Hamstring grafts are tenogenic constructs for ACL reconstruction and Pulsed Electromagnetic Fields improve tendon specific markers expression. An in-vitro study.

Hamstring tendons represent one of the commonest autologous graft used during ACL reconstruction. The harvest of the tendon and the time of tendon processing on the operating table, together with the pretensioning maneuvers and the permanence out of the joint during the time of surgery, might impair tendon derived cells (TCs) viability. The aim of the study was: i) to assess the effective viability of the TCs at the end of the surgical procedure; ii) to investigate if TCs viability and the expression of tendon specific markers may be improved through exposure to prolonged pulsed electromagnetic fields (PEMF) similar to that of clinical practice. Remnants of semitendinosus and gracilis tendons (discarded at the end of the ACL reconstruction) were collected from 13 healthy donors. To isolate TCs, the tendon tissue was minced and digested enzymatically with 0.3% type I collagenase in DMEM with continuous agitation for 15 h at 37°C. The isolated nucleated cells were then plated at 5x103 cells/cm2 in a complete medium composed of DMEM, 10% fetal bovine serum, 50 U/ml Penicillin, 50 mg/ml Streptomycin, 2 mM L-glutamine, and supplemented with 5 ng/ml basic fibroblast growth factor (b-FGF). They were maintained at 37 °C in a humidified atmosphere with 5% CO2, changing culture medium every 3 days. When they reached 80-90% of confluence, the cells were detached by incubation with trypsin/EDTA and then cultured at a density of 5x103 cells/cm2. TCs were cultured in complete medium for 7, 14, 21 days (in chamber slides, to optimize the final immunofluorescence analysis). The following cell cultures were set up: i) TCs cultured with differentiation medium + exposure to PEMF 8 h/day; ii) TCs cultured with differentiation medium without exposure to PEMF. The stimulation with PEMF was generated by a pair of electrical coils, connected with the generator of pulsed electromagnetic fields (PEMF generator system IGEA, Carpi, Italy, intensity of magnetic field = 1.5 mT, frequency = 75 Hz). At day 0, day 7, day 14 and day 21 immunofluorescence analysis was performed to evaluate the expression of tendon specific markers (collagen type I, collagen type VI, scleraxis) and proliferative markers (PCNA, beta-catenin). The TCs from the hamstring tendon fragments at the end of the ACL reconstruction were alive and they expressed markers of proliferation and tendon phenotype at the end of the culture period. The TCs in the presence of PEMF 8h/day showed a greater production of collagen type I, collagen type VI and scleraxis than TCs cultured without PEMF (p<0.05). The expression of these markers increased from 7 to 21 days of culture. The expression of proliferative markers in the presence of PEMF stimulus was significantly lower (p<0.05) than that of TCs cultured without PEMF. Hamstring tendons are not simple "tenoconductive" scaffolds but biologic alive tenogenic constructs rich in cells that can sustain tenogenic behavior and tendon matrix synthesis. Prolonged exposure to PEMF improves their phenotype. Thus, from a clinical perspective, the use of PEMF may represent a possible future strategy to positively influence the early phase of graft remodeling and, ultimately, improve the ligamentization process. Following these concepts, further studies might also exploit the anabolic role of PEMF as an adjunctive postoperative strategy in different tendon pathologies.

Biophysical stimulation and the periprosthetic bone: is there a rationale in the use of Pulsed Electromagnetic Fields after a hip or knee implant?

The biophysical stimulation of bone and cartilage, using Pulsed ElectroMagnetic Fields (PEMF), covers many different aspects of bone formation and/or cartilage repair, such as healing of delayed or non-union of fracture, bone necrosis, osteocartilagineous defects. To date there are no specific data on the effects of PEMFs in osteointegration of prosthetic implants but there are some papers that denote clinical advantages, in terms of early recovery, in patients treated with these procedures. Considering these clinical applications, PEMF stimulation around hip or knee joint implants could be useful to reduce the bone oedema, pain and to reduce excessive bone reabsorption around the femoral stems.

CAPACITIVE COUPLING ELECTRIC FIELDS IN THE TREATMENT OF VERTEBRAL COMPRESSION FRACTURES.

