Bone has a number of different functions in the skeleton including the physical roles of support, protection and sound wave conduction. The mechanical properties, required for these different functions varies and can be achieved by compositional adaption of the bone material, in addition to changes in shape and architecture. A number of previous studies have demonstrated the relationship between mechanical function and mineral to collagen ratio in bones from different species.

The aim of this study is to test the hypothesis that the mineral to collagen ratio is higher in bone with a mechanically harder matrix within a species.

The red deer (Cervus elaphus) (n=6) was chosen as a model for studying bone with extreme properties. The mechanical properties of the antler, metacarpal bone and tympanic bulla were defined by indentation using a bench-top indentation platform (Biodent). The mineral to collagen ratio was quantified using Raman spectroscopy. The deposition of mineral was studied at macro-level using pQCT.

The results showed that the hardness (Indentation Distance Increase) was lowest in the metacarpal (8.5µm), followed by the bulla bone (9.4µm) and highest in the antler (14.5µm). Raman spectroscopy showed a mineral:collagen ratio of 1:0.10 (bulla), 1:0.13 (metacarpal) and 1:0.15 (antler) for the different bones. This does not follow the more linear trend previously shown between young's modulus and the mineral:collagen ratio. The location of the mineral appeared to differ between bone types with pQCT revealing locations of concentrated density and banding patterns in antler. Interestingly, Raman spectra showed differences in the amide peaks revealing differences in protein structure.

The results reject the hypothesis but also suggest that the organisation of mineral and collagen has an impact on the hardness modulus. We demonstrate that the red deer provides a good model for studying bone specialisation. This work will provide the basis for further investigation into collagen as a controlling factor in mineral deposition.

Introduction

Energy storing tendons such as the equine superficial digital flexor tendon (SDFT) stretch and recoil with each stride and therefore require a high degree of compliance compared to tendons with a purely positional function, such as the equine common digital extensor tendon (CDET). This extra extensibility is provided by a specialised interfascicular matrix (IFM), which provides greater sliding and recoil between adjacent fascicles in energy storing tendons. However, the composition of the IFM remains largely undefined. We hypothesised that the IFM in the SDFT has a distinct composition, with a greater abundance of proteoglycans and elastin which facilitate extension and recoil.

Introduction

Regular, repeated stretching increases joint range of movement (RoM), however the physiology underlying this is not well understood. The traditional view is that increased flexibility after stretching is due to an increase in muscle length or stiffness whereas recent research suggests that increased flexibility is due to modification of tolerance to stretching discomfort/pain. If the pain tolerance theory is correct the same degree of micro-damage to muscle fibres should be demonstrable at the end of RoM before and after a period of stretch training. We hypothesise that increased RoM following a 3 weeks hamstrings static stretching exercise programme may partly be due to adaptive changes in the muscle/tendon tissue.

Introduction

Whilst all tendons connect muscle to bone, energy storing (ES) tendons, such as the equine superficial digital flexor tendon (SDFT) play an additional role, storing energy to improve locomotion efficiency. ES tendons experience significantly higher strains during locomotion than other positional tendons, such as the common digital extensor tendon (CDET). Our previous work has demonstrated that the interfascicular matrix (IFM) is more extensible in ES tendons, allowing ES tendons to stretch further during use. However, ES tendons must also recoil efficiently to perform their energy storing function. It has not been yet established if the IFM is able to recoil and recover after loading. Thus, this project aimed to determine the recoil capacity of the IFM in both the ES and positional tendons from young and old horses.

Summary Statement

Fatigue loading has an age-specific effect on tendon fascicle micro-mechanics, with greater fibre sliding in aged samples indicating a decreased mechanical integrity, and a reduced ability to withstand cyclic loading, which may partially explain the age-related risk of tendon injury.

