Molecular Engineering of Pericellular Microniche via Biomimetic Proteoglycans Modulates Cell Mechanobiology

Abstract

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.

Keywords: articular cartilage; biomimetic proteoglycan; chondrocyte mechanotransduction; nanomechanics; pericellular matrix.

Figure 1.

Localization of BPG10 in cartilage pericellular matrix (PCM) augments the micromechanics of PCM. a) Molecular architecture of BPG10, containing ~7 CS-GAG side chains (schematic shown for chondroitin-4-sulfate GAG) conjugated to the PAA backbone. b) Immunofluorescence (IF) images of adult bovine cartilage explants infiltrated with fluorescently-labeled BPG10 and co-stained with collagen VI demonstrate the diffusion of BPG10 throughout all zones of the tissue (SZ: superficial zone, MZ: middle zone, DZ: deep zone), and preferred distribution within the PCM and nearby territorial domain (T-ECM). c) Left panel: Representative indentation modulus (E_ind) maps of control and BPG10-treated cartilage in 20 × 20 μm² regions of interest (ROIs) either containing well-defined PCM rings (40 × 40 indents) or interterritorial domains (IT-ECM) further removed from cells (20 × 20 indents). Moduli corresponding to cell remnants were removed (white voids). Right panel: schematic illustration of IF-guided AFM nanomechanical mapping on bovine cartilage cryo-sections using a microspherical tip (R ≈ 2.25 μm), the PCM is immunolabeled with collagen VI. d) Box-and-whiskers plots of the PCM, T-ECM and IT-ECM micromodulus for control and BPG10-treated cartilage (> 600 locations for each region, n = 5 animals).

Figure 2.

Infiltration of BPG10 into bovine cartilage explants promotes intracellular spontaneous calcium signaling, [Ca²⁺]_i, activities of chondrocytes in situ. a) Representative cell live/dead assay images of control and BPG10-treated adult bovine cartilage after 24 hr exposure, with methanol-treated, devitalized cartilage as positive control. Cell viability is quantified on ≥ 1,750 cells per treatment from n = 3 animals (mean ± 95% CI). b) Representative confocal images of chondrocyte [Ca²⁺]_i signaling and corresponding [Ca²⁺]_i oscillation intensity curve of a single cell over a 15-min time frame illustrating the definition of t_peak. Chondrocytes were labeled with Cal-520™ AM and time series images were recorded using a confocal microscope with a 20× objective submerged in DMEM at 37 °C. c-e) Comparison of [Ca²⁺]_i signaling characteristics between BPG10-treated and control cartilage explants in both isotonic and hypotonic media: c) percentage of responding cells, %R_cell (mean ± 95% CI), d) number of peaks within the 15-min testing time frame, n_peak (mean ± 95% CI), and e) duration of each peak, t_peak (box-and-whisker plot, outliers are not shown to increase clarity, red cross represents the mean value). Data represent ≥ 445 responding cells pooled from n = 3 animals for each group.

Figure 3.

BPG10 increases the molecular adhesion of aggrecan. a) Colloidal force spectroscopy for the measurement of molecular adhesion between opposing aggrecan-aggrecan molecules in 1× PBS (control), or 1× PBS with the addition of free CS-GAGs at 3.3 μg/mL or free BPG10 at 3.5 μg/mL. Top panels: schematic illustration of experimental setup. Bottom panels: Representative force-distance curves illustrate the long-range adhesion behaviors between opposing aggrecan molecules at both 0 sec and 20 sec dwell time, as well as the definition of maximum adhesion force, F_ad, and total adhesion energy, E_ad. b,c) The addition of free BPG10 significantly increases b), F_ad, and c) E_ad, between aggrecan-aggrecan molecules at both 0 sec and 20 sec dwell times. In comparison, addition of free CS-GAG does not have a significant impact, except for a mild decrease in F_ad at 0 sec dwell time (n ≥ 180 measurements from three technical repeats for each condition, red cross represents the mean value).

Figure 4.

Comparison of molecular adhesion interactions between BPG10 and aggrecan. a) Colloidal molecular force spectroscopy for the measurement of molecular adhesion between BPG10-aggrecan molecules (aggrecan-coated tips versus BPG10-coated substrates, BPG10-coated tips versus aggrecan-coated substrates), and between BPG10-BPG10 molecules in 1× PBS. Top panels: schematic illustration of experimental setup. Bottom panels: Representative force-distance curves illustrate the long-range adhesion behaviors between aggrecan-aggrecan, BPG10-aggrecan and BPG10-BPG10 at both 0 sec and 20 sec dwell time. b,c) Comparison of b) maximum adhesion force, F_ad, and c) total adhesion energy, E_ad, between aggrecan-aggrecan, BPG10-aggrecan and BPG10-BPG10 molecules in 1× PBS. Insets illustrate the zoom-in comparisons of F_ad and E_ad at 0 sec dwell time. At 0 sec dwell time, BPG10-aggrecan interactions yield lower F_ad but similar E_ad relative to aggrecan-aggrecan and BPG10-BPG10 interactions. At 20 sec dwell time, BPG10-aggrecan interactions show similar F_ad but lower E_ad relative to aggrecan-aggrecan interactions, as well as lower F_ad and mildly lower E_ad relative to BPG10-BPG10 interactions (n ≥ 190 measurements from three technical repeats for each condition, red cross represents the mean value). Different letters indicate significant differences between groups. Data of aggrecan-aggrecan adhesion are re-plotted from the control experiment from Figure 3.

Figure 5.

Schematic illustration of the working hypothesis of biomimetic proteoglycans in mediating pericellular micro-niche mechanics and cell mechanobiology. The biophysical adhesion interactions between BPG10 and aggrecan enables the integration of BPG10 with the aggrecan-enriched cartilage PCM, and thus, the preferred localization of BPG10 in the PCM. Such localization augments the micromechanical properties of cartilage PCM, and thus, promotes chondrocyte mechanotransduction. Therefore, BPG10 could potentially affect downstream cell signaling and metabolic activities through molecularly engineering the pericellular micro-niche.