Role and Effectiveness of Intra-articular Injection of Hyaluronic Acid in the Treatment of Knee Osteoarthritis: A Systematic Review

Osteoarthritis (OA) is a degenerative joint disease that causes persistent joint pain and stiffness of mainly the large peripheral weight-bearing joints. It is a leading cause of functional disability and poor quality of life. Various modalities of therapy are recommended by different research organizations at different stages of OA including non-pharmacological, pharmacological, and surgical interventions. Intra-articular injections of hyaluronic acid (HA) is widely used for over three decades in the treatment of OA. However controversies exist regarding its safety and efficacy, the number of injections and courses, type of preparation, duration of its effects, and combining it with other drugs or molecules. This study aimed to review the most recent data available in the published literature to address these. Electronic databases like Medline, Embase, ProQuest, and Google Scholar were searched for articles using keywords, intraarticular injections, hyaluronic acid, and osteoarthritis knee. The review was carried out as per PRISMA guidelines. Thirty-eight randomized control trials (RCTs) investigating the efficacy and safety of intra-articular injection of HA were included in the systematic review. Out of the 38 studies, 22 (57.9%) were double-blind, eight (21%) single-blind, three (7.9%) non-blind, four (10%) with simple randomization, and one (2.7%) was open-labeled. Total 5,025 patients were included in these studies. The mean age of the patients was 60.28 years and the osteoarthritis grade of the knee joint was 1 to 3. HA was studied as a test preparation in 19 (50%) while in another 19 (50%) it was studied as a control. In 24 (63.2%) studies, HA was used as high molecular weight preparation in eight (21%) as low molecular weight preparation while in six studies the information was not available. HA was used as a standalone preparation in 31 studies, in two studies it was injected with platelet-rich plasma (PRP) and with either low-level laser therapy (LLLT), triamcinolone (TA), betamethasone (CS), poly deoxyribonucleotide (PDRN) or dexamethasone (DX) in one study each. In the majority of the studies, HA was given as a single injection (52.6% studies) or weekly three injections (28.9% studies). In 13.2 %, it was given as weekly 5 injections and in 5.3% as weekly two injections. IA-HA injections have a limited role in the treatment of knee osteoarthritis in those patients who do not have sufficient pain relief with topical or oral medication and physical therapy. It is safe and effective except for minor side effects such as local pain and swelling lasting for a few days. Severe allergic reactions are extremely rare. They provide adequate pain relief and functional improvement for up to six months irrespective of a number of injections and type of preparations used. The combination formulations with corticosteroids or PRP or MSCs show better results than HA alone. Combining HA with newer molecules such as peptides or diclofenac for sustained and disease-modifying effects requires more studies in the future.


Introduction And Background
Osteoarthritis (OA) is a degenerative joint disease that most commonly affects the lower extremities' large peripheral weight-bearing joints [1]. OA is one of the leading causes of functional impairment in the United States, affecting an estimated 22.7 million individuals [2]. It affects the quality of life by causing persistent pain, stiffness, and reduced mobility of the afflicted joints, as well as physical and/or mental co-morbidity [3]. It will boost healthcare spending, which is anticipated to be roughly $ 128 billion in the United States [4].
Because of its vast complexity and interplay of various biological factors such as genetic alterations, sex hormone deficit, and aging, OA is still poorly understood [5]. Many recent studies have focused on molecular markers that have been linked to chondrocyte senescence caused by stress [6]. Chondrosenescence is a term used to describe the age-related decline in chondrocyte function [7]. A core set of evidence-based therapeutic modalities has been created, according to the OA Research Society International (OARSI) Guidelines and recommendations for OA care [8]. These modalities include non-pharmacological approaches such as patient education and awareness, physical activity, and rehabilitation aids; as well as pharmacological modalities such as acetaminophen prescriptions, and non-selective NSAIDs (Nonsteroidal anti-inflammatory drugs), selective COX-2 inhibitors, and even opioid prescription. The most often recommended OA medications are NSAIDs [9]. Despite the fact that NSAIDs', have been shown to be useful in the treatment of pain, long-term usage is linked to the risk of serious side effects. Furthermore, due to pharmacogenomics interactions, their tailored response is quite heterogenous [9].
Glucosamine and chondroitin, intra-articular injections of viscosupplements, corticosteroids, and plateletrich plasma (PRP) are some of the additional non-operative treatment options [8]. Surprisingly, reduction in BMI, physical treatment, such as mind-body exercises, strength training activities, and aerobic workouts, have all showed promise in improving OA prognosis when patients follow their physical therapy routine regularly [10]. Table 1 outlines the current guidelines for OA treatment regimens from several international associations [11,12].  NSAID=non-steroidal anti-inflammatory drug, I-CS=intra-articular corticosteroid, I-HA=intra-articular hyaluronic acid, SNRI=Serotonin and norepinephrine reuptake inhibitors, I-PRP=intra-articular platelet-rich plasma

