Different doses of hyaluronic acid injections in patients with knee osteoarthritis: A multicenter,randomized,prospective,single-blind,clinical study

Abstract

BACKGROUND:

Many formulations and dosing regimens are available for hyaluronic acid (HA).

OBJECTIVE:

To compare different doses of linear, high-molecular weight (HMW) HA injections among patients with knee osteoarthritis (OA).

METHODS:

Hundred patients were included in this randomized, single-blinded trial and randomly divided into three HA injection groups. The first group received five weekly 20 mg HA injections, the second group received three weekly 32 mg HA injections, and the third group received a single 48 mg HA injection. Patients were evaluated at baseline, 1, 3, and 6 months after the last injection for pain, stiffness, and function using the visual analog scale (VAS) and the Western Ontario and McMaster Universities Arthritis Index (WOMAC) questionnaire. Quality of life, patient’s global assessment, and Timed Up and Go (TUG) test scores were also evaluated.

RESULTS:

There was significant improvement in the WOMAC, VAS-pain, quality of life, patient’s global assessment, and TUG test mean scores at all follow-up time points ( $p<$ 0.001). However, the groups showed no significant differences in WOMAC, VAS-activity pain, and patient global scores at any follow-up point.

CONCLUSION:

Intra-articular injections of different doses of linear HMW HA can improve pain, stiffness, function, and quality of life in patients suffering from knee OA over a six-month period.

Keywords

Hyaluronic acid osteoarthritis knee intra-articular injection

1. Introduction

Knee osteoarthritis (KOA) is a common progressive multifactorial joint disease characterized by progressive cartilage degeneration and inflammation [1]. KOA is one of the leading causes of functional impairment in older adults and accounts for approximately four-fifths of the OA burden worldwide [2]. Approximately 654.1 million individuals aged 40 years and older had knee OA worldwide in 2020 [3]. However, the combined effects of ageing and increasing obesity in the global population are making KOA a more prevalent condition.

The optimal conservative management of KOA requires a combination of pharmacological and non-pharmacological treatment modalities tailored to individual patient needs. The main goals of conservative treatment are to reduce pain and improve function and quality of life. Various conservative treatment methods are recommended in the guidelines for KOA [4, 5]. Among these therapies, intra-articular (IA) injections are attractive in patients with KOA and serve as valid therapy approaches by maximizing the drug effects locally within the joint and limiting systemic adverse events [6]. One well-established and useful treatment option for the management of patients with knee OA is viscosupplementation via intra-articular injections with hyaluronic acid (IA-HA) [7].

Hyaluronic acid (HA) is a natural glycosaminoglycan and the main component of the extracellular matrix of articular cartilage and synovial fluid. HA has viscoelastic properties that contribute to its role in maintaining synovial fluid viscosity and the biomechanical integrity of cartilage [7, 8]. IA-HA injections produce beneficial effects on knee OA through several mechanisms of action. Although chondroprotection is the most frequently reported mechanism, IA-HA can also promote proteoglycan and glycosaminoglycan synthesis, and exert anti-inflammatory, mechanical, subchondral, and analgesic effects [9]. The benefits of HA extend beyond its joint cushioning and lubrication effects to possible disease-modifying effects, such as reduction in synovial inflammation, protection against cartilage erosion, and stimulation of natural HA production [8, 9, 10]. HA itself also has indirect and direct analgesic activity in the joints. The indirect effect arises due to the anti-inflammatory properties of HA, while the direct effect is provided by the inhibition of nociceptors and a reduction in bradykinin and substance P synthesis [11].

Numerous studies have examined the efficacy of IA-HA injections in patients with KOA and have compared their effectiveness to that of other IA injections [6]. A Cochrane systematic review, which evaluated the efficacy of viscosupplementation against a placebo, showed that IA-HA injections were effective in pain reduction, increased function, and improved patient global assessment for KOA [12]. Additionally, IA-HA products are well tolerated and have a low incidence of side effects [13, 14]. However, the currently available guidelines and more recent systematic reviews have provided conflicting recommendations regarding the use of IA-HA [4, 5, 9, 15]. Nevertheless, IA-HA is considered a reasonable alternative treatment for patients with KOA who have not responded sufficiently to previous pharmacologic treatments [16]. Moreover, a good evaluation, based on both an accurate analysis of radiological features and symptoms and a careful clinical examination, could improve the chances of success of IA-HA injections [17].

IA-HA preparations differ in many characteristics, including the origin of the HA, its molecular weight (MW) and structure, and the chosen concentration, injection volume, and dosing schedule. Many HA products are now commercially available for the treatment of KOA, creating a need for studies investigating the choice of the viscosupplement and the dosing regimen [17]. The objective of the present study was to analyze the efficacy of using a linear high molecular weight (HMW) HA at different concentrations and to evaluate the comparative effectiveness of multiple-injection versus single-injection formulations for the treatment of symptomatic knee OA.

