The molecular weight of hyaluronic acid influences metabolic activity and osteogenic differentiation of periodontal ligament cells

Human immortalized periodontal ligament cells (PDL-hTERT) were established as previously described [24]. PDL-hTERT cells were maintained at 5% CO2 and 37°C in Dulbecco's Modified Eagle Medium (DMEM, high glucose, +Glutamine, +Pyruvate) enriched with 10% fetal bone serum, 1% Pen-Strep solution and 2mM L-glutamine (all Sigma Aldrich, Munich, Germany). Medium change was performed twice a week.

A control group of PDL-hTERTs was cultured in DMEM enriched as described above, without any hyaluronan supplements. Another group was constituted of PDL-hTERTs cultured in osteogenic differentiation medium (OS), which was DMEM with osteogenic supplement: 10% FBS, 1% Pen-Strep, 100nM Dexamethasone, 10mM β-glycerol phosphate and 50μM L-ascorbic acid (Sigma Aldrich, Munich, Deutschland).

Hyaluronan (HA) with different molecular weights was purchased from R&D Systems, Minneapolis, MN, USA. The four groups further used for the experiments were as follows: High Molecular Weight Hyaluronan (HMW), Medium Molecular Weight Hyaluronan (MMW), Low Molecular Weight Hyaluronan (LMW), and Ultra Low Molecular Weight Hyaluronan (ULMW). HMW hyaluronan is provided by the company in a molecular mass range of >950kDa. The specific lot used had a mass of 1510kDa. MMW hyaluronan presents a molecular mass range of 75–350kDa. The lot used had a mass of 229kDa. LMW hyaluronan has a molecular mass range of 15–40kDa. The specific lot used had a mass of 37kDa. ULMW hyaluronan is in a molecular mass range of 4–8kDa. The specific lot used had a mass of 4.6kDa. HA was reconstituted as per manufacturer´s indications in phosphate buffered saline and sterilized by filtration with 0.2-μm syringe filters (VWR International, Radonor, USA). The stimulating concentration of 100μg/ml was further used in the experimental groups, and it was determined based on previous studies [25, 26]. PDL-hTERTs between the 27^th and the 29^th passage were used. PDL-hTERT were cultured in 1 ml medium, at 2x 10⁴ cells/well in 12-well plates for 3 weeks.

The cell morphology was examined daily, and photographs were taken using a phase contrast microscope (Axiovert, Carl Zeiss, Jena, Germany). For each studied group, the experiments were performed in triplicates and repeated three times.

Water-soluble tetrazolium-1 (WST-1) assays were performed. For this assay, PDL-hTERTs were seeded in 24-well plates (1x10⁴ cells/well) and cultured for 7, 14, or 21 days, exposed to the above-mentioned HA solutions (HMW, MMW, LMW and ULMW). The groups with medium alone and OS medium were also cultured under the same conditions. Medium change with 0.5 ml per well was performed every 2 days. At each experiment day, WST-1 (Roche Diagnostics GmbH, Mannheim, Germany) was added according to manufacturer’s protocols in a 1:10 final dilution, incubated for 1 hour (5% CO₂ and 37°C). The medium of each well was transferred into two wells of a 96-well plate, with a volume of 100 μl per well. The optical density of the samples was measured through a multi-detection microplate reader (TECAN, Infinite M200, Männedorf, Switzerland). Data analyses were performed with Magellan™ Software (TECAN). Absorbance readings were recorded at 450nm, with a wavelength correction at 650nm. The experiments were performed in triplicates and repeated three times.

Von Kossa staining was used to analyze mineralization in cell cultures in the six study groups.

PDL-hTERT were seeded at 2x 10⁴ cells/well in 12-well plates and cultured for 21 days in the same media as described above. The respective media were replaced twice a week.

The cells were afterwards fixed for 20 min in methanol at -20°C. The standard protocol was used for the staining with silver nitrate (Merck KGaA, Darmstadt, Germany), pyrogallol acid solution (University Pharmacy, Munich, Germany), sodium hydroxide solution (Merck KGaA, Darmstadt, Germany) and May-Grünwald solution (Merck KGaA, Darmstadt, Germany). Afterwards, pictures were taken in standardized conditions for all groups.

PDL-hTERTs in the 28^th passage were plated at a concentration of 2x 10⁴ cells/well in 12-well plates and divided into 6 groups as above: control; +OS; +OS+ HMW, +OS+ MMW, +OS+ LMW and +OS+ ULMW. The cells were stimulated for 21 days and then harvested with QuantiChrom^TM Calcium Assay Kit (Bioassay Systems, Basel, Switzerland) following manufacturer´s protocol. The calcium quantification was done with a microplate reader (TECAN, Infinite M200, Männedorf, Switzerland). The measurements were performed at 612 nm and the calculations of calcium deposition in mg/dl were done establishing a standard curve as per manufacturer´s indications.

Statistical analyses were performed using SPSS software (Version 26.0, SPSS Statistics, IBM, Chicago, IL, USA). Within each group, data were tested for normality with the Kolmogorov-Smirnov and Shapiro-Wilk tests. Homogeneity of variances between groups was tested with Levene test. Since data were not normally distributed, differences between study groups in calcium deposition for each time point were calculated with the Mann-Whitney U test and with Games-Howell test for WST-1. The p-value threshold was set at 0.05. If not otherwise indicated, all data are given as mean ± standard deviation (±SD).

The single cell morphology remained unaltered in all groups, which presented an elongated spindle shaped form (Fig. 1). The cells grew in a monolayer with increased density until day 7. During the second week in culture, they formed a rolled aggregate with oval-shaped, cell-free areas, typical of osteogenic differentiation. This was observed in all groups stimulated with osteogenic medium, as well as in the ones with hyaluronan supplements but, as expected, not in the control. Hyaluronan did obviously not induce any morphological changes in PDL-hTERT cells irrespectively from the particular molecular weight (Fig. 1).

Considering the changes within the single study groups overtime, in all six groups, the cells´ activity increased with time, so that the highest value was measured for all groups on the 21st day. The osteogenic and the control groups increased equally within the observed time intervals and reached comparable values (Fig. 2). Accordingly, there was no significant difference between the two groups (p> 0.05). The maximum increase in cell activity within the observed 21 days was seen in the cells stimulated with LMW and MMW. While the cells stimulated with MMW showed a higher difference in their metabolic activity from day 7 to day 14, this was more the case between day 14 and 21 for the cells stimulated with LMW. Moreover, it can be observed that the groups stimulated with HMW, MMW and ULMW hyaluronan presented at day 14 significantly higher metabolic activity than the control group.

We next assessed the capacity of PDL-hTERT cells stimulated with different molecular weight hyaluronan to differentiate into osteoblasts capable of forming a mineralized extracellular matrix. Mineral deposits by PDL-hTERTs were identified by von Kossa staining technique in all groups after 21 days of stimulation. Photographs of representative cultures are shown in Fig. 3.

The mineralization was also quantified in parallel experiments by calcium deposition assay (Fig. 4). Spectrophotometric quantification confirmed these observations, revealing a significantly increased absorbance at 450 nm and calculated calcium deposition, for the cells stimulated with LMW. The osteogenic parameters investigated were decreased in the group stimulated with HMW, but this difference did not reach significance.