Collagen Fiber Re-Alignment in a Neonatal Developmental Mouse Supraspinatus Tendon Model

Collagen fiber re-alignment is one postulated mechanism of tendon structural response to load. While collagen fiber distribution has been shown to vary by tendon location in the supraspinatus tendon (SST), changes in local re-alignment behavior have not been examined throughout postnatal development. Postnatal tendons, with immature collagen fibrils, may respond to load in a much different manner than collagen fibers with mature fiber–fiber and fiber–matrix connections. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing in a developmental mouse SST model and corresponding mechanical properties measured. Collagen fiber re-alignment occurred during preconditioning for 28 day old tendons, at the toe-region for 10 day tendons and at the linear-region for 4 day tendon midsubstance. Mechanical properties increased with developmental age. Linear modulus was lower at the insertion site compared to the midsubstance location at all time points. Local differences in collagen fiber distributions were found at 10 and 28 days for all mechanical testing points (except the 10 day transition point). This study found that collagen fiber re-alignment depends on developmental age and suggests that collagen fibrillogenesis may influence the tendon’s ability to structurally respond to load. Additionally, results indicate that the insertion site and tendon midsubstance locations develop differently.     

Decreasing inflammatory response of injured patellar tendons results in increased collagen fibril diameters

Tissue inflammation is essential in the healing process, but its effect on the quality of the healing tissue is not clear. This study determines the effect of decreasing early inflammation during wound healing in genetic deficient mice on collagen fibril diameter. Two strains of mice were used: three C3H/HeJ mice and three C3H/HeN mice for each of two time points (7 and 14 days postinjury). C3H/HeJ mice have a genetic deficiency in the production of tumor necrosis factor by macrophages and other cytokines in response to endotoxin, and C3H/HeN mice have no genetic deficiency. The right patellar tendon of both mouse strains was transversely transected, whereas the left patellar tendon was left intact for control. After 7 and 14 days, both right and left patellar tendons were harvested, and tendon samples were examined with transmission electron microscopy. We found that at 7 days, transected tendons of C3H/HeJ mice exhibited on average 1.6 times larger collagen fibril diameters than transected C3H/HeN tendons, whereas at 14 days, collagen fibril diameters of the C3H/HeJ mice were 1.3 times that of C3H/HeN mice. Also, at both 7 days and 14 days, collagen fibrils in C3H/HeJ mice appeared more organized than C3H/HeN mice. In addition, control tendons in both mouse strains showed no significant differences in collagen fibril diameter and organization. Therefore, these results suggest that decreasing the inflammatory response in the early stages of tendon wound healing enhances the quality of the healing tendon through increased collagen fiber diameter and better organization.     

Tensile force transmission in human patellar tendon fascicles is not mediated by glycosaminoglycans

Correct mechanical function of tendons is essential to human physiology and therefore the mechanical properties of tendon have been a subject of research for many decades now. However, one of the most fundamental questions remains unanswered: How is load transmitted through the tendon? It has been suggested that the proteoglycan-associated glycosaminoglycans (GAGs) found on the surface of the collagen fibrils may be an important transmitter of load, but existing results are ambiguous and have not investigated human tendons. We have used a small-scale mechanical testing system to measure the mechanical properties of fascicles from human patellar tendon at two different deformation rates before and after removal of GAGs by treatment with chondroitinase ABC. Efficiency of enzyme treatment was quantified using dimethylmethylene blue assay. Removal of at least 79% of the GAGs did not significantly change the tendon modulus, relative energy dissipation, peak stress, or peak strain. The effect of deformation rate was not modulated by the treatment either, indicating no effect on viscosity. These results suggest that GAGs cannot be considered mediators of tensile force transmission in the human patellar tendon, and as such, force transmission must either take place through other matrix components or the fibrils must be mechanically continuous at least to the tested length of 7?mm.     

