Matteo Berni

Introduction: Degenerative alterations in proteoglycans content and collagen structure compromise the mechanical integrity of articular cartilage and, therefore, its viscoelastic properties. Early detection of these subtle alterations is a prerequisite for enabling intervention with promising treatments under development. Although magnetic resonance imaging provides valuable compositional information, its limited spatial resolution restricts its effectiveness in evaluating thin, soft tissues such as articular cartilage. Contrast-enhanced high-resolution peripheral quantitative computed tomography (HR-pQCT) offers an optimal compromise between spatial resolution and radiation exposure, enabling high-resolution evaluation of the cartilage layer and potentially overcoming this limitation. This study investigated the feasibility of using HR-pQCT enhanced with the cationic contrast agent CA4+ to determine the proteoglycan gradient and thickness of articular cartilage from bovine stifle joints. Methods: The accuracy of thickness measurements was assessed by comparison with micro-computed tomography (μCT) measurements. It also investigated whether the local CT number (mean HU value of the articular cartilage, either uncorrected or corrected to the HU value of CA4+ bath) correlates with tissue mechanical competence. The latter was evaluated in terms of instantaneous, stress-relaxation and equilibrium responses by indenting the cartilage layer with a spherical indenter up to 15%, a value representative of the level of tissue deformation occurring in vivo . Results and discussion: A depth-wise gradient of CT number was distinguishable within the articular cartilage in contrast-enhanced HR-pQCT, allowing in post-processing the subdivision of cartilage into its three main layers. Additionally, contrast-enhanced imaging enabled the accurate quantification of articular cartilage thickness. Significant relationships of varying strength, assessed using Spearman’s rank correlation coefficient (ρ), were found between thickness and instantaneous elastic response (ρ = −0.85), stress-relaxation behaviour (ρ = −0.61), and equilibrium elastic response (ρ = −0.38) of articular cartilage. The same relationship with CT number showed comparable strength. These results, achieved through investigation of healthy bovine articular cartilage, support extending the proposed approach to human articular cartilage. The proposed approach, based on high-resolution X-ray scanner imaging enhanced with a cationic contrast agent, may have the potential to discriminate early-degenerated tissue by capturing subtle local alterations in thickness or CT number gradients. This could reveal changes in the relative thicknesses of the superficial, middle, and deep layers, across the articular surface, while also providing an indication of local alterations to the mechanical competence of the articular cartilage.

X-ray imaging of articular cartilage could be a breakthrough for the early diagnosis of tissue degeneration. This approach relies on radiopaque contrast agents to enhance the visualization of soft tissues. The potential impact of contrast agents on the mechanical response of articular cartilage should be considered in the frame of both clinical and research applications. Attention has been drawn to a solution containing molecules with six iodine atoms and four positive charges (CA4+), which has been shown to improve the X-ray visibility of articular cartilage. This study aimed to determine the effects of a CA4+ solution on tissues’ mechanical properties. An experimental pipeline based on indentation tests was applied to paired samples of articular cartilage before and after the immersion in either CA4+ or phosphate-buffered saline solution, maintained at a temperature of 22 ± 2 °C, for 22 h to determine the differences in instantaneous, viscous, and equilibrium responses between the articular cartilage of the two groups. The 22 h immersion of articular cartilage in either CA4+ or phosphate-buffered saline solution had a significant detrimental effect on the overall response, including the instantaneous, viscous, and equilibrium responses, of the articular cartilage. However, this detrimental effect was greater with exposure to the CA4+ solution. Specifically, the articular cartilage was found to be less stiff in both the instantaneous response (approximately −25%) and the equilibrium response (approximately −38%). The softening effect could be attributable to an alteration of the interaction between the proteoglycans of articular cartilage, induced by the positive charges within the CA4+ contrast agent. Further investigations are needed to elucidate whether this hypothesized mechanism is reversible.

Understanding the microstructure of fibrous tissues, like ligaments, is crucial due to their nonlinear stress-strain behavior from unique fiber arrangements. This study introduces a new method to analyze the relationship between the microstructure and function of anterior cruciate ligaments (ACL). We tested the procedure on two ACL samples, one from a healthy individual and one from an osteoarthritis patient, using a custom tensioning device within a micro-CT scanner. The samples were stretched and scanned at various strain levels (namely 0%, 1%, 2%, 3%, 4%, 6%, 8%) to observe the effects of mechanical stress on the microstructure. The micro-CT images were processed to identify and map fibers, assessing their orientations and volume fractions. A probabilistic mathematical model was then proposed to relate the geometric and structural characteristics of the ACL to its mechanical properties, considering fiber orientation and thickness. Our feasibility test indicated differences in mechanical behavior, fiber orientation, and volume distribution between ligaments of different origins. These indicative results align with existing literature, validating the proposed methodology. However, further research is needed to confirm these preliminary observations. Overall, our comprehensive methodology shows promise for improving ACL diagnosis and treatment and for guiding the creation of tissue-engineered grafts that mimic the natural properties and microstructure of healthy tissue, thereby enhancing integration and performance in biomedical applications.

