In situ Imaging of Collagen Synthesis by Osteoprogenitor Cells in Microporous Bacterial Cellulose Scaffolds

Scaffold production

A protocol for production of microporous BC scaffolds based on particle leaching has been previously described in detail. ¹⁸ Briefly, paraffin wax microspheres having diameters in the range of 300–500 μm were added to the cellulose synthesis by G. Xylinus subsp. sucrofermentas BPR2001, trade number: 700178^™ (LGC Promochem AB). A volume of 2.5 mL of 3.7×10⁶ cfu mL⁻¹ bacteria in culture medium was seeded to each bioreactor. During fermentation, the bacteria extruded cellulose fibers into a mesh imbedding the wax microspheres. After 7 days of growth, the obtained cellulose structures were purified from bacteria, and the wax particles were leached out from the cellulose matrix as previously described. ¹⁸ The obtained microporous cellulose scaffolds were placed in deionized water and steam sterilized (1 bar, 121°C) for 20 min. After cooling to room temperature, the scaffolds were placed in an osteoprogenitor cell growth medium (details in the following text) for pretreatment over night to facilitate cell adhesion. The growth medium was removed just before cell seeding.

Cultivation of MC3T3-E1 osteoprogenitor cells

Cells of the MC3T3-El mouse calvaria-derived cell line (subclone 4. ATCC) were seeded onto the scaffolds (10 μL/scaffold at 2×10⁵ cells mL⁻¹), and allowed to attach for 4 h in the incubator before the addition of growth medium containing alpha-minimum essential medium (αMEM) (Invitrogen), 10% fetal bovine serum (Gemini Bio-Products), and 1% antibiotic/antimycotic solution (Invitrogen). The next day, the growth medium was replaced with differentiation medium (growth medium supplemented with 0.13 mM L-ascorbic acid 2-phosphate, 2 mM β-glycerophosphate, and 10 nM dexamethasone (Sigma Aldrich)) for a set of eight samples, in the following text referred to as sample category I. A group of four samples were cultivated using only growth medium, in the following text referred to as sample category II. Before SHG/CARS microscopy, these samples were fixed in 3.7% formaldehyde (Polyscience) and stored in PBS medium (Fisher). Cells were grown in an incubator at 37°C in an atmosphere containing 5% CO₂ at 95% relative humidity. Growth and differentiation media were changed every 2–3 days.

Histology

Four scaffold-cell samples cultivated in differentiation medium were harvested 1 and 7 days after cell seeding for histological analysis. The sample preparation followed by collagen type I staining was carried out at HistoCenter AB. The scaffold-cell constructs were placed in 4% buffered formaldehyde for dehydration, embedded in paraffin, and sliced along the vertical plane of the scaffolds into 5 μm thick sections. After deparaffinization, the sections to be stained for collagen type I were sequentially incubated in citric buffer pH 6.0 2×5 min, 3 vol% H₂O₂ for 5 min, 1% BSA (Sigma) for 20 min, streptavidin/biotin for 15 min, primary antibody (goat anti-collagen type I, Chemicon) diluted 1:20 in antibody dilutent (Dako) for 60 min, and Streptavidin HRP for 30 min. Incubated sections were then stained for collagen type I with DAB (Vector) for 5 min. Sample analysis was carried using an inverted microscope (Olympus CKX 41) at 20× magnification. Histology sections were also prepared without collagen staining to be used for comparative SHG microscopy.

Nonlinear microscopy

The setup for nonlinear microscopy has been previously described in detail. ²⁶ Briefly, two laser beams of synchronized pico-second pulse trains with near-infrared wavelengths are coupled into an inverted microscope and focused on a mounted sample. Sample illumination with laser beams at wavelengths of 817 nm and 1064 nm simultaneously induces the SHG and CARS processes in the sample. This allows for combined imaging of SHG-active cellulose and collagen fibers simultaneously with the seeded cells (CARS), the latter by probing a vibration of hydrocarbon CH₂ groups at frequency (wavenumber) 2845 cm⁻¹.

