Gathering Evidence to Leverage Musculoskeletal Magnetic Stimulation Towards Clinical Applicability

Musculoskeletal disorders are among the main causes of disease‐associated disability. Moreover, the incidence and prevalence of osteoporosis and osteoarthritis, as well as the risk of bone fractures and the need for joint replacements, are expected to increase with longer life expectancy. New approaches based on electromagnetic stimulation have been developed, aiming to shorten bone healing time, attenuate osteoporosis and osteoarthritis, and increase implants' osseointegration. Inductive coupling (IC), a non‐invasive methodology to deliver magnetic stimuli, has reached clinical trials and some clinical practices but is not yet considered a standard procedure. Indeed, its feasibility in clinical use is still under discussion, and optimal stimulation parameters are fairly undefined. This comprehensive review describes the research trends and applicability of IC‐based therapeutics for musculoskeletal disorders, and starts identifying top‐performing magnetic stimulation parameters. Insights into the magnetic stimuli setups that promote osteogenesis are provided, based on pre‐clinical and clinical evidence from 117 in vivo studies in animal models and human patients. Potential cellular and molecular biomechanisms mediating IC‐induced effects on osteoblasts and osteoclasts are also explored. The transversal knowledge herein delivered will hopefully support innovative designs and medical devices that will implement IC stimulation as a clinical standard and effective therapeutic for musculoskeletal disorders.


Introduction
The 2019 Global Burden of Disease placed musculoskeletal disorders among the 10 top drivers of increasing burden, given its increasing impact on disability-adjusted life-years (DALYs) from 1990 to 2019. [1]Accordingly, these disorders were also leading causes of years lived with disability (YLDs) during that period. [2]t is thus imperative that health systems rapidly address the needs of individuals affected by musculoskeletal conditions endangering functional health. [3,4]Surgery is indicated for severe musculoskeletal disorders that cause substantial pain and disability, and for symptoms refractory to conservative treatment. [5]Total hip and knee arthroplasties are among the most common and successful surgical procedures, and are projected to grow around 71% and 85% by 2030 in the United States. [6]Despite continuous improvements in surgical techniques and implant designs, revision arthroplasties are still rising due to unsuccessful osseointegration. [7,8]Innovative therapies for musculoskeletal disorders, and strategies to improve the efficacy of ongoing ones, are therefore an urgent healthcare priority. [4]ctive bone healing, key for long-term successful therapies, may be achieved by stimulating bone tissue remodeling via biochemical, mechanical, or electromagnetic stimuli. [9][12][13] Bone and other musculoskeletal tissues may also benefit from these, given their responsiveness to extracorporeal electromagnetic stimuli that mimic natural endogenous bone electromagnetic fields and induce similar biological effects. [14,15]ndeed, bone composition confers specific electromechanical properties to this tissue, activated by mechanical loading through compression or tension. [16,17]Two hypotheses account for the electrical potentials generated in loaded bone: the piezoelectric effect (mainly on dry bone, and attributed to the crystalline micelle of collagen molecules) [16,18,19] and the streaming potentials (that explain the smaller potentials on hydrated bone, which in vivo few piezoelectric responses given its water content). [17,20]32] Several animal models have been used to test the efficacy of electromagnetic stimulation in preventing and even reversing osteoporosis, a major musculoskeletal disorder characterized by low bone mass and structural deterioration due to imbalanced bone remodeling. [33,34]Electromagnetic stimulation is also valuable for bone regeneration upon fracture, promoting bone repair and fracture healing. [35,36]Electromagnetic stimulation may ultimately improve osseointegration, crucial for the long-term survival of implantable orthopedic devices. [8,37,38][45] Three methods have been used to provide electromagnetic stimulation, which includes the delivery of electrical fields (EF) and magnetic fields (MF).[48] However, the invasiveness of DC stimulators is inherently associated with infections, inflammation, soft tissue discomfort, or cathode rupture. [47]ontrariwise, non-invasive stimulation through EF and/or MF has significant advantages, including the absence of toxic chemicals' formation and immune responses in host tissues, besides requiring simpler designs and involving little tissue handling. [49]Further, devices incorporating non-invasive stimulators allow both repeated treatment and monitoring of target regions. [37,43,44,49]The Food and Drug Administration (FDA) has already approved non-invasive bone growth stimulators acting through the other two methods: capacitive coupling (CC) and inductive coupling (IC). [50]Classic CC stimulation architectures require two electrodes placed on opposite sides of the target tissue to deliver sinusoidal EFs, while IC stimulation is mostly achieved through the delivery of pulsed electromagnetic fields (PEMF) or combined magnetic fields (CMF), provided by a device surrounding the body target region. [50]PEMF stimulation involves the flow of alternating electric currents through solenoids or coils, generating MFs that in turn induce electric voltages on bone tissues. [47,48]These induced voltages depend on the MF strengths and the magnetic properties of the target tissues.CMF stimulation combines these coil-generated PEMF with an overlapping static MF provided by permanent magnets. [47]Both CC and IC methods have been proposed as therapeutic strategies to minimize osteoporosis, promote bone fracture healing, and improve osseointegration on bone-implant interfaces. [51]Notwithstanding the wide number of studies on IC applications for bone conditions, it is now crucial to optimize the stimulation elicited by IC in clinical settings by identifying the IC operating parameters that deliver the best bone healing outcomes.58] We here comprehensively overview the most relevant bone healing-related outcomes induced by IC in vivo, both in animal models and human patients, aiming to identify top-performer magnetic stimulation parameters that induce significant improvements in musculoskeletal conditions.A search was carried out on PubMed to find studies published until the end of 2023, focused on the delivery of magnetic stimulation via IC in vivo, in both animal models and humans, to treat bone detrimental conditions, such as fractures or osteoporosis, or to promote bone-implant osseointegration.Relevant parameters were extracted from the retrieved studies, including the type of stimulation, stimulation device, waveform, frequency, periodicity, MF strength, daily exposure time, and assay duration.Magnetic stimuli parameters were uniformized according to the standardized signal characterization (Figure S1, Supporting Information).Data relative to the animal models, alongside the clinical conditions evaluated and supplementary procedures or treatments, were also collected.Focus was placed on the most frequently assessed biological/clinical outcomes: radiographic assessments of bone unions, micro-CT scan analyses of bone microarchitecture, histological analyses of the healing tissue, final biomechanical properties, and osteogenesis-related molecular markers.Data on these biological outcomes were extracted from the articles' text, tables or embedded figures.Unless otherwise specified, indicated changes (incremental or decremental %, or foldchanges) refer to stimulated versus unstimulated conditions.

Overview of IC Pre-Clinical Designs
Our search has retrieved 73 pre-clinical studies using IC stimulators to treat musculoskeletal conditions in animal models (Table S1, Supporting Information).PEMF was by far the most used IC stimulation method (94.7%) and various waveforms (sinusoidal, square, triangular, and sawtooth) have been tested (Figure 1A and Table S1, Supporting Information).As presented in Figure 1B, rats (54.8%; mainly Sprague Dawley, but also Wistar and Fischer Inbred) were the most used animals to test IC stimulation, followed by rabbits (27.4%).Other animal models used were dogs (Beagle and mixed breed), guinea pigs (New Zealand White and Japanese White), mice, and sheep.
The pre-clinical therapeutical efficacy of IC stimulation has been tested for several musculoskeletal conditions, namely bone defects, implants' osseointegration, conditions of low bone   S1, Supporting Information)."Bone defects" include osteotomies, fractures, and bone drills; "Low BMD conditions" are conditions resulting in low bone mineral density (BMD) and bone fragility, such as osteoporosis, diabetes, and glucocorticoid treatments; "Combined conditions" represent 4 studies (out of the 73) in which 2 conditions were simultaneously assessed.D) Illustrative schematic of a typical IC stimulation device used in pre-clinical studies to deliver PEMF to animals.Based on illustrations from Jing et al. [120] and Cai et al. [87] E) Photograph of an actual IC stimulation device used in a pre-clinical study here reviewed.Reproduced and adapted with permission. [102]Copyright 2018, Wiley.CEF: constant electromagnetic field; CMF: combined magnetic field; PEMF: pulsed electromagnetic field; RMF: rotating magnetic field.

