Aayushi Randhawa
Keya Ganguly
Sayan Deb Dutta
Tejal V. Patil
Ki-Taek Lim
Abstract
Traditional bioreactor systems involve the use of three-dimensional (3D) scaffolds or stem cell aggregates, limiting the accessibility to the production of cell-secreted biomolecules. Herein, we present the use a pulse electromagnetic fields (pEMFs)-assisted wave-motion bioreactor system for the dynamic and scalable culture of human bone marrow-derived mesenchymal stem cells (hBMSCs) with enhanced the secretion of various soluble factors with massive therapeutic potential. The present study investigated the influence of dynamic pEMF (D-pEMF) on the kinetic of hBMSCs. A 30-min exposure of pEMF (10V-1Hz, 5.82 G) with 35 oscillations per minute (OPM) rocking speed can induce the proliferation (1 × 105 → 4.5 × 105) of hBMSCs than static culture. Furthermore, the culture of hBMSCs in osteo-induction media revealed a greater enhancement of osteogenic transcription factors under the D-pEMF condition, suggesting that D-pEMF addition significantly boosted hBMSCs osteogenesis. Additionally, the RNA sequencing data revealed a significant shift in various osteogenic and signaling genes in the D-pEMF group, further suggesting their osteogenic capabilities. In this research, we demonstrated that the combined effect of wave and pEMF stimulation on hBMSCs allows rapid proliferation and induces osteogenic properties in the cells. Moreover, our study revealed that D-pEMF stimuli also induce ROS-scavenging properties in the cultured cells. This study also revealed a bioactive and cost-effective approach that enables the use of cells without using any expensive materials and avoids the possible risks associated with them post-implantation.
Introduction
Bone disorders are one of the leading medical conditions requiring innovative therapeutic outcomes. Despite the advent of tissue engineering practices, the most commonly used fracture healing measure includes the use of plasters to restrict bone movement and achieve bone regeneration with or without scaffold implantation. In this scenario, two aspects of the implant play a crucial role in determining the success rate of bone regeneration. One of these is the mechanical property of the implant, while the other involves the distinct bioactivity of the surface chemical property of the implant. Several notable studies have already reported the development of highly strong implants of metal, polymers, and alloys. In contrast, bioactivity has also been reported through the use of biologically derived small molecules, peptides, or functional proteins. Nevertheless, we have not yet accomplished fast bone regeneration. Alternatively, the use of cell-based therapies that may expedite bone regeneration over a short period, ranging from days to weeks after scaffold implantation, has great potential as a faster method for bone regeneration [1,2]. In this context, bone healing can be facilitated using stem cell therapy and tissue engineering techniques that incorporate biomaterials and preconditioned stem cells.
Mesenchymal stem cells (MSCs) have received the greatest research attention in bone regeneration [[3], [4], [5], [6]]. Thousands of patients experience bone-related complications yearly, such as osteosarcoma, osteoarthritis, and fractures, among which each patient requires a cell range of 105 to 1010 cells for therapy [7]. Clinical studies showed that stem cell transplantation is an outstanding technique to combat organ damage. However, the practical application of stem cell-based therapeutics is hindered due to the limitations in the large-scale expansion culture of MSCs [[8], [9], [10]]. Therefore, it is imperative to identify more effective stem cell culture methodologies to meet the criteria for large-scale expansion and osteogenic preconditioning of stem cells [11].
To address this need, recent advancements have focused on exposing cells to externally applied physical stimuli, such as shear stress and magnetic, electric, and electromagnetic fields (EMFs), which induce mechanotransduction-mediated stem cell proliferation and differentiation [[12], [13], [14], [15], [16]]. In the physiological in vivo environment, fluid shear, and matrix strain play a significant role in influencing bone hemostasis by stimulating mechanosensory functions [17]. For instance, osteocytes, the predominant cells found in bone, function as primary mechanosensors and regulators of bone metabolism. Through mechanotransduction, they govern bone development and resorption by secreting various signaling molecules that influence the activity of osteoblasts and osteoclasts [18].
