Hyaluronan Preparations
Three commercially available preparations of the hyaluronan
compounds—Synvisc (Wyeth, Philadelphia, Pennsylvania), Hyalgan
(Sanofi-Synthelabo, New York, NY), and Supartz (Smith and Nephew, Memphis,
Tennessee)—were used for injection. The preparations were obtained from
standard hospital sources and were received in the sterile packaging that
would normally be provided to a surgeon. To increase the average molecular
weight and half-life in the joint, some hyaluronans have been modified to form
hylans by chemical cross-linking and thus are not natural molecules. According
to the package insert, Synvisc is an elastoviscous fluid containing hylan A
(average molecular weight, 6,000,000 Da) and hylan-B hydrated gel in a
buffered physiological sodium chloride solution (pH 7.2). Synvisc is
manufactured from hyaluronan found in rooster combs and is recognized to
contain avian serum
albumin5,6.
According to the package inserts provided by the respective manufacturers,
Hyalgan is a viscous solution consisting of a high-molecular-weight (500,000
to 730,000-Da) fraction of purified natural sodium hyaluronate in buffered
physiological sodium chloride, with a pH of 6.8 to 7.5. The sodium hyaluronate
is extracted from rooster combs and is also recognized to include avian serum
proteins. Supartz is a viscoelastic solution of hyaluronate with a molecular
weight of between 620,000 and 1,170,000 Da. It is dissolved in physiological
saline solution and has a pH of 6.8 to 7.8. Supartz is also manufactured from
the hyaluronan found in rooster combs.
Air Pouch Model
Air pouches were created on each of thirty BALB/c mice with use of
previously described
techniques19-21.
The mice were anesthetized, an area of dorsal skin was cleaned with alcohol
and shaved, and a subcutaneous injection of 3 mL of sterile air was delivered
at a single site with a 25-gauge needle. The air pouches were injected with 1
mL of air every other day to establish a definitive pouch. On day 6, the mice
were divided into five groups of six animals each. The pouches were injected
with 500 µL of saline solution (negative control), 500 µL of a 1%
weight/volume suspension of ultra-high molecular weight polyethylene particles
(mean diameter, 10 µm) in saline solution (a gift of Dr. John Cuckler,
University of Alabama, Birmingham, Alabama) (positive control), 500 µL of
Synvisc, 500 µL of Hyalgan, or 500 µL of Supartz.
Thickness and Cellularity of Air Pouch Membrane
Fourteen days after injection of the compounds, the mice were killed. To
prepare the tissue membranes of the pouches for analysis, the pouch-membrane
sections were fixed in 100% cold ethanol, dehydrated in 100% acetone, and
embedded in paraffin blocks. Sections of 8 µm in thickness were cut along
an axis sagittal with respect to the mouse spine and were mounted on 1 ×
3-in (2.5 × 7.6-cm) glass slides. Sections were stained with hematoxylin
and eosin or with esterase and were examined histologically.
Immunohistological analysis with use of polyclonal antibodies was used to
determine the tissue expression of the macrophage marker CD68, fibroblast
surface protein (FSP), and the T-cell marker Thy-1. Paraffin sections were
incubated with anti-mouse CD68 and Thy-1 antibodies (rabbit [1:250] and goat
[1:250] IgG [immunoglobulin G] antibodies, respectively) for four hours at
room temperature. Control antibodies were isotype-matched antibodies (NF66
anti-mouse IgG1; Pharmingen, San Diego, California), and all antibodies were
diluted in phosphate-buffered saline solution containing 1% bovine serum
albumin. Detection was performed with use of either horseradish peroxidase
conjugate or alkaline phosphatase-conjugated secondary anti-rabbit or
anti-goat antibodies. After development with diaminobenzidine or alkaline
phosphate substrate, positive reactions were visualized with use of bright
field microscopy. The histological sections were counterstained with
hematoxylin to visualize cell nuclei morphology.
