Aims

In the context of tendon degenerative disorders, the need for innovative conservative treatments that can improve the intrinsic healing potential of tendon tissue is progressively increasing. In this study, the role of pulsed electromagnetic fields (PEMFs) in improving the tendon healing process was evaluated in a rat model of collagenase-induced Achilles tendinopathy.

Pulsed Electromagnetic Fields (PEMFs) promote joint tissue anabolic activities, particularly in cartilage and bone. Here we investigated the effect of selected PEMFs (75Hz, 1.5mT, 1.3msec) in a differentiating model of murine myoblasts (C2C12) in vitro. C2C12 were seeded at 5×10³ cells/cm² in 4 well plates and left to adhere for 24h. Subsequently, cells were either maintained in growth medium (GM) or induced towards myogenic differentiation in low-serum conditions, with and without PEMF exposure, for 4 days. Morphological analysis, myotube formation and fusion index (FI) were assessed with fluorescence microscopy techniques. Metabolic activity was determined by MTT; moreover, a multiplex cytokine array (RayBiotech) allowed cell supernatant molecule quantification. Cells exposed to PEMFs in GM acquired a distinctive elongated morphology, with increased bi-nuclear figures (3.2-fold FI increase over PEMF-unexposed cells) and displayed a significantly higher metabolic activity (+31%, p<0.05 over PEMF-unexposed cells). PEMF exposure increased metabolic activity also under myogenic differentiation (+15% over PEMF-unexposed differentiating cells, p<0.05), with the formation of long, thick polynuclear myotubes, suggesting a role of PEMFs in enhancing myogenesis (7.7-fold FI increase over PEMF-unexposed cells). 4-day culture supernatants revealed the presence of several myokines (KC/CXCL1, LIX, MCP-1, TIMP-1). Preliminary analysis showed a 1.16-fold increase (n=2) of LIX and, notably, a 1.91-fold increase (n=2) of TNF-RI, in cell supernatants of PEMF-exposed over PEMF-unexposed cells. Collectively, these results suggest that PEMF may successfully be applied in models of muscle cell trauma to optimise muscle fibre repair, by fine-tuning the release of myokines, promoting myoblast proliferation and myotube formation.

Several studies explored the biological effects of low frequency low energy pulsed electromagnetic fields (PEMFs, Igea Biophysics Laboratory, Carpi, Italy) on human body reporting different functional changes. In the orthopedic field, PEMFs have been shown to be effective in enhancing endogenous bone and osteochondral repair, incrementing bone mineral density, accelerating the process of osteogenic differentiation and limiting cartilage damage. Much research activity has focused on the mechanisms of interaction between PEMFs and membrane receptors such as adenosine receptors (ARs). In particular, PEMF exposure mediates a significant upregulation of A_2A and A₃ARs expressed in various cells or tissues involving a reduction of most of the pro-inflammatory cytokines. In tissue engineering for cartilage repair a double role for PEMFs could be hypothesized: in vitro by stimulating cell proliferation, colonization of the scaffold and production of tissue matrix; in vivo after surgical implantation of the construct by favoring the anabolic activities of the implanted cells and surrounding tissues and protecting the construct from the catabolic effects of inflammation. Of particular interest is the observation that PEMFs, through the increase of ARs, enhance the working efficiency of the endogenous modulator adenosine, producing a more physiological effect than the use of exogenous drugs. This observation suggests the hypothesis that PEMFs could be considered a non-invasive treatment with a low impact on daily life. In conclusion, PEMFs represent an important approach in the pharmacological field providing excellent therapeutic results in various inflammatory diseases and in particular in the functional recovery of the damaged joint tissues.

Tendon-related pathologies such as tendinopathy represent a relevant clinical and socioeconomic issue. The most innovative and conservative therapeutic approaches are meant to stimulate the intrinsic healing capability of the tissue. In this study, the use of pulsed electromagnetic fields (PEMFs) was investigated in a rat model of Achilles tendinopathy as a potential therapy. Achilles tendinopathy was chemically induced in eighty-six Sprague Dawley rats by injecting collagenase Type I within the tendon fibers. Fifty-six of them were stimulated with PEMFs (8 hours/day, 1.5 ± 0.2 mT; 75 Hz), divided in different experimental groups basing on the starting-time of PEMFs exposure (after 0, 7, 15 after Collagenase injection) and its duration (7, 15 or 30 days). Thirty animals were left unstimulated (CTRL group). According to the different time points, explanted tendons were evaluated through histological and immunohistochemical analyses in term of matrix deposition, fiber re-organization, neovascularization and inflammatory reaction. The most effective PEMF stimulation was demonstrated in the 15 days of treatment. However, when PEMF were applied immediately after the collagenase injection, no significant therapeutic results were found. On the contrary, when PEMF were applied after 7 and 15 days from the collagenase injection, they promoted the deposition of extracellular matrix and tendon fiber re-organization, reducing both the inflammatory reaction and vascularization, with significant differences compared to the CTRL group (p<0.05). Therefore, these results suggest an effective activity of PEMFs stimulation that provides a satisfying restoration of the damaged tissue, although the most performing protocol of application still needs to be identified.

Tendinopathies represent the 45% of the musculoskeletal lesions and they are a big burden in clinics. Indeed, despite the relevant social impact, both the pathogenesis and the development of the tendinopathy are still under-investigated, thus limiting the therapeutic advancement in this field. Indeed, current treatment for tendinopathy are mainly symptomatic, and they present a high rate of pathology re-occurrence. In this contest, the development of an efficient in vivo model of acute tendinopathy, focused on the choice of the most appropriate species and strategy to induce the disease, would allow a better understanding of the pathology progression throughout its phases.

