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GDF-5 deficiency in mice delays Achilles tendon healing
Journal of Orthopaedic Research
Journal of Orthopaedic Research 21 (2003) 826-835
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GDF-5 deficiency in mice delays Achilles tendon healing A. Chhabra
a,
D. Tsou ', R.T. Clark
a,
V. Gaschen ', E.B. Hunziker ', B. Mikic
b3*
'' Depurtnient of Orthopedics. University of Virginiu Heulth Sciences Cmter. Churlortesville, V A 22908, U S A Picker Engineering Program, Smith College, 51 College Lune, Nortlzuinpton, M A 01063, USA ITT Rcsrtrrch Institute for Dental and Skeletul Bioiogy, Murtenstrusre 35, Postfach 54, CH-3010 Bern, Switzerlund
Received 1 October 2002; accepted 12 February 2003
Abstract
The aim of this study was to examine the role of one of the growthldifferentiation factors, GDF-5, in the process of tendon healing. Specifically, we tested the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, biochemical, and ultrastructural analyses, we demonstrate that Achilles tendons from 8-week-old male GDF-5 4- mice exhibit a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Mutant animals took longer to achieve peak cell density, glycosaniinoglycan content, and collagen content in the repair tissue, and the time course of changes in collagen fibril size was also delayed. Revascularization was delayed in the mutant mice by 1 week. GDF-5 deficient Achilles tendons also contained significantly more fat within the repair tissue at all time points examined, and was significantly weaker than control tissue at 5 weeks after surgery, but strength differences were no longer detectable by 12-weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating tendon repair, and are consistent with previously posited roles for GDF-5 in cell recruitment, migrationladhesion, differentiation, proliferation, and angiogenesis. 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. K t , y \ i ~ ~ l .Tendon c: repair; GDF-5; Mouse; Growthldifferentiation factors; Bone morphogenetic proteins
Introduction
Over the past several decades, the incidence of tendon and ligament injuries has increased dramatically due largely to an increased participation in athletic activities in the general population [20,30]. The long-term clinical outcome for surgical treatment of such injuries, however, is often quite poor [1,2,5,8,19,23,25,28,31,33,34]. Consequently, recent efforts have been aimed at developing biologically based therapies for tendon and ligament repair. One family of signaling molecules that holds promise for eventual therapeutic use in tendon repair is the TGF-P gene superfamily. The TGF-0's include several growth/differentiation factors (GDFs) which play an important role in regulating embryonic development [ 18,221. TGF-P-related peptides are synthesized as large precursor molecules with a poorly conserved aminoterminal region and a highly conserved C-terminal mature domain [9,15,35]. Based on the amino acid *Corresponding author. Tel.: +I-413-585-7007; fax: +1-413-5857001. E-nwil ut/thss: bmi [email protected] h.edu (B. Mi kic).
similarity within this mature domain, members of the TGF-P superfamily have been organized into related subfamilies. For example, the mouse GDF 5, 6, and 7 peptides share 80-86%1 identity with each other in the mature C-terminal region of the molecule, as compared with 57% or less with other bone morphogenetic protein (BMP) subfamilies [36]. Thus, G D F 5, 6, and 7 were identified as a new subgroup of BMP-related TGF-P signaling molecules. The discovery that the brachjyodism (bp) mutation in mice consists of a mutation in the gene for GDF-5 has provided a unique opportunity to study the role of this molecule in vertebrates [36]. The bp mutation alters the length and number of bones in the limbs of mice, but spares the axial skeleton [16,24]. GDF-5 null mutations alter the number and insertions of numerous tendons in the limbs, as well as the size and morphology of tuberosities to which tendons and ligaments attach [ 16,371. More recently, we have shown that the absence of GDF5 also affects the composition, ultrastructure, and biomechanical integrity of the tendons that d o form in these animals [6,29], thus suggesting that GDF-5 may play an important role in the establishment and maintenance of normal tendon properties.
0736-02666 - see front matter 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved doi: 10.1016/S0736-0266(03)00049-4
A. Clihahra et al. I Journal of Orthopaedic Research 21 (2003) 826-835
Additional evidence supports the hypothesis that GDFs 5, 6, and 7 may play a role in tendon and ligament biology [3,13,17,26,27,38]. In 1997, Wolfman et al. reported that GDFs 5, 6, and 7 possess the unique ability to induce neotendodligament formation when implanted ectopically in rodents [38]. Injection of BMP12/GDF-7 gene transferred murine mesenchymal stem cells into the muscles of nude mice also induced formation of tendon-like tissue [26]. Further, BMP-12 gene transfer into a complete tendon laceration model in the chicken served to augment tendon healing based on mechanical outcome measures [27]. Other investigators have found that BMP- 13/GDF-6 can induce neotendon after intramuscular injection of BMP- 13 adenoviral vector into athymic nude rats [17]. In addition, Forslund and Aspenberg have demonstrated that local injection of GDF-5 or GDF-6 protein into an Achilles tendon gap healing model in rats serves to augment tendon repair based on mechanical testing of the repair tissue 8 days after injection [ 131. Together, these data strongly suggest that the GDF-5, 6, and 7 subfamily of bone morphogenetic proteins may be functionally distinct from other BMPs, with a potential role in tendon analogous to that of traditional BMPs in bone maintenance and repair. Before these factors can be used clinically, however, a more complete understanding of their influence on tendon and ligament is required. Based on the abnormal properties of intact tendons from GDF-5 deficient mice [6,29], as well as the demonstrated ability of GDF-5 to affect cell proliferation [ 14,321, adhesion [ 10,11,14], and differentiation, as well as angiogenesis [39], we hypothesize that GDF-5 may modulate tendon repair. If so, mice deficient in GDF-5 should exhibit an impaired response to tendon injury in comparison to control animals of the same age and gender. The aim of this study was to test the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, compositional, and ultrastructural analyses, we demonstrate that Achilles tendons from 8-week-old male GDF-5 -1- mice exhibit a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Using structural mechanical tests, we show that GDF-5 deficient repair tissue is still significantly weaker than control tissue at 5 weeks after surgery, but strength differences are no longer present at 12-weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating tendon repair.
