Effects of the frequency and duration of cyclic stress on the mechanical properties of cultured collagen fascicles from the rabbit patellar tendon

The effects of frequency or duration of cyclic stress on the mechanical properties of collagen fascicles were studied by means of in vitro tissue culture experiments. Collagen fascicles of approximately 300 microm in diameter were obtained from rabbit patellar tendons. During culture, cyclic stress having the peak stress of approximately 2 MPa was applied to the fascicles at 1 Hz for 1 hour/day (1 Hz-1 h group), at 1 Hz for 4 hours/day (1 Hz-4 h group), or at 4 Hz for 1 hour/day (4 Hz-1 h group). The frequency of 4 Hz and the duration of 1 hour/day are considered to be similar to those of the in vivo stress applied to fascicles in the intact rabbit patellar tendon. After culture for 1 or 2 weeks, the mechanical properties of the fascicles were determined using a microtensile tester, and were compared to the properties of non-cultured, fresh fascicles (control group) and the fascicles cultured under no load condition (non-loaded group). The tangent modulus and tensile strength of fascicles in the 4 Hz-1 h group were similar to those in the control group; however, the fascicles of the 1 Hz-1 h and 1 Hz-4 h groups had significantly lower values than those of the control group. There was no significant difference in the tensile strength between the 1 Hz-1 h and non-loaded groups, although the strength in the 1 Hz-4 h group was significantly higher than that of the non-loaded group. It was concluded that the frequency and duration of cyclic stress significantly affect the mechanical properties of cultured collagen fascicles. If we apply cyclic stress having the frequency and duration which are experienced in vivo, the biomechanical properties are maintained at control, normal level. Lower frequencies or less cycles of applied force induce adverse effects.     

Effects of static stress on the mechanical properties of cultured collagen fascicles from the rabbit patellar tendon

In-vitro tissue culture experiments were performed to study the effects of static stress on the mechanical properties of collagen fascicles obtained from the rabbit patellar tendon. After collagen fascicles having the diameter of approximately 300 microm were cultured for 1 and 2 wk under static stress between 0 and 3 MPa, their mechanical properties and crimp morphology were determined using a micro-tensile tester and a light microscope, respectively. The tensile strength and tangent modulus of the fascicles were significantly decreased by culture under no load compared to control fascicles. A statistically significant correlation, which was described by a quadratic curve, was observed between applied stress and tensile strength. The maximum tensile strength (16.7 MPa) was obtained at the applied stress of 1.2 MPa; the strength was within a range of control values. There was a similar correlation between applied stress and tangent modulus, and the modulus was maintained at control level under 1.3 MPa stress. The stress of 1.2 to 1.3 MPa is equivalent to approximately 50 percent of the peak stress developed in the intact rabbit patellar tendon by running. Strain at failure of cultured collagen fascicles was negatively correlated with applied stress, and that at 1.2 to 1.3 MPa stress was almost the same as the control value. Crimp morphology in the fascicles cultured under about 1.2 MPa stress was similar to that in control fascicles. These results indicate that cultured collagen fascicles change the mechanical properties and structure in response to static tensile stress. In addition, their mechanical properties and structure are maintained at control level if the static stress of 50 percent of in-vivo peak stress is applied.     

Effects of cyclic stress on the mechanical properties of cultured collagen fascicles from the rabbit patellar tendon

Effects of cyclic stress on the mechanical properties of collagen fascicles were studied by in vitro tissue culture experiments. Collagen fascicles (approximately 300 microns in diameter) obtained from the rabbit patellar tendon were applied cyclic load at 4 Hz for one hour per day during culture period for one or two weeks, and then their mechanical properties were determined using a micro-tensile tester. There was a statistically significant correlation between tensile strength and applied peak stress in the range of 0 to 5 MPa, and the relation was expressed by a quadratic function. The maximum strength (19.4 MPa) was obtained at the applied peak stress of 1.8 MPa. The tensile strength of fascicles were within a range of control values, if they were cultured under peak stresses between 1.1 and 2.6 MPa. Similar results were also observed in the tangent modulus, which was maintained at control level under applied peak stresses between 0.9 and 2.8 MPa. The stress of 0.9 to 1.1 MPa is equivalent to approximately 40% of the in vivo peak stress which is developed in the intact rabbit patellar tendon by running, whereas that of 2.6 to 2.8 MPa corresponds to approximately 120% of the in vivo peak stress. Therefore, the fascicles cultured under applied peak stresses of lower than 40% and higher than 120% of the in vivo peak stress do not keep the original strength and modulus. These results indicate that the mechanical properties of cultured collagen fascicles strongly depend upon the magnitude of the stress applied during culture, which are similar to our previous results observed in stress-shielded and overstressed patellar tendons in vivo.     