Effects of IC Stimulation on Small-Sized Animals
Rats (Rattus norvegicus) were the most used animal models to test IC stimulation in vivo (Figure 1).Rats are anatomic and physiologically well-studied, have fast growth and reproductive rates and require little aftercare, being appropriate to study various stimulation parameters at reasonable costs.In contrast, their quite smaller size, different proportions of cortical and trabecular bone, innate bone growth after sexual maturity, and higher healing capabilities, may result in different responses from humans upon similar stimuli. [104]IC stimulation has been used to improve different musculoskeletal conditions in these animals, as detailed in the sections below, divided by types of musculoskeletal conditions, and in Table S1 and S2, Supporting Information.
PEMF have been applied to determine whether bone tissues from healthy rats responded well to electromagnetic stimuli.These studies were focused on major macroscopical and microarchitectural effects on bone and also spotlighted some molecular markers and signaling mechanisms altered upon PEMF stimulation under physiological conditions.PEMF of 1 Hz and 30 mT, delivered by the FDA-approved transcranial stimulator Magstim 220 for 30 min day À1 over 3 weeks, has increased cortical and trabecular bone thickness by nearly þ50%, in all measurements. [105]To understand the PEMF's action mechanism on healthy bones, a study applied PEMF of 50 Hz, 0.6 mT, 1.5 h day À1 over 3 months, and measured molecular markers related to bone homeostasis and to the sAC/cAMP/PKA/ CREB signaling pathway.PEMF stimulation increased BMD (≈þ10%) and bone volume (1.69-fold change), as well as the mechanical properties (up to þ14.3% maximum load), and microarchitecture of bone: increased trabecular thickness (Tb.Th, þ39.0%) and number (Tb.N, þ21.2%), and reduced trabecular separation (Tb.S, À40.5%).Moreover, this therapy significantly increased serum levels of the bone formation marker procollagen type I N-terminal propeptide (P1NP, þ15.0%), and tendentially reduced the bone resorption marker C-terminal cross-linked telopeptides of type I collagen (CTX-I, À19.1%).

Studies of IC Stimulation for Soft Tissue Disorders in Small-Sized Animals
Physio-Stim was also used for soft tissue healing in rats [107][108][109] and PEMF of parameters similar to the ones described above (15 Hz; ≈2 mT) led to radiographical, biomechanical, and histological improvements in teared rotator cuffs. [107,108]When delivered for 1 h day À1 over 4 weeks, it increased the tendon modulus and stiffness and enhanced collagen-I and fibronectin tendon expression. [108]When delivered for 3 h day À1 , such stimuli increased the tendon modulus and maximum stress at 4 weeks, and improved bone quality at 16 weeks. [107]However, another study applying the same stimuli to teared Achilles' tendons, for 1 or 3 h day À1 over 1, 3, or 6 weeks, did not observe major improvements in bone volume or BMD, and even reported impaired tendon mechanical properties (e.g., reduced stiffness and modulus at 3 weeks). [109]

IC Stimulation to Improve Implant Osseointegration in Small-Sized Animals
IC stimulation was also tested on rat implants' osseointegration.A study applying PEMF with the most common parameters (15 Hz, 1.0 mT), has compared the effectiveness of 1 vs 3 h day À1 exposures, for 5 days week À1 over 45 days, in promoting titanium tibial implants' osseointegration. [85]Both exposures increased Tb.N by ≈50% but, although 3 h day À1 exposure preserved more Tb.Th around implants (À55.0%vs. À37.2%), 1 h day À1 exposure generally induced better results: increased bone volume (4.7-fold), BMD (þ54.6%) and bone-implant contact (BIC, 2.2fold), better removal torque tests (þ47%), enhanced cell viability, protein content, and nodules' mineralization. [85]In a relevant study that placed a titanium implant in the rat's right tibial crest, and performed an osteotomy in the contralateral tibial crest, increased peri-implant and osteotomy ossification were observed in the group stimulated for 0.5 h twice-a-day with PEMF of 50 Hz and high MF strength (72 mT), demonstrating that this IC therapy can accelerate both bone-healing and peri-implant bone formation. [70]PEMF of higher frequency and lower MF strength (75 Hz, 2.5 mT), delivered by Biostim for 6 h day À1 over 60 days, also improved osseointegration of intramedullary titanium pins in rat femurs.Namely, increased bone volume (1.52-fold), BIC (2.40-fold), and microhardness (þ19.4%) were detected, as well as reduced cortical width (Ct.Wi, À32.3%), fibrous capsule formation (capsule thickness, À52.8%) and osteoclast number (À72.9%). [90]Inspired by a study on rabbits' femur implants, [82] PEMF stimulation of 100 Hz and 0.2 mT was successfully applied to titanium tibial implants in OVX rats for 4 h day À1 over 2 weeks, resulting in higher peri-implant bone volume (up to 1.75-fold) and increased Tb.S (up to þ31.5%). [84]

Handling Osteoarthritis in Small-Sized Animals with IC Stimulation
In two studies of a rat model mimicking temporomandibular joint osteoarthritis, PEMF of 2 mT and 15 Hz pulses (bursts of 5 kHz), applied for 2 h day À1 over 3 or 6 weeks, partially reversed microarchitecture deterioration, as shown by increased BV/TV (up to 1.4-fold) and Tb.Th (up to þ36.8%); reduced bone surface (BS/BV, 0.83-fold) and Tb.Sp (up to À29%) [124] and reversed the decrease in cartilage thickness (by ≈20.6% on average). [125]This IC stimulation also reversed the upregulated osteoclast activity and related RANKL gene expression, and the abnormal downregulation of osteogenic factors (OPG, ALP, Runx2, and OC), that commonly occur at early osteoarthritis stages. [124]Additionally, these IC stimulation parameters inhibited the up-regulation of pro-inflammatory and degradative proteins (TNF-α, IL-1β, MMP-13, ADAMTS-5, IL-6, MMP-3, MMP-9, and COL-X) in the synovium, that classically characterize osteoarthritis. [125]To induce knee osteoarthritis, rats were administered low-dose monosodium iodoacetate and further treated for 2 h day À1 over 4 weeks with PEMF of higher frequency (75 Hz; 1.6 mT).Improved bone volume (1.18-fold) and microarchitecture (þ18.5% Tb.Th, þ6.8% Tb.N, À13.6% Tb.S) of osteoarthritic knees were recorded, as well as increased serum levels of osteogenic markers (e.g., þ47% OC), and decreased bone and cartilage resorption ones (À7.8% CTX-I; À19.9% CTX-II). [126]A follow-up study using the same model and stimulus reported similar radiological findings (increased BV/TV, Tb.Th, Tb.N; decreased Tb.S), and correlated these with increased OPG/RANKL ratio (þ87.0%) and Wnt/β-catenin pathway activation (increased Wnt3a, LRP5, β-catenin). [127]ther animals used as osteoarthritis models are guinea pigs (Cavia porcellus) of the Dunkin-Hartley breed, that spontaneously develop degenerative joint diseases around 3 months old. [104]mproved cartilage thickness (þ15.2%) and lower subchondral bone thickness (SBT, À14.7%) were detected in guinea pigs receiving the same PEMF (75 Hz, 1.6 mT), for 6 h day À1 over 13 weeks for knee osteoarthritis, although with no significant differences in epiphyseal trabecular bone remodeling. [128]nother study stimulated guinea pigs in late knee osteoarthritis stages with 1.5 mT PEMF for 6 h day À1 over 12 weeks and tested two pulse frequencies: 37 and 75 Hz.Both frequencies lowered Fibrillation Index (FI) and SBT, with the 75 Hz being more beneficial to cartilage (þ20.6% thickness; À18.5% cartilage FI).Nevertheless, both frequencies were detrimental for microarchitecture (averages of À21.2% Tb.N and þ26.5% Tb.S). [129]3.Effects of IC Stimulation on Intermediate-Sized Animals Rabbits (Oryctolagus cuniculus) are the second usual choice to study IC stimulation on bone remodeling and implants' osseointegration (Figure 1).Comparatively to smaller models, rabbits undergo more secondary bone remodeling and have sufficient cortical and cancellous bone, and satisfactory intramedullary space to test various types of bone implants.Compared to larger animals, rabbits reach skeletal maturity earlier, and have more fatty bone marrow and thinner condylar cartilage than humans, besides faster cortical bone remodeling and joint cartilage healing rates at young ages.[104]

