Decorin expression is important for age-related changes in tendon structure and mechanical properties

The aging population is at an increased risk of tendon injury and tendinopathy. Elucidating the molecular basis of tendon aging is crucial to understanding the age-related changes in structure and function in this vulnerable tissue. In this study, the structural and functional features of tendon aging are investigated. In addition, the roles of decorin and biglycan in the aging process were analyzed using transgenic mice at both mature and aged time points. Our hypothesis is that the increase in tendon injuries in the aging population is the result of altered structural properties that reduce the biomechanical function of the tendon and consequently increase susceptibility to injury. Decorin and biglycan are important regulators of tendon structure and therefore, we further hypothesized that decreased function in aged tendons is partly the result of altered decorin and biglycan expression. Biomechanical analyses of mature (day 150) and aged (day 570) patellar tendons revealed deteriorating viscoelastic properties with age. Histology and polarized light microscopy demonstrated decreased cellularity, alterations in tenocyte shape, and reduced collagen fiber alignment in the aged tendons. Ultrastructural analysis of fibril diameter distributions indicated an altered distribution in aged tendons with an increase of large diameter fibrils. Aged wild type tendons maintained expression of decorin which was associated with the structural and functional changes seen in aged tendons. Aged patellar tendons exhibited altered and generally inferior properties across multiple assays. However, decorin-null tendons exhibited significantly decreased effects of aging compared to the other genotypes. The amelioration of the functional deficits seen in the absence of decorin in aged tendons was associated with altered tendon fibril structure. Fibril diameter distributions in the decorin-null aged tendons were comparable to those observed in the mature wild type tendon with the absence of the subpopulation containing large diameter fibrils. Collectively, our findings provide evidence for age-dependent alterations in tendon architecture and functional activity, and further show that lack of stromal decorin attenuates these changes.     

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.     

Biomechanical studies of the rabbit patellar tendon after removal of its one-fourth or a half.

Effects of the overstressing induced by the harvest of grafts from the patellar tendon on the mechanical properties and morphometry of remaining tendon were studied using a rabbit model. The width of the patellar tendon was reduced by one-fourth or one-half equally removing the medial and lateral portions; by this surgery, the cross-sectional area was decreased by 25 or 50 percent from the original area. After all the rabbits were allowed unrestricted activities in cages for 3 to 12 weeks, their patellar tendons were harvested for mechanical and histological studies. The one-fourth removal induced no significant changes in the mechanical properties, but significantly increased the cross-sectional area. In the case of one-half removal, tensile strength and tangent modulus did not change in some tendons, although the cross-sectional area increased significantly. In the other central half tendons, mechanical strength decreased markedly, while the cross-sectional area increased; hypercellular areas and breakage of collagen bundles were observed in these tendons. These results indicate that the patellar tendon has an ability of functionally adapting to overstressing by changing the cross-sectional area, while keeping the mechanical properties unchanged, if the extent of overstressing is less than 30 percent.     

Age-related changes in the biomechanics of healing patellar tendon

By 2030, there will be 70 million people in the United States over the age of 65, and by 2050, 22% of the US population will be considered elderly. It is generally believed that injuries in the elderly heal slower and less completely than in adolescents or young adults. To evaluate aging effects on tissue repair a surgical injury was created in the middle third of one patellar tendon in 1- and 4-5-year-old New Zealand White rabbits. The biomechanical properties of the isolated repair tissues and contralateral normal tendon tissues were compared at 6, 12 and 26 weeks post-injury. We hypothesized that repair tissues would exhibit age-related reductions in biomechanical properties at all time intervals of healing, both based on raw data and when normalized to values from contralateral tendons. Repairs from both age groups were similar, with no significant increase in maximum stress, strain at maximum stress, or modulus between 6 and 12 weeks. At 26 weeks, the repairs in the 4-year-old rabbits had higher maximum stress values than repairs in the 1-year-old rabbits (p=0.03). There were no significant differences in the strain at maximum stress or modulus. When repair tissue properties were normalized to values in the contralateral normal tendon, the maximum stress of the patellar tendon repair tissue from the 4 year old was significantly greater than the corresponding value from the 1 year old at the 26 week time point (p=0.04). In conclusion, these findings do not support the presence of age-related declines in the biomechanics of healing tendon.     

