Effect of the delegation of GP-home visits on the development of the number of patients in an ambulatory healthcare centre in Germany

Neeltje Van Den Berg
Romy Heymann
†
Claudia Meinke
Sebastian E Baumeister
Steffen Fleßa
Wolfgang Hoffmann
Effect of the delegation of GP-home visits on the development of the number of patients in an ambulatory healthcare centre in Germany
R E S E A R C H A R T I C L E Open Access
Background: The AGnES-concept (AGnES: GP-supporting, community-based, e-health-assisted, systemic intervention) was developed to support general practitioners (GPs) in undersupplied regions. The project aims to delegate GP-home visits to qualified AGnES-practice assistants, to increase the number of patients for whom medical care can be provided. This paper focuses on the effect of delegating GP-home visits on the total number of patients treated. First, the theoretical number of additional patients treated by delegating home visits to AGnES-practice assistants was calculated. Second, actual changes in the number of patients in participating GP-practices were analyzed. Methods: The calculation of the theoretical increase in the number of patients was based on project data, data which were provided by the Association of Statutory Health Insurance Physicians, or which came from the literature. Setting of the project was an ambulatory healthcare centre in the rural county Oberspreewald-Lausitz in the Federal State of Brandenburg, which employed six GPs, four of which participated in the AGnES project. The analysis of changes in the number of patients in the participating GP-practices was based on the practices' reimbursement data. Results: The calculated mean capacity of AGnES-practice assistants was 1376.5 home visits/year. GPs perform on average 1200 home visits/year. Since home visits with an urgent medical reason cannot be delegated, we included only half the capacity of the AGnES-practice assistants in the analysis (corresponding to a 20 hour-work week). Considering all parameters in the calculation model, 360.1 GP-working hours/year can be saved. These GP-hours could be used to treat 170 additional patients/quarter year. In the four participating GP-practices the number of patients increased on average by 133 patients/quarter year during the project period, which corresponds to 78% of the theoretically possible number of patients. Conclusions: The empirical findings on the potential to increase the number of patients in GP-practices through delegation of tasks come close to the theoretical calculations. Differences between the calculated and the real values may be due to differences in the age and mortality distribution of the patients. The results indicate that a support system based on practice assistants can alleviate the consequences of GP-shortages in rural areas. van den Berg et al. BMC Health Services Research 2012, 12:355  * These practices participated in the AGnES-Brandenburg project. † During these quarter years, these practices were closed due to illness of the GPs. These quarters were not considered in the analyses. ¥ Difference between the means before and during the intervention. van den Berg et al.

Background

In Germany, general practitioners (GPs) have a leading role in primary care. Traditionally, other medical professions, including nurses, do not have a structural role in the organization of primary healthcare. GP-practice assistants mainly conduct non-medical tasks such as administration of appointments and coordination of the work flow of the practice. During the last years, however, the organization of GP-practices has changed towards a more team oriented approach involving the entire practice-staff. Practice assistants can now qualify for more specialized tasks (e.g. case management and other medical tasks [1,2]).

The Association of Statutory Health Insurance Physicians is responsible by law to guarantee an adequate number of physicians providing ambulatory care for each administrative district in Germany. The supply of ambulatory care is based on a fixed ratio between the numbers of physicians in practices and inhabitants of a defined region (usually at the county-level). This ratio is dependent on the speciality of the physician and differs somewhat between rural and urban regions. In Germany, a region is undersupplied with GPs if the ratio is below 75% of the calculated number of GPs for this region. Between 75% and 100%, the care situation in a region is categorized as imminently undersupplied [3].

About 20% of the GPs in Germany are 60 years of age or older and will retire in the next few years [4]. Few younger physicians are willing to start a private practice in a rural area, especially in the eastern part of Germany. In some regions, shortages in primary care are already existent or can be expected within the next years.

At the same time the number of elderly people living in the eastern part of Germany is rapidly increasing, due to demographic changes and migration to urban areas. This leads to a higher absolute number of age-associated chronic diseases and multimorbidity [5,6]. Elderly patients receive a significantly higher number of GP-home visits than younger patients (under 65-years old patients: on average 2.7 home visits/year, 65-79-years old patients: on average 6.3 home visits/year, 80+ year old patients: on average 8.5 home visits/year) [7].

However, the overall frequeny of GP-home visits decreases in Germany as well as in other European countries. A study of van den Berg et al. based on data of 183 Dutch GP-practices shows that the proportion of home visits decreased from 14.1% of all patient contacts in 1987 to 7.4% in 2001 [8]. The decrease has two main causes: fewer home visits involving children and involving patients with less urgent diagnoses. Obviously, a GP having to deal with a large number of patients has to weigh medical and non-medical factors (e.g. workload, availability in his practice) and will more often decide against conducting a home visit [8].

A qualitative study of Theile et al. among 24 GPs in Germany shows that GPs frequently conduct home visits with little or no medical indication. Home visits with vulnerable, elderly people, however, were often imperative [9]. Hence while the need for home visits will probably increase as a consequence of the growing number of older patients, the workload of GPs, particulary in rural areas, is expected to rise in the near future due to an undersupply of physicians.

The delegation of home visits to qualified GP-practice assistants could provide one possibility to support GPs. To examine the option of delegating GP-home visits, we developed and implemented the AGnES concept (AGnES = GP-supporting, community-based, e-healthassisted, systemic intervention) [10,11].

The main aim of the AGnES concept is to decrease the workload of GPs, especially in sparsely populated regions with an imminent or already existing undersupply of GPs. The concept enables the available GPs to provide care for more patients in a larger area [10].

All AGnES projects were conducted in "daily-routinesettings" with minimum intervention. The participating patients were selected by their treating GPs. The only inclusion criterion was that the patient, in the opinion of the GP, needed one or more home visits that could be delegated to a qualified practice assistant. The GP decided about the frequency and type of care provided during the home visit.

All together, 53 GPs and 40 AGnES-practice assistants participated in seven field studies between 2006 and 2008 in four federal states in Germany. The participating AGnES-practice assistants all received training specifically developed for this project [10,12].

In total, 11,228 home visits were carried out involving 1,430, mostly multimorbid patients with a mean age of 78.6 years (standard deviation: 10.7 years). 89% of the patients had limited or no mobility [10].

A previous analysis showed that the home visits conducted by the GPs significantly decreased, especially the number of medically urgent home visits. However, the overall rate of home visits (conducted by the GPs and the AGnES-practice assistants together) did not change significantly after implementation of the AGnES-concept. Thus, the additional possibility to provide home visits did not lead to a supply-driven increase of the total number of home visits [13].

The main aim of the AGnES projects was to support GPs in underserved regions and to enable GPs to increase their number of patients. To examine whether this aim was achieved, participating GPs were interviewed. About 90% of the participating GPs reported a reduced workload due to the AGnES-practice assistants. In an evaluation, the GPs stated that for more than 92% of the participating patients the quality of care provided by the AGnES-practice assistants was comparable to the usual care [10].