Tackling Soft Tissue Disorders in Intermediate-Sized Animals via IC Stimulation
Regarding soft tissue healing applications, rabbits subjected to patellectomy and further exposed for 0.5 h day À1 for 8-16 weeks to CMF (alternating stimuli of 76.6 Hz, 0.04 mT; constant stimuli of 0.02 mT), experienced significant histological benefits for newly formed bone (þ99.2%) and regenerated fibrocartilage zone (þ41.9%).Significant biomechanical benefits were also observed but only at week 16: load to failure (þ25.9%),ultimate strength (þ23.7%), and energy to failure (þ67.3%). [130]4.Effects of IC Stimulation on Medium-Sized Animals Dogs (Canis lupus familiaris) were not widely used to assess IC stimulation (Figure 1) but can offer some advantages when studying the bone response to injury and treatments.
Their skeleton is similar to humans in bone weight, density, organic and inorganic composition, water fraction, secondary osteons, epiphyseal fusion after maturity, intracortical remodeling activity, age-associated bone loss, and poor ability to regenerate cartilage defects.However, dogs' skeleton differs from humans' by retaining plexiform bone on cortical periosteal and endosteal surfaces, having thinner articular cartilage and higher annual trabecular turnover rates, which translates into higher rates of solid bony fusion and lower rates of non-union, comparatively to humans. [104]

Managing Bone Defects in Medium-Sized Animals Through IC Stimulation
The earliest evidence of PEMF effects on bone dates back to 1974 with a study on bone defects, when dog fibular osteotomies were stimulated for 24 h day À1 with PEMF of 1 or 65 Hz over 4 weeks. [73]Although both settings accelerated bone repair, the higher pulse frequency (65 Hz), above the naturally occurring 1 Hz and 2 mV cm À1 biologic events, yielded better outcomes, namely predominantly parallel orientation of the callus fiber bundles, accelerated ossification patterns, and reduced cartilage.These histological features translated into improved mechanical strength (þ114.3%load-bearing) at 28 days post-fracture. [73]ther findings in dogs also support the use of PEMF to repair bone defects and improve the success rate of bone (spine) fusion. [62,131]Exposure of osteotomized tibiae for 1 h day À1 to PEMF of 0.2 mT and 1.5 Hz (asymmetrical pulses), has enhanced callus formation by 53%, and its maturation in the bone healing late-phase.Increased formation of new bone was histologically demonstrated, with more bone (þ1.63-fold) and fibrous (þ1.33-fold) tissues, and less cartilage (0.71-fold) in the maturating callus.Also, stimulated tibiae had faster recovery upon load-bearing (þ1.46-fold) and higher mechanical strength (þ20.4% torque and þ54.6% torsional stiffness). [62]Dogs that have undergone spine fusion and received 1.5 Hz PEMF for 6 h day À1 over 24 weeks, had significantly increased BMD of the anterior vertebral body (þ12%), and slightly increased flexion and bending stiffness. [131]

IC Stimulation of Medium-Sized Animals with Osteoporosis and Cartilage Defects
In OVX osteoporosis dog models, PEMF of 1.5 Hz for 1 h day À1 during 12 weeks reduced bone loss from 23.1% to 9.5%, but had no effect on bone remodeling within the bone cortex. [132]For combined therapies studies, a PEMF of higher frequency and strength (75 Hz, 1.5 mT) was applied for 6 h day À1 over 13 weeks to dogs with articular cartilage defects on the stifle joint (knee), treated with tissue-engineered osteochondral grafts. [133]The PEMF-stimulated group exhibited improved cartilage growth and repair and was less likely to develop osteochondral pathologies (À80% and À60% probabilities of proteoglycan and chondrocyte pathologies, respectively; À70% probability of worse Osteoarthritis Research Society International (OARSI) score).Slightly improved grafts' integration was observed, although not statistically significant probably due to changes in PEMF orientation derived from animals' movement. [133]

IC Musculoskeletal Studies on Medium-Sized Animals Showing No Improvement
Studies that did not report any IC-driven improvements in dogs' bone metabolism have tested the effects of i) a PEMF of 1.5 Hz, 0.1 mT, 0.5 or 1 h day À1 for 12 weeks, on spine fusion rate and bone properties, [134] and ii) a PEMF of 40 kHz pulses (and 1.5 MHz bursts), 0.8 mT, 20 min day À1 for 2 weeks, on osseointegration of dental implants placed in the dog's mandible. [89]oteworthy, PEMF of 1.06-2.56mT have been also applied for 24 h day À1 over 2 or 6 weeks to tibial osteotomies in sheep (Ovis aries), but no significant differences in healing time or bony callus composition were observed with these stimulation parameters. [75]