2. Methods

2.1 Study design and participants

This multicenter, randomized, prospective, single-blind interventional trial recruited 168 consecutive patients with KOA. The consecutive recruitment was conducted at the outpatient physical medicine and rehabilitation departments of three different centers from November 2020 to April 2021. Among the 168 patients, 100 patients with KOA who met the eligibility criteria were included in the study. They were randomly allocated to one of three groups by an independent blinded researcher using computer-generated random numbers and an allocation ratio of 1:1:1. The three groups were a Reviscon Mono 48 mg 2.0% group (48 mg HA, $n=$ 35), a Reviscon Plus 32 mg 1.6% group (32 mg HA, $n=$ 32), and a Reviscon 20 mg 1.0% group (20 mg HA, $n=$ 33). All the products were provided free of charge by VSY Biotechnology.

The inclusion criteria were as follows: adults aged 45–75 years with bilateral symptomatic knee pain for at least three months, grade 2 or 3 (mild or moderate) KOA according to Kellgren-Lawrence classification, and able to walk independently. The exclusion criteria were as follows: IA injection to the knee during the past six months; a history of knee trauma or severe meniscus or ligament injuries that could lead to knee pain or surgery; a previous diagnosis of a neuromuscular, infectious, or inflammatory disease; a body mass index above 35 kg/m²; and patients on anticoagulant drugs (low molecular weight heparin, warfarin) or with bleeding disorders. The patients were informed verbally and in writing about the purpose, duration, and methodology of the study, and all patients provided written informed consent. The study protocol was approved by the institutional review board and was registered on ClinicalTrials.gov (NCT04786613).

2.2 Interventions

In the first group, a linear 20 mg HA (1.0%) as a 2.4 mL solution with a viscosity of 35,000 mPa $\cdot$ s (Reviscon^®, VSY Biotechnology GMBH, Germany) was administered as five injections at one-week intervals. In the second group, patients received injections of a linear 32 mg HA (1.6%) as a 2 mL solution with a viscosity of 250,000 mPa $\cdot$ s (Reviscon Plus^®, VSY Biotechnology GMBH, Germany) at three weekly sessions. The third group received a linear 48 mg HA (2.0%) as a 2.4 mL solution with a viscosity of 900,000 mPa $\cdot$ s (Reviscon Mono^®, VSY Biotechnology GMBH, Germany) was administered as a single IA knee injection. Each product consists of a solution of viscoelastic sodium HA produced by bacterial fermentation, and the purified material has a molecular weight of 3.0M Da.

Figure 1.

Participant flow and study profile.

Under sterile conditions, the HA product was injected into the patellofemoral joint space with a 20 G needle using a supero-lateral approach, with the patient in the supine position and the knee joint in 20 degrees of flexion. Before injection of HA, 5 cc of saline (0.9% sodium chloride) solution was injected, and back-flow was observed. After the needle was placed in the IA position, the HA was applied. The treatments were 20 mg HA repeated once a week, 5 times in total; 32 mg HA once a week, 3 times in total; or 48 mg HA received as a single injection. A period of 24 h rest, along with 15-minute cold therapy 3 times a day and restricted weight-bearing over knee joints, were recommended. Hamstring and quadriceps stretching, quadriceps isometric strengthening, and short arc terminal extension exercises as 3 sets of 10 repetitions at least three days a week were performed by all patients included in the study as a home exercise program. The exercise program was followed up with weekly phone calls. During the follow-up period, the patients were asked to continue their current medical treatments, not to make any changes in their daily life activities, and not to start any other treatment related to the knee area. The Consolidated Standards of Reporting Trials flow diagram proposed for randomized controlled trials of non-pharmacologic treatment, including the reasons for withdrawals/dropouts in the randomized groups, is presented in Fig. 1.

2.3 Outcome measures

Demographic information of the patients was recorded. Rest and activity pain were evaluated using a Visual Analogue Scale (VAS); pain; stiffness and physical function were assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC); functional mobility was measured using Timed Up and Go Test (TUG); and quality of life and patient global assessment were determined using numeric rating scale at baseline and at 1, 3, and 6 months after the treatment. Treatment related adverse events were also questioned. All assessments were made by the same investigator blinded to randomization.

The mean pain level rated by the patients during the last week during activity and at rest was evaluated with the VAS [18], with the number zero indicating no pain and the number 10 referring to severe pain. The quality of life was evaluated with a numerical scale between 0 and 10. Zero indicates that the knee pain complaint has no effect on daily activities, while 10 indicates that the effect is evident in daily living activities. Patient global assessment was evaluated using the numeric scale and scored between 0 and 10, where 0 indicates the best global health and 10 indicates the worst global health. Pain, stiffness, and physical functions were evaluated using the WOMAC questionnaire, one of the most commonly used assessment methods for patients with KOA. The patients answered the questions in the three sections using a 0–4 point Likert scale, and the WOMAC total score was calculated by summing the scores obtained for each section. Higher scores indicated worse pain, more stiffness, and greater functional limitations [19]. The national validity and reliability of the WOMAC have been confirmed. [20]. The Timed Up and Go (TUG) test requires that the person get up from a standard chair, walk 3 m at a comfortable pace to a marked place on the floor, turn around, walk backward to the starting point, and return to the sitting position in the chair [21]. The amount of time needed for a patient to complete the test was measured in seconds with a stopwatch, and the average of the three recorded measurements was used for the analysis. TUG is a sensitive and specific measure of functional mobility.

