Insulin-dependent diabetes impairs the inflammatory response and delays angiogenesis following Achilles tendon injury

Although impaired wound healing associated with type 1 diabetes mellitus has been well studied in skin tissue, the influence of this metabolic disorder on tendon healing and recovery has not been extensively investigated. Because tendons are known to have limited repair potential, we studied the tendon-healing process by using a diabetic rat tendonitis model. We tested the hypothesis that diabetes influences the inflammatory response, cell proliferation, and angiogenesis in injured Achilles tendons. Diabetes was induced by injecting streptozotocin at 45 mg/kg body wt. Non-diabetic rats as well as diabetic and insulin-treated diabetic animals were then injected with collagenase. The accumulation of inflammatory cells was quantified in transversal sections of Achilles tendon by using immunohistochemical staining at days 0, 1, 3, 7, 14, and 28 posttrauma. The number of proliferative cells and the extent of neovascularization was also quantified in the paratenon and the core of the tendon at days 0, 3, 7, 14, and 28 posttrauma. Relative to nondiabetic and insulin-treated diabetic animals, the numbers of accumulated neutrophils and ED1(+) and ED2(+) macrophages in diabetic rats decreased by 46, 43, and 52%, respectively, in the first 3 days after injury compared with levels in nondiabetic and insulin-treated diabetic animals. The density of newly formed blood vessels decreased by 35 and 29% in the paratenon and the core of tendon, respectively, at days 3 and 7 after injury. Lastly, the concentration of proliferative cells decreased by 34% in the paratenon at day 7 posttrauma in injured tendons from diabetic rats relative to nondiabetic rats. These results indicate that alterations in inflammatory, angiogenic, and proliferative processes occurred in the diabetic state that might eventually perturb tendon healing and remodeling.     

Inflammation and tendon healing

Tendons are extracellular matrix rich structures allowing the transmission of forces generated by skeletal muscles to bones in order to produce movements. Some intrinsic characteristics of tendons, namely hypovascularity and hypocellularity, may explain their slow rate of healing. A growing body of evidence suggests that the inflammatory process, essential for pathogen clearance and injury scavenging, may play opposite functions in tendon healing. For instance, inflammation can lead to degradation of intact collagen and to viable cell death, thereby increasing the functional deficit and recovery period. Paradoxically, many cellular and subcellular events occurring during the inflammatory response lead to the release of a plethora of growth factors that trigger the healing phase. Prostaglandins are implicated in the inflammatory process and may also contribute to the primary steps of tendon healing. Prolonged administration of non steroidal anti-inflammatory drugs (NSAIDs) is a common practice following musculoskeletal injuries. However, there is no clear consensus on the effect of NSAIDs on tendon healing. This review presents a contemporary vision of the inflammatory process following tendon injury and examines the roles of the constitutive and inducible COX-derived prostaglandins. The effect of COX inhibitors will be addressed and special attention will be taken to describe COX-independent effects of these pharmacological inhibitors. Together, this review is an attempt to guide readers toward a more conscientious use of NSAIDs following tendon injuries.     

Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1−/− versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the Col6a1−/− tendon. In Col6a1−/− tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1−/− tendons compared to controls. An analysis of fibril density (number/μm²) demonstrated a ~ 2.5 fold increase in the Col6a1−/− versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1−/− tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1−/− tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.     

Quadriceps tendon allografts as an alternative to Achilles tendon allografts: a biomechanical comparison

Quadriceps tendon with a patellar bone block may be a viable alternative to Achilles tendon for anterior cruciate ligament reconstruction (ACL-R) if it is, at a minimum, a biomechanically equivalent graft. The objective of this study was to directly compare the biomechanical properties of quadriceps tendon and Achilles tendon allografts. Quadriceps and Achilles tendon pairs from nine research-consented donors were tested. All specimens were processed to reduce bioburden and terminally sterilized by gamma irradiation. Specimens were subjected to a three phase uniaxial tension test performed in a custom environmental chamber to maintain the specimens at a physiologic temperature (37 ± 2 °C) and misted with a 0.9 % NaCl solution. There were no statistical differences in seven of eight structural and mechanical between the two tendon types. Quadriceps tendons exhibited a significantly higher displacement at maximum load and significantly lower stiffness than Achilles tendons. The results of this study indicated a biomechanical equivalence of aseptically processed, terminally sterilized quadriceps tendon grafts with bone block to Achilles tendon grafts with bone block. The significantly higher displacement at maximum load, and lower stiffness observed for quadriceps tendons may be related to the failure mode. Achilles tendons had a higher bone avulsion rate than quadriceps tendons (86 % compared to 12 %, respectively). This was likely due to observed differences in bone block density between the two tendon types. This research supports the use of quadriceps tendon allografts in lieu of Achilles tendon allografts for ACL-R.     

