Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.
© 2011 OSA
Tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs and tissues for transplantation purposes . Three-dimensional tissue engineering aims at mimicking the characteristics of natural tissue in matrix composition and morphology, as well as cellular distribution and orientation. For cardiac tissue engineering, the following physiological properties are essential for the generation of functional transplantable tissue surrogates: i) spontaneous and synchronous contractility, ii) development of sufficient systolic (contraction) and diastolic (passive) forces to support the heart’s pumping function and to withstand the hydrodynamic pressures in the heart, respectively. Several groups have shown the possibility to generate functional artificial cardiac tissue and its transplantation in animal models with beneficial effects to heart function after acute myocardial infarction [2–4].
Mechanical properties of the artificial tissue are mostly influenced by the initial seeding number and composition of cells, providing stabilization via their cytoskeleton and the production of extracellular matrix (ECM) proteins. By adjusting the initial addition of ECM components and/or composition of other biomaterials as scaffolds with different intrinsic mechanical properties, tissue mechanics can be defined in a wide range of requirements [5,6]. Vice versa, scaffold mechanical stiffness was found to have a significant influence on cardiomyocyte morphology and function, both when seeded as monolayer cultures  or in three-dimensional engineered cardiac constructs . One way to modulate tissue properties after tissue generation is in vitro maturation and remodeling, e.g. by application of mechanical stretch stimulation in a bioreactor [8, 9]. Another way is cross-linking of matrix components. In a transgenic approach, over-expression of lysyl oxidase was utilized to enzymatically cross-link ECM proteins, particularly collagen and elastin, to enhance the mechanical integrity of the ECM and thereby impart mechanical strength to the engineered tissue . Alternatively, glycation, i.e. the non-enzymatic cross-linking of proteins by reducing sugars, was demonstrated to stiffen and strengthen tissue equivalents in a matter of a few weeks of incubation . By contrast, chemical methods allow for fast and efficient cross-linking and might enable direct spatial control of tissue stiffness.
Chemical cross-linking has been established as an efficient method to stabilize collagen-based biomaterials, such as porcine heart valves  and blood vessels . In ophthalmology, it is used to increase the human corneal stiffness to stop the progression of keratoconus . Collagen cross-linking is generally induced by either aldehyde-reactions or UV-A irradiation with photosensitizers, such as riboflavin. Aldehyde cross-linking is presumed to result from covalent bond formation of aldehyde groups with amino groups or peptides. To avoid inflammatory reactions, cytotoxicity and calcification after implantation of cross-linked tissue into the patient, thorough removal or inactivation of excess aldehyde molecules is necessary . UV-A irradiation of photosensitizers catalyzes carbonyl-based cross-linking reactions via one-photon photosensitized singlet oxygen production [16, 17]. However, the positive effect of both methods on the tissue stiffness is accompanied by cell apoptosis in the treated area. In cornea, this negative side effect is compensated by subsequent repopulation of treated areas with viable cells in vivo . In case of cardiac tissue, repopulation is impaired by the non-proliferative phenotype of cardiomyocytes and most probably would result in the formation of a fibrous scar. Therefore, to apply cross-linking to cardiac tissue engineering, novel methods must be developed and evaluated which do not alter cell viability.
Compared to continuous UV-A illumination, the interaction of femtosecond (fs) laser pulses with biological tissue is based on nonlinear excitation. This enables higher penetration depths and impedes out-of-focus absorption and photodamage . Therefore, fs laser in the near infrared (NIR) wavelength range are extensively used for therapeutic applications on a sub-cellular level, such as chromophore assisted laser inactivation of proteins , intracellular nanodissection  and laser uncaging . All these applications have in common that biological samples are treated with a light-sensitive probe (e.g. fluorophore, caged compound) with a high multiphoton absorption cross-section prior to irradiation. These photosensitizers enhance the yield of chemical reactions with surrounding molecules leading to bond cleavage or rearrangements [22,23]. For example, Frederiksen et al. showed that singlet oxygen is largely produced by two-photon excitation of different photosensitizers . Great care has to be taken in the photosensitizer selection, as two-photon absorption of intrinsic fluorophores induces severe photodamage including impaired cell division or apoptosis [25, 26]. At even higher laser intensities, higher-order effects become more important causing photobleaching of photosensitizers, oxidative stress and the formation of a low-density plasma [27, 28]. As the two-photon absorption cross-section scales super-linearly with the one-photon absorption cross-section, differences between extrinsic and intrinsic fluorophores are much more pronounced in the former case . Consequently, nonlinear excitation with NIR wavelengths may be suitable for collagen cross-linking without compromising cell viability.
