Many efforts have been undertaken to improve matrix-based tissue engineering of hyaline cartilage, including manipulation of physical parameters like oxygen supply, improvements of matrix systems or stimulation with growth factors. Until now, only few studies have focused on the effects of stimulating articular chondrocytes with current treatment. Current stimulation of soft tissue for pain relief is a well established method since the 1980 [11–14]. Normally, it is combined with conventional methods like massage, heat or physical manipulation. While previous studies stimulated with current ranging from 20 μA to 600 μA [11, 15, 16], we applied a current of 3 Milliampere. This corresponds to a protocol which was successfully applied in pain treatment and post-operative rehabilitation by the manufacturer of the Algonix-device. Therefore, this slightly enhanced current was used in our study. Frank et al artificially damaged bovine articular cartilage by trypsin digestion and applied impedance analysis to the treatment site. They found that tissue impedance directly correlated with the degree of degradation .
To at least partly avoid cellular dedifferentiation, we chose to carry out the examinations on human chondrocytes embedded in a collagen type-I gel. Collagen gels are used as a matrix system for the cultivation of chondrocytes for many years and are of growing importance for tissue engineering of hyaline cartilage [18, 19]. Additionally, osteochondral explants were subject for millicurrent stimulation.
Only little is known of the effects of current treatment of chondrocytes on the biochemical level. We found that cell metabolism of human chondrocytes is affected by current treatment in the milliampere range. We applied a current density of 10.6 mA/cm2, which should allow cellular stimulation. Investigating important genes of cartilage turnover, with respect to chondrocytes cultivated in a 3 D matrix in-vitro, we found a more universal up-regulation rather than a specific one. Aggrecan and collagen type-II gene expression, which is characteristic for hyaline cartilage, stayed elevated, while expression of the collagen type-I gene, indicating dedifferentiated chondrocytes, was slowly decreasing during cultivation. Immunohistological examination of protein production did not show any obvious enhancements with regard to col-II, col-I and proteoglycan production. This may be due to the relatively short cultivation period of 3 weeks. In contrast, when we treated osteochondral explants, we observed a specific pattern of gene expression, with some genes being stimulated (col-I, aggrecan), some genes being repressed (IL-6, MMP-13, TNF-α) and some left unaffected (col-II, IL-1β). One may speculate that the mechanisms of signal transduction are different with regard to 3 D gel culture and osteochondral explant culture, eventually because of missing ECM components in the collagen gel. On the other hand, we recently demonstrated a changed expression profile of a subset of integrins involved in signal transduction when we investigated chondrocytes with different states of differentiation . Therefore, a different expression profile of signal-transducers of chondrocytes cultured in 3 D or explant culture may be involved.
Todd et al investigated the effects of electrical microcurrents generated by the ACE Stimulator on human dermal fibroblasts in-vitro . They found that cell growth and viability were not influenced. When we were stimulating human articular chondrocytes with millicurrent, proliferation and apoptosis were unaffected, confirming these observations. They also observed an up-regulation of TGF-β1, which is an important regulator of cell-mediated inflammation and tissue regeneration. TGF-β1 secretion level was increased about 20-30%. We addressed the question of an inflammatory response to millicurrent therapy by monitoring the gene expression of Il-1β, IL-6 and TNFα.
Interleukin 1β plays a central role in the pathophysiology of cartilage damage and degradation in arthritis. It promotes the resolution system of cartilage matrix turnover through an increase in inflammatory cytokine and matrix metalloproteinase production by chondrocytes [22, 23]. We found Il-1β gene expression to be elevated in chondrocytes cultivated in a 3 D matrix, while osteochondral explants did not show any differences.
When investigating Interleukin-6, Todd et al found no effect of microcurrent stimulation on dermal fibroblasts, while our results revealed a down-regulation following millicurrent treatment of osteochondral explants. IL-6 is not found in normal adult articular chondrocytes. It is strongly induced by the action of Il-1β . In the presence of soluble IL-6 receptor, IL-6 has been shown to activate osteoclasts to induce bone resorption in vitro, suggesting that IL-6 may be involved in osteoporosis .
The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) is not only critical for host defense against microbial agents but also plays an important role in joint inflammation and cartilage destruction in various forms of arthritis. TNF-α is expressed in the synovial lining cells and is present in the synovial fluid from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) . TNF-α is capable of activating the three subgroups of mitogen-activated protein (MAP) kinases in synovial fibroblasts and chondrocytes i.e. extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinase (JNK), and p38 [27, 28]. It has been reported that one or all of these MAP kinases are involved in regulation of IL-1β and MMP gene expression . Regarding our results, TNFα gene expression was down-regulated following millicurrent treatment of osteochondral explant.
Additionally, we found gene expression of matrix metalloproteinase 13 (collagenase 3) to be down-regulated in osteochondral explants following millicurrent treatment. MMP13 levels are increased in cartilage and synovium of patients with arthritis [30–32]. Forsyth et al demonstrated that MMP13 expression is increased in aging human chondrocytes and could contribute to cartilage catabolism in osteoarthritis .
In summary, important genes of matrix degradation and inflammation are down-regulated in human osteochondral explants stimulated by millicurrent treatment in-vitro. Unfortunately, we found a high variation in gene expression between different donors, resulting in rarely significant gene expression data. Although we used cartilage tissue only from those patients with advanced osteoarthritis and undergoing total knee arthroplasty and from macroscopically unaffected areas, there was certainly some variability between the different donors. Therefore, strictly speaking, our results have to be interpreted as a strong indication rather than a proof. They have to be confirmed by further studies with a larger patient number.
In general, millicurrent therapy seems not only to suppress pain, but to directly influence cell metabolism in a selective way. This view is supported by our data. As no specific influence of millicurrent treatment on chondrocytes grown in 3 D gel culture could be demonstrated, the use in cartilage tissue engineering might be limited. The present work is limited to a phenomenological description of the millicurrent influence on chondrocyte metabolism. The underlying mechanisms have to be elucidated in future studies.