Early voluntary exercise does not promote healing in a rat model of Achilles tendon injury.

Mechanical stress is an important modulator of connective tissue repair. However, the effects on tendon healing are very poorly defined, preventing optimal use of mechanical stress. We hypothesized that early voluntary exercise initially retards tendon repair but results in a faster recovery rate at longer term. Male Wistar rats were injured by a collagenase injection in the Achilles tendon, and exercise was voluntarily performed on a running wheel. We observed the persistent presence of neutrophils in injured tendons of rats that began exercise immediately after the trauma [injured + early exercise (Inj+EEx)]. Early exercise also increased the concentration of ED1(+) macrophages in injured tendons after 3 and 7 days compared with ambulatory injured rats (Inj). Similar results were obtained with the subset of ED2(+) macrophages in the tendon core 3 days after the collagenase injection. Furthermore, collagen content returned to normal values more rapidly in the Inj+EEx tendons than in the Inj group, but this was not associated with an increase in cell proliferation. Surprisingly, Inj+EEx tendons roughly displayed lower stiffness and force at rupture point relative to Inj tendons at day 28. Injured tendons of rats that began exercise only from day 7 had better mechanical properties than those of early-exercised rats 28 days postinjury. We speculate that the persistence of the inflammatory response and undue mechanical loading in the Inj+EEx tendons led to fibrosis and a loss of tendon function.     

Cross-Linking in Collagen by Nonenzymatic Glycation Increases the Matrix Stiffness in Rabbit Achilles Tendon

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174–180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young''s modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young''s modulus of elasticity, and toughness (r values ranging from .61 to .94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from .22 to .84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross-linking is directly associated with the increased matrix stiffness and other mechanical attributes of the tendon.     

Whole body vibration increases area and stiffness of the flexor carpi ulnaris tendon in the rat

Whole body vibration (WBV) has been extensively studied as an anabolic stimulus for bone and muscle. Therapeutic WBV delivers low magnitude, high frequency vibrations to tissues, eliciting biological and structural responses. This study investigated the effect of 0.3G (Peak-to-Peak), 30Hz sinusoidal vibration on intact flexor carpi ulnaris tendons in rats. Experimental rats were subjected to twenty minutes of WBV daily for five days a week for a total of five weeks. The tendon cross-sectional area and the structural properties of the muscle-tendon-bone unit under tensile loading to failure were evaluated. Initial body weights were similar between the groups and the mean change in body weight of the animals of each group did not differ. The cross-sectional area of the tendons of the vibrated animals was found to be 32% greater (P<0.05) than the controls and the structural stiffness of the vibrated tendons was found to be 41% greater (P<0.05) than the controls. For specimens that failed in the midsubstance of the tendon, a trend (P=0.087) for increased ultimate load was observed in the vibrated tendons compared to the controls. No differences in material properties were observed except for the strain to ultimate load, which was reduced 22% in the vibrated group. These initial findings suggest that vibration may serve as an anabolic stimulus to tendon similar to its effects on bone and muscle. These findings are important as they open the potential that low magnitude, high frequency vibration might serve as a means to accelerate tendon healing.     

Organization of collagen bundles during tendon healing in rats treated with L-NAME

The Achilles tendon can support high tension forces and may experience lesions. The damaged tissue does not regenerate completely, with the organization and mechanical properties of the repaired tendon being inferior to those of a healthy tendon. Nitric oxide (NO) plays an important role in wound repair. We have examined the structural reorganization and repair in Achilles tendon after injury in rats treated with the NO synthase inhibitor L-NAME. The right Achilles tendon of male Wistar rats was partially transected. One group of rats was treated with L-NAME (~300 mg/kg per day, given in drinking water) for 4 days prior to tendon sectioning and throughout the post-operative period. Control rats received water without L-NAME. The tendons were excised at 7, 14, and 21 days post-injury and used to quantify hydroxyproline and for mechanical tests. Tendons were also processed for histomorphological analysis by polarized light microscopy, which showed that the collagen fibers were disorganized by day 7 in non-treated and L-NAME-treated rats. In non-treated rats, the organization of the extracellular matrix was more homogeneous by days 14 and 21 compared with day 7, although this homogeneity was less than that in normal tendon. In contrast, in injured tendons from L-NAME-treated rats, the collagen fibers were still disorganized on day 21. Tendons from treated rats had more hydroxyproline but lower mechanical properties compared with those from non-treated rats. Thus, NO modulates tendon healing, with a reduction in NO biosynthesis delaying reorganization of the extracellular matrix, especially collagen. T.C.T. and W.R.N were supported by studentships from CAPES, and S.H. was supported by a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).     