Materials and methods Experir?ienfal design The experimental model used in this study was the GDF-5 deficient h ~ a c h y p ~ d (bp) r s ~ mouse obtained from Jackson Laboratories (Bar Harbor, ME). The hp mutation arises from a frameshift mutation in
827
the gene for GDF-5, resulting in a translational stop codon prior to the mature signaling region of the protein, thereby rendering a functional null mutation in -/- mice [36]. Mutant animals are readily discernable at birth based on the small size of their paws. Control animals consisted of phenotypically normal littermates (note that genotyping was not performed to distinguish between +/- and +I+ littermates). Eightweek-old male animals were examined to provide consistency with earlier studies characterizing the intact properties of tendons from animals of this age and gender. Skeletal maturity was comparable in the mutants and controls, based on prior histologic comparison of the proximal tibia1 growth plates in this model. Approval for all components of the study was obtained from the appropriate Institutional Animal Care and Use Committee. A total of 80 mutant and 80 control animals were used for this study. Surgical procedure All surgical procedures were performed by a single surgeon to provide consistency in technique. Each mouse received an anesthetic mixture of 150 mg/kg ketamine, 10 mg/kg xylazine in sterile saline via intramuscular injection in the right thigh. After appropriate anesthesia was obtained, a vertical incision was made approximately I cm in length just medial to the posterior mid-line slightly above the calcaneus. The Achilles tendon was isolated from the underlying muscle using blunt dissection and the plantaris tendon was left intact. On the leli side, a mid-substance tenotomy was created and repaired using a 6-0 monofilament polypropylene suture (Owens-Minor #SP- 1697) using a modified Kessler technique. As an internal control, sham operations were performed on the right side in which a skin incision was made, the tendon was isolated and irrigated, and the skin closed in a manner identical to the contralateral side. Following copious irrigation, the skin was closed using a 6-0 nylon suture (Owens-Minor #SP-1696), and wounds dressed with betadine ointment. At various timepoints determined by the assay being performed, animals were sacrificed via C 0 2 inhalation and healing tissue was dissected free from all extraneous soft tissue and further processed as described in the sections below. Biocheinicrrl composition Biochemical composition was examined in the healing and sham tissues at 3, 5, 7, 9, 11, 14, 28, and 42 days post-operatively ( n = 4 per group per time point). After sacrifice, tissue was immediately digested in 500 pl of sterile papain solution (125 pg/ml in 1X PBE, pH 6.5) at 60 "C for 18 h. D N A content was determined using the Hoechst 33258 dye assay with calf thymus DNA as a standard [21]. Glycosaminoglycan (GAG) content was determined using the dimethylmethylene blue (DMMB) colorimetric assay with dermatan sulfate as a standard [12]. As an indicator of total collagen content, hydroxyproline content was determined subsequent to acid hydrolysis in 6 N HCI for 24 h at 110 "C using the dimethylaminobenzaldehyde (DMBA) colorimetric assay with purified hydroxyproline as a standard [7]. For each assay, all samples and standards were processed in duplicate in a single experiment. Each assay was repeated twice on separate occasions to verify results, and average values were used for analysis. Data were expressed as a ratio of repair side values normalized to the sham side values because we have previously shown that intact Achilles tendons from 8-week-old GDF-5 4- male animals contain significantly less hydroxyproline per microgram of DNA compared to control tendons ~91. Histologicul cl~uracterizrrtron At 3, 5, 7, 9, 11, 14, 28, and 42 days after surgery, the calcaneus Achilles-muscle complex was harvested and immediately fixed in 4% PFA (paraformaldehyde) for 24 h at 4 "C ( n = 4 per group per time point). Samples were decalcified in 0.5 M EDTA (ethylenediaminetetraacetic acid), and the solution was changed every other day until the bone was sufficiently decalcified for sectioning. Tissue was then either processed for standard cryo-sectioning or dehydrated and embedded in paraffin, and 6-pm, serial, saggital sections were cut. Several sections containing the mid-portion of the healing tissue were stained
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using both hematoxylin and eosin staining and trichrome staining (Weigert's Hematoxylin, Fast Green, and Saffranin-0) on alternate sections to visualize cellularity, vascularization, the presence of adipocytes, and collagen fiber orientation. On five mid-saggital sections, point-counting methods were used to quantify trends in the percentage of healing tissue occupied by vasculature and fatty tissue (based on histologic appearance) using five non-overlapping images per tissue section. Ultrasrructurul u n u l w v
Using previously described methods [29], collagen fibril ultrastructure was characterized at 5, 7, and 14 days post-operatively on one representative animal per genotype per timepoint to document trends in ultrastructural changes as a function of time post-op. Coilagen fibril size distribution was quantified, as were the following dependent variables: (1) mean value of collagen fibril diameter; (2) collagen area fraction (percentage of a given area occupied by collagen); and ( 3 ) number of fibrils per unit area. Ten photos per animal were examined, with 500-1000 fibrils analyzed per mouse.
Mechanical hehucior At 5 and 12 weeks post-operatively, the hindlimbs from six mice per genotype were dissected at the hip joint after sacrifice, wrapped in sterile-saline-soaked gauze, and stored in hermetically sealed bags at -20 "C until the day of testing. Immediately prior to testing, individual limbs were thawed at room temperature. Using a mid-line incision on the anterior surface of the ankle, the skin was removed and in situ tendon gage length measured with the foot at 45" of plantar-flexion from the distal calcaneal insertion to the proximal muscle-tendon junction using sliding dkal calipers. The muscle-tendon-bone complex (gastrocnemiuslsoleus, Achilles, calcaneus) was carefully dissected free from the limb and the tendon was wrapped in sterile-saline-soaked filter paper to maintain adequate hydration. Any remaining soft tissue adhering to the calcaneus was carefully removed. Using the blunt end of a scalpel handle, the gastrocnemius/soleus muscle fibers were completely removed from the intramuscular tendinous fibers. After dissection, the tendon was mounted as previously described for tensile mechanical testing with care taken to ensure vertical anterio-posterior and medial-lateral alignment [?9]. All mechanical testing was performed on an Instron Micromechanical testing machine (Model 5542, Instron Corp., Canton. MA). The room temperature was 25 "C and tendons were kept moist with PBS throughout the test. Tendons were pre-loaded to 0.02'%1 body weight, gage length measured with sliding calipers, and the tendon loaded at 1 00'X) strainls until failure (no pre-cycling was performed). The structural strength and stiffness were obtained for all specimens, and data expressed as a ratio of repair side values normalized to sham side values. Stuti.c licwl ertrluarion
All dependent variables were analyzed statistically using a twofactor ANOVA with genotype and time post-op as the two independent factors. When appropriate, post-hoc analyses were also performed (Fisher's PLSD), and a value o f p < 0.05 was chosen as the cutoff for statistical significance.
Results
At the time of harvest, it was noted for all time points that the majority of sutures had pulled out, although exactly when this occurred is unknown. Wounds showed no signs of infection, and subcutaneous scar tissue was easily discernable and separated from the underlying healing tendon.
Biochemical composition
Based on all biochemical parameters (normalized to internal sham values), GDF-5 deficient mice exhibited a delay of 5-9 days in attaining peak values of normalized DNA, glycosaminoglycan (GAG), and hydroxyproline (Hypro) levels in healing Achilles tendon tissue when compared to control littermates (Fig. 1). Statistically, the time-dependent changes in all three biochemical measures were significantly affected by the GDF-5 mutation (p < 0.001 for the interaction of genotype and time). Control tissue reached a peak value of DNA content at 5 days after surgery, whereas mutant animals did not reach a peak in cellularity until Day 14 (Fig. 1A and B). In addition, the magnitude of the peak in healing/sham DNA content was approximately 30% lower in mutants compared to controls. Time-dependent changes in GAG content were similar: control animals reached a peak in healinghham G A G at 5 days post-op, whereas in GDF-5 deficient animals this peak was delayed until 11 days post-op (Fig. 1C and D). Collagen content peaked at Day 9 after surgery in control animals, but was delayed until Day 14 in mutant animals (Fig. 1E and F). In addition, the magnitude of peak hydroxyproline (healingkham) levels in GDF-5 4- mice was more than twice that of control animals. Histological characterization
Based on histological appearance, GDF-5 deficient Achilles tendons also exhibited a delay in peak areal cell density and collagen reorganization (qualitatively assessed) (Fig. 2). At 3 days after surgery, numerous cells were evident at the repair site of control animals (Fig. 2C). The presence of some fatty tissue within the bulk of the repair site was also readily apparent. Mutant repair tissue contained fewer repair cells which appeared to be clustered in an uneven distribution throughout the repair site (Fig. 2D). Overall, the tissue was highly disorganized in the GDF-5 deficient animals, with an abundance of fatty cells present within the midsubstance of the tendon regenerate. By Day 7 post-op, the repair tissue of control animals continued to increase in cell density, and the cells began to reorient themselves along the long axis of the tendon (Fig. 2G). Revascularization was readily apparent. By contrast, mutant tissue was still highly disorganized (Fig. 2H): while the cell density had increased from Day 3, it was still reduced compared to control tissue, and a large number of fatty cells were still evident. At 2 weeks, mutant tissue began to appear more organized, with some realignment of cells along the long axis of the tendon (Fig. 2L). Fat content appeared to be significantly reduced, but was still evident, and cell density had increased relative to Day 7. Overall, the appearance of the mutant tissue on Day 14 resembled that of control tissue of Day 7
A. Chhabra et al. I Journal of’ Orthopaedic Research 21 (2003) 826435
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Fig. 1. Biochemical composition of healing Achilles tendons in cohtrol (A, C, E) and GDF-5 deficient (B, D, F) mice. Data represent repair side values normalized to sham side values of DNA (A and B), glycosaminoglycan (C and D), and hydroxyproline as an indicator of total collagen (E and F). (Data are depicted as mean fSD.) The time-dependent changes in all biochemical content indicators were significantly affected by genotype.
