Contactless mechanical stimulation of tissue engineered constructs: Development and validation of an air-pulse device.

OBJECTIVE: Tissue engineering (TE) is the study and development of biological substitutes to restore, maintain or improve tissue function. Tissue engineered constructs (TECs) still present differences in mechanical and biological properties compared to native tissue. Mechanotransduction is the process through which mechanical stimulation triggers proliferation, apoptosis, and extracellular matrix synthesis, among other cell activities. Regarding that aspect, the effect of in vitro stimulations such as compression, stretching, bending or fluid shear stress loading modalities have been extensively studied. A fluid flow used to produce contactless mechanical stimulation induced by an air pulse could be easily achieved in vivo without altering the tissue integrity. METHODS: A new air-pulse device for contactless and controlled mechanical simulation of a TECs was developed and validated in this study conducted in the following three phases: 1) conception of the controlled air-pulse device combined with a 3D printed bioreactor; 2) experimental and numerical mechanical characterization of the air-pulse impact by digital image correlation; and 3) achieving sterility and noncytotoxicity of the air-pulse and of the 3D printed bioreactor using a novel dedicated sterilization process. RESULTS: We demonstrated that the treated PLA (polylactic acid) was noncytotoxic and did not influence cell proliferation. An ethanol/autoclaved sterilization protocol for 3D printed objects in PLA has been developed in this study, enabling the use of 3D printing in cell culture. A numerical twin of the device was developed and experimentally characterized by digital image correlation. It showed a coefficient of determination R2 = 0.98 between the numerical and averaged experimental surface displacement profiles of the TEC substitute. CONCLUSION: The results of the study assessed the noncytotoxicity of PLA for prototyping by 3D printing the homemade bioreactor. A novel sterilization process for PLA was developed in this study based on a thermochemical process. A numerical twin using fluid-structure interaction method has been developed to investigate the micromechanical effects of air pulses inside the TEC, which cannot all be measured experimentally, for instance, wave propagation generated during the air-pulse impact. The device could be used to study the cell response to contactless cyclic mechanical stimulation, particularly in TEC with fibroblasts, stromal cells and mesenchymal stem cells, which have been shown to be sensitive to the frequency and strain level at the air-liquid interface.

Deep learning in veterinary medicine, an approach based on CNN to detect pulmonary abnormalities from lateral thoracic radiographs in cats.

Thoracic radiograph (TR) is a complementary exam widely used in small animal medicine which requires a sharp analysis to take full advantage of Radiographic Pulmonary Pattern (RPP). Although promising advances have been made in deep learning for veterinary imaging, the development of a Convolutional Neural Networks (CNN) to detect specifically RPP from feline TR images has not been investigated. Here, a CNN based on ResNet50V2 and pre-trained on ImageNet is first fine-tuned on human Chest X-rays and then fine-tuned again on 500 annotated TR images from the veterinary campus of VetAgro Sup (Lyon, France). The impact of manual segmentation of TR's intrathoracic area and enhancing contrast method on the CNN's performances has been compared. To improve classification performances, 200 networks were trained on random shuffles of training set and validation set. A voting approach over these 200 networks trained on segmented TR images produced the best classification performances and achieved mean Accuracy, F1-Score, Specificity, Positive Predictive Value and Sensitivity of 82%, 85%, 75%, 81% and 88% respectively on the test set. Finally, the classification schemes were discussed in the light of an ensemble method of class activation maps and confirmed that the proposed approach is helpful for veterinarians.

Micromechanical properties of the healthy canine medial meniscus.

