Embryos at embryonic day (E) 18.5 were harvested from time-mated wild-type C57Bl6/J mice obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mouse experiments were approved by the Purdue Animal Care and Use Committee (PACUC protocol 1209000723). PACUC ensures that all Purdue University animal programs, procedures, and facilities adhere to USDA and United States Public Health Service (USPHS) policies, recommendations, guidelines, and regulations as required by law. Animal Welfare and Purdue Animal Welfare Assurance. Mothers were euthanized via CO2 inhalation, which was confirmed by cervical dislocation, and the embryos were removed from the uterine horns in sterile phosphate-buffered saline (PBS; Life Technologies, Grand Island, NY, USA).
To compare the mechanical properties of viable, fresh (vibratomized) and frozen (cryotomized) tissue, forelimbs from the same embryo were matched for each dataset. For sectioning the vibratome, the forelimbs were carefully removed and coated with tissue adhesive (Electron Microscopy Sciences, Hatfield, PA, USA), then embedded in 4% low-melting agarose ( BIO-RAD, Hercules, CA, USA) (Fig. 1A). Forelimbs embedded in agarose were cut into 200 μm sections (Fig. 1B) by a Leica VT-1000S vibratome (Leica Microsystems Inc., Germany). To maintain agarose rigidity and hydration during vibratoming, embedded specimens were placed in a PBS-filled water bath surrounded by ice and the cut sections were kept on ice prior to testing to preserve cellular and matrix integrity. For AFM assays, slices were washed thoroughly with PBS and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies, Grand Island, NY, USA) with calcein for 20 min to identify live cells. . Calcein AM (Life Technologies, Grand Island, NY, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to a concentration of 1 μg/μL, then the solution was added to DMEM at a ratio of 1:1000. The calcein-stained section was then placed on a coverslip coated with a thin layer of tissue adhesive. The coverslip was placed in a liquid cell filled with PBS and ready for AFM testing. Care was taken to keep the samples hydrated at all times.
For cryotome sectioning, embryonic forelimbs were embedded in clear frozen sectioning compound (VWR International, Radnor, PA, USA), frozen in dry ice-cooled isopentane, and stored at -80 ° C up to section. A Shandon Cryotome™ FE cryostat (Thermo Scientific Inc., Waltham, MA, USA) was used to transversely slice 5 μm thick sections of embryonic forelimbs. Sections were then collected on a glass coverslip and washed thoroughly with PBS to remove clear frozen section compound prior to AFM testing. It is important to note that even though cryotomized sections are much thinner than vibratomized sections, they are thick enough to overcome any influence of substrate stiffness.25 since we used a very small indentation force of only 11.5 nN (corresponding to an average indentation depth of ~600 nm, or only ~12% of the sample thickness).
Articular cartilage was harvested from juvenile bovine knee joints obtained from a local abattoir regulated by the United States Department of Agriculture (USDA) (Dutch Valley Foods, Holland, IL, USA ) within 36 hours of slaughter. As these joints were classified as inedible, no additional regulatory compliance was required. The joints were opened under aseptic conditions to expose the tibial and femoral condyles and the trochlear groove. Osteochondral plugs (cartilage-bone) were removed from the bearing region of the distal femoral condyles with an 8 mm diameter reamer. The plugs were then sliced perpendicular to the articular surface into 30 μm thick sections by a Leica VT-1000S vibratome within 2 hours of harvesting, as described above. Cartilage sections were washed thoroughly with PBS and incubated in culture medium consisting of DMEM with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA), 1% bovine serum albumin (Sigma -Aldrich, St. Louis, MO, USA) and 1% penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C and 5% CO2 before AFM testing. Cartilage sections were then pre-stained with calcein to identify live cells and fixed to a coverslip by putting a small drop of cyanoacrylate (Loctite, Westlake, OH, USA) at the ends of the sections but not at the test regions (Fig. 1B). Again, care was taken to keep the samples hydrated at all times.
