ELECTROMAGNETIC MODELING FOR THE DEVELOPMENT AND OPTIMIZATION OF DIFFERENT DEVICES TO SUPPORT BONE REGENERATION

Tissue healing is in general a complex process, which involves both local and systemic responses, and bone regeneration in particular is much slower than repair in any other human tissue. Thus, it exhibits a great challenge in clinical practice and in the field of research. Bone regeneration is comprised of a series of biological events, involving a number of cell types and intracellular and extracellular molecular- signaling pathways, with a definable temporal and spatial sequence, in an effort to optimize the skeletal repair and restore its functionality. Photobiomodulation (PBM) therapy has been shown to be effective in modulating both local and systemic responses, by enhancing cellular activities resulting in an increase in function, especially in injured tissues, leading to optimization of tissue repair and regeneration. In bone tissue, application of the photonic energy leads to bone healing by the activation of osteoblasts, leading to proliferation and differentiation, as well as osteoclast inhibition and, consequently, neoformation of bone matrix. The process of the in vitro pre-osteoblasts maturation, mimicking their in vivo behavior, passes through three distinct stages of development: proliferation, early differentiation (maturation) and late differentiation (mineralization). Despite the extensive research on the effects of photobiomodulation (PBM) light on bone regeneration, the current outcomes ranging from positive to negative effect remain controversial. These contradictory data are thought to be due to; incomplete knowledge and understanding of the mechanistic effects of laser light on cells, lack of standardized laser dosimetry, inefficient laser beam profile, improper study design and varied methods of investigation. The literature is hindered by a considerable heterogeneity of the irradiation parameters of PBM, as well as, the methods utilized to evaluate the results and the type of osteoblast-like cells irradiated. This has led to a need of standardization. Moreover, heterogeneity of the current studies and their limitations could be due to study designs and inefficient beam profile, resulting in undesirable effects and accounting for negative and inconclusive outcomes. Ultimately, lack of intimate knowledge and understanding of the PBM light behavior impinging on the target tissue, as well as the optical tissue properties, can compromise optimization of the therapeutic outcomes. Thus, an evidence-based decision for definite therapeutic application of PBM in bone regeneration is required. In this thesis, we addressed the above issues and challenges via two elements, the electromagnetic (EM) modeling experiments and the molecular and cellular impact of PBM on bone regeneration (In vitro studies). The Electromagnetic models In my PhD proposal, I intended to both create an EM model, for the first time, and examine the mechanism of interaction of the electromagnetic fields (EMFs) with cells/tissues and establish the link that can be utilized in my cellular experiments. As the project evolved, it became clear this work was breaking new grounds and was significantly more complex than initially envisaged. As it is a small part of a much larger exciting project undertaken by University of Genoa, it has meant that I need to coordinate my work with the overall timetable of this larger project. As a result, I decided to defer, the interaction of the EMF with cells of interest part, to my Post doctorate study. We developed, for the first time, a set of simple models to examine the behavior of the local electromagnetic field (EMF), determining the PBM effects on mitochondria. This set of models was tested and crosschecked for its validity by evaluating various variables in terms of, polarization, absorption and scattering coefficient, dissipated energy density and irradiance, as well as the refractive index. Ultimately, our model and preliminary data are the first stepping-stone for further experiments, in order to understand the mechanism of interaction between electromagnetic fields and cells or tissues. Our conclusions showed that when these set of models are utilized, for the phenomenon of interest, the incident field polarization had small effects on the electromagnetic field and negligible consequences on the average energy, as well as, on the dissipated power densities. The same was shown to hold true for different orientations that the mitochondria can assume. The analogous conclusions were obtained by taking into account the possible changes in the dimensions or of the real part of the refractive index of the considered organelles. The variations of the absorption coefficient were shown to have significant effects on the average dissipated power density in the mitochondria but these effects can be predicted in a surprisingly simple way. It was proved that the numerical analysis, of the problems of interest, could be computed by using three-dimensional models, involving only a few mitochondria in the plane, which was transverse to the direction of propagation of the illuminating light that generated a uniform distribution of the energy over 1cm2 area. The one- dimensional models provided significant information on the EMF, utilized to stimulate the mitochondria. Mitochondria behaved like weak scatterers. Therefore, it was not necessary to analyze large extension of such organelles to understand what happen inside one of them. The molecular and cellular impact of 980nm PBM on osteoblast maturation: in vitro studies Our pilot study data, on the bone marrow stromal cells (BMSCs), strongly suggested that the high fluence concept (over 60J/cm2 in continuous emission mode (CW)) delivered by flattop beam profile device (FT) can promote BMSCs differentiation towards osteogenesis. Moreover, the results showed an increase in cytokines synthesis with potent anti-inflammatory properties and a decrease in the release of proinflammatory mediators. This provided me with a platform, demonstrating the validity of high fluence in facilitating osteoblasts differentiation through BMSCs. Based on this; I formulated three PBM protocols for 980nm to be tested on pre- osteoblast cell line in my definitive in vitro studies. The first phase of in vitro studies aimed to evaluate the 980nm bio-stimulatory effects on osteoblasts maturation, optimise the PBM effects on bone healing with various beam profiles delivery devices, and establish protocol/protocols of 980nm PBM in bone regeneration. The primary objective was to determine the optimal 980nm dosimetry, which exerts bio- stimulatory effects to accelerate and enhance the bone regenerative process. The secondary objective was to evaluate the intra-cellular pathways of the photon-cell interaction across the metabolic proliferative and differentiation changes, which ultimately lead to bone healing and repair. The results of this study validated the contribution of PBM in bone regeneration and elucidated the biochemical effects at a cellular level. Moreover, the role of different dosages of 980nm PBM irradiation delivered by FT; in comparison to the Gaussian beam profiles (Standard (ST)) on bone regeneration were highlighted. The setup of the power outputs on the laser device was 1.1Watt (W) for the ST and 1W for the FT. However, the real (the threshold) power output reaching the target, measured by power meter, was as ∼0.9 W, (Irradiance ∼ 0.9W/cm2, Exposure time 60 seconds, energy ∼55 J (Joule), fluence ∼55 J/cm2) delivered with the FT beam profile in CW in comparison to the ST, on MC3T3-E1 pre-osteoblast maturation. The protocol was based on 60 seconds exposure time for two consecutive weeks, which employed for all the groups. The laser grouping and their associated irriadtied energies were as follows: Group 1- Irradiation once per week (Total enrgy 110J). Group 2- Irradiation three times per week (Alternate day) (Total energy 330J). Group 3 - irradiation five- times per week (Total energy 550 J). The control cultures were processed in identical conditions except that the laser device was kept off all the time. The total energy was 0J.