Inflammatory cells do not decrease the ultimate tensile strength of intact tendons in vivo and in vitro: protective role of mechanical loading.

Although inflammatory cells and their products are involved in various pathological processes, a possible role in tendon dysfunction has never been convincingly confirmed and extensively investigated. The goal of this study was to determine whether or not an acute inflammatory process deprived of mechanical trauma can induce nonspecific damages to intact collagen fibers. To induce leukocyte accumulation, carrageenan was injected into rat Achilles tendons. We first tested the effect of leukocyte recruitment on the concentrations or activities of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. Second, we analyzed at the biochemical, histological, and biomechanical levels the impact of leukocyte invasion on tendons. Finally, collagen bundles isolated from rat-tail tendons were exposed in vitro to mechanical stress and/or inflammatory cells to determine if mechanical loading could protect tendons from the leukocyte proteolytic activity. Carrageenan-induced leukocyte accumulation was associated with an increased matrix metalloproteinase activity and a decreased content of tissue inhibitors of matrix metalloproteinases. However, hydroxyproline content and load to failure did not change significantly in these tendons. Interestingly, mechanical stress, when applied in vitro, protected collagen bundles from inflammatory cell-induced deterioration. Together, our results suggest that acute inflammation does not induce damages to intact and mechanically stressed collagen fibers. This protective effect would not rely on increased tissue inhibitors of matrix metalloproteinases content but would rather be conferred to the intrinsic resistance of mechanically loaded collagen fibers to proteolytic degradation.     

ATP modulates load-inducible IL-1beta,COX 2, and MMP-3 gene expression in human tendon cells

Tendon cells receive mechanical signals from the load bearing matrices. The response to mechanical stimulation is crucial for tendon function. However, overloading tendon cells may deteriorate extracellular matrix integrity by activating intrinsic factors such as matrix metalloproteinases (MMPs) that trigger matrix destruction. We hypothesized that mechanical loading might induce interleukin-1beta (IL-1beta) in tendon cells, which can induce MMPs, and that extracellular ATP might inhibit the load-inducible gene expression. Human tendon cells isolated from flexor digitorum profundus tendons (FDPs) of four patients were made quiescent and treated with ATP (10 or 100 microM) for 5 min, then stretched equibiaxially (1 Hz, 3.5% elongation) for 2 h followed by an 18-h-rest period. Stretching induced IL-1beta, cyclooxygenase 2 (COX 2), and MMP-3 genes but not MMP-1. ATP reduced the load-inducible gene expression but had no effect alone. A medium change caused tendon cells to secrete ATP into the medium, as did exogenous UTP. The data demonstrate that mechanical loading induces ATP release in tendon cells and stimulates expression of IL-1beta, COX 2, and MMP-3. Load-induced endogenous IL-1beta may trigger matrix remodeling or a more destructive pathway(s) involving IL-1beta, COX 2, and MMP-3. Concomitant autocrine and paracrine release of ATP may serve as a negative feedback mechanism to limit activation of such an injurious pathway. Attenuation or failure of this negative feedback mechanism may result in the progression to tendinosis.     

Ultrastructural immunolocalization of cartilage oligomeric matrix protein (COMP) in relation to collagen fibrils in the equine tendon.