The Tendon Injury Response is Influenced by Decorin and Biglycan

Defining the constituent regulatory molecules in tendon is critical to understanding the process of tendon repair and instructive to the development of novel treatment modalities. The purpose of this study is to define the structural, expressional, and mechanical changes in the tendon injury response, and elucidate the roles of two class I small leucine-rich proteoglycans (SLRPs). We utilized biglycan-null, decorin-null and wild type mice with an established patellar tendon injury model. Mechanical testing demonstrated functional changes associated with injury and the incomplete recapitulation of mechanical properties after 6 weeks. In addition, SLRP deficiency influenced the mechanical properties with a marked lack of improvement between 3 and 6 weeks in decorin-null tendons. Morphological analyses of the injury response and role of SLRPs demonstrated alterations in cell density and shape as well as collagen alignment and fibril structure resulting from injury. SLRP gene expression was studied using RT-qPCR with alterations in expression associated with the injured tendons. Our results show that in the absence of biglycan initial healing may be impaired while in the absence of decorin later healing is clearly diminished. This suggests that biglycan and decorin may have sequential roles in the tendon response to injury.     

Distributions of types I,II and III collagen by region in the human supraspinatus tendon

Abstract

The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and may reflect an important adaptive response to its complex, multiaxial loading environment. However, these functional properties are associated with a location-dependent structure and composition that have not been fully elucidated. Therefore, the objective of this study was to determine the concentrations of types I, II and III collagen in six distinct regions of the SST and compare changes in collagen concentration across regions with local changes in mechanical properties. We hypothesized that type I collagen content would be high throughout the tendon, type II collagen would be restricted to regions of compressive loading and type III collagen content would be high in regions associated with damage. We further hypothesized that regions of high type III collagen content would correspond to regions with low tensile modulus and a low degree of collagen alignment. Although type III collagen content was not significantly higher in regions that are frequently damaged, all other hypotheses were supported by our results. In particular, type II collagen content was highest near the insertion while type III collagen was inversely correlated with tendon modulus and collagen alignment. The measured increase in type II collagen under the coracoacromial arch provides evidence of adaptation to compressive loading in the SST. Moreover, the structure-function relationship between type III collagen content and tendon mechanics established in this study demonstrates a mechanism for altered mechanical properties in pathological tendons and provides a guideline for identifying therapeutic targets and pathology-specific biomarkers.     

The adaptability of tendon to loading differs in men and women

The reason why women sustain more soft tissue injury than men during physical activity is unknown. Connective tissue properties and extracellular matrix adaptability in human tendon were investigated in models that addressed biochemical, physiological and biomechanical aspects of tendon connective tissue in response to mechanical loading. Habitual training resulted in a larger patellar tendon in men but not in women. Following an acute bout of exercise, men had an elevated tendon collagen synthesis rate and this effect was less pronounced or absent in women. Moreover, levels of circulating oestrogen affected the acute exercise-related increase in collagen synthesis. Finally, the mechanical strength of isolated tendon collagen fascicles in men surpassed that of women. Thus, compared to men, women have (i) an attenuated tendon hypertrophy response to habitual training; (ii) a lower tendon collagen synthesis rate following acute exercise; (iii) a rate of tendon collagen synthesis which is further attenuated with elevated estradiol levels; and (iv) a lower mechanical strength of their tendons. These data indicate that tendons in women have a lower rate of new connective tissue formation, respond less to mechanical loading, and have a lower mechanical strength, which may leave the tissue more susceptible to injury.     

Canine tendon studies. II. Biomechanical evaluation of normal and regrown canine tendons.

Some of the mechanical properties of regrown canine tendons are compared to those of normal tendons of young and mature animals. Patellar and Achilles tendons from 12 beagle dogs were removed and studied with their bone origin and insertions. Mechanical tests were performed within 24 hr and test conditions simulated the physiological function of the tendon in vivo at room temperature. Specimens were soaked in Ringers solution and mounted in an Instron testing machine with load deflection curves plotted automatically. The parameters used for analysis were load extension, stress relaxation, elastic limit, and strain rate dependence. The regrown tendons in young animals appeared to quickly adjust in dimension and structure so that their properties were not significantly different from those of normal tendons on a load extension basis. The normal tendons were stiffer than regrown ones but the modulus of elasticity increased with age. The Achilles were stiffer than patellar tendons. Cyclic loading with 25 kg did not affect reconstructed tendon models, although some increase in stiffness was noted. The elastic modulus decreased with an increase in ambient temperature and increasing strain rate.     

