The effects of hyaluronic acid on articular chondrocytes

Isolation of chondrocytes.

Bovine articular cartilage was harvested from the metacarpophalangeal joints of six 18-month-old calves. The cartilage was obtained aseptically within six hours of slaughter and the entire cartilage from each joint was removed. Extracted articular cartilage slices were placed immediately into Earl’s Balanced Salt Solution (EBSS) (Sigma, Poole, UK). The tissues were finely diced and washed in EBSS. Cells were released from the tissue by sequential enzyme digestion, firstly in 10 mg/mL pronase E (Merck, Poole, UK) in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technology, Paisley, UK) + 20% Fetal Calf Serum (FCS) (Life Technology) for one hour, followed by overnight digestion in 0.07 mg/mL collagenase type XI (Sigma) at 37°C on a rolamixer. The supernatant containing released chondrocytes was then filtered through a sterile 70 μm nylon sieve (Falcon, Oxford, UK) to remove undigested cartilage, washed twice in DMEM + 20% FCS and finally resuspended in culture medium (DMEM, supplemented with 20% FCS, 1 m HEPES buffer, 1% penicillin/streptomycin, 1% L-glutamine and 50 μg/mL L-ascorbic acid). The number of cells and their viability was determined by trypan blue staining and a haemacytometer.

Cell culture.

The isolated cells were resuspended in 2% low-viscosity alginate hydrogel (Kelco Neutrosweet, Tadworth, UK) containing Rooster comb hyaluronic acid (Sigma) at a density of 5 x 10⁶ cells/mL. The alginate gels were made to four different concentrations of hyaluronic acid: 0.1, 1.0, 2.0, and 3.0 mg/mL. The control specimens contained alginate that was free from hyaluronic acid.

The alginate/chondrocyte/hyaluronic acid suspensions were then gently expressed through a 22-gauge needle attached to a 5 mL syringe into a 100 mm CaCl₂ solution in a sterile Petri dish and each drop polymerised to form an alginate bead containing approximately 100 000 chondrocytes (Syringe droplet technique).¹⁷ Twenty minutes of incubation was allowed for further polymerisation. The newly-formed beads were then washed twice in EBSS to remove excess CaCl₂. Five sets of beads containing hyaluronic acid at the four different concentrations, and a control set of beads containing chondrocytes without hyaluronic acid, were formed and were used for the culture experiments.

Each set of alginate beads was then transferred into sterile 24-well culture plates, with eight randomly chosen beads placed in each well containing 1 mL of culture medium supplemented with hyaluronic acid at the same concentration as that in the respective alginate beads. The hydrogel constructs were cultured for a period of 14 days in the humidified atmosphere of a standard incubator at 37°C and 5% CO₂. The culture medium was replaced by fresh culture medium every three days.

Biochemical analysis.

Six replicates of eight alginate beads were removed randomly from each of the five cultures at 0, 3, 6, 9, and 14 days for analysis. In order to release the cells and matrix from alginate, the chondrocyte/alginate beads were dissolved in a buffer containing 55 mm sodium citrate (BDH, Poole, UK), 150 mm sodium chloride (BDH), 5 mm cysteine hydrochloride (Sigma), 5 mm EDTA (BDH) and 0.56 mg/mL of papain (Sigma), at 60°C for 24 hours.

The total DNA was determined using the Hoechst method described by Rao and Otto¹⁸ and Rago, Mitchen and Wilding.¹⁹ Calf thymus DNA, in a 1:1 solution of saline sodium citrate + 0.4% (v/v) sodium dodecylsulphate and papain digest buffer, was used for preparation of the standard dilution curve ranging from 0 to 20 μg/mL. We added 100 μL aliquots of standards or samples to appropriate wells in a fluorimetric well plate. We also added 100 μL Hoechst 33258 (1 μg/mL) in saline sodium citrate to each well and the plates were read on a fluorimeter (Fluoroskan Ascent, LabSystems, Cheshire, UK) with a 365 nm excitation filter and emission set at 460 nm.

The measurement of total sulphated GAG in the chondrocyte cultures was performed using the 1,9-dimethyl-methylene blue (DMB) dye method, which had been modified for use with alginate systems at a pH of 1.5.^20–22 Chondroitin sulphate A from shark fin (Sigma) was diluted with distilled water to prepare a standard curve (0 to 100 μg/mL). We added 40 μL of the papain-digested samples or GAG standards to 250 μL of DMB solution in wells of an absorbance plate. GAG content was determined using a spectrophotometer (Biorad Plate-Reader, Bio-Rad Laboratories, Hemel Hempstead, UK) at a wavelength of 595 nm.

