Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system.

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

An AFM study of the nanostructural response of New Zealand white rabbit Achilles tendons to cyclic loading.

The nanostructural response of New Zealand white rabbit Achilles tendons to a fatigue damage model was assessed quantitatively and qualitatively using the endpoint of dose assessments of each tendon from our previous study. The change in mechanical properties was assessed concurrently with nanostructural change in the same non-viable intact tendon. Atomic force microscopy was used to study the elongation of D-periodicities, and the changes were compared both within the same fibril bundle and between fibril bundles. D-periodicities increased due to both increased strain and increasing numbers of fatigue cycles. Although no significant difference in D-periodicity lengthening was found between fibril bundles, the lengthening of D-periodicity correlated strongly with the overall tendon mechanical changes. The accurate quantification of fibril elongation in response to macroscopic applied strain assisted in assessing the complex structure-function relationship in Achilles tendons.

The influence of glycosaminoglycan proteoglycan side chains on tensile force transmission and the nanostructural properties of Achilles tendons.

This study investigates the nanostructural mechanisms that lie behind load transmission in tendons and the role of glycosaminoglycans (GAGs) in the transmission of force in the tendon extracellular matrix. The GAGs in white New Zealand rabbit Achilles tendons were enzymatically depleted, and the tendons subjected to cyclic loading at 6% strain for up to 2 hr. A nanoscale morphometric assessment of fibril deformation under strain was linked with the decline in the tendon macroscale mechanical properties. An atomic force microscope (AFM) was employed to characterize the D-periodicity within and between fibril bundles (WFB and BFB, respectively). By the end of the second hour of the applied strain, the WFB and BFB D-periodicities had significantly increased in the GAG-depleted group (29% increase compared with 15% for the control, p < .0001). No statistically significant differences were found between WFB and BFB D-periodicities in either the control or GAG-depleted groups, suggesting that mechanical load in Achilles tendons is uniformly distributed and fairly homogenous among the WFB and BFB networks. The results of this study have provided evidence of a cycle-dependent mechanism of damage accumulation. The accurate quantification of fibril elongation (measured as the WFB and BFB D-periodicity lengths) in response to macroscopic applied strain has assisted in assessing the complex structure-function relationship in Achilles tendon.

A multiscale study of morphological changes in tendons following repeated cyclic loading.

The response of white New Zealand rabbit Achilles tendons to load was assessed using mechanical measures and confocal arthroscopy (CA). The progression of fatigue-loading-induced damage of the macro- (tenocyte morphology, fiber anisotropy and waviness), as well as the mechanical profile, were assessed within the same non-viable intact tendon in response to prolonged cyclic and static loading (up to four hours) at different strain levels (3%, 6% and 9%). Strain-mediated repeated loading induced a significant decline in mechanical function (p < 0.05) with increased strain and cycles. Mechanical and structural resilience was lost with repeated loading (p < 0.05) at macroscales. The lengthening of D-periodicity correlated strongly with the overall tendon mechanical changes and loss of spindle shape in tenocytes. This is the first study to provide a clear concurrent assessment of form (morphology) and function (mechanics) of tendons undergoing different strain-mediated repeated loading at multiple-scale assessments. This study identifies a variety of multiscale properties that may contribute to the understanding of mechanisms of tendon pathology.

Contribution of glycosaminoglycans to the structural and mechanical properties of tendons - A multiscale study.

Tendinopathy of the Achilles tendon contributes to a large range of disorders, including mechanical damage and degenerative diseases. Glycosaminoglycans (GAGs), are thought to play a role in the mechanical strength of tendons by forming cross-links between collagen molecules and allowing the transmission of forces between fibrils. This study assessed the response of GAG-depleted tendons to damage induced by fatigue loading, investigating the mechanical damage (stiffness, hysteresis and maximum load), macrostructural changes (tenocyte morphology, fiber anisotropy and waviness) assessed by confocal imaging and nanostructural changes (fibril D-periodicity length) within the same non-viable intact tendons. Changes in fiber waviness and tenocyte shape are strongly correlated to mechanical and nano-structural (D-periodicity elongation) properties in both Control and GAG-depleted tendons. This study supports firstly, the vital role GAGs play as mechanical connectors facilitating the load transfer between the fibrils and their hydrophilic role in facilitating fibril sliding. Secondly, that observed changes in tenocyte shape and fiber waviness correlate with tendon stiffness and other mechanical profiles.

Confocal arthroscopic assessment of osteoarthritis in situ.

