Riccardo Gottardi

Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy

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.

Enhancement of Tenogenic Differentiation of Human Adipose Stem Cells by Tendon-Derived Extracellular Matrix

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.

From single fiber to macro-level mechanics: A structural finite-element model for elastomeric fibrous biomaterials.

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.

Stem cell-based microphysiological osteochondral system to model tissue response to interleukin-1β.

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.

Three-dimensional osteochondral microtissue to model pathogenesis of osteoarthritis

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.

Three-dimensional osteogenic and chondrogenic systems to model osteochondral physiology and degenerative joint diseases.

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.

Carbon nanotubes as a novel tool for vaccination against infectious diseases and cancer.

Due to their unusual properties, carbon nanotubes have been extensively employed in electronics, nanotechnology and optics, amongst other. More recently, they have also been used as vehicles for drug and antigen delivery, the latter being a novel immunization strategy against infectious diseases and cancer. Here we discuss the potential of carbon nanotubes as an antigen delivery tool and suggest further directions in the field of vaccination.

The scope and sequence of growth factor delivery for vascularized bone tissue regeneration

Bone regeneration is a complex process, that in vivo, requires the highly coordinated presentation of biochemical cues to promote the various stages of angiogenesis and osteogenesis. Taking inspiration from the natural healing process, a wide variety of growth factors are currently being released within next generation tissue engineered scaffolds (in a variety of ways) in order to heal non-union fractures and bone defects. This review will focus on the delivery of multiple growth factors to the bone regeneration niche, specifically 1) dual growth factor delivery signaling and crosstalk, 2) the importance of growth factor timing and temporal separation, and 3) the engineering of delivery systems that allow for temporal control over presentation of soluble growth factors. Alternative methods for growth factor presentation, including the use of gene therapy and platelet-rich plasma scaffolds, are also discussed.

Strategies to Direct the Enrichment, Expansion, and Recruitment of Regulatory Cells for the Treatment of Disease

Disease and injury perturb the balance of processes associated with inflammation and tissue remodeling, resulting in positive feedback loops, exacerbation of disease and compromised tissue repair. Conversely, under homeostatic healthy conditions, these processes are tightly regulated through the expansion and/or recruitment of specific cell populations, promoting a balanced steady-state. Better understanding of these regulatory processes and recent advances in biomaterials and biotechnology have prompted strategies to utilize cells for the treatment and prevention of disease through regulation of inflammation and promotion of tissue repair. Herein, we describe how cells that regulate these processes can be increased in prevalence at a site of disease or injury. We review several relevant cell therapy approaches as well as new strategies for directing endogenous regulatory cells capable of promoting environmental homeostasis and even the establishment of a pro-regenerative micro-environment. Collectively, these examples may provide a blueprint for next-generation “medicine” that spurs the body’s own cells to action and replaces conventional drugs.

Anatomical region-dependent enhancement of 3-dimensional chondrogenic differentiation of human mesenchymal stem cells by soluble meniscus extracellular matrix

Extracellular matrix (ECM) derived from decellularized tissues has been found to promote tissue neogenesis, most likely mediated by specific biochemical and physical signaling motifs that promote tissue-specific differentiation of progenitor cells. Decellularized ECM has been suggested to be efficacious for the repair of tissue injuries. However, decellularized meniscus contains a dense collagenous structure, which impedes cell seeding and infiltration and is not readily applicable for meniscus repair. In addition, the meniscus consists of two distinct anatomical regions that differ in vascularity and cellular phenotype. The purpose of this study was to explore the region-specific bioactivity of solubilized ECM derived from the inner and outer meniscal regions as determined in 2D and 3D cultures of adult mesenchymal stem cells (MSCs). When added as a medium supplement to 2D cultures of MSCs, urea-extracted fractions of the inner (imECM) and outer meniscal ECM (omECM) enhanced cell proliferation while imECM most strongly upregulated fibrochondrogenic differentiation on the basis of gene expression profiles. When added to 3D cultures of MSCs seeded in photocrosslinked methacrylated gelatin (GelMA) hydrogels, both ECM fractions upregulated chondrogenic differentiation as determined by gene expression and protein analyses, as well as elevated sulfated glycosaminoglycan sGAG content, compared to ECM-free controls. The chondrogenic effect at day 21 was most pronounced with imECM supplementation, but equivalent between ECM groups by day 42. Despite increased cartilage matrix, imECM and omECM constructs possessed compressive moduli similar to controls. In conclusion, soluble meniscal ECM may be considered for use as a tissue-specific reagent to enhance chondrogenesis for MSC-based 3D cartilage tissue engineering. STATEMENT OF SIGNIFICANCE The inner region of the knee meniscus is frequently injured and possesses a poor intrinsic healing capacity. Solubilized extracellular matrix (ECM) derived from decellularized meniscus tissue may promote homologous differentiation of progenitor cells, thereby enhancing fibrocartilage formation within a meniscal lesion. However, the meniscus possesses regional variation in ultrastructure, biochemical composition, and cell phenotype, which may affect the bioactivity of soluble ECM derived from different regions of decellularized menisci. In this study, we demonstrate that urea-extracted fractions of ECM derived from the inner and outer regions of menisci enhance chondrogenesis in mesenchymal stem cells seeded in 3-dimensional photocrosslinkable hydrogels and that this effect is more strongly mediated by inner meniscal ECM. These findings suggest region-specific bioactivity of decellularized meniscal ECM.