Positive effects of Capacitive Coupling Electric Field (CCEF) stimulation are described for several orthopedic indications such as the healing of recent fractures, non-unions and spinal fusion, due to the capacity to involve the up-regulation of osteopromotive factors. In vitro studies on MC3T3-E1 bone cells showed that CCEF acts opening the plasma membrane voltage gated calcium channels, thus increasing the cytosolic calcium concentration and the phospholipase A2 (PLA2) activity. Cytosolic calcium activates the calmodulin pathway, thus resulting in an up-regulated expression of osteogenic genes, such as transforming growth factor-ß superfamily genes (TGF-ß1, -ß2 -ß3, bone morphogenetic protein-2 and -4), fibroblast growth factor (FGF)-2, osteocalcin (BGP) and alkaline phosphatase (ALP). PLA2 acts increasing the synthesis of Prostaglandin E2 (PGE2), which promotes osteogenesis by raising the cellular L-ascorbic acid uptake through the membrane carrier sodium vitamin C transporter-2 (SVCT-2). In vivo, Brighton et al. in a castration-induced osteoporosis animal model, demonstrated that CCEF was able to restore bone mass/unit volume in the rat vertebral body. To investigate the role of CCEF stimulation in vertebral bone marrow edema (VBME) its percentage was assessed in 24 patients with 25 acute vertebral compression fractures (VCFs) conservatively treated with CCEF (group A) or without CCEF (group B) using serial MR imaging follow-up at 0, 30, 60, 90 days. Pain and quality of life were assessed by visual analog scale (VAS) and Oswestry Low Back Disability Index (ODI) in the same periods. At 90 day follow-up the complete resolution of VBME was found only in group A (p=0.0001). A significant improvement of VAS (p=0.007) and ODI (p=0.002) was also observed in group A. This preliminary observational study shows that patients treated with CCEF stimulation present an improvement of clinical symptoms with faster fracture healing and a complete VBME resolution.

In vitro functional response of human tendon cells to different dosages of low-frequency pulsed electromagnetic field.

PURPOSE: Chronic tendinopathy is a degenerative process causing pain and disability. Current treatments include biophysical therapies, such as pulsed electromagnetic fields (PEMF). The aim of this study was to compare, for the first time, the functional in vitro response of human tendon cells to different dosages of PEMF, varying in field intensity and duration and number of exposures. METHODS: Tendon cells, isolated from human semitendinosus and gracilis tendons (hTCs; n = 6), were exposed to different PEMF treatments (1.5 or 3 mT for 8 or 12 h, single or repeated treatments). Scleraxis (SCX), COL1A1, COL3A1 and vascular endothelial growth factor-A (VEGF-A) expression and cytokine production were assessed. RESULTS: None of the different dosages provoked apoptotic events. Proliferation of hTCs was enhanced by all treatments, whereas only 3 mT-PEMF treatment increased cell viability. However, the single 1.5 mT-PEMF treatment elicited the highest up-regulation of SCX, VEGF-A and COL1A1 expression, and it significantly reduced COL3A1 expression with respect to untreated cells. The treated hTCs showed a significantly higher release of IL-1ß, IL-6, IL-10 and TGF-ß. Interestingly, the repeated 1.5 mT-PEMF significantly further increased IL-10 production. CONCLUSIONS: 1.5 mT-PEMF treatment was able to give the best results in in vitro healthy human tendon cell culture. Although the clinical relevance is not direct, this investigation should be considered an attempt to clarify the effect of different PEMF protocols on tendon cells, in particular focusing on the potential applicability of this cell source for regenerative medicine purpose, both in surgical and in conservative treatment for tendon disorders.

Does exposure to extremely low frequency magnetic fields produce functional changes in human brain?

Behavioral and neurophysiological changes have been reported after exposure to extremely low frequency magnetic fields (ELF-MF) both in animals and in humans. The physiological bases of these effects are still poorly understood. In vitro studies analyzed the effect of ELF-MF applied in pulsed mode (PEMFs) on neuronal cultures showing an increase in excitatory neurotransmission. Using transcranial brain stimulation, we studied noninvasively the effect of PEMFs on several measures of cortical excitability in 22 healthy volunteers, in 14 of the subjects we also evaluated the effects of sham field exposure. After 45 min of PEMF exposure, intracortical facilitation produced by paired pulse brain stimulation was significantly enhanced with an increase of about 20%, while other parameters of cortical excitability remained unchanged. Sham field exposure produced no effects. The increase in paired-pulse facilitation, a physiological parameter related to cortical glutamatergic activity, suggests that PEMFs exposure may produce an enhancement in cortical excitatory neurotransmission. This study suggests that PEMFs may produce functional changes in human brain.