Osteoarthritis is associated with changes to the matrix composition of subchondral bone. Raman spectroscopy has the potential to detect in vivo the molecular changes in osteoarthritic subchondral bone. The objectives were to determine the levels of mineralisation, carbonate accumulation and bone remodelling in osteoarthritic subchondral bone, which we defined as within 3mm of articular cartilage. This was compared to the proximal-compartment (10mm distal to articular cartilage) and the head-neck junction. Five osteoarthritic (average age: 76 years) and five normal cadaveric femoral heads (average age: 72 years) were scanned using peripheral quantitative computed tomography and then sectioned coronally. Raman spectroscopy was then used to scan the femoral heads. All scans were done in the plane of the longitudinal axis of the diaphysis. Cores were subsequently extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis performed to determine the levels of homotrimeric collagen. The phosphate-to-amide I ratio, from the Raman spectra, in osteoarthritic subchondral bone was significantly greater than controls (p=0.023). Within osteoarthritic specimens, the phosphate-to-amide I ratio increased proximally. The density in osteoarthritic subchondral bone was 89mg/cm3 higher than controls (p=0.022), and 494mg/cm3 higher than the osteoarthritic proximal-compartment (p<0.001). Moreover, carbonate substitution into the apatite crystals decreased in osteoarthritic specimens. The carbonate-to-amide I ratio was highest in osteoarthritic subchondral bone. Furthermore, the median α1-to-α2-chain ratio in osteoarthritic specimens was 2:1. The changes found in subchondral bone are important in the pathogenesis of osteoarthritis. This study shows that Raman spectroscopy can detect differences between osteoarthritic specimens and controls, further supporting its potential use in diagnosing bone disorders.

Osteoarthritis (OA) is a common, debilitating joint disease involving degeneration of cartilage and bone. It has been suggested that subtle changes in the molecular structure of subchondral bone may precede cartilaginous changes in the osteoarthritic joint. To explore these changes Raman spectroscopy was employed as a diagnostic tool. Raman spectroscopy measures inelastic scattered laser light produced when photons interact with chemical materials. Resultant changes in wavelength form spectra relative to the chemical composition of the given sample: with bone this includes the mineral and matrix components, unlike conventional X-rays. The aim of our study is to explore the hypothesis: Changes in matrix composition of osteoarthritic subchondral bone can be detected with Raman spectroscopy. pQCT and Raman spectroscopy were employed to determine the bone mineral density (BMD) and bone quality, respectively. Ten medial compartment OA and five control (non-OA) tibial plateaus were interrogated and analysis performed to compare OA to control, and medial to lateral compartments. The subchondral bone of the medial OA compartments had higher BMD (p=0.05) and thickness compared to lateral and control samples. Spectral analysis revealed there is no difference between the medial and lateral compartments within either cohort. However, there is a statistically significant (p=0.02) spectral difference between the OA and control specimens. The detection of bone matrix changes in osteoarthritis using Raman spectroscopy contributes to the understanding of the biochemical signature of subchondral bone across diseased and control tibial plateaus. This technique has potential to shed light on the role of bone in osteoarthritis.

Tendons and ligaments are similar in composition but differ in function. Simple anatomical definitions do not reflect the fact individual tendons and ligaments have unique properties due to their adaptation to a specific role. The patellar tendon is a structure of particular clinical interest. A null hypothesis was declared stating that the patellar tendon is not significantly different in terms of matrix composition and collagen fibril diameter to other tendons.

The lateral and medial collateral ligaments (LCL, MCL), anterior and posterior cruciate ligaments (ACL, PCL), together with the long digital extensor, superfi-cial digital extensor and patellar tendons (LDET, SDFT, PT) were harvested from 3 cadaveric ovine hindlimbs. The extracellular matrix was assessed in terms of water, collagen and total sulphated glycosaminoglycan (GAG) content. The organisation of the collagen component was determined by an ultrastructural analysis of collagen fibril diameter distributions using electron microscopy, together with values for the collagen fibril index (CFI) and mass-average diameter (MAD).