Both fixated whole cellulose scaffolds and sectioned samples were investigated using nonlinear microscopy. The intact scaffolds were contained in glass-bottom Petri dishes (WillCo-dish^® GWSt-5030, thickness 0.17 mm), whereas the sectioned scaffolds were mounted using a microscope slide and a 0.17 mm thick cover slip. The laser beams were focused on the sample (objective 40× Nikon Plan Fluor N.A. 1.3, working distance 0.21 mm), and forward propagating SHG and CARS signals transmitted through the sample were collected, separated by a dichroic beamsplitter, and registered by single-photon-counting photomultiplier tubes. Bandpass filters were used to isolate the SHG and CARS signals, generated at 409 nm and 663 nm, respectively, from background radiation. Typical average excitation powers at the sample were 40 mW per laser beam. SHG microscopy images were acquired in sectioned samples of category I for days 1 and 7, the image area was 200×200 μm², and the acquisition time was 60 s. Image data from the intact scaffolds of sample category II were acquired as simultaneous SHG and CARS microscopy z-stacks covering horizontal planes of area 100×100 μm² distributed over vertical ranges of 5–100 μm (vertical step size 0.5–2 μm). For the z-stack measurements, with multiple images collected at each lateral position, the image acquisition time was either 20 s or 60 s depending on the signal at the measurement location. Measurements were carried out on days 1, 4, 6, and 8 after cell seeding, one sample for each day. Merged SHG and CARS images and volume views showing the cell/scaffold system in three dimensions were generated using the software ImageJ. A measure on collagen content was evaluated from the image data as an area percentage of collagen fibers in the cell regions. The number of collagen signal pixels, isolated by manual thresholding of the SHG images, was divided by the area of the cell region identified from the corresponding CARS image. Collagen content was quantified for data measured at 1 (day 1), 5 (day 4), 2 (day 6), and 3 (day 8) different lateral positions and up to six slices, separated 5–10 μm, were evaluated in each z-stack.

Sample category I

Histology sections of samples from category I, that is, cellulose scaffolds with osteoprogenitor cells grown on differentiation medium, clearly showed ECM collagen type I as identified by DAB-stained regions in brown color (Fig. 1a, c) and expected for the investigated cell type. ³⁴

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FIG. 1.

Collagen type I imaging on microporous cellulose scaffolds one (a and b) and seven (c and d) days after MC3T3-E1 cell seeding. (a) Histology cross section of a one-day-old sample shows brown DAB-stained collagen regions along the upper edge corresponding to the seeded surface (arrows). (b) SHG microscopy image of corresponding unstained section. Image contrast is mainly due to SHG signal of the cellulose scaffold, and the contour corresponds well to the right part of the histology image. Regions of stronger SHG signal can be seen at the boundaries in agreement with stained regions in (a). (c) Histology cross section of seven-day-old scaffold shows a thicker cell layer and an increased amount of stained collagen across the scaffold surface compared with (a). (d) SHG image of unstained section showing a much higher amount of strongly SHG-generating structures compared with (b), signifying an increase in collagen on the scaffold. Fibrillar collagen segments (indicated by arrows) can be identified in the magnified image in the inset. SHG, second harmonic generation. Color images available online at www.liebertonline.com/tec

After one day of growth, regions of stained collagen can, in particular, be seen along the upper edge, where regions of cells can be identified, indicating the seeded surface (Fig. 1a, arrows). An SHG microscopy image measured on an unstained section cut from the same one-day-old scaffold mainly shows a relatively low SHG signal obtained from the cellulose fibers (Fig. 1b, blue color). The contours in the SHG image of the scaffold correspond well to the shape of the stained scaffold section (Fig. 1a). In addition, narrow regions of higher SHG signal can be found on the edges of the structure in the SHG image, in good agreement with the brown collagen staining seen along the edges in the histology image (Fig. 1a).

In contrast to the relatively low amount of DAB-stained structures after one day of growth (Fig. 1a), substantial amounts of stained collagen can be found in a seven-day-old sample (Fig. 1c). Stained fibers can be seen in the multiple cell layers built up across the entire scaffold and essentially bridging over the open pores (Fig. 1c, arrows). In addition, rather dense stained regions can be seen along the pore edges and at the bottom of the pores, indicating a downward cell migration along the pore edges.