Albert Wojciech Adamkiewicz (1850-1921): unsung hero behind the eponymic artery.

The artery of Adamkiewicz is an important radiculomedullary artery supplying the spinal cord, especially the lumbar enlargement. Anatomical knowledge of this artery is important for avoiding serious neurological complications during surgery performed in this region--for neurosurgeons and interventional radiologists treating intramedullary tumors and spinal arteriovenous malformations, traumatologists performing spinal fusions, thoracic surgeons treating aortic aneurysms, and urologists and pediatric surgeons conducting retroperitoneal dissections. However, the biography of the talented Polish pathologist Albert Adamkiewicz, after whom the landmark artery is named, has not been described adequately in the existing neurosurgical literature. The authors bring to light the historical perspective of the eponymic artery and provide a recapitulation of other significant contributions made by Adamkiewicz, mostly involving the nervous system. His research papers on the histology of neuronal tissues and neurodegenerative diseases had high scientific merit, but the discovery of the anticancer antitoxin "cancroin" and his postulation of a cancer-causing parasite he named "Coccidium sarcolytus" met with harsh criticism and eventually led to his ill fame. The biography is supplemented with a brief overview of the important surgical implications of the artery of Adamkiewicz.

The legendary contributions of Thomas Willis (1621-1675): the arterial circle and beyond.

Thomas Willis established neurology as a distinct discipline and made significant original contributions to many related fields including anatomy, pathology, cardiology, endocrinology, and gastroenterology. He is most remembered for his work in elucidating the function and anatomy of the circle of Willis. Willis' accomplishments and research methods can be credited in large part to his unconventional medical education which did not include traditional teachings, but rather emphasized learning through clinical practice. Although Willis was not the first to describe the arterial circle, he was the first to describe its function and provide a complete, undisputed illustration through his own innovative use of dye studies. The Willis classification of cranial nerves was still in use over 100 years after its original description. He has also described several disease entities and named many brain structures. Willis' accomplishments in comparative anatomy and understanding the pathophysiology of various diseases through original multidisciplinary experimental work in a clinical setting reveal that he was a true pioneer in translational research.

Pulsed electromagnetic fields reduce knee osteoarthritic lesion progression in the aged Dunkin Hartley guinea pig.

An experimental in vivo study was performed to test if the effect of Pulsed Electromagnetic Fields (PEMFs) on chondrocyte metabolism and adenosine A2a agonist activity could have a chondroprotective effect on the knee of Dunkin Hartley guinea-pigs of 12 months with spontaneously developed osteoarthritis (OA). After a pilot study, 10 animals were randomly divided into two groups: PEMF-treated group (6 h/day for 3 months) and Sham-treated group. Microradiography and histomorphometry were performed on the entire articular surface of knee joints used in evaluating chondropathy severity, cartilage thickness (CT), cartilage surface Fibrillation Index (FI), subchondral bone plate thickness (SBT) and histomorphometric characteristics of trabecular epiphyseal bone. The PEMF-treated animals showed a significant reduction of chondropathy progression in all knee examined areas (p<0.05). CT was significantly higher (p<0.001) in the medial tibia plateaus of the PEMF-treated group when compared to the Sham-treated group. The highest value of FI was observed in the medial tibia plateau of the Sham-treated group (p<0.05). Significant lower values were observed in SBT of PEMF-treated group in comparison to Sham-treated group in all knee examined areas (p<0.05). The present study results show that PEMFs preserve the morphology of articular cartilage and slower the progression of OA lesions in the knee of aged osteoarthritic guinea pigs. The chondroprotective effect of PEMFs was demonstrated not only in the medial tibial plateau but also on the entire articular surface of the knee.

Effects of pulsed electromagnetic fields on articular hyaline cartilage: review of experimental and clinical studies.

