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Rheumatology 2001; 40: 158-169
© 2001 British Society for Rheumatology

Morphological analysis of knee synovial membrane biopsies from a randomized controlled clinical study comparing the effects of sodium hyaluronate (Hyalgan®) and methylprednisolone acetate (Depomedrol®) in osteoarthritis

I. Pasquali Ronchetti, D. Guerra1, F. Taparelli1, F. Boraldi1, G. Bergamini1, G. Mori1, F. Zizzi1 and L. Frizziero1

Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena and
1 Department of Internal Medicine, Rheumatology Unit, Ospedale Maggiore, Bologna, Italy


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. The study was part of a randomized open-label clinical trial designed to evaluate the effects of intra-articular injections of hyaluronan (Hyalgan®) (HY) in osteoarthritis (OA) of the human knee. Data were compared with those obtained after treatment with methylprednisolone acetate (Depomedrol®) (MP).

Methods. Synovial membranes from patients with OA of the knee, primary or secondary to a traumatic event and classified according to the American College of Rheumatology criteria, were examined by arthroscopy and by light and electron microscopy before and 6 months after local injection of HY (2 ml of 500–730 000 MW hyaluronan, 10 mg/ml in saline, one injection per week for 5 weeks) or MP (1 ml of methylprednisolone acetate, 40 mg/ml, one injection per week for 3 weeks).

Results. Arthroscopy revealed a significant decrease in inflammatory score after both treatments. Histology showed that HY treatment was effective (P<=0.05) in reducing the number and aggregation of lining synoviocytes, as well as the number and calibre of the vessels. MP treatment significantly reduced the number of mast cells in primary OA. Both treatments tended to decrease the number of hypertrophic and to increase the number of fibroblast-like lining cells, to decrease the numbers of macrophages, lymphocytes, mast cells and adipocytes, and to decrease oedema, especially in primary OA, and to increase the number of fibroblasts and the amount of collagen. These phenomena were evident throughout the thickness of the synovial tissue.

Conclusion. At least in the medium term, both HY and MP modified a number of structural variables of the synovial membrane of the osteoarthritic human knee towards the appearance of that of normal synovium. The effect was more evident in primary OA than in OA secondary to a traumatic event. This is the first evidence that local hyaluronan injections modify the structural organization of the human knee synovium in OA.

KEY WORDS: Corticosteroid, Human knee, Hyaluronan, Hyaluronic acid, Joint cavity, Osteoarthritis, Synovium, Synovium structure, Therapeutic trial.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
In normal conditions, high molecular weight hyaluronan represents the major component of the synovial fluid within the joint cavities. It is well known that in pathological processes such as osteoarthritis (OA) and rheumatoid arthritis, its molecular weight can be reduced by an order of magnitude and its concentration is reduced, mainly by the effect of the accumulation of liquid derived from the inflamed synovial vessels [1–3].

Injection of hyaluronan into the joint cavity started in the late 1960s in horses with joint injury followed by arthritis, and it was observed that local hyaluronan injections reduced pain and improved functional joint mobility [4, 5]. Rydell and Balazs [6] and Peyron and Balazs [7] published the first studies of the use of hyaluronan as a therapeutic agent in OA of the human knee. The first results were encouraging from the clinical point of view, as it was shown that hyaluronan treatment led to statistically significant improvement in almost all functional variables of the articular joint. Since then, a large number of studies have been published on the effects of hyaluronan in human and animal joint diseases [8–11] and on the best therapeutic schedule to obtain the most lasting results [11–14]. The great majority of these studies were clinical observations [6, 8, 15] or were performed on animal models of OA, which are similar but not identical to spontaneous human OA [16, 17]. In the beginning, the rationale for the use of intra-articular injection of hyaluronan was to replace the depolymerized endogenous hyaluronan and to improve joint lubrication. However, subsequent studies clearly demonstrated that injected hyaluronan is quite rapidly degraded and removed from the joint cavity [18–20]. Therefore, other biological mechanisms would seem to be responsible for the effectiveness of intra-articular hyaluronan therapy in OA, and for the duration of beneficial effects for months after the cessation of treatment [8, 11, 13, 21]. In recent years, hyaluronan has been shown to have several biological activities, some of which may be important for its effect in the treatment of OA [22]. Several cell types have been shown to have hyaluronan receptors [23–25]. Moreover, hyaluronan was observed to modulate leukocyte functions [26–30] and to inhibit the release of arachidonic acid from human synovial fibroblasts [31] and the production of prostaglandin E2 [32, 33]; it was also shown to have analgesic activity [34, 35]. Therefore, hyaluronan might be able to reduce the inflammatory reaction associated with OA.

