer sections were prepared for hematoxylin and eosin staining (magnification, ×200).

The magnitude of the difference in strength that we observed in rat Achilles tendons is comparable with the results following use of BMP-12 gene therapy to enhance healing of flexor tendons in chickens¹⁵. Although the rat model for Achilles tendon injury was well established in several previous studies^9,10,12,13, the comparison of our results with the chicken flexor tendon model is confounded by two factors. The presence of a comparatively large plantaris tendon may act as an internal splint during nascent healing of the adjacent Achilles tendon, especially during quadrupedal gait. Also, the lack of a true tenosynovial envelope around the Achilles tendon may alter the reparative environment as compared with that of an injured flexor tendon. However, preservation of the plantaris tendon during healing may better represent the conditions associated with a rupture of the Achilles tendon in the clinical setting.

The formation of bone or cartilage has been a concern with the use of BMPs to augment tendon-healing. The induction of bone or cartilage formation within the tendon has been observed primarily with recombinant-protein-injection models^10,11,13. Investigators using adenoviral gene-therapy models have not reported ectopic bone or cartilage formation^14,15. In the present study, histological examination of one tendon in each group at all time-points revealed no evidence of bone or cartilage. Thus, the results of this study suggest that adenoviral delivery of BMP-14 does not induce ectopic bone or cartilage formation in injured and repaired Achilles tendons of rats.

Limitations associated with the healing and testing of rat tendons should be considered when the results of this investigation are interpreted. A lacerated rat Achilles tendon heals within three weeks. This robust reparative process and short time-interval in which to observe alterations in healing may have diminished our capacity to observe the full effect of gene therapy on tensile strength. The inclusion of additional time-points spaced at shorter intervals during healing may have permitted a more extensive characterization of the effects of AdBMP-14 on the biomechanical properties of a repaired Achilles tendon laceration. Also, we employed a continuous load to failure to test the freshly harvested tendons. Although a cyclical load would have been the preferred method of testing, difficulties encountered with mounting the specimens in a consistent manner to maintain a firm grip without slippage or tearing during testing prevented the use of a cyclical load. In addition, we did not precondition our fresh specimens before testing, as preconditioning was not included in the continuous-loading protocol described by Forslund and Aspenberg¹². The testing of fresh tendons that have not undergone a freeze-thaw under a slow continuous load mitigates the effect of viscoelasticity on the ultimate load to failure. Although the use of continuous, rather than a cyclical, loading and the omission of preconditioning may alter the load at which a tendon fails, this effect was presumably uniform throughout the population of specimens tested in our study. Under these conditions, the testing protocol that we employed in our study is unlikely to have affected the differences observed among the groups during biomechanical testing.

The results of this study indicate that adenoviral-mediated BMP-14 transgene expression improves tendon-healing at the site of the repair. Future studies of more advanced animal models are warranted to further our understanding of the effects of BMP-14 with regard to the biologic augmentation of tendon repair. ?

References

Materials and Methods | Results | Discussion | References

Bhandari M, Guyatt GH, Siddiqui F, Morrow F, Busse J, Leighton RK, Sprague S, Schemitsch EH. Treatment of acute Achilles tendon ruptures: a systematic overview and metaanalysis. Clin Orthop Relat Res. 2002;400: 190-200.400190 2002 [PubMed][CrossRef]

Cetti R, Christensen SE, Ejsted R, Jensen NM, Jorgensen U. Operative versus nonoperative treatment of Achilles tendon rupture. A prospective randomized study and review of the literature. Am J Sports Med. 1993;21: 791-9.21791 1993 [PubMed][CrossRef]

Kocher MS, Bishop J, Marshall R, Briggs KK, Hawkins RJ. Operative versus nonoperative management of acute Achilles tendon rupture: expected-value decision analysis. Am J Sports Med. 2002;30: 783-90.30783 2002

Lo IK, Kirkley A, Nonweiler B, Kumbhare DA. Operative versus nonoperative treatment of acute Achilles tendon ruptures: a quantitative review. Clin J Sport Med. 1997;7: 207-11.7207 1997 [PubMed][CrossRef]

Wills CA, Washburn S, Caiozzo V, Prietto CA. Achilles tendon rupture. A review of the literature comparing surgical versus nonsurgical treatment. Clin Orthop Relat Res. 1986;207: 156-63.207156 1986 [PubMed]

Wong J, Barrass V, Maffulli N. Quantitative review of operative and nonoperative management of achilles tendon ruptures. Am J Sports Med. 2002;4: 565-75.4565 2002

Luo J, Sun MH, Kang Q, Peng Y, Jiang W, Luu HH, Luo Q, Park JY, Li Y, Haydon RC, He TC. Gene therapy for bone regeneration. Curr Gene Ther. 2005;52: 167-79.52167 2005 [CrossRef]

Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, Dube JL, DiBlasio-Smith E, Nove J, Song JJ, Wozney JM, Rosen V. Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family. J Clin Invest. 1997;100: 321-30.100321 1997 [PubMed][CrossRef]

Aspenberg P, Forslund C. Enhanced tendon healing with GDF 5 and 6. Acta Orthop Scand. 1999;70: 51-4.7051 1999 [PubMed][CrossRef]