Mechanical response of individual collagen fibrils in loaded tendon as measured by atomic force microscopy

A precise analysis of the mechanical response of collagen fibrils in tendon tissue is critical to understanding the ultrastructural mechanisms that underlie collagen fibril interactions (load transfer), and ultimately tendon structure–function. This study reports a novel experimental approach combining macroscopic mechanical loading of tendon with a morphometric ultrascale assessment of longitudinal and cross-sectional collagen fibril deformations. An atomic force microscope was used to characterize diameters and periodic banding (D-period) of individual type-I collagen fibrils within murine Achilles tendons that were loaded to 0%, 5%, or 10% macroscopic nominal strain, respectively. D-period banding of the collagen fibrils increased with increasing tendon strain (2.1% increase at 10% applied tendon strain, p < 0.05), while fibril diameter decreased (8% reduction, p < 0.05). No statistically significant differences between 0% and 5% applied strain were observed, indicating that the onset of fibril (D-period) straining lagged macroscopically applied tendon strains by at least 5%. This confirms previous reports of delayed onset of collagen fibril stretching and the role of collagen fibril kinematics in supporting physiological tendon loads. Fibril strains within the tissue were relatively tightly distributed in unloaded and highly strained tendons, but were more broadly distributed at 5% applied strain, indicating progressive recruitment of collagen fibrils. Using these techniques we also confirmed that collagen fibrils thin appreciably at higher levels of macroscopic tendon strain. Finally, in contrast to prevalent tendon structure–function concepts data revealed that loading of the collagen network is fairly homogenous, with no apparent predisposition for loading of collagen fibrils according to their diameter.     

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.     

Mechanical properties of human patellar tendon at the hierarchical levels of tendon and fibril

Tendons are strong hierarchical structures, but how tensile forces are transmitted between different levels remains incompletely understood. Collagen fibrils are thought to be primary determinants of whole tendon properties, and therefore we hypothesized that the whole human patellar tendon and its distinct collagen fibrils would display similar mechanical properties. Human patellar tendons (n = 5) were mechanically tested in vivo by ultrasonography. Biopsies were obtained from each tendon, and individual collagen fibrils were dissected and tested mechanically by atomic force microscopy. The Young's modulus was 2.0 ± 0.5 GPa, and the toe region reached 3.3 ± 1.9% strain in whole patellar tendons. Based on dry cross-sectional area, the Young's modulus of isolated collagen fibrils was 2.8 ± 0.3 GPa, and the toe region reached 0.86 ± 0.08% strain. The measured fibril modulus was insufficient to account for the modulus of the tendon in vivo when fibril content in the tendon was accounted for. Thus, our original hypothesis was not supported, although the in vitro fibril modulus corresponded well with reported in vitro tendon values. This correspondence together with the fibril modulus not being greater than that of tendon supports that fibrillar rather than interfibrillar properties govern the subfailure tendon response, making the fibrillar level a meaningful target of intervention. The lower modulus found in vitro suggests a possible adverse effect of removing the tissue from its natural environment. In addition to the primary work comparing the two hierarchical levels, we also verified the existence of viscoelastic behavior in isolated human collagen fibrils.     

Membrane-bound intracellular collagen fibrils in fibroblasts and myofibroblasts of regenerating rabbit calcaneal tendons.

The ultrastructures of 33 rabbit calcaneal tendons were studied to determine (1) whether vacuolar fibrils are present in three regions of tendons undergoing normal healing after tenotomy and repair, and (2) to stimulate collagen synthesis via functional loading, and hence determine the effect of loading on the presence of vacuolar fibrils in healing tendons. In all the loaded tendons, electron microscopy revealed membrane-bound collagen fibril equivalents in sections of neotendon obtained from the site of tenotomy, and in sections of tendon segments proximal and distal to the site of surgery. Similar vacuolar fibrils were visualized in sections of the proximal and distal segments of the non-loaded regenerating tendons, and also in sections of neotendons formed at the site of tenotomy after 12 and 15 days of healing without functional loading. No such fibrils were visualized in the non-tenotomized normal control tendons. These findings indicate that chemical agents and disease are not necessary to induce the appearance of intracytoplasmic fibrils in vivo and that functional loading augments the presence of fibril-bearing vacuoles in regenerating tendons.     