In order to further study the effect of hyaluronan on joint tissues in OA, we carried out a histological and electron microscope study of synovial membranes sampled from osteoarthritic human knees of the same individuals before and 6 months after local injections of Hyalgan® (Fidia, Abano Terme, Italy). Unfortunately, proper control observations, such as the effect of time on spontaneous modifications of the OA synovial membranes during the 6 months of observation or the effect of simple arthroscopic inspection and saline washing of the joint cavity, could not be carried out for ethical reasons. Therefore, the effect of hyaluronan was compared with that of a corticosteroid that is accepted to have beneficial effects in OA [36, 37]. Data from patients, before and after treatment, were compared with those we have already published for the same structural variables in subjects who underwent arthroscopy for knee pain but did not show any structural joint disorder [38].


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
This study was part of a large open-label clinical study carried out on patients fulfilling the criteria of the American College of Rheumatology for the diagnosis of OA of the knee [39]. Patients judged not controllable or unreliable, those with concomitant diseases, those who had been under therapy during the previous 3 months (apart from oral administration of non-steroidal anti-inflammatory drugs), pregnant women and women who were breast-feeding were excluded from the trial.

Consecutive out-patients with clinically and radiologically diagnosed OA of the knee (Kellgren–Lawrence grade II–III) were admitted to the study. They had been referred to the Rheumatology Unit (Department of Internal Medicine, Maggiore Hospital in Bologna), usually by the family doctor, and fulfilled the above-mentioned criteria. The diagnosis was confirmed by arthroscopy. When the OA was bilateral, the most severely affected knee was selected for treatment. Patients were recruited during a period of 3 yr and divided into those with primary OA and those with secondary OA. The total number of patients recruited into the study was determined on the assumption of a 50% dropout for the second arthroscopy and/or sampling. Recruitment stopped at 50 individuals with primary OA and 49 with secondary OA. These two patient groups were further divided into two subgroups according to a randomization scheme generated by computer. One subgroup received local injections of hyaluronan and the other local injections of methylprednisolone acetate.

Data from these patients were compared with those we had obtained, using the same criteria, on the knee synovial membrane from 19 subjects who had undergone arthroscopy for pain but had not shown any arthroscopic sign of OA or rheumatoid arthritis [38]; these data will be referred to here as control data. The trial was approved by the Ethics Committee of the Maggiore Hospital of Bologna. Written informed consent was obtained from all patients for treatment, arthroscopic inspections and sampling.

Treatment
After clinical and radiological evaluation, patients judged eligible for the study underwent an initial arthroscopy during which biopsy samples of the synovial membrane were taken; 2–3 weeks later, they had a baseline visit at which blood and urine were collected for laboratory analysis. Two subgroups of patients, one with primary OA and the second with secondary OA, received an intra-articular injection of 2 ml of Hyalgan® (Fidia) (10 mg/ml MW 500–730 000 hyaluronan in saline) (HY) once per week for 5 consecutive weeks. The other two subgroups of patients, affected by primary and secondary OA, respectively, received an intra-articular injection of 1 ml of Depomedrol® (Pharmacia & Upjohn, Puurse, Belgium) (40 mg/ml methylprednisolone acetate) (MP) per week for 3 consecutive weeks. On days 7, 14, 21, 28, 35, 60, 120 and 180 after the last injection, clinical variables were assessed and blood and urine samples were collected for laboratory analysis. On day 180, a new arthroscopic examination was performed and biopsies for morphological analysis were taken from patients who came to the final assessment and agreed to have a biopsy taken (Table 1Go).