To obtain a more objective indication for the influence of the AGnES-concept on the workload of the GPs, we analyzed the development of the total number of patients treated in participating GP-practices in one of the project regions, the county Oberspreewald-Lausitz in the Federal State of Brandenburg. We choose this region for two reasons:

Methods

The data needed to calculate the capacity of the AGnESpractice assistants was retrieved from the standardized project documentation which was identical in all AGnES projects (duration of the home visits, driving times, time needed for consultation with the GP and other activities). Information about the capacity of the GPs were taken both from published literature [14,15] and from data from the Associations of Statutory Health Insurance Physicians from Saxony and Mecklenburg-Western Pomerania.

The changes in the number of patients in GP-practices were analyzed using data of an ambulatory healthcare centre in the rural county Oberspreewald-Lausitz in the Federal State of Brandenburg (Eastern Germany). This healthcare centre participated in the AGnES-Brandenburg project from July 2006 until December 2008.

Six GPs had their practices in the ambulatory healthcare centre; four of these participated in the project and delegated home visits to AGnES-practice assistants. In total, three AGnES-practice assistants, all of them qualified nurses with extended professional experience, were working full-time in the AGnES-project. The GPs decided whether or not to delegate home visits to the AGnES-practice assistants.

The number of treated patients in the participating GP-practices was calculated based on reimbursement data for all patients of the GPs of the ambulatory healthcare centre, who were insured with one of the German statutory health insurances. This was true for more than 95% of the patients of the ambulatory healthcare centre. These data were available for each of the GP-practices of the ambulatory healthcare centre. To analyze the changes in the sizes of the practices, we used absolute numbers rather than relative changes. As the same relative increase in a small and a large practice involves a larger number of patients in the large practice, and since GPs have to treat real numbers of patients, we used the absolute numbers of patients.

All participating patients gave written informed consent. The study was approved by the ethics committee of the University Medicine Greifswald, Germany (Reg.-Nr. III UV 61/06).

Results

To answer the first research question "How many additional patients can a GP theoretically include in his practice due to delegation under ideal conditions?" data of all seven AGnES-projects were used.

The main element of this analysis was the calculation of the mean capacity of AGnES-practice assistants. To calculate the real working time of the AGnES-practice assistants, 2008 was used as the reference year. This year had 104 Saturdays and Sundays, and 9 bank holidays. We assumed 31.1 vacation days, 7.3 days of sick leave [16] and 5.0 days for vocational training of the AGnESpractice assistants. Additionally, 5.0 days for administration and quality management within the GP-practice were subtracted from the total number of working days. In total, 203.6 net working days per year were calculated. Assuming a work-week of 40 hours, the average yearly working time of an AGnES-practice assistant amounts to 1,628.8 hours [17].

To calculate the yearly number of home visits that an AGnES-practice assistant can theoretically conduct, we took the mean length of all documented home visits within the AGnES-projects, which was 33.4 minutes (range 29-40.6 minutes). Additionally, we added:

driving time for each home visit (mean time 16.1 minutes; range 11.7-22.7 minutes), this includes associated drives, e.g. to the pharmacist or laboratory; consultation with the GP (for each home visit: mean time 4.6 minutes; SD 3.2; range 3.6-6.7 minutes)

time for organization and documentation (for each home visit: mean time 17.3 minutes) [17].

In total, an average of 71 minutes was needed for each home visit. With reference to the calculated annual net working time of 1628.8 hours, the mean yearly capacity of an AGnES-practice assistant corresponds to 1,376.5 home visits.

GPs conduct, on average, 1,200 home visits a year [7]. A proportion of these home visits cannot be delegated to AGnES-practice assistants, e.g. due to a medical emergency or unclear situation of a patient. Hence the full capacity of an AGnES-practice assistant (1,376.5 home visits per year) exceeds by far the number of theoretically delegable home visits. Consequently, we assumed only a halftime position of the AGnES-practice assistants (which is a 20 hours working week) corresponding to 50% of their capacity (688 home visits per year).

The mean length of a home visit, conducted by a GP in Germany is 20.6 minutes (calculated form reimbursement data of the Associations of Statutory Health Insurance Physicians from Saxony and Mecklenburg-Western Pomerania). 12.4 minutes driving time are added (based on the driving times of the AGnES-practice assistants, without considering associated drives) [17] and 3.0 minutes of documentation, giving instructions to the practice team etc. In total, a GP needs 36.0 minutes for each home visit. Assuming that the AGnES-practice assistant can conduct 688 delegated GP-home visits per year, the GP would save 412.8 working hours a year. However, according to the data from the AGnES projects the GP invests on average 4.6 minutes to consult with the AGnES-practice assistant for each delegated home visit. Consequently, 52.8 hours have to be subtracted from the GP's theoretical time gain. This leaves 360.1 GP working hours yearly that can be saved by implementing the AGnES-concept in a practice.

In this model we assume that the time a GP can save by delegating home visits to an AGnES-practice assistant is used to extend the number of treated patients per practice. In Germany, the mean time for each patient contact in a GP-practice is 7.6 minutes [18]. Assuming a mean additional time for documentation and administration of 3.0 minutes, every patient contact in a GP practice requires 10.6 minutes. Assuming an investment of all saved working time in additional patient contacts, the GP could conduct 2,038 additional patient contacts per year. On average, patients visit the GP-practice 2-3 times each quarter year [7]. This means the GP could extend his patient base by 170 additional patients per quarter year for each half time AGnES-practice assistant.

The second research question focused on the actual increase in number of patients in the participating GPpractices. We examined this question for the AGnES Brandenburg project which was conducted in the ambulatory healthcare centre with six GP-practices [10].

The four participating GPs delegated in total 4,454 home visits to 379 patients (mean age: 76.8 ± 12.7 years, range 21-100 years) to the AGnES-practice assistants. Table 1 shows the number of patients of all GPpractices of the ambulatory healthcare centre per quarter. We considered four quarter years before and eight quarter years after the implementation of AGnES. Comparing the mean numbers of patients before and after the implementation of the AGnES-project, all six GPpractices increased the number of treated patients over the project period ( Figure 1, Table 1).

In the four participating GP-practices, the mean number of patients before implementation of the AGnES-concept was 1,245 / quarter year. After implementation of the project, the mean number of patients increased to 1,378 patients / quarter year, an increase of 133, corresponding to about 78% of the theoretically possible increase of 170 patients / quarter year. In the two non-participating GPpractices, the mean number of patients before the start of the project was 415 / quarter year, during the project the mean number of patients increased to 519 / quarter year which is a plus of 104 patients / quarter year.

Discussion and conclusion

The analyses described in this paper address the eligibility of the AGnES-concept for implementation into the regular health care system in Germany. The calculation of the number of additional patients a GP can treat in his practice due to delegation of home visits to AGnES practice-assistants was modelled, using parameters from the project documentation, and from the literature. In reality every GP-practice is different, regarding both the structure of the patient base and the practice organization. Our calculation therefore is meant to provide a numerical basis for the analysis, rather than to reflect the actual situation in the participating practices.

The results of the analysis are not transferable to any region but only to regions with equal preconditions, which are rural regions with an equal level of general practitioner care, an equal age distribution of the population, and an equal distribution of the morbidity of the population.

