The effects of pulsed electromagnetic field on bone regeneration

Pulsed electromagnetic field (PEMF) is known for its bone healing ability in non-union fractures and spinal fusions. PEMF therapy is reported to be relatively pain-free and non-invasive. However, there is lack of consistency in investigating the effects of PEMF on test subjects. Various types of PEMF-emitting devices were used to test different subjects (cells and animals). The optimal set of PEMF parameters for bone healing application is still an ongoing research topic. In addition, currently there is also a lack of research done on the effects of PEMF flux direction and movement since most of the studies conducted used PEMF that is in stationary condition. The objective of this thesis is to study the effect of PEMF on osteogenesis. Due to the inconsistencies of existing commercially available PEMF devices, we specially designed our own PEMF-emitting device. PEMF parameters such as duration of exposure, intensity, and dynamic movement were tested. The studies were carried out on: (a) cellular (MC3T3-E1 and mesenchymal stem cells), (b) 3D tissue scaffolds (3D-printed from Osteopore International Pte Ltd.), and (c) animal (chicken embryo) models. Three different PEMF configurations were designed and built. They were classified into: (a) Stationary PEMF emitted by solenoid coils for MC3T3-E1, mesenchymal stem cells (MSCs, from monkey and rabbit), and chicken embryos, (b) PEMF emitted by two Helmholtz coils which moved in two directions using a specially designed biaxial bioreactor (manufactured by Quintech) for MC3T3-E1 cells, (c) PEMF emitted by two Helmholtz coils with perfusion flow and biaxial rotation using bioreactor for the 3D-printed scaffolds. The results showed that the PEMF emitted from these devices was constantly uniform throughout the duration of exposure on the test subjects. They were modelled with COMSOL Multiphysics software and were consistent with the on-site measurement. Different lengths of exposure and intensities of PEMF were tested on both immortalized pre-osteoblastic cell line, MC3T3-E1, and primary rabbit MSCs. The results showed that 0.6mT, 50 Hz, 30 minutes daily PEMF exposure improved the proliferation of both types of cells regardless of the culture media types. MSCs displayed higher metabolic activity as compared to MC3T3-E1. However, as the intensity of PEMF increased, the effects of PEMF on MSCs were lessened. The oscillating movement of PEMF improved both cell proliferation and calcium deposition of MC3T3-E1, especially those cultured in differentiating media. It was hypothesized that PEMF could affect the opening of calcium ion channel and enhance the release of intracellular calcium ions which might trigger downstream pathways that led to increasing cell proliferation. The dynamic motion of oscillating PEMF might intensify this occurrence since PEMF flux would cut through the cells, producing eddy currents. There were also indications that window of efficacy of PEMF treatment existed. The first week of PEMF treatment elicited the highest cell proliferation. In addition, the longer exposure to PEMF and the higher the intensity did not result in better cell response, proving that the efficacy of PEMF treatment could be maximized within this window. This optimized set of parameters (0.6mT, 50 Hz, 30 minutes) were then applied to both chicken embryo and 3D-tissue models. Chicken embryo was chosen because PEMF could penetrate inside without going through muscles, bones, and other tissues before reaching to the embryo, which may dampen the effects of exposure. The results showed that PEMF improved the bone volume fraction (BV/TV) of chicken embryo. This was measured on day 14 of the embryonic age which corresponded to the increase of calcium ions content in amniotic fluid. However, prolonged PEMF exposure resulted in lesser value on day 18. It was hypothesized that the increase in calcium ions on day 18 was directed towards the development of internal organ since PEMF-exposed chicken embryo had larger value of stomach and heart dried weight as compared to the non-exposed group. This was also an indication of the existence of window of efficacy of PEMF to affect a specific stage in chicken embryo development. The optimized set of PEMF was paired with multimodal bioreactor to culture primary MSC-laden 3D scaffold. The bioreactor moved in two directions and the culture media was perfused into the scaffolds-containing chamber. Results showed that PEMF-exposed 3D scaffolds had more than twice the cell numbers as compared to those that were not exposed to PEMF on both day 7 and 14. Compared with those cultured in stationary condition with only perfusion, the combination of PEMF and biaxial rotation stimulations improved both cell proliferation and calcium deposition in differentiating media on both day 7 and 14. This thesis provided specially designed devices that emitted uniform PEMF exposure to test different subjects. The configuration parameters were also tunable, which allowed versatile usage of PEMF depending on the focus of the study. The chicken embryo model for osteogenesis study of PEMF also laid a foundation for further developmental biology studies involving the effects of man-made EMFs. The optimized set of parameters for osteogenesis could enhance cell proliferation and mineralization on 3D scaffolds. These scaffolds could be potentially used as bone grafts and benefit patients who have less MSCs count as PEMF could help in improving the cell numbers.Doctor of Philosoph

