In vitro development of a muscle-tendon junction construct using decellularised extracellular matrix: Effect of cyclic tensile loading.

The muscle tendon junction (MTJ) plays a crucial role in transmitting the force generated by muscles to the tendon and then to the bone. Injuries such as tears and strains frequently happen at the MTJ, where the regenerative process is limited due to poor vascularization and the complex structure of the tissue. Current solutions for a complete tear at the MTJ have not been successful and therefore, the development of a tissue-engineered MTJ may provide a more effective treatment. In this study, decellularised extracellular matrix (DECM) derived from sheep MTJ was used to provide a scaffold for the MTJ with the relevant mechanical properties and differentiation cues such as the relase of growth factors. Human mesenchymal stem cells (MSCs) were seeded on DECM and 10 % cyclic strain was applied using a bioreactor. MSCs cultured on DECM showed significantly higher gene and protein expression of MTJ markers such as collagen 22, paxillin and talin, than MSCs in 2D culture. Although collagen 22 protein expression was higher in the cells with strain than without strain, reduced gene expression of other MTJ markers was observed when the strain was applied. DECM combined with 10 % strain enhanced myogenic differentiation, while tenogenic differentiation was reduced when compared to static cultures of MSCs on DECM. For the first time, these results showed that DECM derived from the MTJ can induce MTJ marker gene and protein expression by MSCs, however, the effect of strain on the MTJ development in DECM culture needs further investigation.

Evaluation of Corrosion Performance of AZ31 Mg Alloy in Physiological and Highly Corrosive Solutions.

Resorbable Mg and Mg alloys have gained significant interest as promising biomedical materials. However, corrosion of these alloys can lead to premature reduction in their mechanical properties, and therefore their corrosion rate needs to be controlled. The aim of this study is to select an appropriate environment where the effects of coatings on the corrosion rate of the underlying Mg alloy can be discerned and measured in a relatively short time period. The corrosion resistance of uncoated AZ31 alloy in different solutions [Hank's Balanced Salt Solution, 1× phosphate buffered solution (PBS), 4× PBS, 0.9%, 3.5%, and 5 M sodium chloride (NaCl)] was determined by measuring the weight loss over a 2 week period. Upon exposure to physiological solutions, the uncoated AZ31 alloys exhibited a variable weight increase of 0.4 ± 0.4%. 3.5% and 5 M NaCl solutions led to 0.27 and 9.7 mm/year corrosion rates, respectively, where the compositions of corrosion products from AZ31 in all saline solutions were similar. However, the corrosion of the AZ31 alloy when coated by electrochemical oxidation with two phosphate coatings, one containing fluorine (PF) and another containing both fluorine and silica (PFS), showed 0.3 and 0.25 mm/year corrosion rates, respectively. This is more than 30 times lower than that of the uncoated alloy (7.8 mm/year), making them promising candidates for corrosion protection in severe corrosive environments. Cross-sections of the samples showed that the coatings protected the alloy from corrosion by preventing access of saline to the alloy surface, and this was further reinforced by corrosion products from both the alloy and the coatings forming an additional barrier. The information in this paper provides a methodology for evaluating the effects of coatings on the rate of corrosion of magnesium alloys.

Full-Field Strain Measurements of the Muscle-Tendon Junction Using X-ray Computed Tomography and Digital Volume Correlation.

We report, for the first time, the full-field 3D strain distribution of the muscle-tendon junction (MTJ). Understanding the strain distribution at the junction is crucial for the treatment of injuries and to predict tear formation at this location. Three-dimensional full-field strain distribution of mouse MTJ was measured using X-ray computer tomography (XCT) combined with digital volume correlation (DVC) with the aim of understanding the mechanical behavior of the junction under tensile loading. The interface between the Achilles tendon and the gastrocnemius muscle was harvested from adult mice and stained using 1% phosphotungstic acid in 70% ethanol. In situ XCT combined with DVC was used to image and compute strain distribution at the MTJ under a tensile load (2.4 N). High strain measuring 120,000 µÎµ, 160,000 µÎµ, and 120,000 µÎµ for the first principal stain (εp1), shear strain (γ), and von Mises strain (εVM), respectively, was measured at the MTJ and these values reduced into the body of the muscle or into the tendon. Strain is concentrated at the MTJ, which is at risk of being damaged in activities associated with excessive physical activity.

