Due to the thin cartilage layer in rats the isolation of adult rat articular chondrocytes derived from the knee joint is challenging and only low cell numbers can be harvested. Since it is well known that human and also rat derived chondrocytes lose their specific differentiated phenotype during cell expansion and further culturing in monolayer a process called dedifferentiation [
13‐
15], the expression of cartilage markers was evaluated in the present study. Cartilage-specific markers such as type II collagen and sox9 could be demonstrated indicating that the primary rat chondrocytes introduced in the experiments exhibited still differentiated functions. Previous studies indicated that chondrocytes can maintain their differentiated phenotype for up to four passages in monolayer culture [
13,
14]. Cultured rat chondrocytes easily took dPGS up as an important precondition for using dPGS in the joint. In another study were viable human cartilage chips were tested for penetration by dPGS we found that dPGS could also penetrate the cartilage extracellular matrix to some degree (50 μm distance). The results of the FACS analysis suggest that dPGS is not only taken up by the cells over the course of the experiment but might also be actively segregated again back in the culture supernatant. However, this assumption requires further experimental support. An instability OA model of the rat knee was selected in the present study to test the effect of dPGS on OA progression. This OA model has already been used by various research groups before [
16‐
18]. Since OA needs several weeks for manifestation in this model, the daily dPGS treatment was started after 6 weeks and lasted for two weeks. The treatment had no effect on overall behavior, body weight and gait of the rats indicating no clinical signs of OA. To exclude toxic effects of the nanoparticular compounds during this rather long treatment course we analyzed the histology of organs involved in metabolism and elimination of toxic compounds such as liver, spleen and kidney. The AB staining was used here to detect negatively charged dPGS particles [
19] and therefore, to monitor a potential accumulation of dPGS within these organs. It is known that administered nanoparticular compounds can accumulate in secondary target organs [
20,
21]. Some cross reaction with heparin can usually be found using this staining procedure due to its negative charges and similar chemical composition like dPGS. Therefore, mast cells which contain cytoplasmic heparin inclusions were also stained positively blue with AB and could be detected in the connective tissue of the liver surrounding the Glisson trias vessels in all animals. In addition to these mast cells, the dPGS treated animals but not the controls revealed also dPGS positive Kupffer cells. This observation is supported by the study of Holzhausen, Gröger et al., [
19]. Apart from this observation, the dPGS treatment did not induce any histopathological changes in the investigated organs suggesting that it might not impair organ function at the administered dose and time course. In agreement, some macrophages in the spleen revealed dPGS in the dPGS treated rats, but not in the controls. However, dPGS could not be shown in the kidneys. This observation suggests that the parenchymal cells are able to eliminate the dPGS in response to a daily treatment and a longer treatment with dPGS could be possible. The onset of OA could be proven in all animals included in this study, since there was a highly significant difference in the Mankin Score and also in the Glasson score values between the operated right and unoperated left knee joints. The difference between the right knee joints of the group treated with dPGS
versus the controls was clearly detectable and supported by histopathology but it did not reach the significance level. Therefore, it suggests that the dPGS treatment led to an improvement in the condition of the knee joints of the treated animals. Furthermore, the fact that the animals treated with dPGS also showed a lower Mankin score value than the untreated animals in their left knees, which were not surgically destabilized underlines chondroprotective capabilities of dPGS. The grade of synovitis was significantly higher in operated compared to the unoperated legs of the animals suggesting that OA progression was generally associated with synovitis in this rat model. Nevertheless, the differences between dPGS treated animals and controls were not significant. However, neither clinical signs of OA such as pain, joint swelling, warmness and fluctuation nor macroscopical alterations could be found during joint explantation. The mode of action of dPGS on joint inflammation remains a matter of debate. Studies in other inflammatory models revealed a suppressive effect of dPGS on NF-κB and AP-1 activation. These both transcription factors are involved in pathways leading to the release of inflammatory cytokines [
22,
23] and other mediators or enzymes such as matrix-metalloproteinases [
24]. Apart from its modulatory effect on IL-10 and TNFα [
16], dPGS affects also complement factors e.g. C5a and selectins thereby impairing leukodiapedesis [
5]. Several tissues interact with each other under OA conditions [
25]: apart from articular cartilage, synovial membrane, infrapatellar joint fat pad and subchondral bone cross-talk [
26,
27]. Therefore, the mode of chondroprotective action of dPGS requires further investigations.
This study has some limitations. Since we did not detect clinical features of OA one should consider a more sensitive testing system for gait analysis such as the so called “cat walk” which is an automated quantitative gait analysis system for rodents in the future. Furthermore, a sensitive pain tests could be included. Since dPGS treatment led generally to less severe histopathological OA in the rats, more effective treatment profiles should be selected (earlier start or longer duration of dPGS treatment) in the future.