Growth-related changes in the mechanical properties of collagen fascicles from rabbit patellar tendons

Growth-related changes in the mechanical properties of collagen fascicles (approximately 300 microm in diameter) were studied using patellar tendons obtained from skeletally immature 1 and 2 months old and matured 6 months old rabbits. Tensile properties were determined using a specially designed micro-tensile tester. In each age group, there were no significant differences in the properties among cross-sectional locations in the tendon. Tangent modulus and tensile strength significantly increased with age; the rates of their increases between 1 and 2 months were higher than those between 2 and 6 months. The tangent modulus and tensile strength were positively correlated with the body weight of animals. However, growth-related changes in the mechanical properties were different between collagen fascicles and bulk patellar tendons, which may be attributable to such non-collagenous components as ground substances and also to mechanical interactions between collagen fascicles.     

Mechanical properties of collagen fascicles from the rabbit patellar tendon

Tensile and viscoelastic properties of collagen fascicles of approximately 300 microns in diameter, which were obtained from rabbit patellar tendons, were studied using a newly designed micro-tensile tester. Their cross-sectional areas were determined with a video dimension analyzer combined with a CCD camera and a low magnification microscope. There were no statistically significant differences in tensile properties among the fascicles obtained from six medial-to-lateral locations of the patellar tendon. Tangent modulus, tensile strength, and strain at failure of the fascicles determined at about 1.5 percent/s strain rate were 216 +/- 68 MPa, 17.2 +/- 4.1 MPa, and 10.9 +/- 1.6 percent (mean +/- S.D.), respectively. These properties were much different from those of bulk patellar tendons; for example, the tensile strength and strain at failure of these fascicles were 42 percent and 179 percent of those of bulk tendons, respectively. Tangent modulus and tensile strength of collagen fascicles determined at 1 percent/s strain rate were 35 percent larger than those at 0.01 percent/s. The strain at failure was independent of strain rate. Relaxation tests showed that the reduction of stress was approximately 25 percent at 300 seconds. These stress relaxation behavior and strain rate effects of collagen fascicles differed greatly from those of bulk tendons. The differences in tensile and viscoelastic properties between fascicles and bulk tendons may be attributable to ground substances, mechanical interaction between fascicles, and the difference of crimp structure of collagen fibrils.     

Effects of stress shielding on the transverse mechanical properties of rabbit patellar tendons

With the aim of studying mechanisms of the remodeling of tendons and ligaments, the effects of stress shielding on the rabbit patellar tendon were studied by performing tensile and stress relaxation tests in the transverse direction. The tangent modulus, tensile strength, and strain at failure of non-treated, control patellar tendons in the transverse direction were 1272 kPa, 370 kPa, and 40.5 percent, respectively, whereas those of the tendons stress-shielded for 1 week were 299 kPa, 108 kPa, and 40.4 percent, respectively. Stress shielding markedly decreased tangent modulus and tensile strength in the transverse direction, and the decreases were larger than those in the longitudinal direction, which were determined in our previous study. For example, tensile strength in the transverse and longitudinal direction decreased to 29 and 50 percent of each control value, respectively, after 1 week stress shielding. In addition, the stress relaxation in the transverse direction of stress-shielded patellar tendons was much larger than that of nontreated, control ones. In contrast to longitudinal tensile tests for the behavior of collagen, transverse tests reflect the contributions of ground substances such as proteoglycans and mechanical interactions between collagen fibers. Ground substances provide lubrication and spacing between fibers, and also confer viscoelastic properties. Therefore, the results obtained from the present study suggest that ground substance matrix, and interfiber and fiber-matrix interactions have important roles in the remodeling response of tendons to stress.     

Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice

Evaluations of tendon mechanical behavior based on biochemical and structural arrangement have implications for designing tendon specific treatment modalities or replacement strategies. In addition to the well studied type I collagen, other important constituents of tendon are the small proteoglycans (PGs). PGs have been shown to vary in concentration within differently loaded areas of tendon, implicating them in specific tendon function. This study measured the mechanical properties of multiple tendon tissues from normal mice and from mice with knock-outs of the PGs decorin or biglycan. Tail tendon fascicles, patellar tendons (PT), and flexor digitorum longus tendons (FDL), three tissues representing different in vivo loading environments, were characterized from the three groups of mice. It was hypothesized that the absence of decorin or biglycan would have individual effects on each type of tendon tissue. Surprisingly, no change in mechanical properties was observed for the tail tendon fascicles due to the PG knockouts. The loss of decorin affected the PT causing an increase in modulus and stress relaxation, but had little effect on the FDL. Conversely, the loss of biglycan did not significantly affect the PT, but caused a reduction in both the maximum stress and modulus of the FDL. These results give mechanical support to previous biochemical data that tendons likely are uniquely tailored to their specific location and function. Variances such as those presented here need to be further characterized and taken into account when designing therapies or replacements for any one particular tendon.     

Glycation-induced matrix stability in the rabbit achilles tendon

Connective tissue susceptibility to nonenzymatic glycation was examined following 0, 2, 4, 6, 8, and 10 weeks of incubating the rabbit Achilles tendon in phosphate-buffered saline containing ribose (glycated). The biomechanical integrity of the glycated tendons was then compared to control tendons incubated in phosphate-buffered saline (non-glycated) at each time interval, while the biochemical stability of both groups of tendons was determined by examining collagen extractability and the formation of pentosidine at 8 weeks. Whereas there were no significant biomechanical differences between control and glycated tendons at 0- and 2-week intervals (P > 0.05), moderately significant increases in maximum load, energy to yield, and toughness of glycated tendons were observed at 4 weeks. Beyond 4 weeks of incubation, the differences between glycated and non-glycated tendons became highly significant, as glycated tendons withstood more load and tensile stress (P < 0.01 for each variable), attained significantly higher modulus of elasticity (P < 0.01), absorbed more energy (P < 0.01), and became tougher (P < 0.01) than controls. These differences in the biomechanical indices of the effects of glycation were stable between the 6th and 10th week of glycation. The maximum increases in the biomechanical measurements as a result of glycation were 29% for maximum load, 125% for stress, 19% for strain, 106% for Young's modulus of elasticity, 14% for energy to yield, and 57% for toughness. Biochemical analysis showed a 61% reduction in the extractability of neutral salt-soluble collagen, a 48% decrease in acid-soluble collagen, and a 29% decline in pepsin-soluble collagen in glycated tendons (P < 0.01). In contrast, there was a 28% increase in the amount of insoluble collagen and significantly higher amounts of pentosidine (P < 0.01) in glycated tendons. Collectively, these biomechanical and biochemical results suggest that nonenzymatic glycation may explain the altered stability of connective tissue matrix induced by the processes of diabetes and aging.     

Collagen fibril diameter distribution does not reflect changes in the mechanical properties of in vitro stress-deprived tendons

The purpose of this study was to determine if an association exists between the tensile properties and the collagen fibril diameter distribution in in vitro stress-deprived rat tail tendons. Rat tail tendons were paired into two groups of 21 day stress-deprived and 0 time controls and compared using transmission electron microscopy (n = 6) to measure collagen fibril diameter distribution and density, and mechanical testing (n =6) to determine ultimate stress and tensile modulus. There was a statistically significant decrease in both ultimate tensile strength (control: 17.95+/-3.99 MPa, stress-deprived: 6.79+/-3.91 MPa) and tensile modulus (control: 312.8+/-89.5 MPa, stress-deprived: 176.0+/-52.7 MPa) in the in vitro stress-deprived tendons compared to controls. However, there was no significant difference between control and stress-deprived tendons in the number of fibrils per tendon counted, mean fibril diameter, mean fibril density, or fibril size distribution. The results of this study demonstrate that the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons is not correlated with the collagen fibril diameter distribution and, therefore, the collagen fibril diameter distribution does not, by itself, dictate the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons.     