Osteoarthritis (OA) is the most common disorder of the musculoskeletal system and is a consequence of mechanical and biological events that destabilize tissue homeostasis in articular joints. Controlling chondrocyte death and apoptosis, function, response to anabolic and catabolic stimuli, matrix synthesis or degradation and inflammation is the most important target of potential chondroprotective treatment, aimed to retard or stabilize the progression of OA. Although many drugs or substances have been recently introduced for the treatment of OA, the majority of them relieve pain and increase function, but do not modify the complex pathological processes that occur in these tissues. Pulsed electromagnetic fields (PEMFs) have a number of well-documented physiological effects on cells and tissues including the upregulation of gene expression of members of the transforming growth factor beta super family, the increase in glycosaminoglycan levels, and an anti-inflammatory action. Therefore, there is a strong rationale supporting the in vivo use of biophysical stimulation with PEMFs for the treatment of OA. In the present paper some recent experimental in vitro and in vivo data on the effect of PEMFs on articular cartilage were reviewed. These data strongly support the clinical use of PEMFs in OA patients.

Bone morphogenetic proteins: basic concepts.

The cellular and molecular events governing bone formation in the embryo, healing of a fractured bone, and induced bone fusion follow a similar pattern. Discovery, purification, and recombinant synthesis of bone morphogenetic proteins (BMPs) constitute a major milestone in the understanding of bone physiology. In this review the author discusses the mechanism of action, clinical applications, dosage, and optimum carriers for BMPs. The roles played by other growth factors are also discussed.

Reduction of spondylolisthesis.

In this article the authors discuss the syndrome of isthmic spondylolisthesis occurring at the lumbosacral junction in adults, providing a description of the clinical syndrome, altered biomechanics, and imaging characteristics. The authors pose arguments in favor of reduction and instrument-assisted fusion. Their surgical technique is detailed. They describe transsacral interbody fusion in which fibular allograft and in situ fixation are used, which they consider the second-best alternative technique when attempts at reduction fail.

Black disc disease: a commentary.

Internal disc disruption associated with axial back pain but not radicular pain is a disease entity that was recognized about two decades ago as a disorder that could potentially be treated by spinal fusion. In this article the authors describe the clinical syndrome, magnetic resonance imaging and discography findings of pathophysiological pain generation, and the available surgical options. Based on the current understanding of this disease entity, the optimum surgical procedure entails radical discectomy, anterior column support, adequate amounts of auto- or allograft bone, bone extenders and enhancers, and rigid stabilization of the motion segment.

Bone healing and spinal fusion.

Bone is a tissue that constantly undergoes deposition, resorption of stromal matrix, and remodeling. These processes may be altered by a variety of chemical, mechanical, cellular, and pathological mechanisms. Understanding the physiology of bone healing and the mechanisms affecting this process is important not only when evaluating normal skeletal development but also when initiating fracture repair. Because the ultimate success of spinal fusions involves creation of an osseous union, we focus this review on the anatomy and physiology of bone under physiological conditions, normal bone healing and mechanisms that alter it, and available adjuvant therapies that may enhance healing potential in a clinical setting.

Current trends in the enhancement of biomaterial osteointegration: biophysical stimulation.

To enhance bone implant osteointegration, many strategies for improving biomaterial properties have been developed which include optimization of implant material, implant design, surface morphology and osteogenetic coatings. Other methods that have been attempted to enhance endogenous bone healing around biomaterials are different forms of biophysical stimulations such as pulsed electromagnetic fields (PEMFs) and low intensity pulsed ultrasounds (LIPUS), which were initially developed to accelerate fracture healing. To aid in the use of adjuvant biophysical therapies in the management of bone-implant osteointegration, the present authors reviewed experimental and clinical studies published in the literature over the last 20 years on the combined use of biomaterials and PEMFs or LIPUS, and summarized the methodology, and the possible mechanism of action and effectiveness of the different biophysical stimulations for the enhancement of bone healing processes around bone implanted biomaterials.

In vivo effect of two different pulsed electromagnetic field frequencies on osteoarthritis.

Osteoarthritis (OA) is a joint pathology characterized by fibrillation, reduced cartilage thickness and subchondral bone sclerosis. There is evidence that pulsed electromagnetic fields (PEMFs) counteract OA progression, but the effect of two different PEMF frequencies has not yet been shown. The aim of this study was to test the effectiveness of PEMFs at two different frequencies (37 and 75 Hz) in a late OA stage in 21-month-old Guinea pigs. After 3 months of 6 h/day PEMF stimulation, histological and histomorphometric analyses of the knees were performed. At both frequencies, PEMFs significantly reduced histological cartilage score, fibrillation index (FI), subchondral bone thickness (SBT) and trabecular number (Tb.N) and increased trabecular thickness (Tb.Th) and separation (Tb.Sp) in comparison to the not treated SHAM group. However, PEMFs at 75 Hz produced significantly more beneficial effects on the histological score and FI than 37 Hz PEMFs. At 75 Hz, PEMFs counteracted cartilage thinning as demonstrated by a significantly higher cartilage thickness values than either those of the SHAM or 37 Hz PEMF-treated groups. Although in severe OA both PEMF frequencies were able to limit its progression, 75 Hz PEMF stimulation achieved the better results.