Findings from IC Pre-Clinical Studies
Pre-clinical studies in non-human animal models have gathered evidence on the benefits of IC stimulation to treat bone and cartilage disorders, as illustrated in Figure 3.These included faster callus formation, bridging and bone maturation; improved bone volume and microarchitecture (higher trabecular number, area, and/or thickness, Figure 3A); increased BMD and prevention of bone volume loss, associated to conditions like osteoporosis; increased osseointegration (enhanced peri-implant bone volume and bone-implant contact, Figure 3C,D).Histological techniques revealed IC-induced increases in callus fibrous content, mineral apposition rate, faster cartilage-to-bone transition, and better cellular and tissue organization (Figure 3B). .Reproduced and adapted with permission. [120]Copyright 2014, Wiley.B) Histological assessment of osteochondral repair in dogs upon 3-month PEMF treatment: general overview of the tissue with hematoxylin and eosin (H&E, 1a-1b); toluidine blue (2a-2b) for proteoglycan visualization; picrosirius red (3a-3b) for collagen staining and immunohistochemical staining of collagen-II (4a-4b).Better tissue structure and increased content on proteoglycans and type II collagen were observed in the PEMF-treated tissues.Reproduced and adapted with permission. [133]Copyright 2020, Wiley.C) Histological evaluation of the peri-implant bone in rabbits, 2 weeks post-implantation surgery, using acid fuchsine and toluidine: newly formed bone is seen around the implant in both control and PEMF-treated groups, but in this last the bone tissue is better organized and trabeculae are present on the implant's surface.Reproduced and adapted with permission. [86]Copyright 2016, Wiley.D) Histological analysis with H&E of the peri-implant bone tissue in rats, showing a better tissue organization and increased formation of bone trabeculae around the implant in PEMF-treated tissues.Reproduced and adapted with permission. [90]Copyright 2018, Elsevier.
properties, including bone bending stiffness, callus/bone elastic modulus, torque and load bearing, have also improved upon IC stimulation.At a molecular/biochemical level, IC stimulation activated osteogenic pathways as the Wnt/β-catenin signaling, increased molecules key to bone mineralization (e.g., deposition of collagen-I and Ca 2þ ), [135] as well as increased serum levels of osteogenic markers (e.g,.ALP, Ca 2þ , OC, OPG, CTXs) while lowering osteoclastogenesis ones, like RANKL and TRACP5b.
A general overview of IC stimulation efficacy in animal studies is presented in Figure 2, which presents improvements versus no improvements according to stimuli frequency, MF strength or exposure time.Overall, stimuli with pulse frequencies >7.5 Hz performed better, with success rates of 93.4% (57 out of 60 assays), against a success rate of 53.8% for pulse frequencies ≤7.5 Hz (7 out of 13 assays).Accordingly, studies comparing different frequencies inferred the same: higher frequencies usually outperformed lower ones, [69,73] and studies on rats and dogs using quite low frequencies were non-effective. [61,69,73,134]The same occurred for MF strengths ≥1.2 mT (93.3% success rate, corresponding to 42 out of 45 assays) when compared to weaker fields (56.5%, 26 out of 46 assays).Exposure to stimuli for at least 3.5 h day À1 led to higher success rates (89.3%, 25 out of 28 assays), compared to shorter exposures (78.0%, 39 out of 50 assays).Indeed, when comparing different daily exposure times (0.5, 3, and 6 h per day), longer exposure enhanced the effects of early treatments. [67]Importantly, the application of IC stimulation at earlier phases of the injury/condition may improve healing effects and prevent BMD loss.
Overall, whenever biological outcomes were assessed, IC stimulation induced improvements in more than 75% of the assays, regardless of the assessed outcome (Figure 4A).Nevertheless, radiological and biomechanical outcomes were not assessed as widely as histological and biochemical ones.A deeper analysis of the improvement effect sizes according to the combination of frequencies and MF strengths, is presented in Figure 4B-D which shows various effective pairs of frequency/MF combinations in green (e.g., 15 Hz/2.0 mT, 75 Hz/1.6mT).However, these graphs may include some bias derived from directly comparing effect sizes of studies using different animal models, heterogenous designs and assessments.For example, tibial bone conditions seem to overall respond less to PEMF than femoral ones, in intermediate-and medium-sized animal models. [65,66,77]or comparison purposes, efficacy analyses separated per small-sized, intermediate-sized, and medium-sized animal models are presented in Figure S2-S4, Supporting Information.These analyses reveal that IC stimulation induced much higher improvement rates (in 64-72% of the assays) in small and intermediate-sized animals like rats and rabbits.The most effective (and most tested) frequency/MF combinations for these animal models were 15 Hz/1.6-2.0 mT (Figure S2-S3, Supporting Information).Conversely, IC stimulation in medium-sized animals such as dogs and sheep, induced improvements in 44% (at most) of the biological outcomes analyzed.Surprisingly, a combination of relatively low frequency and MF (1.5 Hz, 0.2 mT) was quite effective in the medium-sized animals (Figure S4, Supporting Information).Also to highlight, some studies did not observe any benefits of applying IC stimuli. [59,61,74,75,89,95,103,109,134,136]This absence of improvement upon IC stimulation may be ascribed to the use of novel IC modalities that still require optimization, like RMF, or to the relatively lower frequencies, field strengths, or exposure times used (Table S1, Supporting Information).Of note, CMF and PEMF were directly compared in some studies and presented similar efficacies, [67] except when CMF was applied intra-medullary. [68]iven its invasiveness, intra-medullar CMF may be more effective if combined with traditional orthopedic internal fixations.
In general, the studies applying IC stimulation to animal models revealed promising success rates, although radiological and biomechanical outcomes should be more thoroughly assessed.
The available data reinforce the potential of IC stimulation as a safe and effective non-invasive method to improve several musculoskeletal conditions.Notwithstanding, "size matters", since the success rate of IC stimulation decreases with the increase of the target animal, highlighting the importance to test several stimuli parameters when scaling up an IC intervention to bigger animal models, until reaching humans.

Overview of IC Clinical Designs
The clinical application of IC stimulation to treat multiple complications has been under development since the middle of the 20 th century.This therapeutic is gaining increasing popularity in clinical practice, due to its non-invasive nature and the wide range of clinical applications of the delivered magnetic fields.[139] Delving deeper into the clinical application of IC stimulation, our search retrieved 44 studies on the therapeutic potential of the IC stimulation to treat musculoskeletal conditions in humans (Table S3, Supporting Information).
IC clinical studies mainly used devices delivering PEMF (in 88.6% of the studies) (Figure 5A).FDA-approved stimulators are the most used devices to test the efficacy of IC musculoskeletal therapy in human patients (used in 56.8% of the studies), as well as the stimuli parameters and protocols recommended by their manufacturers (Figure 5B).Most popular models include the "Biomet EBI Bone Healing System", the "Orthofix Cervical-Stim", the "Orthofix Physio-Stim" (Figure 5D,E) and the "Orthofix Spinal-Stim" (Figure 5F).Several of these devices allow patients to wear PEMF devices over surgical dressings, placed on the skin over the fracture site.Still, 15.9% of studies used custom-made devices, while 27.3% did not specified the device used.By far, the most frequent application of IC stimulation on human patients is to heal non-unions and delayed unions, mainly deriving from fractures (Figure 5C "bone defects", 61.4% of the studies).Spine fusion has been the second target application, although it only corresponds to 11.4% of the studies.
Regarding the IC stimuli parameters applied in clinical studies (Figure 6A-D), there is first to highlight that the clinical studies reported less frequently the stimuli parameters applied than the pre-clinical ones, with only 75% of the studies having disclosed at least one parameter.Some studies only mentioned the device used, while others did not present any setup information.In the fewer clinical studies that could be analyzed here, the stimuli pulse frequencies ranged from 1.5-80 Hz, with a high prevalence of 15 Hz, the frequency used in 44.1% of the studies that have provided the stimuli specifications.A much higher frequency (1 kHz pulses; 27 MHz bursts) was also tested on soft tissue after tooth removal. [140]Only half of the studies (22 out of 44) provided the values for the applied MF strengths.
These highly varied from 6 μT to 5 mT (except for a higher MF strength of 105 mT), although 45.4% (10 out of 22 studies) applied MF strengths between 1.0 and 3.0 mT.The daily exposure time ranged from 2 min to a full day exposure, with an average and 3 rd quartile value of 7.9 and 10 h day   S3, Supporting Information)."Bone defects" include non-unions and delayed unions mainly from traumatic etiology (fractures).D,E) Photographs of the FDA-approved PEMF device Orthofix Physio-Stim used in the clinical practice for scaphoid and tibial non-unions, respectively.Reproduced with permission. [168]Copyright 2017, Springer.Reproduced with permission. [138]Copyright 2012, BioMed Central.F) Photographs of the FDA-approved PEMF device Orthofix Spinal-Stim used in clinical practice for spinal fusion.Taken from Orthofix Spinal-Stim Instruction Manual (Model 5212), available online at htps://orthofix.com.
durations varied from 6 days to 18 months, with a median of 3 months and an average of 5.4 months.

Evidence of IC Clinical Efficacy in Humans
Regarding the type of non-invasive biological/clinical outcome measures used to assess the efficacy of IC stimulation, radiological imaging outcomes were the ones mainly assessed in almost all the clinical studies (40 out of 44 studies; Figure 7A).In contrast, the influence of IC stimulation on serum biochemical markers was only addressed in four studies.Other clinical outcomes, namely biomechanical ones, were reported in 50% of the studies (Figure 7A).42][143][144][145][146][147][148]