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.     

Antibody neutralization of TGF-beta enhances the deterioration of collagen fascicles in a tissue-cultured tendon matrix with ex vivo fibroblast infiltration

A tissue-cultured tendon matrix infiltrated with cultured fibroblasts can be regarded as an ideal tissue-engineered tendon model. To clarify the role of TGF-beta in a tissue-cultured tendon matrix during ex vivo cellular infiltration, the present ex vivo study was conducted to test the following hypothesis that antibody neutralization of TGF-beta enhances weakening of the collagen fascicles of the patellar tendon matrix in response to ex vivo fibroblast infiltration. In skeletally mature female rabbits, fibroblasts were isolated from the right patellar tendons using an explant culture technique, and the left patellar tendons underwent multiple freeze/thaw treatment with liquid nitrogen to obtain an acellular tendon matrix. Each acellular tendon was placed in a collagen gel containing cultured fibroblasts and then incubated with or without anti-TGF-beta1 antibody for 6 weeks. We found that antibody neutralization of TGF-beta enhanced the decrease in the tensile strength and tensile modulus of the collagen fascicles of the patellar tendon matrix in response to ex vivo fibroblast infiltration. The present study indicates a possibility that TGF-beta may have a role in suppressing the material deterioration of the fascicles in the tendon during ex vivo cellular infiltration.     

A quantitative investigation of structure-function relationships in a tendon fascicle model

These studies sought to investigate quantitative relationships between the complex composite structure and mechanical properties of tendon. The isolated mouse tail tendon fascicle was chosen as an appropriate model for these so-called "structure-function" investigations. Specifically, collagen fibril diameters and mechanical properties were measured in fascicles from immature (3 week) control, adult (8 week) control, and adult (8 week) MovI3 transgenic mice. Results demonstrated a moderate correlation between mean fibril diameter and fascicle stiffness (r = 0.73, p = 0.001) and maximum load (r = 0.75, p < 0.001), whereas a weak correlation with fascicle modulus (r = 0.39, p = 0.11) and maximum stress (r = 0.48, p = 0.04). An analysis of pooled within-group correlations revealed no strong structure-function trends evidenced at the local or group level, indicating that correlations observed in the general structure-function analyses were due primarily to having three different experimental groups, rather than significant correlations of parameters within the groups.     

Changes in the morphology and synthetic activity of cultured rat tail tendon

Summary Isolated single fascicles from tail tendons of young rats were freed of epitenon cells and cultured in vitro for up to 7 days. The tissue remained viable, as judged by the structural integrity of cell organelles and the ability to synthesize DNA and glycosaminoglycans (GAG). The rate of DNA synthesis peaked after 2 days in culture and decreased slowly thereafter. Concomitantly, an increase in cell number was noted at the periphery of the fascicle. GAG production also increased during culture, sulphated GAG being increased proportionately more than hyaluronic acid. Dermatan sulphate was the predominant sulphated GAG in freshly isolated fascicles, but in cultured tissue, the newly synthesized sulphated GAG was more sensitive to degradation by chondroitinase AC and had an increased electrophoretic mobility. Fine structural changes were observed in cultured tissues such as the retraction of cell processes. rounding up of cell bodies and the appearance of gaps between collagen fibrils. Cultured tenocytes also frequently contained apparently phagocytized collagen fibrils which were not seen in freshly isolated fascicles, and this appearance was suggestive of collagen degradation occurring in vitro, although no change in the total hydroxyproline content was noted. The data show that when individual fascicles are cultured in vitro they undergo a process of matrix remodelling which has features in common with events occurring in vivo when tendons have been surgically manipulated.     

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.     

