MATERIALS AND METHODS
This review also contains some original new data presented in chapter 4 for which the methods are
given:
Cell culture
The human MG-63 cell line (osteoblast-like, osteosarcoma-derived) was cultured at 37° C, 5 % CO2
and 95 % relative humidity. The culture medium was DMEM supplemented with 10 % FBS and 1 %
penicillin/streptomycin (Sigma, Germany). Each passage of cells was cultured for 48 h before
splitting. After harvesting by trypsination, cells were counted and diluted with medium to the desired
concentration. Cells were cultured in 2D (films) and 3D environments (capsules, prints) for up to 6
weeks.
Preparation of biomaterials
Alginate (Alginic acid sodium salt from brown algae; Sigma) with medium molecular weight
(100,000-200,000 g/mol, guluronic acid content 65-70 %) was dissolved in PBS to 3.33 % (w/v). For
encapsulating cells in alginate-RGD, pure alginate-RGD (Novatach G RGD; FMC BioPolymer, USA)
was mixed with high molecular weight alginate (Sigma) to achieve a final concentration of 100 μM
RGD (total alginate concentration: 1.5 %). RGD, a tripeptide that mediates cell attachment via integrin
receptors, was covalently linked to the alginate. Gelatin (porcine, Type A; Sigma) was dissolved in
deionized water at 40° C to 5 % (w/v). Stock solutions were sterilized by filtration.
Production of alginate-gelatin blends
Alginate-gelatin blends were prepared by mixing alginate and gelatin stock solutions in an 80:20 ratio
by shaking and incubating at 40° C for at least 1 h prior to use. Final concentrations (w/v) were 2 %
(alginate) and 0.5 % (gelatin).
Preparation of alginate crosslinked with gelatin
For the crosslinking procedure alginate first needs to be oxidized. Oxidation was performed by
reacting alginate with sodium meta-periodate in a water-ethanol mixture according to13. The degree of
oxidation, which depends on the amount of sodium meta-periodate, was kept at 30 %. After reaction
stop with ethylene glycol, the resulting dispersion was left to sediment and the supernatant was
dialyzed against deionized water (6-8 kDa cut-off) for 7 days with daily change of water. The resulting
alginate di-aldehyde (ADA) solution was lyophilized. ADA was crosslinked with gelatin in a 50:50
ratio. The final concentrations of both ADA and gelatin were 2 % (w/v). ADA was dissolved in PBS
and sterilized by filtration. The gelatin solution was added while stirring. Due to fast increase in
viscosity, the crosslinked material (which has increased mechanical stability) had to be used in the
next 10-30 min for production of films or capsules.
Both blend and crosslinked biomaterials have been tested for biocompatibility (viability tests,
mitochondrial activity).
Film production and cell encapsulation and printing
Preparation of biomaterial films
Stock solutions of alginate and gelatin were combined to the desired ratio of 80:20 (blend). The
solutions were poured into Petri dishes and left to dry for 30 min to create a superficial, dried layer,
and to ensure a homogenous hydrogel surface. When dried, a sterile solution of 0.1 M CaCl 2 was
added to completely cover the hydrogel film and to initiate the gelation of alginate. After 20 min the
hydrogel was washed three times with ionized water to remove the CaCl2. Films were punched out
using a cylindrical stainless steel device (diameter: 0.9 cm, film thickness: 1-2 mm), transferred into
24-well plates and incubated in DMEM medium at least 1 h before cell seeding (with 1 ml of cells at
105 cells/ml).
Cell encapsulation
About 5x106 cells were resuspended in 50 µl DMEM medium and mixed carefully with 5ml of the
preheated (37° C) biomaterial solutions (blend - 80:20, crosslinked - 50:50, or pure alginate). The cell
suspension was 1 % of the total volume (106 cells/ml biomaterial) in order to prevent changes in
material concentration and viscosity. Additionally, alginate-RGD solution was either used pure or
mixed with nano-sized Bioglass 45S5 (0.1% w/v, particle size: 20-60 nm). The mixtures were poured
immediately into an extrusion cartridge which was connected to a high-precision fluid dispenser
(Ultimus V, Nordson EFD, Germany). After pressure adjustment, the mixture was extruded from a
specially designed nozzle (diameter: 200 µm) forming droplets of defined size into a beaker containing
sterile 0.1 M CaCl2 (gelation of alginate by Ca2+ complexation). After 10 min the capsules (diameter:
450±100 µm) were collected using stainless steel sieves (pore size: 100 µm), washed three times with
medium, transferred into 24-well plates and cultured. Capsules typically contained ~100-150 cells.