The measurement of the hydroxyapatite content of the alginate beads was performed using an oxidation assay²³ which was run in the Cobas Bio centrifugal analyser (Roche, Rotkruez, Switzerland) at 40°C. The assay involves the oxidation of the amino acid hydroxyproline in the samples by chloramine T. The chromogens formed (pyrrole and pry-2-carboxylic acid) are coupled with p-dimethylamino-benzaldehyde (DMAB) to produce a chromophore whose absorbance can be measured at 570 nm. Standard curves were used to determine the hydroxyproline concentrations in the sample range 0 to 100 μmol L.

Histological analysis.

Specimens were collected on days 0, 3, 6, 9 and 14 for the five alginate bead sets and were fixed at 4°C in buffer consisting of 4% paraformaldehyde (AGAR Scientific Ltd, Stansted, UK), 100 mm sodium cacodylate (AGAR Scientific Ltd) and 10 mm CaC1₂ (BDH) at pH 7.4 for 24 hours. Samples were then washed overnight at 4°C in 0.1 m sodium cacodylate and 50 mm BaCl₂ (Merck) at pH 7.4. The fixed beads were then dehydrated through a series of ethanol concentrations, cleared with xylene and embedded in paraffin wax. Six sections were cut at a thickness of 5 μm, using a sledge microtome, mounted onto glass slides, rehydrated and deparaffinised. The sections were stained with Harris’s acid alum haematoxylin and eosin in order to demonstrate cell nuclei and extracellular matrix. The sections were mounted in a synthetic mounting medium (DPX − dibutylphthalate + xylene) for microscopic examination (Model, BH2, Olympus, UK).

Statistical analysis.

Graphs were plotted to represent the mean (sem) of six replicate samples. Two-way analysis of variance was carried out to assess the effects of dose, day and the statistical interaction between these two factors. Post hoc Dunnet’s T3 tests were carried out to assess the difference between each dose and the control (zero) dose. Checks on assumptions of normality and constant variance were also performed. Assumptions of constant variance held for GAG and hydroxyproline data, but did not hold for DNA values. Therefore, analysis was repeated on log-transformed DNA values, but gave essentially similar results. Consequently, analyses on untransformed data have been reported.

Biochemistry.

The chondrocytes exposed to 0.1 μg/mL hyaluronic acid showed the greatest levels of DNA proliferation and sulphated GAG and collagen production. Significantly higher levels of DNA and extracellular matrix components were recorded on days 9 and 14 in the cultures exposed to both 0.1 μg/mL and 1.0 μg/mL hyaluronic acid compared with the controls (p = 0.01). As the concentration of hyaluronic acid was increased to 2.0 and 3.0 mg/mL, the level of DNA and extracellular matrix synthesis declined considerably, although it remained higher than in the controls.

DNA analysis.

The DNA content of the control and hyaluronic acid-treated chondrocyte groups is shown in Figure 1 and Table I. An overall increase in DNA content occurred in all study groups during the culture period, which indicated an increase in cell numbers. In all conditions there appeared to be an initial decrease in DNA content during the first three days of culture. At days 9 and 14 there was a statistically significant mean increase in content for cultures exposed to 0.1 and 1.0 mg/mL hyaluronic acid (p = 0.006 for both, compared with zero dose), with the greatest increase occurring with 0.1 mg/mL hyaluronic acid. By day 14 there was a 76% increase in the 0.1 mg/mL hyaluronic acid group compared with a 49% increase in the control group. At an hyaluronic acid concentration of 2.0 mg/mL the increase was more modest (p = 0.027) and with a concentration of 3.0 mg/mL the increase in DNA content was not significant.

Table I.

Mean DNA content (μg/mL) of cell-seeded alginate bead constructs cultured in medium containing varying concentrations of hyaluronic acid for up to 14 days

		Hyaluronic acid concentration
Day	Control	0.1 mg/ml	1.0 mg/ml	2.0 mg/ml	3.0 mg/ml
0	2.13	2.20	2.13	2.12	2.14
3	2.04	2.14	2.11	2.09	2.06
6	2.51	2.66	2.62	2.52	2.51
9	3.07	3.96	3.45	3.18	3.11
14	3.19	3.89	3.72	3.38	3.22

Fig. 1

Bar chart showing DNA content of cell-seeded alginate bead constructs cultured in medium containing varying concentrations of hyaluronic acid (HA) for up to 14 days (sem bars) (n = 6). * statistically significant difference from the control mean with p < 0.05.

Sulphated GAG analysis.