In this paper, we show the great potential of fs laser pulses for collagen cross-linking in bioartificial cardiac tissue. Collagen cross-linking was induced by raster-scanning the laser beam over riboflavin treated tissue. Subsequent stress-strain measurements were done to evaluate tissue stiffness and contractility while fluorescence microscopy was used to assess cell viability.
2. Materials and methods
2.1. Tissue preparation
Two cell sources were used for artificial tissue preparation: i) for proof-of-concept, murine embryonic fibroblasts (MEF) were used to generate a model system of collagen-rich artificial tissue, ii) neonatal rat cardiomyocytes (NRCM) were used to generate bioartificial cardiac tissue (BCT). Three-dimensional tissue was prepared as described earlier . In brief, either 6·105 gamma-irradiated MEFs from CD1-ICR mice, or 1·106 freshly isolated cardiomyocytes (NRCMs) from Sprague-Dawley neonatal rats (enriched by discontinuous Percoll gradient centrifugation) were mixed with liquid extracellular matrix (consisting of 0.9 mg/ml collagen type I (R&D Systems), 10% Matrigel (BD Biosciences)) and poured into custom-made Teflon molds (220 μl). The mixture was covered with 5 ml Dulbecco’s modified Eagle’s medium (DMEM) containing 12% horse serum, 2 mM L-glutamin (all Gibco), 2% chicken embryo extract (US Biological), 10 μg/ml insulin (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories) and cultured in a standard incubator at 5% CO2, 37°C and 80% humidity with daily medium exchange. Within the molds, the solidified tissue was suspended between two titanium rods and had an average cross-sectional area of 0.8 mm2 on day 7 (see Fig. 1a).
All experiments were performed in accordance with the principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985) as well as the Animal Welfare Law of Lower Saxony, Germany.
2.2. Optical Setup
The optical setup is shown in Fig. 1b. The laser source was a regeneratively amplified Ti:Sapphire laser system producing femtosecond pulses, either a Spectra Physics Spitfire Pro (120 fs, 800 nm, 5 kHz) or a Thales Bright (120 fs, 780 nm, 5 kHz). At the used wavelengths, riboflavin has a two-photon action cross-section of about 0.5 GM at 780 nm and 0.45 GM at 800 nm . The laser beam was demagnified by a two-lens telescope to match the diameter of the following optics and attenuated by a variable neutral density (ND) filter. Two high-speed galvanometer mirrors (Litrack) were used to scan the laser beam in the x-y plane. Focusing into the tissue was achieved either by a 140-mm or 400-mm focal length lens. This resulted in a spot diameter of 130 and 380 μm as well as a rayleigh length of 17 and 145 mm, respectively, being much larger than the average tissue depth of 0.8 mm.
2.3. Two-photon induced collagen cross-linking
After six days of cultivation, the photosensitizer riboflavin (RF, Sigma Aldrich) was added to one part of the tissue constructs at 0.01% (0.27 mM) concentration in the culture medium. This RF concentration is one order below that used for UV-A collagen cross-linking . However, solutions with higher concentrations could not be made because of its limited solubility in the culture medium. On day seven, each tissue was transferred within the Teflon mold to a 35 mm glass bottom dish (ibidi GmbH) with a thickness of 170 μm. Collagen cross-linking was induced by raster scanning the laser beam over the tissue with a constant scanning speed and line separation (see Fig. 1c). The line separation Δy corresponded to the focal spot radius for both focusing lenses. Depending on the scanning parameters, the cross-linking procedure lasted approximately 30 seconds to 5 min. The experiments were done at room temperature and normal atmosphere unless otherwise stated. Immediately after laser irradiation, each tissue was washed in culture medium without RF and further incubated for 24 hours at 37°C and 5% CO2 humidified atmosphere.
2.4. Stress-strain measurements
Forces were measured in a bioreactor system that allows for continuous increase in strain and on-line force measurement in a standard incubator . For MEF-based and bioartificial cardiac tissue (BCT), the increase in passive force as a function of increase in strain (33.25 ± 1.75 μm/s) along the longitudinal axis of the tissue constructs was measured for each tissue in culture medium until the preload reached 20%. The engineering stress was calculated as force divided by the initial cross-sectional area. The slope of the linear region of the stress-strain curve (”Young’s modulus”) was determined by linear regression. To assess the active contractility of treated and untreated control groups of cardiac tissue, BCTs were electrically paced five times with no preload at 25 V with rectangular pulses (5 ms) prior to stress-strain measurements.