Table 1
Homogeneity of demographic and clinical variables between three groups at baseline

	Group A ( $n=$ 31)	Group B ( $n=$ 28)	Group C ( $n=$ 34)	$P$
Age (years)
Mean $\pm$ SD	59.2 $\pm$ 7.3	63.5 $\pm$ 7.2	62.2 $\pm$ 8.5	0.096^†
Median (min-max)	57 (48–77)	63.5 (52–75)	64 (44–74)
BMI (kg/m²)
Mean $\pm$ SD	29.9 $\pm$ 3.7	30.3 $\pm$ 3.8	29.2 $\pm$ 4.5	0.551^†
Median (min-max)	30.1 (23.7–34.8)	30.2 (24.0–35.1)	28.9 (18.1–35.0)
	n (%)	n (%)	n (%)
Sex
Women	19 (61.3)	21 (75)	25 (73.5)	0.438^††
Men	12 (38.7)	7 (25)	9 (26.5)
Education level
Illiterate	8 (25.8)	5 (17.9)	4 (11.8)	0.055^††
Primary/Junior high school	8 (25.8)	9 (32.1)	15 (44.1)
High school	10 (32.3)	7 (25)	2 (5.9)
University	5 (16.1)	7 (25)	13 (38.2)
Job
Unemployed	13 (41.9)	15 (53.6)	16 (47.1)	0.241^††
Active employee	8 (25.8)	1 (3.6)	6 (17.6)
Retired	10 (32.3)	12 (42.9)	12 (35.3)
Marital status
Maried	28 (90.3)	20 (71.4)	27 (79.4)	0.302^††
Alone	3 (9.7)	8 (28.6)	7 (20.6)
Kellgren-Lawrence grading
II	14 (45.2)	7 (25.0)	16 (47.1)	0.159^††
III	17 (54.8)	21 (75.0)	18 (52.9)

Group A, 20 mg HA; Group B, 32 mg HA; Group C, 48 mg HA; SD, Standard Deviation. ^†Kruskal-Wallis test ( $\alpha=$ 0.05). ^††Chi-Squared test ( $\alpha=$ 0.05).

2.4 Statistical analysis

The primary outcome was the WOMAC pain subscale, and all the remaining measures were considered secondary outcomes. A total sample size of 30 patients in each group would be needed to have a power of 90% at a significance level of 5%, based on the expected differences (20 mm) and standard deviations (25 mm) between the groups. Assuming a dropout rate of 10%, 100 patients were included. All analyses were conducted using SPSS software, version 23.0 for Windows (SPSS Inc., Chicago, IL, USA). A $p$ -value of $<$ 0.05 was considered as the significance level. The descriptive data were presented as mean (SD) and median (range). The normality of distribution was determined by the Kolmogorov-Smirnov test. The Wilcoxon signed-rank test was used for within-group comparisons. The between-group differences were compared using the Kruskal-Wallis test. The pairwise comparisons were done using the Mann-Whitney U test. The significance level for the multiple comparison test was determined to be 0.017 using the Bonferroni correction ( $p$ value $=$ 0.05/number of pair-wise comparisons).

Table 2
Homogeneity of outcome variables between three groups at baseline

	Group A ( $n=$ 31)	Group B ( $n=$ 28)	Group C ( $n=$ 34)	$P^{{\dagger}}$
WOMAC-pain (0–20)
Mean $\pm$ SD	11,4 $\pm$ 3,3	12,2 $\pm$ 3,6	11,5 $\pm$ 3,6	0.681
Median (min-max)	11.0 (7–18)	12 (6–20)	12 (2–20)
WOMAC-stiffness (0–8)
Mean $\pm$ SD	4,3 $\pm$ 1,6	4,5 $\pm$ 1,9	4,0 $\pm$ 2,4	0.719
Median (min-max)	4.0 (1–7)	4.0 (1–8)	4.5 (1–8)
WOMAC- physical function (0–68)
Mean $\pm$ SD	43,4 $\pm$ 6,0	42,1 $\pm$ 13,2	40,4 $\pm$ 14,0	0.827
Median (min-max)	45.0 (33–55)	43 (20–68)	45 (20–68)
WOMAC-total (0–100)
Mean $\pm$ SD	59,1 $\pm$ 8,0	58,8 $\pm$ 18,0	55,9 $\pm$ 18,0	0.898
Median (min-max)	58 (47–77)	57 (32–96)	58 (5–96)
Pain-activity (VAS, 0–10)
Mean $\pm$ SD	7.1 $\pm$ 1,1	7.8 $\pm$ 1.0	7.5 $\pm$ 0.9	0.028
Median (min-max)	7.0 (6.0–9.0)	8.0 (6.0–9.0)	7.0 (6.0–9.0)
Pain-rest (VAS, 0–10)
Mean $\pm$ SD	6.7 $\pm$ 2.0	5.8 $\pm$ 1.6	5.8 $\pm$ 1.4	0.036
Median (min-max)	7.0 (3.0–9.0)	6.0 (3.0–9.0)	6.0 (3.0–8.0)
Patient global (VAS, 0–10)
Mean $\pm$ SD	7.0 $\pm$ 1.2	7.1 $\pm$ 1.2	6.7 $\pm$ 1.3	0.457
Median (min-max)	7.0 (5.0–9.0)	7.0 (5.0–9.0)	6.0 (5.0–9.0)
Quality of life (VAS, 0–10)
Mean $\pm$ SD	7.2 $\pm$ 1.5	7.1 $\pm$ 1.2	7.2 $\pm$ 1.3	$0.996$
Median (min-max)	7.0 (5.0–9.0)	7.0 (5.0–9.0)	7.0 (5.0–9.0)
TUG
Mean $\pm$ SD	14.3 $\pm$ 2.3	14.1 $\pm$ 1.7	13.1 $\pm$ 2.5	0.202
Median (min-max)	13.0 (11.0–21.0)	14.0 (12.0–17.0)	13.0 (9.0–18.0)

Group A, 20 mg HA; Group B, 32 mg; Group C, 48 mg; SD, Standard Deviation. VAS, Visual Analogue Scale; WOMAC, Western Ontario and McMaster Universities Arthritis Index; TUG, Time Up and Go Test. †Kruskal-Wallis’s test ( $\alpha=$ 0.05).