Comparison of two contrast-enhancing staining agents for use in X-ray imaging and digital volume correlation measurements across the cartilage-bone interface.

OBJECTIVE: The pathogenesis of osteoarthritis (OA) is associated with subchondral bone changes, which is linked to abnormal strain distribution in the overlying articular cartilage. This highlights the importance of understanding mechanical interaction at the cartilage-bone interface. The aim of this study is to compare solutions of two contrast-enhancing staining agents (CESA) for combining high-resolution Contrast-Enhanced X-ray microfocus Computed Tomography (CECT) with Digital Volume Correlation (DVC) for full-field strain measurements at the cartilage-bone interface. DESIGN: Bovine osteochondral plugs were stained with phosphotungstic acid (PTA) in 70% ethanol or 1:2 hafnium-substituted Wells-Dawson polyoxometalate (Hf-WD POM) in PBS. Mechanical properties were assessed using micromechanical probing and nanoindentation. Strain uncertainties (from CECT data) were evaluated following two consecutive unloaded scans. Residual strains were computed following unconfined compression (ex situ) testing. RESULTS: PTA and Hf-WD POM enabled the visualisation of structural features in cartilage, allowing DVC computation on the CECT data. Residual strains up to ∼10,000 µÉ› were detected up to the tidemark. Nanoindentation showed that PTA-staining caused an average ∼6-fold increase in articular cartilage stiffness, a ∼19-fold increase in reduced modulus and ∼7-fold increase in hardness, whereas Hf-WD POM-stained specimens had mechanical properties similar to pre-stain tissue. Micromechanical probing showed a 77% increase in cartilage surface stiffness after PTA-staining, in comparison to a 16% increase in stiffness after staining with Hf-WD POM. CONCLUSION: Hf-WD POM is a more suitable CESA solution compared to PTA for CECT imaging combined with DVC as it allowed visualisation of structural features in the cartilage tissue whilst more closely maintaining tissue mechanical properties.

Early Degenerative Changes in a Spontaneous Osteoarthritis Model Assessed by Nanoindentation.

Understanding early mechanical changes in articular cartilage (AC) and subchondral bone (SB) is crucial for improved treatment of osteoarthritis (OA). The aim of this study was to develop a method for nanoindentation of fresh, unfixed osteochondral tissue to assess the early changes in the mechanical properties of AC and SB. Nanoindentation was performed throughout the depth of AC and SB in the proximal tibia of Dunkin Hartley guinea pigs at 2 months, 3 months, and 2 years of age. The contralateral tibias were either histologically graded for OA or analyzed using immunohistochemistry. The results showed an increase in the reduced modulus (Er) in the deep zone of AC during early-stage OA (6.0 ± 1.75 MPa) compared to values at 2 months (4.04 ± 1.25 MPa) (*** p < 0.001). In severe OA (2-year) specimens, there was a significant reduction in Er throughout the superficial and middle AC zones, which correlated to increased ADAMTS 4 and 5 staining, and proteoglycan loss in these regions. In the subchondral bone, a 35.0% reduction in stiffness was observed between 2-month and 3-month specimens (*** p < 0.001). The severe OA age group had significantly increased SB stiffness of 36.2% and 109.6% compared to 2-month and 3-month-old specimens respectively (*** p < 0.001). In conclusion, this study provides useful information about the changes in the mechanical properties of both AC and SB during both early- and late-stage OA and indicates that an initial reduction in stiffness of the SB and an increase in stiffness in the deep zone of AC may precede early-stage cartilage degeneration.