Society
In the last few decades, there has been an increasing trend to use intra-articular injections of corticosteroids, analgesics/anti-inflammatory drugs, polymerized collagen, anti-cytokine drugs, or hyaluronic acid as alternative modalities to maximize the local effect while minimizing systemic side effects [13]. Since its approval in Japan and Italy in 1987-1988, intra-articular (IA) injections of hyaluronic acid (HA) have been frequently utilized. Even after successful use of IA-HA for over three decades, controversies still exist regarding its safety, efficacy, number of injections, type of preparation, duration of its effects and combining it with other drugs or molecules. This systematic review aims to appraise the most recent clinical data available on I-HA therapy in patients with KOA. The objective of this systematic review is, 1) does I-HA has any role in the treatment of Knee OA? 2) is it safe and effective?

Study Design and Search Strategy
The review was performed as per the PRISMA guidelines. The search was carried out in the databases such as Medline, Embase ProQuest and Google scholar. Keywords like hyaluronic acid, hyaluronate injection, and osteoarthritis knee were used individually and in combination for the search.

Study Criteria
All randomized control trials (RCTs) investigating the efficacy and safety on intra-articular injection of HA from January 2015 to December 2021 were included in the review. Articles published in languages other than English, review articles, meta-analyses, book chapters, and editorials were not included in the review. The study selection process is represented in Figure 1.

Search Results
The literature search yielded 1,146 articles. Out of which 225 articles removed, as they were duplicate articles. Remaining 921 articles screened further for appropriateness. Further 677 articles excluded from the study as, they were not relevant or not published in English language. Out of remaining 244 articles, 206 articles excluded from the study, as these were review articles or book chapters or meta-analysis. Thirtyeight studies met our inclusion criteria were included in the review. Quality assessment of the trials included in the review was done using Cochrane risk-of-bias tool for randomized trials (version 2).
Out of 38 studies, HA was studied as a test preparation in 19(50%) while in another 19(50%) it was studied as a control. In 24(63.2%) studies HA was used as high molecular weight preparation in eight (21%) as low molecular weight preparation while in six studies the information was not available. HA was used as a standalone preparation in 31 studies, in two studies it was injected with platelet rich plasma (PRP) and with either low-level laser therapy (LLLT), triamcinolone (TA), betamethasone (CS), poly deoxyribonucleotide (PDRN) or dexamethasone (DX) in one study each. In majority of the studies, HA was given as a single injection (52.6% studies) or weekly three injections (28.9% studies). In 13.2 %, it was given as weekly 5 injections and in 5.3% as weekly two injections ( Table 2). The details of characteristics of studies included in the systematic review are represented in Table 2. All of the studies reportedly concluded that intra-articular injections of HA have resulted in clinical improvement over baseline pain, stiffness and function up to three to six months. The improvement was more rapid when HA was combined with corticosteroid and more sustained when combined with PRP or BM-MSC. The most common AEs reported were arthralgia, join swelling, joint stiffness and back pain.