Numerous bioreactor systems have been developed in conjunction with physical stimulation techniques to elicit specific responses in stem cells, representing a significant advancement for translational applications in bone regeneration and therapeutics. These sophisticated bioreactors stimulate stem cell proliferation, differentiation, and functional maturation by mechanical and physical stimulation [19]. For instance, electrical stimulation in bioreactors enhances stem cell osteogenic development, resulting in bone-like structures with improved bone matrix production [20]. Pulsed electromagnetic fields (pEMFs) similarly increase the expression of osteogenic markers and enhance mineralization [21]. Additionally, the mechano-sensitivity of MSCs cultured in a pulsatile-pressure bioreactor system has been shown to promote osteogenic differentiation on soft matrices [22]. Moreover, external mechanical forces such as compression, tension, and fluid shear are often used to promote stem cell growth and osteogenic differentiation [23].
Despite such advancements, we are yet to achieve a bioreactor stimulation system that can enhance stem cell proliferation and differentiation for advanced therapeutic applications. We hypothesized that while each type of stimulation individually has been shown to enhance stem cell proliferation and therapeutic applications, their combined application could offer several unique advantages.
Considering the abovementioned conditions, in the present study, we devised a custom-built rocking bioreactor system for expanding human bone-marrow-derived mesenchymal stem cells (hBMSCs) under combined effects of wave motion and pEMFs to monitor proliferation and osteogenic behavior for cell-based therapeutics. Scheme 1 represents the stem cell-based therapeutic bioreactor setup, and the study design is aimed at hBMSCs’ regulation and therapeutic outcomes. To the best of our knowledge, this investigation has not been previously reported. The primary goal of our study was to develop a cost-effective scaffold-free stimuli-responsive expansion of the hBMSCs, triggering the secretion of biologically active molecules to encourage rapid bone regeneration in a matter of days and weeks. We chose wave motion stimulation to mimic the natural mechanical forces that cells experience in vivo, while pEMFs can activate various cellular signaling pathways to achieve cell proliferation. Given the current constraints in bone regeneration, including low proliferation rate, poor bioactivity, and lengthy healing processes, we envision that our study will significantly advance pEMF-stimuli-based therapeutics by promoting rapid stem cell proliferation, enhanced cell secretion, rapid osteogenic differentiation for stem cell-based therapeutic applications. Furthermore, it can help in the development of pEMF-based wearable devices, facilitating more efficient bone repair in the future.
Section snippets
Materials
Human bone marrow-derived mesenchymal stem cells (hBMSCs, PromoCell C-12974) supplemented with Fetal bovine serum (FBS) (Welgene Inc., Republic of Korea), Dulbecco Modified Eagle Medium (DMEM), 1 % antibiotics (P/S), and phosphate buffer saline (PBS) were purchased from Welgene, Republic of Korea. The Live-Dead staining kit, osteo-inductive media, RNAzol, F-actin probe, osteo-inductive media, ALP, ARS detection kit, and 4,6-diamino-2-phenylindole dihydrochloride (DAPI) were obtained from
Bioreactor design and performance
A digital photograph of the custom-built pEMF-wave motion bioreactor system is shown in Fig. 1a. The bioreactor chamber consisted of an incubator with an integrated rocking platform and two additional Helmholtz coils to generate the desired wave flow (seesaw motion) and pEMF simultaneously.
The rocker platform induced an oscillatory wave motion of the culture media onto adhered hBMSCs. This wave motion of the cells can be controlled with the help of the rocker controller unit, which allows for
Discussion
An alternate strategy that has recently gained attraction is called bone tissue engineering (BTE), which uses an external scaffold to supply regulatory growth hormones that stimulate cell proliferation. These implanted scaffolds must have the same osteoinductivity, osteoconductivity, biocompatibility, and appropriate mechanical strength as the original tissues [[51], [52], [53], [54], [55], [56]]. For bone tissue remodeling, a variety of synthetic implantable scaffolds are produced, such as
Conclusion
Recent research reveals that pEMF is typically safe and can cause a variety of apparent actions in cultured bone cells. However, the mechanism still needs to be better comprehended, and optimization is needed depending on the cell type, disease stage, developmental stage, tissue microenvironment, and pEMF parameters. The main aim of this research was to outline the essential procedural conditions for the large-scale production of hBMSCs in a wave motion bioreactor system using pEMF stimulation.
