OA is a common chronic musculoskeletal disease that causes structural alterations in the cartilage, the subchondral bone, the ligaments, the capsule, the synovium and the periarticular muscles destroying the entire joint structure [1]. OA causes pain, inflammation and finally results in significant disability [2]. Thus it is crucial to understand which treatment can offer the best improvement in quality of life. A variety of conservative therapies has been proposed to provide viable long-term results. Hyaluronic acid, also referred to as “visco-supplementation”, is one of the most used infiltrative therapies to relieve pain in knee osteoarthritis.

Role of Hyaluronic Acid in OA

Hyaluronic acid is a glycosaminoglycan (GAG) with negatively-charged polysaccharide compound. It consists of sequentially repeated glucuronic acid and N-acetyl glucosamine unit. It is also the major component of synovial fluid. Because of its negative charge, it is highly hydrophilic and therefore highly soluble. These properties ensure the hydration of the tissue maintaining the articular matrix viscosity, joint lubrication and shock absorption [3].

During OA, the dynamic mechanisms involve the release of inflammatory cytokines, such as interleukin 1 beta (IL-1β), which leads to the mitochondrial dysfunction. The consequence is the increase of the Reactive Oxygen Species (ROS) production and the triggering of an apoptosis cascade causing a persistent oxidative stress environment. One of the properties of HA is anti-inflammatory effect by the capacity of decreasing in ROS synthesis and the prevention of IL-1β-induced oxidative stress. In this mechanism, cytokines cause the deregulation and loss of crucial macromolecules from the extracellular matrix (ECM) such as type II collagen and proteoglycan biosynthesis. HA has an anabolic effect, so it stimulates glycosaminoglycans and type II collagen formation.3 In addition, HA reduces the formation of peroxide hydrogen (H₂O₂) and oxygen species in the synovial fluid causing the suppression of the cell death induced by H₂O₂ [4].

Niacinamide

Nicotinamide (NAM) and nicotinic acid (NA), known as vitamin B3 (or niacin), is water-soluble vitamin of the B complex vitamins. NAM and NA are components of the enzyme cofactors nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which play important physiological roles, e.g. in various redox processes [5,6]. Niacin is classified as a semi-essential vitamin due to the endogenous formation from the amino acid tryptophan, with approximately 60 mg of tryptophan being equivalent to 1 mg NA [7]. The structure of NAM consists of a pyridine ring to which a primary amide group is attached in the meta position (Figure 1). It is an amide of nicotinic acid [8]. As an aromatic compound, it undergoes electrophilic substitution reactions and transformations of its two functional groups.

A product based on high molecular weight hyaluronic acid and niacinamide (pre-filled syringe with 2 ml of product with 40 mg of high molecular weight hyaluronic acid) has been tested for safety in pre-clinical studies [9-11]. High molecular weight combined with high concentration of HA (2%) creates a product with high viscosity that makes it suitable for visco-supplementation. The most important thing is that niacinamide provides protection from hyaluronidaseactivity. Giardina in his in vitro study showed that HA combined with niacinamide had improved resistance to hyaluronidase enzyme degradation when compared to standard HA and showed a longer activity. Thus the sample containing niacinamide showed a 22% amount of hydrolyzed hyaluronic acid in the reaction mix after 60 minutes, while the control 65,6% (P<0.05) [12].

Patients with persistent symptomatic OA of the knee received N-HA or standard HA. The Consolidated Standards of Reporting Trials (CONSORT) Statement was followed [13]. The study was performed according to the ethical standards outlined in the 2013 revision of the 1975 Declaration of Helsinki, approved, and monitored by our institutional review board. Additionally, niacinamide hyaluronate is marketed under Directive 93/42/EEC; it is compliant with medical device documents (MEDDEV 2.7/1 Rev. 4 Clinical evaluation: guide for manufacturers and notified bodies.)

Participants

The patients were recruited at a single clinical institution by the chairman of the orthopedic department. Patients aged 18 to 80 years, with symptomatic knee OA, grade II to III according to Kellgren-Lawrence classification were included in the study groups. Every patient had to sign the informed consent before enrollment; the potential benefits and risks of hyaluronic acid injections were explained and understood by all. The main exclusion criteria were any recent intra-articular injection therapy, knee instability, significant axial deviation, systemic disorders such as rheumatoid arthritis, coagulopathies, or infections. All inclusion and exclusion criteria have been listed in Table 1.

Sixty patients who met our inclusion criteria were enrolled and divided into two groups of 30 patients each.

Statistical methods

Normal distribution was determined by the Shapiro-Wilk test for all pre-injections scores in every category between patients treated with N-HA and those treated with NN-HA. Wilcoxon-Mann was used for variables non-normally distributed while one sample t test was chosen for variables normally distributed.

Treatment

The manufacturer provided the products with the same syringe, color (transparent), texture, and quantity (2 mL). One of the two infiltration products used was HA composed of medium-weight sodium hyaluronate (1.0-1.5 x 106 Da) that is a non–animal source, obtained by bacterial fermentation (Streptococcus Equi). The other one was HA composed of high-weight sodium hyaluronate (1800-2600 kDa) plus the addition of 0,8% niacinamide. Both came in the dosage presentation of 2 mL, 40 mg/2 mL hyaluronate.

After basal clinical scores were recorded, an intra articular injection with either of the products was performed always by the same orthopedic physician in a sterile environment. The knee was held in extension, and a suprapatellar approach was used. Precisely, 2 mL of hyaluronate were injected with a 20 gauge after which ice was applied for 5 minutes. Then patients were instructed to avoid intense exercise for 48 hours and to apply ice for 15 minutes 3 times a day. Each patient received 3 doses of the same product in 15 days intervals. Afterward, they were invited for follow-up at 3 and 6 months. Follow up for clinical evaluation was by the same senior author. None was lost at final follow up. The use of medication patients was not recorded.