Effects of freeze-thaw on the biomechanical and structural properties of the rat Achilles tendon

Rodent models are commonly used to investigate tendon healing, with the biomechanical and structural properties of the healed tendons being important outcome measures. Tendon storage for later testing becomes necessary when performing large experiments with multiple time-points. However, it is unclear whether freezing rodent tendons affects their material properties. Thus the aim of this study was to determine whether freezing rat Achilles tendons affects their biomechanical or structural properties. Tendons were frozen at either −20 °C or −80 °C directly after harvesting, or tested when freshly harvested. Groups of tendons were subjected to several freeze-thaw cycles (1, 2, and 5) within 3 months, or frozen for 9 months, after which the tendons were subjected to biomechanical testing. Additionally, fresh and thawed tendons were compared morphologically, histologically and by transmission electron microscopy. No major differences in biomechanical properties were found between fresh tendons and those frozen once or twice at −20 °C or −80 °C. However, deterioration of tendon properties was found for 5-cycle groups and both long-term freezing groups; after 9 months of freezing at −80 °C the tear resistance of the tendon was reduced from 125.4 ± 16.4N to 74.3 ± 18.4N (p = 0.0132). Moreover, tendons stored under these conditions showed major disruption of collagen fibrils when examined by transmission electron microscopy. When examined histologically, fresh samples exhibited the best cellularity and proteoglycan content of the enthesis. These properties were preserved better after freezing at −80 °C than after freezing at −20 °C, which resulted in markedly smaller chondrocytes and less proteoglycan content. Overall, the best preservation of histological integrity was seen with tendons frozen once at −80 °C. In conclusion, rat Achilles tendons can be frozen once or twice for short periods of time (up to 3 months) at −20 °C or −80 °C for later testing. However, freezing for 9 months at either −20 °C or −80 °C leads to deterioration of certain parameters.     

Regional stiffening with aging in tibialis anterior tendons of mice occurs independent of changes in collagen fibril morphology

The incidence of tendon degeneration and rupture increases with advancing age. The mechanisms underlying this increased risk remain unknown but may arise because of age-related changes in tendon mechanical properties and structure. Our purpose was to determine the effect of aging on tendon mechanical properties and collagen fibril morphology. Regional mechanical properties and collagen fibril characteristics were determined along the length of tibialis anterior (TA) tendons from adult (8- to 12-mo-old) and old (28- to 30-mo-old) mice. Tangent modulus of all regions along the tendons increased in old age, but the increase was substantially greater in the proximal region adjacent to the muscle than in the rest of the tendon. Overall end-to-end modulus increased with old age at maximum tendon strain (799 ± 157 vs. 1,419 ± 91 MPa) and at physiologically relevant strain (377 ± 137 vs. 798 ± 104 MPa). Despite the dramatic changes in tendon mechanical properties from adulthood to old age, collagen fibril morphology and packing fraction remained relatively constant in all tendon regions examined. Since tendon properties are influenced by their external loading environment, we also examined the effect of aging on TA muscle contractile properties. Maximum isometric force did not differ between the age groups. We conclude that TA tendons stiffen in a region-dependent manner throughout the life span, but the changes in mechanical properties are not accompanied by corresponding changes in collagen fibril morphology or force-generating capacity of the TA muscle.     

Strain-rate sensitive mechanical properties of tendon fascicles from mice with genetically engineered alterations in collagen and decorin

Tendons have complex mechanical behaviors that are nonlinear and time dependent. It is widely held that these behaviors are provided by the tissue composition and structure. It is generally thought that type I collagen provides the primary elastic strength to tendon while proteoglycans, such as decorin, play a role in failure and viscoelastic properties. This study sought to quantify such structure-function relationships by comparing tendon mechanical properties between normal mice and mice genetically engineered for altered type I collagen content and absence of decorin. Uniaxial tensile ramp to failure experiments were performed on tail tendon fascicles at two strain rates, 0.5%/s and 50%/s. Mutations in type I collagen led to reduced failure load and stiffness with no changes in failure stress, modulus or strain rate sensitivity. Fascicles without decorin had similar elastic properties to normal fascicles, but reduced strain rate sensitivity. Fascicles from immature mice, with increased decorin content compared to adult fascicles, had inferior elastic properties but higher strain rate sensitivity. These results showed that tendon viscoelasticity is affected by decorin content but not by collagen alterations. This study provides quantitative evidence for structure-function relationships in tendon, including the role of proteoglycan in viscoelasticity.     