(Fig. 2G), although with a qualitatively greater abundance of fatty inclusions. By 6 weeks after surgery, both the control and mutant tissue exhibited a marked improvement in the histologic appearance of the tissue (Fig. 2 0 and P). Although cell density appeared qualitatively higher than that of sham-operated tissue, control animal histology was otherwise similar to the sham side in appearance. The organization of mutant tissue at this stage was dramatically improved and resembled that of control animal repair tissue with the noted exception that a significant number of fatty cells still remained within the tissue proper (Fig. 2P). No evidence of cartilage or bone was detected in any of the mutant or control tendon samples at any of the time-points examined. Because of the qualitative trends observed in the healing tissue, the degree of vasculature and fatty cells were further quantified in mutant and control tissue using point-counting techniques. GDF-5 deficient tissue exhibited a delay of 1 week in revascularization compared to controls, with a statistically significant interaction between genotype and time (Fig. 3A). In ad-
dition, GDF-5 deficient tissue contained a significantly greater proportion of fatty tissue compared to controls throughout the time-period examined (Fig. 3B). Sham values of the percentage of vascular and fatty tissue were not significantly different between mutants and controls (not shown). Ultr-astructurcrlanalyses The time-dependent changes in collagen fibril ultrastructure during Achilles tendon healing were also noticeably affected by GDF-5 deficiency (Figs. 4 and 5). During the first 2 weeks of healing, mutant tendons had a lower collagen fibril area fraction (the percentage of a given area occupied by collagen) (Fig. 5A). By 2 weeks post-op, these differences were no longer apparent. The difference in fibril area fraction was due primarily to smaller fibrils at Days 5 and 7 (Fig. 4): by Day 14, GDF-5 deficient mice had a collagen area fraction comparable to that of control mice, and fibril diameter distributions were also comparable (Fig. 5C and D-F). The approximate 2-week delay in changes in ultrastructural
A. Chhabra et a/. I Journal of Orthopueciic Research 21 (2003) 826-835
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Fig. 2. Healing and sham operated Achilles tendons from GDF-5 deficient and control animals at 3 (A-D), 7 (E-H). 14 (I- L), and 42 (M-P) days post-tenotomy. For each time-point shown, top panels depict sham tendons, and bottom panels depict repair tissue, while left panels are from control animals and right panels are from GDF-5 deficient animals.
characteristics is consistent with the observed 1-2 week delay in changes in biochemical composition and tissue organization at the light microscope level.
repair tendons failed in the mid-substance, whereas only 50% of control tendons did.
Mechanical behavior
Discussion
Structurally, GDF-5 deficient healing tissue (normalized to individual sham values) was significantly weaker and more compliant than control tissue at 5 weeks, but by 12 weeks, no significant difference was detected between genotypes in either structural strength or stiffness (Fig. 6A and B). Because cross-sectional area measurements were not obtained, it cannot be definitively concluded that structural differences were solely due to material property (rather than geometric) differences, although by 5 weeks post-op, no distinction could be made between healing and sham tendons at the time of dissection based on gross appearance. Consistent with our previous findings in intact GDF-5 -1- Achilles tendons, the majority of all mutant sham tendons failed in the tendon mid-substance, whereas the majority of control sham tendons failed via avulsion from the calcaneus. At both 5 and 12 weeks, 100% of GDF-5 -1-
The aim of this study was to test the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, compositional, and ultrastructural analyses on healing tendons from 8-week-old male brachypodism mice, we found that GDF-5 deficient animals exhibited a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Structurally, GDF-5 deficient repair tissue was still significantly weaker and more compliant (after normalization to internal sham values) than control tissue at 5 weeks after surgery, but differences in structural parameters were no longer detected at 12 weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating early tendon repair. One of the most interesting effects of GDF-5 deficiency observed in this study was the delay in achieving
A . Clihuhru et ul. I Journul qf Orthopurdic Rrseurcli 21 (2003) 826435
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Fig. 3. (A) Time-dependent change in the percentage of healing Achilles tendon tissue occupied by vasculature in GDF-5 deficient (- - -) vs. control animals (-). (B) Time-dependent change in the percentage of healing Achilles tendon tissue occupied by fat in GDF-5 deficient (- - -) vs. control animals (-). (Data are depicted as mean f SD.) The time-dependent changes in both parameters were significantly affected by genotype.
peak cell content (as measured by DNA content) at the Achilles tendon repair site. One interpretation of this delay is that GDF-5 may be affecting cell recruitment to the repair site. Indeed, it has been demonstrated that GDF-5 is capable of inducing the migration of certain cell types in a chemotactic (directional) manner [39]. Whether GDF-5 is capable of inducing a chemotactic response in the tenocytes and fibroblasts involved in tendon repair has yet to be examined. The delay in peak DNA content seen in GDF-5 deficient repair tissue could also be explained by altered cell recruitment if the ability of cells to migrate to the repair site is somehow affected by GDF-5 deficiency. An essential component of cell migration is cell adhesion. Duke and Elmer [lo] examined the adhesive characteristics of limb mesenchymal cells from 12-day old normal and GDF-5 deficient, brachypod mouse embryos in rotation culture and found that GDF-5 deficient cells were more adhesive than control mesenchyme cells. In a subsequent study, these same authors used fusion studies of normal and mutant limb-bud fragments to test adhesion, and again found evidence to support the conclusion that limb bud mesenchymal cells from 12day old GDF-5 deficient mouse embryos were more
Fig. 4. Representative electron microscope images from GDF-5 deficient and control mice. (A and B) Collagen fibrils from sham (A) and 5-day post-op (B) tissue of a control animal. Panels C and D show comparable images from a GDF-5 deficient mouse. Bar represents approximately 100 nm.
adhesive than control mesenchyme [ 1 11. These studies support a role for GDF-5 in cell adhesion, which, if also a factor in tendon, could serve to delay the early stages of the healing process by slowing the migration of cells to the repair site. An alternative explanation for the observed delay in the early stages of tendon repair could be that, in the absence of GDF-5, cell proliferation may be impaired. In overexpression studies, Francis-West et al. used pulse-labeling of chick cartilage to show that GDF-5 can increase chondrocyte proliferation [ 141. In their in vitro micromass experiments using normal chick mesenchyme, however, exogenous GDF-5 protein was unable to stimulate cell proliferation. Yamashita et al. similarly found that GDF-5 was unable to stimulate cell proliferation (assessed via [3H]thymidine incorporation) in bovine endothelial cells [39]. Consistent with the mitogenic effects of GDF-5 on chondrocytes,
A. Chliuhra et aI. I Journal qf Orthopurdic Reseurch 21 (2003) 826435
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Fig. 5. Ultrastructural parameters from healing Achilles tendons of GDF-5 deficient and control mice. (A) Collagen fibril area fraction. (B) Mean collagen fibril diameter. (C) Number of fibrils per unit area. Error bars indicate the standard deviation of the mean of values obtained from ten photos analyzed per mouse per genotype per timepoint. Panels (D-F) depict collagen fibril distributions for mutant and control animals on Days 5, 7, and 14 post-op (note that differences in scale were required to best capture the distribution of fibril size at each time point shown). Mutant data are shown with dark bars, control data by white bars.
Nakamura et al. used thymidine autoradiography on hp growth plates from 21-day old mice, and demonstrated a significant reduction in the rate of chondrocyte cell division in GDF-5 deficient growth plates [32]. Clearly, the mitogenic potential of GDF-5 appears to depend on (at the very least), cell type. Whether the division of cells involved in tendon repair is affected by GDF-5 remains to be determined. Aspenberg and Forslund have reported that GDF-6 has strong proliferative effects on tendon in vivo [4], thus it is likely that GDF-5 may as well. It would be interesting to determine whether transfecting tendon cells with GDF-5, 6, or 7 could rescue the effects of GDF-5 deficiency in the early stages of the repair process. The results of our study might also be interpreted to suggest that GDF-5 is involved in cell differentiation at the repair site. It is well known that pluripotent cells of musculoskeletal origin are capable of differentiating along multiple pathways, including cartilage, bone, muscle, fat, and fibrous tissue such as tendon and ligament. One striking feature of the healing process in GDF-5 deficient Achilles tendons is the greater abundance of adipocytes in the healing tissue at all time
points examined in GDF-5 deficient animals. These observations present the intriguing possibility that, in the absence of GDF-5, some portion of the repair cell population may be receiving abnormal signals to differentiate into fat. Interestingly, in the steady-state situation of non-operated tendons, no appreciable fat is present within the tendon proper in either mutants or controls. Although the amount of fat present in mutant tissue declines with post-operative time, there is still significantly more present in mutant tissue compared to controls at 6 weeks post-op. It is likely that the presence of these fatty inclusions serve to weaken the material substance of the tendon, thereby contributing to the lower structural strength in GDF-5 deficient tendons (normalized to sham values) at 5 weeks of healing. Although material properties could not be calculated because cross-sectional area measurements were not made, the gross appearance of the operated and sham Achilles tendons was indistinguishable in both groups of animals at 5 weeks. Histologic sections were not taken at 12 weeks post-op, but we hypothesize that the mutant tissue would no longer contain significantly more fatty cells than control tissue at this timepoint, based on the
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Fig. 6. Normalized (repair sidekham side) values of structural strength (A) and stiffness (B) in control (white bars) and GDF-5 deficient (dark bars) Achilles tendons at 5 and 12 weeks post-op. (Data are depicted as mean C! SD.)