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  		TABLE 1. Numbers of patients recruited and receiving the different treatments, and numbers followed up

 

Arthroscopic examination and sampling
Apparatus
We used Hamou-Storz and Microview-Wolf arthroscopes, 20 cm long and 4 mm in diameter, with a range of magnification of 1x to 150x and automatic focus control. Light intensity was variable from 150 to 1000 W, as described [38, 40]. Photographs of synovial membrane and cartilage were taken with an Olympus OM2 camera; an Ikegami ITC-370 M video camera connected to a Sony U-Matic V 0-5800 PS video recorder was also used.

Assessment and sampling
Local anaesthesia was obtained by injection of 2 ml of 2% mepivacaine HCl without a vasoconstrictor. The endoscope was introduced anterolaterally, keeping the knee flexed at an angle of 30°. The anterolateral point of insertion is the best for routine initial examination of the knee. The point of insertion for the anterolateral approach lies 2 mm above the tibial edge and close to the patellar tendon. This point can be identified by flexing the knee to 30–50° and pushing the thumbnail into the small depression just lateral to the patellar tendon [40]. After a detailed examination, 10 ml of 2% mepivacaine HCl was injected, followed by injection of 3 ml of 1% methylene blue in aqueous solution (pH 4.5). Intermittent irrigation with Ringer's acetate was used, regulating the infusion pressure to optimize the distension of the joint cavity. The intra-articular pressure was maintained around 80 mmHg. Photographs and video recordings of synovial membrane and cartilage were carried out at 20x, 60x and 150x magnification. Detailed arthroscopic data will be reported in a separate paper. Statistical analysis of structural variables of the synovial membrane was performed on micrographs of the same areas taken before and after treatment.

Keeping the knee flexed at an angle of 30°, two synovial samples were taken, one from the suprapatellar pouch and one from the medial tibiofemoral compartment, using basket forceps. Samples were kept separate until the end of the study. The areas of sampling were identified carefully by transillumination and marked on the skin above. The second samplings, 6 months after treatments, were taken from areas about 1–2 cm distant from the previous ones. The patient was discharged from the hospital the next day. No complications were observed, with the exception of a little effusion. Arthroscopic assessment was performed by investigators who were not involved in the intra-articular injections or the clinical evaluation.

Specimen preparation and structural analysis
Samples were numbered, processed and examined, with no information other than an identification number, by investigators who were not involved in the clinical or arthroscopic examinations. Synovial specimens were immersed immediately in 1% glutaraldehyde (Fluka, Buchs, Switzerland) in Tyrode's physiological solution (pH 7.2) and then cut under a stereo microscope into small blocks, less than 0.5 mm wide, taking care to maintain the whole thickness of the synovial membrane in each fragment. Six to eight fragments were obtained from each biopsy sample. Fragments were then fixed in 2.5% glutaraldehyde in Tyrode's solution (pH 7.2) in the presence of 0.1% toluidine blue O (Serva Feinbiochemica, Heidelberg, Germany) for 24 h at 4°C, followed by 1% osmium tetroxide (Fluka) in Tyrode's solution (pH 7.2) containing 0.05% toluidine blue O for 2 h at room temperature. After dehydration in ethanol and propylene oxide, specimens were embedded in Spurr resin (Polysciences, Warrington, PA, USA), taking care to orientate them so as to have the whole thickness of the synovial membrane on the same section. Sections (1 µm) obtained from each tissue fragment were stained with 0.1% toluidine blue O in 1% Na2B4O7 and were observed by light microscopy (Axiophot; Carl Zeiss, Oberkochen, Germany); the data obtained were used in the statistical analysis. Ultrathin sections (70–80 nm thick), prepared from three fragments from each biopsy sample, were stained with uranyl acetate and lead citrate, and observed in a Siemens IA or a JEM 1200 EXII transmission electron microscope.