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.     

The influence of indomethacin on collagen synthesis during tendon healing in the rabbit

The influence of indomethacin on collagen synthesis in intact and healing plantaris longus tendons in the rabbit was investigated. Forty-four male New Zealand White rabbits were subjected to a standardized trauma (tenetomy + repair) on the left hindlimb. Half of the animals were subsequently treated with indomethacin, 10 mg/kg per day orally, and the other half with placebo. After 2 and 4 weeks the rabbits were injected intravenously with ³H-proline and killed 18 h later. Indomethacin affected the collagen metabolism differently depending on whether the tendons were involved in wound healing or not. In intact tendons the drug caused a small general inhibition of collagen synthesis. In the healing tendon there was a shift towards the synthesis of more insoluble collagen with little effect on the total synthesis. After 4 weeks there was also a slight but significant decrease in the amount of hydroxyproline in the most soluble collagen fraction from the tenotomized, indomethacin treated tendons.     

The influence of indomethacin on collagen synthesis during tendon healing in the rabbit

The influence of indomethacin on collagen synthesis in intact and healing plantaris longus tendons in the rabbit was investigated. Forty-four male New Zealand White rabbits were subjected to a standardized trauma (tenetomy + repair) on the left hindlimb. Half of the animals were subsequently treated with indomethacin, 10 mg/kg per day orally, and the other half with placebo. After 2 and 4 weeks the rabbits were injected intravenously with 3H-proline and killed 18 h later. Indomethacin affected the collagen metabolism differently depending on whether the tendons were involved in wound healing or not. In intact tendons the drug caused a small general inhibition of collagen synthesis. In the healing tendon there was a shift towards the synthesis of more insoluble collagen with little effect on the total synthesis. After 4 weeks there was also a slight but significant decrease in the amount of hydroxyproline in the most soluble collagen fraction from the tenotomized, indomethacin treated tendons.     

Comparative anatomy of rabbit and human achilles tendons with magnetic resonance and ultrasound imaging

We sought to describe the comparative anatomy of the Achilles tendon in rabbits and humans by using macroscopic observation, magnetic resonance imaging, and ultrasonography. The calcaneus-Achilles tendon-gastrocnemius-soleus complexes from 18 New Zealand white rabbits underwent ultrasound (US) and magnetic resonance (MR) imaging and gross anatomic sectioning; these results were compared with those from a cadaveric gastrocnemius-soleus-Achilles tendon-calcaneus specimen from a 68-y-old human male. The medial and lateral gastrocnemius muscle tendons merged 5.2 +/- 0.6 mm proximal to the calcaneal insertion macroscopically, at 93% of their course, different from the gastrocnemius human tendons, which merged at 23% of their overall course. The rabbit flexor digitorum superficialis tendon, corresponding to the flexor digitorum longus tendon in human and comparable in size with the gastrocnemius tendons, was located medial and anterior to the medial gastrocnemius tendon proximally and rotated dorsally and laterally to run posterior to the Achilles tendon-calcaneus insertion. In humans, the flexor digitorum longus tendon tracks posteriorly to the medial malleolus. The soleus muscle and tendon are negligible in the rabbit; these particular comparative anatomic features in the rabbit were confirmed on the MR images. Therefore the rabbit Achilles tendon shows distinctive gross anatomical and MR imaging features that must be considered when using the rabbit as a research model, especially for mechanical testing, or when generalizing results from rabbits to humans.     