Development of a method to investigate strain distribution across the cartilage-bone interface in guinea pig model of spontaneous osteoarthritis using lab-based contrast enhanced X-ray-computed tomography and digital volume correlation.

OBJECTIVE: Strain changes at the cartilage-bone interface play a crucial role in osteoarthritis (OA) development. Contrast-Enhanced X-ray Computed Tomography (CECT) and Digital Volume Correlation (DVC) can measure 3D strain changes at the osteochondral interface. Using lab-based CT systems it is often difficult to visualise soft tissues such as articular cartilage without staining to enhance contrast. Contrast-Enhancing Staining Agents (CESAs), such as Phosphotungstic Acid (PTA) in 70% ethanol, can cause tissue shrinkage and alter tissue mechanics. The aims of this study were, firstly, to assess changes to the mechanical properties of osteochondral tissue after staining with a PTA/PBS solution, and secondly, to visualise articular cartilage during loading and with CECT imaging in order to compare strain across the interface in both healthy and OA joints using DVC. DESIGN: Nanoindentation was used to assess changes to mechanical properties in articular cartilage and subchondral bone before and after staining. Hindlimbs from Dunkin-Hartley guinea pigs were stained with 1% PTA/PBS at room temperature for 6 days. Two consecutive CECT datasets were acquired for DVC error analysis. In-situ compression with a load corresponding to 2x body weight was applied, the specimen was re-imaged, and DVC was performed between the pre- and post-load tomograms. RESULTS: Nanoindentation before and after PTA/PBS staining showed similar cartilage stiffness (p < 0.05), however, staining significantly decreased the stiffness of subchondral bone (∼9-fold; p = 0.0012). In severe OA specimens, third principal/compressive (εp3) strain was 141.7% higher and shear strain (γ) was 98.2% higher in tibial articular cartilage compared to non-OA (2 - month) specimens. A 23.1% increase in third principal stain strain and a 54.5% significant increase in the shear (γ) strain (p = 0.0027) was transferred into the mineralised regions of calcified cartilage and subchondral bone in severe OA specimens. CONCLUSIONS: These results indicate the suitability of PTA in PBS as a contrast agent for the visualisation of cartilage during CECT imaging and allowed DVC computation of strain across the cartilage-bone interface. However, further research is needed to address the reduction in stiffness of subchondral bone after incubation in PBS.

Effect of radiation-induced damage of trabecular bone tissue evaluated using indentation and digital volume correlation.

Exposure to X-ray radiation for an extended amount of time can cause damage to the bone tissue and therefore affect its mechanical properties. Specifically, high-resolution X-ray Computed Tomography (XCT), in both synchrotron and lab-based systems, has been employed extensively for evaluating bone micro-to-nano architecture. However, to date, it is still unclear how long exposures to X-ray radiation affect the mechanical properties of trabecular bone, particularly in relation to lab-XCT systems. Indentation has been widely used to identify local mechanical properties such as hardness and elastic modulus of bone and other biological tissues. The purpose of this study is therefore, to use indentation and XCT-based investigative tools such as digital volume correlation (DVC) to assess the microdamage induced by long exposure of trabecular bone tissue to X-ray radiation and how this affects its local mechanical properties. Trabecular bone specimens were indented before and after X-ray exposures of 33 and 66 h, where variation of elastic modulus was evaluated at every stage. The resulting elastic modulus was decreased, and micro-cracks appeared in the specimens after the first long X-ray exposure and crack formation increased after the second exposure. High strain concentration around the damaged tissue exceeding 1% was also observed from DVC analysis. The outcomes of this study show the importance of designing appropriate XCT-based experiments in lab systems to avoid degradation of the bone tissue mechanical properties due to radiation and these results will help to inform future studies that require long X-ray exposure for in situ experiments or generation of reliable subject-specific computational models.

Poly-ε-Caprolactone 3D-Printed Porous Scaffold in a Femoral Condyle Defect Model Induces Early Osteo-Regeneration.