Managing Bone Defects in Humans with IC Stimulation
][150][151][152][153][154][155][156][157][158][159] Most of these bone defect studies used commercial devices like the Biomet EBI Bone Healing System, the EBI Bone Healing System Model 420, the Orthopulse I or II, and the OrthoLogic 1000 device.Overall, our analysis of IC stimulation efficacy in treating bone defects, which includes studies from 1982 to 2023, agrees with previous revision studies.According to Heckman et al. [160] (1981), 64% of 149 non-unions healed properly upon PEMF stimulation, while Bassett et al. [161] (1982) calculated this stimulation was successful for 76-81% of  S3 and S4, Supporting Information).A) Pulse frequencies used (in Hz); of note, 12 studies had non-defined pulse frequencies and 1 study tested 2 different pulse frequencies, totalizing the 33 pulse frequencies here presented.B) List of the applied magnetic field (MF) strengths (in mT); of note, 23 studies had non-defined MF strengths and 1 study tested 2 different MF strengths, totalizing the 22 MF strengths here presented.C) Daily exposure time to the stimulus (in hours day -1 ); of note, 4 studies had non-defined exposure times, 1 study tested 3 different exposure times, and 1 study tested patient-dependent exposure times, totalizing the 42 exposure times here presented.D) Box-violin plot with the distribution and summary statistics of the total duration of the IC stimulation assays, in months.
1007 ununited fractures.Similar success rates were obtained when comparing PEMF against surgery with bone grafts, to treat ununited tibial fractures.Aaron et al. [162] (2004) concluded that both treatments performed equally, with success rates of 82% for bone grafts (569 cases from 14 studies), and 81% for PEMF treatment (1718 cases from 28 studies).This means that therapeutic intervention could have evolved more over the last decades, still highlighting the lack of setup optimization and/or personalized medicine studies.
Examples of setups with positive osteogenic effects include the EBI Bone Healing System Model 420 (15 Hz pulses, 4.5 kHz bursts, 8-10 h day À1 ) to treat scaphoid non-unions, which Outcomes from clinical measurements   S3 and S4, Supporting Information, respectively).The number of studies is indicated in the graph."NA", not applicable (outcome not assessed).B) Radiological (green), Histological/biochemical (blue) and Biomechanical (yellow) outcomes, according to the stimulus' pulse frequency and MF strength used in each study.Each circle represents one study, and its diameter is directly proportional to the amplitude of the effect that IC stimulation had on that outcome category.Values of AE0.1 and zero (0) were chosen to represent non-stated outcomes and unknown pulse frequencies or MFs strengths, respectively.
resulted in better grip and wrist motion unto 83% and 89% of normal levels, respectively. [141]However, in a follow-up study 6 years later, using the same settings, results slightly decreased to 77% and 80%. [152]Authors suggested that the less successful cases may derive from low compliance to the non-weight-bearing restriction while using the device. [141]Biochemical outputs, such as significant increases in growth factors like PIGF, BDNF, BMP-5 and -7, were also observed after stimulation of metatarsal non-unions with the Biomet EBI Bone Healing System using similar parameters (15 Hz pulses, 4.5 kHz bursts, 1.8 mT, 10 h day À1 ). [163]PEMF stimulation also proved effective in treating congenital tibial pseudarthrosis, [164,165] although still needing additional interventions in more severe cases. [165,166]Noticeably, less successful outcomes were also observed when using commercial devices.In another study using EBI Bone Healing System, PEMF stimulation (15 Hz, 1.8 mT) of delayed unions resultant from ankle arthrodesis, with immobilization and limited weight-bearing, only 26% of cases achieved union. [167]o improvements in the carpal scaphoid non-union healing rate occurred using Physio-Stim (1.5 Hz, 0.2 mT, 3 h day À1 ), with its late application (6 weeks post-fixation surgery) being suggested as the main cause of failure. [168]EMF efficacy was also tested in acute bone fractures.PEMF of 40-72 Hz, 1 mT, applied 2-6 h day À1 after mandibular fracture, complemented with maxillo-mandibular fixation, was able to return BMD to basal levels, or even increasing it by ≈16.7% after 1-month post-surgery. [143,146,169]Authors also reported increased stability in mouth opening, with less pain, for the stimulated patients. [146][172] Also, no significant differences in the recovery of wrist movement were observed for patients with fractured scaphoids stimulated with Orthopulse PEMF of 15 Hz pulses, for 24 h day À1 over 1.5 [170,171] or 3 [170] months.Nevertheless, application of PEMF of 16 Hz, 0.006-0.282mT, for 7 min day À1 on osteotomized tibia, increased serum ALP levels and accelerated osseous consolidation in stimulated patients, compared with the placebo group, although this later effect was only significative for patients over 50 years old. [173]Also, radius fractures stimulated for 24 h day À1 during ≈3 months with PEMF of 10 Hz pulses/20 kHz bursts and 0.05-0.5 mT, delivered by the "Fracture Healing Patch" (Pulsar Medtech Ltd), accelerated bone healing (þ29.6% union bridging, þ104.3%hand grip strength) and improved wrist function (up to þ1.3-fold). [174]egarding spine fusion, some studies state that both PEMF (4-8 h day À1 ) and CMF (30 min day À1 ) can improve the surgery's success rate by up to 48.8%. [145,175,176]In contrast, a comparative study observed that both PEMF of 15 Hz pulses, 0.68 mT, 2 min day À1 , and CMF of 76.6 Hz, 40.0 AE 8.0 μT AC þ 20.0 AE 2.0 μT DC, 30 min day À1 , performed worse than the unstimulated group, which achieved solid fusions in 100% of the treated patients, against 68.8% for PEMF and 87.5% for CMF. [177]evertheless, when comparing these stimuli parameters with others reviewed in this section, we can denote that PEMF/ CMF daily exposure times and CMF-induced MF strength are much lower than the average of all clinical studies (7.9 h day À1 and 6.3 mT; Figure 6 and Table S3, Supporting Information), what may explain the worse success rates.In line with this hypothesis, a recent multicenter study on the use of PEMF as an adjuvant therapy to lumbar spine fusion in 142 patients at risk for pseudarthrosis (Orthofix SpinalStim, 1.5 Hz pulses, 3.85 kHz bursts, 0.4 mT, 2 h day À1 over 6 months), reported that 88.0% of patients exhibited successful fusions and significant improvements in pain, function, and quality of life at a 12 months follow-up appointment. [178]Interestingly, a study that computationally compared the electric current densities induced by the FDA-approved PEMF stimulator (Orthofix SpinalStim), the CMF stimulator (SpinaLogic) and the CC stimulator (Biomet SpinalPak), reported that PEMF induces the strongest maximum electric field and current density amplitudes in spinal vertebrae, besides generating local micromechanical forces that were more similar to the micromechanical oscillations naturally generated by EMF in the bone. [179]

Tackling Osteoporosis in Humans Through IC Stimulation
In post-menopausal women prone to osteoporosis and taking calcium and vitamin D supplements, a 40 min day À1 thrice-a-week treatment with PEMF of 8 Hz pulses and 3.82 mT only slightly increased vertebral, femoral neck, and hip BMD (þ≈1.5-2.0%) at the end of the 6-week treatment, and significantly reduced bone marrow fat fraction (À4.81%). [180]Osteoporotic patients who suffered bone fractures and were stimulated with the same PEMF parameters showed improvements in bone microarchitecture (average of þ7.2% BV/TV, þ24.8% cortical thickness (Ct.Th), þ19.0%Tb.Th, þ13.4% Tb.N), as well as in health scores related to body functions, quality of life and back pain. [181]In other study on osteoporotic patients under calcium supplementation, a 10 h day À1 (overnight) PEMF stimulation of the non-dominant forearm with higher frequencies (72 Hz, 2.85 mT), resulted in increased radii BMD, that further decreased upon 36 weeks after the last stimulation.Interestingly, the radii from the unexposed forearm experienced a similar pattern, suggesting a systemic "cross-talk" or close proximity of both radii during asleep stimulation. [182]Bone-related biochemical markers altered in osteoporotic patients treated with PEMF of lower frequency but higher MF (8 Hz, 3.82 mT), including serum ALP (þ3.23%) and CTX-I levels (À9.12%). [180]Additionally, a study on women with postmenopausal osteoporosis, testing PEMF rotating every 4 min in frequencies varying between 16, 18, 20, and 22 Hz, and MF strengths between 3.0, 3.2, 3.4, and 3.6 mT, for 0.8 h day À1 during 2 weeks, reported significant changes in serum markers related to the Wnt/β-catenin and the OPG/RANKL pathways, which can explain the positive effects of this IC stimulation on bone metabolism. [183]

