The Effect of Photobiomodulation Therapy on the Differentiation, Proliferation, and Migration of the Mesenchymal Stem Cell: A Review

Introduction: The purpose of this study is to investigate the effect of a low-power laser on the proliferation, migration, differentiation of different types of mesenchymal stem cells (MSCs) in different studies.Methods: The relevant articles that were published from 2004 to 2019 were collected from the sources of PubMed, Scopus, and only the articles specifically examining the effect of a low-power laser on the proliferation, differentiation, and migration of the MSCs were investigated.Results: After reviewing the literature, only 42 articles were found relevant. Generally, most of the studies demonstrated that different laser parameters increased the proliferation, migration, and differentiation of the MSCs, except the results of two studies which were contradictory. In fact, changing the parameters of a low-power laser would affect the results. On the other hand, the source of the stem cells was reported as a key factor. In addition, the combination of lasers with other therapeutic approaches was found to be more effective.Conclusion: The different parameters of lasers has been found to be effective in the proliferation, differentiation, and migration of the MSCs and in general, a low-power laser has a positive effect on the MSCs, helping to improve different disease models

Lasers, stem cells, and COPD

The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, non-thermal intervention that has the potential to modulate regenerative processes is worthy of attention when searching for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a "photoceutical" for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis, and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous stem cells, as well as post-mobilization directing of stem cells will be discussed

Steering the multipotent mesenchymal cells towards an anti-inflammatory and osteogenic bias via photobiomodulation therapy: How to kill two birds with one stone

The bone marrow-derived multipotent mesenchymal cells (MSCs) have captured scientific interest due to their multi-purpose features and clinical applications. The operational dimension of MSCs is not limited to the bone marrow reservoir, which exerts bone-building and niche anabolic tasks; they also meet the needs of quenching inflammation and restoring inflamed tissues. Thus, the range of MSC activities extends to conditions such as neurodegenerative diseases, immune disorders and various forms of osteopenia. Steering these cells towards becoming an effective therapeutic tool has become mandatory. Many laboratories have employed distinct strategies to improve the plasticity and secretome of MSCs. We aimed to present how photobiomodulation therapy (PBM-t) can manipulate MSCs to render them an extraordinary anti-inflammatory and osteogenic instrument. Moreover, we discuss the outcomes of different PBM-t protocols on MSCs, concluding with some perplexities and complexities of PBM-t in vivo but encouraging and feasible in vitro solutions

Neuronal differentiation of adipose derived stem cells: progress so far

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Alternative Strategies for Stem Cell Osteogenic Differentiation

Discovering strategies that increase the osteogenic differentiation potential of mesenchymal stem cells (MSCs) can lead to new perspectives for bone disease treatments. The possibility to associate the mesenchymal stem cells with scaffolds and to use them in bone regeneration as well as the number of studies to understand the signaling pathway of osteogenesis are increasing. Identifying osteogenic induction factors is extremely important and crucial for the success of bone regeneration. Studies have shown that proteins, such as bone morphogenetic proteins (BMPs), trichostatin A and IGF-1, can be efficiently used for osteogenic regeneration. However, the use of these proteins increases the treatment cost. Fortunately, low-level laser therapy (LLLT) may be a new alternative for adjuvant therapy to treat bone regeneration because it has biostimulatory effects on the conversion of mesenchymal stem cells into osteoblasts and on the induction of ex vivo ossification. The principle of tissue photobiomodulation with LLLT was first described in dermatology for healing wounds; however, other applications have been described, with anti-inflammatory and anti-edema effects and cellular proliferation and differentiation. Following this way, we will discuss some alternative strategies for osteogenic differentiation and suggest that the low-power lasers can be an innovative instrument for cell differentiation

Photobiomodulation Therapy in the Proliferation and Differentiation of Human Umbilical Cord Mesenchymal Stem Cells: An In Vitro Study