The statistical significance was tested using one-way analysis of variance (ANOVA) followed by multiple comparisons against the untreated control group using Dunnett’s test. Differences were considered significant at P < 0.05.
2.5. Fluorescence microscopy
One day prior to cross-linking experiments, the tissue was incubated with 25 nM tetramethylrhodamine methyl ester (TMRM; Invitrogen). TMRM is a cationic fluorescent dye being rapidly and reversibly taken up by live cells and sequestered to active mitochondria, and therefore can be used to monitor the cellular metabolic activity. Re-staining with TMRM immediately after laser irradiation was performed to compensate for photobleaching. Images of the tissue cross-sectional area were obtained while sandwiched between two cover glasses. To analyze the volumetric cell density one day after cross-linking, tissues were fixed for 20 minutes in acetone and nuclei were stained with DAPI (Invitrogen). Fluorescence images were either captured with a commercial AxioObserver Z1 microscope (Carl Zeiss AG) or a custom made scanning laser optical tomograph (SLOT) described elsewhere . In brief, SLOT is a highly efficient 3D fluorescence microscopy technique capable of imaging specimens with sizes up to several millimeters. For imaging, the specimen was mounted in a glass capillary filled with 100% glycerol for optical clearing.
3.1. Collagen cross-linking in MEF-based tissue
The first set of experiments was done with the Spitfire Pro laser system. The focal spot radius and scanning speed were determined to 65 μm and 300 μm/s, respectively, corresponding to an irradiation time of approximately 5 minutes. In preliminary experiments, tissues were irradiated with different pulse energies to identify suitable parameters. The laser fluence was thereby defined as the pulse energy, divided by the focal area, times the number of pulses. Two laser fluences were chosen empirically for two-photon induced collagen cross-linking : 160 and 320 J/cm2. For MEF-based tissue as our model system of collagen-rich artificial tissue, samples were divided into five groups: (1) untreated control, (2) RF treatment, (3) 160 J/cm2 laser fluence with RF treatment and 320 J/cm2 laser fluence (4) with and (5) without RF treatment.
All tissues exhibited similar stress-strain relationships 24 hours after irradiation. The stress-strain curves showed an initial nonlinear (”toe”) region followed by a linear region from about 15% strain (see Fig. 2a). RF treatment alone did not influence the stress-strain relation. By contrast, irradiation of RF treated tissues at 160 J/cm2 resulted in an increased stiffness of 35% compared to untreated controls at 20% strain (see Fig. 2b). A similar rise was observed for the slope of the stress-strain relation in the linear region, the so-called Young’s modulus (46.6±0.3 vs. 34.8±2.3 kPa). When the laser fluence was doubled to 320 J/cm2, the positive effect on tissue stiffness was no longer observed, independent of RF treatment.
At the lower fluence, the TMRM and DAPI fluorescence intensities of irradiated and non-irradiated areas were comparable. Therefore, laser irradiation at these parameters had no detrimental effects on cell metabolic activity and density. By contrast, the metabolic activity was markedly reduced at the higher fluence (see Fig. 4a), independent of RF treatment. At the same time, no influence on cell density was found as indicated by unchanged DAPI fluorescence intensities (data not shown).
3.2. Collagen cross-linking in bioartificial cardiac tissue (BCT)
As a next step, collagen cross-linking in BCTs was evaluated under the same experimental conditions as in Sec. 3.1. BCTs were divided into four groups: (1) untreated control, 160 J/cm2 laser fluence (2) with and (3) without RF treatment and (4) 320 J/cm2 laser fluence with RF treatment.
As with MEF-based tissue, the tensile stress showed a nonlinear dependence on the strain amplitude, which changes into a linear dependence at 15% strain. However, the stiffness and Young’s modulus of BCTs were about a factor three lower (data not shown). Irradiation of untreated BCTs at 160 J/cm2 did not change the stress-strain relation. Using the same irradiation parameters for RF treated tissues resulted in an increased stiffness by 25% compared to untreated controls at 20% strain, while Young’s modulus only increased by 6% (14.9±1.7 vs. 14.1±1.4 kPa). At the higher fluence of 320 J/cm2, mechanical properties of tissues were no longer influenced. Because of the high variation of stiffness in all treatment groups, the measured differences were not statistically significant (P > 0.25).
Similar to the previous section, irradiation at 160 J/cm2 laser fluence had no influence on cell metabolic activity and density, as indicated by strong TMRM and DAPI fluorescence (see Fig. 3a and c). At the higher fluence, reduced metabolic activity was observed in irradiated areas, but without any change in volumetric cell density (see Figs. 3b and 5). TMRM fluorescence images of the BCT cross-sectional area in this parameter regime revealed that the depth of laser treatment decreased with increasing incident angle of the laser beam on the curved tissue surface (see Fig. 4b). The maximum depth of laser treatment was measured at an incident angle of 0° to about 520 μm, being more than half the tissue depth.