PURPOSE: This study aimed to assess the ability of the laser scanning confocal arthroscope (LSCA) to evaluate cartilage microstructure, particularly in differentiating stages of human osteoarthritis (OA) as classified by the International Cartilage Repair Society (ICRS) OA grade definitions. METHODS: Ten tibial plateaus from total knee arthroplasty patients were obtained at the time of surgery. Cartilage areas were visually graded based on the ICRS classification, imaged by use of a 7-mm-diameter LSCA (488-nm excitation with 0.5% [wt/vol] fluorescein, 20-minute staining period), and then removed with underlying bone for histologic examination with H&E staining. The 2 imaging techniques were then compared for each ICRS grade to ascertain similarity between the methods and thus gauge the techniques' diagnostic resolution. Cartilage surface degeneration was readily imaged and OA severity accurately gauged by the LSCA and confirmed by histology. RESULTS: LSCA and histologic images of specimens in the late stages of OA were seen to be mutually related even though they were imaged in planes that were orthogonal to each other. Useful and comparable diagnostic resolution was obtained in all imaged specimens from subjects with various stages of OA. CONCLUSIONS: This study showed the LSCA's ability to image detailed cartilage surface morphologic features that identify grade 1 through 4 of the ICRS OA grading system. The LSCA's imaging potential was best shown by its ability to resolve the fine collagen network present under the lamina splendens. The incorporation of high-magnification confocal technology within the confines of an arthroscopic probe has proved to provide the imaging requirements necessary to perform detailed cartilage condition assessment. CLINICAL RELEVANCE: In comparison to video arthroscopy, LSCA provides increased magnification along with improved contrast and resolution.

Confocal arthroscopy-based patient-specific constitutive models of cartilaginous tissues--I: development of a microstructural model.

Current development of a laser scanning confocal arthroscope within our school will enable 3D microscopic imaging of joint tissues in vivo. Such an instrument could be useful, for example, in assessing the microstructural condition of the living tissues without physical biopsy. It is envisaged also that linked to a suitable microstructural constitutive formulation, such imaging could allow non-invasive patient-specific estimation of tissue mechanical performance. Such a procedure could have applications in surgical planning and simulation, and assessment of engineered tissue replacements, where tissue biopsy is unacceptable. In this first of two papers the development of a suitable constitutive framework for generating such estimates is reported. A microstructure-based constitutive formulation for cartilaginous tissues is presented. The model extends existing fibre composite-type models and accounts for strain-rate sensitivity of the tissue mechanical response through incorporation of a viscoelastic fibre phase. Importantly, the model is constructed so as to allow direct incorporation of structural data from confocal images. A finite element implementation of the formulation suitable for incorporation within commercial codes is also presented.

Confocal arthroscopy-based patient-specific constitutive models of cartilaginous tissues - II: prediction of reaction force history of meniscal cartilage specimens.

The theoretical framework developed in a companion paper (Part I) is used to derive estimates of mechanical response of two meniscal cartilage specimens. The previously developed framework consisted of a constitutive model capable of incorporating confocal image-derived tissue microstructural data. In the present paper (Part II) fibre and matrix constitutive parameters are first estimated from mechanical testing of a batch of specimens similar to, but independent from those under consideration. Image analysis techniques which allow estimation of tissue microstructural parameters form confocal images are presented. The constitutive model and image-derived structural parameters are then used to predict the reaction force history of the two meniscal specimens subjected to partially confined compression. The predictions are made on the basis of the specimens' individual structural condition as assessed by confocal microscopy and involve no tuning of material parameters. Although the model does not reproduce all features of the experimental curves, as an unfitted estimate of mechanical response the prediction is quite accurate. In light of the obtained results it is judged that more general non-invasive estimation of tissue mechanical properties is possible using the developed framework.

Pull-out strength comparison of a novel expanding fastener against an orthopaedic screw in an ovine vertebral body: an ex-vivo study.

The purpose of this study was to mechanically test a novel Unthreaded Expandable Fastener (UEF), manufactured using Selective Laser Melting, which was designed for fixation in the cervical lateral mass. The pull-out strength and stiffness of the prototype UEFs was evaluated in a non-osteoporotic ovine bone model against equivalent screws. The prototype UEF demonstrated a 41% increase in failure force and a 60% reduction in failure force standard deviation compared to the screws. All bone samples were micro CT-scanned and no significant differences in bone microstructural properties was found between the screw and UEF sample sets, indicating that the UEFs may be less sensitive to bone quality variation. This increased performance can potentially translate into improved surgical outcome and reduced surgical risk for lateral mass fixation. With further design optimisation, additional improvement in performance over screws may be possible in future studies.

Cyclic mechanical stimulation rescues achilles tendon from degeneration in a bioreactor system.

