Layout 1 Thematic Section: Advances in Musculoskeletal and Neuromuscular Rehabilitation | Maccarone & Masiero Eur J Transl Myol 34 (3) 12413, 2024 doi: 10.4081/ejtm.2024.12413 Introduction Osteoarthritis Osteoarthritis (OA) is a debilitating chronic degenerative disease that affects over 300 million patients worldwide. OA is characterized by joint pain and dysfunction, pro- gressive loss of autonomy in Activities of Daily Living (ADL), and worsening of Quality of Life (QoL).1,2 In particular, knee osteoarthritis has a prevalence of 10% and 13% respectively in men and women aged above 60 years.3 Joints affected by OA show a progressive degra- dation of articular cartilage, a thickening and sclerosis of subchondral bone, formation of pseudocysts and osteophytes, inflammation of synovium or bursa, the hy- pertrophy of joint capsule, and possible associated dege- neration of ligaments and menisci.4 Articular cartilage consists of 95% of water and extracellular matrix and only 5% of chondrocytes, the cellular elements respon- sible for proteoglycans and glycosaminoglycans synthe- sis.5 OA starts with the alteration of the normal process of remodeling of articular cartilage, with a consequent increase in the content of proteoglycans and subsequent increase of catabolic cytokines, including interleukin-1β, which promotes the increase of synthesis of metallopro- teases.6 Synovial damage is often secondary to cartilage and bone damage and consists of a reactive inflamma- tory thickening resulting from increased activity of sy- noviocytes; this process leads to an increase in the synthesis of low molecular weight hyaluronic acid, with subsequent alteration of the synovial fluid.4 The clinical manifestations of osteoarthritis are represented by pain and functional limitation with variable characteristics de- pending on the joint involved. Arthritic pain often begins insidiously; it is usually localized and accentuated with joint load, while it tends to recede during the night hours Abstract Osteoarthritis (OA) is a disabling disease that causes pain and functional limitation. OA symptoms can be treated with intra-articular injections of anti-inflammatory, viscosupplementary, or viscoinductive products. Non-responders to these approaches have limited options, often surgical (e.g. knee replacement). This retrospective study aims to evaluate the efficacy of a single injection of Carboxymethyl-Chitosan for advanced (Kellgren-Lawrence ≥3) and symptomatic knee OA in non-responders to hyaluronic acid. We enrolled 10 patients (5 female, 5 male). Treatment efficacy was assessed through the Visual Analogue Scale (VAS, pain) and the Knee Injury and Osteoarthritis Outcome Score (KOOS, knee function). Data are acquired from rating scales administered at the time of injection (T0), one month (T1), three months (T2), and six months (T3) after treatment as for clinical practice. Results showed a significant improvement in pain and function at T1, with a subsequent gradual resumption of symptoms. In conclusion, the treatment showed a better outcome in the short term (i.e. up to 1 month after treatment); however, raw values of VAS and KOOS did not return to baseline levels showing a maintenance of improvement albeit not statistically significant. Key Words: knee osteoarthritis, intra-articular injections, Carboxymethyl-Chitosan, hyaluronic acid, quality of life. Eur J Transl Myol 34 (3) 12413, 2024 doi: 10.4081/ejtm.2024.12413 Intra-articular injections with Carboxymethyl-Chitosan in patients affected by knee osteoarthritis non-responders to hyaluronic acid: a pilot study Nicola Manocchio,1 Concetta Ljoka,1,2 Nicolò Piacentini,1 Roberto Sorge,3 Giulia Vita,1 Calogero Foti1,2 1Physical and Rehabilitation Medicine, Clinical Sciences and Translational Medicine Department, University of Rome Tor Vergata, Italy; 2Physical and Rehabilitation Medicine Unit, Tor Vergata University Hospital, Rome, Italy; 3Systems Medicine, Biometric Unit, University of Rome Tor Vergata, Italy. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. - 31 - Non -co mmerc ial us e o nly Intra-articular injections with CMC in patients affected by knee OA in non-responders to hyaluronic acid Eur J Transl Myol 34 (3) 12413, 2024 doi: 10.4081/ejtm.2024.12413 to reappear during the day. Morning stiffness may coe- xist, but it is usually resolved with joint mobilization. Pain is associated with functional limitation of various entities depending on the stage of the disease.7-9 OA dia- gnosis is based on clinical detection of pain, functional limitation, bone swelling and radiographic detection of osteophytes, reduction of joint interline and subchondral sclerosis.10 Nowadays there is no definitive treatment for OA but only a series of strategies for pain control, and improvement of joint function and mobility, to lead the patient to the reco- very of autonomy in ADL and the improvement of QoL. The pharmacological approach mainly makes use of non- steroidal anti-inflammatory drugs (NSAIDs), corticoste- roids, additional analgesics (paracetamol, opioids), and vis coinductive/viscosupplementary drugs, administered orally or inside the affected joints.11-13 Chitosan Chitosan is a linear biocompatible and biodegradable po- lymer obtained from the N-deacetylation of chitin, with mucoadhesive, antioxidant, and antimicrobial properties, which make it useful in various medical fields. Due to its physical, chemical, and biological characteristics, chi- tosan and its derivatives have been extensively studied for many medical applications, including wound healing, drug administration, and tissue engineering.14-19 Various studies conducted in vitro and ex vivo have shown that intra-articular administration of this polymer could pre- vent the degradation of articular cartilage, inducing chondrogenic differentiation of mesenchymal stem cells, triggering the production of type I and II collagen and reducing the production of inflammatory and catabolic mediators by chondrocytes.15,20-22 The Carboxylated and Methylated form of Chitosan (Carboxymethyl-chitosan, CM-C) is extracted from the fungus Agaricus bisporus. If applied to the biological tissue, the degradation of CM-C occurs through a physiological macrophage reab- sorption process, in which granuloma formation has not been observed and no cytotoxic potentials have been de- monstrated in vivo. However, macrophage activation may present with a transient and reversible post-injection inflammatory reaction that responds well to treatment with oral NSAIDs.23 Experimental studies conducted on intra-articular administration of this macromolecule in animal models have shown a low incidence of post-ad- ministration side effects, limited to minimal local tissue reactions.23,24 Further studies recently conducted in vitro and ex-vivo found a higher lubricating capacity by CM- C, with a significant reduction in coefficient of friction, compared to traditional formulations of cross-linked hyaluronic acid (HA), with a more significant recovery of joint mobility.25,26 Aim This study aims to evaluate the efficacy of a single intra- articular knee injection with CM-C in non-responders to HA with advanced OA (KL≥3) on pain and functional outcomes. Materials and Methods The study has a retrospective design. Data were collected from patients attending the Physical Medicine and Rehabilitation outpatient clinic at the Tor Ver- gata University Hospital, Rome. The study analyzed data from patients treated with intra-ar- ticular CM-C (a compound of CM-C (60 mg/3 ml) consi- sting of 2% (w/w) CM-C in phosphate buffer supplemented with 3.5% sorbitol) who met the inclusion criteria within the period from September 2022 to October 2023. Data were acquired from rating scales administered by a physiatrist with several years of experience in knee OA and injection therapy at the time of injection (T0), one month (T1), three months (T2), and six months (T3) after treatment as for clinical practice. The clinical protocol was conducted, recorded, and reported by Good Clinical Practice guidelines and the Declaration of Helsinki and approved by the the Territorial Ethics Com- mittee “Lazio Area 2” (173.24). Before collecting the data, an informed consent form was signed by all the participants.27 Inclusion and exclusion criteria Subjects were enrolled according to the following criteria. Inclusion criteria: i) male and female patients of all ages with advanced and symptomatic gonarthrosis [radiographic Kellgren-Lawrence (KL) grade ≥ 3];28 ii) patients previou- sly unsuccessfully treated with intra-articular HA injections in the knee and subsequently treated at the same level with CM-C; iii) patients with a minimum 6-month follow-up who underwent scheduled clinical assessments at 1, 3, and 6 months. Exclusion criteria: i) patients not treated with CM-C; ii) patients for whom KOOS and VAS were not completed. Rating scales For this study, two rating scales were considered: the Visual Analogue Scale (VAS) for pain measurement and the Knee Injury and Osteoarthritis Outcome Score (KOOS) as a fun- ctional outcome. VAS is a pain rating scale developed by Scott and Huskis- son29 that consists of a straight line, generally 100mm long, at the extremes of which it is