3. Results

Of the 100 patients included in the study, seven participants withdrew due to attendance failure and insufficient follow-up; therefore, those patients were not included in the statistical analysis (Fig. 1). The demographic and clinical characteristics of the participants are shown in Table 1. No significant difference was noted in the demographic and clinical characteristics among the groups ( $p>$ 0.05). As shown in Table 2, a significant difference was detected in the VAS activity-pain ( $p=$ 0.028) and VAS rest-pain scores ( $p=$ 0.036) between the groups at the baseline, but no significant differences were observed for the other measures ( $p>$ 0.05).

Table 3
Changes in outcome measure values between three groups from the baseline to after treatment 1^th, 3^th and 6^th month

Outcome measures		Baseline	1-Month	3-Month	6-Month	Friedman	Change	Wilcoxon S rank	Kruskal- wallis	Pairwise	Mann– whitney U	Change	Wilcoxon S rank	Kruskal- wallis	Pairwise	Mann– whitney U	Change	Wilcoxon S rank	Kruskal- wallis	Pairwise	Mann– whitney U
							(1-Month) vs (Baseline)					(3-Month) vs (Baseline)					(6-Month) vs (Baseline)
		Measures (Mean $\pm$ SD)					Within group		Between groups			Within group		Between groups			Within group		Between groups
WOMAC-	GA	11.4 $\pm$ 3.3	6.2 $\pm$ 3.1	6.1 $\pm$ 3.5	7.1 $\pm$ 4.7	$<$ 0.001	$-$ 5.2	$<$ 0.001	$0.825$	GA vs GC	N/A	$-$ 5.3	$<$ 0.001	0.912	GA vs GC	N/A	$-$ 4.3	$<$ 0.001	0.867	GAvsGC	N/A
pain (0–20)	GB	12.2 $\pm$ 3.6	6.9 $\pm$ 5.0	6.6 $\pm$ 4.0	7.6 $\pm$ 5.2	$<$ 0.001	$-$ 5.3	$<$ 0.001		GB vs GC	N/A	$-$ 5.2	$<$ 0.001		GB vs GC	N/A	$-$ 4.6	$<$ 0.001		GB vs GC	N/A
	GC	11.5 $\pm$ 3.6	6.6 $\pm$ 4.0	7.0 $\pm$ 4.8	7.5 $\pm$ 4.1	$<$ 0.001	$-$ 4.9	$<$ 0.001		GA vs GB	N/A	$-$ 5.4	$<$ 0.001		GA vs GB	N/A	$-$ 4.1	$<$ 0.001		GA vs GB	N/A
WOMAC-	GA	4.3 $\pm$ 1.6	1.4 $\pm$ 1.4	1.4 $\pm$ 1.3	2.4 $\pm$ 2.4	$<$ 0.001	$-$ 2.9	$<$ 0.001	0.106	GA vs GC	N/A	$-$ 3.0	$<$ 0.001	$0.410$	GA vs GC	N/A	$-$ 2.0	$<$ 0.001	0.734	GA vs GC	N/A
stiffness	GB	4.5 $\pm$ 1.9	2.0 $\pm$ 2.3	1.9 $\pm$ 1.7	2.2 $\pm$ 2.2	$<$ 0.001	$-$ 2.5	$<$ 0.001		GB vs GC	N/A	$-$ 2.6	$<$ 0.001		GB vs GC	N/A	$-$ 2.2	$<$ 0.001		GB vs GC	N/A
(0–8)	GC	4.0 $\pm$ 2.4	1.8 $\pm$ 1.9	1.5 $\pm$ 1.8	2.1 $\pm$ 2.1	$<$ 0.001	$-$ 2.2	$<$ 0.001		GA vs GB	N/A	$-$ 2.5	$<$ 0.001		GA vs GB	N/A	$-$ 2.0	$<$ 0.001		GA vs GB	N/A
WOMAC-	GA	43.4 $\pm$ 6.0	22.3 $\pm$ 8.2	23.0 $\pm$ 11.0	25.1 $\pm$ 14.7	$<$ 0.001	$-$ 21.1	$<$ 0.001	0.562	GA vs GC	N/A	$-$ 20.4	$<$ 0.001	0.670	GA vs GC	N/A	$-$ 18.3	$<$ 0.001	0.955	GA vs GC	N/A
physical	GB	42.1 $\pm$ 13.2	23.2 $\pm$ 15.8	22.8 $\pm$ 15.2	23.9 $\pm$ 15.4	$<$ 0.001	$-$ 18.9	$<$ 0.001		GB vs GC	N/A	$-$ 19.3	$<$ 0.001		GB vs GC	N/A	$-$ 18.2	$<$ 0.001		GB vs GC	N/A
function	GC	40.4 $\pm$ 14.0	22.7 $\pm$ 11.6	22.0 $\pm$ 11.2	22.8 $\pm$ 11.6	$<$ 0.001	$-$ 17.6	$<$ 0.001		GA vs GB	N/A	$-$ 18.4	$<$ 0.001		GA vs GB	N/A	$-$ 17.6	$<$ 0.001		GA vs GB	N/A
(0–68)
WOMAC-	GA	59.1 $\pm$ 8.0	29.9 $\pm$ 11.0	30.4 $\pm$ 14.9	34.5 $\pm$ 20.9	$<$ 0.001	$-$ 29.2	$<$ 0.001	0.629	GA vs GC	N/A	$-$ 28,7	$<$ 0.001	0.787	GA vs GC	N/A	$-$ 24.6	$<$ 0.001	0.977	GA vs GC	N/A