Then, the purpose of this study was to evaluate the dose-dependent and time-related tissue-level changes occurring in a collagenase-induced tendinopathy in rat Achilles tendons, in order to establish a standardized model for future pre-clinical studies.

40 Sprague Dawley rats were randomly divided into two groups, treated by injection of collagenase type I within the Achilles tendon at 1 mg/mL (low dose, LD) or 3 mg/mL (high dose, HD). Tendon explants were histologically evaluated at 3, 7, 15, 30 and 45 days by H&E staining.

Our results showed that both the collagenase doses induced a disorganization of collagen fibers and increased the number of rounded resident cells. In particular, the high dose treatment determined a greater fatty degeneration and neovascularization with respect to the lower dose. These changes are time-dependent, thus resembling the tendinopathy development in humans. Indeed, the acute phase occurred from day 3 to day 15, while from day 15 to 45 it progressed towards the proliferative phase, displaying a degenerative appearance associated with a precocious remodeling process.

The model represents a good balance between feasibility, in terms of reproducibility and costs, and similarity with the human disease. Moreover, the present model contributes to improve the knowledge about tendinopathy development, and then it could be useful to design further pre-clinical studies, in particular in order to test innovative treatments for tendinopathy.

Summary

In an in vitro tendon cell model, the tendon-specific gene expression up-regulation induced by PEMF negatively correlates with field intensity; moreover repeated lower-intensity PEMF treatments (1.5 mT) provokes a higher release of anti-inflammatory cytokines respect to the single treatment.

A multicenter retrospective analysis of patients treated for tibial fracture was conducted to develop a score that correlates with fracture healing time and, ultimately, to identify the risk gradient of delayed healing.

The clinical records of 93 patients treated for tibial fracture at three orthopaedic centers were evaluated. Patients were considered healed when full weight bearing was allowed and no further controls were scheduled. For the purpose of our analysis, we separated patients healed within or after 180 days.

Patient's risk factors known to be associated to delay healing, as well as fracture morphology and orthopaedic treatment were recorded in an electronic Case Report Form (e-CRF). Information available in the literature was used to weight the relative risk (RR) associated to each risk factor; values were combined to calculate a score to be correlated to the fracture healing time: L-ARRCO (Literature-Algoritmo Rischio Ritardo Consolidazione Ossea). Among all information collected in e-CRFs, we identified other risk factors, associated to delayed healing, that were used to calculate a new score: ARRCO. Univariate logistic analysis was used to determine a correlation between the score and healing time. Analysis by receiver operating characteristic (ROC) and calculation of the area under the curve (AUC) were used for sensitivity and specificity.

Complete information was available for 53 patients. The mean value of the L-ARRCO score among patients healed within 180 days was 5.78 ± 1.59 and 7.05 ± 2.46 among those healed afterwards, p=0.044. The mean value of the ARRCO score of patients healed within 180 days was 5.92 ± 1.78 and 9.03 ± 2.79 among those healed afterwards, p<0.0001. The ROC curve shows an AUC of 0.62±0.09 for L-ARRCO and an AUC of 0.82±0.07 for ARRCO, (p<0.0001).

We have shown that the ARRCO score value is significantly correlated to fracture healing time. The score may be used to identify fractures at risk of delayed healing, thus allowing surgeon's early intervention to stimulate osteogenesis.

The employment of biophysical therapy to accelerate the healing of tissues is by now a well-established practice in many orthopaedic situations, indicated mainly for osteogenesis and chondrogenesis. Assessments of the effects of biophysical stimuli on joint cartilage (CRES, Cartilage Repair & Elecromagnetic Stimulation) performed with pre-clinical studies and clinical studies (in operations to reconstruct LCA and microfractures) have shown how biophysical stimulation controls the microambience, and have suuplied the rationale for passing to an evaluation of the effects also in the case of joint replacement.

We launched a randomized prospective clinical study of 30 patients aged between 60 and 85 years, afflicted with gonarthrosis and undergoing operation for prosthesis. The randomization involved subdividing them into two homogeneous groups: the first with biophysical treatment with I-ONE therapy (Igea-Clinical Biophysics) (experimental group); the second not undergoing the biophysical treatment (control group). In the experimenal group, the I-ONE therapy was commenced at 3–7 days from the operation, administered for 4 hours per day and maintained for 60 days consecutively. The clinical evaluations were performed by compiling functional reports (swelling of the knee, Knee Score, SF-36 and VAS) in the pre-operative period and postoperatively at 1, 2, 6 and 12 months. The data processing was subjected to statistical evaluation by an independent observer using Student’s two-tail t test and the Generalized Linear Mixed Effects Model.

The preliminary results showed that at the baseline there are no differences between the groups either for the KNEE score, nor the VAS, or the SF-36. Already after 1 month the differences between the groups are statistically significant (p< 0.05 for KNEE score, p< 0.001 for swelling, p< 0.0001 for VAS and SF-36). At 2 months the differences between the groups are highly significant (p< 0.0001). The study entails a long-term evaluation with monitoring of the patients at one year from operation.

The results of this study supply the basis for clinical employment of biophysical treatment with I-ONE immediately following joint surgery, enabling inflammation to be controlled and increasing anabolic activity and protecting its microambience.