Discussion
Osteoarthritis (OA) is a degenerative joint disease that most commonly affects the lower extremities' mainly large peripheral weight bearing joints. The current multimodal knowledge of OA incorporates trauma, mechanical forces, biochemical cartilage degradation, inflammation, and metabolic derangements in contrast to the idea that OA is just a degenerative process [14].
We may continue to target our treatments on stopping OA at the cause rather than relieving pain with analgesic, as we understand more about the pathophysiology of OA. Various evidence-based methods of therapy, including non-pharmacological, pharmacological and surgical are advised by various research bodies at various phases of osteoarthritis. In individuals, who do not react well to oral or topical medicine and physical therapy, intra-articular injectable treatments with corticosteroids, viscosupplements, platelet rich plasma (PRP) and stem cells are recommended. Intra articular HA has been utilized widely to treat symptomatic knee OA over three decades.

Hyaluronic acid physiology in synovial fluid of joint
Hyaluronate is a high molecular weight, widely distributed substance found in the cartilage and synovial fluid. It's made up of alternating N-acetyl d-glucosamine and d-glucuronic acid residues connected by ß(1-4) and ß(1-3) bonds and has a molecular mass of 6,500 to 10,900 kDa [15]. It functions as a lubricant, a scavenger for free radicals, and a regulator of cellular activity including protein binding. The endogenous HA in the joint depolymerizes from a high molecular weight (6,500-10,900 kDa) to a lower molecular weight (2,700-4,500 kDa) as OA progresses, reducing the mechanical and viscoelastic properties of the synovial fluid in the affected joint [15,16]. Exogenous HA injections have therefore been employed clinically to attenuate the macerated activities of OA patients' depolymerized endogenous HA. Although the exogenous HA may not fully repair and replace the characteristics and activities of the synovial fluid's depolymerized endogenous HA, it may provide enough pain relief through a variety of methods [16]. Synthesis of proteoglycan and/or glycosaminoglycan, anti-inflammatory action, and viscoelasticity maintenance are among these processes [16]. Exogenous HA has a varied reaction; one possible reason for the varying efficacy of HA therapies on OA patients is the levels of hyaluronidases in a patient's synovial fluid. Hyaluronidases are a group of enzymes that degrade hyaluronic acid by cleaving the β(1-4) linkages of HA and fragmenting it into smaller fragments before degradation [17].

Hyaluronic acid and its preparations for treatment of OA
HA is given to OA patients in one of the two ways, by mouth or by local injection. Synvisc and Synvisc-One (Genzyme); Gel-One (Zimmer); Hyalgan (Fidia); Spartz FX (Bioventus); Orthovisc (Anika); Euflexxa (Savient); Monovisc (Anika Therapeutics) and Gel-Syn (Institute Biochimique SA) are some of the injectable HA formulations available for clinical usage [18]. They differ in many ways; mean molecular weight, molecular structure (linear, cross-linked or both), source (animal versus bacterial bio-fermentation using modified organisms), method of crosslinking, volume of injection (0.5-6.0 mL), concentration (0.8-30 mg/mL) and posology [19]. For many years, animal source was regarded a conventional source; however, modified bacterial source is now the primary source since it is connected with cheaper costs and fewer adverse effects [20]. The pain relieving action of the injected HA is assumed to be produced through a number of different mechanisms. These include increasing extracellular matrix protein synthesis, modifying inflammatory mediators to prevent degradation, limiting lymphocyte motility, and preserving cartilage thickness, area and surface smoothness [21]. However, further study is needed to comprehend the physiological effect of HA.

Human clinical studies involving hyaluronic acid
Several clinical trials have developed numerous HA formulations and investigated their effectiveness and safety throughout the last decade. Despite the fact that several studies have shown that intra-articular HA injection can be a good non-surgical treatment for OA and can delay the need for joint replacement, there is still debate over its clinical efficacy and long-term viability.
Altman et al. concluded in 2004 that Durold in the affected joint duct with very high molecular weight HA(100,000 kDa) is not beneficial and has no superiority over placebo treated groups in terms of The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score and other efficacy parameters [22]. There is a wide range of responses to different injectable HA preparations; Johan et al. discovered that using of high molecular weight I-HA preparations resulted in clinically significant pain reduction [22]. In patients with knee OA, Lundsgaard et al. found no significant difference between the intra-articular injection of HA (Hylagan®; 6000 kDa) and injection of physiological saline [23]. When compared to I-CS, which offered the highest pain reduction during first four weeks after injection, Rodriquez-Merchan found that when compared to I-CS, which provided the most pain relief during the first 4 weeks post-injection, I-HA produced superior results at five and thirteen weeks, and that the enhanced impact lasted until 26 weeks [24]. Knee OA appears to be treated well and safely with repeated rounds of I-HA. The most prevalent adverse effect of repeated I-HA was joint swelling and arthralgia, according to a 2018 systemic evaluation of patients who had recurrent cycles of I-HA for up to 25 months [25].