The GAG content of the control and hyaluronic acid-treated chondrocyte groups is shown in Figure 2 and Table II. Again, a significant interaction was observed between day and dose (p < 0.001); the effect of all doses, particularly at 0.1 mg/mL and 1.0 mg/mL, was seen more clearly for days 9 and 14 when compared with the earlier timepoints. There was an overall increase in GAG content within the alginate beads in all five cultures. The increase in GAG content was greater in all chondrocyte cultures exposed to hyaluronic acid compared with the control group and there was a dose-dependent response to hyaluronic acid. A steady increase in GAG content in all culture groups occurred up to day six, the content between the groups being very similar. At days 9 and 14 the mean GAG content in chondrocytes cultured in 0.1 mg/mL and 1.0 mg/mL hyaluronic acid was significantly greater (p = 0.005) than the control group. The largest increase (307%), occurred in the 0.1 mg/mL hyaluronic acid group. At hyaluronic acid concentrations of 2.0 mg/mL and 3.0 mg/mL no significant difference in GAG content was found when compared with the control groups.

Table II.

Mean sulphated glycosaminoglycan content (μg/mL) of cell-seeded alginate bead constructs cultured for up to 14 days in medium containing varying concentrations of hyaluronic acid

		Hyaluronic acid concentration
Day	Control	0.1 mg/ml	1.0 mg/ml	2.0 mg/ml	3.0 mg/ml
0	8.12	8.09	8.08	8.08	8.09
3	12.89	13.09	13.00	12.95	12.90
6	15.71	16.10	15.96	15.89	15.85
9	25.98	28.98	27.32	26.30	26.17
14	30.85	32.95	32.61	32.00	31.34

Fig. 2

Bar chart showing sulphated glucosaminoglycan (GAG) content of cell-seeded alginate bead constructs cultured for up to 14 days in medium containing varying concentrations of hyaluronic acid (HA) (sem bars) (n = 6).

Hydroxyproline analysis.

The level of hydroxyproline is a marker for collagen production by the chondrocytes. Figure 3 and Table III show the concentration of hydroxypro-line in the chondrocyte/alginate composite beads exposed to the different concentrations of hyaluronic acid. All the culture groups demonstrated a significant increase by day 14 (p = 0.007) compared with day 0. A significant interaction was observed between day and dose (p = 0.003); the effect of doses at 0.1 mg/mL and 1.0 mg/mL hyaluronic acid were, once again seen more clearly for days 9 and 14 than for earlier days. The mean increases in hydroxyproline concentration were significantly greater in the chondrocytes exposed to 0.1, 1.0, and 2.0 mg/mL of hyaluronic acid compared with the control groups (p = 0.03) at days 9 and 14. The increase in hydroxyproline concentration in the hyaluronic acid groups demonstrated a dose-dependent response, with doses greater than 0.1 mg/mL hyaluronic acid showing a smaller increase in hydroxyproline.

Fig. 3

Bar chart showing hydroxyproline content of cell-seeded alginate bead constructs cultured for up to 14 days in medium containing varying concentrations of hyaluronic acid (HA) (sem bars) (n = 6)

Histology and immunolocalisation.

Histology demonstrated changes in cellular morphology, behaviour and extracellular matrix that were consistent with the biochemical findings. As a marker for cell proliferation, Ki-67 antigen immunostaining was strongly positive in the low-dose hyaluronic acid groups at day 14.

There was increased cellularity in the 0.1 mg/mL hyaluronic acid (low-dose) and 3 mg/mL hyaluronic acid (high-dose) cultures compared with the control. Greatest cellularity occurred in the 0.1 mg/mL hyaluronic acid specimens. The cells were either actively dividing or were present in small clusters of three to four cells. In the 3 mg/mL hyaluronic acid specimens, the cells had divided but fewer clusters were identified compared with the low-dose hyaluronic acid specimens. The results from the 2 mg/mL hyaluronic acid specimens showed intermediate findings. The majority of cells in the control group remained singular with very few mitotic figures.

The quality of the synthesised extracellular matrix was assessed by immunolocalisation of key articular cartilage matrix components. Increased levels of chondroitin-6-sulphate and collagen type II staining were present within culture specimens containing hyaluronic acid, compared with the controls. The presence of extracellular matrix rich in type II collagen and chondroitin-6-sulphate, combined with the absence of type I collagen, indicates that the chondrocytic phenotype was maintained throughout the culture period. Although the control specimens showed small increases in these components, the levels were by no means as marked as for the hyaluronic acid cultures. The most intense staining for collagen type II and chondroitin-6-sulphate was seen within beads containing 0.1 mg/mL and 1 mg/mL. Hyaluronic acid staining was less within beads containing 2 mg/mL and 3 mg/mL hyaluronic acid, although all were greater than the control beads.

The detection of chondroitin-6-sulphate indicated the presence of sulphated GAG. There was intense chondroitin-6-sulphate immunostaining around chondrocytes (in the territorial matrix) cultured in low-dose hyaluronic acid. Staining around the cells cultured in the control group, and with high concentration of hyaluronic acid, was less intense at day 14. Cells cultured in low concentrations of hyaluronic acid also stained more intensely for collagen type II. All chondrocytes cultured with hyaluronic acid stained negatively for collagen type I, whereas there was collagen type I staining within the control group.