The pathological changes in osteoarthritis--a degenerative joint disease prevalent among older people--start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ.

Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering.

Osteoarthritis (OA) is a chronic degenerative disease of the articular joint that involves both bone and cartilage degenerative changes. An engineered osteochondral tissue within physiological conditions will be of significant utility in understanding the pathogenesis of OA and testing the efficacy of potential disease-modifying OA drugs (DMOADs). In this study, a multichamber bioreactor was fabricated and fitted into a microfluidic base. When the osteochondral construct is inserted, two chambers are formed on either side of the construct (top, chondral; bottom, osseous) that is supplied by different medium streams. These medium conduits are critical to create tissue-specific microenvironments in which chondral and osseous tissues will develop and mature. Human bone marrow stem cell (hBMSCs)-derived constructs were fabricated in situ and cultured within the bioreactor and induced to undergo spatially defined chondrogenic and osteogenic differentiation for 4 weeks in tissue-specific media. We observed tissue specific gene expression and matrix production as well as a basophilic interface suggesting a developing tidemark. Introduction of interleukin-1β (IL-1β) to either the chondral or osseous medium stream induced stronger degradative responses locally as well as in the opposing tissue type. For example, IL-1β treatment of the osseous compartment resulted in a strong catabolic response in the chondral layer as indicated by increased matrix metalloproteinase (MMP) expression and activity, and tissue-specific gene expression. This induction was greater than that seen with IL-1β application to the chondral component directly, indicative of active biochemical communication between the two tissue layers and supporting the osteochondral nature of OA. The microtissue culture system developed here offers novel capabilities for investigating the physiology of osteochondral tissue and pathogenic mechanisms of OA and serving as a high-throughput platform to test potential DMOADS.

Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.

In the present work, we demonstrate that the mesoscopic in-plane mechanical behavior of membrane elastomeric scaffolds can be simulated by replication of actual quantified fibrous geometries. Elastomeric electrospun polyurethane (ES-PEUU) scaffolds, with and without particulate inclusions, were utilized. Simulations were developed from experimentally-derived fiber network geometries, based on a range of scaffold isotropic and anisotropic behaviors. These were chosen to evaluate the effects on macro-mechanics based on measurable geometric parameters such as fiber intersections, connectivity, orientation, and diameter. Simulations were conducted with only the fiber material model parameters adjusted to match the macro-level mechanical test data. Fiber model validation was performed at the microscopic level by individual fiber mechanical tests using AFM. Results demonstrated very good agreement to the experimental data, and revealed the formation of extended preferential fiber orientations spanning the entire model space. We speculate that these emergent structures may be responsible for the tissue-like macroscale behaviors observed in electrospun scaffolds. To conclude, the modeling approach has implications for (1) gaining insight on the intricate relationship between fabrication variables, structure, and mechanics to manufacture more functional devices/materials, (2) elucidating the effects of cell or particulate inclusions on global construct mechanics, and (3) fabricating better performing tissue surrogates that could recapitulate native tissue mechanics.

The production of veritable in-vitro models of bone tissue is essential to understand the biology of bone and its surrounding environment, to analyze the pathogenesis of bone diseases (e.g., osteoporosis, osteoarthritis, osteomyelitis, etc.), to develop effective therapeutic drug screening, and to test potential therapeutic strategies. Dysregulated interactions between vasculature and bone cells are often related to the aforementioned pathologies, underscoring the need for a bone model that contains engineered vasculature. Due to ethical restraints and limited prediction power of animal models, human stem cell-based tissue engineering has gained increasing relevance as a candidate approach to overcome the limitations of animals and to serve as preclinical models for drug testing. Since bone is a highly vascularized tissue, the concomitant development of vasculature and mineralized matrix requires a synergistic interaction between osteogenic and endothelial precursors. A number of experimental approaches have been used to achieve this goal, such as the combination of angiogenic factors and three-dimensional scaffolds, prevascularization strategies, and coculture systems. In this review, we present an overview of the current models and approaches to generate in-vitro stem cell-based vascularized bone, with emphasis on the main challenges of vasculature engineering. These challenges are related to the choice of biomaterials, scaffold fabrication techniques, and cells, as well as the type of culturing conditions required, and specifically the application of dynamic culture systems using bioreactors.

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Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) three-dimensional (3D) bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold, which underwent osteogenic differentiation for 2 weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during 2 more weeks of culture. We tested three experimental conditions: condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM): endothelial medium (EM); condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just 4 weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.