Precision Medicine for Nasopharyngeal Cancer—A Review of Current Prognostic Strategies

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV) driven malignancy arising from the nasopharyngeal epithelium. Current treatment strategies depend on the clinical stage of the disease, including the extent of the primary tumour, the extent of nodal disease, and the presence of distant metastasis. With the close association of EBV infection with NPC development, EBV biomarkers have shown promise in predicting treatment outcomes. Among the omic technologies, RNA and miRNA signatures have been widely studied, showing promising results in the research setting to predict treatment response. The transformation of radiology images into measurable features has facilitated the use of radiomics to generate predictive models for better prognostication and treatment selection. Nonetheless, much of this work remains in the research realm, and challenges remain in clinical implementation

Effects of electromagnetic field on proliferation, differentiation, and mineralization of MC3T3 cells

The steep increasing incidence of bone diseases and fractures provides a commanding impetus and growing demands for bone tissue engineering research. Pulsed electromagnetic fields (PEMFs) have been documented to promote bone fracture healing in nonunions and to enhance the maturation of osteoblastic cell, which is the key element in bone tissues. However, the optimal parameters for PEMF stimulation are still being explored. In this study, we investigated the effects of PEMF treatment on the proliferation, differentiation, and mineralization of osteoblast precursor cells MC3T3-E1 to explore the cell growth profile under different PEMF exposure durations (15, 30, and 60 min daily) with a magnetic field strength of 0.6 mT, at a frequency of 50 Hz, and cultured in media with or without osteogenic supplements for 28 days. Cell viability and metabolic activity were accessed by confocal microscopy, and alamarBlue time-course measurements and results indicated that there were no adverse effects under designated PEMF condition. After 7 days of PEMF exposure, in comparison with negative controls, cell numbers increased when exposed to PEMF in culture medium and were independent of osteogenic supplements. However, PEMF might not have significant impact on cellular mineralization as observed from calcium deposition analysis, even though osteogenic gene expression was upregulated for cells with PEMF exposure. Von Kossa and Alizarin Red staining indicated that extracellular matrix mineralization occurred at day 28 with osteogenic supplements only, and no significant differences were found among those samples with different PEMF treatment durations. In summary, our results suggested that PEMF stimulation for as short as 15 min could improve cell proliferation but not mineralization in vitro. Thus, this study highlights the importance of choosing appropriate PEMF parameters to achieve the desired effect on target cells. The optimization of PEMFs will enhance the efficiency of its usage as a clinical, adjuvant therapeutic treatment for bone defect regeneration. Impact Statement We present the study about how the parameters of pulsed electromagnetic field (PEMF) stimulus affected calvarial osteoblast precursor cell in terms of growth, viability, and differentiation. This research provides insight and foundation to clinical application of noninvasive therapy using PEMF to improve bone regeneration.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted versio

Self-assembled nanofibrous marine collagen matrix accelerates healing of full-thickness wounds