The number of patients varies over the quarter years by up to 100% in the established practices. In some instances, the study doctors provided explanations for this, e.g. sick leaves of the GP in the 3rd and  (Table 1). Other variations may be due to a variety of reasons including sick leave of other GPs in the region, and, since the region attracts many tourists, seasonal variations in the population size.

On average, a GP-practice in Germany provides medical care for about 900 patients / quarter year. The results of our analysis show that GPs with large practices quickly adapted to the possibility to delegate home visits, which is an indication of ready acceptance of the AGnES-concept by the most important target group.

One of the four participating practices (practice 6 in Table 1), with an increase of 190 patients during the project period, exceeds the calculated possible expansion of 170 patients. Since during the project three AGnESpractice assistants were available for the six GPs in the ambulatory healthcare centre but only worked for four of them, they were more flexible with their working time, which could be an explanation for the high increase of patients in practice 6.  Although the GP-practices participating in our study had already numbers of patients above the national average before the start of the AGnES-Brandenburg project, they increased the sizes of their practices on average by 133 patients / quarter year. With this increase they reached on average 78% of the possible expansion during the project period. Various factors could be responsible for not reaching the calculated maximum increase. The model uses mean values for the included variables that can be different in the analyzed GP-practices. Also regional structures with respect to the numbers of patients in the different sectors of the health care system may be important. The age and morbidity distribution of the population are major determinants of the need for medical care.

Other reasons may be the time required to adjust to this new concept, and its integration into the work-flow of the practice. Also the fact that the GPs did not have a guarantee of continuing the delegation-concept after finishing the project could possibly have kept them from exploiting this concept to its full extent.

Delegated home visits are not identical to GP-home visits. First of all, not all GP-home visits can be delegated. Home visits with an urgent medical reason usually have to be conducted by the GP himself. And secondly, the AGnES-practice assistants recorded more driving time needed for the home visits, because they had to drive to the pharmacist, the laboratory etc. This could be an indication for delegating particularly elaborate home visits. Furthermore, the AGnES-practice assistants had to complete some tasks which are normally delegated to the practice team, such as those belonging to the preparation for or finishing of a home visit, by themselves.

This study has several methodological limitations. Most importantly, participating practices self-selected themselves into the intervention or control group. Because the allocation did not follow a randomization procedure, we cannot rule out the possibility that other factors apart from the measured factors are partly or fully responsible for the observed effects. A descriptive comparison revealed some structural differences including size and working experience of the GPs in the nonparticipating practices. Hence, the non-participating practices do not fulfil the criteria for a control group and can not readily be used to estimate the secular trend of patient numbers for the four participating practices.

In theory, it would be easier for small practices than for larger practices to increase their numbers of patients. An interesting question is why the non-participating practices are small in a region with an imminent undersupply of GPs. The GP of practice 5 had just started her practice. This would explain why her number of patients increased steadily during the observation time and reached the average numbers of patients in the other practices only in the final observed quarter year. The GP of practice 3 was absent during the first two quarter years for medical reasons. After returning to work, she had inconstant and less working hours as the other GPs and was not interested to participate in the AGnES project.

Based on the positive results and high degree of acceptance of the AGnES-concept among GPs, practice assistants, and patients [10], in 2008 a legislation amendment was passed on the federal level that explicitly allowed the delegation of GP-home visits. Since 2009, the reimbursement of delegated home visits is possible in regions that are (imminently) underserved.

The results of this explorative analysis may support the hypothesis that an increase of the number of patients after implementing the AGnES-concept is possible. To provide more conclusive evidence, a prospective clusterrandomised study with a larger number of GP-practices in comparable regions would be preferable; alternatively reimbursement data of GPs, using the new legislation, could be analyzed to examine the effects of the AGnESconcept on the health care situation in selected underserved regions.

1 .
1The county Oberspreewald-Lausitz has an imminent undersupply of GPs: in the year 2008 the degree of supply with GPs in this county was 95%. In 2011, this percentage decreased to 87%, indicating an imminent undersupply of primary medical care. A shortage of GPs is necessary to increase the number of patients not at the expense of other practices in the region. 2. The ambulatory healthcare centre in which all four participating GPs and the two non-participating GPs had their practices provided detailed data for the analysis. The research questions are: 1. How many additional patients can a GP theoretically include in his practice under ideal conditions? 2. Did the implementation of the AGnES-concept actually result in an increase in the number of patients in the participating GP-practices in the county Oberspreewald-Lausitz in the Federal State of Brandenburg?

4th quarter year of 2005, and in the first quarter year of 2007 in practice 3, and in the 3rd quarter year of 2007 in practice 2. The increase of about 1000 patients in the first quarter year of 2007 in practice 6 was caused by the presence of an additional "GP in training"

Figure 1
1Numbers of patients in the GP-practices of the ambulatory healthcare centre, before and after the implementation of AGnES-Brandenburg.

Table 1
1Numbers of patients in the GP-practices of the ambulatory healthcare centre, before and after the implementation of AGnES-BrandenburgPractice 1*
Practice 2*
Practice 3
Practice 4*
Practice 5
Practice 6*

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AcknowledgementWe would like to thank the ambulatory healthcare centre in Brandenburg for providing temporary access to their reimbursement data for this analysis.Competing interestsThe authors declare that they have no competing interests.Authors' contributions NB, CM, SF, and WH participated in the design of the study. CM and RH participated in the acquisition of data. RH performed the modelling of the capacity of the AGnES-practice assistants. NB, RH and SB performed statistical analysis. All authors participated in the interpretation of the results. NB drafted the manuscript, all authors revised it critically. All authors read and approved the final manuscript.Author information * Romy Heymann shared first authorship.
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Open Access Intra-articular injections of high-molecular-weight hyaluronic acid have biphasic effects on joint inflammation and destruction in rat antigen-induced arthritis
Published: 31 Mar 2005

Andreas Roth
Department of Orthopaedics
'Rudolf-Elle' Hospital
Friedrich Schiller University Jena
EisenbergGermany

Jürgen Mollenhauer
Department of Orthopaedics
'Rudolf-Elle' Hospital
Friedrich Schiller University Jena
EisenbergGermany

Department of Biochemistry
Rush Medical College Head
ChicagoIllinoisUSA

Andreas Wagner
Department of Orthopaedics
'Rudolf-Elle' Hospital
Friedrich Schiller University Jena
EisenbergGermany

Reneè Fuhrmann
Department of Orthopaedics
'Rudolf-Elle' Hospital
Friedrich Schiller University Jena
EisenbergGermany

Albrecht Straub
Department of Orthopaedics
'Rudolf-Elle' Hospital
Friedrich Schiller University Jena
EisenbergGermany

Rudolf A Venbrocks
Department of Orthopaedics
'Rudolf-Elle' Hospital
Friedrich Schiller University Jena
EisenbergGermany

Peter Petrow
Institute of Pathology
Friedrich Schiller University Jena
Germany

Rolf Bräuer
Institute of Pathology
Friedrich Schiller University Jena
Germany

Harald Schubert
Institute of Animal Studies
Friedrich Schiller University Jena
Germany