Introduction: Since photobiomodulation therapy (PBMT) favors in vitro mesenchymal stem cell (MSC) preconditioning before MSC transplantation, increasing the proliferation of these cells without molecular injuries by conserving their characteristics, in the present in vitro study we analyzed the effect of PBMT on the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs).Methods: Irradiation with an InGaAIP Laser (660 nm, 10 mW, 2.5 J/cm2, 0.08 cm2 spot size, and 10 s) was carried out. The cells were divided into four groups: CONTROL [cells grown in Dulbecco’s Modified Eagle Medium (DMEM)], OSTEO (cells grown in an osteogenic medium); PBMT (cells grown in DMEM+PBMT), and OSTEO+PBMT (cells grown in an osteogenic medium-plus PBMT). The cell proliferation curve was obtained over periods of 24, 48 and 72 hours using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Osteogenic differentiation was analyzed by the formation of calcium nodules over periods of 7, 14 and 21 days. Morphometric analysis was performed to quantify the total area of nodular calcification.Results: The highest cell proliferation and cell differentiation occurred in the OSTEO+PBMT group, followed by the PBMT, OSTEO, and CONTROL groups respectively, at the observed times (P < 0.05).Conclusion: PBMT enhanced the osteogenic proliferation and the differentiation of hUCMSCs during the periods tested, without causing damage to the cells and preserving their specific characteristics, a fact that may represent an innovative pretreatment in the application of stem cells

CELL SURFACE COATINGS FOR MAMMALIAN CELL-BASED THERAPEUTIC DELIVERY

The cell plasma membrane is an interactive interface playing an important role in regulating cell-to-cell, cell-to-tissue contact, and cell-to-environment responses. This environment-responsive phospholipid layer consisting of multiple dynamically balanced macromolecules, such as membrane proteins, carbohydrate and lipids, is regarded as a promising platform for various surface engineering strategies. Through different chemical modification routes, we are able to incorporate various artificial materials into the cell surface for biomedical applications in small molecule and cellular therapeutics. In this dissertation, we establish two different cell coating techniques for applications of cell-mediated drug delivery and the localization of cell-based therapies to specific tissues. The first part of this dissertation establishes a membrane-associated hydrogel patch for drug delivery. The crosslinking of a grafted polymeric patch from a mammalian cell membrane is achieved through surface-mediated photolithographic polymerization. With the use of photomask, the formation of nanoparticle-loaded PEGDA hydrogel is controlled to deposit various geometric features on photoinitiator-immobilized surfaces. Through microarray patch patterning, we analyzed the influence of processing parameters on the accuracy of polymer patterning on a microarray. We then optimized the patterning approach for the formation of PEGDA patches on live A549 cells. In the second part of this dissertation, we study the use of tissue-adhesive coatings to improve the retention of therapeutic mesenchymal stem cells (MSCs) in the heart following intramyocardial or intravenous injection. MSCs were coated with antibodies against ICAM1 to adhere to CAM-overexpressed endothelium present in the heart following MI. Through intramyocardial or intravenous delivery, we observe higher number of coated cells retained in the heart over uncoated ones, supporting enhanced affinity for the inflamed endothelium near the infarct. We correlate the detachment force of antigen-interacted MSCs by a parallel laminar flow assay with the density of ICAM on the substrate and the density of anti-ICAM on the MSC surface. MSC retention on CAMmodified surfaces or activated HUVECs was significantly increased on antibody-coated groups (~90%) under physiologically hemodynamic forces (\u3c 30dyne/cm2), compared to uncoated MSCs (~20%). Moreover, a dramatic reduction of immune cell quantity was observed after intravenous injection, indicating the enhanced immunoregulatory efficacy by systemically delivering ICAM-adhesive MSCs to the site of inflammation

The effects of combined low level laser therapy and mesenchymal stem cells on bone regeneration in rabbit calvarial defects

Abstract: This study evaluated the effect of Low Level Laser Therapy (LLLT) and Mesenchymal Stem Cells (MSCs) on bone regeneration. Background data: Although several studies evaluated the effects of MSCs and LLLT, there is little information available regarding in vivo application of LLLT in conjunction with MSCs. Methods: Forty-eight circular bone defects (6 mm in diameter) were prepared in the calvaria of 12 New- Zealand white rabbits. The defects of each animal were randomly assigned to 4 groups: (C) no treatment; (L) applying LLLT; (SC) filled with MSCs; (SCL) application of both MSCs and LLLT. LLL was applied on alternate days at wavelength of 810 nm, power density of 0.2 W/cm2 and a fluency of 4 J/cm2 using a Gallium–Aluminum–Arsenide (GaAlAs) diode laser. The animals were sacrificed after 3 weeks and then histological samples were evaluated to determine the amount of new bone formation and the remaining scaffold and inflammation. Results: The histological evaluation showed a statistically significant increase in new bone formation of LLLT group relative to the control and the other two experimental groups (p < 0.05). There was no significant difference in bone formation of the control group compared to experimental groups filled with MSCs. Laser irradiation had no significant effect on resorption of the scaffold material. In addition, inflammation was significantly reduced in LLLT group compared to the control defects and the other two experimental groups. Conclusion: Low level laser therapy could be effective in bone regeneration but there is no evidence of a synergistic effect when applied in conjunction with MSCs