Specimen evaluation and statistical analysis
Arthroscopy
Structural parameters of the synovial membrane were evaluated on micrographs of the same areas before and after treatments, by using the scale score system already described [38, 40]. Briefly, macroscopic characteristics were graded from 0 to 50, cellularity from 0 to 30 and vascularity from 0 to 20, where 0 was assigned to normal features and increasing values were attributed to increasing inflammatory aspects, up to a picture of typical rheumatoid synovitis [38].

Histology and electron microscopy
Sections were examined blind by two independent investigators following criteria and scores discussed and established previously [38, 40]. Primary or secondary OA, the basal/final sequence and the type of treatment were matched at the end of the study. Only data from patients who underwent both basal and final sampling of synovium were considered (Table 1Go). Data obtained from the suprapatellar pouch samples were kept separate from those from the mediolateral femorotibial areas. However, these data were very similar and were later combined. Only tissue fragments including the whole synovial membrane were considered, and sections were always perpendicular to the synovial surface. The synovium was divided arbitrarily into three regions (Fig. 1Go): (i) the synovial lining, the most superficial layer facing the joint cavity, the thickness of which varied according to the number of cell layers and, in the great majority of samples, was about 200 µm below the free surface of the synovium; (ii) the sublining, a highly vascular region extending down to about 500–700 µm from the synovium surface; and (iii) the subsynovium region, down to about 2 mm from the free surface of the synovial membrane.



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  		FIG. 1. For image analysis, the synovial membrane was divided into three regions: the lining, about 200 µm deep, facing the joint cavity; the subintima, including the vessel region, about 500–700 m thick; and the subsynovium, the deepest region of the synovial membrane.

 
The following variables were evaluated: the arrangement of the lining cells into monolayers, bilayers or multilayers; the clustering of the lining cells (aggregated or sparse); the appearance of the cells (size, shape, vacuolization); cell type (fibroblast, macrophage, lymphocyte, adipocyte, mast cell); matrix features (fibrosis, oedema, necrosis, fibrin); and the number and appearance of vessels. Intracellular organelles, such as the Golgi apparatus, rough endoplasmic reticulum, mitochondria, lysosomes and cytoskeleton filaments, were also considered. Variables were evaluated using the following criteria. Cell arrangement and clustering in the synovial lining was observed at 40x (objective lens) and the variable (monolayered/bilayered, aggregated, etc.) was considered to be positive when present on more than 70% of the specimens; when opposite features (i.e. aggregated and sparse) were almost equally represented on the same section the sample was considered to be positive for both features; cell types were identified at 63 x and the specimen was considered positive when at least five cells of the given type were present in a field. Fibrosis and oedema were considered positive when the feature occupied at least a quarter of the field at 63x. Necrosis and fibrin were considered to be positive when present in the field. In all these cases, data were expressed as the percentage of positive samples for that specific variable. In secondary OA, which was always the consequence of mechanical injury, macrophages and fibroblasts were very numerous and were scored (at 63x) on a semiquantitative scale from 0 to 5 (macrophages: score 0=absent; score 1=from 1 to 4; score 2=from 5 to 8; score 3=from 9 to 11; score 4=from 12 to 14; score 5=more than 15 in a field; fibroblasts: score 0=absent; score 1=from 1 to 5; score 2=from 6 to 10; score 3=from 11 to 15; score 4=from 16 to 20; score 5=more than 20 in a field). Vessel variables were graded on a semiquantitative scale from 0 to 3 (score 0=absent; score 1=scarce; score 2=moderately represented; score 3=strongly represented). Cytoplasmic organelles of B synoviocytes, evaluated on electron micrographs at 5000x, were graded as for vessels.

