Biomimetic proteoglycans as a tool to engineer the structure and mechanics of porcine bioprosthetic heart valves.

The utility of bioprosthetic heart valves (BHVs) is limited to certain patient populations because of their poor durability compared to mechanical prosthetic valves. Histological analysis of failed porcine BHVs suggests that degeneration of the tissue extracellular matrix (ECM), specifically the loss of proteoglycans and their glycosaminoglycans (GAGs), may lead to impaired mechanical performance, resulting in nucleation and propagation of tears and ultimately failure of the prosthetic. Several strategies have been proposed to address this deterioration, including novel chemical fixatives to stabilize ECM constituents and incorporation of small molecule inhibitors of catabolic enzymes implicated in the degeneration of the BHV ECM. Here, biomimetic proteoglycans (BPGs) were introduced into porcine aortic valves ex vivo and were shown to distribute throughout the valve leaflets. Incorporation of BPGs into the heart valve leaflet increased tissue overall GAG content. The presence of BPGs also significantly increased the micromodulus of the spongiosa layer within the BHV without compromising the chemical fixation process used to sterilize and strengthen the tissue prior to implantation. These findings suggest that a targeted approach for molecularly engineering valve leaflet ECM through the use of BPGs may be a viable way to improve the mechanical behavior and potential durability of BHVs.

Nanomechanics of Aggrecan: A New Perspective on Cartilage Biomechanics, Disease and Regeneration.

Articular cartilage is a hydrated macromolecular composite mainly composed of type II collagen fibrils and the large proteoglycan, aggrecan. Aggrecan is a key determinant of the load bearing and energy dissipation functions of cartilage. Previously, studies of cartilage biomechanics have been primarily focusing on the macroscopic, tissue-level properties, which failed to elucidate the molecular-level activities that govern cartilage development, function, and disease. This chapter provides a brief summary of Dr. Alan J. Grodzinsky's seminal contribution to the understanding of aggrecan molecular mechanics at the nanoscopic level. By developing and applying a series of atomic force microscopy (AFM)-based nanomechanical tools, Grodzinsky and colleagues revealed the unique structural and mechanical characteristics of aggrecan at unprecedented resolutions. In this body of work, the "bottle-brush"-like ultrastructure of aggrecan was directly visualized for the first time. Meanwhile, molecular mechanics of aggrecan was studied using a physiological-like 2D biomimetic assembly of aggrecan on multiple fronts, including compression, dynamic loading, shear, and adhesion. These studies not only generated new insights into the development, aging, and disease of cartilage, but established a foundation for designing and evaluating novel cartilage regeneration strategies. For example, building on the scientific foundation and methodology infrastructure established by Dr. Grodzinsky, recent studies have elucidated the roles of other proteoglycans in mediating cartilage integrity, such as decorin and perlecan, and evaluated the therapeutic potential of biomimetic proteoglycans in improving cartilage regeneration.

Impacts of aging on murine cartilage biomechanics and chondrocyte in situ calcium signaling.

Aging is the most prominent risk factor for osteoarthritis onset, but the etiology of aging-associated cartilage degeneration is not fully understood. Recent studies by Guilak and colleagues have highlighted the crucial roles of cell-matrix interactions in cartilage homeostasis and disease. This study thus quantified aging-associated changes in cartilage biomechanics and chondrocyte intracellular calcium signaling, [Ca2+]i, activities in wild-type mice at 3, 12 and 22 months of age. In aged mice, articular cartilage exhibits reduced staining of sulfated glycosaminoglycans (sGAGs), indicating decreased aggrecan content. On cartilage surface, collagen fibrils undergo significant thickening while retaining their transverse isotropic architecture, and exhibit signs of fibril crimping in the 22-month group. These compositional and structural changes contribute to a significant decrease in cartilage modulus at 22 months of age (0.55 ± 0.25 MPa, mean ± 95 % CI, n = 8) relative to those at 3 and 12 months (1.82 ± 0.48 MPa and 1.45 ± 0.46 MPa, respectively, n ≥ 8). Despite the decreases in sGAG content and tissue modulus, chondrocytes do not exhibit significantly demoted [Ca2+]i activities in situ, in both physiological (isotonic) and osmotically instigated (hypo- and hypertonic) conditions. At 12 months of age, there exists a sub-population of chondrocytes with hyper-active [Ca2+]i responses under hypotonic stimuli, possibly indicating a phenotypic shift of chondrocytes during aging. Together, these results yield new insights into aging-associated biomechanical and mechanobiological changes of murine cartilage, providing a benchmark for elucidating the molecular mechanisms of age-related changes in cell-matrix interactions.

Targeting cell-matrix interface mechanobiology by integrating AFM with fluorescence microscopy.

Mechanosensing at the interface of a cell and its surrounding microenvironment is an essential driving force of physiological processes. Understanding molecular activities at the cell-matrix interface has the potential to provide novel targets for improving tissue regeneration and early disease intervention. In the past few decades, the advancement of atomic force microscopy (AFM) has offered a unique platform for probing mechanobiology at this crucial microdomain. In this review, we describe key advances under this topic through the use of an integrated system of AFM (as a biomechanical testing tool) with complementary immunofluorescence (IF) imaging (as an in situ navigation system). We first describe the body of work investigating the micromechanics of the pericellular matrix (PCM), the immediate cell micro-niche, in healthy, diseased, and genetically modified tissues, with a focus on articular cartilage. We then summarize the key findings in understanding cellular biomechanics and mechanotransduction, in which, molecular mechanisms governing transmembrane ion channel-mediated mechanosensing, cytoskeleton remodeling, and nucleus remodeling have been studied in various cell and tissue types. Lastly, we provide an overview of major technical advances that have enabled more in-depth studies of mechanobiology, including the integration of AFM with a side-view microscope, multiple optomicroscopy, a fluorescence recovery after photobleaching (FRAP) module, and a tensile stretching device. The innovations described here have contributed greatly to advancing the fundamental knowledge of extracellular matrix biomechanics and cell mechanobiology for improved understanding, detection, and intervention of various diseases.

Molecular Engineering of Pericellular Microniche via Biomimetic Proteoglycans Modulates Cell Mechanobiology.

Molecular engineering of biological tissues using synthetic mimics of native matrix molecules can modulate the mechanical properties of the cellular microenvironment through physical interactions with existing matrix molecules, and in turn, mediate the corresponding cell mechanobiology. In articular cartilage, the pericellular matrix (PCM) is the immediate microniche that regulates cell fate, signaling, and metabolism. The negatively charged osmo-environment, as endowed by PCM proteoglycans, is a key biophysical cue for cell mechanosensing. This study demonstrated that biomimetic proteoglycans (BPGs), which mimic the ultrastructure and polyanionic nature of native proteoglycans, can be used to molecularly engineer PCM micromechanics and cell mechanotransduction in cartilage. Upon infiltration into bovine cartilage explant, we showed that localization of BPGs in the PCM leads to increased PCM micromodulus and enhanced chondrocyte intracellular calcium signaling. Applying molecular force spectroscopy, we revealed that BPGs integrate with native PCM through augmenting the molecular adhesion of aggrecan, the major PCM proteoglycan, at the nanoscale. These interactions are enabled by the biomimetic "bottle-brush" ultrastructure of BPGs and facilitate the integration of BPGs within the PCM. Thus, this class of biomimetic molecules can be used for modulating molecular interactions of pericellular proteoglycans and harnessing cell mechanosensing. Because the PCM is a prevalent feature of various cell types, BPGs hold promising potential for improving regeneration and disease modification for not only cartilage-related healthcare but many other tissues and diseases.