Variations in collagen fibril structure in tendons

Variation of collagen fibril structure in tendon was investigated by x-ray diffraction. Anatomically distinct tendons from single species, as well as tendons from different species, were examined to determine the variations that exist in both the axial and lateral structure of the collagen fibrils. The meridional diffraction is derived from the axial collagen fibril structure. Anatomically distinct tendons of a particular species give meridional patterns that are indistinguishable within experimental error. The meridional diffraction patterns from tendons of different mammals are similar but show small species-specific variations, most noticeably in the 14th–18th orders. Tendons of birds also give meridional patterns that are similar to each other, but the avian patterns differ considerably from the mammalian ones. Avian tendons give stronger odd and weaker even low orders, a feature consistent with a reduced gap:overlap ratio, and have a distinctive intensity pattern for the higher meridional orders. Interpretation of these differences has been approached using biochemical data, diffraction by reconsituted fibers of purified collagen, and Fourier transform analysis. From these methods, it appears that the variations observed in the lower orders (2nd–8th) and in the higher orders (29th–52nd) are probably related to differences in the primary structure of the Type I collagen found in the different species. The variations observed in the 14th–18th orders appear not to be related to features within the triple-helical domain of the molecule. Equatorial diffraction yields information on the lateral packing of collagen molecules in the fibrils, and considerable variation was seen in different tendons. Rat tail tendon gives sharp Bragg reflections, demonstrating the presence of a crystalline lateral arrangement of molecules in the fibril. For the first time, sharp lattice reflections similar to those in rat tail tendon have been observed in nontail tendons, including rat achilles tendon, rabbit leg tendon, and wing and leg tendons of quail. In the rabbit and quail tendons, one of the strong equatorial reflections characteristic of the rat tendon pattern, at 1.26 nm, was absent. The positions of the equatorial maxima, which are a measure of intermolecular spacing, varied considerably, being smallest in the specimens displaying crystalline packing. The intermolecular distance in chiken and turkey leg tendons is longer than that found in mammalian tendons, or in avian wing tendons, which supports the hypothesis that a larger intermolecular spacing is characteristic of tendons that calcify. Thus, x-ray diffraction indicates there are reproducible differences in both the axial and lateral structure of collagen fibrils among different tendons. This work on tendon, a tissue containing almost exclusively Type I collagen as its major component, should serve as a basis for analyzing the structure of other connective tissues, which contain different genetic types of collagen and larger amounts of noncollagenous components.     

Influence of decorin on the mechanical, compositional, and structural properties of the mouse patellar tendon

The interactions of small leucine-rich proteoglycans (SLRPs) with collagen fibrils, their association with water, and their role in fibrillogenesis suggests that SLRPs may play an important role in tendon mechanics. Some studies have assessed the role of SLRPs in the mechanical response of the tendon, but the relationships between sophisticated mechanics, assembly of collagen, and SLRPs have not been well characterized. Decorin content was varied in a dose dependent manner using decorin null, decorin heterozygote, and wild type mice. Quantitative measures of mechanical (tension and compression), compositional, and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was increased in the decorin heterozygous tendons compared to wild type. These tendons also had a significant decrease in total collagen and no structural changes compared to wild type. Decorin null tendons did not have any mechanical changes; however, a significant decrease in the average fibril diameter was found. No differences were seen between genotypes in elastic or compressive properties, and all tendons demonstrated viscoelastic mechanical dependence on strain rate and frequency. These results suggest that decorin, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. In addition, reductions in decorin do not cause large changes in indentation compressive properties, suggesting that other factors contribute to these properties. Understanding these relationships may ultimately help guide development of tissue engineered constructs or treatment modalities.     

Frog tendon structure and its relationship with locomotor modes

Tendon collagen fibrils are the basic force‐transmitting units of the tendon. Yet, surprisingly little is known about the diversity in tendon anatomy and ultrastructure, and the possible relationships between this diversity and locomotor modes utilized. Our main objectives were to investigate: (a) the ultra‐structural anatomy of the tendons in the digits of frogs; (b) the diversity of collagen fibril diameters across frogs with different locomotor modes; (c) the relationship between morphology, as expressed by the morphology of collagen fibrils and tendons, and locomotor modes. To assess the relationship between morphology and the locomotor modes of the sampled taxa we performed a principal component analysis considering body length, fibrillar cross sectional area (CSA) and tendon CSA. A MANOVA showed that differences between species with different locomotor modes were significant with collagen fibril diameter being the discriminating factor. Overall, our data related the greatest collagen fibril diameter to the most demanding locomotor modes, conversely, the smallest collagen fibril CSA and the highest tendon CSA were observed in animals showing a hopping locomotion requiring likely little absorption of landing forces given the short jump distances.     