comparable structural behavior of GDF-5 deficient and control Achilles at 12 weeks. One final possibility that could explain the short-term delay in healing observed in GDF-5 deficient tendons is the known angiogenic capability of this molecule. Our results demonstrate that the absence of GDF-5 significantly affects the time-course of revascularization in our Achilles tendon model, with a delay of approximately 1 week seen in bp mice. These data are consistent with the work of Yamashita et al. [39] who showed that GDF-5 induced angiogenesis in vivo in both chick chorioallanotoic membrane and rabbit cornea assays. These data thus suggest that the observed effects of GDF-5 deficiency on tendon healing could be explained in part by a decreased angiogenic capacity in these animals. One of the interesting results of this study is the collagen fibril size disparity observed between mutant and control tendons during the first 2 weeks of Achilles tendon repair. It is well known that the small leucine rich proteoglycans decorin, lumican, and fibromodulin help regulate collagen fibril diameter. Collagen V has also been shown to play a similar role. Our data suggest
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the possibility that GDF-5 4- animals may have altered levels of such fibril size regulating proteins, although we did not look beyond bulk compositional measures in the present study. Despite regaining comparable fibril size by 2 weeks post-op, mutant tendons still exhibit significantly lower structural strength and stiffness 5 weeks after surgery. As collagen cross-linking is known to affect tendon strength, it will be important to examine this parameter in future studies, in addition to the expression levels of molecules known to serve cross-linking functions for collagen type I such as the FACIT collagen XII. Despite the clear effect of GDF-5 deficiency on the time-course of Achilles tendon healing shown in the present study, several limitations exist. First, the age of the animals was 8 weeks, which represents young adolescents. Many mutant mouse strains as well as transgenic mouse lines exhibit delayed development and maturation. Consequently, it is possible that mutants and controls, although the same age, could be at different maturational stages, and this could conceivably affect the time-course of repair. To rule out this possibility, older animals (e.g. older than 16 weeks of age) would have to be examined. A second limitation of this study is inherent to the bruchypodism mouse model. Many of these animals exhibit a mild degree of knee joint subluxation which can affect their gait, resulting in a more crouched posture. It is important to note, however, that mobility is not impaired in hp mice, and these animals are just as active as their control littermates, both before and after surgery. No differences were noted in the time it took for mutant animals to begin walking after surgery compared to their littermates. Nonetheless, with this animal model it is not possible to separate the effects of possible altered loading superimposed on the effect of GDF-5 deficiency. As with all knockout mouse models or naturally occurring mutations, it is also possible that the absence of one growth factor may result in over-compensation of other, related family members. For example, it is not known whether GDF-6 and/or GDF-7 expression is altered in the absence of GDF-5. In fact, to date, no one has reported on the expression (or lack of expression) of GDFs 5,6, or 7 during normal tendon repair. Additional, more minor limitations with the present study include the fact that no specific markers were used to identify vasculature or fat; rather, these tissues were identified based solely on histologic appearance. Further, although bulk biochemical composition data such as GAG and hydroxyproline provide valuable information, such aggregate data do not allow us to determine whether synthesis, degradation, or both have been affected. Lastly, the lack of determination of cross-sectional geometric data (or equivalent gravimetric measures such as Hypro/gage length) prevent us from determining whether the observed structural differences in strength and stiffness are due to material property
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differences (as we hypothesize), size differences, or some combination of the two. In conclusion, the results of this study demonstrate for the first time that GDF-5 deficiency in 8-week-old male mice leads to a delay in the Achilles tendon repair process of 1-2 weeks, based on compositional, histological, and ultrastructural measures. These data are consistent with previously documented roles for GDF-5 in cell recruitment, differentiation, adhesionlmigration, proliferation, as well as angiogenesis. In combination with prior studies from other investigators in which GDF-5 has been shown to augment tendon repair [3], our data suggest that GDF-5 may play an important role in tendon healing and might someday be of clinical use to enhance the repair process of tendon injuries in humans.
Acknowledgements
We would like to thank Dr. Rashard Dacus, M.D. for his assistance with the histomorphometric analyses, and Dr. Gary Balian, Ph.D. for helpful discussion. This work was funded in part by a grant from the NIH to BM (AR45828).
References [I] Amiel D, Kleiner JB. Akeson WH. The natural history of the anterior cruciate ligament autograft of patellar tendon origin. Am J Sports Med 1986:14:449-62. [2] Amiel D, Kilper S, Akeson WH. Cruciate ligaments. Response to injury. In: Daniel D, Akeson WH, O’Connor J, editors. Knee Ligaments: Structure. Function, Injury and Repair. New York: Raven Press; 1990. p. 365-77. [3] Aspenberg P, Forslund C. Enhanced tendon healing with G D F 5 and 6. Acta Orthop Scand 1999;70:514. [4] Aspenberg P, Forslund C. Improved tendon healing in the rabbit Achilles tendon after a single injection of CDMP-2. Trans ORS 2002;27:626. [5] Caubaud JE, Rodkey WG, Feagin JA. Experimental studies of acute anterior cruciate ligament injury and repair. Am J Sports Med 1979;7:18-22. [6] Clark RT, Johnson TL, Schalet BJ, Davis L, Gashen V, Hunziker EB, et al. GDF-5 deficiency in mice leads to disruption of tail tendon form and function. Conn Tiss Res 2001;42:175-86. [7] Creemers LB. Jansen DC, van Veen-Reurings A, van den Bos T, Everts V. Microassay for the assessment of low levels of Hydroxyproline. Biotechniques 1997;22:656-8. [8] Crenshaw Jr AH. Shoulder and elbow injuries. In: Crenshaw AH, editor. Campbells’ Operative Orthopaedics. St. Louis: Mosby Year Book; 1992. p, 1733-66. [9] Daopin S, Piez KA, Ogawa J, Davies DR. Crystal structure of transforming growth Factor-beta 2: an unusual fold for the superfamily. Science 1992;257:369-73. [lo] Duke J. Elmer WA. Effect of the brachypod mutation on cell adhesion and chondrogenesis in aggregates of mouse limb mesenchyme. J Embryol Exp Morph 1977;42:209-17. [ I I ] Duke J, Elmer WA. Cell adhesion and chondrogenesis in brachypod mouse limb mesenchyme: fragment fusion studies. J Embryol Exp Morph 1978;48:161-8.