Physiotherapy is one of the effective treatments for tendinopathy, whereby symptoms are relieved by changing the biomechanical environment of the pathological tendon. However, the underlying mechanism remains unclear. In this study, we first established a model of progressive tendinopathy-like degeneration in the rabbit Achilles. Following ex vivo loading deprivation culture in a bioreactor system for 6 and 12 days, tendons exhibited progressive degenerative changes, abnormal collagen type III production, increased cell apoptosis, and weakened mechanical properties. When intervention was applied at day 7 for another 6 days by using cyclic tensile mechanical stimulation (6% strain, 0.25 Hz, 8 h/day) in a bioreactor, the pathological changes and mechanical properties were almost restored to levels seen in healthy tendon. Our results indicated that a proper biomechanical environment was able to rescue early-stage pathological changes by increased collagen type I production, decreased collagen degradation and cell apoptosis. The ex vivo model developed in this study allows systematic study on the effect of mechanical stimulation on tendon biology.

Rotated Hough filtering for automatically distinguishing the collagen bundles in the most superficial layer of articular cartilage.

The structure of the collagen matrix in the most superficial layer of articular cartilage (AC) is particularly critical to the wear and tensile resistance of AC. Disruption of the collagen network leads to rapid wear of the articular surface, which is a major contributory factor of osteoarthritis. Interwoven fiber bundles have been observed in the most superficial layer of healthy AC under confocal microscopy, but gradually disappear with age and pathological change of AC. The image analysis techniques developed in this paper aimed to provide a quantitative description of the relationship between the presence of the fiber bundles in the lamina splendens and health status of AC. The result of this quantitative study confirmed the existence of fiber bundles in healthy AC, and the accuracy of the identified fiber bundles was up to 90%. With the development of confocal arthroscopy for imaging microstructure of AC without biopsy, the image analysis technique can aid to efficiently assess the physiological status of AC for orthopedic clinics.

Bioreactor design for tendon/ligament engineering.

Tendon and ligament injury is a worldwide health problem, but the treatment options remain limited. Tendon and ligament engineering might provide an alternative tissue source for the surgical replacement of injured tendon. A bioreactor provides a controllable environment enabling the systematic study of specific biological, biochemical, and biomechanical requirements to design and manufacture engineered tendon/ligament tissue. Furthermore, the tendon/ligament bioreactor system can provide a suitable culture environment, which mimics the dynamics of the in vivo environment for tendon/ligament maturation. For clinical settings, bioreactors also have the advantages of less-contamination risk, high reproducibility of cell propagation by minimizing manual operation, and a consistent end product. In this review, we identify the key components, design preferences, and criteria that are required for the development of an ideal bioreactor for engineering tendons and ligaments.

Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique.

INTRODUCTION: The collagen structure in the superficial zone of articular cartilage is critical to the tissue's durability. Early osteoarthritis is often characterized with fissures on the articular surface. This is closely related to the disruption of the collagen network. However, the traditional histology can not offer visualization of the collagen structure in articular cartilage because it uses conventional optical microscopy that does not have insufficient imaging resolution to resolve collagen from proteoglycans in hyaline articular cartilage. This study examines the 3D collagen network of articular cartilage scored from 0 to 2 in the scoring system of International Cartilage Repair Society, and aims to develop a 3D histology for assessing early osteoarthritis. METHODS: Articular cartilage was visually classified into five physiological groups: normal cartilage, aged cartilage, cartilage with artificial and natural surface disruption, and fibrillated. The 3D collagen matrix of the cartilage was acquired using a 3D imaging technique developed previously. Traditional histology was followed to grade the physiological status of the cartilage in the scoring system of International Cartilage Repair Society. RESULTS: Normal articular cartilage contains interwoven collagen bundles near the articular surface, approximately within the lamina splendens. However, its collagen fibres in the superficial zone orient predominantly in a direction spatially oblique to the articular surface. With age and disruption of the articular surface, the interwoven collagen bundles are gradually disappeared, and obliquely oriented collagen fibres change to align predominantly in a direction spatially perpendicular to the articular surface. Disruption of the articular surface is well related to the disappearance of the interwoven collagen bundles. CONCLUSION: A 3D histology has been developed to supplement the traditional histology and study the subtle changes in the collagen network in the superficial zone during early physiological alteration of articular cartilage. The fibre confocal imaging technology used in this study has allowed developing confocal arthroscopy for in vivo studying the chondrocytes in different depth of articular cartilage. Therefore, the current study has potential to develop an in vivo 3D histology for diagnosis of early osteoarthritis.