Cell printing
The 80:20 blend solution was filled into a syringe and mixed with MG-63 cells to 106 cells/ml.
Cylindrical 3D scaffolds (diameter 15 mm, 2 layers of configuration 0/90) with spacing between fibers
of 600 µm and a layer thickness of 200 µm were extruded (1.15 bar, 60 mm/s) with a nanoplotter 2.1
(GeSIM, Germany) in sterile Petri dishes. Prints were then treated for 10 min with 0.1 M CaCl2 to
induce fixation by gelation), washed with DMEM, covered with medium and cultured.
Preparation of collagen I – Bioglass composite scaffolds
For hydrogels, collagen I (calf skin, 0.5 % in 0.01 M HCl; MATRIX BioScience, Germany) was
diluted to 2 mg/ml with DMEM and neutralized to pH 7. Micron-sized bioactive glass (Bioglass 45S5,
mBG) with a mean particle size of 5 µm (commercially available) was solubilized by sonification,
mixed with collagen I to 1 mg/ml and incubated for 2 h at 37 ° C to induce gelation. A small amount
of hydrogel was applied to a coverslip and imaged with SHG. For scaffolds, collagen I (horse,
Kollagen resorb M; RESORBA, Germany) was dissolved to 3 mg/ml in DMEM and either mixed with
1.5 mg/ml mBG or left unmodified. After 4 h at 37° C, a gel was formed which was freeze-dried
(ALPHA 1-2 LD plus; Christ, Germany) and imaged with SHG.
Cell labeling
DAPI and calcein AM staining of encapsulated cells was performed in accordance to manufacturer
(Sigma) recommendations.
Multiphoton microscopy (MPM)
MPM was performed on two different laser scanning microscopes. The first system was TriMScope II,
a multiphoton microscope (LaVision BioTec, Germany) based on a Nikon eclipse Ti-U inverted
microscope, which was equipped with a femtosecond-pulsed titanium:sapphire (Ti:Sa) laser
(Chameleon Vision II, Coherent, USA) with dispersion precompensation and tuneable wavelength in
the range of 710-980 nm (pulse duration: <150 fs, repetition rate: 80 MHz). Images were collected
using a water immersion LD C-Apochromat objective (40×/NA 1.1/UV-VIS-IR/WD 0.62; Zeiss,
Germany) and detected with a high-sensitivity photomultiplier tube (H 7422-40, Hamamatsu
Photonics, Japan) mounted in non-descanned configuration close to the back aperture of the objective.
The second system was a Leica TCS MP5 (Leica Microsystems, Germany). This system was equipped
with a Mai Tai HP tunable Ti:Sa laser system (690-1040 nm, <100 fs, 80 MHz; Spectra-Physics,
USA), 2 NDD photomultiplier detectors and objectives with different magnification (HC PL Fluotar:
10x/dry, HCX PL APO CS objectives: 20x/IMM/NA 0.7 and 63x/Glyc/NA 1.3/UV; Leica
Microsystems).
Two-photon fluorescence and transmission imaging (figures 5A & 6D) and SHG experiments
(figures 7B & C) were performed on the MP5 system. Two-photon autofluorescence (AF) imaging
occurred at 800 nm and imaging wavelength of stained samples was dependent on the individual stains
(DAPI: 700 nm, calcein: 900 nm). SHG imaging was carried out in backscattering mode at 800 nm
with signal detection at 400±10 nm. Three-dimensional reconstructions from image stacks (z-distance:
9 µm) were calculated using the plugin 3D viewer in Image J (NIH, USA). Transmission images were
created by applying Ar+ laser light and detection in transmission mode. AF imaging experiments from
figures 5B & C (films and prints) were performed on the TriMScope. Depending on the individual
experiment according dichroic mirrors and blocking filters were applied.