[12] Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochem Biophys Acta 1986:883: 173-7. [13] Forslund C, Aspenberg P. Tendon healing stimulated by injected CDMP-2. Med Sci Sports Exer 2001:685-867. [I41 Francis-West P H , Abdelfattah A, Chen P, Allen C , Parish J, Ladher R, et al. Mechanisms of GDF-5 action during skeletal development. Development 1999:126: 1305-15. [I51 Griffith DL, Keck PC, Sampath TK, Rueger DC, Carlson WD. Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily. Proc Natl Acad Sci 1996;93:878-83. [I61 Grunenberg H, Lee AJ. The anatomy and development of brachypodism in the mouse. J Embryol Exp Morph 1973:30:11941. [I 71 Helm GA, Li JZ, Alden T D , Hudson SB, Beres EJ, Cunningham M, et al. A light and electron microscopic study of ectopic tendon and ligament formation induced by bone morphogenetic protein12 adenoviral gene therapy. J Neurosurg 2001;95(2):298-307. [I 81 Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev 1996; 10:1580-94. [I91 Jarvinen M. Epidemiology of tendon injuries in sports. Clin Sports Med 1992;11:493-504. [20] Jozsa L, Kvist M, B a h t BJ, Reffy A, Jarvinen M, Lehto M, et al. The role of recreational sport activity in Achilles tendon rupture. A clinical, pathoanatomical, and sociological study of 292 cases. Am J Sports Med 1989;17(3):33843. [21] Kim YJ, Sah RL, Doong JY. Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988:174:168-76. [22] Kingsley DM. The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 1 9 9 4 813346. [23] Kvist M. Achilles tendon injuries in athletes. Ann Chir Gynaecol l991;80: 188-201. [24] Landauer W. Brachypodism. a recessive mutation of house mice. J Heredity 1952;43:293-8. [25] Leddy JP. Avulsions of the flexor digitorum profundus. Hand Clin 1985;1:77-83. [26] Lou J, Tu F, Ludwig FJ, Zhang J, Manske PR. Effect of bone morphogenetic protein-12 gene transfer on mesenchymal progenitor cells. Clin Orth Re1 Res 1999;369:333-9. [27] Lou J. Tu Y, Burns M, Silva MJ, Manske P. BMP-I2 gene transfer augmentation of lacerated tendon repair. J Orthop Res 200 1 ;19: I 199-202. [28] Lubovitz JH, Grauer J D . Arthroscopic treatment of anterior cruciate ligament avulsion. Clin Orthop 1993:294:242-6. [29] Mikic B, Schalet BJ, Clark RT, Gaschen V, Hunziker EB. GDF-5 deficiency in mice alters the ultrastructure, mechanical properties and composition of the Achilles tendon. J Orthop Res 2001;19: 365-7 I . [30] Moller A, Astron M, Westlin N. Increasing incidence of Achilles tendon rupture. Acta Orthop Scand 1996;67(5):479-8 I . [31] Morberg P, Jerre R, Sward L, Karlsson J. Long-term results after surgical management of partial Achilles tendon ruptures. Scand J Med Sci Sports 1997;7(5):299-303. [32] Nakamura K, Hirosawa K, Hama K, Oda S-I. Defects in the growth plate of brachypodism mice--light and electron microscopic findings and cell proliferation by the method of tritiated thymidine autoradiography. J Jpn Orthop Assoc 1984;58:835-45. [33] Nobuhara K, Hata Y , Komai M. Surgical procedure and results of repair of massive tears of the rotator cuff. Clin Orthop 1994;304:549. [34] Norman WH. Repair of avulsion of insertion of biceps brachii tendon. Clin Orthop 1985:193:189-94. [35] Schlunegger MP, Grutter MG. An unusual feature revealed by the crystal structure at 2.2 A resolution of transforming growth factor-beta 2. Nature 1992;358:4304.
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[36] Storm EA, Huynh TV, Copeland NG, Jenkins NA, Kingsley DM. Lee S-J. Limb alterations in brachypodism mice due to mutations in a new member of the TGF-beta superfamily. Nature 1994; 36863943. [37] Storm EE, Kingsley DM. Joint patterning defects caused by single and double mutations in members of the bone morphogenetic proteins (BMP) family. Development 1996;122( 12):3969-79.
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[38] Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, et al. Ectopic induction of tendon and ligament in rats by growth/differentiation factors 5 , 6, and 7, members of the TGF-fi gene family. J Clin Invest 1997;100(2):321-30. [39] Yamashita H. Shimizi A, Kato M, Nishitoh H, Ichijo H, Hanyu A, et al. Growthldifferentiation factor-5 induces angiogenesis in vivo. Exp Cell Res 1997;235:218-26.
Journal of Orthopaedic Research 21 (2003) 826-835
ELSEVIER
www.elsevier.com/locate/orthres
GDF-5 deficiency in mice delays Achilles tendon healing A. Chhabra
a,
D. Tsou ', R.T. Clark
a,
V. Gaschen ', E.B. Hunziker ', B. Mikic
b3*
'' Depurtnient of Orthopedics. University of Virginiu Heulth Sciences Cmter. Churlortesville, V A 22908, U S A Picker Engineering Program, Smith College, 51 College Lune, Nortlzuinpton, M A 01063, USA ITT Rcsrtrrch Institute for Dental and Skeletul Bioiogy, Murtenstrusre 35, Postfach 54, CH-3010 Bern, Switzerlund
Received 1 October 2002; accepted 12 February 2003
Abstract
The aim of this study was to examine the role of one of the growthldifferentiation factors, GDF-5, in the process of tendon healing. Specifically, we tested the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, biochemical, and ultrastructural analyses, we demonstrate that Achilles tendons from 8-week-old male GDF-5 4- mice exhibit a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Mutant animals took longer to achieve peak cell density, glycosaniinoglycan content, and collagen content in the repair tissue, and the time course of changes in collagen fibril size was also delayed. Revascularization was delayed in the mutant mice by 1 week. GDF-5 deficient Achilles tendons also contained significantly more fat within the repair tissue at all time points examined, and was significantly weaker than control tissue at 5 weeks after surgery, but strength differences were no longer detectable by 12-weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating tendon repair, and are consistent with previously posited roles for GDF-5 in cell recruitment, migrationladhesion, differentiation, proliferation, and angiogenesis. 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. K t , y \ i ~ ~ l .Tendon c: repair; GDF-5; Mouse; Growthldifferentiation factors; Bone morphogenetic proteins
Introduction
Over the past several decades, the incidence of tendon and ligament injuries has increased dramatically due largely to an increased participation in athletic activities in the general population [20,30]. The long-term clinical outcome for surgical treatment of such injuries, however, is often quite poor [1,2,5,8,19,23,25,28,31,33,34]. Consequently, recent efforts have been aimed at developing biologically based therapies for tendon and ligament repair. One family of signaling molecules that holds promise for eventual therapeutic use in tendon repair is the TGF-P gene superfamily. The TGF-0's include several growth/differentiation factors (GDFs) which play an important role in regulating embryonic development [ 18,221. TGF-P-related peptides are synthesized as large precursor molecules with a poorly conserved aminoterminal region and a highly conserved C-terminal mature domain [9,15,35]. Based on the amino acid *Corresponding author. Tel.: +I-413-585-7007; fax: +1-413-5857001. E-nwil ut/thss: bmi [email protected] h.edu (B. Mi kic).
similarity within this mature domain, members of the TGF-P superfamily have been organized into related subfamilies. For example, the mouse GDF 5, 6, and 7 peptides share 80-86%1 identity with each other in the mature C-terminal region of the molecule, as compared with 57% or less with other bone morphogenetic protein (BMP) subfamilies [36]. Thus, G D F 5, 6, and 7 were identified as a new subgroup of BMP-related TGF-P signaling molecules. The discovery that the brachjyodism (bp) mutation in mice consists of a mutation in the gene for GDF-5 has provided a unique opportunity to study the role of this molecule in vertebrates [36]. The bp mutation alters the length and number of bones in the limbs of mice, but spares the axial skeleton [16,24]. GDF-5 null mutations alter the number and insertions of numerous tendons in the limbs, as well as the size and morphology of tuberosities to which tendons and ligaments attach [ 16,371. More recently, we have shown that the absence of GDF5 also affects the composition, ultrastructure, and biomechanical integrity of the tendons that d o form in these animals [6,29], thus suggesting that GDF-5 may play an important role in the establishment and maintenance of normal tendon properties.
