Age and anatomical region-related differences in vascularization of the porcine meniscus using microcomputed tomography imaging.

Meniscal lesions in vascularized regions are known to regenerate while lack of vascular supply leads to poor healing. Here, we developed and validated a novel methodology for three-dimensional structural analysis of meniscal vascular structures with high-resolution microcomputed tomography (µCT). We collected porcine medial menisci from 10 neonatal (not-developed meniscus, n-) and 10 adults (fully developed meniscus, a-). The menisci were cut into anatomical regions (anterior horn (n-AH and a-AH), central body (n-CB and a-CB), and posterior horn (n-PH and a-PH). Specimens were cut in half, fixed, and one specimen underwent critical point drying and µCT imaging, while other specimen underwent immunohistochemistry and vascularity biomarker CD31 staining for validation of µCT. Parameters describing vascular structures were calculated from µCT. The vascular network in neonatal spread throughout meniscus, while in adult was limited to a few vessels in outer region, mostly on femoral side. n-AH, n-CB, and n-PH had 20, 17, and 11 times greater vascular volume fraction than adult, respectively. Moreover, thickness of blood vessels, in three regions, was six times higher in adults than in neonatal. a-PH appeared to have higher vascular fraction, longer and thicker blood vessels than both a-AH and a-CB. Overall, neonatal regions had a higher number of blood vessels, more branching, and higher tortuosity compared to adult regions. For the first time, critical point drying-based µCT imaging allowed detailed three-dimensional visualization and quantitative analysis of vascularized meniscal structures. We showed more vascularity in neonatal menisci, while adult menisci had fewer and thicker vascularity especially limited to the femoral surface.

Osteological profiling of femoral diaphysis and neck in aquatic, semiaquatic, and terrestrial carnivores and rodents: effects of body size and locomotor habits.

The increased limb bone density documented previously for aquatic tetrapods has been proposed to be an adaptation to overcome buoyancy during swimming and diving. It can be achieved by increasing the amount of bone deposition or by reducing the amount of bone resorption, leading to cortical thickening, loss of medullary cavity, and compaction of trabecular bone. The present study examined the effects of locomotor habit, body size, and phylogeny on the densitometric, cross-sectional, and biomechanical traits of femoral diaphysis and neck in terrestrial, semiaquatic, and aquatic carnivores, and in terrestrial and semiaquatic rodents (12 species) by using peripheral quantitative computed tomography, three-point bending, and femoral neck loading tests. Groupwise differences were analyzed with the univariate generalized linear model and the multivariate linear discriminant analysis supplemented with hierarchical clustering. While none of the individual features could separate the lifestyles or species adequately, the combinations of multiple features produced very good or excellent classifications and clusterings. In the phocid seals, the aquatic niche allowed for lower femoral bone mineral densities than expected based on the body mass alone. The semiaquatic mammals mostly had high bone mineral densities compared to the terrestrial species, which could be considered an adaptation to overcome buoyancy during swimming and shallow diving. Generally, it seems that different osteological properties at the levels of mineral density and biomechanics could be compatible with the adaptation to aquatic, semiaquatic, or terrestrial niches.

Clinical Super-Resolution Computed Tomography of Bone Microstructure: Application in Musculoskeletal and Dental Imaging.

PURPOSE: Clinical cone-beam computed tomography (CBCT) devices are limited to imaging features of half a millimeter in size and cannot quantify the tissue microstructure. We demonstrate a robust deep-learning method for enhancing clinical CT images, only requiring a limited set of easy-to-acquire training data. METHODS: Knee tissue from five cadavers and six total knee replacement patients, and 14 teeth from eight patients were scanned using laboratory CT as training data for the developed super-resolution (SR) technique. The method was benchmarked against ex vivo test set, 52 osteochondral samples are imaged with clinical and laboratory CT. A quality assurance phantom was imaged with clinical CT to quantify the technical image quality. To visually assess the clinical image quality, musculoskeletal and maxillofacial CBCT studies were enhanced with SR and contrasted to interpolated images. A dental radiologist and surgeon reviewed the maxillofacial images. RESULTS: The SR models predicted the bone morphological parameters on the ex vivo test set more accurately than conventional image processing. The phantom analysis confirmed higher spatial resolution on the SR images than interpolation, but image grayscales were modified. Musculoskeletal and maxillofacial CBCT images showed more details on SR than interpolation; however, artifacts were observed near the crown of the teeth. The readers assessed mediocre overall scores for both SR and interpolation. The source code and pretrained networks are publicly available. CONCLUSION: Model training with laboratory modalities could push the resolution limit beyond state-of-the-art clinical musculoskeletal and dental CBCT. A larger maxillofacial training dataset is recommended for dental applications.