The structure and organisation of the extracellular matrix, and in particular the axial alignment of type I collagen fibrils, are essential for the tensile strength of tendons. The resident tenocytes synthesize and maintain the composition of the extracellular matrix, which changes with age and maturation. Other components of the extracellular matrix include less abundant collagen types II, III, V, VI, XII, proteoglycans and glycoproteins. Cartilage oligomeric matrix protein (COMP) is an abundant non-collagenous pentameric glycoprotein in the tendon, which can bind to collagen types I and II. The function of COMP in the tendon is not clear, but it may act as a catalyst in fibrillogenesis. Its concentration changes with age, maturation and load. The present study delineates the ultrastructural distribution of COMP and its correlation to collagen fibril thickness in different compartments in two flexor tendons from horses of different ages (foetus, 8 months, 3 years, 12 years). The immunolabeling for COMP was higher in the superficial digital flexor tendon compared with the deep digital flexor tendon and it increased with the age of the animal, with the highest concentration in the 3-year-olds. Fibril diameter differed between age groups and a more homogenous fibril population was found in the fetal tendons. A positive correlation between high COMP immunolabeling and the percentage of small fibrils (<60 nm) were present in the SDFT. COMP immunolabeling was enriched at the gap region of the collagen fibril. In situ hybridization revealed the strongest expression in tendons from the 3-year-old horses whereas there was no expression in foetal tendon.     

Comparison of viscoelastic, structural, and material properties of double-looped anterior cruciate ligament grafts made from bovine digital extensor and human hamstring tendons

Due to ready availability, decreased cost, and freedom from transmissible diseases in humans such as hepatitis and AIDS, it would be advantageous to use tendon grafts from farm animals as a substitute for human tendon grafts in in vitro experiments aimed at improving the outcome of anterior cruciate ligament (ACL) reconstructive surgery. Thus the objective of this study was to determine whether an anterior cruciate ligament (ACL) graft composed of two loops of bovine common digital extensor tendon has the same viscoelastic, structural, and material properties as a graft composed of a double loop of semitendinosus and gracilis tendons from humans. To satisfy this objective, grafts were constructed from each tissue source. The cross-sectional area was measured using an area micrometer, and each graft was then pulled using a materials testing system while submerged in a saline bath. Using two groups of tendon grafts (n = 10), viscoelastic tests were conducted over a three-day period during which a constant displacement load relaxation test was followed by a constant amplitude, cyclic load creep test (first day), a constant load creep test (second day), and an incremental cyclic load creep test (third day). Load-to-failure tests were performed on two different groups of grafts (n = 8). When the viscoelastic behavior was compared, there were no significant differences in the rate of load decay or the final load (relaxation test) and rates of displacement increase or final displacements (creep tests) (p > 0.115). To compare both the structural and material properties in the toe region (i.e., < 250 N) of the load-elongation curve, the tangent stiffness and modulus functions were computed from parameters used in an exponential model fit to the load (stress)-elongation (strain) data. Although one of the two parameters in the functions was different statistically, this difference translated into a difference of only 0.03 mm in displacement at 250 N of load. In the linear region (i.e., 50-75 percent of ultimate load) of the load-elongation curve, the linear stiffness of the two graft types compared closely (444 N/mm for bovine and 418 N/mm for human) (p = 0.341). At failure, the ultimate loads (2901 N and 2914 N for bovine and human, respectively) and the ultimate stresses (71.8 MPa and 65.6 MPa for bovine and human, respectively) were not significantly different (p > 0.261). The theoretical effect of any differences in properties between these two grafts on the results of two types of in vitro experiments (i.e., effect of surgical variables on knee laxity and structural properties of fixation devices) are discussed. Despite some statistical differences in the properties evaluated, these differences do not translate into important effects on the dependent variables of interest in the experiments. Thus the bovine tendon graft can be substituted for the human tendon graft in both types of experiments.     

Autophagy guards tendon homeostasis

Tendons are vital collagen-dense specialized connective tissues transducing the force from skeletal muscle to the bone, thus enabling movement of the human body. Tendon cells adjust matrix turnover in response to physiological tissue loading and pathological overloading (tendinopathy). Nevertheless, the regulation of tendon matrix quality control is still poorly understood and the pathogenesis of tendinopathy is presently unsolved. Autophagy, the major mechanism of degradation and recycling of cellular components, plays a fundamental role in the homeostasis of several tissues. Here, we investigate the contribution of autophagy to human tendons’ physiology, and we provide in vivo evidence that it is an active process in human tendon tissue. We show that selective autophagy of the endoplasmic reticulum (ER-phagy), regulates the secretion of type I procollagen (PC1), the major component of tendon extracellular matrix. Pharmacological activation of autophagy by inhibition of mTOR pathway alters the ultrastructural morphology of three-dimensional tissue-engineered tendons, shifting collagen fibrils size distribution. Moreover, autophagy induction negatively affects the biomechanical properties of the tissue-engineered tendons, causing a reduction in mechanical strength under tensile force. Overall, our results provide the first evidence that autophagy regulates tendon homeostasis by controlling PC1 quality control, thus potentially playing a role in the development of injured tendons.Subject terms: Physiology, Cell biology     

