Kinetics and efficacy of photo-activated crosslink for tissue-engineering

We will review the kinetics, applications and efficacy of light-activated crosslink. The crosslink efficacy is influenced by multiple factors including: the applied light intensity, exposure period and dose, the initial concentration profiles of photosensitizers (PS) and oxygen, the quantum yield of the PS triplet state, the kinetic rate constants of PS (in type-I) and oxygen concentration (in typeII). Lower light intensity (I0) and higher PS concentration (C0) provides higher efficacy, but also suffers more toxicity to the cells. Higher light intensity (I0) may accelerate the crosslink but has lower efficacy (for the same dose). Therefore, optimal ratio of C0/ I0 is required for accelerated and safe crosslink. Safety issue also limits the maximum dose can be applied to the collagen, besides the limitation of PS concentration. Three important issues are presented: improvement of the crosslink speed (via higher light intensity) and efficacy of crosslink, and the role of photosensitizer (photo initiator) concentration.

Fundamentals and applications of medical lasers for various clinical procedures were reviewed recently by Lin [3]. Laser spectra of UV (190-400) um, visible (400-700) nm, near-IR (700-2900) nm, and mid-IR (3)(4)(5) um having various penetration depths (from determine the procedures are thermal or non-thermal or combined effect. invasive or noninvasive. Diode lasers have been widely used in many surgical procedures including soft tissue cutting, coagulation and cancer thermal therapy. Combining the nanoparticles and photosensitizers, diode lasers have been also used for cancer diagnosis and therapy [4,5]. Tissue-engineering using scaffold-based procedures have been attracting increasing attention [13][14][15][16][17][18][19], where chemical modification of gelatin has been reported to improve its mechanical properties by crosslinking or polymerization with UV [14][15][16] or visible light [17] to produce gels or high-molecular-weight polymers. A scaffold provides an environment for cell attachment, proliferation, differentiation, and drug delivery with high-loading efficiency at specific sites [13,14]. The goal of a photo-click hydrogel system is to identify key reaction parameters to enable fast gelation (<2 minutes), minimal photoinitiator-induced toxic response, and tunable hydrogel elasticity. In this mini-review article, we will focus on the kinetics of cross-linking to stabilize collagen in hydrogel biomaterials for tissue engineering [13][14][15][16][17][18][19]. we will present 3 important issues: improvement of the crosslink speed (via higher light intensity) and efficacy of crosslink, and the role of photosensitizer (photo initiator) concentration.

Method and Materials
The selection of photosensitizers Photodynamic therapy (PDT) uses photosensitizers activated by lights (lasers or LEDs) for various clinical procedures. As shown in Table 1

Mini Review
Kinetics and efficacy of photo-activated crosslink for tissue-engineering Photochemical Kinetics  [10].
As shown in Figure 1, the photochemical kinetics has three pathways [10,11] interacts with the ground oxygen (O 2 ) to form a reactive singlet oxygen (O*) [16]. The PS excited triplet can undergo two kinds of reactions. In type I reaction, it can react directly with a substrate (cell membrane or a molecule) and transfer a proton or an electron to form a radical anion or radical action, respectively. These radicals may further react with oxygen to produce reactive oxygen species (ROS). Alternatively, in a type II reaction, the triplet RF can transfer its energy directly to molecular oxygen (triplet in the ground state), to form excited-state singlet oxygen. As show by Figure   1, both type-I and type-II reactions can occur simultaneously, and the ratio between these processes depends on the types and the concentrations of PS, substrate and oxygen, the kinetic rates involved in the process.

Applications of crosslinking
As shown in Table 2, photodynamic therapy (PDT) offers many applications in dermatology, ophthalmology and cancer treatments in various parts of human body. Photo-polymerization activated collagen crosslinking offers corneal diseases treatment and tissue engineering, such as repair of osteochondral injuries. The design of regenerative devices using three-dimensional (3D) scaffolds may provide the appropriate environment, mechanical support and an initial cell anchorage site for the regeneration of tissues and organs [13]. Hydrogels provide a 3D hydrated framework with tissue-like elasticity for culturing cells.
They also possess the ability to encapsulate cells and molecules prior to gelation, thus affording a minimally invasive avenue to deliver cells and bioactive factors [13]. Recently, [14,15] developed a thiol-Ene reaction incorporated with collagen to form novel photo-click collagen-PEG hybrid hydrogels. The goal of a photo-click hydrogel system is to identify key reaction parameters to enable fast gelation (in seconds), minimal photo-initiator-induced toxic response, and tunable hydrogel elasticity. In the following, we will present 3 important issues: improvement of the speed and efficacy of crosslink; and the role of photosensitizer (PS) concentration, where lower PS concentration secures the cell viability, but has lower efficacy. Therefore, an optimal concentration and protocol are required.

Crosslink efficacy and safety
The crosslink efficacy is influenced by multiple factors including: the applied light intensity, exposure period and dose, the initial concentration profiles of photosensitizers (PS) and oxygen, the quantum yield of the RF triplet state, the kinetic rate constants of PS (in type-I) and oxygen (in type-II). Besides, the control of PS concentration during the light exposure are also important. Higher PS concentration provides higher efficacy, but also suffers more toxicity to the cells. Therefore, optimal ratio of C 0 /I 0 is required for accelerated and safe crosslink. Safety issue also limits the maximum dose can be applied to the collagen, besides the limitation of PS concentration. For a type-I dominated process, the corssolink efficacy is given by Eff=1 -exp(-S) and the photoinitiation rate S-function is given by [9,10]. (1), we will investigate the roles of C 0 , I 0 and D on the spatial (z) and temporal (t) profiles of S1, for both uniform and non-uniform cases.    e) As shown by Fig. 5, higher PS concentration leads to higher S, or efficacy; and optimal PS concentration (for maximum S) exits for z>0, but not for z=0. Lower PS concentration secures the cell viability but has lower efficacy. Therefore, an optimal concentration and protocol are required.

Conclusion
Lower light intensity (I 0 ) and higher PS concentration (C 0 ) provides higher efficacy, but also suffers more toxicity to the cells.
Higher light intensity (I 0 ) may accelerate the crosslink but has lower efficacy (for the same dose). Therefore, optimal ratio of C 0 / I 0 is required for accelerated and safe crosslink. Safety issue also limits the maximum dose can be applied to the collagen, besides the limitation of PS concentration.