The non-parametric signed rank test was used for statistical analysis. Data were analysed by a computer program (SAS Institute, Cary, NC, USA). P<=0.05 was considered to indicate a statistically significant difference between final and baseline values.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Clinical and laboratory data
The data reported here refer to the patients who underwent arthroscopy and sampling at baseline and 6 months after intra-articular injections (Table 1Go). Twenty-five patients did not finish the treatments and/or did not attend the final evaluation, 19 refused the second arthroscopy and seven refused the second biopsy (Table 1Go). No cases of adverse reactions were noted with either treatment. Laboratory variables of blood and urine were not significantly affected by treatments.

Complete clinical and arthroscopic findings will be the subject of a separate publication. Data on the arthroscopic assessments are briefly summarized in Table 2Go and indicate an improvement in macroscopic characteristics, cellularity and the number of vessels after both treatments, in both primary and secondary OA.


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  		TABLE 2. Arthroscopy evaluation before (baseline) and 6 months after (final) treatment with HY or MP

 

Structural analysis
Synovial lining
Tables 3Go and 4Go report the cell arrangement in the superficial lining facing the joint cavity, before and 6 months after treatment in both primary and secondary OA. Considerable heterogeneity was observed among fragments of the same biopsy and among patients. In both primary and secondary untreated OA, specimens exhibited a different distribution of lining cells compared with normal synovium. This variable was evaluated in two ways, which gave similar results: the percentage of specimens in which lining cells were organized into multilayers and the percentage of specimens presenting a monolayer/bilayer cell distribution. Compared with controls, in both primary and secondary untreated OA there was a significant increase in the percentage of samples with cells forming multilayers (P<0.0001 compared with controls) and a concomitant decrease in samples with cells in monolayers/bilayers (P<0.03, primary OA vs control). By contrast, after both treatments, an increase in the percentage of specimens showing monolayer/bilayer lining cells was observed; the increase was statistically significant after HY treatment, especially in primary OA. Correspondingly, multilayer lining cells decreased significantly after HY treatment, becoming more similar to controls (Table 3Go). In untreated patients with primary or secondary OA, lining cells were significantly more aggregated than in controls (P<0.001); 6 months after either treatment, cells were less aggregated than before treatment and were more similar to controls; HY was more active than MP in reducing cell clustering (Table 3Go). This feature was confirmed by an independent analysis of samples showing sparse cells. The number of specimens with cells scattered within the extracellular matrix was lower in untreated OA than in controls and increased after both treatments, especially in primary OA, in which HY was more effective than MP (Table 3Go).


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  		TABLE 3. Features of lining cells in the synovial membrane of the human knee in primary and secondary OA before (baseline) and 6 months after (final) treatment with HY or MP

 

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  		TABLE 4. Cytoplasmic organelles in B synoviocytes of the synovial membrane of the human knee in primary and secondary OA before (baseline) and 6 months after (final) treatment with HY or MP

 
As already described in the literature, synoviocytes appeared larger and more spherical in OA than in controls (P<0.03); after both treatments, in both primary and secondary OA, they appeared less hypertrophic and more spindle-shaped, and became more similar to those in controls (Table 3Go). In normal synovium, 27% of specimens showed vacuolized lining cells. The percentage increased to about 38% in both primary and secondary OA. Treatment with either HY or MP induced a decrease in vacuolized cells towards normal values (data not shown).

In controls, some specimens showed necrotic areas (14% of specimens) and deposition of fibrin (23% of specimens) on the top of the lining layer. These features increased significantly in both primary and secondary OA (P<0.0001) and became more similar to controls after both treatments. MP was more active than HY in reducing necrosis in primary OA (P<0.01).