Digital photomicrographs were made with use of an Axiophot light microscope
(Carl Zeiss, Thornwood, New York) equipped with a Toshiba camera (Tokyo,
Japan). These images were analyzed for cell counts and morphology with use of
the Image-Pro Plus software package (Media Cybernetics, Silver Spring,
Maryland). The thickness of the pouch membrane was analyzed in eight different
areas of each pouch. A uniform 100-µm longitudinal pouch area was selected
at random to establish the total cell count (hematoxylin and eosin stained),
and three different areas were analyzed in each pouch. Since fibroblasts are
spindle-shaped and have different nuclei aspect ratios than the more round
nuclei observed in mononuclear cells (monocytes and lymphocytes), nuclear
aspect and area ratios were used to differentiate these various cell types.
Cells were classified according to the aspect ratio, with a single variable
setting of aspect ratio = 1.8. The area of interest was set to an
approximately square region sized to include the entire width of the
inflammatory pouch. The analysis applied false color to all nuclei counted;
round (mononuclear cell) nuclei appeared red, while fibroblastic cell nuclei
appeared green. The area of interest was visually inspected, and the data were
accepted if the software criteria appeared consistent with the visual
determination of cell morphology and >90% of the cells within the area of
interest were included in the analysis. Immunohistological analysis (with
Thy-1, CD68, and FSP) and histochemical staining (with esterase) were used to
confirm the accuracy of the cell morphological determination.
Antibody Responses to Hyaluronan Preparations
Serum samples were obtained from mice by retro-orbital bleeding five days
prior to experimentation and at the termination of the experiment. Sera were
separated and were stored at -80°C prior to measurement with the ELISA
(enzyme-linked immunosorbent assay) technique. Nunc-Immuno II ninety-six-well
plates (Rochester, New York) were coated overnight at 4°C with either the
commercial hyaluronan preparation or purified hyaluronic acid (Sigma-Aldrich,
St. Louis, Missouri) at 10 µg/well. After washing with phosphate-buffered
saline solution/Tween, the plates were blocked with phosphate-buffered saline
solution/bovine serum albumin for twenty-four hours at 4°C. The plates
were again washed, and 100 µL of serum samples diluted 1/100 in
phosphate-buffered saline solution/bovine serum albumin were dispensed in
trip-licate. For each sample, a test (hyaluronan) and a control (bovine serum
albumin)-coated well were run in parallel. Plates were incubated overnight at
4°C and washed, and 100 µL of goat anti-mouse immunoglobulin, labeled
with alkaline phosphatase (Fisher-Southern Biotech, Birmingham, Alabama), was
added to each well. Following incubation for one hour at 37°C, plates were
washed and were developed with 1 M paranitrophenyl phosphate (Sigma), and the
absorbance was read at 405 nm with use of a microplate photometer (Model 340;
Molecular Devices, Sunnyvale, California). Values for specific antibody
binding were obtained by subtracting background bovine serum albumin binding
from hyaluronan binding and were expressed as OD (optical density) units. Sera
were considered positive for hyaluronan antibodies if the specific binding
exceeded three times the total uncorrected prebleed binding, determined
concomitantly. In order to evaluate any variations in antigen coating of the
ELISA plate among the commercial preparations, bound hyaluronan levels were
assessed by the addition of Hyaluronic Acid Binding Protein coupled to Biotin
(Sigma-Aldrich H9910) at 10 µg/mL in phosphate-buffered saline solution
containing 5% bovine serum albumin. After washing with phosphate-buffered
saline solution/Tween, avidin labeled with alkaline phosphatase
(Fisher-Southern Biotech) was added to each well and the plates were developed
as described above.
Statistical Analysis
The measurements in each experimental group were evaluated with analysis of
variance with use of the statistical software package SPSS (Chicago,
Illinois). Post hoc analysis was performed with the least significant
difference formula. Power for significant determinations was calculated with
use of the software PS Power and Sample Size Calculation
().
A p value of <0.05 was considered to be significant.
Histological Analysis
Representative photomicrographs of the fourteen-day pouch membranes from
the five different groups of mice are shown in
Figures 1-A through E. The
increase in membrane thickness (Figs.