To obtain the primary endpoint, patients were clinically evaluated using KOOS [14] (percentage score obtained from the evaluation of 5 separately scored subscales: pain, other symptoms, functions of daily living, function in sport and recreation, and knee-related quality of life. Scoring systems were recorded through questionnaires filled by the same patients before the first injection (T0), at 3 months (T1) and 6 months (T2). Patients were also assessed for any adverse reaction such as effusion, flare or pain during the clinical evaluation. After clinical evaluation and score recording, an independent researcher archived the data into a database.

The mean age of the 30 study patients treated with N-HA was 58.8 ± 16.1 years (BMI: mean 24.5 ± 4.5). Patients treated with HA had a mean age of 56.4 ± 15.6.

N-HA group showed a statistically significant improvement at 3 and 6 months when compared to basal score with KOOS Final Score (P < 0.05) (Figure 1).

HA group revealed an improvement at 3 months (P < 0.01) but at 6 months there was a deterioration of the results and KOOS score was similar to pre-treatment (P > 0.05) (Figure 2).

At 3 months a statistically significant improvement was seen in KOOS SPORT, ADL, PAIN and SYMPTOMS scores for N-HA group (P < 0.05), furthermore SPORT scores maintained the improvement from month 3 to 6 (P < 0.05) (Figure 3,4,5,6).

When comparing both groups at 6 months, N-HA reported significant clinical outcomes for QOL (P value = 0.0081), ADL (P value = 0.0091), SYMPTOMS (P value = 0.0081) when compared to HA (Figure 7,8,9).

Finally, at 6 months N-HA resulted to have significant better results (P < 0.01) (Figure 10).

To our knowledge, this is the first prospective randomized controlled trial comparing the effects of a niacinamide hyaluronate formulation used as an infiltrative therapy for knee OA.

Our results are in line with other studies in the literature regarding the effectiveness of hyaluronic acid as a treatment for symptomatic OA of the knee, as both compounds of hyaluronic acid demonstrated a statistically significant improvement in pain and function from the basal time point [15,16].

The background of niacinamide is not new; some studies indicate that niacinamide reduces inflammation and some OA symptoms [17,18].

Another study by Hadjab et al. has shown that niacinamide has a dual positive effect: a major resistance to oxidative degradation and greater protection from the hyaluronidase. Furthermore, niacinamide provides more stability of HA against thermal degradation preserving its viscosity [19].

Protein-based compounds such as hyaluronic acid are easily degraded by strong acids, bases, inorganic salts and organic solvents. The active end groups of these proteins, generally associated with hydrogen-bonded water, can bind to other molecules, leading to its denaturation and loss of function. Niacinamide helps preserving the structure and function of proteins giving more stability.

In this setting, the new product does not react with the hyaluronidase enzyme and thus extends its effects, as previously demonstrated in vitro.

This fact is highly relevant in orthopaedics because further inflammatory damage could be prevented by stabilizing chemical reactions in the knee joint. Due to the niacinamide, hyaluronic acid resulted to have more resistance to hyaluronidase

Our therapy was performed with high-weight hyaluronic acid using sodium hyaluronate HW (1800-2600 KDa). The high concentration of HA (2,0 %) maintains an high viscosity suitable enough for supplementation.

Some studies show that high-weight hyaluronic acid has a positive effect for the treatment of knee OA improving pain and function [20]. However, the disadvantage of high viscosity hyaluronate is that the product is more difficult to administrate.

Overall, there isn’t an evidence that supports the superiority of one kind of HA preparation over another [21]. In a randomised double-blind controlled trial, a slightly lower efficacy for low weight HA preparations versus intermediate and high weight hyaluronic acid was demonstrated [22]. This fact also correlates with our findings as with our control, we did not find significant adverse reactions proving it to be safe to administer.

The new niacinamide hyaluronic acid resulted a viable treatment improving the clinical outcomes at 3 and 6 months when compared to standard HA products.

Our study demonstrated that the difference of clinical outcomes was statistically significant at 6 months when the standard products markedly returned to basal scores at the same time.

Comparing both hyaluronic acids at 3 months, niacinamide-HA resulted to have a statistically significant advantage in KOOS symptoms; furthermore, the difference was statistically significant with KOOS activity daily life and patients treated with niacinamide-HA nearly returned back to their normal life (P<0.05).

At 6 months niacinamide-HA resulted to be more effective with KOOS quality of life when compared with standard infiltrative therapy, however, similar effects were shown in terms of pain and sport.

Limitations

Our study is not exempt from limitations. The small number of patients should be taken into consideration. Furthermore, the short clinical follow-up doesn’t allow determining the total lasting effects of N-HA. Another limitation is the lack of recording of the rescue medicine by the patients during the follow-up period. Additionally compared hyaluronates were different, used with niacinamide was a high molecular weight HA compared to sole medium-weight sodium hyaluronate.

Hyaluronic acid therapy with or without niacinamide is a safe and effective treatment of early arthritis; however, our study demonstrated that a particular hyaluronic acid at high molecular weight with niacinamide offers long lasting effects.

Further studies with longer follow up are needed to determine the complete duration of symptom relief of niacinamide-hyaluronic acid.

Funding: This research received no external funding.

Institutional review board statement: Not applicable.

Informed consent statement: Not applicable.

Data availability statement: Not applicable.

Conflicts of interest: The authors declare that they have no conflict of interest.