0736-02666 - see front matter 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved doi: 10.1016/S0736-0266(03)00049-4
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Additional evidence supports the hypothesis that GDFs 5, 6, and 7 may play a role in tendon and ligament biology [3,13,17,26,27,38]. In 1997, Wolfman et al. reported that GDFs 5, 6, and 7 possess the unique ability to induce neotendodligament formation when implanted ectopically in rodents [38]. Injection of BMP12/GDF-7 gene transferred murine mesenchymal stem cells into the muscles of nude mice also induced formation of tendon-like tissue [26]. Further, BMP-12 gene transfer into a complete tendon laceration model in the chicken served to augment tendon healing based on mechanical outcome measures [27]. Other investigators have found that BMP- 13/GDF-6 can induce neotendon after intramuscular injection of BMP- 13 adenoviral vector into athymic nude rats [17]. In addition, Forslund and Aspenberg have demonstrated that local injection of GDF-5 or GDF-6 protein into an Achilles tendon gap healing model in rats serves to augment tendon repair based on mechanical testing of the repair tissue 8 days after injection [ 131. Together, these data strongly suggest that the GDF-5, 6, and 7 subfamily of bone morphogenetic proteins may be functionally distinct from other BMPs, with a potential role in tendon analogous to that of traditional BMPs in bone maintenance and repair. Before these factors can be used clinically, however, a more complete understanding of their influence on tendon and ligament is required. Based on the abnormal properties of intact tendons from GDF-5 deficient mice [6,29], as well as the demonstrated ability of GDF-5 to affect cell proliferation [ 14,321, adhesion [ 10,11,14], and differentiation, as well as angiogenesis [39], we hypothesize that GDF-5 may modulate tendon repair. If so, mice deficient in GDF-5 should exhibit an impaired response to tendon injury in comparison to control animals of the same age and gender. The aim of this study was to test the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, compositional, and ultrastructural analyses, we demonstrate that Achilles tendons from 8-week-old male GDF-5 -1- mice exhibit a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Using structural mechanical tests, we show that GDF-5 deficient repair tissue is still significantly weaker than control tissue at 5 weeks after surgery, but strength differences are no longer present at 12-weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating tendon repair.
Materials and methods Experir?ienfal design The experimental model used in this study was the GDF-5 deficient h ~ a c h y p ~ d (bp) r s ~ mouse obtained from Jackson Laboratories (Bar Harbor, ME). The hp mutation arises from a frameshift mutation in
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the gene for GDF-5, resulting in a translational stop codon prior to the mature signaling region of the protein, thereby rendering a functional null mutation in -/- mice [36]. Mutant animals are readily discernable at birth based on the small size of their paws. Control animals consisted of phenotypically normal littermates (note that genotyping was not performed to distinguish between +/- and +I+ littermates). Eightweek-old male animals were examined to provide consistency with earlier studies characterizing the intact properties of tendons from animals of this age and gender. Skeletal maturity was comparable in the mutants and controls, based on prior histologic comparison of the proximal tibia1 growth plates in this model. Approval for all components of the study was obtained from the appropriate Institutional Animal Care and Use Committee. A total of 80 mutant and 80 control animals were used for this study. Surgical procedure All surgical procedures were performed by a single surgeon to provide consistency in technique. Each mouse received an anesthetic mixture of 150 mg/kg ketamine, 10 mg/kg xylazine in sterile saline via intramuscular injection in the right thigh. After appropriate anesthesia was obtained, a vertical incision was made approximately I cm in length just medial to the posterior mid-line slightly above the calcaneus. The Achilles tendon was isolated from the underlying muscle using blunt dissection and the plantaris tendon was left intact. On the leli side, a mid-substance tenotomy was created and repaired using a 6-0 monofilament polypropylene suture (Owens-Minor #SP- 1697) using a modified Kessler technique. As an internal control, sham operations were performed on the right side in which a skin incision was made, the tendon was isolated and irrigated, and the skin closed in a manner identical to the contralateral side. Following copious irrigation, the skin was closed using a 6-0 nylon suture (Owens-Minor #SP-1696), and wounds dressed with betadine ointment. At various timepoints determined by the assay being performed, animals were sacrificed via C 0 2 inhalation and healing tissue was dissected free from all extraneous soft tissue and further processed as described in the sections below. Biocheinicrrl composition Biochemical composition was examined in the healing and sham tissues at 3, 5, 7, 9, 11, 14, 28, and 42 days post-operatively ( n = 4 per group per time point). After sacrifice, tissue was immediately digested in 500 pl of sterile papain solution (125 pg/ml in 1X PBE, pH 6.5) at 60 "C for 18 h. D N A content was determined using the Hoechst 33258 dye assay with calf thymus DNA as a standard [21]. Glycosaminoglycan (GAG) content was determined using the dimethylmethylene blue (DMMB) colorimetric assay with dermatan sulfate as a standard [12]. As an indicator of total collagen content, hydroxyproline content was determined subsequent to acid hydrolysis in 6 N HCI for 24 h at 110 "C using the dimethylaminobenzaldehyde (DMBA) colorimetric assay with purified hydroxyproline as a standard [7]. For each assay, all samples and standards were processed in duplicate in a single experiment. Each assay was repeated twice on separate occasions to verify results, and average values were used for analysis. Data were expressed as a ratio of repair side values normalized to the sham side values because we have previously shown that intact Achilles tendons from 8-week-old GDF-5 4- male animals contain significantly less hydroxyproline per microgram of DNA compared to control tendons ~91. Histologicul cl~uracterizrrtron At 3, 5, 7, 9, 11, 14, 28, and 42 days after surgery, the calcaneus Achilles-muscle complex was harvested and immediately fixed in 4% PFA (paraformaldehyde) for 24 h at 4 "C ( n = 4 per group per time point). Samples were decalcified in 0.5 M EDTA (ethylenediaminetetraacetic acid), and the solution was changed every other day until the bone was sufficiently decalcified for sectioning. Tissue was then either processed for standard cryo-sectioning or dehydrated and embedded in paraffin, and 6-pm, serial, saggital sections were cut. Several sections containing the mid-portion of the healing tissue were stained
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using both hematoxylin and eosin staining and trichrome staining (Weigert's Hematoxylin, Fast Green, and Saffranin-0) on alternate sections to visualize cellularity, vascularization, the presence of adipocytes, and collagen fiber orientation. On five mid-saggital sections, point-counting methods were used to quantify trends in the percentage of healing tissue occupied by vasculature and fatty tissue (based on histologic appearance) using five non-overlapping images per tissue section. Ultrasrructurul u n u l w v
Using previously described methods [29], collagen fibril ultrastructure was characterized at 5, 7, and 14 days post-operatively on one representative animal per genotype per timepoint to document trends in ultrastructural changes as a function of time post-op. Coilagen fibril size distribution was quantified, as were the following dependent variables: (1) mean value of collagen fibril diameter; (2) collagen area fraction (percentage of a given area occupied by collagen); and ( 3 ) number of fibrils per unit area. Ten photos per animal were examined, with 500-1000 fibrils analyzed per mouse.
Mechanical hehucior At 5 and 12 weeks post-operatively, the hindlimbs from six mice per genotype were dissected at the hip joint after sacrifice, wrapped in sterile-saline-soaked gauze, and stored in hermetically sealed bags at -20 "C until the day of testing. Immediately prior to testing, individual limbs were thawed at room temperature. Using a mid-line incision on the anterior surface of the ankle, the skin was removed and in situ tendon gage length measured with the foot at 45" of plantar-flexion from the distal calcaneal insertion to the proximal muscle-tendon junction using sliding dkal calipers. The muscle-tendon-bone complex (gastrocnemiuslsoleus, Achilles, calcaneus) was carefully dissected free from the limb and the tendon was wrapped in sterile-saline-soaked filter paper to maintain adequate hydration. Any remaining soft tissue adhering to the calcaneus was carefully removed. Using the blunt end of a scalpel handle, the gastrocnemius/soleus muscle fibers were completely removed from the intramuscular tendinous fibers. After dissection, the tendon was mounted as previously described for tensile mechanical testing with care taken to ensure vertical anterio-posterior and medial-lateral alignment [?9]. All mechanical testing was performed on an Instron Micromechanical testing machine (Model 5542, Instron Corp., Canton. MA). The room temperature was 25 "C and tendons were kept moist with PBS throughout the test. Tendons were pre-loaded to 0.02'%1 body weight, gage length measured with sliding calipers, and the tendon loaded at 1 00'X) strainls until failure (no pre-cycling was performed). The structural strength and stiffness were obtained for all specimens, and data expressed as a ratio of repair side values normalized to sham side values. Stuti.c licwl ertrluarion
All dependent variables were analyzed statistically using a twofactor ANOVA with genotype and time post-op as the two independent factors. When appropriate, post-hoc analyses were also performed (Fisher's PLSD), and a value o f p < 0.05 was chosen as the cutoff for statistical significance.
Results
At the time of harvest, it was noted for all time points that the majority of sutures had pulled out, although exactly when this occurred is unknown. Wounds showed no signs of infection, and subcutaneous scar tissue was easily discernable and separated from the underlying healing tendon.