There were significant differences between ligaments and tendons. The PT had a bimodal collagen fibril diameter distribution with CFI72.9%, MAD 202nm, water content 53.1%, GAG content 2.3 g/mg and collagen content 73.7%, which was not significantly different from the other tendons.

The results of this study support the null hypothesis suggesting that the patellar tendon is similar to other tendons and demonstrate that tendons have different characteristics to ligaments.

Introduction Epidemiological studies have revealed that the incidence of Achilles tendon rupture is increasing and is especially high in middle age. Similarly, in horses, the superficial digital flexor tendon (SDFT) is often injured with older horses being most at risk. Tendons which play a role in elastic energy storage, such as the human Achilles tendon and equine SDFT, are much more susceptible to degenerative change and subsequent rupture than non-energy storing positional tendons, such as the human anterior tibialis tendon and the equine common digital extensor tendon (CDET). These energy storing tendons are required to operate with small safety margins and are likely therefore to incur high levels of micro-damage. The ability to repair micro-damage depends on the capacity for matrix turnover which requires both the capability to synthesise and degrade matrix components. In a previous study we have shown that the levels of matrix degrading enzymes (matrix metalloproteinases) differ significantly between the SDFT and CDET (Faram et al., 2004, Proc. BORS, Bristol) and that some matrix metalloproteinases (MMP-3) increase significantly with increasing age (Eissa et al., 2004, Proc. BSMB, Bristol). The aim of this study was to test the hypothesis that MMP derived fragments of collagen resulting from collagen breakdown are present at higher levels in the energy storing SDFT than the CDET and increase significantly with increasing age.

Methods The SDFT and CDET were harvested from the left forelimb of horses (n=20) ranging in age from skeletal maturity to senescence (5 – 30 years) and tissue from the mid-metacarpal level of each tendon analysed. A commercially available radioimmunoassay kit (Oxford Biosystems) was used to measure levels of the C-terminal telopeptide of type I collagen (ICTP). In addition, DNA levels were measured by a fluorometric assay using Hoechst 33258 dye to give an indication of tissue cellularity and collagen-linked fluorescence was measured to give an indication of the age of the collagen in the matrix. Statistical significance (p = 0.05) was evaluated using a general linear model (SPSS software) to compare tendons (SDFT and CDET) and to determine changes with age.

Results The levels of ICTP were approximately four times higher (p=0.001) in the CDET compared to the SDFT and in both tendons appeared to decrease with increasing age. DNA levels were significantly (p< 0.001) higher in the SDFT than the CDET and these levels did not change significantly with age. The collagen-linked fluorescence was significantly (p< 0.001) higher in the SDFT than the CDET and decreased significantly (p=0.006) with age in both tendons.

Discussion The results demonstrate that the SDFT is more cellular than the CDET and may therefore be expected to be more metabolically active. Contrary to this, collagen-linked fluorescence is higher in the SDFT suggesting that the matrix is older and furthermore the levels of collagen fragments are much lower in the SDFT suggesting that the collagen within the matrix is turned over more rapidly in the CDET than the SDFT. The changes in collagen-linked fluorescence and ICTP levels suggest than collagen turnover decreases with ageing and low turnover may be responsible for SDFT degneration.

Introduction: Energy storing tendons, such as the human Achilles tendon, suffer a much higher incidence of rupture than non- energy storing positional tendons, such as the anterior tibialis tendon. Similarly, in the horse partial rupture of the energy storing superficial digital flexor tendon (SDFT) and suspensory ligament (SL) occurs much more frequently than to the deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET) which are not involved in energy storage. In order to function effectively, energy storing tendons experience strains during high speed locomotion which are much closer to failure strain than non-energy storing tendons. Therefore, these tendons are likely to sustain high levels of microdamage, hence cell metabolism may also be higher in order to repair damage and maintain matrix integrity. Maintenance of the matrix requires not only synthesis of new matrix components but also degradation of matrix macromolecules which is achieved, in part, by a family of matrix metalloproteinase enzymes (MMPs). In this study we test the hypothesis that the energy storing equine SDFT and SL which are prone to degenerative changes have higher levels of MMP2 and MMP9 than the positional DDFT and CDET that are rarely injured.