Timing and duration of injection
One of the most contentious aspects of HA injection is when and how long it should be given, and if this has an influence on its efficacy and long-term viability. Cubukuc et al. compared Hylan G-F 20 and saline intraarticular injections three times a week in OA patients. They found that the HA group experienced optimal pain alleviation as early as the third week, and that functional improvement occurred by the eighth week [26]. Patrella et al. conducted a randomized controlled trial in 2006 to compare the effects of three vs six consecutive weekly HA injections. They showed that there are no variations in pain, function or patient satisfaction between 3 and 6 HA injections [27]. Huskisson et al. demonstrated in 1999 that 5 weekly intraarticular injections of sodium hyaluronate (Hyalgan A) resulted in a 6-month clinical improvement [28]. Another randomized controlled clinical trial found that that 5 weekly IA injections of HA (Hyalgan) in patients with knee OA offered long-term pain relief and improved patient function, and were at least as effective as continuous naproxen therapy for 26 weeks with fewer side effects [29].
Despite its success in KOA management, I-HA is contested, and there is no consensus among international recommendations on how to utilize it. We have tried to find the answer to these controversies by reviewing the most recent data of randomized controlled clinical trials of the last five years reporting the safety and efficacy of I-HA in KOA.

Efficacy and Safety
These clinical studies have investigated the safety and efficacy of I-HA in general [30][31][32].
and in different dose regimens of I-HA [33]. The preparations were used from various sources of I-HAanimal and non-animal [33,34] of low molecular weight (LMW) and high molecular weight (HMW) [35] of different structures -cross-linked and linear [36]. The different formulations of I-HA [37] were studied for safety and efficacy of I-HA in combination or in comparison with corticosteroids [37,38] collagen [39] bone marrow-derived mononuclear cells [40] umbilical cord-derived mesenchymal stromal cells [41] bone marrow mesenchymal stem cells [42] ozone therapy [43] polydeoxyribonucleotide [44] platelet-rich plasma [45,46] plasma rich in growth factor [47] amniotic suspension allograft [48] chondroitin [49] and N-acetyl cysteine [50]. All these trials conclude that I-HA is both safe and effective when used alone and in conjunction with other viscosupplements. Furthermore, they support I-HA's therapeutic efficacy in reducing pain and improving function in patients with KOA. In trials comparing the safety and efficacy of I-HA to other viscosupplements, the vast majority of the studies found I-HA to be non-inferior to its competitors.

Single/Multiple Injections
A variety of I-HA formulations are currently on the market for the treatment of KOA. These preparations differ in origin, structure, molecular weights, concentrations and injected volume. The majority of I-HA preparations on the market today are multiple injection regimens; however, single injection I-HA has also been developed and is becoming more widely used in clinical practice. Zhang H et al. examined the effects of single and multiple injections of I-HA in KOA in a randomized multicenter study and found that single injections were non-inferior to multiple injections of I-HA in alleviating pain, knee stiffness, and increasing physical function over 18 and 26 weeks [33]. Baron [36]. These results indicate that number of I-HA injections may not have a significant difference in terms of effectiveness of the therapy.

HMW or LMW Preparations and Cross Linking
Over the years, several studies on I-HA with different molecular weights for the treatment of KOA have been published, with contradictory results, but favoring HMW HA. A similar study that compared the effectiveness of a HMW HA with a LMW HA preparation, found that both reduced pain, stiffness and function in patients with KOA in a similar way [35].
Another study comparing the efficacy of chemically cross-linked HA and avian-derived HA in KOA patients found that the chemically cross-linked HA was superior to avian-derived HA in lowering the VAS pain score and total Western Ontario and McMaster Universities Arthritis Index (WOMAC) score as well as in decreasing the Lequesne index score [34]. These findings imply that the source of I-HA may have an impact on the therapy's efficacy.

