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.

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.

Osteoarthritis (OA), the most prevalent form of arthritis, affects up to 15% of the adult population and is principally characterized by degeneration of the articular cartilage component of the joint, often with accompanying subchondral bone lesions. Understanding the mechanisms underlying the pathogenesis of OA is important for the rational development of disease-modifying OA drugs. While most studies on OA have focused on the investigation of either the cartilage or the bone component of the articular joint, the osteochondral complex represents a more physiologically relevant target because the disease ultimately is a disorder of osteochondral integrity and function. In our current investigation, we are constructing an in vitro three-dimensional microsystem that models the structure and biology of the osteochondral complex of the articular joint. Osteogenic and chondrogenic tissue components are produced using adult human mesenchymal stem cells derived from bone marrow and adipose seeded within biomaterial scaffolds photostereolithographically fabricated with defined internal architecture. A three-dimensional-printed, perfusion-ready container platform with dimensions to fit into a 96-well culture plate format is designed to house and maintain the osteochondral microsystem that has the following features: an anatomic cartilage/bone biphasic structure with a functional interface; all tissue components derived from a single adult mesenchymal stem cell source to eliminate possible age/tissue-type incompatibility; individual compartments to constitute separate microenvironment for the synovial and osseous components; accessible individual compartments that may be controlled and regulated via the introduction of bioactive agents or candidate effector cells, and tissue/medium sampling and compositional assays; and compatibility with the application of mechanical load and perturbation. The consequences of mechanical injury, exposure to inflammatory cytokines, and compromised bone quality on degenerative changes in the cartilage component are examined in the osteochondral microsystem as a first step towards its eventual application as an improved and high-throughput in vitro model for prediction of efficacy, safety, bioavailability, and toxicology outcomes for candidate disease-modifying OA drugs.

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.

Tissue engineered constructs have the potential to function as in vitro pre-clinical models of normal tissue function and disease pathogenesis for drug screening and toxicity assessment. Effective high throughput assays demand minimal systems with clearly defined performance parameters. These systems must accurately model the structure and function of the human organs and their physiological response to different stimuli. Musculoskeletal tissues present unique challenges in this respect, as they are load-bearing, matrix-rich tissues whose functionality is intimately connected to the extracellular matrix and its organization. Of particular clinical importance is the osteochondral junction, the target tissue affected in degenerative joint diseases, such as osteoarthritis (OA), which consists of hyaline articular cartilage in close interaction with subchondral bone. In this review, we present an overview of currently available in vitro three-dimensional systems for bone and cartilage tissue engineering that mimic native physiology, and the utility and limitations of these systems. Specifically, we address the need to combine bone, cartilage and other tissues to form an interactive microphysiological system (MPS) to fully capture the biological complexity and mechanical functions of the osteochondral junction of the articular joint. The potential applications of three-dimensional MPSs for musculoskeletal biology and medicine are highlighted.