The metabolic activity and the osteoblasts maturation were analyzed by alkaline phosphatase assay, alizarin red S histological staining, immunoblot and/or double immunolabeling analysis for Bcl2, Bax, Runx-2, Osx, Dlx5, osteocalcin, and collagen Type 1. Our data, for the first time, prove that laser irradiation of 980 nm wavelength with flattop beam profile delivery system, compared to standard-Gaussian profile, has improved photobiomodulatory efficacy on pre-osteoblastic cells differentiation. Mechanistically, the irradiation enhances the pre-osteoblast differentiation through activation of Wnt signaling as well as the Smads 2/3-βcatenin pathway. Our results indicated and valued the intra-cellular pathways of the photon-cell interaction across the metabolic, proliferative and differentiation changes in the cells. Additionally, our data showed that the cells irradiated THREE times a week (Total energy of 330 J) and ONCE a week (Total energy of 110 J) for two consecutive weeks protocols have increased the proliferation and differentiation of the osteoblasts in both ST & FT hand-pieces but the data showed increasingly statistical significant in the FT group. The only Runx2 was detected when the cells were irradiated with the ST hand-piece. Therefore, total energy of 110 J when either of the hand-pieces utilized, has influenced early differentiation markers. Interestingly, when the process was carried out, until the mineralization and maturation (Late osteogenesis), the ST hand-piece irradiation failed to induce an effective process, and did not lead to matrix deposition, while the FT profile showed a significant effect. In conclusion, our data, for the first time, prove that laser irradiation of 980 nm wavelength with the FT beam profile delivery system in comparison to the ST profile has a great photobiomodulatory efficacy on pre-osteoblastic cells differentiation, which would assist in accelerating bone regeneration, due to its homogeneous energy distribution at each point of its cross-section. Moreover, the irradiation protocols of three times a week and once a week for two consecutive weeks were able to increase the pre-osteoblasts and osteoblasts transcription factors, which were strongly and statistically significantly increased when the FT hand-piece was utilized. Therefore, the 980 nm laser irradiation protocol was able to promote the MC3T3-E1 cell differentiation. Researchers have demonstrated that the major barrier for an effective biological healing is insufficient laser photonic energy delivered to the injured site. PBM can modify the cell metabolism by increasing the mitochondria's ATP production. Currently, the challenge is to understand the target tissues optical properties and its cellular pathway when irradiated with laser phonic energy. In this way, modification of various energy exposure values can influence clinical outcomes predictability. Therefore, in the second phase of my in vitro study, we evaluated the effect of 980nm irradiation delivered with ST and FT beam profile hand-pieces on monolayer cell, at various power outputs; 0.8W, 0.5W and 0.25W. However, the exact power output values reaching the target, measured by power meter, were as follows: 0.75W, 0.45W, and 0.20W respectively. The MC3T3-E1 cells irradiated for two consecutive weeks, according to the following protocols: once a week (Total energy 90, 54, 24 J), respectively); three times a week (total energy 270, 162, 72J, respectively); five times a week (total energy 450, 270, 120 J, respectively). Metabolic activity of viable cells evaluated as follows: Hoechst staining; Western blotting for Runx-2, Bcl2, Bax, Osx, Dlx5, β-catenin, Smads 2/3, TGFβ, p.PI3K, PI3K, p.AKt, AKt, and p.ERK. Our data, for the first time, prove that the 980 nm irradiation at power output setting at 0.75W (0.75W/cm2) for 60 seconds in CW stimulated the MC3T3-E1 pre- osteoblasts viability, by affecting the critical pre-survival markers such as p.PI3K, p.Akt, Bcl2 and Bclxl. Moreover, we concluded that 980nm PBM delivered with FT at 0.75W power output was comparable to results with the ST. However, 0.45W and 0.20W did not modulate the cell metabolic features. Additionally, none of the laser protocols delivered with FT or ST had any influence on the cell differentiation process. In summary, our in vitro studies data, for the first time, have demonstrated the potential of utilizing the FT beam profile with our established protocols in bone regeneration, as a therapeutic tool for future pre-clinical and clinical applications. Moreover, these studies have shown the mechanistic effects of the PBM light on intracellular pathway across the metabolic and differentiation of the osteoblasts towards bone regeneration.