Alone or in Combination With Other Drugs or Molecules
I-HA, alone or in conjunction with corticosteroids has been shown in studies to be safe and effective for the management of KOA. Petrella et al. showed that a single I-HA injection alone or in combination of triamcinolone was well tolerated and decreased pain associated with KOA in a prospective, multicenter, randomized study [37].
Maia et al. undertook another prospective randomized study to assess patient outcomes with intra-articular HA infiltration in conjunction with dexamethasone in treatment of KOA. The WOMAC overall score and sub scores for pain, stiffness, and function improved significantly with I-HA alone and in combination with dexamethasone. It also resulted in improvement of knee extensor and flexor strength [38].
In patients with KOA, a multicenter study testing the efficacy of a novel intra-articular combination of sodium hyaluronate-chondroitin sulfate found that the combination significantly improved pain severity and reduced analgesic use [49]. In a prospective, randomized research done by Yoon et al. combining intraarticular injection of polydeoxyribonucleotide with HA improved the efficiency of HA in enhancing knee joint functioning. All of these trials show that combination therapies with HA significantly enhances clinical outcomes in terms of pain and function in patients with KOA [44].

In Comparison With Other Injectable Therapies
In the majority of trials comparing I-HA to other viscosupplements in KOA, I-HA had similar results to its competitors. Martin et al. compared the safety and effectiveness of I-HA to MD-Knee, a collagen-containing product. The study's findings revealed at three and six months after treatment, both I-HA and MD-knee groups had equal clinical outcomes in terms of improved function and pain reduction [39].
A six-month randomized clinical research comparing the benefits of ozone treatment with I-HA in KOA found that both groups saw equal reductions in total WOMAC score and improvements in pain, stiffness and function in both the groups [43]. A double-blind randomized controlled clinical trial comparing the intraarticular injections of Platelet-Rich Plasma (PRP) with HA in the treatment of KOA found that both arms saw similar pain and functional improvements [46]. Another study comparing the effectiveness of Plasma Rich in Growth Factor (PRGF) vs HA patients with symptomatic mild to moderate KOA found that both PRGF and HA, produced significant reductions in pain evaluated by VAS and significant improvement in WOMAC and Lequesne scores at six-month follow-up [47].
Ozcamdalli et al. evaluated intra-articular injections of HA and N-acetyl cysteine (NAC) in the treatment of KOA and found that both were equally efficient in reducing pain and enhancing function in KOA patients. In addition, combination preparations consisting of HA with diclofenac [51] and HA with polynucleotides have been found to significantly improve pain in patients with KOA [52]. All of these trials show that I-HA is not inferior to other viscosupplements in the treatment of KOA [50].

Duration of Symptomatic Relief and Disease Modifying Effect
Recently the disease modifying effects of I-HA in KOA have also been studied [30,53]. Henrotin et al. conducted an open labelled multicenter study to investigate the effect of I-HA on cartilage degradation biomarkers and found that I-HA had a favorable effect on type II collagen turnover and cartilage volume, indicating that I-HA may have a potential structure-modifying effect in KOA patients [30].

Limitations of this study
Studies included are of specific time period as we wanted to look for more recent data ( Table 3). Studies published in languages other than English are not included.

Conclusions
The IA-HA injections have a limited role in treatment of knee osteoarthritis. It is recommended to use in mild to moderate knee osteoarthritis in patients who do not have sufficient pain relief with topical or oral medication and physical therapy. Not used as first line therapy as it's expensive and has no data to shows improved efficacy. It is safe and effective except minor side effects such as local pain and swelling lasting for few days. Severe allergic reactions to the preparations are extremely are. They provide adequate pain relief and functional improvement but for limited period of time up to six months irrespective of number of injections and type of preparations used. The combination formulations with corticosteroids or PRP shows better results than HA alone. Combining HA with newer molecules such as peptides or diclofenac for sustained and disease-modifying effects, requires more studies in the future.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.