Enhancing Implant Osseointegration in Humans via IC Stimulation
To our knowledge, PEMF efficacy on implants' osseointegration in humans was only tested in dental and hip prosthesis implants. [142,184]For dental implants, PEMF was implemented through caps that delivered continuous daily stimulation (no parameters were provided).From 1.6 months onward, the active caps promoted overall higher implant stability (average þ10.2% in stability scores vs. unstimulated controls). [184]On hip prosthesis, BMD levels at 0 and 90 post-operative days, were measured on different zones following stimulation with PEMF of 75 Hz, 2.0 mT (6 h day À1 ).Although no significant differences were found in the average BMD levels at both timepoints, the percentage of "responder patients" (with >3.5% increase in BMD) was bigger upon PEMF stimulation (66% and 93% in PEMF-stimulated patients, for 2 measured zones, against 40% and 40% in the non-stimulated group), proving that PEMF is indeed helpful in clinical recovery and in bone stock restoration. [142]

Handling Osteoarthritis in Humans Through IC Stimulation
Only two studies tested the efficacy of PEMF on (knee) osteoarthritis.One study used the FDA-approved MAGCELL ARTHRO device to deliver PEMF of 8 Hz, 105 mT for 5 min twice-a-day, for 15 days, and atients reported a reduction in pain, stiffness, and disability in carrying out daily activities, overall improving their health condition. [147]Another study assessed the efficacy of PEMF of 50 Hz, 5 mT, for 30 min day À1 over 2 months, with and without combination of progressive resistance exercise (PRE), to improve function and pain in patients with knee osteoarthritis.Both PEMF with and without combination of PRE were equally effective in decreasing pain and improving function (in 60-80% of patients), suggesting that optimal PEMF parameters to improve the effects of PRE in knee OA are yet to be determined. [185]

Addressing Soft Tissue Disorders in Humans with IC Stimulation
In the only study on IC clinical application for soft tissues reported in the literature, PEMF stimuli of very high frequency (1 kHz pulses, with 27 MHz bursts) could improve soft tissue healing, pain management and reduce the risk of dehiscence 1 week post oral surgery, when applied for 24 h day À1 . [140]

Findings from IC Clinical Studies
The relative efficacy of the IC clinical studies, according to the applied stimuli parameters, is presented in Figure 6 (global overview for each parameter) and Figure 7, which associates pairs of frequencies and MFs of stimuli with their therapeutic efficacy, based on the three categories of outcome measures.
First, no clinical study has reported major adverse effects from the use of IC stimulation devices.Studies assessing radiographic outcomes (examples illustrated in Figure 8) have reported effects driven by IC stimulation that include faster osseous consolidation and healing of non-unions and delayed unions (Figure 8A,B), prevention of BMD loss immediately after injury and, in some cases, increases in BMD upon prolonged treatment (Figure 8C).The few studies that measured serum markers observed increases in osteogenic-associated markers, such as ALP and specific growth factors.Overall, human studies with IC simulation could benefit from analyzing biochemical outcomes (e.g., osteogenic markers in serum) more frequently, as such analyses may help to understand the signaling effects of IC stimulation.In general, the IC stimulation setups used had low efficacy for treating scaphoid non-unions and delayed unions after foot and ankle arthrodesis, and effectiveness on bone fractures highly varied.Indeed, various setups did not significantly improve the bone-associated main endpoint. [171,177,186]This may be related to the stimuli parameters applied, but also to the tested condition, since several unstimulated control groups have achieved quasi-optimum recoveries in the assay duration time.Nevertheless, IC stimulation improved patient condition in secondary endpoints, like recovery time, pain management or prevention of bone mass loss. [171,186]any authors did not report the IC parameters used in their studies, making it difficult to draw more assertive conclusions regarding effective magnetic stimuli settings.However, most of them used clinically approved devices, mainly Biomet EBI Bone Healing System, which generally deliver PEMF of 15 Hz quasi-rectangular pulses, followed by a sharper reverse form, generating MF strengths up to 1.8-2.0mT.Most studies using this device and respective setups have achieved significant positive outcomes on non-unions and delayed unions, bone fractures, and consolidation of spine fusions.With two exceptions, all setups using >15 Hz stimuli presented positive osteogenic effects, and stimuli generating MFs ≥1 mT presented improvements in 80% of cases (Figure 6A and 7B).Regarding the exposure time, 11 out of 43 studies (25.6%) applied the stimuli for the average recommended 8-10 h interval of daily stimulation, with 9 of them reporting osteogenic improvements.Altogether, the use of IC stimulation seems clinically feasible to treat non-unions, to reduce its risk after fracture and accelerate healing when combined with standard procedures, and to increase the bone fusion success rate. [138,139]Further, although still few, the promising results here presented for implants' osseointegration, stress the need to further investigate the benefits of IC stimulation on this musculoskeletal condition, a main societal challenge associated with high numbers of primary and revision surgeries.Although IC efficacy has been proved in animal models of osteoporosis, few clinical trials have tested it, most likely due to the burden associated with the prolonged use of the device.Lastly, studies on IC's potential beneficial effects on osteoarthritis are still lacking and are highly demanded.
Overall, this study indicates that it would be desirable to develop more attractive devices for daily use, with the ability to provide personalized stimuli, and highlights the need to comprehensively optimize the stimuli setups to increase IC clinical performance.