Effect of chemical treatments on tendon cellularity and mechanical properties

Removal of cells may decrease the antigenicity and risk of disease transmission associated with tendon allografts and xenografts. An ideal cell removal method would not compromise graft structure and mechanical properties. This study compared the effects of three extraction chemicals [t-octyl-phenoxypolyethoxyethanol (Triton X-100), tri(n-butyl)phosphate (TnBP), and sodium dodecyl sulfate (SDS)] on tendon cellularity, structure, nativity, and mechanical properties. Rat tail tendons were soaked in extraction solutions for various time periods (12-48 h) and concentrations (0.5-2%), then they were rinsed with distilled water and ethyl alcohol. Histological analysis and tensile tests were performed on control and chemically treated tendons. Changes in collagen nativity were estimated by mechanical testing following incubation in a trypsin solution. Treatment of tendons with 1% Triton X-100 for 24 h disrupted the collagen fiber structure and did not remove cells. Treatment with 1% SDS for 24 h or 1% TnBP for 48 h resulted in an acellular tendon matrix with retention of near normal structure and mechanical properties. Consistent with previous studies demonstrating cell removal from other tissue types using SDS and TnBP, our preliminary results suggest these treatments are potentially useful for removing cells from tendon allografts or xenografts without compromising the graft structure or mechanical properties.     

Reconstruction of the canine Achilles and patellar tendons using dacron mesh silicone prosthesis. I. Clinical and biocompatibility evaluation.

Surgical replacement of the Achilles and patellar tendons using Dacron mesh silicone prostheses was performed in 15 mature beagle dogs. This part of the study includes clinical evaluation, gross inspection at autopsy of regrown tendons, and histological determination of biocompatibility of prosthetic implants. Functional continuity and integrity of prosthetic patellar tendons have been established by evaluating the biologic-prosthetic interface and the mechanical properties of regrown tendon tissue around and through the Dacron silicone replacements. Results of prosthetic Achilles tendons were less satisfactory because of difficulties in suturing to a muscle and its fascia. Although the prosthetic tendon did not regrow through the tube, it provided a structure for regrowth around it. The regrown tendons became nearly ten times the normal cross-sectional area after three to four months and decreased to two to three times after 13 months. Histological studies indicate that in the absence of infection, the Dacron mesh silicone tendon was well tolerated for periods up to 13 months. Overall results are encouraging and warrant further investigation although the regenerative capacity of human patellar and Achilles tendons is unknown.     

Fetal and postnatal development of the patella, patellar tendon and suprapatella in the rabbit; changes in the distribution of the fibrillar collagens

The development of the patella, its associated tendons, and suprapatella of the rabbit knee joint is described from the 17 d fetus to the mature adult. The patellar tendon (ligament) with the patella on its posterior surface is seen in the 17 d fetus and is fully developed by 1 postnatal wk. It is composed of bundles of types I and V collagens separated by endotenons of types III and V collagens. Anteriorly there is an epitenon of types III and V collagens while synovium and a fat pad cover its posterior surface. In the 25 d fetus, the patella is cartilaginous and is separated from the femoral condyles. The cartilage contains type II collagen, but types I, III and V collagens are found along the articular surface. Ossification starts 1 postnatal wk and at 6 wk only the articular cartilage remains. In addition to type II, types III and V collagens are located around the chondrocyte lacunae. The long anterior junction between the patella and its tendon is fibrocartilaginous at 1 wk, but as ossification proceeds this is replaced by bone. Types I and V collagens are found in this region. The suprapatella on the posterior surface of the quadriceps tendon is first seen 1 wk postnatally as an area of irregularly organised fibres and chondrocyte-like cells. Types I, II, III and V collagens are present from 3 wk onwards. It is compared with the fibrocartilage of other tendons that are under compression. The arrangement of the collagens in the patellar tendon is discussed in relation to its use as a replacement for injured anterior cruciate ligaments. It is suggested that the structural differences between the patellar tendon and anterior cruciate ligament preclude the translocated tendon acquiring mechanical strength similar to that of a normal cruciate ligament. The designation 'patellar ligament' as opposed to 'patellar tendon' is questioned. It is argued that the term patellar tendon reflects its structure more accurately than patellar ligament.     