Jörg Ozegowski
Institute of Biochemistry 2
Friedrich Schiller University Jena
Germany

Gundela Peschel
Hans Knoell Institute for Natural Products Research
JenaGermany

Peter J Müller
Hans Knoell Institute for Natural Products Research
JenaGermany

Raimund W Kinne
Experimental Rheumatology Unit
Friedrich Schiller University Jena
Germany

Open Access Intra-articular injections of high-molecular-weight hyaluronic acid have biphasic effects on joint inflammation and destruction in rat antigen-induced arthritis
73Published: 31 Mar 200510.1186/ar1725)Received: 6 Dec 2004 Revisions requested: 23 Feb 2005 Revisions received: 23 Feb 2005 Accepted: 1 Mar 2005Available online R677 Research article Corresponding author: Andreas Roth, email@example.com
To assess the potential use of hyaluronic acid (HA) as adjuvant therapy in rheumatoid arthritis, the anti-inflammatory and chondroprotective effects of HA were analysed in experimental rat antigen-induced arthritis (AIA). Lewis rats with AIA were subjected to short-term (days 1 and 8, n = 10) or long-term (days 1, 8, 15 and 22, n = 10) intra-articular treatment with microbially manufactured, high-molecular-weight HA (molecular weight, 1.7 × 10 6 Da; 0.5 mg/dose). In both tests, 10 buffertreated AIA rats served as arthritic controls and six healthy animals served as normal controls. Arthritis was monitored by weekly assessment of joint swelling and histological evaluation in the short-term test (day 8) and in the long-term test (day 29). Safranin O staining was employed to detect proteoglycan loss from the epiphyseal growth plate and the articular cartilage of the arthritic knee joint. Serum levels of IL-6, tumour necrosis factor alpha and glycosaminoglycans were measured by ELISA/ kit systems (days 8 and 29). HA treatment did not significantly influence AIA in the short-term test (days 1 and 8) but did suppress early chronic AIA (day 15, P < 0.05); however, HA treatment tended to aggravate chronic AIA in the long-term test (day 29). HA completely prevented proteoglycan loss from the epiphyseal growth plate and articular cartilage on day 8, but induced proteoglycan loss from the epiphyseal growth plate on day 29. Similarly, HA inhibited the histological signs of acute inflammation and cartilage damage in the short-term test, but augmented acute and chronic inflammation as well as cartilage damage in the long-term test. Serum levels of IL-6, tumour necrosis factor alpha, and glycosaminoglycans were not influenced by HA. Local therapeutic effects of HA in AIA are clearly biphasic, with inhibition of inflammation and cartilage damage in the early chronic phase but with promotion of joint swelling, inflammation and cartilage damage in the late chronic phase.

Introduction

Rheumatoid arthritis (RA), a chronic systemic disease primarily affecting the joints, is characterised by progressive destruction of cartilage and bony structures of the joints [1,2]. Its social impact results from the personal suffering of patients as well as from medical and indirect costs [3]. AIA = antigen-induced arthritis; ELISA = enzyme-linked immunosorbent assay; GAG = glycosaminoglycan; HA = hyaluronic acid; HL = HA-treated AIA rats, long-term test; HS = HA-treated AIA rats, short-term test; IL = interleukin; mBSA = methylated bovine serum albumin; PBS = phosphatebuffered saline; RA = rheumatoid arthritis; TNF-α = tumour necrosis factor alpha; UL = untreated AIA rats, long-term test; US = untreated AIA rats, short-term test.

R678

Hyaluronic acid (HA) is a large linear glycosaminoglycan composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine, linked via the 1-4 position of the sugar rings [4]. The synovial fluid in the joint consists of ultrafiltrated plasma and HA, the latter being produced by type-B synoviocytes of the lining layer [5]. Inflammatory changes lead to depolymerisation of HA, resulting in a decrease of its molecular weight and its concentration [6]. Its lubricant properties decrease, contributing to the destruction of cartilage and bone [7].

HA protects cells and anatomical structures against mechanical overloading due to its viscoelastic characteristics [8]. The viscosity of the synovial fluid is reduced in patients with RA [9], a deficit that can be balanced by the supply of exogenous HA [10]. In addition, the production of endogenous synovial HA is stimulated via the supply of exogenous HA [11].

RA is characterised by a loss of proteoglycans in the affected joints [12,13]. HA possesses chondroprotective effects [10,14] and is reported to inhibit the loss of proteoglycans from the matrix of joint cartilage [15,16]. HA also blocks the loss of proteoglycans caused by the addition of catalytic cytokines to cultivated cartilage [17,18] and suppresses the degradation of cartilage matrix mediated by fibronectin fragments [19,20]. HA is also reported to protect the cartilage against proteoglycan loss, against chondrocyte cell death caused by free oxygen radicals, IL-1, or mononuclear-cellenriched medium, and against other alterations [14,15,[21][22][23][24]. Cartilage degradation induced by neutrophil leukocytes is also reduced by HA in vitro [25]. Injection of exogenous HA induces a decrease of inflammatory and proliferative processes within the synovium [26]. Also, HA inhibits the proliferation [27] and migration of white blood cells [28], and affects their adherence, chemotaxis, and phagocytosis properties [11,29,30]. Degradation of HA by reactive oxygen species, on the other hand, may reduce the protective properties of HA [14,31].

In spite of the known potential benefits of HA on a number of pathological features of RA, a general estimate of its validity for the treatment of RA is still lacking, particularly in terms of experimental studies in animal models of arthritis. The present study was therefore designed to examine the effects of HA in rat antigen-induced arthritis (AIA). This experimental monoarticular arthritis shares some characteristics of RA; for example, hyperplasia of the synovial membrane, inflammatory infiltration of the joints, and destruction of cartilage [32]. This model is also useful to characterise treatment responses; for example, the reduction of inflammation or changes in the synovial connective tissue [33].

Materials and methods

Animals

Female Lewis rats (10-12 weeks of age) were obtained from the Institute of Animal Studies, Friedrich Schiller University Jena, Germany. The rats were housed under standard conditions, in a 12-hour light/dark cycle. The animals were fed with standard rodent chow and water ad libitum. The rats were divided into two groups: non-arthritic animals (n = 6) and arthritic animals (n = 40). The latter were subdivided into the following groups (each n = 10): untreated AIA rats, short-term test (US); untreated AIA rats, long-term test (UL); HA-treated AIA rats, short-term test (HS); and HA-treated AIA rats, longterm test (HL). All animal studies were approved by the governmental committee for animal protection.

Hyaluronic acid

Pyrogen-free, sterile-filtered HA with a molecular weight of 1.7 × 10 6 Da was used, obtained by biotechnological fermentation from Streptococcus equisimilis ssp. zooepidemicus V 2541. This bacterial HA, also called non-animal-source hyaluronan, is completely identical to human HA. The content of pyrogen was minimised to less than 0.05 IE/ml HA by cleaning steps, therefore fulfilling the demands of the European Pharmacopeia (Supplement 2001, page 1472). The zero-viscosity of the purified 1.0% high-molecular-weight HA (molecular weight, 1.7 × 10 6 Da) in 0.9% NaCl solution amounted to h0 = 10.74 Pa s. The injection units contained 10 mg HA in 1 ml of 0.9% NaCl.