In the development of bone graft substitutes, a fundamental step is the use of scaffolds with adequate composition and architecture capable of providing support in regenerative processes both on the tissue scale, where adequate resistance to mechanical stress is required, as well as at the cellular level where compliant chemical–physical and mechanical properties can promote cellular activity. In this study, based on a previous optimization study of this group, the potential of a three-dimensional construct based on polycaprolactone (PCL) and a novel biocompatible Mg- and Sr-containing glass named BGMS10 was explored. Fourier-transform infrared spectroscopy and scanning electron microscopy showed the inclusion of BGMS10 in the scaffold structure. Mesenchymal stem cells cultured on both PCL and PCL-BGMS10 showed similar tendencies in terms of osteogenic differentiation; however, no significant differences were found between the two scaffold types. This circumstance can be explained via X-ray microtomography and atomic force microscopy analyses, which correlated the spatial distribution of the BGMS10 within the bulk with the elastic properties and topography at the cell scale. In conclusion, our study highlights the importance of multidisciplinary approaches to understand the relationship between design parameters, material properties, and cellular response in polymer composites, which is crucial for the development and design of scaffolds for bone regeneration.

The damping system ensured by the osteochondral (OC) unit is essential to deploy the forces generated within load-bearing joints during locomotion, allowing furthermore low-friction sliding motion between bone segments. The OC unit is a multi-layer structure including articular cartilage, as well as subchondral and trabecular bone. The interplay between the OC tissues is essential in maintaining the joint functionality; altered loading patterns can trigger biological processes that could lead to degenerative joint diseases like osteoarthritis. Currently, no effective treatments are available to avoid degeneration beyond tissues’ recovery capabilities. A thorough comprehension on the mechanical behaviour of the OC unit is essential to (i) soundly elucidate its overall response to intra-articular loads for developing diagnostic tools capable of detecting non-physiological strain levels, (ii) properly evaluate the efficacy of innovative treatments in restoring physiological strain levels, and (iii) optimize regenerative medicine approaches as potential and less-invasive alternatives to arthroplasty when irreversible damage has occurred. Therefore, the leading aim of this review was to provide an overview of the state-of-the-art—up to 2022—about the mechanical behaviour of the OC unit. A systematic search is performed, according to PRISMA standards, by focusing on studies that experimentally assess the human lower-limb joints’ OC tissues. A multi-criteria decision-making method is proposed to quantitatively evaluate eligible studies, in order to highlight only the insights retrieved through sound and robust approaches. This review revealed that studies on human lower limbs are focusing on the knee and articular cartilage, while hip and trabecular bone studies are declining, and the ankle and subchondral bone are poorly investigated. Compression and indentation are the most common experimental techniques studying the mechanical behaviour of the OC tissues, with indentation also being able to provide information at the micro- and nanoscales. While a certain comparability among studies was highlighted, none of the identified testing protocols are currently recognised as standard for any of the OC tissues. The fibril-network-reinforced poro-viscoelastic constitutive model has become common for describing the response of the articular cartilage, while the models describing the mechanical behaviour of mineralised tissues are usually simpler (i.e., linear elastic, elasto-plastic). Most advanced studies have tested and modelled multiple tissues of the same OC unit but have done so individually rather than through integrated approaches. Therefore, efforts should be made in simultaneously evaluating the comprehensive response of the OC unit to intra-articular loads and the interplay between the OC tissues. In this regard, a multidisciplinary approach combining complementary techniques, e.g., full-field imaging, mechanical testing, and computational approaches, should be implemented and validated. Furthermore, the next challenge entails transferring this assessment to a non-invasive approach, allowing its application in vivo, in order to increase its diagnostic and prognostic potential.