Osteochondral plugs were obtained as described above. The superficial zone (~200 μm) and the deep zone (near the subchondral bone) were carefully removed from the plugs with a custom cutting template. Then, the remaining cartilage from the middle zone (~300 μm thick) was washed twice in sterilized PBS and cut into fine fragments. These fragments were collected and immersed in 0.1% (w/v) collagenase P (Roche Diagnostics GmbH, Mannheim, Germany) in DMEM at 37°C with shaking. After 2–3 hours of digestion, cell solutions were filtered through a 70 μm nylon cell strainer (Falcon, Waltham, MA, USA), then centrifuged and resuspended twice in PBS. Final cell populations were seeded on a poly-L-lysine (PLL) coated Petri dish (Sigma-Aldrich, St. Louis, MO, USA). After 2 hours of incubation at 37°C, the chondrocytes formed a strong attachment to the Petri dish while maintaining a rounded morphology and were ready for AFM testing. Note that this test was intentionally performed on cells before they had a chance to fully propagate15.28 and take on a more physiologically irrelevant fibroblastic phenotype38to mimic the rounded morphology of chondrocytes on the spot.
Mechanical characterization via AFM stiffness mapping
A Keysight 5500 AFM system (Keysight Technologies Inc., Santa Rosa, CA, USA) was combined with a Nikon Eclipse Ti widefield inverted microscope (Nikon Instruments Inc., Melville, NY, USA), enabling AFM scanning and simultaneous fluorescence microscopy. For tissue sections, AFM was used in force-volume mode, in which an array (32 × 32 or 64 × 64 points) of force-distance (F–Z) curves was collected over the entire area of the tissue. scanning. For isolated chondrocytes, the AFM probe was carefully placed over each individual cell for testing and three sets of F–Z curves were collected for each cell. The force trigger was set at approximately 11.5 nN indicating the point at which the cantilever approach was stopped and then retracted. By fitting the F–Z curves to a contact mechanics model (i.e.39), the compression module has been extracted. Thus, force-volume mode imaging provides a material property map of the sample simultaneously with the topographic map (Fig. 1C). To ensure an accurate model fit, a known AFM tip geometry is essential. A cantilever with a 5 μm borosilicate glass sphere attached to the free end (NovaScan, Ames, IA, USA) was used. The cantilever stiffness has been pre-calibrated at 0.07 N/m by the thermal fluctuation method40.
Hyaluronidase and cytochalasin D Treatment of bovine cartilage
Hyaluronidase (Hyal; Worthington Biochemical Corp., Lakewood, NJ, USA) was diluted to 7500 U/mL in sterile PBS. Sections of bovine cartilage were incubated in 50 μL of Hyal solution at 7500 U/mL for 30 min. Hyaluronic acid (HA) digestion was confirmed by fluorescent localization of HA using biotinylated hyaluronic acid-binding protein (HABP; Calbiochem, Billerica, MA, USA) . Control and Hyal incubated sections were labeled with HABP (1:300) and streptavidin-488 (1:500; Life Technologies, Grand Island, NY, USA) to visualize HA and DAPI content (1:500; Roche, Mannheim, Germany) to identify nuclei.
Cytochalasin D (Cyto D; 5 mg/mL in dimethylsulfoxide, Sigma-Aldrich, St. Louis, MO, USA) was diluted to 0.5 mg/mL in PBS. 50 μL of diluted CytoD was used to treat bovine cartilage for 30 min. Disruption of filamentous actin was confirmed by fluorescence microscopy for phalloidin-555 (1:100; Life Technologies, Grand Island, NY, USA) on undigested control and digested sections, in addition to DAPI ( 1:500) to identify cell locations.
Control and treated sections were imaged with a Zeiss LSM 710 microscope at Purdue University’s Life Sciences Fluorescence Imaging Facility using a 63× oil objective. All imaging parameters were exactly the same for control and treated sections.
For statistical analyses, each treatment was performed on three animals and each animal was evaluated at 3 different locations. For each location, a 5 × 5 pixel2 The matrix was extracted from the AFM stiffness map in the middle of a cell and die region, respectively, to calculate the average stiffness of the cell and the ECM. The p-value was calculated using IBM SPSS Statistics (IBM Corp., Armonk, New York, USA).