An SHG image measured on a seven-day-old sample (Fig. 1d) contains many compact SHG-active structures generating signals at least one order of magnitude stronger than the cellulose, as identified in the SHG image of Figure 1b. Since collagen generally generates much stronger SHG signal than cellulose^21,26 the higher signals compared with the one-day-old sample are in agreement with the increased amount of collagen expressed on this scaffold as identified by histology (Fig. 1c). Similar to the histology image, the collagen regions identified in the SHG image appear rather compact and homogeneous; however, fiber segments can also be identified, as shown in the insert of Figure 1d. Thus, the ability to monitor collagen production on cellulose scaffolds by means of SHG microscopy was well confirmed by comparison with established technology.

Sample category II

SHG-imaging of cellulose and collagen in scaffolds seeded with osteoprogenitor cells cultivated in growth medium, sample category II, was combined with simultaneous CARS microscopy for visualization of cell proliferation and morphology (Figs. 2 and 4). Merged SHG and CARS microscopy images measured on cellulose scaffolds one day after cell seeding show that the spindle-shaped osteoprogenitor cells (orange color) have proliferated on the cellulose (blue fibers) and started forming a confluent single-cell layer on the surface (Fig. 2b). SHG data measured at this point of time essentially show the cellulose matrix (Fig. 2a, b, blue fibers); however, SHG-active fiber segments, 10–20 μm long and around 1 μm thick, located in the immediate vicinity of some of the imaged cells, and with 5–10 times stronger signals than the cellulose, could also be identified (Fig. 2c, arrows). The position of cells (orange color) and collagen fibers (bright blue color) above the cellulose scaffold (dark blue region) can be seen in the volume views of Figure 2d and e, a more three-dimensional arrangement can be observed compared with Figure 2b.

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FIG. 2.

Early cell proliferation and collagen production. SHG/CARS overlay images and rendered volume views of MC3T3-E1 cells grown on cellulose scaffold using standard growth medium for one day. (a) Image of cells (CARS, orange color) proliferating on a homogenous cellulose fiber matrix (SHG, blue color). (b) Volume view showing the cells arranged in a planar layer 5 μm thick. (c) Fiber segments of higher SHG signal (arrows) located in the vicinity of cells indicate early collagen production. (d) Overlay volume view (e) SHG volume view, depth 30 μm. Cells (CARS, orange color) and collagen fibers (SHG, blue color) are positioned on top of the cellulose (SHG, dark blue color) in a three-dimensional configuration compared with the planar cell arrangement in (a). CARS, coherent anti-Stokes Raman scattering. Color images available online at www.liebertonline.com/tec

This indicates an initiated collagen production at this early stage, also observed for the samples of category I (Fig. 1a, b). Additionally, the detection of individual fiber segments highlights the feasibility of SHG microscopy for detailed studies of collagen production. Collagen synthesis is characteristic for osteoprogenitor cells in their mature stage and, in particular, for cells differentiated into osteoblasts. ³⁴ The generation of an extracellular collagen matrix has been identified as a prerequisite for osteoblast differentiation of MC3T3-E1 cells, and initial collagen production during the first two days of growth was followed by increasing expression of the osteoblast phenotype and further collagen synthesis. ³⁵ In addition, ECM collagen production after one day of growth has previously been noted for MC3T3-E1 cells grown on collagen scaffolds. ³⁶

Gradually, during growth, denser multi-cell regions developed and could be observed in the cellulose micropores (Fig. 3) in agreement with previous observations ¹⁹ and indicating proliferation characteristic for the osteoprogenitor phenotype. ³⁴ Simultaneous SHG and CARS measurements in these dense regions clearly show developed networks of collagen fibers within the cellular matrix (Fig. 4a–c) at depths ranging from 10 to 80 μm below the upper cell layer. Thus, SHG/CARS microscopy allows for noninvasive imaging under native conditions below the surface of the tissue construct without need of the sectioning carried out for samples of category I (Fig. 1). Based on excitation using near-infrared radiation, nonlinear techniques have a distinct advantage compared with microscopy using visible light, which generally shows higher extinction during tissue penetration. The maximum attainable imaging depth depends on experimental parameters such as excitation intensity, focusing optics, and extinction of laszr beam and propagating signal in the sample. In the presented measurements, scanning depths down to 100 μm was adequate to cover the cell regions at the probed positions. Under appropriate experimental conditions, nonlinear microscopy techniques allow for imaging deep inside tissue, demonstrated for collagen down to a depth of 550 μm using femtosecond laser pulses for excitation, and an objective with long working distance for focusing. ²¹

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FIG. 3.