Temporal response of canine flexor tendon to limb suspension

Tendon disuse, or stress deprivation, frequently accompanies clinical disorders and treatments, yet the metabolism of tendons subject to stress deprivation has rarely been investigated systematically. The effects of stress deprivation on canine flexor tendon were investigated in this study. One adult canine forepaw was suspended for 21 or 42 days. Control forepaws were collected from dogs that had no intervention on their limbs and paws. The expression of collagen I and III was not significantly altered in the tendons disused for 21 days but was significantly decreased at 42 days (P < 0.03). The expression of collagen II, aggrecan, decorin, and fibronectin was significantly decreased in the tendons in the suspended limbs at 21 days (P < 0.002) and further reduced at 42 days. With stress deprivation, the expression of matrix metalloproteinase 2 (MMP2) was significantly increased (P < 0.004) at 21 and 42 days. The expression of MMP3 was significantly decreased at 21 and 42 days (P < 0.03). The expression of MMP13 was not altered with stress deprivation at 21 and 42 days. The expression of MMP14 was significantly increased at 21 days (P = 0.0015) and returned to the control level at 42 days. Tissue inhibitor of metalloproteinase 1 (TIMP1) expression was decreased after the limbs were suspended for 42 days (P = 0.0043), but not 21 days. However, TIMP2 expression was not significantly different from control at 21 or 42 days. Furthermore, the cross-sectional area of the stress-deprived tendons at 42 days was decreased compared with the control group (P < 0.01). The intervention method in this study did not result in any alteration of stiffness of the tendon. Our study demonstrated that stress deprivation decreases the anabolic process and increases the catabolic process of extracellular matrix in flexor tendon.     

Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system

Identification of functional programmable mechanical stimulation (PMS) on tendon not only provides the insight of the tendon homeostasis under physical/pathological condition, but also guides a better engineering strategy for tendon regeneration. The aims of the study are to design a bioreactor system with PMS to mimic the in vivo loading conditions, and to define the impact of different cyclic tensile strain on tendon. Rabbit Achilles tendons were loaded in the bioreactor with/without cyclic tensile loading (0.25 Hz for 8 h/day, 0–9% for 6 days). Tendons without loading lost its structure integrity as evidenced by disorientated collagen fiber, increased type III collagen expression, and increased cell apoptosis. Tendons with 3% of cyclic tensile loading had moderate matrix deterioration and elevated expression levels of MMP‐1, 3, and 12, whilst exceeded loading regime of 9% caused massive rupture of collagen bundle. However, 6% of cyclic tensile strain was able to maintain the structural integrity and cellular function. Our data indicated that an optimal PMS is required to maintain the tendon homeostasis and there is only a narrow range of tensile strain that can induce the anabolic action. The clinical impact of this study is that optimized eccentric training program is needed to achieve maximum beneficial effects on chronic tendinopathy management. Biotechnol. Bioeng. 2013; 110: 1495–1507. © 2012 Wiley Periodicals, Inc.     

An in vitro model of fibroplasia: simultaneous quantification of fibroblast proliferation, migration, and collagen synthesis

Previous studies of fibroblast proliferation, migration, and collagen synthesis have been limited in their ability to define the interrelationship among these events in response to various inflammatory mediators. We have now defined an in vitro tissue culture model for the synchronous quantification of these parameters of fibroplasia. Biopsies (2 mm) of chicken flexor tendons are embedded in a fibrin matrix and exposed to various factors for 5 days in tissue culture. The availability of the fibrin matrix surrounding the tendon biopsy satisfies the need for a solid support medium for fibroblast migration. Multiple measurements of tendon fibroblast proliferation, migration into the fibrin matrix, and relative collagen synthesis are then made on these preparations. Fetal calf serum stimulated tendon fibroblast proliferation and migration in a dose responsive fashion, whereas the selective expression of collagen synthesis was decreased. Platelet lysate stimulated fibroblast proliferation at low concentration, but migration only at high concentration and was without effect on relative collagen synthesis. This model now provides a means of more clearly defining the influence of various inflammatory factors on the events of fibroplasia.     