Induction of AIA

All experimental animals were immunised by two subcutaneous injections (days -21 and -14) of 0.5 g methylated bovine serum albumin (mBSA), dissolved in 0.5 ml saline and emulsified with 0.5 ml complete Freund's adjuvant [32,34]. Knee monoarticular arthritis was induced 2 weeks after the second immunisation via a single joint injection of 0.5 mg mBSA (50 µl of 10 mg/ml mBSA dissolved in 0.9% NaCl) into the right knee joint (day 0 of AIA). The left knee remained without injection.

Treatment with HA

On day 1 of AIA, all 40 arthritic animals received an intra-articular injection into the right inflamed knee joint. HA-treated AIA rats (groups HS and HL) received in each case 0.5 mg HA (50 µl of 10 mg/ml HA in 0.9% NaCl), whereas the untreated AIA rats (groups US and UL) received 50 µl PBS. The AIA rats of the long-term test received further injections at the beginning of each subsequent week (days 8, 15, and 22): the HL group received 50 µl HA, and the UL group received 50 µl PBS.

The short-term test (groups US and HS) was terminated 1 week after the first injection of HA or PBS (day 8). The longterm test (groups UL and HL) was terminated 1 week after the fourth injection (day 29).

In all cases, the contralateral (left) knee joint remained untreated. The group of six non-arthritic animals without AIA (12 weeks of age) served for the collection of normal values.

All injections (including those necessary to induce immunisation and knee AIA) were performed under ether anaesthesia. At the end of the experiment, the animals were sacrificed using an overdose of CO 2 and cervical dislocation.

Collection of samples

Blood samples were collected by heart puncture after opening the thorax. The blood was centrifuged for 10 min at 3000 × g and ambient temperature. The serum was divided into three portions of at least 250 µl and was frozen at -80°C until analysis.

The knee joints were disconnected from the long bones and stored in 6% formaldehyde. In order to ensure an optimal impregnation with formaldehyde, the adhering remainders of the long bones were kept very short (approximately 1.0 cm above and below the joint space) and the dorsal joint capsule was opened.

Evaluation of arthritis

Joint swelling, body weight, and the general state of the animals were regularly monitored. The measurements of weight and mediolateral joint diameter took place on days 0, 1, 4, 8, 15, 22, and 29. The mediolateral joint diameter was measured using a vernier caliper [32,34].

Histological analyses

All preparations were stored in 6% formaldehyde for 24 hours. Decalcification in ethylenediamine tetraacetic acid subsequently took place and the preparations were embedded in paraffin. After the removal of paraffin, 5-µm thick sections were cut [35].

For the assessment of the histological arthritis scores, the sections were stained with haematoxylin and eosin. All slides were evaluated by an independent observer who was blinded to the design and details of the study. In all cases, three sections per knee joint were examined and scored using a semiquantitative scale.

The extent of acute joint inflammation -as defined by the degree of infiltration of the synovial membrane by polymorphonuclear leukocytes, and defined by the exudation of granulocytes in the joint space -was evaluated in each case with 0 = no changes, 1 = mild changes, 2 = moderate changes, and 3 = severe changes. In addition, the presence (score 1) or absence (score 0) of fibrin exudation in the joint space and periarticular inflammation was assessed, resulting in a maximum total score of 8 for acute inflammation.

Chronic joint inflammation -based on the parameters hyperplasia of synovial lining cells, infiltration by mononuclear cells, and fibrosis of synovial membrane or periarticular tissue -was evaluated with a score of 0-3, resulting in a maximum total score of 9.

The extent of the damage to articular cartilage and adjacent bone structures (cell necrosis, structural bone, and cartilage defects) was evaluated with score 0 = no damage, score 1 = <5% of the cartilage surface affected, score 2 = 5-10% of the cartilage surface affected, score 3 = 10-50% of the cartilage surface affected, and score 4 = >50% of the cartilage surface affected (maximal total score of 4).

Safranin O staining was performed to estimate the proteoglycan content in the cartilage [36][37][38]. In order to obtain comparable histological results, all slides were stained using exactly the same procedure [39]. The preparations were analysed under defined conditions using a Zeiss microscope Axiovert 200 M (20 × magnification) (Carl Zeiss, Göttingen, Germany)] and the results were stored as pixel pictures. The staining intensity was determined in 175 × 25 mm 2 areas, using Scion Image software (Scion Corporation, Frederick, MD, USA).

First, the staining intensity (red) at the epiphyseal growth plate of the femoral condyle of non-arthritic and arthritic animals was measured (maximum value 255). The arithmetic mean obtained from these values was used as a reference value (232 [= 100%]). The measurements of articular cartilage took place at the most distal point of the curvature of the femoral condyle. In each case, values were obtained for the superficial layer, middle layer, and deep layer of the hyaline cartilage, as well as for the calcified cartilage layer (Fig. 1). Data were expressed as a percentage of the reference value. Subsequently, the values of the contralateral, non-arthritic knee joint (left) were subtracted from the arthritic knee (right), resulting in negative values in the case of proteoglycan loss.

Cytokine and serum glycosaminoglycan evaluation

The serum levels of IL-6, tumour necrosis factor alpha (TNF-α) and glycosaminoglycan (GAG) were determined at the end point of the short-term test (day 8) and at the endpoint of the long-term test (day 29).

The serum levels of IL-6 and TNF-α were determined using a commercial sandwich ELISA kits for rats according to the manufacturer's instructions (Biosource International, Camalliro, CA, USA). The detection limits were 8 pg/ml for IL-6 and 4 pg/ml for TNF-α. According to the manufacturer, there was no cross-reactivity with other rat cytokines.

The serum levels of total GAG were measured in non-diluted serum with a commercially available kit. The standard values for healthy rats were 10.8-17.4 mg/l (Glycane T Labor + Diagnostica, Freital, Germany).

Statistics

Statistical evaluations were carried out using the programme SigmaStat 2.0. Since nearly all data were not normally distributed, the non-parametric Mann-Whitney U test was used. Data were expressed as means and standard errors of the means. P ≤ 0.05 was considered statistically significant for α.

In cases in which P values for α were at the limits of significance (0.05 ≤ P ≤ 0.1; joint swelling day 29, cartilage damage day 8), the statistical power of the U test was determined using the actual difference at a given time point as delta.

Because for the time period from day 0 to day 8 the procedure and results did not differ between the US and UL groups or between the HS and HL groups, respectively, the values from the short-term test and the long-term test were pooled for statistical evaluation of this period in both cases.

Results

Body weight

At baseline, the body weight was 188 ± 29 g (untreated AIA rats) and 197 ± 22 g (HA-treated AIA rats). After a plateau between day 0 and day 8 in both untreated rats and HAtreated AIA rats, the body weight rose in concomitance with the decrease of arthritis severity. At the end of the long-term test (day 29), the animals weighed 213 ± 15 g (untreated AIA rats) and 230 ± 13 g (HA-treated AIA rats). The differences between the groups did not reach statistical significance at any time point.