Biochemical and biomechanical properties of tendons in two commercial types of chickens

The quality of the attachment of meat to bone is often reported to be insufficient by more and more poultry's consumers. This is particularly true for thigh meat in broilers. The aim of this study was to compare muscle to bone attachment (namely, tendons) from a biomechanical and a biochemical point of view in 50 standard (S) and 50 Label Rouge (LR) chickens. Carcasses weighted around 1.7 kg in the two groups. Two tendons were harvested and proceeded for passive stretch tests, prior to cooking or not, to determine main mechanical characteristics (maximum load, stiffness and longitudinal strain). Biochemical parameters such as dry matter percentage, total collagen content, collagen solubility and sulphated glycosaminoglycans (sGAGs) content were also determined. Results showed that biomechanical values differ largely between the two studied tendons. For a given tendon, the values were also different between the two groups of chickens mainly after cooking. The results clearly showed that, mainly after cooking, the mechanical resistance of tendon to stretch was better in LR than in S chickens. LR chickens were reported to have tendons with higher collagen and sGAGs contents associated with a lower collagen solubility. These differences may explain biomechanical differences observed for the two types of tendons and could be due to increased age and/or higher physical activity of LR chickens.     

Cyclic strain increases fibroblast proliferation, matrix accumulation, and elastic modulus of fibroblast-seeded polyurethane constructs

Rapid induction of matrix production and mechanical strengthening is essential to the development of bio-artificial constructs for repair and replacement of load-bearing connective tissues. Toward this end, we describe the development of a mechanical bioreactor and its application to investigate the influence of cyclic strain on fibroblast proliferation, matrix accumulation, and the mechanical properties of fibroblast-seeded polyurethane constructs (FSPC). Human fibroblasts were cultured in 10% serum-containing conditions within three-dimensional, porous elastomeric substrates under static conditions and a model regime of cyclic strain (10% strain, 0.25 Hz, 8 h/day), with and without ascorbic acid supplementation. After one week, the combination of cyclic strain and ascorbic acid resulted in significantly increased construct elastic modulus (>110%) relative to either condition alone. In contrast, cyclic strain alone was sufficient to stimulate significant increases in fibroblast proliferation. Mechanical strengthening of FSPCs was accompanied by increased type I collagen and fibronectin matrix accumulation and distribution, and significantly increased gene expression for type I collagen, TGFbeta-1, and CTGF. These results suggest that strain-induced conditioning in vitro leads to mechanical strengthening of fibroblast/material constructs, most likely resulting from increased collagen matrix deposition, secondary to strain-induced increases in cytokine production.     

Specimen dimensions influence the measurement of material properties in tendon fascicles

Stress, strain and modulus are regularly used to characterize material properties of tissue samples. However, when comparing results from different studies it is evident the reported material properties, particularly failure strains, vary hugely. The aim of our study was to characterize how and why specimen length and cross-sectional area (CSA) appear to influence failure stress, strain and modulus in fascicles from two functionally different tendons. Fascicles were dissected from five rat tails and five bovine foot extensors, their diameters determined by a laser micrometer, and loaded to failure at a range of grip-to-grip lengths. Strain to failure significantly decreased with increasing in specimen length in both rat and bovine fascicles, while modulus increased. Specimen length did not influence failure stress in rat tail fascicles, although in bovine fascicles it was significantly lower in the longer 40 mm specimens compared to 5 and 10 mm specimens. The variations in failure strain and modulus with sample length could be predominantly explained by end-effects. However, it was also evident that strain fields along the sample length were highly variable and notably larger towards the ends of the sample than the mid-section even at distances in excess of 5 mm from the gripping points. Failure strain, stress and modulus correlated significantly with CSA at certain specimen lengths. Our findings have implications for the mechanical testing of tendon tissue: while it is not always possible to control for fascicle length and/or CSA, these parameters have to be taken into account when comparing samples of different dimensions.