Forslund C. BMP treatment for improving tendon repair. Studies on rat and rabbit Achilles tendons. Acta Orthop Scand Suppl. 2003;74:I, 1-30.741 2003

Forslund C, Aspenberg P. Improved healing of transected rabbit Achilles tendon after a single injection of cartilage-derived morphogenetic protein-2. Am J Sports Med. 2003;31: 555-9.31555 2003 [PubMed]

Forslund C, Aspenberg P. Tendon healing stimulated by injected CDMP-2. Med Sci Sports Exerc. 2001;33: 685-7.33685 2001 [PubMed]

Forslund C, Rueger D, Aspenberg P. A comparative dose-response study of cartilage-derived morphogenetic protein (CDMP)-1, -2 and -3 for tendon healing in rats. J Orthop Res. 2003;21: 617-21.21617 2003 [PubMed][CrossRef]

Lou J. In vivo gene transfer into tendon by recombinant adenovirus. Clin Orthop Relat Res. 2000;379 Suppl: S252-5.379S252 2000 [PubMed][CrossRef]

Lou J, Tu Y, Burns M, Silva MJ, Manske P. BMP-12 gene transfer augmentation of lacerated tendon repair. J Orthop Res. 2001;19: 1199-202.191199 2001 [CrossRef]

Lou J, Manske PR, Aoki M, Joyce ME. Adenovirus-mediated gene transfer into tendon and tendon sheath. J Orthop Res. 1996;14: 513-7.14513 1996 [CrossRef]

Mehta V, Kang Q, Luo J, He TC, Haydon RC, Mass DP. Characterization of adenovirus-mediated gene transfer in rabbit flexor tendons. J Hand Surg [Am]. 2005;30: 136-41.30136 2005 [PubMed][CrossRef]

He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998;95: 2509-14.952509 1998 [PubMed][CrossRef]

He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science. 1998;281: 1509-12.2811509 1998 [PubMed][CrossRef]

Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, Szatkowski JP, Park JY, He TC. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am. 2003;85: 1544-52. Erratum in: J Bone Joint Surg Am. 2004;86:141.851544 2003 [PubMed]

Kang Q, Sun MH, Cheng H, Peng Y, Montag AG, Deyrup AT, Jiang W, Luu HH, Luo J, Szatkowski JP, Vanichakarn P, Park JY, Li Y, Haydon RC, He TC. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther. 2004;11: 1312-20.111312 2004 [PubMed][CrossRef]

Aspenberg P, Virchenko O. Platelet concentrate injection improves Achilles tendon repair in rats. Acta Orthop Scand. 2004;75: 93-9.7593 2004 [CrossRef]

Orhan Z, Ozturan K, Guven A, Cam K. The effect of extracorporeal shock waves on a rat model of injury to tendo Achillis. A histological and biomechanical study. J Bone Joint Surg Br. 2004;86: 613-8.86613 2004

Wieloch P, Buchmann G, Roth W, Rickert M. A cryo-jaw designed for in vitro tensile testing of the healing Achilles tendons in rats. J Biomech. 2004;37: 1719-22.371719 2004 [PubMed][CrossRef]

Yeung CK, Guo X, Ng YF. Pulsed ultrasound treatment accelerates the repair of Achilles tendon rupture in rats. J Orthop Res. 2006;24: 193-201.24193 2006 [PubMed][CrossRef]

Zhang F, Liu H, Stile F, Lei MP, Pang Y, Oswald TM, Beck J, Dorsett-Martin W, Lineaweaver WC. Effect of vascular endothelial growth factor on rat Achilles tendon healing. Plast Reconstr Surg. 2003;112: 1613-9.1121613 2003 [CrossRef]

Topics

adenovirus infections ; gene therapy ; adenoviruses ; achilles tendon ; lacerations ; tendon ; tensile strength ; achilles tendon injury

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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. Rickertâ€™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. Rickertâ€™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. Rickertâ€™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 â€œcartilage and bone formation is essential during rat Achilles tendon healing.â€ 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 Youngâ€™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 â€œimprecise and unconventional;â€ however, in a study by Gelberman et al.(7), gaps in the repair tendons were found to correlate with â€œthe ultimate force, repair- site rigidity, and repair-site strainâ€ 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 â€œno evidence of an acute inflammatory reaction to the adenoviral vectors.â€ 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. Rickertâ€™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, â€œBMP-14 Gene Therapy Increases Tendon Tensile Strength in a Rat Model of Achilles Tendon Injuryâ€(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 â€œgappingâ€ of the healing tendon seems to be an imprecise and unconventional method to determine structural and biomechanical data in this model. Why didnÂ´t the authors measure the cross -sectional area ( e.g. using a non-contact laser method) and why didnÂ´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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Patrick Bolt, Avnish Neil Clerk, Hue H. Luu, Quan Kang, Jennifer L. Kummer, Zhong-Liang Deng, Kirstina Olson, Frank Primus, Anthony G. Montag, Tong-Chuan He, Rex C. Haydon, Brian C. Toolan; BMP-14 Gene Therapy Increases Tendon Tensile Strength in a Rat Model of Achilles Tendon Injury. The Journal of Bone & Joint Surgery. 2007 Jun;89(6):1315-1320.

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