GDF-5 deficiency in mice alters the ultrastructure, mechanical properties and composition of the Achilles tendon.

Acromesomelic dysplasia of the Hunter-Thompson and Grebe types are rare human disorders based on growth/differentiation factor (GDF)-5/CDMP-1 genetic mutations. Numerous skeletal abnormalities are present in these individuals, including shortened limb bones and severe dislocations of the knee. In the GDF-5 deficient brachypodism mouse, similar, although less severe, phenotypes are observed. It is unknown whether the joint dislocations observed in these disorders are due to a defect in the original formation of joints such as the knee, or to abnormalities in the tendons and ligaments themselves. We hypothesized that tendons from GDF-5 deficient mice would exhibit altered composition, mechanical properties, and ultrastructure when compared with heterozygous control littermates. GDF-5 deficient Achilles tendons were structurally weaker than controls, and structural strength differences appeared to be caused by compromised material properties: after normalizing by collagen per unit length, mutant tendons were still 50% weaker (P < 0.0001) and 50% more compliant (P < 0.001) than controls. Despite comparable levels of skeletal maturity in the two cohorts, the majority of mutant tendon failures occurred in the mid-substance of the tendon (64% of all failures), whereas the majority of control failures occurred via avulsion (92% of all failures). Mutant Achilles tendons contained 40% less collagen per microgram of DNA when compared to controls (P = 0.004). No significant difference in glycosaminoglycan (GAG)/DNA was detected. Ultrastructural analyses indicated a slight trend toward increased frequency of small diameter (30-100 nm) collagen fibrils in the mutant Achilles. Our findings suggest that increased tendon and ligament laxity may be the cause of the joint dislocations seen in patients with Hunter-Thompson and Grebe type dysplasia, rather than developmental abnormalities in the joints themselves.     

GDF-5 deficiency in mice delays Achilles tendon healing.

The aim of this study was to examine the role of one of the growth/differentiation 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 -/- 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, glycosaminoglycan 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, migration/adhesion, differentiation, proliferation, and angiogenesis.     

Sexual dimorphism in the effect of GDF‐6 deficiency on murine tendon

Three members of the growth/differentiation factor (GDF) subfamily of bone morphogenetic proteins (BMPs), GDFs‐5, ‐6, and ‐7, have demonstrated the potential to augment tendon and ligament repair. To gain further insight into the in vivo role of these molecules, previous studies have characterized intact and healing tendons in mice with functional null mutations in GDF‐5 and ‐7. The primary goal of the present study was to perform a detailed characterization of the intact tendon phenotype in 4‐ and 16‐week‐old male and female GDF6?/? mice and their +/+ littermates. The results demonstrate that GDF6 deficiency was associated with an altered tendon phenotype that persisted into adulthood. Among males, GDF6?/? tail tendon fascicles had significantly less collagen and glycosaminoglycan content, and these compositional differences were associated with compromised material properties. The effect of GDF6 deficiency on tendon was sexually dimorphic, however, for among female GDF6?/? mice, neither differences in tendon composition nor in material properties were detected. The tendon phenotype that was observed in males appeared to be stronger in the tail site than in the Achilles tendon site, where some compositional differences were present, but no material property differences were detected. These data support existing in vitro studies, which suggest a potential role for BMP‐13 (the human homologue to GDF‐6) in tendon matrix modeling and/or remodeling. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1603–1611, 2009     

Effect of GDF‐7 deficiency on tail tendon phenotype in mice

The subfamily of growth/differentiation factors (GDFs) known as GDFs 5, 6, and 7 appears to be involved in tendon maintenance and repair, although the precise nature of this role has yet to be elucidated. The aim of the present study was to examine the role of GDF‐7 in tendon maintenance by studying tail tendon fascicle gene expression, composition, and material property strain rate dependency in 16‐week‐old male and female GDF‐7 deficient mice. GDF‐7 deficiency did not affect the biochemical composition of tail tendon fascicles, nor did it significantly affect the tensile material properties obtained at either slow (5%/s) or fast (50%/s) strain rates. Further, no difference was found between genotypes in the strain rate sensitivity of any tensile material property. Consistent with the compositional analyses, QRT‐PCR data did not reveal any differences of twofold or greater in the gene expression levels of collagens I, III, V, nor in the proteoglycans decorin, fibromodulin, lumican, biglycan, versican, or aggrecan. Gdf5 expression was upregulated twofold in GDF‐7 deficient tail tendons, and Bmp7 expression was downregulated twofold. No notable differences in expression levels for Bmp1‐6 or Gdf6 were detected. GDF‐5 protein levels were 50% higher in GDF‐7 deficient tail tendon compared to wild type tail tendon. The results of this study support the intriguing possibility that compensation by Gdf‐5 may be at least in part responsible for the absence of a strong phenotype in GDF‐7 deficient mice. © 2008 Orthopaedic Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:834–839, 2008     

Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5

The present study examined the effect of ADAMTS5 (TS5) knockout on the properties of murine flexor digitorum longus (FDL) and Achilles tendons. FDL and Achilles tendons were analyzed using biomechanical testing, histology, and immunohistochemistry; further characterization of FDL tendons was conducted using transmission electron microscopy (collagen fibril ultrastructure), SDS-PAGE (collagen content and type), fluorescence-assisted carbohydrate electrophoresis for chondroitin sulfate and hyaluronan, and Western blotting for aggrecan, versican, and decorin abundance and distribution. FDL tendons of TS5(-/-) mice showed a 33% larger cross-sectional area, increased collagen fibril area fraction, and decreased material properties relative to those of wild type mice. In TS5(-/-) mice, aggrecan accumulated in the pericellular matrix of tendon fibroblasts. In Achilles tendons, cross-sectional area, stress relaxation, and structural properties were similar in TS5(-/-) and wild type mice; however, the TS5(-/-) tendons exhibited a higher tensile modulus and a weakened enthesis. These results demonstrate that TS5 deficiency disturbs normal tendon collagen organization and alters biomechanical properties. Hence, the role of ADAMTS5 in tendon is to remove pericellular and interfibrillar aggrecan to maintain the molecular architecture responsible for normal tissue function.     

Mechanical Load and BMP Signaling During Tendon Repair: A Role for Follistatin?

Healing of the rat Achilles tendon is sensitive to mechanical loading, and the callus strength is reduced by 3/4 after 14 days, if loading is prevented. Exogenous GDFs stimulate tendon healing. This response is influenced by loading: without loading, cartilage and bone formation is initiated. This implies BMP signaling is crucial during tendon healing and influenced by mechanical loading. We therefore asked if mechanical loading influences the gene expression of the BMP signaling system in intact and healing tendons, and how the BMP signaling system changes during healing. The genes were four BMPs (OP-1/BMP-7, GDF-5/CDMP-1/BMP-14, GDF-6/CDMP-2/BMP-13, and GDF-7/CDMP-3/BMP-12), two receptors (BMPR1b and BMPR2), and the antagonists follistatin and noggin. The Achilles tendon was transected in rats and left to heal. Half of the rats had one Achilles tendon unloaded by injection of Botox in the calf muscles. Ten tendons were analyzed before transection and for each of four time points. All genes except noggin were expressed at all time points, but followed different patterns during healing. Loading strongly decreased the expression of follistatin, which could lead to increased signaling. The BMP system appears involved in tendon maintenance and healing, and may respond to mechanical loading.     

Complete stress shielding of the Achilles tendon: ultrastructure and level of interleukin-1 and TGF-β

Few studies emphasize the collagen metabolism-related cytokines and ultrastructure of the completely stress-shielded Achilles tendon. In this study, we used a rat model with complete stress shielding of the Achilles tendon to observe the changes in the ultrastructure of the Achilles tendon and concentration of IL-1 and TGF-β 3 weeks after stress shielding. The model group comprised 12 male Sprague-Dawley rats. The stress of the Achilles tendon of the left hind limb was shielded through tendon cerclage combined with sciatic nerve transection, and the right served as a normal control. Three weeks later, the ultrastructure of the Achilles tendon was observed under electron microscopy and IL-1 and TGF-β levels were determined by enzyme-linked immunosorbent assay. Compared with the control side, collagen fibrils of the shielded Achilles tendons were irregularly arranged and loose. The number of small-diameter collagen fibrils increased significantly with the decrease of the average diameter of collagen fibrils. At the same time, IL-1 concentrations increased significantly in the model group as compared to that in the control group, but no significant difference was found in TGF-β levels. These results suggest that IL-1 may play an important role in the change of ultrastructure after stress shielding.     

Experimental treatment of tendon injury with heparin

The effect of heparin on the progress of healing in the injured tendon was examined after a standard injury had been created in the Achilles tendons of New Zealand White rabbits. Half of the rabbits were then treated by the intratendinous injection of heparin. The diameters of the injured right hind and the uninjured left hind Achilles tendon and overlying skin were measured using vernier calipers before injury and at one month after injury. At this time histological analysis was carried out on all Achilles tendons and tendon dry weight, collagen concentration and DNA concentration were measured. Orientation of collagen fibres was more pronounced in treated than untreated tendons while cellularity and blood vessel density were less pronounced. Analysis by Students t-test demonstrated a statistically significant difference between treated and untreated tendons in terms of leg diameter (p less than 0.005), dry weight (p less than 0.01) and DNA concentration (p less than 0.01).     