In case of BCTs, tissue functionality can be directly assessed by measurement of contractile forces, which will be exerted by viable cardiomyocytes only. All BCT groups exhibited spontaneous active contraction with no preload both before and 24 hours after irradiation. Electrical stimulation in the bioreactor induced contraction forces up to 0.8 mN with no significant difference between the experimental groups (P > 0.3, data not shown).
3.3. Optimization of two-photon induced collagen cross-linking in BCTs
For the treatment of cardiac tissue, protocols were further optimized to reduce the stress applied to the cells and to improve the enhancement of mechanical properties. First, an infrared lamp was integrated into the setup and HEPES buffer was added to the culture medium to maintain optimal culture conditions outside the incubator (37°C and 5% CO2). Second, the focal spot diameter and scanning speed were increased from 130 to 380 μm and from 300 to 1300 μm/s, respectively, to reduce the irradiation time to 30 s. Third, repetitive raster scanning of the tissue enabled the use of lower pulse energies for collagen cross-linking as multiphoton-induced photochemical effects accumulate over multiple pulses [23, 28]. However, to maintain a high sample throughput, the scanning pattern was only applied five times. Fourth, the experiments were done with the Thales Bright laser system at 780 nm at which the two-photon action cross-section of RF is about 10% higher .
Using optimized conditions, stress-strain measurements revealed a strong influence of the laser fluence on the mechanical properties (see Fig. 6). Significant tissue stiffening by 40% at 20% strain was only observed in a small window of laser fluences around 50 J/cm2 (P < 0.05). The corresponding Young’s modulus increased by about 15% (30.6±0.9 vs. 26.5±1.9 kPa). Compared to Sec. 3.2, the enhancement of mechanical properties was improved by about a factor two. At the same time, no significant influence on active contraction force and cell metabolic activity was observed (see Fig. 4a), independent of the laser fluence up to 95 J/cm2 (P > 0.1).
4. Discussion and conclusion
The presented results indicate the great potential of femtosecond (fs) laser pulses for fast, efficient and non-toxic cross-linking of bioartificial collagenous tissue.
To induce collagen cross-linking, we irradiated riboflavin treated collagenous tissue with fs laser pulses in the NIR wavelength range at low pulse energies generally used for multiphoton-induced photochemistry . The resulting maximum treatment depth was about 520 μm, corresponding to 65% of the average tissue depth (see Fig. 4b). In contrast to existing methods, raster scanning of the laser beam enabled full spatial control of selective cross-linking in three-dimensional tissue [14, 15]. Owing to the surface curvature, the coupling efficiency of laser energy into tissue continuously decreased towards the edges of the tissue, as described by the Fresnel reflectivity equations and Snell’s Law . Consequently, only half the tissue volume was irradiated with about the same fluence. The achievable precision with our setup was in the order of 100 μm, equivalent to the focal spot diameter. By applying tight focusing with high numerical aperture objectives, the focal spot diameter can be reduced to the sub-micrometer range . In combination with a rotating stage for the tissue, homogeneous energy deposition and hence selective cross-linking within a sub-femtoliter volume is feasible in future experiments.
In our experiments, we used MEF-based and bioartificial cardiac tissue (BCT), containing fibroblasts and enriched primary cardiomyocytes with a lower proportion of fibroblasts, respectively, to examine the influence of different cell compositions. One-day post irradiation, the stress-strain relations of both tissue types exhibited a nonlinear ”toe” region followed by a linear region from about 15% strain, characteristic of collagenous tissue [33, 34]. At a fluence of 160 J/cm2, considerable tissue stiffening and increase in Young’s modulus by 35% was observed in MEF-based tissues. Using the same parameters, the enhancement of mechanical properties was lower in BCTs (compare Figs. 2a and 6a). The higher increase in MEF-based tissues can be explained by the higher initial fibroblast content and hence a higher content and/or turnover of extracellular matrix (ECM) molecules, such as collagen, over cultivation time .
As riboflavin is known to induce UV-A collagen cross-linking via one-photon photosensitized singlet oxygen production [16, 17], the observed increase in stiffness most likely resulted from similar processes upon two-photon excitation. This is underlined by two facts: i) riboflavin fluorescence was observed during fs laser beam raster scanning and ii) irradiation of untreated tissues did not influence the tissue stiffness. Although TMRM was present in the culture medium with a much higher two-photon action cross-section than riboflavin , it had no detectable influence on collagen cross-linking owing to the 10,000 times lower concentration and the selective accumulation in mitochondria.