Magnetic-Responsive Carbon Nanotubes Composite Scaffolds for Chondrogenic Tissue Engineering.

The demand for engineered scaffolds capable of delivering multiple cues to cells continues to grow as the interplay between cell fate with microenvironmental and external cues is revealed. Emphasis has been given to develop stimuli-responsive scaffolds. These scaffolds are designed to sense an external stimulus triggering a specific response (e.g., change in the microenvironment, release therapeutics, etc.) and then initiate/modulate a desired biofunction. Here, magnetic-responsive carboxylated multi-walled carbon nanotubes (cMWCNTs) are integrated into 3D collagen/polylactic acid (PLA) scaffold via a reproducible filtration-based method. The integrity and biomechanical performance of the collagen/PLA scaffolds are preserved after cMWCNT integration. In vitro safety assessment of cMWCNT/collagen/PLA scaffolds shows neither cytotoxicity effects nor macrophage pro-inflammatory response, supporting further in vitro studies. The cMWCNT/collagen/PLA scaffolds enhance chondrocytes metabolic activity while maintaining high cell viability and extracellular matrix (i.e., type II collagen and aggrecan) production. Comprehensive in vitro study applying static and pulsed magnetic field on seeded scaffolds shows no specific cell response in dependence with the applied field. This result is independent of the presence or absence of cMWCNT into the collagen/PLA scaffolds. Taken together, these findings provide additional evidence of the benefits to exploit the CNTs outstanding properties in the design of stimuli-responsive scaffolds.

Micro-computed Tomography-Based Collagen Orientation and Anisotropy Analysis of Rabbit Articular Cartilage.

The collagen network is the highly organized backbone of articular cartilage providing tissue tensile stiffness and restricting proteoglycan bleaching out of the tissue. Osteoarthritis (OA) diminishes proper collagen network adaptation. Our aim was to provide quantitative three-dimensional (3D) information of the cartilage collagen network adaptation in early osteoarthritis using high resolution micro-computed tomography (µCT)-imaging. Osteochondral samples from the femoral condyles were collected from healthy (N = 8, both legs) and experimental OA rabbit model with anterior cruciate ligament transection (N = 14, single leg). Samples were processed for cartilage µCT-imaging and histological evaluation with polarized light microscopy (PLM). Structure tensor analysis was used to analyse the collagen fibre orientation and anisotropy of the µCT-images, and PLM was used as a validation for structural changes. Depth-wise comparison of collagen fibre orientation acquired with µCT-imaging and PLM correlated well, but the values obtained with PLM were systematically greater than those measured with µCT-imaging. Structure tensor analysis allowed for 3D quantification of collagen network anisotropy. Finally, µCT-imaging revealed only minor differences between the control and experimental groups.

Bone disturbances and progression of atherosclerosis in ApoE knockout mice.

OBJECTIVES: Epidemiological evidence supports a link between atherosclerosis and osteoporosis. These conditions might share common pathophysiological mechanisms, with inflammation being one of the hypotheses.Apolipoprotein E deficient mice (ApoE-/-) develop atherosclerotic lesions spontaneously, further aggravated by a high-fat diet. Their bone remodelling is also disturbed. We hypothesised that a proinflammatory state could be a common contributive factor for vessel and bone disturbances observed in this animal model. METHODS: We evaluated vessels and bones of ApoE-/- and control C57BL/6 (B6) female mice fed a high-fat diet in five time-points (8, 16, 20, 24 and 28 weeks of age) and quantified the development of atherosclerotic lesions, analysed gene expression of inflammatory and bone remodelling proteins (IL-1ß, IL-6, IL-17A, TNF, RANKL, and OPG), measured serum bone turnover markers (P1NP and CTX-I), performed bone (L3-L4 vertebras) histomorphometric analysis and evaluated biomechanical properties of bones. RESULTS: We compared the outcomes of B6 and ApoE-/- groups at each time-point and, within each group, over time. Atherosclerotic lesions developed as previously described for ApoE-/- mice, but no significant differences were found in bone histomorphometry or biomechanical properties between ApoE-/- and B6 mice. Also, gene expression (either in bones or aortas) and serum biomarkers were similar in both groups. When considering over time evaluations we found that bone histomorphometry changes were similar between ApoE-/- and B6 mice, but CTX-I/P1NP ratio was significantly increased (meaning higher resorption than bone formation) in ApoE-/- as compared to B6 mice. CONCLUSIONS: Our study suggests that inflammation is not the principal driver for atherosclerosis progression and bone disturbances in this animal model.

