Fig 1.
Cell-seeded compressed hydrogels contract with physiological kinetics.
Porcine conjunctival fragments and plastic-compressed gels containing primary human conjunctival fibroblasts (HTF0748-2; HTF1785R; HTF9154) were placed in 12 well-plates in medium with 10% serum, and contraction was monitored at least twice a week for 3 weeks. A) Porcine conjunctiva (left) and compressed collagen gels (right) on days 1, 7 and 21 in culture. B) Contraction kinetics of porcine conjunctiva (n = 6, 8–12 pieces per time point), and collagen gels seeded with human conjunctival fibroblasts (n = 6, pooled data for all 3 cell lines). C) Individual contraction kinetics for compressed hydrogels made with each primary fibroblast line (n = 2 per cell line, 3 gels per repeat). Graphs show mean ± SEM.
Fig 2.
Cell-seeded compressed hydrogels and porcine conjunctiva display a similar architecture.
Porcine conjunctival fragments and plastic compressed gels containing primary human conjunctival fibroblasts (HTF9154) were placed in medium with 10% serum and left to contract for 10 days. The tissues were then fixed, stained for F-actin using Rhodamine-labelled phalloidin, and imaged using confocal microscopy using fluorescence (F-actin, red) and reflection microscopy (collagen matrix, white). Shown are representative extended views (maximum intensity projection; A and B) and 3D reconstruction (A’ and B’) of porcine conjunctiva (A and A’) and compressed collagen tissues (B, B’). Scale bar, 90 μm. 3D view grid: one unit, 62.33 μm.
Fig 3.
Compressed collagen gels maintain a high cellularity throughout contraction.
Plastic compressed gels containing primary human conjunctival fibroblasts (HTF0748-2) were placed in medium with 10% serum and the gels were left to contract for 3 weeks. Cell viability and proliferation were assessed on day 7, 14 and 21, using LDH and Alamar Blue assays respectively, and confirmed visually using a Live/Dead assay at day 21. A) LDH assay: cell viability is expressed with reference to the 100% death control (cells lysed with RIPA buffer). Cell death was minimal throughout the 3 weeks of contraction. B) Live/Dead assay: 3-week-old tissues were well populated, with almost 100% viable cells (green) and only a few dead (red) cells. Scale bar, 200 um. C) Alamar Blue assay: there was no significant change in metabolic activity during contraction. Graphs show mean ± SEM for 3 independent experiments, with 2 replicates each.
Fig 4.
Using engineered conjunctiva stroma to screen potential anti-scarring drugs.
Plastic compressed gels containing primary human conjunctival fibroblasts (HTF0748-2) were placed in medium with 10% serum with/without anti-scarring drugs, and monitored over the period of 21 days. A, B, C) Contraction kinetics of gels exposed to NSC23766 [50 μM], Ehop-016 [10 μM] and doxycycline [416 μM] respectively for 24hrs, and their corresponding controls without drug. D) Toxicity assay: on day 7, 14, and 21, cell death was evaluated using the LDH assay. Graph shows LDH levels normalised to untreated control. E) Alamar blue assay: on day 7, 14, and 21 the metabolic activity in the gels was examined using the Alamar Blue assay. F) Coomassie Blue assay: on day 21, the amount of protein in the gels was examined using Coomassie Blue assay. Matrix degradation was reduced in gels exposed to Ehop-016 and doxycycline hyclate. Graphs show mean ± SEM for 3 independent experiments, each in triplicate.
Fig 5.
Macrophages can be successfully incorporated in compressed collagen gels with maintenance of the contraction potential and structure.
A) Contraction kinetics of compressed gels with/without macrophages. Compressed gels with the addition of macrophages maintain a tissue-like contraction pattern. Porcine conjunctiva contraction from Fig 1B was replotted for reference. B) 3D reconstruction of compressed gel with fibroblasts (thin arrow) and macrophages (thick arrow) on day 21. 3D grid: 1 unit = 62.45 μm.
Fig 6.
Heterogenous bilayers mimic bulbar conjunctiva/Tenon capsule interface.
A) 3D confocal microscopy image of the interface of the two layers on day 21, stained for F-actin using Rhodamine-labelled phalloidin, and imaged using confocal microscopy using fluorescence (F-actin, red) and reflection microscopy (collagen matrix, white). Dense collagen layer can be seen in compressed gel (bottom, white), whereas the more hydrated uncompressed layer of collagen is invisible in the image (top, only “floating” cells are visible). 1 grid unit = 62.45 μm. A’) Compressed gels retain a visibly higher cell density. B) 3D images of a bilayer gel with HTF and macrophages taken at day 25 using second harmonic generation microscopy. Green: collagen signal, blue: autofluorescent cells. The image shows low-density cell layout/loose collagen matrix in standard gel layer (top layer) and high-density layout/dense collagen matrix in compressed gel (bottom layer). 1 grid unit = 59.39 μm. C) 3D Images of a bilayer gel taken at day 1 using second harmonic generation microscopy. Green: collagen signal; blue: autofluorescent cells (HTF0748-2, selected cell indicated with an arrow); 1 grid unit = 33.58 μm. D) Higher magnification of the same cell imaged using confocal reflection and D’) SHG microscopy, respectively, reveals the collagen network around the cell Scale bar = 33 μm. E) 3D confocal microscopy image of a bilayer gel with fibroblasts and macrophages at day 25; 1 grid unit = 62.45 μm. F) Contraction pattern of bilayer gels with human conjunctival fibroblasts and with/without macrophages (n = 3, mean± SEM). Porcine conjunctiva contraction from Fig 1B was replotted for reference.
Fig 7.
Mechanical properties of engineered biomimetic and porcine conjunctiva.
A) Blueprint of a standard hourglass-shaped specimen for tensile test. A’) Porcine conjunctiva was dissected to approximate the blueprint and its mechanical properties were measured within 24 hours of animal sacrifice. A”) Engineered conjunctiva was created as previously, then cut to match the blueprint and its mechanical properties were measured within 24 hours. B, B’) Porcine and engineered conjunctiva seen sidewise, with a coin as a reference for the thickness measurements. C, C’) The typical stress-strain curve of the porcine and engineered conjunctiva obtained using a dynamic biomechanical analyser at room temperature in uniaxial tension mode with a stretching force at an extension rate of 0.5 mm/s until sample rupture. The slope of the curve in the linear deformation region was calculated to obtain the elastic modulus of the samples. D) Elastic modulus [kPa] as measured by dynamic biomechanical analysis for porcine conjunctiva and engineered conjunctiva. Graph shows mean and SEM for 8 independent experiments.
Fig 8.
Bilayer gels can be used to test local drug delivery.
A) Representative image of cell-seeded bilayer gel with a hydrated (thin arrow) and compressed (thick arrow) layer, and an acellular insert in between the two layers. For illustrative purpose, the two cell-seeded layers were made to differ in size and the insert was coloured with haematoxylin. B) Contraction kinetics of bilayer gels without an insert, with a control insert and with an insert soaked in a10 mM doxycycline solution. Doxycycline-soaked insert inhibited contraction, whereas control insert has no effect on the contraction pattern of the bilayer. N = 3, mean and SEM.