Bright field image of microporous cellulose scaffold after six days of growth with dense clusters of MC3T3-E1 cells located in the pores (arrows).

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FIG. 4.

Collagen production in cellulose micropores. Synthesized collagen fibers (SHG, blue color) can be distinguished embedded in compact cells clusters (CARS, orange color) established in the scaffold pores. Measurements on six- (a and b) and eight- (c) day-old samples shown as SHG/CARS overlay images (left), SHG/CARS overlay volume views (middle), and SHG box volume views (right). (a) Region with alignment between cells and fibers; however, the clusters of randomly oriented cells in the micropores generally result in random collagen fiber orientation (c). (b) Fiber network in vicinity of a pore boundary, indicated by the dashed white line and the cellulose SHG signal in the upper right corners of the corresponding volume view and box view. (c) Collagen network in the pore center. A thicker cell region and arrangement of collagen fibers ∼70 μm deep (SHG box volume view, right) can be seen compared with (a) and (b). Note the random fiber orientation in the box volume view compared with (a). Lateral image size 100×100 μm, box view depths 36 μm (a), 42 μm (b) and 100 μm (c). Color images available online at www.liebertonline.com/tec

Evaluated collagen fiber diameters were typically 1 μm, although some fiber bundles a few microns wide could also be observed. Since high collagen expression is a hallmark of the osteoblast phenotype and differentiation of MC3T3-E1 cells grown on cell cultivation dishes have been observed after four days of growth, ³⁵ it is possible that category II samples of the later time points also contain cells differentiated into osteoblasts.

An alignment of ECM collagen fibers along the long axis of MC3T3-E1 cells has previously been observed for cells grown on synthesized collagen scaffolds, and a similar arrangement could be seen in some of the measured SHG/CARS images, as exemplified in Figure 4a. Moreover, other studies of MC3T3-E1 cells have reported an alignment of ECM collagen fibers in the main migration direction of the cells.^36,37 However, the dense structures of randomly oriented cells found in many micropores make such fiber alignment less apparent (Fig. 4c, box view). Furthermore, networks of ECM collagen fibers can be found close to pore boundaries (Fig. 4b) as well as in centers (Fig. 4c), showing collagen build-up within the entire volume of a cellulose micropore and confirming supply of nutrition and oxygen.

For the images of Figure 4a and b, measured in a 6-day-old sample, collagen fibers are located in regions around 25 μm thick, whereas the image in Figure 4c, taken in an 8-day-old sample, shows an entangled collagen network covering a depth of around 70 μm. The development in these samples indicates a first step toward matrix mineralization and confirms the functionality of BC as a scaffold for osseous tissue.

Production of ECM collagen by osteoprogenitor cells has been reported to depend on multiple parameters such as scaffold material structure, ³⁸ cell density, ³⁹ mechanical cues, ⁴⁰ and oxygen availability.^28,29 A dependence on local cell density could be observed in our data when evaluating the cell matrix collagen content from SHG images. Figure 5 shows the percentage of imaged cell area covered by collagen fibers versus the thickness of the cell region. Highest values are obtained for SHG images measured in thick regions of multiple cell layers, that is, of high local cell density.

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FIG. 5.

Percentage collagen fiber area relative to cell region area, evaluated from SHG/CARS images, plotted versus cell region thickness. Bars represent averages, whereas (+) and (−) indicate maximum and minimum values, respectively. Highest amounts of collagen were obtained in images from thick cell regions, indicating a coupling to local cell density.

In general, there is potential for control of the ECM collagen synthesis by modification of scaffold structure and composition as well as via cell cultivation conditions. Recent technical developments allowing for improved control of cellulose scaffold architecture, cell seeding, and growth conditions facilitate the possibility to obtain an optimal cellulose-based bone graft.