No adverse effects on periarticular tissue by intra-articular vitamin D analogue calcipotriol in a reduced-dose zymosan-induced arthritis model in rats.

Calcipotriol, a vitamin D analogue, is an antiproliferative and anti-inflammatory drug currently used in psoriasis. Here, our aim was to analyse the safety of calcipotriol for cartilage and bone in alleviated-dose (0.1 mg instead of usual ≥1mg dose) zymosan-induced arthritis in rats. Theoretically, high doses of vitamin D or analogues could have detrimental effects on bone or cartilage. The rats were divided into four groups: vehicle (n = 9), dexamethasone 0.1 mg/kg (n = 9), calcipotriol 0.1 mg/kg (n = 8) and negative control (n = 10) with no injections. Arthritic rats were given phosphate-buffered saline (PBS) injections to left knees as a control. After euthanasia on day 8, all knees were imaged with micro-computed tomography for surface lesions and decalcified for histological analyses. Contrary to our expectations, no significant changes could be observed in the tomography data and histological scores among the three treatment groups or between the vehicle-treated and non-arthritic group. Calcipotriol did not cause adverse effects on cartilage or subchondral bone within a week, suggesting that it could be safely used in local treatment of arthritis. The alleviated model caused synovitis with local and systemic inflammatory response without cartilage erosions, which might be useful in studying self-limiting synovitis where cartilage or bone effects are not of primary interest.

Adaptation of Fibril-Reinforced Poroviscoelastic Properties in Rabbit Collateral Ligaments 8 Weeks After Anterior Cruciate Ligament Transection.

Ligaments of the knee provide stability and prevent excessive motions of the joint. Rupture of the anterior cruciate ligament (ACL), a common sports injury, results in an altered loading environment for other tissues in the joint, likely leading to their mechanical adaptation. In the collateral ligaments, the patterns and mechanisms of biomechanical adaptation following ACL transection (ACLT) remain unknown. We aimed to characterize the adaptation of elastic and viscoelastic properties of the lateral and medial collateral ligaments eight weeks after ACLT. Unilateral ACLT was performed in six rabbits, and collateral ligaments were harvested from transected and contralateral knee joints after eight weeks, and from an intact control group (eight knees from four animals). The cross-sectional areas were measured with micro-computed tomography. Stepwise tensile stress-relaxation testing was conducted up to 6% final strain, and the elastic and viscoelastic properties were characterized with a fibril-reinforced poroviscoelastic material model. We found that the cross-sectional area of the collateral ligaments in the ACL transected knees increased, the nonlinear elastic collagen network modulus of the LCL decreased, and the amount of fast relaxation in the MCL decreased. Our results indicate that rupture of the ACL leads to an early adaptation of the elastic and viscoelastic properties of the collagen fibrillar network in the collateral ligaments. These adaptations may be important to consider when evaluating whole knee joint mechanics after ACL rupture, and the results aid in understanding the consequences of ACL rupture on other tissues.

Associations of human femoral condyle cartilage structure and composition with viscoelastic and constituent-specific material properties at different stages of osteoarthritis.