Microstructural stress relaxation mechanics in functionally different tendons

Tendons experience widely varying loading conditions in vivo. They may be categorised by their function as either positional tendons, which are used for intricate movements and experience lower stress, or as energy storage tendons which act as highly stressed springs during locomotion. Structural and compositional differences between tendons are thought to enable an optimisation of their properties to suit their functional environment. However, little is known about structure–function relationships in tendon.This study adopts porcine flexor and extensor tendon fascicles as examples of high stress and low stress tendons, comparing their mechanical behaviour at the micro-level in order to understand their stress relaxation response. Stress-relaxation was shown to occur predominantly through sliding between collagen fibres. However, in the more highly stressed flexor tendon fascicles, more fibre reorganisation was evident when the tissue was exposed to low strains. By contrast, the low load extensor tendon fascicles appears to have less capacity for fibre reorganisation or shearing than the energy storage tendon, relying more heavily on fibril level relaxation. The extensor fascicles were also unable to sustain loads without rapid and complete stress relaxation. These findings highlight the need to optimise tendon repair solutions for specific tendons, and match tendon properties when using grafts in tendon repairs.     

In vivo tendon engineering with skeletal muscle derived cells in a mouse model

Engineering a functional tendon with strong mechanical property remains an aim to be achieved for its eventual application. Both skeletal muscle and tendon are closely associated during their development and both can bear strong mechanical loading dynamically. This study explored the possibility of engineering stronger tendons with mouse skeletal muscle derived cells (MDCs) and with mouse tenocytes as a control. The results demonstrated that both MDCs and tenocytes shared the gene expression of growth differentiation factor-8 (GDF-8), collagens I, III, VI, scleraxis and tenomodulin, but with MyoD gene expression only in MDCs. Quantitatively, MDCs expressed higher levels of GDF-8, collagens III and VI (p < 0.05), whereas tenocytes expressed higher levels of collagen I, scleraxis and tenomodulin (p < 0.05). Interestingly, MDCs proliferated faster with more cells in S + G2/M phases than tenocytes (p < 0.05). After been seeded on polyglycolic acid (PGA) fibers, MDCs formed better quality engineered tendons with more mature collagen structure and thicker collagen fibrils as opposed to tenocyte engineered tendons. Biochemically, more collagen VI and decorin were produced in the former than in the later. Functionally, MDC engineered tendons exhibited stronger mechanical properties than tenocyte engineered tendons, including maximal load, stiffness, tensile strength and Young's modulus (p < 0.05). Furthermore, with the increase of implantation time, MDCs gradually lost their expression of myogenic molecules of MyoD and desmin and gained the expression of tenomodulin, a marker for tenocytes. Collectively, these results indicate that MDCs may serve as a desirable alternative cell source for engineering functional tendon tissue.     

Tensile Tests of Collagen Fibers Obtained from the Rabbit Patellar Tendon

Tensile properties of collagen fibers of approximately 1 m in diameter were determined using a newly developed micro tensile test system for cells and fine fibrous biological tissues. The test system consists of a thermostatic test chamber, an inverted microscope, micromanipulators, a direct drive linear actuator, a cantilever-type load cell, and a video dimension analyzer (VDA). The fibers were isolated with a mechanical method from collagen fascicles (approximately 300 m in diameter) cut out from the rabbit patellar tendon. The ends of each fiber were attached to the tips of a pair of glass microtubes (15 to 20 m in outer diameter) using a cyanoacrylate adhesive. One of the microtubes was attached to the load cell; the other one was connected to the linear actuator which was utilized to stretch the fiber. Load applied to the fiber was measured with the load cell, while its elongation was determined with the VDA using the images of the edges of the adhesive as markers. Tangent modulus, tensile strength, and strain at failure of the tested fibers were 54.3± 25.1 MPa, 8.5± 2.6 MPa, and 21.6± 3.0%, respectively. These values were much different from those of collagen fascicles (300 m in diameter) cut out from the rabbit patellar tendon and also from those of the bulk patellar tendon (Trans. ASME, J. Biomech. Eng. 121, 124–294, 1999); for example, tensile strength and strain at failure of the fibers were approximately 50 and 200% of those of the fascicles, respectively. These results suggest that the mechanical interactions between fibers and between fibers and ground substances contribute much to the mechanical properties of collagen fascicles and bulk tendons.     

Ligament repair: a molecular and immunohistological characterization

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during "ligamentization". Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction.     