total	GB	58.8 $\pm$ 18.0	32.1 $\pm$ 22.8	31.7 $\pm$ 21.6	33.7 $\pm$ 22.4	$<$ 0.001	$-$ 26.6	$<$ 0.001		GB vs GC	N/A	$-$ 27.1	$<$ 0.001		GB vs GC	N/A	$-$ 25.1	$<$ 0.001		GB vs GC	N/A
(0–100)	GC	55.9 $\pm$ 18.0	31.2 $\pm$ 16.7	29.6 $\pm$ 15.8	32.3 $\pm$ 16.9	$<$ 0.001	$-$ 24.7	$<$ 0.001		GA vs GB	N/A	$-$ 26.2	$<$ 0.001		GA vs GB	N/A	$-$ 23.6	$<$ 0.001		GA vs GB	N/A
Pain-	GA	7.1 $\pm$ 1.1	3.5 $\pm$ 2.0	3.3 $\pm$ 1.8	4.3 $\pm$ 2.6	$<$ 0.001	$-$ 3.6	$<$ 0.001	0.714	GA vs GC	N/A	$-$ 3.9	$<$ 0.001	0.962	GA vs GC	N/A	$-$ 2.8	$<$ 0.001	0.610	GA vs GC	N/A
activity	GB	7.8 $\pm$ 1.0	3.9 $\pm$ 1.7	4.1 $\pm$ 1.7	4.3 $\pm$ 2.0	$<$ 0.001	$-$ 3.9	$<$ 0.001		GB vs GC	N/A	$-$ 3.7	$<$ 0.001		GB vs GC	N/A	$-3.5$	$<$ 0.001		GB vs GC	N/A
(VAS,	GC	7.5 $\pm$ 0.9	3.8 $\pm$ 1.9	3.6 $\pm$ 1.8	4.3 $\pm$ 1.9	$<$ 0.001	$-$ 3.6	$<$ 0.001		GA vs GB	N/A	$-$ 3.8	$<$ 0.001		GA vs GB	N/A	$-$ 3.1	$<$ 0.001		GA vs GB	N/A
0–10)
Pain-rest	GA	6.7 $\pm$ 2.0	3.0 $\pm$ 1.7	3.1 $\pm$ 1.8	3.9 $\pm$ 2.7	$<$ 0.001	$-$ 3.7	$<$ 0.001	0.073	GA vs GC	N/A	$-$ 3.7	$<$ 0.001	0.021	GA vs GC	0.097^∗	$-$ 2.8	$<$ 0.001	0.452	GA vs GC	N/A
(VAS,	GB	5.8 $\pm$ 1.6	3.1 $\pm$ 1.9	3.5 $\pm$ 2.0	3.7 $\pm$ 2.4	$<$ 0.001	$-$ 2.7	$<$ 0.001		GB vs GC	N/A	$-$ 2.4	$<$ 0.001		GB vs GC	0.249^∗	$-$ 2.0	$<$ 0.001		GB vs GC	N/A
0–10)	GC	5.8 $\pm$ 1.4	3.0 $\pm$ 1.4	2.9 $\pm$ 1.5	3.6 $\pm$ 1.9	$<$ 0.001	$-$ 2.8	$<$ 0.001		GA vs GB	N/A	$-$ 2.8	$<$ 0.001		GA vs GB	0.006^∗	$-$ 2.1	$<$ 0.001		GA vs GB	N/A
Patient	GA	7.0 $\pm$ 1.2	3.4 $\pm$ 1.6	3.4 $\pm$ 1.8	4.6 $\pm$ 3.1	$<$ 0.001	$-$ 3.5	$<$ 0.001	0.895	GA vs GC	N/A	$-$ 3.5	$<$ 0.001	0.253	GA vs GC	N/A	$-$ 2.3	$<$ 0.001	0.740	GA vs GC	N/A
global	GB	7.1 $\pm$ 1.2	3.9 $\pm$ 2.0	4.1 $\pm$ 2.0	4.4 $\pm$ 2.2	$<$ 0.001	$-$ 3.2	$<$ 0.001		GB vs GC	N/A	$-$ 3.0	$<$ 0.001		GB vs GC	N/A	$-$ 2.7	$<$ 0.001		GB vs GC	N/A
(VAS,	GC	6.7 $\pm$ 1.3	3.3 $\pm$ 1.7	3.3 $\pm$ 1.7	4.4 $\pm$ 2.3	$<$ 0.001	$-$ 3.4	$<$ 0.001		GA vs GB	N/A	$-$ 3.4	$<$ 0.001		GA vs GB	N/A	$-$ 2.3	$<$ 0.001		GA vs GB	N/A
0–10)
Life	GA	7.2 $\pm$ 1.5	3.2 $\pm$ 1.3	3.2 $\pm$ 1.7	4.7 $\pm$ 3.1	$<$ 0.001	$-$ 4.0	$<$ 0.001	0.523	GA vs GC	N/A	$-$ 4.0	$<$ 0.001	0.040	GA vs GC	0.364^∗	$-$ 2.5	$<$ 0.001	0.961	GA vs GC	N/A
quality	GB	7.1 $\pm$ 1.2	3.8 $\pm$ 2.0	4.1 $\pm$ 2.0	4.3 $\pm$ 2.3	$<$ 0.001	$-$ 3.3	$<$ 0.001		GB vs GC	N/A	$-$ 3.0	$<$ 0.001		GB vs GC	0.115^∗	$-$ 2.8	$<$ 0.001		GB vs GC	N/A
(VAS,	GC	7.2 $\pm$ 1.3	3.7 $\pm$ 1.8	3.5 $\pm$ 1.7	4.5 $\pm$ 2.3	$<$ 0.001	$-$ 3.5	$<$ 0.001		GA vs GB	N/A	$-$ 3.7	$<$ 0.001		GA vs GB	0.010^∗	$-$ 2.6	$<$ 0.001		GA vs GB	N/A
0–10)
TUG	GA	14.3 $\pm$ 2.3	13.3 $\pm$ 2.6	13.0 $\pm$ 2.5	13.5 $\pm$ 2.3	$<$ 0.001	$-1.0$	$<$ 0.001	0.092	GA vs GC	N/A	$-1.2$	$<$ 0.001	0.049	GA vs GC	0.011^∗	$-$ 0.8	$<$ 0.001	0.730	GA vs GC	N/A
	GB	14.1 $\pm$ 1.7	13.3 $\pm$ 2.1	13.5 $\pm$ 1.6	13.5 $\pm$ 1.7	0.001	$-$ 0.8	0.002		GB vs GC	N/A	$-$ 0.6	0.011		GB vs GC	0.940^∗	$-$ 0.6	0.023		GB vs GC	N/A
	GC	13.1 $\pm$ 2.5	12.5 $\pm$ 2.4	12.3 $\pm$ 2.2	12.5 $\pm$ 2.0	$<$ 0.001	$-$ 0.6	$<$ 0.001		GA vs GB	N/A	$-$ 0.8	$<$ 0.001		GA vs GB	0.089^∗	$-$ 0.6	0.001		GA vs GB	N/A