The relationships between structure and function in human knee femoral cartilage are not well-known at different stages of osteoarthritis. Thus, our aim was to characterize the depth-dependent composition and structure (proteoglycan content, collagen network organization and collagen content) of normal and osteoarthritic human femoral condyle cartilage (n = 47) and relate them to their viscoelastic and constituent-specific mechanical properties that are obtained through dynamic sinusoidal testing and fibril-reinforced poroelastic material modeling of stress-relaxation testing, respectively. We characterized the proteoglycan content using digital densitometry, collagen network organization (orientation angle and anisotropy) using polarized light microscopy and collagen content using Fourier transform infrared spectroscopy. In the superficial cartilage (0-10 % of thickness), the collagen network disorganization and proteoglycan loss were associated with the smaller initial fibril network modulus - a parameter representing the pretension of the collagen network. Furthermore, the proteoglycan loss was associated with the greater strain-dependent fibril network modulus - a measure of nonlinear mechanical behavior. The proteoglycan loss was also associated with greater cartilage viscosity at a low loading frequency (0.005 Hz), while the collagen network disorganization was associated with greater cartilage viscosity at a high loading frequency (1 Hz). Our results suggest that proteoglycan loss and collagen network disorganization reduce the pretension of the collagen network while proteoglycan degradation also increases the nonlinear mechanical behavior of the collagen network. Further, the results also highlight that proteoglycan loss and collagen disorganization increase the viscosity of femoral cartilage, but their contribution to increased viscosity occurs in completely different loading frequencies.

Assessment of Ligament Viscoelastic Properties Using Raman Spectroscopy.

Injuries to the ligaments of the knee commonly impact vulnerable and physically active individuals. These injuries can lead to the development of degenerative diseases such as post-traumatic osteoarthritis (PTOA). Non-invasive optical modalities, such as infrared and Raman spectroscopy, provide means for quantitative evaluation of knee joint tissues and have been proposed as potential quantitative diagnostic tools for arthroscopy. In this study, we evaluate Raman spectroscopy as a viable tool for estimating functional properties of collateral ligaments. Artificial trauma was induced by anterior cruciate ligament transection (ACLT) in the left or right knee joint of skeletally mature New Zealand rabbits. The corresponding contralateral (CL) samples were extracted from healthy unoperated joints along with a separate group of control (CNTRL) animals. The rabbits were sacrificed at 8 weeks after ACLT. The ligaments were then harvested and measured using Raman spectroscopy. A uniaxial tensile stress-relaxation testing protocol was adopted for determining several biomechanical properties of the samples. Partial least squares (PLS) regression models were then employed to correlate the spectral data with the biomechanical properties. Results show that the capacity of Raman spectroscopy for estimating the biomechanical properties of the ligament samples varies depending on the target property, with prediction error ranging from 15.78% for tissue cross-sectional area to 30.39% for stiffness. The hysteresis under cyclic loading at 2 Hz (RMSE = 6.22%, Normalized RMSE = 22.24%) can be accurately estimated from the Raman data which describes the viscous damping properties of the tissue. We conclude that Raman spectroscopy has the potential for non-destructively estimating ligament biomechanical properties in health and disease, thus enhancing the diagnostic value of optical arthroscopic evaluations of ligament integrity.

Reduced Bone Mass in Collagen Prolyl 4-Hydroxylase P4ha1 +/-; P4ha2 -/- Compound Mutant Mice.

Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4-hydroxylases (C-P4Hs) hydroxylate proline residues in the -X-Pro-Gly- repeats of all known collagen types. Their product, 4-hydroxyproline, is essential for correct folding and thermal stability of the triple-helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse P4ha1 gene, which codes for the catalytic α subunit of the major C-P4H isoform, is embryonic lethal, whereas inactivation of the P4ha2 gene produced only a minor phenotype. Instead, mice with a haploinsufficiency of the P4ha1 gene combined with a homozygous deletion of the P4ha2 gene present with a moderate chondrodysplasia due to transient cell death of the growth plate chondrocytes. Here, to further characterize the bone phenotype of the P4ha1 +/-; P4ha2 -/- mice, we have carried out gene expression analyses at whole-tissue and single-cell levels, biochemical analyses, microcomputed tomography, histomorphometric analyses, and second harmonic generation microscopy to show that C-P4H α subunit expression peaks early and that the C-P4H deficiency leads to reduced collagen amount, a reduced rate of bone formation, and a loss of trabecular and cortical bone volume in the long bones. The total osteoblast number in the proximal P4ha1 +/-; P4ha2 -/- tibia and the C-P4H activity in primary P4ha1 +/-; P4ha2 -/- osteoblasts were reduced, whereas the population of osteoprogenitor colony-forming unit fibroblasts was increased in the P4ha1 +/-; P4ha2 -/- marrow. Thus, the P4ha1 +/-; P4ha2 -/- mouse model recapitulates key aspects of a recently recognized congenital connective tissue disorder with short stature and bone dysplasia caused by biallelic variants of the human P4HA1 gene. Altogether, the data demonstrate the allele dose-dependent importance of the C-P4Hs to the developing organism and a threshold effect of C-P4H activity in the proper production of bone matrix. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Mineral Crystal Thickness in Calcified Cartilage and Subchondral Bone in Healthy and Osteoarthritic Human Knees.