Joint swelling

On day 1, AIA developed as a significant swelling of the right knee joint in all animals (Fig. 2). The swelling increased up to day 4 in untreated AIA rats (P < 0.001, n = 20), significantly decreasing on day 8 (P < 0.001, n = 20). The swelling then continued to slowly decrease until day 29 (P < 0.001, n = 10). At all time points after initiation of AIA, the swelling remained significantly higher compared with the baseline levels on day 0 (Fig. 2).

Intra-articular treatment with HA did not significantly affect the degree of joint swelling on days 1, 4, and 8 ( Fig. 2). On day 15 (groups UL and HL, n = 10 each) there was a significant reduction of joint swelling in the HA-treated AIA group compared with the untreated AIA group (P < 0.05). On day 22 the swelling was no longer significantly different from the untreated AIA group (P = 0.37); in fact, it was even somewhat higher. On day 29 (end of the long-term test) the small increase of joint swelling in the HA-treated AIA group persisted (as compared with the untreated AIA group), although without reaching statistical significance (power 1 β = 0.851).

In general, therefore, HA seemed to positively affect the early chronic phase of AIA (day 15), but did not have an influence on the acute or late chronic phases of AIA, at least in terms of joint swelling. Measurement frames for Safranin O staining of the knee joint cartilage Measurement frames for Safranin O staining of the knee joint cartilage. After elimination of green tones and transformation of all red tones into grey tones, the staining intensity (a measure of the proteoglycan content) was determined in the following layers: S, superficial layer; M, middle layer; D, deep layer; and C, calcified cartilage.

Figure 2

Time course of knee joint swelling Time course of knee joint swelling. Joint swelling (difference between the bilateral diameter of the right knee and the left knee) in untreated antigen-induced arthritis (AIA) rats and in hyaluronic acid (HA)-treated AIA rats. V, end of the short-term test (day 8) and end of the long-term test (day 29). The arrows indicate the days of intra-articular injection of HA (days 1, 8, 15, and 22). In the short-term test there was no significant difference between HA-treated rats and untreated AIA rats. In the long-term test HA-treated AIA rats showed significantly reduced values on day 15 (* P < 0.05). On day 29 there were no longer differences between the two groups; if at all, the swelling in the HA-treated group was somewhat higher than in untreated AIA group.

Loss of proteoglycans from the epiphyseal growth plate of the femoral condyle and the articular cartilage

In the short-term test (day 8), the untreated AIA group was characterised by a significant decrease of the proteoglycan content in the epiphyseal growth plate of the arthritic right knee compared with the contralateral knee or with the right knee joint of non-arthritic animals (in both cases, P < 0.05; Fig.  3). Treatment with HA prevented this loss, maintaining proteoglycan levels close to those of non-arthritic animals.

In terms of individual zones of the articular cartilage, the untreated AIA rats underwent a change of -37% in the superficial layer, -26% in the middle layer, -13% in the deep layer, and -15% in the calcified cartilage layer (Figs 4b,d and 5). At this time point, treatment with HA was significantly effective in preventing the proteoglycan loss in the superficial layer (P < 0.01), the middle layer (P < 0.05), and the calcified cartilage layer (P < 0.05; Figs 4b,f and 5). In all layers, the proteoglycan content reached normal levels.

In the long-term test (day 29) there was no significant loss of proteoglycan content in the epiphyseal growth plate of untreated AIA rats (see Fig. 3). However, treatment with HA was characterised by a significant proteoglycan loss in the growth plate of the arthritic right knee compared with the con-tralateral knee or with the right knee joint of non-arthritic animals (P < 0.001; Fig. 3).

In the different layers of the articular cartilage, the untreated AIA rats no longer showed any significant proteoglycan loss; that is, there were no significant differences between the right knee joints and left knee joints of AIA rats, or between the right knee joint of AIA rats and the right knee joint of non-arthritic animals ( Fig. 5; Safranin O staining data not shown). Treatment with HA did not significantly affect the proteoglycan content in any layer of the articular cartilage. Safranin O staining intensity in the epiphyseal growth plate of the femo-ral condyle Safranin O staining intensity in the epiphyseal growth plate of the femoral condyle. The reference value of 232 (100%; continuous line) was obtained by computing all available values from both non-arthritic rats and antigen-induced arthritis (AIA) rats. In untreated AIA rats, the right (arthritic) joint showed a significant reduction of proteoglycan content of the epiphysis in the short-term test (day 8; *P < 0.05). This loss was not observed following hyaluronic acid (HA) treatment (day 8). The latter values were comparable with non-arthritic animals and with the contralateral joint (data not shown). Long-term treatment with HA (day 29) induced a significant loss of proteoglycans in the epiphyseal growth plate (*** P < 0.001). In contrast, the arthritic joints of untreated AIA rats showed values comparable with non-arthritic rats and contralateral joints (not shown).

Histological scores of arthritis and cartilage damage

In the short-term test (day 8) there was a strong acute inflammation in untreated AIA rats (Fig. 6a). Treatment with HA significantly reduced the acute inflammation compared with the untreated AIA group (P < 0.05; Figs 4a,c,e and 6a). Notably, the untreated AIA group underwent a complete, spontaneous remission of the acute inflammation score from day 8 to day 29 (acute inflammation score nearly 0 in the long-term test; Figs 4c,g and 6a). The HA-treated AIA group in the long-term test clearly improved compared with the short-term test (P < 0.05), but it still showed significantly higher, residual acute inflammation than the untreated AIA group (P < 0.05; Figs 4e,g,h and 6a).

In the short-term test (day 8) a clear score for chronic inflammation was also observed (Figs 4a,c,e and 6b), without significant differences between untreated and HA-treated AIA groups. The chronic inflammation significantly decreased from day 8 to day 29 in untreated and HA-treated AIA (group US versus group UL, P < 0.001; group HS versus group HL, P < 0.01; Figs 4c,e,g,h and 6b). Unexpectedly, however, on day 29 the chronic inflammation score was more pronounced in Comparisons were made for different layers (S, superficial layer; M, middle layer; D, deep layer; and C, calcified cartilage) between nonarthritic rats, untreated antigen-induced arthritis (AIA) rats, and hyaluronic acid (HA)-treated AIA rats in terms of relative differences between right (arthritic) and left (contralateral) joints. In the short-term test (day 8) untreated AIA rats showed a reduced proteoglycan content in the superficial layer (** P = 0.01), middle layer (* P < 0.05), and calcified cartilage (* P < 0.05). HA-treated AIA rats showed proteoglycan contents comparable with those of non-arthritic rats in all layers. In the long-term test (day 29), untreated AIA rats also showed reduced proteoglycan contents in all layers compared with non-arthritic rats, but no statistical significance was attained. HA-treated AIA rats showed proteoglycan contents comparable with those of non-arthritic rats. Safranin O staining intensity of the articular cartilage untreated AIA rats HA-treated AIA rats nonarthritic rats

Figure 6

Histological scores Histological scores. In the short-term test, hyaluronic acid (HA)-treated antigen-induced arthritis (AIA) rats showed a significant reduction of (a) the acute inflammation score compared with untreated AIA rats (* P < 0.05). The score of (b) chronic inflammation and (c) cartilage damage did not show significant differences between HA-treated rats and untreated AIA rats. In the long-term test, (a) the acute inflammation was reduced in both AIA groups compared with that in the short-term test (HA-treated AIA rats, P < 0.05; untreated AIA rats, P < 0.001; significance not indicated); nonetheless, the HA-treated AIA rats showed significantly higher scores than untreated AIA rats on day 29 (* P < 0.05).