Table 4Go shows the scores reached by cytoplasmic organelles of B synoviocytes. The rough endoplasmic reticulum of B synoviocytes was not modified in OA and was not affected by treatment. By contrast, differences were found for the Golgi complex, which appeared less pronounced than in controls (untreated patients with either primary or secondary OA), and was shown to increase towards normal values after both treatments. HY was more effective than MP in secondary OA. A similar phenomenon was observed for intermediate filaments: they were less represented in OA than in controls, and increased after both treatments. Rather surprisingly, the amount of lysosomes in both groups of untreated patients appeared similar to or slightly lower than that in controls. Their number tended to diminish after HY treatment in primary and secondary OA. Myofilaments, such as those described in myofibroblasts, were scarcely represented in B synoviocytes of controls and of patients with primary OA, whereas they were rather well represented in patients with secondary OA. In both cases, treatment with either HY or MP induced a slight increase in the number of cells with myofilaments in the cytoplasm (data not shown). Another interesting feature was the presence of pinocytic vesicles decorating the plasma membrane of B synoviocytes. These vesicles were always very numerous in normal B synoviocytes, where they formed an almost continuous decoration on the cytoplasmic side of the cell membrane (Fig. 2aGo) [38]. Their number decreased drastically in patients with untreated OA (Fig. 2bGo) and increased towards normal values in patients treated with HY (Fig. 2cGo) (Table 4Go).



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  		FIG. 2. Typical type B synoviocytes in normal synovium (left), in the synovial lining of untreated osteoarthritis (middle) and 6 months after HY treatment (right). In normal subjects, the plasma membrane of type B synoviocytes is almost completely decorated by pinocytic vesicles, which disappear in osteoarthritis and become visible again after HY treatment. Scale bar=1 µm.

 

Sublining region
Table 5Go reports data on the principal cell types in the sublining region. In the control group, in which there was considerable variability among subjects, fibroblasts were present in more than 90% of specimens, macrophages in fewer than 60%, mast cells and lymphocytes in about 10%, and adipocytes in more than 25% of samples. In both subgroups of patients with primary OA, the percentage of specimens with fibroblasts and adipocytes was almost the same as in controls, whereas macrophages were present in about 80% of samples and mast cells and lymphocytes were present in up to 2–3 times more samples than in controls. Treatment with either HY or MP induced an increase in fibroblasts and a decrease in macrophages, mast cells, lymphocytes and adipocytes. The sublining region in untreated OA secondary to a traumatic event was characterized by a very high number of fibroblasts and macrophages; there was also an increase in fibroblasts and a decrease in macrophages after both treatments.


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  		TABLE 5. Sublining region of the synovial membrane of the human knee in primary and in secondary OA before (baseline) and 6 months after (final) treatment with HY or MP

 
Table 6Go illustrates matrix parameters of the sublining region. In normal synovium, more than 75% of specimens showed fibrosis and fewer than 50% showed clear signs of oedema. No specimen showed necrosis and fibrin deposition. In both subgroups of patients with untreated primary OA, fibrosis was similar to that in controls, whereas oedema increased to 65–70% of specimens; moreover, necrosis and fibrin deposition were observed in some specimens (3–8%). After both treatments, fibrosis tended to increase, whereas oedema and fibrin deposition decreased. In untreated secondary OA, matrix variables were similar to those in controls. As in primary OA, the treatments induced an increase in fibrosis and a decrease in oedema and fibrin deposition (Table 6Go).


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  		TABLE 6. Matrix variables in the sublining region of the synovial membrane of the human knee in primary and secondary OA before (baseline) and 6 months after (final) treatment with HY or MP

 
Vessel number, permeability and basement membrane thickness were also considered (Table 7Go). The last variable was seen by electron microscopy to be related to the number of concentric basement membranes surrounding small-vessel endothelial cells. Compared with normal synovium, the synovium of untreated primary OA was found to have more vessels and to decrease towards normal values after treatment with HY; vessel permeability decreased after both treatments; the thickness of basement membranes was greater in untreated secondary OA than in controls and was not modified by the treatments.