Magnetic carbon nanotubes: a new tool for shepherding mesenchymal stem cells by magnetic fields

We investigated the interaction between magnetic carbon nanotubes (CNTs) and mesenchymal stem cells (MSCs), and their ability to guide these intravenously injected cells in living rats by using an external magnetic field. MATERIALS & METHODS: Multiwalled CNTs were used to treat MSCs derived from rat bone marrow. Cytotoxicity induced by nanotubes was studied using the WST-1 proliferation and Hoechest 33258 apoptosis assays. The effects of nanotubes on MSCs were evaluated by monitoring the effects on cellular growth rates, immunophenotyping and differentiation, and on the arrangement of cytoskeletal actin. MSCs loaded with nanotubes were injected in vivo in the portal vein of rats driving their localization in the liver by magnetic field. An histological analysis was performed on the liver, lungs and kidneys of all animals. RESULTS: CNTs did not affect cell viability and their ability to differentiate in osteocytes and adipocytes. Both the CNTs and the magnetic field did not alter the cell growth rate, phenotype and cytoskeletal conformation. CNTs, when exposed to magnetic fields, are able to shepherd MSCs towards the magnetic source in vitro. Moreover, the application of a magnetic field alters the biodistribution of CNT-labelled MSCs after intravenous injection into rats, increasing the accumulation of cells into the target organ (liver). CONCLUSION: Multiwalled CNTs hold the potential for use as nanodevices to improve therapeutic protocols for transplantation and homing of stem cells in vivo. This could pave the way for the development of new strategies for the manipulation/guidance of MSCs in regenerative medicine and cell transplantation