In the context of a large animal model of early osteoarthritis (OA) treated by orthobiologics, the purpose of this study was to reveal relations between articular tissues structure/composition and cartilage viscoelasticity. Twenty-four sheep, with induced knee OA, were treated by mesenchymal stem cells in various preparations—adipose-derived mesenchymal stem cells (ADSCs), stromal vascular fraction (SVF), and amniotic endothelial cells (AECs)—and euthanized at 3 or 6 months to evaluate the (i) biochemistry of synovial fluid; (ii) histology, immunohistochemistry, and histomorphometry of articular cartilage; and (iii) viscoelasticity of articular cartilage. After performing an initial analysis to evaluate the correlation and multicollinearity between the investigated variables, this study used machine learning (ML) models—Variable Selection Using Random Forests (VSURF) and Extreme Gradient Boosting (XGB)—to classify variables according to their importance and employ them for interpretation and prediction. The experimental setup revealed a potential relation between cartilage elastic modulus and cartilage thickness (CT), synovial fluid interleukin 6 (IL6), and prostaglandin E2 (PGE2), and between cartilage relaxation time and CT and PGE2. SVF treatment was the only limit on the deleterious OA effect on cartilage viscoelastic properties. This work provides indications to future studies aiming to highlight these and other relationships and focusing on advanced regeneration targets.

The in-vivo quantification of knee motion in physiological loading conditions is paramount for the understanding of the joint’s natural behavior and the comprehension of articular disorders. Dynamic MRI (DMRI) represents an emerging technology that makes it possible to investigate the functional interaction among all the joint tissues without risks for the patient. However, traditional MRI scanners normally offer a reduced space of motion, and complex apparatus are needed to load the articulation, due to the horizontal orientation of the scanning bed. In this study, we present an experimental and computational procedure that combines an open, weight-bearing MRI scanner with an original registration algorithm to reconstruct the three-dimensional kinematics of the knee from DMRI, thus allowing the investigation of knee deep-flexion under physiological loads in space. To improve the accuracy of the procedure, an MR-compatible rig has been developed to guide the knee flexion of the patient. We tested the procedure on three volunteers. The overall rotational and positional accuracy achieved are 1.8° ± 1.4 and 1.2 mm ± 0.8, respectively, and they are sufficient for the characterization of the joint behavior under load.

Tissue engineering aims at developing complex composite scaffolds for articular cartilage repair. These scaffolds must exhibit a mechanical behavior similar to the whole osteochondral unit. In situ spherical indentation allows us to map the mechanical behavior of articular cartilage, avoiding removal of the underlying bone tissue. Little is known about the impact of grid spacing, indenter diameter, and induced deformation on the cartilage response to indentation. We investigated the impact of grid spacing (range: a to 3a, where a is the radius of the contact area between cartilage and indenter), indenter diameter (range: 1 to 8 mm), and deformation induced by indentation (constant indentation depth versus constant nominal deformation) on cartilage response. The bias induced by indentations performed in adjacent grid points was minimized with a 3a grid spacing. The cartilage response was indenter-dependent for diameters ranging between 1 and 6 mm with a nominal deformation of 15%. No significant differences were found using 6 mm and 8 mm indenters. Six mm and 8 mm indenters were used to map human articular cartilage with a grid spacing equal to 3a. Instantaneous elastic modulus E₀ was calculated for constant indentation depth and constant nominal deformation. E₀ value distribution did not change significantly by switching the two indenters, while dispersion decreased by 5–6% when a constant nominal deformation was applied. Such an approach was able to discriminate changes in tissue response due to doubling the indentation rate. The proposed procedure seems to reduce data dispersion and properly determine cartilage mechanical properties to be compared with those of complex composite scaffolds.

Background: Anterior cruciate ligament (ACL) tear represents a common orthopedic traumatic issue that often leads to an early development of osteoarthritis. To improve the diagnostic and prognostic techniques involved in the assessment of the joint after the trauma and during the healing process, the present work proposes a multi-parametric approach that aims to investigate the relationship between joint function and soft tissue status before and after ACL reconstruction. Methods: Thirteen consecutive patients who underwent ACL reconstruction were preliminarily enrolled in this study. Joint laxity assessment as well as magnetic resonance imaging with T2 mapping were performed in the pre-operative stage, at four and 18 months after surgery to acquire objective information to correlate knee function and soft tissue condition. Results: Correlations were found between graft and cartilage T2 signal, suggesting an interplay between these tissues within the knee joint. Moreover, graft maturation resulted in being connected to joint laxity, as underlined by the correlation between the graft T2 signal and the temporal evolution of knee function. Conclusions: This preliminary study represents a step forward in assessing the effects of ACL graft maturation on knee biomechanics, and vice versa. The presented integrated framework underlines the possibility to quantitatively assess the impact of ACL reconstruction on trauma recovery and cartilage homeostasis. Moreover, the reported findings—despite the preliminary nature of the clinical impacts—evidence the possibility of monitoring the surgery outcomes using a multi-parametric prognostic investigation tool.