肌腱及其末端结构的断裂特性实验观察

对兔髌骨－髌腱－胫骨结节和跟腱－跟骨试件的组织结构观察,应力－应变关系本构方程和力学特性的测定,发现肌腱的力学强度明显高于牵拉屈折型和滑车型腱末端,而与牵拉型末端无明显差异。     

Mechanical,Compositional, and Structural Properties of the Post-natal Mouse Achilles Tendon

During post-natal development, tendons undergo a well orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue. Conversely, during the repair response to injury, structural and compositional changes occur, but in this case, a mechanically inferior tendon is produced. As a result, the process of development has been postulated as a potential paradigm through which improved adult tissue healing may occur. In this study we measured the mechanical, compositional, and structural properties in the post-natal mouse Achilles tendon at 4, 7, 10, 14, 21, and 28 days old. Throughout post-natal development, the mechanical properties, collagen content, fibril diameter mean, and fibril diameter standard deviation increased. Biglycan expression decreased and decorin expression and fiber organization were unchanged. This study provides a new mouse model that can be used to quantitatively examine mechanical development, as well as compositional and structural changes and biological mechanisms, during post-natal tendon development. This model is advantageous due to the large number of genetically modified mice and commercially available assays that are not available in other animal models. A mouse model therefore allows future mechanistic studies to build on this work.     

Effects of mechanical stimulation on the biomechanics and histology of stem cell-collagen sponge constructs for rabbit patellar tendon repair

The objective of this study was to determine how mechanical stimulation affects the biomechanics and histology of stem cell-collagen sponge constructs used to repair central rabbit patellar tendon defects. Autogenous tissue-engineered constructs were created for both in vitro and in vivo analyses by seeding mesenchymal stem cells from 10 adult rabbits at 0.14x10(6) cells/construct in type I collagen sponges. Half of these constructs were mechanically stimulated once every 5 min for 8 h/day to a peak strain of 4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. Samples allocated for in vitro testing revealed that mechanically stimulated constructs had 2.5 times the linear stiffness of nonstimulated constructs. The remaining paired constructs for in vivo studies were implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Twelve weeks after surgery, repair tissues were assigned for biomechanical (7 pairs) and histologic (3 pairs) analyses. Maximum force, linear stiffness, maximum stress, and linear modulus for the stimulated (vs. nonstimulated) repairs averaged 70% (vs. 55%), 85% (vs. 55%), 70% (vs. 50%), and 50% (vs. 40%) of corresponding values for the normal central third of the patellar tendons. The average force-elongation curve for the mechanically stimulated repairs also matched the corresponding curve for the normal patellar tendons, up to 150% of the peak in vivo force values recorded in a previous study. Construct and repair linear stiffness and linear modulus were also positively correlated (r = 0.6 and 0.7, respectively). Histologically both repairs showed excellent cellular alignment and mild staining for decorin and collagen type V, and moderate staining for fibronectin and collagen type III. This study shows that mechanical stimulation of stem cell-collagen sponge constructs can significantly improve tendon repair biomechanics up to and well beyond the functional limits of in vivo loading.     

Comparative multi‐scale hierarchical structure of the tail,plantaris, and Achilles tendons in the rat

Rodent tendons are widely used to study human pathologies such as tendinopathy and repair, and to address fundamental physiological questions about development, growth, and remodeling. However, how the gross morphology and multi‐scale hierarchical structure of rat tendons, such as the tail, plantaris, and Achilles tendons, compare with that of human tendons are unknown. In addition, there remains disagreement about terminology and definitions. Specifically, the definitions of fascicle and fiber are often dependent on diameter sizes, not their characteristic features, and these definitions impair the ability to compare hierarchical structure across species, where the sizes of the fiber and fascicle may change with animal size and tendon function. Thus, the objective of the study was to select a single species that is commonly used for tendon research (rat) and tendons with varying mechanical functions (tail, plantaris, Achilles) to evaluate the hierarchical structure at multiple length scales using histology, SEM, and confocal imaging. With the exception of the specialized rat tail tendon, we confirmed that in rat tendons there are no fascicles and the fiber is the largest subunit. In addition, we provided a structurally based definition of a fiber as a bundle of collagen fibrils that is surrounded by elongated cells, and this definition was supported by both histologically processed and unprocessed samples. In all rat tendons studied, the fiber diameters were consistently between 10 and 50 μm, and this diameter range appears to be conserved across larger species. Specific recommendations were made highlighting the strengths and limitations of each rat tendon as a research model. Understanding the hierarchical structure of tendon can advance the design and interpretation of experiments and development of tissue‐engineered constructs.