1 and
2) seen after the injections of
Synvisc and Hyalgan was not significant when compared with the membrane
thickness in the negative control group (injected with phosphate-buffered
saline solution). The membrane thickness in the mice injected with Supartz was
significantly less (p < 0.02) than that in the mice injected with Synvisc
but not significantly different from that in the mice injected with Hyalgan
(Fig. 2). The membrane
thickness in the positive control group (injected with the suspension of
ultra-high molecular weight polyethylene) was significantly increased compared
with the membrane thickness in the negative control group (p < 0.005) and
compared with all three groups injected with the hyaluronan products (p <
0.05).
All of the hyaluronan products significantly increased (p < 0.001 to p
< 0.03) the total cell count within the membrane compared with that in the
negative control group (Fig.
3), but no single hyaluronan product resulted in more membrane
cellularity than did the other hyaluronan products. The positive control group
had significantly increased cellularity compared with the three groups treated
with the hyaluronan products (p < 0.001).
Significant differences between the groups were seen with respect to the
morphology of cells within the membrane tissues. Patterns of staining with
esterase, CD68, and FSP confirmed the differentiation of the cells into
fibroblast-like and monocyte-like cells on the basis of nuclear morphology.
The dense cellular accumulation within the pouch tissue prevented the image
analysis from identifying individual cells with surface markers. We used
conventional histo-pathological observational techniques to address whether
cells identified by image analysis as either fibroblasts or monocytes on the
basis of nuclear morphology exhibited confirmatory (or contradictory) chemical
or immunohisto-logical staining. Only one cell in the entire analysis with an
aspect ratio of 2.1 (defined as a fibroblast) expressed CD68. Between 5% and
15% of the cells were null for the expected marker (i.e., defined as monocytes
but CD68-negative or defined as fibroblasts but FSP-negative). The ratio of
macrophages/monocytes to fibroblasts in the membrane tissue was influenced by
the source of the stimulus (Fig.
3). While cells in the negative control pouch (injected with
phosphate-buffered saline solution) exhibited a 66:34-ratio
monocyte-tofibroblast morphology, stimulation with ultra-high molecular weight
polyethylene raised the percentage of monocytes to 81%. Similar increases in
the monocyte population were seen in membranes exposed to Supartz, while no
appreciable increase was observed in those exposed to Hyalgan. However,
Synvisc exposure resulted in 90% of the cells expressing a monocyte
morphology. That increase was significantly different from the increases
resulting from all other stimuli, including ultra-high molecular weight
polyethylene (p < 0.05). All significant values achieved a power value
>83%. The overall increase in total membrane cellularity resulting from
stimulation with hyaluronan products can be attributed to an inflammatory cell
influx rather than an accumulation of fibroblasts. The frequency of T
lymphocytes in the control tissue and in the pouches stimulated by Hyalgan and
Supartz was low (<1%), but the frequency was significantly increased (p
< 0.05) by exposure to ultra-high molecular weight polyethylene (3.14%) and
Synvisc (3.88%).
Antibodies to Hyaluronan Products and Purified Hyaluronic Acid
When the serum from mice injected with the different hyaluronan
preparations and harvested after fourteen days was assayed against the
hyaluronan preparations and the purified hyaluronic acid, only the sera from
the mice injected with Synvisc showed evidence of specific antibodies. Sera
from the mice injected with Synvisc bound to Synvisc in the ELISA, and these
sera exhibited no binding to either Hyalgan or Supartz. It appears that the
antibody response was not directed against the hyaluronic acid itself, since
binding to purified hyaluronic acid was not observed in any group
(Fig. 4). This suggests that
the antibody response may be directed against either a contaminant in the
preparation (such as the avian serum albumin) or a novel antigenic structure
created during the cross-linking procedures used during manufacture. No
variation in binding of hyaluronan products was detected with use of labeled
Hyaluronic Acid Binding Protein. This suggests that Synvisc, Hyalgan, and
Supartz were equivalent with regard to their capacity to bind to the ELISA
plate. Injection of Hyalgan or Supartz did not result in any relevant level of
antibody binding to the preparation or binding to the purified hyaluronic
acid. These data suggest that Synvisc alone was antigenic when it was injected
into the murine air pouch.