(b) The scores of chronic inflammation were reduced in both AIA groups compared with the short-term test (untreated AIA rats, P < 0.001; HA-treated AIA rats, P < 0.01; significance not indicated); nonetheless, the HA-treated rats showed significantly higher scores than untreated AIA rats on day 29 (* P < 0.05). (c) The cartilage damage was relatively low in both untreated rats and HA-treated AIA rats, but HA-treated AIA rats showed a significantly higher damage (* P < 0.05). In terms of cartilage damage, the untreated AIA group was characterised by a maximum individual score of 3; that is, the maximal possible score of 4 was not observed (Fig. 6c). On day 8, the mean cartilage damage was somewhat more pronounced in the untreated AIA group, but without significant differences in comparison with the HA-treated AIA group (power 1 -β = 0.821). From day 8 to day 29, the cartilage damage decreased significantly in untreated rats and HA-treated AIA rats (group US versus group UL, P < 0.001; group HS versus group HL, P < 0.01). In the long-term test (day 29), however, the cartilage damage was significantly higher in the animals treated with HA than in the untreated AIA group (P < 0.05; Fig.  6c).

Systemic cytokine levels

In non-arthritic animals, the serum IL-6 levels were below the detection limit of the assay (Fig. 7a). Untreated AIA rats had significantly elevated IL-6 levels both in the short-term test and in the long-term test (P < 0.001 in both cases; significance not indicated in Fig. 7). Treatment with HA did not significantly influence IL-6 levels at either time point (Fig. 7a).

As for TNF-α, non-arthritic animals had mean serum levels of 5.45 ± 4.56 pg/ml (Fig. 7b). In the short-term test these values were increased both in the untreated and in the HA-treated AIA groups, but not to a significant degree. In the long-term test, the mean TNF-α levels were very similar to those of nonarthritic animals. Treatment with HA did not significantly influence TNF-α levels at either time point (Fig. 7b).

Serum GAG levels

In non-arthritic animals, the mean serum levels of GAG were 12.70 ± 3.30 µg/ml (Fig. 7c). Untreated AIA rats had significantly higher GAG levels than non-arthritic animals in the short-term test and in the long-term test (P < 0.05 and P < 0.001, respectively; significance not indicated in Fig. 7). Treatment with HA had no influence on this parameter at either time point (Fig. 7c).

Discussion

Clinical parameters of arthritis

The time course of AIA was similar to that described by other authors [32,34], confirming that the present results were representative of previous studies.

Treatment with HA did not reduce joint swelling in the acute phase, as significant reduction of joint swelling was found only on day 15 (i.e. in the early chronic phase of AIA). The temporary reduction of joint swelling may be a result of the reduced acute inflammation observed histologically at an earlier time point (day 8). This anti-inflammatory effect of HA is consistent Serum levels of IL-6, tumour necrosis factor alpha (TNF-α) and glycosaminoglycan Serum levels of IL-6, tumour necrosis factor alpha (TNF-α) and glycosaminoglycan. During the course of antigen-induced arthritis (AIA), (a) IL-6 levels in non-arthritic rats showed values below the detection limit of the assay. Untreated and hyaluronic acid (HA)-treated AIA rats showed significantly increased values in the short-term test and in the long-term test compared with non-arthritic rats (all P < 0.001; data not shown), but HA treatment resulted in IL-6 levels comparable with those of untreated AIA. (b) Regarding TNF-α levels, the only change was a short-term, non-significant increase in all AIA rats, whether HA-treated or untreated. (c) Serum values of glycosaminoglycan increased significantly in all AIA rats compared with non-arthritic animals (non-arthritic animals versus untreated AIA rats [short-term test], P < 0.05, nonarthritic animals versus HA-treated AIA rats, untreated AIA rats [longterm test] and HA-treated AIA rats [long-term test], P < 0.001; significance not indicated). There were no differences between short-term tests and long-term tests, or between HA-treated rats and untreated AIA rats. with the effects previously reported in collagen-induced arthritis [22,40] and human RA [41,42].

Interestingly, however, while the joint swelling continued to progressively and spontaneously decrease in untreated AIA, it persisted in HA-treated animals after day 15, although the difference remained at the limits of significance (P = 0.06).

The persistence of inflammation after prolonged application of HA (day 29) is further substantiated by significant proteoglycan loss in the epiphyseal growth plate, by significant persistence of both acute and chronic inflammation, and by significantly increased histological signs of articular cartilage damage. This biphasic course regarding joint inflammation and destruction after repeated application of high-molecularweight HA has not been previously described. HA therefore probably shows only a limited temporal window of anti-inflammatory activity in arthritis.

Adverse reactions to HA have been described in human osteoarthritis, either after the first injection and subsequent intraarticular injections or at the beginning of a new treatment course. These adverse events consisted of pain and/or transient swelling of the injected joint, mostly mild or moderate in intensity [43][44][45]. The adverse reactions observed upon intraarticular treatment of human osteoarthritis are not comparable with the biphasic effects in the present study, since they were only short-lasting and limited to a period immediately following injection.

Proteoglycans in the epiphyseal growth plate of the femoral condyle and the articular cartilage AIA was accompanied by a significant loss of proteoglycans in the epiphyseal growth plate of untreated AIA rats in the shortterm test (significantly ameliorated by HA; day 8). This proteoglycan loss is probably caused by the inflammatory micromilieu in the arthritic joint [46] and the adjacent periarticular bone marrow [47], in analogy to the alterations of the primary and secondary spongiosa in AIA [48]. The significant prevention of proteoglycan loss from the epiphyseal growth plate by HA at this time point may be due to the clear anti-arthritic effects of HA, as also documented by the significant decrease of acute inflammation on day 8 (Fig. 6a). Whether intra-articular treatment with HA indirectly influences the inflammatory changes in the bone marrow, thereby preventing proteoglycan loss in the epiphyseal growth plate, remains to be investigated.

Unexpectedly, repeated intra-articular HA treatment induced proteoglycan loss in the epiphyseal growth plate in the longterm test (day 29). This late loss of proteoglycans in the epiphysis under prolonged HA treatment suggests late proinflammatory effects of HA, as also indicated by significantly elevated levels of acute and chronic inflammation, as well as cartilage damage (Fig. 6).