There is an urgent clinical need for wound dressings to treat skin injuries, particularly full-thickness wounds caused by acute and chronic wounds. Marine collagen has emerged as an attractive and safer alternative due to its biocompatibility, diversity, and sustainability. It has minimum risk of zoonotic diseases and less religious constraints as compared to mammalian collagen. In this study, we reported the development of a self-assembled nanofibrous barramundi (Lates calcarifer) collagen matrix (Nano-BCM), which showed good biocompatibility for full-thickness wound-healing applications. The collagen was extracted and purified from barramundi scales and skin. Thereafter, the physicochemical properties of collagen were systematically evaluated. The process to extract barramundi skin collagen (BC) gave an excellent 45% yield and superior purity (∼100%). More importantly, BC demonstrated structural integrity, native triple helix structure, and good thermal stability. BC demonstrated its efficacy in promoting human primary dermal fibroblast (HDF) and immortalized human keratinocytes (HaCaT) proliferation and migration. Nano-BCM has been prepared via self-assembly of collagen molecules in physiological conditions, which resembled the native extracellular matrix (ECM). The clinical therapeutic efficacy of the Nano-BCM was further evaluated in a full-thickness splinted skin wound mice model. In comparison to a clinically used wound dressing (DuoDerm), the Nano-BCM demonstrated significantly accelerated wound closure and re-epithelization. Moreover, Nano-BCM nanofibrous architecture and its ability to facilitate early inflammatory response significantly promoted angiogenesis and differentiated myofibroblast, leading to enhanced wound healing. Consequently, Nano-BCM demonstrates great potential as an economical and effective nonmammalian substitute to achieve skin regeneration.Agency for Science, Technology and Research (A*STAR)This work was funded by the Agency for Science, Technology and Research (A*STAR) RIE2020 Advanced Manufacturing and Engineering (AME) programmatic grant (A18A8b0059) Additive Manufacturing for Biological Materials (AMBM) project SP1.1

Synergistic Effect of PVDF-Coated PCL-TCP Scaffolds and Pulsed Electromagnetic Field on Osteogenesis

Bone exhibits piezoelectric properties. Thus, electrical stimulations such as pulsed electromagnetic fields (PEMFs) and stimuli-responsive piezoelectric properties of scaffolds have been investigated separately to evaluate their efficacy in supporting osteogenesis. However, current understanding of cells responding under the combined influence of PEMF and piezoelectric properties in scaffolds is still lacking. Therefore, in this study, we fabricated piezoelectric scaffolds by functionalization of polycaprolactone-tricalcium phosphate (PCL-TCP) films with a polyvinylidene fluoride (PVDF) coating that is self-polarized by a modified breath-figure technique. The osteoinductive properties of these PVDF-coated PCL-TCP films on MC3T3-E1 cells were studied under the stimulation of PEMF. Piezoelectric and ferroelectric characterization demonstrated that scaffolds with piezoelectric coefficient d(33) = -1.2 pC/N were obtained at a powder dissolution temperature of 100 degrees C and coating relative humidity (RH) of 56%. DNA quantification showed that cell proliferation was significantly enhanced by PEMF as low as 0.6 mT and 50 Hz. Hydroxyapatite staining showed that cell mineralization was significantly enhanced by incorporation of PVDF coating. Gene expression study showed that the combination of PEMF and PVDF coating promoted late osteogenic gene expression marker most significantly. Collectively, our results suggest that the synergistic effects of PEMF and piezoelectric scaffolds on osteogenesis provide a promising alternative strategy for electrically augmented osteoinduction. The piezoelectric response of PVDF by PEMF, which could provide mechanical strain, is particularly interesting as it could deliver local mechanical stimulation to osteogenic cells using PEMF.Funding Agencies|Ministry of Education-SingaporeMinistry of Education, Singapore [MOE2016-T2-2-108]; Nanyang Technological UniversityNanyang Technological University; Agency for Science, Technology, and ResearchAgency for Science Technology &amp; Research (ASTAR) [A18A8b0059]</p