Biochemical composition
Based on all biochemical parameters (normalized to internal sham values), GDF-5 deficient mice exhibited a delay of 5-9 days in attaining peak values of normalized DNA, glycosaminoglycan (GAG), and hydroxyproline (Hypro) levels in healing Achilles tendon tissue when compared to control littermates (Fig. 1). Statistically, the time-dependent changes in all three biochemical measures were significantly affected by the GDF-5 mutation (p < 0.001 for the interaction of genotype and time). Control tissue reached a peak value of DNA content at 5 days after surgery, whereas mutant animals did not reach a peak in cellularity until Day 14 (Fig. 1A and B). In addition, the magnitude of the peak in healing/sham DNA content was approximately 30% lower in mutants compared to controls. Time-dependent changes in GAG content were similar: control animals reached a peak in healinghham G A G at 5 days post-op, whereas in GDF-5 deficient animals this peak was delayed until 11 days post-op (Fig. 1C and D). Collagen content peaked at Day 9 after surgery in control animals, but was delayed until Day 14 in mutant animals (Fig. 1E and F). In addition, the magnitude of peak hydroxyproline (healingkham) levels in GDF-5 4- mice was more than twice that of control animals. Histological characterization
Based on histological appearance, GDF-5 deficient Achilles tendons also exhibited a delay in peak areal cell density and collagen reorganization (qualitatively assessed) (Fig. 2). At 3 days after surgery, numerous cells were evident at the repair site of control animals (Fig. 2C). The presence of some fatty tissue within the bulk of the repair site was also readily apparent. Mutant repair tissue contained fewer repair cells which appeared to be clustered in an uneven distribution throughout the repair site (Fig. 2D). Overall, the tissue was highly disorganized in the GDF-5 deficient animals, with an abundance of fatty cells present within the midsubstance of the tendon regenerate. By Day 7 post-op, the repair tissue of control animals continued to increase in cell density, and the cells began to reorient themselves along the long axis of the tendon (Fig. 2G). Revascularization was readily apparent. By contrast, mutant tissue was still highly disorganized (Fig. 2H): while the cell density had increased from Day 3, it was still reduced compared to control tissue, and a large number of fatty cells were still evident. At 2 weeks, mutant tissue began to appear more organized, with some realignment of cells along the long axis of the tendon (Fig. 2L). Fat content appeared to be significantly reduced, but was still evident, and cell density had increased relative to Day 7. Overall, the appearance of the mutant tissue on Day 14 resembled that of control tissue of Day 7
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Fig. 1. Biochemical composition of healing Achilles tendons in cohtrol (A, C, E) and GDF-5 deficient (B, D, F) mice. Data represent repair side values normalized to sham side values of DNA (A and B), glycosaminoglycan (C and D), and hydroxyproline as an indicator of total collagen (E and F). (Data are depicted as mean fSD.) The time-dependent changes in all biochemical content indicators were significantly affected by genotype.
(Fig. 2G), although with a qualitatively greater abundance of fatty inclusions. By 6 weeks after surgery, both the control and mutant tissue exhibited a marked improvement in the histologic appearance of the tissue (Fig. 2 0 and P). Although cell density appeared qualitatively higher than that of sham-operated tissue, control animal histology was otherwise similar to the sham side in appearance. The organization of mutant tissue at this stage was dramatically improved and resembled that of control animal repair tissue with the noted exception that a significant number of fatty cells still remained within the tissue proper (Fig. 2P). No evidence of cartilage or bone was detected in any of the mutant or control tendon samples at any of the time-points examined. Because of the qualitative trends observed in the healing tissue, the degree of vasculature and fatty cells were further quantified in mutant and control tissue using point-counting techniques. GDF-5 deficient tissue exhibited a delay of 1 week in revascularization compared to controls, with a statistically significant interaction between genotype and time (Fig. 3A). In ad-
dition, GDF-5 deficient tissue contained a significantly greater proportion of fatty tissue compared to controls throughout the time-period examined (Fig. 3B). Sham values of the percentage of vascular and fatty tissue were not significantly different between mutants and controls (not shown). Ultr-astructurcrlanalyses The time-dependent changes in collagen fibril ultrastructure during Achilles tendon healing were also noticeably affected by GDF-5 deficiency (Figs. 4 and 5). During the first 2 weeks of healing, mutant tendons had a lower collagen fibril area fraction (the percentage of a given area occupied by collagen) (Fig. 5A). By 2 weeks post-op, these differences were no longer apparent. The difference in fibril area fraction was due primarily to smaller fibrils at Days 5 and 7 (Fig. 4): by Day 14, GDF-5 deficient mice had a collagen area fraction comparable to that of control mice, and fibril diameter distributions were also comparable (Fig. 5C and D-F). The approximate 2-week delay in changes in ultrastructural
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Fig. 2. Healing and sham operated Achilles tendons from GDF-5 deficient and control animals at 3 (A-D), 7 (E-H). 14 (I- L), and 42 (M-P) days post-tenotomy. For each time-point shown, top panels depict sham tendons, and bottom panels depict repair tissue, while left panels are from control animals and right panels are from GDF-5 deficient animals.
characteristics is consistent with the observed 1-2 week delay in changes in biochemical composition and tissue organization at the light microscope level.
repair tendons failed in the mid-substance, whereas only 50% of control tendons did.
Mechanical behavior
Discussion
Structurally, GDF-5 deficient healing tissue (normalized to individual sham values) was significantly weaker and more compliant than control tissue at 5 weeks, but by 12 weeks, no significant difference was detected between genotypes in either structural strength or stiffness (Fig. 6A and B). Because cross-sectional area measurements were not obtained, it cannot be definitively concluded that structural differences were solely due to material property (rather than geometric) differences, although by 5 weeks post-op, no distinction could be made between healing and sham tendons at the time of dissection based on gross appearance. Consistent with our previous findings in intact GDF-5 -1- Achilles tendons, the majority of all mutant sham tendons failed in the tendon mid-substance, whereas the majority of control sham tendons failed via avulsion from the calcaneus. At both 5 and 12 weeks, 100% of GDF-5 -1-
The aim of this study was to test the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, compositional, and ultrastructural analyses on healing tendons from 8-week-old male brachypodism mice, we found that GDF-5 deficient animals exhibited a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Structurally, GDF-5 deficient repair tissue was still significantly weaker and more compliant (after normalization to internal sham values) than control tissue at 5 weeks after surgery, but differences in structural parameters were no longer detected at 12 weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating early tendon repair. One of the most interesting effects of GDF-5 deficiency observed in this study was the delay in achieving
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Fig. 3. (A) Time-dependent change in the percentage of healing Achilles tendon tissue occupied by vasculature in GDF-5 deficient (- - -) vs. control animals (-). (B) Time-dependent change in the percentage of healing Achilles tendon tissue occupied by fat in GDF-5 deficient (- - -) vs. control animals (-). (Data are depicted as mean f SD.) The time-dependent changes in both parameters were significantly affected by genotype.
peak cell content (as measured by DNA content) at the Achilles tendon repair site. One interpretation of this delay is that GDF-5 may be affecting cell recruitment to the repair site. Indeed, it has been demonstrated that GDF-5 is capable of inducing the migration of certain cell types in a chemotactic (directional) manner [39]. Whether GDF-5 is capable of inducing a chemotactic response in the tenocytes and fibroblasts involved in tendon repair has yet to be examined. The delay in peak DNA content seen in GDF-5 deficient repair tissue could also be explained by altered cell recruitment if the ability of cells to migrate to the repair site is somehow affected by GDF-5 deficiency. An essential component of cell migration is cell adhesion. Duke and Elmer [lo] examined the adhesive characteristics of limb mesenchymal cells from 12-day old normal and GDF-5 deficient, brachypod mouse embryos in rotation culture and found that GDF-5 deficient cells were more adhesive than control mesenchyme cells. In a subsequent study, these same authors used fusion studies of normal and mutant limb-bud fragments to test adhesion, and again found evidence to support the conclusion that limb bud mesenchymal cells from 12day old GDF-5 deficient mouse embryos were more
Fig. 4. Representative electron microscope images from GDF-5 deficient and control mice. (A and B) Collagen fibrils from sham (A) and 5-day post-op (B) tissue of a control animal. Panels C and D show comparable images from a GDF-5 deficient mouse. Bar represents approximately 100 nm.