Regarding the proteoglycan content within the articular cartilage, HA completely prevented the proteoglycan loss in the short-term test. This applied to the severe loss of proteoglycans in the superficial layer -the layer most strongly affected in the present study (37%) and also that most strongly affected in the fibronectin-mediated arthritis model [19]. This prevention also applied to deeper layers of the calcified cartilage, however, indicating either deep-reaching effects of intraarticular HA (up to 100 µm; see Fig. 1) or an indirect effect on the micromilieu in the epiphyseal bone core. Such profound chondroprotective effects of HA have not previously been reported in an in vivo arthritis model. Previous in vitro studies, in which the proteoglycan release from the cell matrix of bovine chondrocyte cultures was inhibited by HA, have suggested a covering of the matrix of as a mechanism for the chondroprotective capacity of HA [19].

Acute and chronic inflammation, and cartilage damage

HA reduced the histological signs of acute inflammation in the short-term test (day 8). At this point, HA also showed a tendency (P = 0.06) to protect the joint again against cartilage damage (Fig. 6c). The tendency for decreased cartilage damage in the short-term test supports the assumption that HA forms a temporary protecting barrier over the cartilage, and thereby protects it against degradation [16,19,49]. The known reduction of free oxygen radicals [31], as well as the reduction of cytokines and other mediators of acute inflammation by HA [15,21], could contribute to both its chondroprotective capacity (Fig. 6c) and its anti-inflammatory effects (Fig. 6a) in early chronic AIA (day 8).

In the long-term test (day 29) HA treatment was associated with significantly higher histological signs of acute and chronic inflammation and, more importantly, with more severe cartilage damage. The late proinflammatory effects of repeated HA application, in parallel to the late loss of epiphysis proteoglycans and late damage of articular cartilage (see earlier), point to an interdependence of inflammation and damage in HAtreated AIA rats at this stage.

Significantly higher histological signs of articular cartilage damage after repeated HA application (day 29) are in contrast to a non-altered proteoglycan content of the articular cartilage at this time (Fig. 5). A seemingly normal proteoglycan content may therefore not be sufficient to exclude structural damage of the articular cartilage in arthritis. Consequently, more sensitive in vivo procedures will have to be established to reliably assess functional or structural cartilage alterations before irreversible damage occurs [50].

Serum levels of cytokines and GAG

In the present study, the systemic levels of IL-6, TNF-α and GAG were not significantly influenced by intra-articular administration of high-molecular-weight HA, indicating that both anti-inflammatory effects (day 8) and proinflammatory effects (day 29) are locally restricted.

Conclusion

In conclusion, HA appears to have a limited therapeutic window for local treatment of arthritis, as shown by amelioration of clinical signs (day 15), by prevention of proteoglycan loss in the articular cartilage and epiphyseal growth plate, and by prevention of structural cartilage damage, as well as by the reduction of acute inflammation in the arthritic joint (day 8).

Late aggravation of clinical signs (not significant, day 29), proteoglycan loss from the epiphyseal growth plate, and acute/ chronic inflammation and structural cartilage damage at this time point strongly indicate biphasic effects of local HA treatment.

Whether these biphasic effects are due to accumulation of HA beyond pathological levels (which may be avoidable by single injection instead of repeated injection) or whether certain phases of the clinical course render the animals sensitive to the proinflammatory effects of HA remains the subject of future research, both in animal models and in human RA.

Figure 1
1Figure 1

Figure 3
3Figure 3

Figure 4
4Histological findings in synovial tissue and articular cartilage: Histological findings in synovial tissue and articular cartilage: haematoxylin and eosin (HE) staining (a, c, e, g, and h) for acute inflammation (arrowheads), chronic inflammation (*), and cartilage damage (arrows), as well as Safranin O staining (b, d, and f) for proteoglycan depletion (arrows). Images are shown for non-arthritic rats (a and b), untreated antigen-induced arthritis (AIA) rats (day 8, c and d; day 29, g), and hyaluronic acid hyaluronic acid (HA)-treated AIA rats (day 8, e and f; day29, h

Figure 5 Safranin
5O staining intensity in different layers of the articular cartilage Safranin O staining intensity in different layers of the articular cartilage.

Figure 7
7Figure 7

). The bar indicates the distance in the histological section.SM, synovial membrane; P, patella; FE, femur. Safranin O staining: S,
superficial layer; M, middle layer; D, deep layer; and C, calcified carti-
lage. N, non-arthritic rats; US, untreated AIA, short-term test; HS, HA-
treated AIA, short-term test; UL, untreated AIA, long-term test; HL, HA-
treated AIA, long-term test.

AcknowledgementsThe authors are grateful to Dr Frank Brand, Mrs K Neumann, and Mrs K Axt (Department of Clinical Chemistry and Laboratory Diagnostics, Rudolf-Elle Hospital) for determination of laboratory values, and to Cordula Müller and Jana Schömburg (Research Department, Rudolf-Elle Hospital) for preparation of the histological sections. They are also grateful to Dr A Notni, Dr K Bergmann, and Dr R Winter (Department of Orthopaedics, Rudolf-Elle Hospital), as well as to Dr C Wicher and Mrs P Dobermann (Institute of Animal Studies, Friedrich-Schiller University Jena) for assistance in the animal experiments.Competing interestsThe co-authors J-H Ozegowski, P-J Müller, S Möller, G Peschel, A Roth, and RA Venbrocks published a patent for the use of sulfated hyaluronic acid for the prevention of inflammatory arthritis in 2000. This substance is not the same as that used for the experiments in the present study. A Roth has to publish papers in peer-reviewed journals as a part of the process to finish his thesis (habilitation in Germany). This is a non-financial academic interest.Authors' contributionsAR carried out all experiments, the measurements and evaluation of the data, as well as the statistics, and drafted, revised, finalised, and submitted the manuscript. JM developed the experimental basis for the measurements of proteoglycans in the serum and supervised the analysis, trained and supervised AR in the analysis of proteoglycan loss from cartilage, actively participated in the analysis and evaluation of the data, and reviewed and contributed to the final version of the manuscript. AW participated in the preparation of the animals, fixation of the specimens, and blood collection, and reviewed the manuscript. RF actively participated in the experimental and organisational design of the study, gave valuable advice for the evaluation and interpretation of the experimental results, and reviewed the manuscript. AS reviewed all experimental data, gave valuable advice for the evaluation and interpretation of the experimental results and the subsequent conclusions, and reviewed the manuscript. RAV participated in the experimental and organisational design of the study, supervised the evaluation and interpretation of the experimental results, reviewed the manuscript, and organised financial support and laboratory space for the experiments. PP performed and summarised all histological analyses, and reviewed the manuscript. RB actively participated in the underlying animal experiments and the evaluation of the experimental results, and reviewed and contributed to the final version of the manuscript. HS actively participated in the underlying animal studies, provided and monitored all experimental animals, and reviewed the manuscript. JO, GP, and PJM developed the method for the production of HA, produced, purified, and quality-controlled the microbially manufactured, high-molecular-weight HA, and reviewed the manuscript. RWK participated in the experimental design of the study and the interpretation of the results, and reviewed and contributed to the initial and final version of the manuscript.
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