Large bone reconstruction following trauma poses significant challenges for reconstructive surgeons, leading to a healthcare burden for health systems, long-term pain for patients, and complex disorders such as infections that are difficult to resolve. The use of bone substitutes is suboptimal for substantial bone loss, as they induce localized atrophy and are generally weak, and unable to support load. A combination of strong polycaprolactone (PCL)-based scaffolds, with an average channel size of 330 µm, enriched with 20% w/w of hydroxyapatite (HA), ß-tricalcium phosphate (TCP), or Bioglass 45S5 (Bioglass), has been developed and tested for bone regeneration in a critical-size ovine femoral condyle defect model. After 6 weeks, tissue ingrowth was analyzed using X-ray computed tomography (XCT), Backscattered Electron Microscopy (BSE), and histomorphometry. At this point, all materials promoted new bone formation. Histological analysis showed no statistical difference among the different biomaterials (p > 0.05), but PCL-Bioglass scaffolds enhanced bone formation in the center of the scaffold more than the other types of materials. These materials show potential to promote bone regeneration in critical-sized defects on load-bearing sites.

Effect of Demineralized Bone Matrix, Bone Marrow Mesenchymal Stromal Cells, and Platelet-Rich Plasma on Bone Tunnel Healing After Anterior Cruciate Ligament Reconstruction: A Comparative Micro-Computed Tomography Study in a Tendon Allograft Sheep Model.

BACKGROUND: The effect of demineralized bone matrix (DBM), bone marrow-derived mesenchymal stromal cells (BMSCs), and platelet-rich plasma (PRP) on bone tunnel healing in anterior cruciate ligament reconstruction (ACLR) has not been comparatively assessed. HYPOTHESIS: These orthobiologics would reduce tunnel widening, and the effects on tunnel diameter would be correlated with tunnel wall sclerosis. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 20 sheep underwent unilateral ACLR using tendon allograft and outside-in interference screw fixation. The animals were randomized into 4 groups (n = 5 per group): Group 1 received 4mL of DBM paste, group 2 received 10 million BMSCs in fibrin sealant, group 3 received 12 mL of activated leukocyte-poor platelet-rich plasma, and group 4 (control) received no treatment. The sheep were euthanized after 12 weeks, and micro-computed tomography scans were performed. The femoral and tibial tunnels were divided into thirds (aperture, midportion, and exit), and the trabecular bone structure, bone mineral density (BMD), and tunnel diameter were measured. Tunnel sclerosis was defined by a higher bone volume in a 250-µm volume of interest compared with a 4-mm volume of interest surrounding the tunnel. RESULTS: Compared with the controls, the DBM group had a significantly higher bone volume fraction (bone volume/total volume [BV/TV]) (52.7% vs 31.8%; P = .020) and BMD (0.55 vs 0.47 g/cm3; P = .008) at the femoral aperture and significantly higher BV/TV at femoral midportion (44.2% vs 32.9%; P = .038). There were no significant differences between the PRP and BMSC groups versus controls in terms of trabecular bone analysis or BMD. In the controls, widening at the femoral tunnel aperture was significantly greater than at the midportion (46.7 vs 41.7 mm2; P = .034). Sclerosis of the tunnel was common and most often seen at the femoral aperture. In the midportion of the femoral tunnel, BV/TV (r = 0.52; P = .019) and trabecular number (r S = 0.50; P = .024) were positively correlated with tunnel widening. CONCLUSION: Only DBM led to a significant increase in bone volume, which was seen in the femoral tunnel aperture and midportion. No treatment significantly reduced bone tunnel widening. Tunnel sclerosis in the femoral tunnel midportion was correlated significantly with tunnel widening. CLINICAL RELEVANCE: DBM might have potential clinical use to enhance healing in the femoral tunnel after ACLR.

Micromechanical evaluation of cortical bone using in situ XCT indentation and digital volume correlation.