Effects of supercritical fluid CO<Subscript>2</Subscript> and 25 kGy gamma irradiation on the initial mechanical properties and histological appearance of tendon allograft

Tendon allografts, when autograft options are limited or when obtaining an autograft is not aligned with the patients’ best interest, play an important role in tendon and ligament reconstruction. To minimize the risk of infectious disease transmission tissue banks perform screening tests and the allografts cleaned are sterilized. The current study examines and compares the initial mechanical properties and histological appearance of supercritical CO₂ (SCCO₂)-treated and gamma-irradiated porcine extensor tendons. Thirty intact porcine forelimb extensor tendons randomized equally into three groups: control group, gamma-irradiation group, and SCCO₂-treated group. Once treated, histological assessment and histomorphologic measurements were made on the histological sections obtained from each tendon while stiffness and ultimate failure loads were evaluated from tensile testing. Histological evaluation of gamma-irradiated tendons showed significant disruption to the hierarchical morphology of the fascicle bundles, which was not evident in SCCO₂-treated specimens. Histomorphologic measurements showed a significant increase for measured dead space (void) between tendon fibrils of the gamma-irradiated group comparing to both control and SCCO₂ treated groups (p?<?0.01). There was a significant reduction in the ultimate failure load for tendons treated by gamma-irradiation compared to the control group (p?<?0.05). No statistically significant difference was detected between control and SCCO₂-treated tendons in the ultimate failure load. Stiffness values were not significantly different between three-study groups. This study suggests that while gamma-irradiation has a deleterious effect on mechanical properties of tendon tissue, SCCO₂ does not alter the biomechanical properties and the histological structure of porcine extensor tendons.     

Murine patellar tendon biomechanical properties and regional strain patterns during natural tendon-to-bone healing after acute injury

Tendon-to-bone healing following acute injury is generally poor and often fails to restore normal tendon biomechanical properties. In recent years, the murine patellar tendon (PT) has become an important model system for studying tendon healing and repair due to its genetic tractability and accessible location within the knee. However, the mechanical properties of native murine PT, specifically the regional differences in tissue strains during loading, and the biomechanical outcomes of natural PT-to-bone healing have not been well characterized. Thus, in this study, we analyzed the global biomechanical properties and regional strain patterns of both normal and naturally healing murine PT at three time points (2, 5, and 8 weeks) following acute surgical rupture of the tibial enthesis. Normal murine PT exhibited distinct regional variations in tissue strain, with the insertion region experiencing approximately 2.5 times greater strain than the midsubstance at failure (10.80±2.52% vs. 4.11±1.40%; mean±SEM). Injured tendons showed reduced structural (ultimate load and linear stiffness) and material (ultimate stress and linear modulus) properties compared to both normal and contralateral sham-operated tendons at all healing time points. Injured tendons also displayed increased local strain in the insertion region compared to contralateral shams at both physiologic and failure load levels. 93.3% of injured tendons failed at the tibial insertion, compared to only 60% and 66.7% of normal and sham tendons, respectively. These results indicate that 8 weeks of natural tendon-to-bone healing does not restore normal biomechanical function to the murine PT following injury.     

Adaptation of bone and tendon to prolonged hindlimb suspension in rats

The rat hindlimb suspension model was used to ascertain the importance of ground reaction forces in maintaining bone and tendon homeostasis. Young female Sprague-Dawley rats were randomly assigned to either a suspended or a nonsuspended group. After 28 days, femur bones and patellar tendons were obtained for morphological and biochemical analyses. Prolonged suspension induced a significant change in the geometric configuration of the femur middiaphysis by increasing the minimum diameter (12%) without any significant alterations in cortical area, density, mineral, and collagen concentrations. Femur wet weight, length, DNA, and uronic acid concentrations of suspended animals were not significantly different from bones of nonsuspended rats. However, the collagen and proteoglycan concentrations in patellar tendons of suspended rats were 28% lower than the concentrations of matrix proteins in tissues obtained from nonsuspended animals. These data suggest that elimination of ground reaction forces induces alterations in tendon composition and femur diaphyseal shape by changing regional rates in bone remodeling and localized tendon strain. Therefore it appears that ground reaction forces are an important factor in the maintenance of cortical bone and patellar tendon homeostasis during weight-bearing conditions.