In vitro effects of hyaluronic acid on human periodontal ligament cells

In the present study, the effect of high molecular weight (HMW) HA_ncl and HA_cl were evaluated on in vitro periodontal ligament cell behavior. It was first found that both HA_ncl and HA_cl did not elucidate PDL cell apoptosis even at high concentrations (Fig. 1). Both compositions of HA were shown to be biocompatible on PDL cells without any noticeable differences between their in vitro conditions (diluted in culture media versus pre-coated onto TCP).

We then sought to investigate the inflammatory response of HA on PDL cells. It has previously been shown that low molecular weight (LMW, 100–500 kDa) HA, but not the native HMW HA molecules (~4,000 kDa), stimulated inflammatory cells [34]. Nakatani et al. reported that the expression of MMP-1 in cultured human PDL cells was enhanced by the treatment with HA-oligo through p38MAPK signaling pathway, suggesting that the degradation of the periodontal tissues under pathologic conditions may involve MMP-1 induction by HA-oligo [35]. On the other hand, the majority of products used in connection with periodontal therapy contain HMW HA [8]. The mechanism of anti-inflammatory effects of HMW HA has been widely investigated previously due to the pronounced impact and desire for treatment modalities in the field of osteoarthritis and periodontitis. HA has been shown to modulate inflammation in articular chondrocytes and synoviocytes, due to the specific inhibition of MMPs [19, 36] and down-regulation of TNF-?, IL-8, and inducible nitric oxide synthase [37]. In the present study, HMW HA and HA_cl did not affect the mRNA expressions of inflammatory cytokines, including MMP-2 and IL-1 at 1 day post seeding in healthy PDL cells (Fig. 2). The present study was assessed only in utilizing healthy human PDL cells, whereas in the presence of inflammation, HMW HA within these tissues is reportedly broken down to LMW HA by reactive oxygen species or by bacterial hyaluronidase [8, 38–40]. Therefore, these combined results suggest that although HA demonstrated the little influence on the inflammation in the current study utilizing healthy PDL cells, a more pronounced effect could be observed in diseased tissues due to gingival tissue inflammation in periodontitis.

Thereafter, the effects of HA demonstrated an increase in PDL cell proliferation (Fig. 3). During the granulation phase of periodontal tissue repair, HA has been shown to be a key protein highly expressed in various tissues responsible for promoting cell proliferation, migration, and granulation tissue organization [19]. In non-mineralized tissues, HA is transiently elevated during the formation of tissue repair and helps with the re-establishment of the epithelium [38]. In our study, both 1:100 and 1:10 dilution of HA_ncl, as well as HA_cl, promoted cell proliferation at 3 and 5 days post PDL cell seeding (Fig. 3). It was previously shown by Takeda et al. in the same manner that HMW HA enhanced cell adhesion and proliferation of human periodontal ligament cells [9]. In our study, both diluted HA compositions and pre-coated HA promoted PDL cell proliferation, which suggests a positive effect for periodontal tissue regeneration.

The effect of HMW HA on cell differentiation has however had controversial findings. To date, most studies reported that cell differentiation was increased by low-MW HA [16, 41–43]. Huang et al. [41] reported an osteogenic cell behavior of HMW HA in a concentration-dependent manner on rat mesenchymal stem cells. Moreover, it was demonstrated that sulfated HMW HA could enhance the osteogenic differentiation of human mesenchymal stem cells [44]. On the other hand, studies have also shown that cell differentiation was not affected by HMW HA [16, 45–48]. For example, Kaneko et al. demonstrated that HA inhibited BMP-induced osteoblastic differentiation through the CD44 receptor in osteoblasts [45]. Noteworthy, in a pure bone defect model, it was demonstrated that HA-gelatin hydrogels loaded into biphasic calcium phosphate (BCP) ceramics in rabbit femurs promoted the new bone formation and collagen mineralization [46]. HA gel has further been shown to accelerate the healing process in the tooth sockets of rats, stimulating the expression of osteogenic proteins such as bone morphogenetic protein (BMP)-2 and osteopontin (OPN) in vivo [47]. On the other hand, Atilgan et al. reported that the demineralized bone matrix (DBM)?+?tricalcium phosphate?+?HA combination showed more new bone formation without HA [48]. Therefore, there remains great interest to determine what may be causing the reported variability in the literature.

In the present study, the expression of osteogenic markers stimulated by HA_ncl and HA_cl was investigated with and without ODM. Both HA_ncl and HA_cl down-regulated Runx2, COL1a2 and OCN mRNA levels, suggesting that HA treatment of PDL cells inhibits osteogenesis in a regular cell culture medium (Fig. 4). Moreover, it was observed that HA significantly decreased ALP activity at 7 days, COL1 and OCN immunofluorescent staining at 14 days when compared to control samples (Figs. 5a, 6). Interestingly, however, when ODM was added to promote osteoblast differentiation, it was found that both HA_ncl or HA_cl in 1:10 diluted condition demonstrated a 3-fold and 20-fold increase in COL1a2 and ALP mRNA levels respectively in ODM (Fig. 4d, f). Moreover, HA_ncl at a dilution of 1:10 demonstrated a 5-fold significant increase in ALP staining compared to control samples in the presence of ODM (Fig. 5b). Therefore, it may be concluded that if granted the right culture conditions, the HA may be used to promote early osteogenic differentiation. Interestingly, however, was the finding that irrespective of the addition of ODM, both HA compositions consistently down-regulated late osteoblast differentiation as assessed by OCN mRNA expression as well as alizarin red staining (Fig. 7).

For periodontal regeneration, HMW HA has previously been investigated in a dog intrabony defect model and neither significant periodontal tissue nor bone tissue regeneration was observed [9]. Interestingly, in another canine model, Kim et al. reported that HA improved wound healing and bone formation of hemisection performed extraction sockets with communication of the periodontal lesion [49]. The effect of HMW-HA on periodontal regeneration remains somewhat controversial, however, its contradiction might be explained due to the variety of HA molecular weight, modification methods, concentration, existence of inflammation and also cell types. It may be that HA requires an osteoconductive matrix during periodontal regeneration to improve the osteogenic phase of PDL regeneration, however, this hypothesis certainly requires further investigation.