The overall mechanical behaviour of cortical bone is strongly dependant on its microstructure. X-ray computed tomography (XCT) has been widely used to identify the microstructural morphology of cortical tissue (i.e. pore network, Haversian and Volkmann's canals). However, the connection between microstructure and mechanics of cortical bone during plastic deformation is unclear. Hence, the purpose of this study is to provide an in-depth evaluation of the interplay of plastic strain building up in relation to changes in the canal network for cortical bone tissue. In situ step-wise XCT indentation was used to introduce a localised load on the surface of the tissue and digital volume correlation (DVC) was employed to assess the three-dimensional (3D) full-field plastic strain distribution in proximity of the indent. It was observed that regions adjacent to the imprint were under tensile strain, whereas the volume underneath experienced compressive strain. Canal loss and disruption was detected in regions of higher compressive strains exceeding -20000 µÎµ and crack formation occurred in specimens where Haversian canals were running parallel to the indentation tip. The results of this study outline the relationship between the micromechanical and structural behaviour of cortical bone during plastic deformation, providing information on cortical tissue fracture pathways.

Full-Field Strain Uncertainties and Residuals at the Cartilage-Bone Interface in Unstained Tissues Using Propagation-Based Phase-Contrast XCT and Digital Volume Correlation.

A deeper understanding of the cartilage-bone mechanics is fundamental to unravel onset and progression of osteoarthritis, enabling better diagnosis and treatment. The aim of this study is therefore to explore the capability of X-ray computed (XCT) phase-contrast imaging in a lab-based system to enable digital volume correlation (DVC) measurements of unstained cartilage-bone plugs from healthy adult bovines. DVC strain uncertainties were computed for both articular cartilage and mineralized tissue (calcified cartilage and subchondral bone) in the specimens at increasing propagation distances, ranging from absorption up to four times (4× such effective distance. In addition, a process of dehydration and rehydration was proposed to improve feature recognition in XCT of articular cartilage and mechanical properties of this tissue during the process were assessed via micromechanical probing (indentation), which was also used to determine the effect of long X-ray exposure. Finally, full-field strain from DVC was computed to quantify residual strain distribution at the cartilage-bone interface following unconfined compression test (ex situ). It was found that enhanced gray-scale feature recognition at the cartilage-bone interface was achieved using phase-contrast, resulting in reduced DVC strain uncertainties compared to absorption. Residual strains up to ~7000 µÎµ in the articular cartilage were transferred to subchondral bone via the calcified cartilage and micromechanics revealed the predominant effect of long phase-contrast X-ray exposure in reducing both stiffness and hardness of the articular cartilage. The results of this study will pave the way for further development and refinement of the techniques, improving XCT-based strain measurements in cartilage-bone and other soft-hard tissue interfaces.

Preservation of Bone Tissue Integrity with Temperature Control for In Situ SR-MicroCT Experiments.

Digital volume correlation (DVC), combined with in situ synchrotron microcomputed tomography (SR-microCT) mechanics, allows for 3D full-field strain measurement in bone at the tissue level. However, long exposures to SR radiation are known to induce bone damage, and reliable experimental protocols able to preserve tissue properties are still lacking. This study aims to propose a proof-of-concept methodology to retain bone tissue integrity, based on residual strain determination using DVC, by decreasing the environmental temperature during in situ SR-microCT testing. Compact and trabecular bone specimens underwent five consecutive full tomographic data collections either at room temperature or 0 °C. Lowering the temperature seemed to reduce microdamage in trabecular bone but had minimal effect on compact bone. A consistent temperature gradient was measured at each exposure period, and its prolonged effect over time may induce localised collagen denaturation and subsequent damage. DVC provided useful information on irradiation-induced microcrack initiation and propagation. Future work is necessary to apply these findings to in situ SR-microCT mechanical tests, and to establish protocols aiming to minimise the SR irradiation-induced damage of bone.