Osteoarthritis (OA) is the most common joint disease, where articular cartilage degradation is often accompanied with sclerosis of the subchondral bone. However, the association between OA and tissue mineralization at the nanostructural level is currently not understood. In particular, it is technically challenging to study calcified cartilage, where relevant but poorly understood pathological processes such as tidemark multiplication and advancement occur. Here, we used state-of-the-art microfocus small-angle X-ray scattering with a 5-µm spatial resolution to determine the size and organization of the mineral crystals at the nanostructural level in human subchondral bone and calcified cartilage. Specimens with a wide spectrum of OA severities were acquired from both medial and lateral compartments of medial compartment knee OA patients (n = 15) and cadaver knees (n = 10). Opposing the common notion, we found that calcified cartilage has thicker and more mutually aligned mineral crystals than adjoining bone. In addition, we, for the first time, identified a well-defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32 nm thicker crystals compared to the rest of calcified cartilage. Finally, we found 0.2 nm thicker mineral crystals in both tissues of the lateral compartment in OA compared with healthy knees, indicating a loading-related disease process because the lateral compartment is typically less loaded in medial compartment knee OA. In summary, we report novel changes in mineral crystal thickness during OA. Our data suggest that unloading in the knee might be involved with the growth of mineral crystals, which is especially evident in the calcified cartilage. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Subchondral bone plate thickness is associated with micromechanical and microstructural changes in the bovine patella osteochondral junction with different levels of cartilage degeneration.

The influence of joint degeneration on the biomechanical properties of calcified cartilage and subchondral bone plate at the osteochondral junction is relatively unknown. Common experimental difficulties include accessibility to and visualization of the osteochondral junction, application of mechanical testing at the appropriate length scale, and availability of tissue that provides a consistent range of degenerative changes. This study addresses these challenges. A well-established bovine patella model of early joint degeneration was employed, in which micromechanical testing of fully hydrated osteochondral sections was carried out in conjunction with high-resolution imaging using differential interference contrast (DIC) optical light microscopy. A total of forty-two bovine patellae with different grades of tissue health ranging from healthy to mild, moderate, and severe cartilage degeneration, were selected. From the distal-lateral region of each patella, two adjacent osteochondral sections were obtained for the mechanical testing and the DIC imaging, respectively. Mechanical testing was carried out using a robotic micro-force acquisition system, applying compression tests over an array (area: 200 µm × 1000 µm, step size: 50 µm) across the osteochondral junction to obtain a stiffness map. Morphometric analysis was performed for the DIC images of fully hydrated cryo-sections. The levels of cartilage degeneration, DIC images, and the stiffness maps were used to associate the mechanical properties onto the specific tissue regions of cartilage, calcified cartilage, and subchondral bone plate. The results showed that there were up to 20% and 24% decreases (p < 0.05) in the stiffness of calcified cartilage and subchondral bone plate, respectively, in the severely degenerated group compared to the healthy group. Furthermore, there were increases (p < 0.05) in the number of tidemarks, bone spicules at the cement line, and the mean thickness of the subchondral bone plate with increasing levels of degeneration. The decreasing stiffness in the subchondral bone plate coupled with the presence of bone spicules may be indicative of a subchondral remodeling process involving new bone formation. Moreover, the mean thickness of the subchondral bone plate was found to be the strongest indicator of mechanical and associated structural changes in the osteochondral joint tissues.

Changes in subchondral bone structure and mechanical properties do not substantially affect cartilage mechanical responses - A finite element study.