Optimization of the experimental conditions and cross-linking protocol resulted in a significantly increased tissue stiffness by 40% at 20% strain in BCTs (see Fig. 6b). At the same time, Young’s modulus increased by 15%. Compared to the first set of experiments, this was a two-fold improvement. In comparison, ”biologic” cross-linking by over-expression of lysyl oxidase in smooth muscle cells seeded in collagen gels resulted in a doubling of Young’s modulus for these constructs. However, this process was slow and required genetic modification . Previous studies using UV-A irradiation for collagen cross-linking of riboflavin treated human corneas reported an even higher 4.5-fold enhancement of Young’s modulus . This can be mainly attributed to the much higher collagen content, much longer irradiation time of 30 minutes and 10-fold higher riboflavin concentration. As multiphoton-induced photochemical effects such as photosensitized singlet oxygen production accumulate over multiple pulses, we expect to further increase the tissue stiffening by using prolonged irradiation times .
UV-A cross-linking protocols for corneas are usually associated with cell death within the treated area followed by in-vivo repopulation . In our approach, cell viability was maintained both in MEF-based tissue and BCT, as indicated by strong TMRM fluorescence in the irradiated area (see Figs. 3a and 4a). Furthermore, their volumetric density was not influenced and DAPI fluorescence was still intense (see Figs. 5 and 3c). As active contraction forces of irradiated BCTs were comparable to untreated controls (see Fig. 6b), we can conclude that cardiomyocytes remained fully functional after cross-linking. This could indicate that intracellular free radical scavengers like glutathione were able to neutralize the oxidative stress induced by fs laser irradiation [25, 37]. To the best of our knowledge, similar observations have only been made after one-photon induced cross-linking of fibrin-based engineered connective tissue . Therefore, our experimental setup provides for the first time a fast and effective method to increase the stiffness of collagenous tissue without impairing cell viability. However, great care has to be taken in adjusting the laser fluence, as cellular metabolic activity markedly decreased and no positive effect on tissue stiffness was observed at very high fluences (see Fig. 3b and 2a). This is likely a result of excessive oxidative stress and/or progressive heat accumulation within the tissue during the scanning procedure . Nevertheless, active contraction forces of those BCTs were comparable to untreated controls (see Fig. 6b)), providing evidence that cardiomyocytes were still functional.
Our results suggest a ”window” of laser fluences, in which two-photon induced collagen cross-linking can be achieved while maintaining cell viability. To the best of our knowledge, this was not yet observed using one-photon absorption. To obtain a safe process window, the difference of the absorption cross-sections of riboflavin compared to intrinsic fluorophores, such as NAD(P)H, has to be sufficiently high . Other groups have shown that the two-photon absorption cross-section scales super-linearly with the one-photon absorption cross-section . Therefore, we assume that nonlinear excitation of riboflavin with NIR wavelengths is the only mechanism providing a large enough process window for minimally invasive collagen cross-linking. Future studies have to further optimize the laser fluence within the process window to enhance the achievable increase in stiffness.
In conclusion, we demonstrated for the first time the fast and efficient cross-linking of riboflavin treated collagenous tissues with fs laser pulses in the NIR wavelength range. In contrast to existing methods, the laser-tissue interaction is based on nonlinear (two-photon) absorption. Our experimental data suggest that this provides a process window, in which collagen cross-linking is achieved while maintaining cell viability and functionality. The nonlinear laser-tissue interaction in combination with high spatial precision in the micrometer range offers the possibility for selective cross-linking of arbitrarily defined patterns in three-dimensional tissue engineered constructs.
Patterning of artificial cardiac tissue could be implemented to recapitulate the intrinsic anisotropy of native myocardial tissue. As tissue stiffness plays an important role in remodeling and regeneration after myocardial infarction, artificial tissue with precisely defined mechanical properties using two-photon induced cross-linking could provide new therapeutic strategies to mechanically support heart function. For instance, it was proposed that most of the benefit could be achieved by selectively stiffening the longitudinal direction without altering the circumferential direction . Therefore, our fast, efficient and cell-compatible method for controlling the physical properties of the ECM is a valuable tool for studying and improving engineered cardiac tissue for regenerative therapies.
We would like to thank David Skvorc for excellent technical assistance. This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Cluster of Excellence ”REBIRTH” (From Regenerative Biology to Reconstructive Therapy).
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