AIMS: Knowledge of the micromechanical characteristics of the menisci is required to better understand their role within the stifle joint, improve early diagnosis of meniscal lesions, and develop new treatment and/or replacement strategies. The aim of the study was to determine the mechanical properties of the healthy medial canine meniscus and to evaluate the effect of regional (caudal, central, and cranial) and circumference (axial and abaxial) locations on these properties. METHODS: To study the micromechanical properties of the medial menisci in healthy (Beagle) dogs, the influence of regional (caudal, central, and cranial) and circumference (axial and abaxial) locations were evaluated. Nanoindentation-relaxation tests were performed to characterize the local stiffness and the viscoelastic properties at each region and specific circumference. Linear interpolation onto the indentation points was performed to establish a map of the micromechanical property heterogeneities. RESULTS: The results indicate that the cranial region was significantly stiffer and less viscous than the central and caudal regions. Within the central region the inner part (axial) was significantly stiffer than the periphery (abaxial). Within the caudal region the inner part was significantly less viscous than the periphery. CONCLUSION: Significant regional and radial variations were observed for both the stiffness and the viscoelastic properties. Moreover, a viscous behavior of the entire medial meniscus was observed (elastic fraction <0.5). These results deter the use of average elastic modulus to study the regional mechanical properties of healthy meniscus.

Mechanical alterations of the bone-cartilage unit in a rabbit model of early osteoarthrosis.

OBJECTIVE: The purpose of this study was to assess mechanical properties along with microstructural modifications of the hyaline cartilage (HC), calcified cartilage (CC) and cortical plate (Ct.Pt), in an anterior cruciate ligament transection (ACLT) model. Medial femoral condyles of six healthy rabbits (control group) and of six ACLT rabbits 6 weeks after OA induction were explanted. The zone of interest (ZOI) for all experiments was defined as the weight bearing areas of the samples. Biomechanical properties were measured using nanoindentation and morphological changes were evaluated using biphotonic confocal microscopy (BCM). RESULTS: All rabbits of the ACLT group displayed early PTOA. The results indicate an overall decrease in the mechanical properties of the HC, CC and Ct.Pt in the ACLT group. The average equilibrium modulus and elastic fraction of the HC decreased by 42% and 35%, respectively, compared with control group. The elastic moduli of the CC and Ct.Pt decreased by 37% and 16%, respectively, compared with control group. A stiffness gradient between CC and Ct.Pt appeared in the ACLT group. The irregularity of the cement line, quantified by its tortuosity in BCM images, was accentuated in the ACLT group compared with the control group. CONCLUSIONS: In the ACLT model, weight-bearing stress was modified in the ZOI. This disruption of the stress pattern induced alterations of the tissues composing the bone-cartilage unit. In term of mechanical properties, all tissues exhibited changes. The most affected tissue was the most superficial: hyaline cartilage displayed the strongest relative decrease (42%) followed by calcified cartilage (37%) and cortical plate was slightly modified (16%). This supports the hypotheses that PTOA initiates in the hyaline cartilage.

Efficacy study of two novel hyaluronic acid-based formulations for viscosupplementation therapy in an early osteoarthrosic rabbit model.

Viscosupplementation (VS) is a therapy for osteoarthrosis (OA) consisting of repetitive intra-articular injections of hyaluronic acid (HA). It is known to be clinically effective in relieving pain and increasing joint mobility by restoring joint homeostasis. In this study, the effects of two novel HA-based VS hydrogel formulations were assessed and challenged against a pure HA commercial formulation for the first time and this in a rabbit model of early OA induced by anterior cruciate ligament transection (ACLT). The first formulation tested was a hybrid hydrogel composed of HA and reacetylated chitosan, a biopolymer considered to be chondroprotective, assembled thanks to an ionic shielding. The second formulation consisted of a novel HA polymer grafted with antioxidant molecules (HA-4AR) aiming at decreasing OA oxidative stress and increasing HA retention time in the articulation. ACLT was performed on rabbits in order to cause structural changes comparable to traumatic osteoarthrosis. The protective effects of the different formulations were observed on the early phase of the pathology in a full randomized and blinded manner. The cartilage, synovial membrane, and subchondral bone were evaluated by complementary investigation techniques such as gross morphological scoring, scanning electron microscopy, histological scoring, and micro-computed tomography were used. In this study, ACLT was proven to successfully reproduce early OA articular characteristics found in humans. HA and HA-4AR hydrogels were found to be moderately protective for cartilage as highlighted by µCT. The HA-4AR was the only formulation able to decrease synovial membrane hypertrophy occurring in OA. Finally, the hybrid HA-reacetylated chitosan hydrogel surprisingly led to increased subchondral bone remodeling and cartilage defect formation. This study shows significant effects of two innovative HA modification strategies in an OA rabbit model, which warrant further studies toward more effective viscosupplementation formulations.