Low frequency pulsed electromagnetic field affects proliferation, tissue-specific gene expression, and cytokines release of human tendon cells.

Low frequency pulsed electromagnetic field (PEMF) has proven to be effective in the modulation of bone and cartilage tissue functional responsiveness, but its effect on tendon tissue and tendon cells (TCs) is still underinvestigated. PEMF treatment (1.5 mT, 75 Hz) was assessed on primary TCs, harvested from semitendinosus and gracilis tendons of eight patients, under different experimental conditions (4, 8, 12 h). Quantitative PCR analyses were conducted to identify the possible effect of PEMF on tendon-specific gene transcription (scleraxis, SCX and type I collagen, COL1A1); the release of pro- and anti-inflammatory cytokines and of vascular endothelial growth factor (VEGF) was also assessed. Our findings show that PEMF exposure is not cytotoxic and is able to stimulate TCs' proliferation. The increase of SCX and COL1A1 in PEMF-treated cells was positively correlated to the treatment length. The release of anti-inflammatory cytokines in TCs treated with PEMF for 8 and 12 h was significantly higher in comparison with untreated cells, while the production of pro-inflammatory cytokines was not affected. A dramatically higher increase of VEGF-A mRNA transcription and of its related protein was observed after PEMF exposure. Our data demonstrated that PEMF positively influence, in a dose-dependent manner, the proliferation, tendon-specific marker expression, and release of anti-inflammatory cytokines and angiogenic factor in a healthy human TCs culture model.

Conservative treatment of spontaneous osteonecrosis of the knee in the early stage: pulsed electromagnetic fields therapy.

HYPOTHESIS: pulsed electromagnetic fields treatment might improve symptoms in the early stage of spontaneous osteonecrosis of the knee. METHODS: Twenty-eight patients (19M/9F, age 49.8±16.4 years) suffering from symptomatic (pain) Koshino stage I spontaneous osteonecrosis of the knee, confirmed by magnetic resonance imaging (MRI) were treated with local pulsed electromagnetic fields therapy (6 h daily for 90 days). Clinical evaluation: baseline, 6- and 24-month follow-up by VAS for pain, knee society score (KSS), Tegner and EQ-5D scales. MRI evaluation: baseline and 6-month follow-up, measuring bone marrow lesion's areas and grading these lesions by WORMS score. Failures: patients undergoing knee arthroplasty. RESULTS: Pain significantly reduced at 6 months (from 73.2±20.7 to 29.6±21.3, p<0.0001), which remained almost unchanged at final follow-up (27.0±25.1). KSS significantly increased in first 6 months (from 34.0±13.3 to 76.1±15.9, p<0.0001) and was slightly reduced at final follow-up (72.5±13.5, p=0.0044). Tegner median level increased from baseline to 6-month follow-up (1(1-1) and 3(3-4), respectively, p<0.0001) and remained stable. EQ-5D improved significantly throughout the 24 months (0.32±0.33, baseline; 0.74±0.23, 6-month follow-up (p<0.0001); 0.86±0.15, 24-month follow-up (p=0.0071)). MRI evaluation: significant reduction of total WORMS mean score (p<0.0001) and mean femoral bone marrow lesion's area (p<0.05). This area reduction was present in 85% and was correlated to WORMS grading both for femur, tibia and total joint (p<0.05). Four failures (14.3%) at 24-month follow-up. CONCLUSIONS: Pulsed electromagnetic fields stimulation significantly reduced knee pain and necrosis area in Koshino stage I spontaneous osteonecrosis of the knee already in the first 6 months, preserving 86% of knees from prosthetic surgery at 24-month follow-up. No correlation was found between MRI and clinical scores. LEVEL OF EVIDENCE: Level IV; case series.