adhesive than control mesenchyme [ 1 11. These studies support a role for GDF-5 in cell adhesion, which, if also a factor in tendon, could serve to delay the early stages of the healing process by slowing the migration of cells to the repair site. An alternative explanation for the observed delay in the early stages of tendon repair could be that, in the absence of GDF-5, cell proliferation may be impaired. In overexpression studies, Francis-West et al. used pulse-labeling of chick cartilage to show that GDF-5 can increase chondrocyte proliferation [ 141. In their in vitro micromass experiments using normal chick mesenchyme, however, exogenous GDF-5 protein was unable to stimulate cell proliferation. Yamashita et al. similarly found that GDF-5 was unable to stimulate cell proliferation (assessed via [3H]thymidine incorporation) in bovine endothelial cells [39]. Consistent with the mitogenic effects of GDF-5 on chondrocytes,
A. Chliuhra et aI. I Journal qf Orthopurdic Reseurch 21 (2003) 826435
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Fig. 5. Ultrastructural parameters from healing Achilles tendons of GDF-5 deficient and control mice. (A) Collagen fibril area fraction. (B) Mean collagen fibril diameter. (C) Number of fibrils per unit area. Error bars indicate the standard deviation of the mean of values obtained from ten photos analyzed per mouse per genotype per timepoint. Panels (D-F) depict collagen fibril distributions for mutant and control animals on Days 5, 7, and 14 post-op (note that differences in scale were required to best capture the distribution of fibril size at each time point shown). Mutant data are shown with dark bars, control data by white bars.
Nakamura et al. used thymidine autoradiography on hp growth plates from 21-day old mice, and demonstrated a significant reduction in the rate of chondrocyte cell division in GDF-5 deficient growth plates [32]. Clearly, the mitogenic potential of GDF-5 appears to depend on (at the very least), cell type. Whether the division of cells involved in tendon repair is affected by GDF-5 remains to be determined. Aspenberg and Forslund have reported that GDF-6 has strong proliferative effects on tendon in vivo [4], thus it is likely that GDF-5 may as well. It would be interesting to determine whether transfecting tendon cells with GDF-5, 6, or 7 could rescue the effects of GDF-5 deficiency in the early stages of the repair process. The results of our study might also be interpreted to suggest that GDF-5 is involved in cell differentiation at the repair site. It is well known that pluripotent cells of musculoskeletal origin are capable of differentiating along multiple pathways, including cartilage, bone, muscle, fat, and fibrous tissue such as tendon and ligament. One striking feature of the healing process in GDF-5 deficient Achilles tendons is the greater abundance of adipocytes in the healing tissue at all time
points examined in GDF-5 deficient animals. These observations present the intriguing possibility that, in the absence of GDF-5, some portion of the repair cell population may be receiving abnormal signals to differentiate into fat. Interestingly, in the steady-state situation of non-operated tendons, no appreciable fat is present within the tendon proper in either mutants or controls. Although the amount of fat present in mutant tissue declines with post-operative time, there is still significantly more present in mutant tissue compared to controls at 6 weeks post-op. It is likely that the presence of these fatty inclusions serve to weaken the material substance of the tendon, thereby contributing to the lower structural strength in GDF-5 deficient tendons (normalized to sham values) at 5 weeks of healing. Although material properties could not be calculated because cross-sectional area measurements were not made, the gross appearance of the operated and sham Achilles tendons was indistinguishable in both groups of animals at 5 weeks. Histologic sections were not taken at 12 weeks post-op, but we hypothesize that the mutant tissue would no longer contain significantly more fatty cells than control tissue at this timepoint, based on the
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Fig. 6. Normalized (repair sidekham side) values of structural strength (A) and stiffness (B) in control (white bars) and GDF-5 deficient (dark bars) Achilles tendons at 5 and 12 weeks post-op. (Data are depicted as mean C! SD.)
comparable structural behavior of GDF-5 deficient and control Achilles at 12 weeks. One final possibility that could explain the short-term delay in healing observed in GDF-5 deficient tendons is the known angiogenic capability of this molecule. Our results demonstrate that the absence of GDF-5 significantly affects the time-course of revascularization in our Achilles tendon model, with a delay of approximately 1 week seen in bp mice. These data are consistent with the work of Yamashita et al. [39] who showed that GDF-5 induced angiogenesis in vivo in both chick chorioallanotoic membrane and rabbit cornea assays. These data thus suggest that the observed effects of GDF-5 deficiency on tendon healing could be explained in part by a decreased angiogenic capacity in these animals. One of the interesting results of this study is the collagen fibril size disparity observed between mutant and control tendons during the first 2 weeks of Achilles tendon repair. It is well known that the small leucine rich proteoglycans decorin, lumican, and fibromodulin help regulate collagen fibril diameter. Collagen V has also been shown to play a similar role. Our data suggest
833
the possibility that GDF-5 4- animals may have altered levels of such fibril size regulating proteins, although we did not look beyond bulk compositional measures in the present study. Despite regaining comparable fibril size by 2 weeks post-op, mutant tendons still exhibit significantly lower structural strength and stiffness 5 weeks after surgery. As collagen cross-linking is known to affect tendon strength, it will be important to examine this parameter in future studies, in addition to the expression levels of molecules known to serve cross-linking functions for collagen type I such as the FACIT collagen XII. Despite the clear effect of GDF-5 deficiency on the time-course of Achilles tendon healing shown in the present study, several limitations exist. First, the age of the animals was 8 weeks, which represents young adolescents. Many mutant mouse strains as well as transgenic mouse lines exhibit delayed development and maturation. Consequently, it is possible that mutants and controls, although the same age, could be at different maturational stages, and this could conceivably affect the time-course of repair. To rule out this possibility, older animals (e.g. older than 16 weeks of age) would have to be examined. A second limitation of this study is inherent to the bruchypodism mouse model. Many of these animals exhibit a mild degree of knee joint subluxation which can affect their gait, resulting in a more crouched posture. It is important to note, however, that mobility is not impaired in hp mice, and these animals are just as active as their control littermates, both before and after surgery. No differences were noted in the time it took for mutant animals to begin walking after surgery compared to their littermates. Nonetheless, with this animal model it is not possible to separate the effects of possible altered loading superimposed on the effect of GDF-5 deficiency. As with all knockout mouse models or naturally occurring mutations, it is also possible that the absence of one growth factor may result in over-compensation of other, related family members. For example, it is not known whether GDF-6 and/or GDF-7 expression is altered in the absence of GDF-5. In fact, to date, no one has reported on the expression (or lack of expression) of GDFs 5,6, or 7 during normal tendon repair. Additional, more minor limitations with the present study include the fact that no specific markers were used to identify vasculature or fat; rather, these tissues were identified based solely on histologic appearance. Further, although bulk biochemical composition data such as GAG and hydroxyproline provide valuable information, such aggregate data do not allow us to determine whether synthesis, degradation, or both have been affected. Lastly, the lack of determination of cross-sectional geometric data (or equivalent gravimetric measures such as Hypro/gage length) prevent us from determining whether the observed structural differences in strength and stiffness are due to material property
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differences (as we hypothesize), size differences, or some combination of the two. In conclusion, the results of this study demonstrate for the first time that GDF-5 deficiency in 8-week-old male mice leads to a delay in the Achilles tendon repair process of 1-2 weeks, based on compositional, histological, and ultrastructural measures. These data are consistent with previously documented roles for GDF-5 in cell recruitment, differentiation, adhesionlmigration, proliferation, as well as angiogenesis. In combination with prior studies from other investigators in which GDF-5 has been shown to augment tendon repair [3], our data suggest that GDF-5 may play an important role in tendon healing and might someday be of clinical use to enhance the repair process of tendon injuries in humans.
Acknowledgements
We would like to thank Dr. Rashard Dacus, M.D. for his assistance with the histomorphometric analyses, and Dr. Gary Balian, Ph.D. for helpful discussion. This work was funded in part by a grant from the NIH to BM (AR45828).
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[38] Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, et al. Ectopic induction of tendon and ligament in rats by growth/differentiation factors 5 , 6, and 7, members of the TGF-fi gene family. J Clin Invest 1997;100(2):321-30. [39] Yamashita H. Shimizi A, Kato M, Nishitoh H, Ichijo H, Hanyu A, et al. Growthldifferentiation factor-5 induces angiogenesis in vivo. Exp Cell Res 1997;235:218-26.