Methods: Tendons (SDFT, DDFT, SL, CDET) were harvested from the distal part of the forelimbs of 18 month old Thoroughbred horses (n = 12). Tissue from the mid-metacarpal region of each tendon was snap frozen, lyophilised, powdered and MMPs extracted. Gelatin zymography was used to determine levels of the pro and active forms of the gelatinase enzymes, MMP2 and MMP 9. Proteolytic activity (units per mg dry weight tissue) was quantified based on densitometry measurements and standardised between gels using an equine neutrophil MMP extract. Statistical significance was evaluated using a general linear model (SPSS software).

Results: The main activity observed in all tendon samples was that of proMMP2. Quantification showed that the energy storing SDFT (23.4 ± 10.95) and SL (18.9 ± 5.3) had significantly higher levels than the non-energy storing DDFT (2.90 ± 0.99) and CDET (4.06 ± 2.06). Active MMP2 levels were lower than the pro form and were not sufficient to quantify. However, there appeared to be more in the energy storing structures compared with the non energy storing structures. MMP9 activity was detected in some samples. A higher number of the CDET extracts contained MMP9 activity compared to extracts from the other structures.

Discussion: The results of this study show higher levels of MMPs in energy storing structures than in non-energy storing structures. This suggests that there may be an increased demand for repair of micro-damage in these tendons and hence an increased capacity for matrix degradation. Previous studies on energy storing structures in the horse have shown that they do not undergo adaptive hypertrophy or a change in structural architecture in response to mechanical demand, unlike non-energy storing structures. The results of this study indicate that this lack of adaptation in energy storing structures is not due to a general deficiency in cell activity but may be a means of preventing increase in tendon stiffness and a subsequent decrease in efficiency. In order to maintain tendon integrity MMP activity must be matched by mechanisms to inhibit activity and/or to synthesize new matrix components. Degeneration may therefore occur when there is an imbalance between these processes.

Introduction: Different tendons and ligaments have a specific elasticity which relates to their role in joint movement and locomotion. To ensure an optimal functional outcome it is essential that this mechanical property is restored following surgical procedures to repair or replace damaged tendons and ligaments. This demands appropriate selection of an autograft or artificial construct aided by an understanding of how molecular composition and morphology determines the stiffness of the material. This study tests the hypothesis that tendons with a higher elastic modulus (stiffer) have larger collagen fibril diameters and lower water and sulphated glycosaminoglycan (GAG) contents.

Methods: The superficial digital flexor tendon (SDFT, 30 pairs), deep digital flexor tendon (DDFT, 6 pairs), suspensory ligament (SL, 6 pairs) and common digital extensor tendon (CDET, 6 pairs) were collected from the forelimbs of horses aged 2–23 years destroyed for reasons other than tendon injuries. Left limb tendons were tested to failure in a hydraulic materials testing machine (Dartec) following measurement of cross sectional area. Collagen fibril diameters, water content and sulphated GAG content were measured in tendon tissue from the right limb. Statistical significance was evaluated using Spearman’s correlation and a general linear model (SPSS software).

Results: The elastic modulus was significantly (p< 0.001) different between the different structures and showed a significant positive correlation with the mass average collagen fibril diameter (MAFD) for the different structures and within the SDFT (FIG. I). The water content showed a significant negative correlation with elastic modulus and significant positive correlation with GAG content.

Discussion and Conclusion: Tendons composed of a stiffer material have larger collagen fibril diameters which are associated with lower water and GAG contents. These characteristics should be considered when choosing suitable replacements in tendon reconstruction procedures. Future work to determine the mechanisms that control collagen fibril diameters and water content will aid in the design of bioengineered constructs.