GA, Group A, 20 mg 5 injection; GB, Group B, 32 mg 3 injection; GC, Group C, 48 mg 1 injection ; SD, Standard Deviation. VAS, Visual Analogue Scale; WOMAC, Western Ontario and McMaster Universities Arthritis Index; TUG, Time Up and Go Test. ^∗Statistically significant in the pair-wise comparisons by Mann-Whitney U tests with Bonferroni correction ( $p<$ 0.0166).

All three groups had a significant decrease in the WOMAC, VAS-pain, patient global, quality of life, and TUG scores at 1, 3, and 6 months after the injection ( $p<$ 0.05). However, the comparative analysis of the treatments showed no significant difference in the WOMAC, activity pain, and patient global scores at the 1, 3, or 6 month follow-ups. The WOMAC pain score in the 20 mg HA group decreased from 11.4 to 6.1 at the 3 month follow-up (RMD $=-$ 5.3, $p<$ 0.01); in the 32 mg HA group, it decreased from 12.2 to 6.6 (RMD $=$ $-$ 5.2, $p<$ 0.01); and in the 48 mg HA group, it decreased from 11.5 to 7.0 (RMD $=-$ 5.4, $p<$ 0.01). The rest pain and quality of life scores showed better improvement in the 20 mg HA group than in the 32 mg HA group ( $p=$ 0.006 and $p=$ 0.010, respectively). Changes in WOMAC, VAS-pain, VAS-quality of life, and TUG mean values for each group are depicted in Table 3.

No serious adverse events related to any of the treatments were observed. Four patients reported local pain in the injection site (two in the 20 mg HA group, one in the in the 32 mg HA group and one in the 48 mg HA group). These adverse events successfully treated with local ice application; none of them needed medication. No post-injection reactions such as effusion or pseudoseptic arthritis were registered.