Development of collagen fibril organization and collagen crimp patterns during tendon healing

Normal tendon comprises coaxially aligned bundles of crimped collagen fibres each of which possesses a fibrillar substructure. In acute traumatic injury this level of organization is disrupted and the mechanical function of the tendon impaired. During repair, a degree of recovery of the fibrillar structure takes place. In this tudy we have assessed the re-establishment of tendon organization after injury on the basis of the collagen fibril diameter distribution and the collagen crimp parameters. Crimp became undetectable following injury but one month later was present throughout the tissue. At this time the periodicity was greatly reduced by comparison with that of the normal tendon and normal values were not re-established within 14 months following injury. Collagen fibril diameters remained abnormally small over this same period of time. In particular, fibrils of diameters in excess of 100 nm, commonly found in normal and contralateral tendons, were totally absent from the observed distributions in the healing tendons. Such large diameter fibrils often account for as much as 50% of the total mass of collagen present in the uninjured tissue. Thus the mechanical properties of the healing tendon may remain significantly different from those of normal tendon for a minimum time of 14 months after injury.     

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.     

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.     

Ultrastructural features of adult human tendon

Summary A variety of human tendons have been studied at the electron microscope level. The fibers of these tendons are composed of collagen fibrils that average 1,750 Å and 600 Å in diameter. A third population that measures 100 Å in diameter may represent immature collagen or filaments that are incorporated into tendon elastic fibers. The larger collagen fibrils vary in ratio with respect to one another, and are connected by interfibrillar bridges which in some cases appear to extend through the substance of the fibril. The collagen fibrils of the paratenon are less-well organized than those of the tendon proper and average 600 Å in diameter. Tendons that exhibit the property of lateral stretch (plantaris and palmaris) were compared at the ultrastructural level with tendons that do not have this property. No differences between the two tendon types could be determined in normal or spread preparations, indicating that the differences in physical characteristics are a result of fiber rather than fibril organization.Supported by Edward G. Schlieder Foundation GrantThe authors wish to thank Mrs. Janell Buck and Mrs. Eunice Schwartz for their excellent technical and secretarial assistance, and Mr. Garbis Kerimian for his excellent photographic work     

Effect of scaffold material, construct length and mechanical stimulation on the in vitro stiffness of the engineered tendon construct

Introducing mesenchymal stem cell (MSC)-seeded collagen constructs into load-protected wound sites in the rabbit patellar and Achilles tendons significantly improves their repair outcome compared to natural healing of the unfilled defect. However, these constructs would not be acceptable alternatives for repairing complete tendon ruptures because they lack the initial stiffness at the time of surgery to resist the expected peak in vivo forces thereafter. Since the stiffness of these constructs has also been shown to positively correlate with the stiffness of the subsequent repairs, improving initial stiffness by appropriate selection of in vitro culture conditions would seem crucial. In this study we examined the individual and combined effects of collagen scaffold type, construct length, and mechanical stimulation on in vitro implant stiffness. Two levels each of scaffold material (collagen gel vs. collagen sponge), construct length (short vs. long), and mechanical stimulation (stimulated vs. non-stimulated) were examined. Our results indicate that all three treatment factors influenced construct linear stiffness. Increasing the length of the construct had the greatest effect on the stiffness compared to introducing mechanical stimulation or changing the scaffold material. A significant interaction was also found between length and stimulation. Of the eight groups studied, longer, stimulated, cell-sponge constructs showed the highest in vitro linear stiffness. We now plan in vivo studies to determine if higher stiffness constructs generate higher stiffness repairs 12 weeks after surgery and if in vitro construct stiffness continues to correlate with in vivo repair parameters like linear stiffness.