A novel approach for therapeutic angiogenesis : low doses of ionizing radiation

Tese de doutoramento, Medicina (Cirurgia Vascular), Universidade de Lisboa, Faculdade de Medicina, 2018Peripheral arterial disease (PAD) is a vascular occlusive disease accompanied by an insufficient angiogenic response, resulting in hypoperfusi on of the affected extremities. When arterial lesions impair blood flow to such an extent that the nutritive requirements of the tissues cannot be met, limb pain is chronically present at rest, frequently with trophic and necrotic skin lesions. This condition corresponds to the most advanced clinical stage of PAD, known as critical limb ischemia (CLI). Although just a minority of PAD patients progress to CLI, as PAD is highly prevalent among diabetics, smokers and people over 70 years, this condition remains very common, with 500 to 1000 new cases per million inhabitants every year, in the developed countries. The goals of CLI treatment are ischemic pain relief, ulcer healing, improvement of muscle function and quality of life, limb loss prevention, and overall survival increase. The most important aspect of CLI is the poor prognosis, no matter what treatment is employed. Any kind of surgical/endovascular revascularization, the therapy of choice in CLI patients, should be done whenever technically possible. Attempts to manipulate and normalize the microcirculatory flow pharmacologically may enhance the results of revascularization or be one option in patients in whom revascularization is impossible or has failed. In patients with CLI not eligible for arterial revascularization, the “non-option” patients, most pharmacological agents have reduced or no real effect. Amputation is often recommended, even if it is associated to morbidity and mortality. The need for alternative treatment in CLI patients is therefore compelling, and therapeutic angiogenesis is an undoubtedly promising tool to treat these patients. There are three major processes that contribute to neovascularization and are essential for the establishment of functional collateral networks, namely angiogenesis, arteriogenesis and vasculogenesis. While angiogenesis describes the process of growth of new blood vessels from pre-existing ones, arteriogenesis is characterized by the enlargement of arteriolar anastomoses to collateral vessels through growth and proliferation and vasculogenesis is de novo formation of blood vessels. These vessels can grow considerably, enough even to take over the role of a large artery when occluded. Several strategies have been pursued to stimulate therapeutic angiogenesis, ranging from recombinant protein and gene transfer therapies to the use of progenitor cell therapies. During this research, in collaboration with the biotechnology company ECBio (Amadora, Portugal), cell based therapeutic angiogenesis was addressed, as we evaluated the possibility of UCX®, a specific population of human umbilical cord derived-mesenchymal stromal cells (UC-MSCs), to induce therapeutic angiogenesis. Using a C57BL/6 mouse model of hindlimb ischemia (HLI), we demonstrated that UCX® delivered via intramuscular injection enhance blood perfusion (evaluated by laser Doppler imaging) by stimulating angiogenesis (capillary density determined by CD31 immunohistochemistry) and arteriogenesis (collateral vessel density determined by diaphonization) in ischemic muscles. This is achieved through a new mechanism in which durable and simultaneous up-regulation of several proangiogenic genes in endothelial cells (ECs) are induced by UCX®. Cryopreservation and subsequent thawing, both in vitro and in vivo, did not impair phenotype, immunomodulatory and angiogenic potencials of this specific UC-MSCs population. These data demonstrate that UCX® improve the angiogenic potency of ECs in the murine ischemic limb, suggesting the potential of UCX® as a new therapeutic tool for CLI. This study also suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of an advanced therapy medicinal product. However, the use of UCX® in patients with CLI is conditioned by the lack of studies to ensure its safety and the absence of significant adverse effects. The scope of our work, in the search for a new therapeutic approach that could overcome the current lack of available medical treatments for a large number of CLI patients, led us to consider therapeutic angiogenesis beyond its cellular component, in a unique perspective based on the use of low-dose ionizing radiation (LDIR). Our previous research has demonstrated that LDIR (<0.8 Gy) induces a proangiogenic phenotype in ECs in vitro, modulating endothelial dysfunction, promoting survival, migration and preventing ECs apoptosis. Likewise, LDIR promotes neovascularization in vivo by inducing angiogenic sprouting in the transgenic fluorescent zebrafish Tg (fli1:EGFP) embryos and by increasing vessel density in adult fli1:EGFP zebrafish after caudal fin regeneration. Therefore, according to our previous results, LDIR induces angiogenesis in vitro and in vivo; however, there is no evidence so far, that it enhances neovascularization in vascular occlusive disease, our overall objective. After surgical induction of unilateral HLI, both hindlimbs of female C57BL/6 mice were sham-irradiated or irradiated with four daily fractions of 0.3 Gy, in consecutive days and limb perfusion, capillary density and collateral vessel formation were measured. We found that LDIR (4 x 0.3 Gy) improves limb perfusion by enhancing arteriogenesis through EPCs recruitment to sites of collateral vessel development, an effect dependent on exposure of the ischemic niche to LDIR, but not on the local recruitment of myeloid cells. Likewise, LDIR also favours angiogenesis through simultaneous activation of a repertoire of pro-angiogenic factors in mature ECs in a mechanism dependent of VEGFR signaling, with no short-term side effects and no effects on resting vasculature, opening a possibility to new therapeutic strategies in lower limb vascular insufficiency. The vasculature in an irradiated non-ischemic bed was not affected and after 52-wk of LDIR exposure no differences in the incidence of morbidity and mortality were noticed. Moreover, it was found that a dose of 0.3 Gy administered during 4 consecutive days does not induce toxicity in C57BL/mice and this dose fraction was identified as the lowest dose that is still able to induce therapeutic angiogenesis. The outcome of these in vivo experiments performed in a mice model suggests that LDIR may have a potential clinical use in the treatment of lower limb vascular insufficiency, emerging as a novel approach in the treatment of CLI patients. Accordingly, we designed an already approved and ongoing clinical trial – Low-dose ionizing radiation modulates the expression of pro-angiogenic genes in Critical Limb Ischemia Patients – and report our preliminary results. To date, 16 “non-option” CLI patients were enrolled in the study, but only 12 patients, corresponding to 13 limbs, were considered for analysis. As the expected amputation number (12 major limb amputations) for analysis of the primary endpoint was not reached, the trial is still ongoing. Concerning the primary endpoint, preliminary results suggest that LDIR induces a pro-angiogenic effect through the modulation of several pro-angiogenic factors in ECs collected from “non-option” CLI gastrocnemius muscles. The primary endpoint is corroborated by the finding that LDIR is associated with an increase in capillary density and a significant increase (P = 0.03) in vessel density (VD), 30 days post-intervention. It is also interesting to report that in the non-amputated patients, 6 months after irradiation, only the one muscle exposed to LDIR showed a persistent and continuous increase of VD. Regarding the surrogate clinical endpoints of ischemia and as expected, no significant differences were found between LDIR limbs and controls. LDIR has a biological rationale widely investigated and discussed in the work developed by our research group. Therefore, based on these preliminary results and considering that up to 40% of CLI patients are not candidates to revascularization, LDIR may be considered as a novel approach for the management of these patients.Quadro de Referência Estratégico Nacional (QREN), LISBOA-01-0202-FEDER-030196: ClinUC