The Achillon achilles tendon repair: is it strong enough?

BACKGROUND: Open repair of the Achilles tendon is associated with wound breakdown, infection and percutaneous methods risk sural nerve injury. The Achillon mini-incision technique can reduce these risks and may provide the opportunity for early active rehabilitation. The aim of this study was to compare the strength of the Achillon method with the commonly used Kessler method and to assess whether the strength of the repair was related to tendon diameter. MATERIALS AND METHODS: Simulated ruptures in sheep Achilles tendons were repaired using either the Achillon method or a two-strand Kessler technique with a No. 2 Ticron suture (Tyco Healthcare, UK). Each tendon diameter was measured, and matched for both groups. Specimens were loaded to failure using an Instron tensile testing machine (Instron Limited, UK). RESULTS: Mean load to failure for the Achillon repair was 153 N+/-60 (range, 65 to 270), and the mean load to failure for the Kessler Repair was 123 N+/-24 (range, 75 to 150). This difference was not statistically significant (p=0.21). There was a statistically significant higher mean load to failure for wider tendons repaired by the Achillon method (p=0.05), however mean load to failure was not related to tendon width in Kessler repairs (p=0.23). CONCLUSION: This is the first study to compare these two methods of repair. The Achillon repair has comparable tensile strength to the Kessler Repair. CLINICAL RELEVANCE: The Achillon repair appears to be a biomechanically sound method of repair for the acutely ruptured Achilles tendon.     

Sex matters in the establishment of murine tendon composition and material properties during growth

The existence of sex‐based differences in tendon and ligament injury rates has led investigators to test the hypothesis that sex plays a significant role in modulating tendon and ligament composition and material properties. To date, no studies have attempted to characterize how such differences develop during the course of normal tissue maturation and growth. Thus, the primary aim of the present study was to use a murine model to test the hypothesis that sex‐based differences in the normal age‐related development of tendon composition and material properties exist by assessing these parameters in the Achilles and tail tendons from 4‐, 6‐, 9‐, 12‐, and 15‐week‐old male and female C57Bl/6J mice. Despite significantly lower levels of total collagen content in females subsequent to sexual maturity (p < 0.0001), as well as a significant effect of sex on glycosaminoglycan content (p < 0.0001), Achilles tendon elastic modulus was not compromised in females. Female Achilles tendons did exhibit a significantly higher failure strain (p = 0.0201) and strain energy density (p = 0.0004) than did males, as well as a trend toward higher ultimate strength (p = 0.0556). In contrast to the high load‐bearing environment of the Achilles tendon site, sex did not have a statistically significant effect on any compositional or material property in the low load‐bearing tendon fascicles of the tail. These data support recent studies by others, which suggest that male and female tendons have a differential adaptational response to their local mechanical loading environment. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:631–638, 2010     

Altered hypertrophic chondrocyte kinetics in GDF-5 deficient murine tibial growth plates.

The growth/differentiation factors (GDFs) are a subgroup of the bone morphogenetic proteins best known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling proteins, GDF-5, exhibit numerous skeletal abnormalities, including shortened limb bones. The primary aim of this study was determine whether GDF-5 deficiency would alter the growth rate in growth plates from the long bones in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-5 -/- mice and control littermates were examined. GDF-5 deficiency resulted in a statistically significant reduction in growth rate (-14%, p=0.03). The effect of genotype on growth rate was associated with an altered hypertrophic phase duration, with hypertrophic cells from GDF-5 deficient mice exhibiting a significantly longer phase duration compared to control littermates (+25%, p=0.006). These data suggest that one way in which GDF-5 might modulate the rate of endochondral bone growth could be by affecting the duration of the hypertrophic phase in growth plate chondrocytes.     