The Potential Biomechanism Behind IC Therapeutic Effects
At the turn of the 21 st century, various groups started attempting to infer the biomechanisms underlying the IC effects on the musculoskeletal system, with this line of research accelerating in the last 5 years.A sum up of these authors' findings is illustrated in Figure 9.It is currently assumed that the electromagnetic fields generated by IC can pass through the cellular membranes and elicit a release of Ca 2þ from intracellular storages, increasing the cytosolic Ca 2þ concentration.[189] This first cellular event induces changes in the membrane polarization, coupled with the activation of voltage-sensitive enzymes, the reorganization of the cytoskeleton, and the activation of Ca 2þ -dependent enzymes, including calmodulin (CaM). [190]n osteoblasts, calcineurin (CaN) becomes active in a Ca 2þ and CaM-dependent manner, [191] and dephosphorylates Nuclear A) Anteroposterior and lateral radiographs of a distal tibial nonunion: i) 10 months after fracture; ii) after PEMF stimulation of the fracture site for 5 months, leading to fracture union.Reproduced and adapted with permission. [138]Copyright 2012, BioMed Central.B) Delayed union of femoral fracture in a patient who received reduction and intramedullary fixation: i) before PEMF treatment; ii) upon 3 months of PEMF treatment, already showing some progress to union; iii) upon 8 months of PEMF treatment, when the fracture became united.Reproduced and adapted with permission. [151]Copyright 2013, BioMed Central.C) Anteroposterior X-rays of patients with hip prosthesis in a control group (top set of images) or a PEMF-treated group (bottom set of images): i) pre-operative; ii) post-operative; iii) 90-day-follow-up.Images illustrate a higher rate of implant osseointegration in the PEMF-treated group.Reproduced and adapted with permission. [142]Copyright 2009, Wiley.
Factor of Activated T Cells 1 (NFATc1), promoting its nuclear translocation and a subsequent increase in the transcription of genes related to the Wnt/β-catenin pathway. [192]3,193] Another widely known osteogenic signaling pathway that cross-talks with the Wnt pathway is the TGF-β/BMP (2/5/7) signaling pathway, which is also found to increase following IC stimulation. [111,124,163,193][196][197] Also observed to be increased upon IC stimulation is the Runt-related transcription factor 2 (Runx2), a target gene of these pathways that induces osteogenesis [198,199] and osteogenesis-related markers (e.g., OC). [98,123,124]Runx2 is also involved in the gene regulation of some G protein-coupled receptors (GPCR), [200] which can be activated by PTH (also increased by IC stimulation [121,193] ) to initiate the sAC/cAMP/PKA/CREB signaling cascade, leading to increased sAC, serum cAMP, p-PKA and p-CREB. [102,121]his sequence of events promotes osteoblastogenic effects, for example by allowing the accumulation of β-catenin [201] and thus cross-talking with the Wnt signaling pathway.124]126,127,173,180] In contrast, IC stimulation can also regulate osteoclast differentiation and activity, by increasing the OPG/RANKL ratio.The increase in OPG expression, together with a decrease in RANKL expression, are thought to be the major events in the IC-induced decrease of osteoclastogenesis. [113,124,127,183]][122][123] Enzymes involved in matrix degradation, such as metalloproteins (MMP-3/9/13) and ADAMTS-5, were observed to be decreased as well. [125]Subsequently to all these osteoclast-related molecular events, there is a reduction Figure 9. Biomechanisms that may underly the observed therapeutical bone-associated effects of inductive coupling (IC) in vivo stimulation.The electromagnetic fields (EMF) delivered by IC pass the cytoplasmic membrane of cells, such as osteoblasts and others, to induce the release of intracellular calcium, alterations in the membranes' potential, and elicit a series of other molecular alterations (e.g., signaling pathways, biological processes) whose interplay culminate in the promotion or repression of the expression of genes associated to increased osteoblastogenesis and osteoblast activity, and decreased osteoclastogenesis and osteoclast activity.The green squares delimit events mostly observed in osteoblasts, while blue squares delimit events mostly observed in osteoclasts.Black bold headings denote pathways and mechanisms reported only in vitro, while colored bold headings denote pathways and mechanisms already observed in vivo; headings surrounded by colored boxes represent the main cellular endpoints observed in most in vivo studies upon IC stimulation.Continuous black arrows represent associations already reported between the indicated cellular events, while dashed black arrows represent indirect associations that have other intermediary steps; dashed grey arrows represent probable associations.
of bone resorption, and thus a decrease in serum CTX. [87,88,102,112,120,121,126,180]Netrin 4 may also play a role in the PEMF-induced decrease of osteoclastogenesis, [202] since PEMF increases the levels of this inhibitor of osteoclast differentiation, [203] and consequently of bone resorption, as other netrin proteins (e.g., netrin 1).PEMF effects on osteoclasts may also be related to the desensitization of osteoblasts to inflammation initiators, such as IL-1β, [204] resulting in a decreased expression and secretion of IL-6 and RANKL, among other cytokines, by these cells. [111,115,125]n vitro experiments suggest two other parallel and interacting mechanisms of action by which IC may lead to decreased osteoclast and increased osteoblast activities.PEMF was observed to induce, at an early phase, an increase in mitochondrial activity and production of reactive oxygen species (ROS, •O 2 À and H 2 O 2 ), resulting in increased non-cytotoxic oxidative stress.This event is important to trigger the cell's antioxidative defense mechanisms, through the increased gene and protein expression and activity of antioxidant enzymes, namely superoxide dismutase 2 (SOD2), catalase (CAT), glutathione peroxidase 3 (GPX3) and glutathione-disulfide reductase (GSR), which consequentially decreases ROS. [187,205]The reduction of ROS may play a role in promoting osteoblastogenesis, as well as decreasing the inflammatory response associated with osteoclastogenesis and osteoclast activity. [205]Potential contributors to the decreased circulating levels of inflammatory cytokines are the decreased levels of angiogenic factors like VEGF. [115]PEMF can also promote other stress-related processes and associated tissue repair cascades, by inducing the expression of heat shock proteins like hsp70, whose gene's promoters have electromagnetic fields (EMF)-responsive sequences. [206,207]Noteworthy, in a peripheral blood mononuclear cells (PBMCs) model of Alzheimer's disease PEMF was reported to decrease the expression of miRNAs, namely miR-107, miR-335-5p, and miR-26b-5p, that regulate the translation of mRNAs associated with transcription factors, growth factors, enzymes, and transporters (e.g., SP1, IGFR1 and MAPK, SLC17A6, respectively). [208]Although not directly related to musculoskeletal disorders, this miRNA-based action mechanism may likely play a role in the clinical outcomes here reported and may be worth studying in the future.
In summary, there are various possible cellular events by which IC stimuli modulate osteogenic and osteoclastic activities to increase bone formation and decrease bone resorption, underscoring IC therapeutic effects in musculoskeletal conditions.A comprehensive study of the less explored mechanisms like the regulation of oxidative and proteostasis stress, of inflammatory responses, and miRNA expression, will deeper our knowledge on IC stimulation mechanisms of action, not only on bone but also on other potential target tissues.Further, it will open new avenues for the potential use of combined therapies, such as PEMF together with a molecular therapy targeting key protein(s) of the core signaling cascades, to boost the IC therapy.

General Conclusions and Future Perspectives
This review analyzed 117 studies (73 in animal models, and 44 in human patients) using magnetic stimulation delivered by IC devices for musculoskeletal therapy purposes.Overall, from the studies here analyzed, IC stimulation is an attractive option for the clinical management of bone and cartilage disorders.IC is a non-invasive therapy, with the MF being able to pass the cellular membranes to elicit various intracellular events culminating in tissue repair via e.g., increased bone deposition and decreased bone resorption.However, clinical evidence is still scarce for each condition tested, particularly for implants' osseointegration, osteoporosis and osteoarthritis in humans.To achieve good clinical evidence, not only a higher number of trials is required, but also more studies comparing different stimuli parameters.The comparative analyses of pre-clinical in vivo data here presented hold the potential to support the design of such advanced studies.Overall, stimuli with pulses' frequencies >7.5 Hz in animal studies and >15 Hz for human patients, performed better, while in both pre-clinical and clinical studies, MF strengths >1.0-1.2 mT were associated with higher success rates.Generally, higher frequencies and MF strengths outperformed the lower ones, as well as longer exposures outperformed shorter ones.Noteworthy, the effect sizes recorded on human patients are generally lower than in animal studies (and in medium-sized animals lower than in small-sized ones), possibly because the regeneration potential decreases with increased animal size and complexity, again strengthening the need for further optimization of stimuli clinical setups.Other major limitations of the current approach to IC therapeutics include the delivery of stimuli based on parameters found empirically (usually by the trial-error method), and the fact that it is statically maintained throughout the treatment.IC stimulation will only be able to achieve its highest impact on bone bioactivity and be fully effective, if it can be used for customized therapy, according to an individual's physiological response. [69]Indeed, both extra and intracorporeal electrical stimulators will most likely provide poor therapeutic effects if multiple patient-related and externally driven factors are not considered, including a wide range of physical, behavioral, social and psychological factors.Optimal personalized stimulation parameters cannot be static: the unpredictability of disorders' dynamics demands that the stimulus parameters dynamically change to fit the idiosyncrasies of each patient.Such advances will most likely require innovative methods and intelligent technologies, extracorporeally controlled by medical specialists using wirelessly mobile applications. [209]oreover, IC devices for bioelectronic implantable applications require electric powering characterized by very high electric currents (usually exceeding 1 A), which is an additional high-risk scenario that can trigger post-surgical complications.Therefore, new magnetic stimulation systems must be designed to deliver targeted magnetic stimuli when electrically supplied by very low current (lower than 100 mA). [210]Another line to which IC therapeutics can evolve comprehends smart magnetic bioactive materials, which may contribute to a more localized application of EMF and ultimately improve bone (and other tissues) regeneration. [211]Tissue engineering strategies applying such materials could further potentiate the effects of IC stimulation.
Hopefully, the transversal knowledge presented herein will serve as a valuable guide as we navigate the path toward optimizing clinical evidence and personalized IC-based treatment strategies.A deepen study of the biomechanisms of IC stimulation, the comprehensive testing of various stimuli parameters in the clinical setup, and the development of innovative medical devices, will be vital for pushing forward the clinical implementation and achieve the full therapeutic potential of IC stimulation for musculoskeletal disorders, toward more effective and tailored patient care.

Figure 1 .
Figure 1.General trends of IC stimulation on pre-clinical studies with animal models of musculoskeletal disorders.A) IC stimulation methods applied; B) Type of animal models used; C) Musculoskeletal conditions assessed in the animal studies retrieved (details in TableS1, Supporting Information)."Bone defects" include osteotomies, fractures, and bone drills; "Low BMD conditions" are conditions resulting in low bone mineral density (BMD) and bone fragility, such as osteoporosis, diabetes, and glucocorticoid treatments; "Combined conditions" represent 4 studies (out of the 73) in which 2 conditions were simultaneously assessed.D) Illustrative schematic of a typical IC stimulation device used in pre-clinical studies to deliver PEMF to animals.Based on illustrations from Jing et al.[120] and Cai et al.[87] E) Photograph of an actual IC stimulation device used in a pre-clinical study here reviewed.Reproduced and adapted with permission.[102]Copyright 2018, Wiley.CEF: constant electromagnetic field; CMF: combined magnetic field; PEMF: pulsed electromagnetic field; RMF: rotating magnetic field.