4. Discussion

Our findings indicated that all three dosing regimens of IA-HA (Reviscon 20 mg 1.0%/ Reviscon Plus 32 mg 1.6%/ Reviscon Mono 48 mg 2.0%) were effective at reducing pain and improving the function and the quality of life in patients with bilateral KOA. Treatment with linear HMW IA-HAs appears to be well tolerated and no serious adverse events related to any of the treatments were registered. Although numerous studies have evaluated the efficacy of different HA products in patients with knee OA, few have compared the efficacy of a single HA injection versus multiple HA injections. A systematic review by McElheny et al. [22] examined the efficacy of HA formulations based on the number of injections when compared head-to-head and found no significant difference between single and multiple injections in patient-reported outcomes, in agreement with our findings. The authors also concluded that 5-injection formulations were not superior to 3-injection formulations [22]. A meta-analysis of single-injection products by Vincent et al. [23] also concluded that the effects of single injections of HA produce results similar to multiple injections of IA-HA in terms of pain relief in the treatment of knee OA. The pooled data of another systematic review revealed that 2–4 and $\geqslant$ 5 injection regimens provided pain relief over IA-saline in patients with knee OA, whereas a single injection did not [24]. However, in the latter review, the number of included studies with a single-injection procedure was limited, and the molecular and rheological properties of the compared HA products had too much heterogeneity. Therefore, the results from previous studies are contradictory regarding the relative effectiveness of single-use viscosupplementation agents.

In the current research, significant pain relief was observed in all groups, as assessed with the VAS-activity pain scale and the WOMAC-pain subscale. This improvement could occur within 4 weeks post-injection and could remain beneficial for up to 6 months. These findings are similar to those reported in a systematic review that found significant pain relief for up to 6 months with IA-HA compared to saline in patients with early-moderate knee OA [25]. The results about the effect of linear HMW IA-HAs on mild and moderate knee OA in our study may be related to the molecular composition and rheological properties of HA. In addition to its short-term effects, it has also shown benefit surpassing its duration of action, which may have been linked to chondroprotective biochemical functions. In this respect, further studies should be designed to elucidate the mechanism of action of different HA products. The VAS-rest pain scores in our study showed a significant decrease in all groups at three months; however, the decrease was only statistically significant for the 20 mg HA group at three months in pairwise comparisons. However, the higher baseline values of the rest pain scores in the 20 mg HA group should be considered.

A limited number of studies have compared the effects of single versus multiple injections of viscosupplements with the same molecular structure, molecular weight, and volume in patients with KOA [22]. Most single-injection HAs were HMW and cross-linked types [26, 27, 28, 29]. Conrozier et al. [30] demonstrated that a single injection (1 $\times$ 6 mL) and three weekly injections (3 $\times$ 4 mL and 3 $\times$ 2 mL) of HMW cross-linked derivative of HA (hylan G-F 20) showed the greatest mean improvements ( $-$ 34.9, $-$ 32.6, and $-$ 36.7 mm, respectively) in the patient-rated knee OA pain assessment VAS. Additionally, a 6 mL single-injection group had the lowest number of retreated patients. This study also achieved significant pain relief and functional improvement with HA injections.

This prospective multicenter, randomized trial evaluated variable concentrations of linear HMW sodium HA over a period of 6 months by comparing a sodium HA 48 mg/2.4 mL single injection with a sodium HA 32 mg/2 mL 3-dose weekly injection series and a 20 mg/2 ml 5-dose weekly injection series. A large number of HA products are commercially available, and the intrinsic properties and characteristics of the IA-HAs used in various studies are different [13, 22]. Therefore, comparing these studies is complicated, and this may partially account for the contradictions in the results. The current study used a highly purified HMW sodium HA obtained from bacteria by fermentation. However, the threshold values of HMW have varied among different studies. HMW IAHAs were defined as products whose MW ranged from $\geqslant$ 3000 kDa to $\geqslant$ 6000 kDa [13, 31]. Similar to our study, Altman et al. [13] demonstrated that Bio-HA products with a MW $\geqslant$ 3000 kDa were the most effective and safest HA products, as their subgroup characteristics consistently showed favorable results in the reported analyses. A recent meta-analysis demonstrated that HMW IA-HAs were statistically significantly superior to placebo in terms of pain management, whereas low molecular weight (LMW) IA-HAs were not. However, in this meta-analysis, the HMW IA-HAs were defined as products whose MW was at least 6000 kDa and the LMW IA-HAs were defined as products whose MW was under 1500 kDa [31].

In the current study, the single-injection HMW-HA formulation achieved similar outcomes in pain and physical function when compared with the multiple injection series. Some studies have shown that single cross-linked HMW-HA can be as effective as multiple linear LMW-HA injections. Dıraçoğlu et al. [32] demonstrated that a single dose of lightly cross-linking HA and three doses of standard linear HA significantly improved pain and functional status in patients with knee OA. However, the improvement in VAS-resting values was statistically better in the standard linear HA group than in the lightly cross-linked HA group at the 6th month. Similarly, a recent study by Bahrami et al. [27] showed similar effects on pain and functional status of knee OA patients after a single dose of lightly cross-linked HA or three doses of standard linear HA during 2 and 6 months of follow-up. Additionally, the improvement in the WOMAC stiffness score was significantly higher in the LMW-HA group at the 2nd month follow-up. Zhang et al. [28] concluded that a single injection of a cross-linked HMW-HA (Durolane) was equivalent to five injections of LMW-HA (Artz) over 18 and 26 weeks in terms of pain, physical function, global self-assessment, and knee stiffness. However, a point to note is that the aforementioned studies evaluated the efficacy of two HA types with different molecular weights and different numbers of injections.