Macrophage depletion reduces cell proliferation and extracellular matrix accumulation but increases the ultimate tensile strength of injured Achilles tendons

Macrophages are present in large numbers and display specific and distinct phenotypes during the various phases of tissue repair. However, their role following tendon injury and during repair has never been investigated. We injected C57BL/6 mice daily for 4 days with liposome‐encapsulated clodronate to deplete circulating monocytes/macrophages. Placebo mice were injected with PBS. The left Achilles tendons of the mice were transversely sectioned and sutured using the 8‐strand technique. Macrophage accumulation and cell proliferation were significantly lower in the tendons of clodronate‐treated mice than in those of PBS‐treated mice on days 3 and 7 post‐injury. TGF‐β1 staining was significantly more intense in the tendons of PBS‐treated mice on day 7 post‐injury. Edema and the dry mass of the Achilles tendons were also higher in the PBS‐treated mice on days 7 and 14 post‐injury. No differences in absolute strength and stiffness were observed, but Young's modulus and maximal stress were significantly greater for tendons from the clodronate‐treated mice than those from PBS‐treated mice after 14 days of tendon repair. Overall, our findings showed that macrophages promote cell proliferation and extracellular matrix accumulation but their presence leads to inferior ultimate tensile strength of the Achilles tendons. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:279–285, 2014.     

A new clamping technique for biomechanical testing of tendons in small animals. aprobst@uni-muenster.de.

When biomechanical properties of tendons are studied, the technique of clamping the tendons in the testing machine presents a methodological challenge, especially when murine tendons are examined. These short tendons tend to rupture at the transition line to the fixation, leading to false interpretations. Therefore a new clamping technique for investigation of healthy murine Achilles tendons (n = 50) was developed, in which the intramuscular tendon fibers were fixed between two paper strips and the calcaneus was wedged into a conical slot in a wooden block and then mounted in the testing machine (n = 20). This technique was compared with the conventional clamping technique that fixes both ends of the tendon by clamps (n = 15) and an earlier described method that used glue or plastic cement for the fixation of the intramuscular tendon fibers and calcaneus in the testing machine (n = 15). When tested by the new clamping technique, 17 tendons ruptured intratendinously at a mean tensile force of 8.4 +/- 1.1 N. Three Achilles tendons (17%) tore at the site of paper fixation and had to be excluded from investigation. Data from 73% of the measurements fixed by gluing had to be excluded because slippage of the proximal tendon fibers and contamination of the tendon with glue occurred. All the conventionally clamped tendons ruptured at the site of fixation at a mean tensile force of 6.1 +/- 2.3 N (p < .05). This was 30% lower than with the new clamping technique. Thus, the newly developed clamping technique enables investigators to obtain more valid biomechanical studies of the murine Achilles tendon.     

Accelerated hypertrophic chondrocyte kinetics in GDF‐7 deficient murine tibial growth plates

The Growth/Differentiation Factors (GDFs) are a subgroup of the Bone Morphogenetic Proteins (BMPs) well known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling molecules, GDF‐5, have recently been shown to exhibit a decreased rate of endochondral bone growth in the proximal tibia due to a significantly longer hypertrophic phase duration. GDF‐7 is a related family member, which exhibits a high degree of sequence identity with GDF‐5. The purpose of the present study was to determine whether GDF‐7 deficiency also alters the endochondral bone growth rate in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5‐week‐old female GDF‐7 ?/? mice and wild type control littermates were examined. GDF‐7 deficiency resulted in a statistically significant increase in growth rate (+26%; p = 0.0084) and rate of cell loss at the chondrosseous junction (+25%; p = 0.0217). Cells from GDF‐7 deficient mice also exhibited a significantly shorter hypertrophic phase duration compared to wild type controls (?27%; p = 0.0326). These data demonstrate that, in the absence of GDF‐7, the rate of endochondral bone growth is affected through the modulation of hypertrophic phase duration in growth plate chondrocytes. These findings further support a growing body of evidence implicating the GDFs in the formation, maturation, and maintenance of healthy cartilage. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:986–990, 2008     

Effects of low-voltage microamperage stimulation on tendon healing in rats

STUDY DESIGN: Randomized controlled prospective experimental study. OBJECTIVES: To examine the effects of transcutaneous low-voltage microamperage stimulation (LVMAS) on the mechanical strength of Achilles tendon repair in rats at 4 weeks after injury. BACKGROUND: Understanding the effect of LVMAS on the healing of injured tendons is hampered by the lack of related experimental studies, especially from the aspect of biomechanical outcome measures. METHODS AND MEASURES: Fourteen 3-month-old male Sprague-Dawley rats received surgical transection to the medial portion of their right Achilles tendon. The rats were divided into a LVMAS group (n = 7) and control group (n = 7). From day 6 postsurgery onwards, the LVMAS group received daily treatment of transcutaneous LVMAS (2.5 V, 100 microA/cm2, 10 pulses per second, positive current) for a total of 22 sessions, while the control group received placebo LVMAS by the same investigator during that period. On day 31, the Achilles tendons were harvested for biomechanical testing for load relaxation, stiffness, and ultimate tensile strength along the longitudinal direction. RESULTS: The normalized Achilles tendon ultimate tensile strength of the LVMAS group (mean +/- SD, 110.5% +/- 25.0%) was higher than that of the control group (75.3% +/- 20.8%) (P = .014), but no significant difference was found in normalized stiffness and load relaxation between the 2 groups (P = .239 and .350, respectively). CONCLUSION: The results of this study suggest that the administration of transcutaneous LVMAS could improve healing and consequently the tensile strength of partially transected Achilles tendons of rats at 4 weeks after injury.     