Ultrastructural analysis of healthy synovial fluids in three mammalian species.

A better knowledge of synovial fluid (SF) ultrastructure is required to further understand normal joint lubrication and metabolism. The aim of the present study was to elucidate SF structural features in healthy joints from three mammalian species of different size compared with features in biomimetic SF. High-resolution structural analysis was performed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and environmental SEM/wet scanning transmission electron microscopy mode complemented by TEM and SEM cryogenic methods. Laser-scanning confocal microscopy (LCM) was used to locate the main components of SF with respect to its ultrastructural organization. The present study showed that the ultrastructure of healthy SF is built from a network of vesicles with a size range from 100 to a few hundred nanometers. A multilayered organization of the vesicle membranes was observed with a thickness of about 5 nm. LCM study of biological SF compared with synthetic SF showed that the microvesicles consist of a lipid-based membrane enveloping a glycoprotein gel. Thus, healthy SF has a discontinuous ultrastructure based on a complex network of microvesicles. This finding offers novel perspectives for the diagnosis and treatment of synovial joint diseases.

Radiographic assessment of the femorotibial joint of the CCLT rabbit experimental model of osteoarthritis.

BACKGROUND: The purposes of the study were to determine the relevance and validity of in vivo non-invasive radiographic assessment of the CCLT (Cranial Cruciate Ligament Transection) rabbit model of osteoarthritis (OA) and to estimate the pertinence, reliability and reproducibility of a radiographic OA (ROA) grading scale and associated radiographic atlas. METHODS: In vivo non-invasive extended non weight-bearing radiography of the rabbit femorotibial joint was standardized. Two hundred and fifty radiographs from control and CCLT rabbits up to five months after surgery were reviewed by three readers. They subsequently constructed an original semi-quantitative grading scale as well as an illustrative atlas of individual ROA feature for the medial compartment. To measure agreements, five readers independently scored the same radiographic sample using this atlas and three of them performed a second reading. To evaluate the pertinence of the ROA grading scale, ROA results were compared with gross examination in forty operated and ten control rabbits. RESULTS: Radiographic osteophytes of medial femoral condyles and medial tibial condyles were scored on a four point scale and dichotomously for osteophytes of medial fabella. Medial joint space width was scored as normal, reduced or absent. Each ROA features was well correlated with gross examination (p < 0.001). ICCs of each ROA features demonstrated excellent agreement between readers and within reading. Global ROA score gave the highest ICCs value for between (ICC 0.93; CI 0.90-0.96) and within (ICC ranged from 0.94 to 0.96) observer agreements. Among all individual ROA features, medial joint space width scoring gave the highest overall reliability and reproducibility and was correlated with both meniscal and cartilage macroscopic lesions (rs = 0.68 and rs = 0.58, p < 0.001 respectively). Radiographic osteophytes of the medial femoral condyle gave the lowest agreements while being well correlated with the macroscopic osteophytes (rs = 0.64, p < 0.001). CONCLUSION: Non-invasive in vivo radiography of the rabbit femorotibial joint is feasible, relevant and allows a reproducible grading of experimentally induced OA lesion. The radiographic grading scale and atlas presented could be used as a template for in vivo non invasive grading of ROA in preclinical studies and could allow future comparisons between studies.