Subchondral bone structure has been observed to change in osteoarthritis (OA). However, it remains unclear how the early-stage OA changes affect the mechanics (stresses and strains) of the osteochondral unit. In this study, we aim to characterize the effect of subchondral bone structure and mechanical properties on the osteochondral unit mechanics. A 3-D finite element model of the osteochondral unit was constructed based on a rabbit femoral condyle µCT data and subjected to creep loading in indentation. Trabecular bone volume fraction, subchondral bone plate thickness, and equilibrium modulus were varied (including experimentally observed changes in early OA) to characterize the effect of these parameters on the osteochondral unit mechanics. At the end of the creep phase, the maximum principal strain at the bone surface of the cartilage-bone interface was decreased by 50% when the trabecular bone volume fraction was reduced from 48% to 28%. The maximum principal stress at the same location was decreased by 36% when plate thickness was reduced by 100 µm (-31%). In cartilage, small changes in the mechanics were seen near the cartilage-bone interface with a considerably thinner (-31%) plate. The changes in trabecular bone volume fraction, subchondral bone thickness and plate equilibrium modulus did not substantially affect the cartilage mechanics. Our results suggest that experimentally observed changes that occur in the subchondral bone structure in early OA have a minimal effect on cartilage mechanics under creep indentation loading; clear changes in the cartilage mechanics were seen only with an unrealistically soft subchondral bone plate.

Near infrared spectroscopic evaluation of biochemical and crimp properties of knee joint ligaments and patellar tendon.

Knee ligaments and tendons play an important role in stabilizing and controlling the motions of the knee. Injuries to the ligaments can lead to abnormal mechanical loading of the other supporting tissues (e.g., cartilage and meniscus) and even osteoarthritis. While the condition of knee ligaments can be examined during arthroscopic repair procedures, the arthroscopic evaluation suffers from subjectivity and poor repeatability. Near infrared spectroscopy (NIRS) is capable of non-destructively quantifying the composition and structure of collagen-rich connective tissues, such as articular cartilage and meniscus. Despite the similarities, NIRS-based evaluation of ligament composition has not been previously attempted. In this study, ligaments and patellar tendon of ten bovine stifle joints were measured with NIRS, followed by chemical and histological reference analysis. The relationship between the reference properties of the tissue and NIR spectra was investigated using partial least squares regression. NIRS was found to be sensitive towards the water (R2CV = .65) and collagen (R2CV = .57) contents, while elastin, proteoglycans, and the internal crimp structure remained undetectable. As collagen largely determines the mechanical response of ligaments, we conclude that NIRS demonstrates potential for quantitative evaluation of knee ligaments.

Elastic, Dynamic Viscoelastic and Model-Derived Fibril-Reinforced Poroelastic Mechanical Properties of Normal and Osteoarthritic Human Femoral Condyle Cartilage.

Osteoarthritis (OA) degrades articular cartilage and weakens its function. Modern fibril-reinforced poroelastic (FRPE) computational models can distinguish the mechanical properties of main cartilage constituents, namely collagen, proteoglycans, and fluid, thus, they can precisely characterize the complex mechanical behavior of the tissue. However, these properties are not known for human femoral condyle cartilage. Therefore, we aimed to characterize them from human subjects undergoing knee replacement and from deceased donors without known OA. Multi-step stress-relaxation measurements coupled with sample-specific finite element analyses were conducted to obtain the FRPE material properties. Samples were graded using OARSI scoring to determine the severity of histopathological cartilage degradation. The results suggest that alterations in the FRPE properties are not evident in the moderate stages of cartilage degradation (OARSI 2-3) as compared with normal tissue (OARSI 0-1). Drastic deterioration of the FRPE properties was observed in severely degraded cartilage (OARSI 4). We also found that the FRPE properties of femoral condyle cartilage related to the collagen network (initial fibril-network modulus) and proteoglycan matrix (non-fibrillar matrix modulus) were greater compared to tibial and patellar cartilage in OA. These findings may inform cartilage tissue-engineering efforts and help to improve the accuracy of cartilage representations in computational knee joint models.

High-resolution infrared microspectroscopic characterization of cartilage cell microenvironment.