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  		TABLE 7. Vessel variables in the sublining region of the synovial membrane of the human knee in primary and secondary OA before (baseline) and 6 months after (final) treatment with HY or MP

 

Subsynovium
Tables 8Go and 9Go show cellular and matrix variables in the deep region of the synovial membrane. In both subgroups of patients, fibroblasts and fibrosis, which were slightly better represented in untreated primary OA than in controls, increased after the treatments, with no difference between HY and MP. On the contrary, macrophages, which were more numerous in primary OA than in controls, decreased after treatment with either HY or MP. As already observed in the sublining region, adipocytes were less represented in both primary and secondary OA than in controls, and were even less frequent after either treatment. Lymphocytes were always very few and tended to decrease after treatment with HY in primary OA and to disappear after either treatment in secondary OA. As already described in the sublining region, oedema decreased and fibrosis increased after treatment (Table 8Go).


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  		TABLE 8. Cell and matrix variables in the subsynovium region of the synovial membrane of the human knee in primary and secondary OA before (baseline) and 6 months after (final) treatment with HY or MP

 

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  		TABLE 9. Vessel variables in the subsynovium region of the synovial membrane of the human knee in primary and secondary OA before (baseline) and 6 months (final) after treatment with HY or MP

 
In the subsynovium, the number of vessels was higher in both primary and secondary OA than in controls, and tended to decrease after both treatments, becoming more similar to controls (Table 9Go); HY was more active than MP in primary OA. By contrast, in both primary and secondary OA, the permeability of the vessels was always lower than in controls and tended to become even lower after both treatments, with an increase in ectasic vessels (not shown).


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
The study was part of an open-label clinical trial designed to compare the effects of intra-articular injections of hyaluronan and methylprednisolone in both primary and secondary osteoarthritis of the human knee. This paper reports the structural variables of the synovial membrane at baseline and 6 months after treatment. At the 6-month evaluation, biopsies could not be collected from about 50% of the recruited patients. About 20% of patients with primary OA and more than 30% of those with secondary OA did not attend the final evaluation. Moreover, as shown in Table 1Go, a number of patients refused the second arthroscopy and/or biopsy. About half of these patients reported that they felt better and judged the practice superfluous. There were no apparent demographic differences between these patients and those who agreed to have the second biopsy. The authors are aware that the differential dropout rate may seriously bias the results; however, this is an insoluble problem in clinical trials.

From the arthroscopic point of view, the HY and MP treatments induced a significant decrease in the inflammatory aspect of the synovial membrane in both primary and secondary OA (Table 2Go).

In order to evaluate the effect of treatments on structural variables of the synovial membrane in OA, we think it is important to compare, in the same experimental conditions, the appearance of the synovium in normal subjects and in patients with OA before any treatment. In agreement with data from the literature and compared with controls, the superficial lining layer of the synovial membrane of the osteoarthritic human knee is characterized by the following structural modifications: increases in the number of cells and in cell aggregation as well as an increase in the number of round-to-oval hypertrophic cells, associated with deposition of necrotic material. Moreover, ultrastructural data on B synoviocytes reveal a reduction in specialized cytoplasmic structures, such as the Golgi complex (primary OA vs control, P<=0.05; secondary OA vs control, P<=0.01), intermediate filaments (primary OA vs control, P<=0.002; secondary OA versus control, P<=0.0002) and pinocytic vesicles on the plasma membrane (primary and secondary OA vs control, P<=0.0001).