Evidence from the literature has shown that patients with knee OA experience losses of proprioception and postural stability, which may result in an increased risk of falling [33, 34, 35]. A randomized controlled trial that examined postural stability and fall risk using the Biodex Balance System and the TUG test reported that patients with bilateral knee OA had impaired balance; moreover, this impairment was more pronounced in patients with moderate knee OA than with mild OA [36]. Therefore, improving the functional mobility in patients with knee OA has become an important challenge. In the present study, functional mobility showed a statistically significant improvement compared to the baseline level, without any marked superiority between the groups. Khalaj et al. [36] also found significant improvements in terms of postural stability and fall risk after administering five weekly IA-HA injections in patients with both unilateral and bilateral knee OA. Another randomized controlled trial demonstrated the effectiveness of IA injections of hyaluronan (Hylan G-F 20) for improving proprioception, isokinetic muscle force, and functional status in patients with bilateral knee OA in the short term [30]. In the current study, a greater improvement in TUG scores was observed at three months in the group receiving five doses of injected HA. The difference between consecutive HA injections and single dose HA injection in terms of TUG scores at three months may be related to the larger number of injections and the lower concentration of HA. However, no significant difference was found in the post-intervention changes in the TUG scores between the groups at the 6-month follow-up.

To the best of our knowledge, this study is the first to determine the effectiveness of alternative dosing regimens of injected linear HMW HA (20 mg HA/32 mg HA/48 mg HA) on various outcome measures in individuals with bilateral knee OA. The use of three different dosing regimens applied with the same technique allowed us to assess the efficacy of the HA products, as well as to compare the effects of the injections themselves. Furthermore, very few studies have assessed the effect of HA injections on functional mobility and quality of life in individuals with knee OA. Improvement in pain, functional status and mobility is accompanied by a concomitant increase in quality of life. When deciding on single versus multiple injections, patient preference is an important issue, and the findings of the present study revealed similar patient global assessment scores among the three groups.

The main limitation of our study was the absence of a control group. Furthermore, the patients were not blinded to their group allocations. For ethical issues, placebo injections were not possible. However, considering that a placebo is also effective at relieving pain in the treatment of OA and this effect is more prominent when a placebo was given through injections [37], this possible treatment bias may have some effect on the internal validity of our study. Nevertheless, all data assessors in this study were completely blinded. All our patients were advised to perform a standard home-based exercise program; however, home-based exercise programs can be evaluated as an active treatment for knee OA and may have enhanced the positive effects of IA-HA injections [38, 39]. Thus, the potential additive or synergistic effects of exercise and IA injections of hyaluronans should be considered when interpreting the results of our study. The use of viscosupplementation recommended in patients with symptomatic, mild to moderate knee OA (Kellgren-Lawrence grade I-III). However, joint space narrowing severity may influence the response of viscosupplementation in knee OA [40]. Future research should clarify the role of the radiological severity in patients receiving linear HMW HA at different concentrations for the treatment of knee OA. Additionally, it should be considered that the small sample size might have affected our results and their variability. Finally, this study does not take into consideration cost-effectiveness. However, single-use viscosupplementation agents are attracting growing consideration because of increased cost-effectiveness and patient convenience, along with decreased exposure to risks associated with knee injection [41].

5. Conclusion

Our findings indicated that the use of linear HMW IA-HAs (20 mg HA-2.0% /32 mg HA-1.6% / 48 mg HA-1.0%) in patients with early-moderate knee OA has positive effects on pain, functional status, quality of life, and functional mobility up to 6 months post-injection. Our data may show that a single-injection regimen provides similar efficacy to regimens using 3 or 5 injections. Further well-designed studies with larger numbers of patients are needed to evaluate the long-term effects of the injection of linear HMW IA-HA on knee OA.

Author contributions

Study concept and design: DD, EIS, SS, BD, MAY; Analysis and interpretation of data: DD, EIS, SS, BD, MAY; Analysis and/or interpretation: DD, EIS, SS; Literature review: EIS, SS, BD, MAY; Writing the article: DD, EIS, BD; Control/supervision, critical review: DD, EIS. All authors read and approved the final manuscript.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethical approval

The study was conducted in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of the Medical Faculty of Istanbul University (2020/207575) and Republic of Turkey Ministry of Health. The trial was prospectively registered on ClinicalTrials.gov (NCT04786613).

Funding

The authors report no funding.

Informed consent

Written informed consent was obtained from all participants.

Footnotes

Acknowledgments

All the products were provided free of charge by VSY Biotechnology. VSY Biotechnology did not play a role in the study design, data collection, analysis and interpretation, and manuscript writing.

Conflict of interest

The authors declare that they have no conflicts of interest.

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Different doses of hyaluronic acid injections in patients with knee osteoarthritis: A multicenter,randomized,prospective,single-blind,clinical study

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Methods

2.1 Study design and participants

2.2 Interventions

Table 1 Homogeneity of demographic and clinical variables between three groups at baseline

Table 2 Homogeneity of outcome variables between three groups at baseline

Table 3 Changes in outcome measure values between three groups from the baseline to after treatment 1th, 3th and 6th month

5. Conclusion

Author contributions

Data availability statement

Ethical approval

Funding

Informed consent

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Homogeneity of demographic and clinical variables between three groups at baseline

Table 2
Homogeneity of outcome variables between three groups at baseline

Table 3
Changes in outcome measure values between three groups from the baseline to after treatment 1^th, 3^th and 6^th month