The lateral resolution of infrared spectroscopy has been inadequate for accurate biochemical characterization of the cell microenvironment, a region regulating biochemical and biomechanical signals to cells. In this study, we demonstrate the capacity of a high-resolution Fourier transform infrared microspectroscopy (HR-FTIR-MS) to characterize the collagen content of this region. Specifically, we focus on the collagen content in the cartilage cell (chondrocyte) microenvironment of healthy and osteoarthritic (OA) cartilage. Human tibial cartilage samples (N = 28) were harvested from 7 cadaveric donors and graded for OA severity (healthy, early OA, advanced OA). HR-FTIR-MS was used to analyze the collagen content of the chondrocyte microenvironment of five distinct zones across the tissue depth. HR-FTIR-MS successfully showed collagen content distribution across chondrocytes and their environment. In zones 2 and 3 (10 - 50% of the tissue thickness), we observed that collagen content was smaller (P < 0.05) in early OA compared to the healthy tissue in the vicinity of cells (pericellular region). The collagen content loss was extended to the extracellular matrix in advanced OA tissue. No significant differences in the collagen content of the chondrocyte microenvironment were observed between the groups in the most superficial (0-10%) and deep zones (50-100%). HR-FTIR-MS revealed collagen loss in the early OA cartilage pericellular region before detectable changes in the extracellular matrix in advanced OA. HR-FTIR-MS-based compositional assessment enables a better understanding of OA-related changes in tissues. This technique can be used to identify new disease mechanisms enabling better intervention strategies. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) is the most common degenerative joint disease causing pain and disability. While significant progress has been made in OA research, OA pathogenesis is still poorly understood and current OA treatments are mainly palliative. This study demonstrates that high-resolution FTIR microspectroscopy (HR-FTIR-MS) can characterize OA-induced compositional changes in the cell microenvironment (pericellular matrix) during the early disease stages before tissue changes in the extracellular matrix become apparent. This technique may further enable the identification of new OA mechanisms and improve our current understanding of OA pathogenesis, thus, enabling the development of better treatment methods.

Bone toxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the retinoid system: A causality analysis anchored in osteoblast gene expression and mouse data.

Dioxin exposures impact on bone quality and osteoblast differentiation, as well as retinoic acid metabolism and signaling. In this study we analyzed associations between increased circulating retinol concentrations and altered bone mineral density in a mouse model following oral exposure to 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD). Additionally, effects of TCDD on differentiation marker genes and genes involved with retinoic acid metabolism were analysed in an osteoblast cell model followed by benchmark dose-response analyses of the gene expression data. Study results show that the increased trabecular and decreased cortical bone mineral density in the mouse model following TCDD exposure are associated with increased circulating retinol concentrations. Also, TCDD disrupted the expression of genes involved in osteoblast differentiation and retinoic acid synthesis, degradation, and nuclear translocation in directions compatible with increasing cellular retinoic acid levels. Further evaluation of the obtained results in relation to previously published data by the use of mode-of-action and weight-of-evidence inspired analytical approaches strengthened the evidence that TCDD-induced bone and retinoid system changes are causally related and compatible with an endocrine disruption mode of action.

Endocrine, metabolic and apical effects of in utero and lactational exposure to non-dioxin-like 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB 180): A postnatal follow-up study in rats.

PCB 180 is a persistent and abundant non-dioxin-like PCB (NDL-PCB). We determined the developmental toxicity profile of ultrapure PCB 180 in developing offspring following in utero and lactational exposure with the focus on endocrine, metabolic and retinoid system alterations. Pregnant rats were given total doses of 0, 10, 30, 100, 300 or 1000 mg PCB 180/kg bw on gestational days 7-10 by oral gavage, and the offspring were sampled on postnatal days (PND) 7, 35 and 84. Decreased serum testosterone and triiodothyronine concentrations on PND 84, altered liver retinoid levels, increased liver weights and induced 7-pentoxyresorufin O-dealkylase (PROD) activity were the sensitive effects used for margin of exposure (MoE) calculations. Liver weights were increased together with induction of the metabolizing enzymes cytochrome P450 (CYP) 2B1, CYP3A1, and CYP1A1. Less sensitive effects included decreased serum estradiol and increased luteinizing hormone levels in females, decreased prostate and seminal vesicle weight and increased pituitary weight in males, increased cortical bone area and thickness of tibial diaphysis in females and decreased cortical bone mineral density in males. Developmental toxicity profiles were partly different in male and female offspring, males being more sensitive to increased liver weight, PROD induction and decreased thyroxine concentrations. MoE assessment indicated that the 95th percentile of current maternal PCB 180 concentrations do not exceed the estimated tolerable human lipid-based PCB 180 concentration. Although PCB 180 is much less potent than dioxin-like compounds, it shares several toxicological targets suggesting a potential for interactions.