Figure 2 .
Figure 2. IC stimuli parameters applied in animal pre-clinical studies.Graphical analysis of the different IC stimulation parameters used in the 73 animal studies reviewed, plotting the number of studies applying such parameters, and the stimulation's relative success (details in TableS1and S2, Supporting Information).A) Pulse frequencies used, in Hz; of note, 5 studies had non-defined pulse frequencies and 6 studies tested 2 different pulse frequencies, totalizing the 75 pulse frequencies here presented.B) Magnetic field (MF) strengths applied, in mT; 10 studies had non-defined MF strengths, while 6, 3, and 1 studies tested 2, 3, and 4 different MF strengths, respectively, and 1 study tested 14 different MF strengths, totalizing the 91 MF strengths presented.C) Daily exposure time to stimulation, in hours day À1 ; 3 studies had non-defined exposure times, while 4 and 2 studies tested 2 and 3 different exposure times, respectively, totalizing the 78 different exposure times presented.Studies reporting positive outcomes upon IC stimulation are colored green, and studies reporting no effect or detrimental outcomes are colored orange.D) Box-violin plot with the distribution and summary statistics of the total duration of the IC stimulation assay, in weeks; 12 and 7 studies tested 2 and 3 different durations, respectively, while 1 study tested 4 different durations and another tested 5 different durations, totalizing the 106 different assay durations presented.

Figure 3 .
Figure3.Examples of biological outcomes commonly assessed in IC stimulation studies using animal models.A) Improved trabecular bone microarchitecture and cortical bone thickness of femurs of rats subjected to hindlimb unloading (HU) when exposed to PEMF for 4 weeks: i) Volume of interest (VOI); ii) 3D μCT images of trabecular bone microarchitecture in the VOI; iii) 2D μCT images of trabecular bone microarchitecture (different observation planes).Reproduced and adapted with permission.[120]Copyright 2014, Wiley.B) Histological assessment of osteochondral repair in dogs upon 3-month PEMF treatment: general overview of the tissue with hematoxylin and eosin (H&E, 1a-1b); toluidine blue (2a-2b) for proteoglycan visualization; picrosirius red (3a-3b) for collagen staining and immunohistochemical staining of collagen-II (4a-4b).Better tissue structure and increased content on proteoglycans and type II collagen were observed in the PEMF-treated tissues.Reproduced and adapted with permission.[133]Copyright 2020, Wiley.C) Histological evaluation of the peri-implant bone in rabbits, 2 weeks post-implantation surgery, using acid fuchsine and toluidine: newly formed bone is seen around the implant in both control and PEMF-treated groups, but in this last the bone tissue is better organized and trabeculae are present on the implant's surface.Reproduced and adapted with permission.[86]Copyright 2016, Wiley.D) Histological analysis with H&E of the peri-implant bone tissue in rats, showing a better tissue organization and increased formation of bone trabeculae around the implant in PEMF-treated tissues.Reproduced and adapted with permission.[90]Copyright 2018, Elsevier.

FrequencyFigure 4 .
Figure 4. Efficacy analysis of IC stimulation parameters (magnetic field and frequency) on pre-clinical studies with animal models, based on the boneassociated outcomes reported.A) Overview of the IC efficacy on the three outcome categories (details in TableS1 and S2, Supporting Information).The number of studies is indicated in the graph."NA", not applicable (outcome not assessed).B) Radiological, C) Histological/biochemical and D) Biomechanical outcomes, according to the combination of stimulus' frequency and MF strength used in each study (when undefined by the authors, the parameter was taken as "0").Each circle represents one study, and its diameter is directly proportional to the amplitude of the IC stimulation effect.FigureS2-S4, Supporting Information, show these efficacy analyses considering the combination of stimulus' frequency and MF strength per animal model.Asterisks (*) represent graph sections that show separately the studies with improvements and no improvements, for better visualization.

Figure 5 .
Figure 5.General trends of IC clinical stimulation in clinical studies.Types of A) IC stimulation applied, and B) Stimulation devices used in human patients."Approved devices" refer to stimulators that are approved for clinical use and available in the market, while "Custom-made" stimulators are devices developed by the research group to test new setups.C) Musculoskeletal conditions evaluated by the 44 clinical studies here reviewed (details in TableS3, Supporting Information)."Bone defects" include non-unions and delayed unions mainly from traumatic etiology (fractures).D,E) Photographs of the FDA-approved PEMF device Orthofix Physio-Stim used in the clinical practice for scaphoid and tibial non-unions, respectively.Reproduced with permission.[168]Copyright 2017, Springer.Reproduced with permission.[138]Copyright 2012, BioMed Central.F) Photographs of the FDA-approved PEMF device Orthofix Spinal-Stim used in clinical practice for spinal fusion.Taken from Orthofix Spinal-Stim Instruction Manual (Model 5212), available online at htps://orthofix.com.

Figure 6 .
Figure 6.IC stimuli parameters applied in clinical studies.Types of IC stimulation parameters used on patients with musculoskeletal disorders, number of studies in which they were used, and their relative success, in the 44 clinical studies here reviewed (details in TableS3and S4, Supporting Information).A) Pulse frequencies used (in Hz); of note, 12 studies had non-defined pulse frequencies and 1 study tested 2 different pulse frequencies, totalizing the 33 pulse frequencies here presented.B) List of the applied magnetic field (MF) strengths (in mT); of note, 23 studies had non-defined MF strengths and 1 study tested 2 different MF strengths, totalizing the 22 MF strengths here presented.C) Daily exposure time to the stimulus (in hours day -1 ); of note, 4 studies had non-defined exposure times, 1 study tested 3 different exposure times, and 1 study tested patient-dependent exposure times, totalizing the 42 exposure times here presented.D) Box-violin plot with the distribution and summary statistics of the total duration of the IC stimulation assays, in months.

Figure 7 .
Figure 7. Analysis of the efficacy of the IC in vivo stimulation parameters (magnetic field and frequency) on human patients, based on the reported boneassociated outcomes.A) Overview of the IC efficacy on the three categories of outcomes (qualitative and quantitative details in TableS3and S4, Supporting Information, respectively).The number of studies is indicated in the graph."NA", not applicable (outcome not assessed).B) Radiological (green), Histological/biochemical (blue) and Biomechanical (yellow) outcomes, according to the stimulus' pulse frequency and MF strength used in each study.Each circle represents one study, and its diameter is directly proportional to the amplitude of the effect that IC stimulation had on that outcome category.Values of AE0.1 and zero (0) were chosen to represent non-stated outcomes and unknown pulse frequencies or MFs strengths, respectively.

Figure 8 .
Figure 8. Examples of biological outcomes assessed in IC stimulation studies in humans.A) Anteroposterior and lateral radiographs of a distal tibial nonunion: i) 10 months after fracture; ii) after PEMF stimulation of the fracture site for 5 months, leading to fracture union.Reproduced and adapted with permission.[138]Copyright 2012, BioMed Central.B) Delayed union of femoral fracture in a patient who received reduction and intramedullary fixation: i) before PEMF treatment; ii) upon 3 months of PEMF treatment, already showing some progress to union; iii) upon 8 months of PEMF treatment, when the fracture became united.Reproduced and adapted with permission.[151]Copyright 2013, BioMed Central.C) Anteroposterior X-rays of patients with hip prosthesis in a control group (top set of images) or a PEMF-treated group (bottom set of images): i) pre-operative; ii) post-operative; iii) 90-day-follow-up.Images illustrate a higher rate of implant osseointegration in the PEMF-treated group.Reproduced and adapted with permission.[142]Copyright 2009, Wiley.