The effects of growth and differentiation factor 5 on bone marrow stromal cell transplants in an in vitro tendon healing model

The effects of growth differentiation factor-5 (GDF-5) and bone marrow stromal cells (BMSCs) on tendon healing were investigated under in?vitro tissue culture conditions. BMSCs and GDF-5 placed in a collagen gel were interpositioned between the cut ends of dog flexor digitorum profundus tendons. The tendons were randomly assigned into four groups: 1) repaired tendon without gel; 2) repaired tendon with BMSC-seeded gel; 3) repaired tendon with GDF-5 gel without cells; and 4) repaired tendon with GDF-5 treated BMSC-seeded gel. At 2 and 4 weeks, the maximal strength of repaired tendons with GDF-5 treated BMSCs-seeded gel was significantly higher than in tendons without gel interposition. However, neither BMSCs nor GDF-5 alone significantly increased the maximal strength of healing tendons at 2 or 4 weeks. These results suggest that the combination of BMSCs and GDF-5 accelerates tendon healing, but either BMSCs or GDF-5 alone are not effective in this model.     

Achilles tendon rupture repair: biomechanical comparison of the triple bundle technique versus the Krakow locking loop technique

This study was designed to compare the tensile strength of ruptured Achilles tendons repaired using either the triple bundle technique or the Krakow locking loop technique. Eight pairs of fresh frozen cadaveric Achilles tendons were harvested. A simulated "Achilles tendon rupture" was created 4 cm from the calcaneal insertion in all sixteen tendons by transversely cutting the tendon with a scalpel. One Achilles tendon "rupture" of a pair was repaired using the triple bundle technique, while the other tendon of the pair was repaired using the Krakow locking loop technique. Then, using a servohydraulic testing machine, each tendon was tested to failure in tension at a displacement rate of 2.54 cm/sec. The average rupture load for the triple bundle technique was 453 N (range 397 n 549 N), while the average rupture load for the Krakow locking loop technique was 161 N (range 121 n 216 N). This difference in averages represents a statistically significant superiority of 2.8 to 1 (p < 0.001) in favor of the triple bundle technique.     

A biomechanical study of Achilles tendon repair augmentation using GraftJacket matrix

BACKGROUND: Ruptured Achilles tendons benefit from primary repair by decreasing re-rupture rates and allowing earlier range of motion. A stronger repair might allow for more aggressive rehabilitation decreasing postoperative stiffness, calf atrophy, and repair site gapping. The hypothesis of this study was that human dermal allograft augmentation of an Achilles repair would significantly increase repair strength and stiffness. This study evaluated strength and stiffness of an Achilles tendon repair augmented with a human dermal allograft (GraftJacket). MATERIALS AND METHODS: Eight matched pairs of human cadaver legs were used. Simulated Achilles tendon ruptures were created 4 cm proximal to the calcaneal insertion. All tendons were repaired with a Krackow locking loop stitch. One of each matched pair was augmented with GraftJacket. Each construct was pre-loaded at 10 N and cyclically loaded (20 cycles) from 2 N to 30 N at a rate of 5 N/sec on an Instron machine. This was followed by testing to failure at a displacement rate of 6 mm/sec. RESULTS: The ultimate failure load in the control group was 217 N +/- 31 compared to 455 N +/- 76.5 in the GraftJacket group (p < 0.001). The mean stiffness in the control group was 4.3 +/- 0.83 N/mm which was significantly less than the 12.99 +/- 5.34; N/mm in the GraftJacket group (p = 0.002). CONCLUSION: The augmentation of an Achilles tendon repair with GraftJacket significantly increased repair strength and stiffness. CLINICAL SIGNIFICANCE: These findings suggest that a GraftJacket augmented Achilles tendon repair could acutely withstand a more aggressive rehabilitation program, potentially decreasing ankle stiffness and allowing earlier return to full activities.