Two-photon induced collagen cross-linking in bioartificial cardiac tissue

Abstract

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.

Nonlinear optical collagen cross-linking and mechanical stiffening: a possible photodynamic therapeutic approach to treating corneal ectasia

“…26 It has also been demonstrated 27 that mechanical stiffening of bioengineered cardiac tissue can be achieved using a 5-KHz FS laser light focused using long focal distance lenses, although the degree of stiffening was less than 35%. The current report demonstrates for the first time that NLO photoactivation of riboflavin using 75-MHz FS laser focused using a high NA lens within a collagen hydrogel induces similar amount of collagen stiffening to that achieved by UVA CXL.…”

“…26 Recently, NLO CXL has been used to stiffen bioengineered cardiac tissue, although the degree of mechanical stiffening was limited to less than 35% and not comparable to the therapeutically utilized UVA CXL of the cornea, which induces the mechanical stiffening of more than 200%. 27 In this study, we have evaluated the effectiveness of NLO CXL to mechanically stiffen compressed type I collagen hydrogels and compared the stiffening effect with that obtained using conventional UVA CXL. ), and the fluorescent signal was collected using the Meta Detector (Carl Zeiss) over the spectral range of 506 to 559 nm to include the peak signal (520 to 530 nm).…”

Biomaterial-based microstructures fabricated by two-photon polymerization microfabrication technology

“…The rigidity of the matrix was increased about 40% and the activity and function of cells were not affected. 87 Cell proliferation and differentiation behaviors are precisely controlled by TPP microfabrication. Shear et al showed that protein matrixes can be functionalized either through direct cross-linking of enzymes such as biotinylated enzymes or by cross-linking biotinylated proteins that were then linked to biotinylated enzymes via an avidin couple.…”

Pulsetrain-burst mode, ultrafast-laser interactions with 3D viable cell cultures as a model for soft biological tissues

“…Ever since they were first introduced for corneal flap creation [1,2] in Laser-Assisted in situ Keratomileusis (LASIK), ultrafast lasers have become a standard resource in corneal refractive surgery [3] and an emerging tool for use in cataract surgery [4]. Extensive research aims to incorporate ultrafast lasers in various surgical procedures such as microsurgery of vocal folds [5], craniofacial osteotomy [6], stapedotomy [7], cardiology [8,9], dentistry [10,11], and sub-cellular nanosurgery [12,13]. Continued increases in average powers for ultrafast fiber lasers [14] should eventually offer an even more compact and cost-effective alternative to solid-state ultrafast-laser systems.…”