In the highly vascular sublining area, primary OA and OA secondary to a traumatic event exhibit different structural modifications compared with controls. Primary OA is characterized by a significant increase in macrophages (P<=0.0002), lymphocytes (P<=0.002), matrix oedema (P<=0.01), necrosis (P<=0.03) and fibrin deposition (P<=0.08), whereas secondary OA is characterized by a very large number of fibroblasts and macrophages (Table 5Go). Similar features and differences between primary and secondary OA, compared with controls, were found in the deepest region of the synovial membrane. In agreement with data from the literature, these findings indicate that both primary and secondary OA are characterized, from the structural point of view, by features suggesting hyperplasia of the lining cells, which is associated with a decrease in B synoviocyte cytoplasmic organelles, such as the Golgi apparatus, pinocytic vesicles and intermediate filaments. In the deepest regions of the synovial membrane, primary OA is characterized by a modest inflammatory process. By contrast, in secondary OA, both sublining and sybsynovial regions are characterized by a more intense inflammatory process associated with reparative fibrotic phenomena, as suggested by the high number of macrophages and fibroblasts, the presence of huge, thick collagen bundles (not shown) and by the high number of ectasic vessels (vs control, P<=0.03; vs primary OA, P<=0.008). A description of these parameters is perhaps redundant but necessary in order to evaluate modifications upon treatment. Six months after both treatments, a number of structural variables had changed and tended to become more similar to controls [38].

In the superficial lining region, there was an increase in the percentage of specimens with sparse cells forming a mono-/bilayer after both treatments; moreover, the cells were less hypertrophic, tended to be more spindle-shaped and partly regained the ultrastructural characteristics of controls, indicating partial regression towards normal. These processes were more evident in primary than in secondary OA. HY was mostly effective in reducing the number and aggregation of synoviocytes.

In the deepest region of the synovial membrane, both treatments induced structural modifications typical of reparative processes, such as increases in the number of fibroblasts and in the amount of fibrosis, and decreases in inflammatory cells (macrophages, lymphocytes and mast cells). Also in these regions, the treatments were more effective in primary than in secondary OA.

Without excluding the possibility that lavage of the joint cavity with saline during arthroscopy may have been beneficial, all these data seem to indicate that both treatments were active in reducing the values of the variables typical of OA. Given the low number of subjects and the heterogeneity observed even among tissue fragments from the same subject, the statistical significance of the effects of these treatments is low for the great majority of variables. However, combining arthroscopic and histological data, data from primary and secondary OA and from the different regions of the synovial membrane, it clearly appears that, 6 months after both treatments, the organization of the synovial membrane had changed towards the appearance of a reparative fibrotic process. Given the low number of subjects, it is not possible to say whether HY is more effective than MP. The two drugs seem to have beneficial effects on the evolution of the disease, perhaps through different mechanisms. The corticosteroid seems to be rather active in reducing the inflammatory process, whereas hyaluronan seems to influence mostly the number and distribution of lining cells and to stimulate reparative processes. These data are in agreement with a recent study showing that hyaluronan treatment induces increases in collagen type I gene expression and in reducible collagen cross-links after partial meniscectomy in a rabbit model of OA compared with vehicle treatment [41].

Several studies point to the beneficial clinical effect of hyaluronan injections in OA, such as pain relief and reduction of functional joint impairment [6–14], and the data of the present study indicate that, given its excellent safety profile, Hyalgan® seems a valuable means of ameliorating the structural variables of the osteoarthritic synovial membrane, at least in the medium term. This statement is supported by recent data showing the positive effect of repeated intra-articular injections of hyaluronan in human knee OA evaluated by X-ray joint-space narrowing and arthroscopy [42, 43]. Moreover, it has been shown recently in rabbits that preservation of the articular cartilage and synovial tissue is better after anterior cruciate ligament transection and repeated intra-articular injections of hyaluronan than in controls [44–46]. To our knowledge, this is the first evidence in humans of modifications of structural variables of the synovial membrane in OA after hyaluronan treatment, suggesting that hyaluronan injected into the joint cavity in OA may exert a biological effect.


   Acknowledgments
 
This work was supported in part by MURST and CNR. We thank Fidia SpA for scholarships and partial financial support, Professor E. Govoni for help in the experimental design, and Dr S. Piva for the statistical analysis.


   Notes
 
Correspondence to: I. Pasquali Ronchetti, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100-Modena, Italy. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 14 July 1999; Accepted 2 September 2000


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