possible to read the indications “absence of pain” and “maximum pain”. The patient has to self-report pain intensity by placing a sign according to his or her current pain level. The proximity of the sign to one of the two extremities indicates more or less intense pain. KOOS is a self-administered questionnaire that aims to assess the reported symptoms in the knee joint.30 The scale consists of 42 items and 5 domains that respectively assess Sym- ptoms, Pain, ADL, Sports and Recreational Activities, and QoL. All items on the scale have the same response mode, using a 5-point Likert scale ranging from 0 (no problems or difficulties) to 4 (problems or high difficulties). The results of each subscale are calculated separately using the formula: 100 − (score obtained x 100) / (maximum score) The score will then be expressed as a percentage for each subscale, ranging from 0 (condition of severe disability) to 100 (excellent condition).31 - 32 - Non -co mmerc ial us e o nly Intra-articular injections with CMC in patients affected by knee OA in non-responders to hyaluronic acid Eur J Transl Myol 34 (3) 12413, 2024 doi: 10.4081/ejtm.2024.12413 Statistical analysis All data were initially entered into an Excel spreadsheet (Microsoft, Redmond, Washington, U.S.A.) and analysis was performed using the statistical package for the social sciences Windows, version 15.0 (SPSS, Chicago, Illinois, U.S.A.). Descriptive statistics shows mean ± standard de- viation (SD) since all variables were normally distributed parameters after confirmation by the Kolgomorov-Smirnov test.32 Range (min; max) is also reported as additional data. Comparisons between variables at different times were per- formed with ANOVA for repeated measures and post-hoc Bonferroni test.33,34 A value of p<0.05 was considered statistically significant. Results According to the inclusion and exclusion criteria, 10 pa- tients were enrolled in this study; male (5, 50%) and fe- male (5, 50%) were equally distributed. The anthropometric data of the sample are reported in Table 1. Table 2 shows the descriptive analysis of the variables over time. VAS (Figure 1) showed statistically significant changes over time at the ANOVA for repeated measures test (p<0.01). At the post-hoc analysis with the Bonferroni test, changes were found between T0 and T1 (p<0.01) representing a significant reduction of pain. However, VAS scores had an ascending trend after T1, with a si- gnificant worsening when comparing this timepoint to T3 (p=0.02) and T6 (p<0.01). All KOOS domains (Figure 2-6) showed statistically si- gnificant changes over time at the ANOVA for repeated measures test (Pain p=0.02; Symptoms p<0.01; ADL p<0.01; QoL p=0.01). The only exception was the Sport and Recreational Activities related domain (p=0.07). Specifically, at the post-hoc analysis with the Bonferroni test all the domains analyzed showed a significant im- provement at T1 compared to T0 (Pain p<0.01; Sym- - 33 - Figure 1. Error-bar of VAS variation during study timeline. Figure 2. Error-bar of the Symptoms Domain of KOOS variation during study timeline. Table 1. Anthropometric data of the sample. N Mean SD Min. Max. Age (Yrs) 10 74.5 4.8 68 83 Weight (kgs) 10 80.6 15.2 56 98 Height (cms) 10 167.9 8.7 159 178 BMI (kg/m2) 10 28.66 5.60 21.88 38.28 KL 10 3.6 0.5 3 4 Non -co mmerc ial us e o nly Intra-articular injections with CMC in patients affected by knee OA in non-responders to hyaluronic acid Eur J Transl Myol 34 (3) 12413, 2024 doi: 10.4081/ejtm.2024.12413 ptoms p=0.02; ADL p<0.01; QoL p=0.02). Similar to VAS, KOOS domain scores showed a deterioration trend after T1, too. However, the worsening wasn’t statistically significant, except for the Symptoms domain (Figure 2) at T6 compared to T1 (p=0.03). Discussion HA is a viable treatment option for advanced knee OA.35 In case of treatment failure, arthroscopic or surgical ap- proaches (i.e. knee replacement) are available. Total knee arthroplasty is a surgical option with a success rate, but - 34 - Table 2. Descriptive analysis of the variables over time. T0 T1 T3 T6 N 10 10 10 10 VAS Mean 72 38.5 58.5 70.6 SD 19.9 21.6 26.1 21.9 Min 40 20 10 30 Max 90 70 85 100 KOOS_PAIN Mean 38.6 60.2 51.7 44.1 SD 17.9 18.1 21.4 17.4 Min 11.1 25 11.1 22 Max 63.9 86.1 88.8 83.3 KOOS_SYM Mean 48.6 63.5 56.7 47.8 SD 14.1 15.7 16.4 17.8 Min 28.6 28.6 25 25 Max 71.4 85.7 85.7 89.3 KOOS_ADL Mean 37.2 63 51.3 47.9 SD 17.6 22.7 15.6 14.5 Min 7.4 8.8 25 30.9 Max 58.8 88.2 79.4 83.4 KOOS SPORT Mean 14.5 35 29.4 15 SD 23.1 32.3 17.1 18.3 Min 0 0 5 0 Max 75 90 55 60 KOOS_QOL Mean 22.5 36.8 31.6 32.8 SD 11.8 15.4 22.1 17.4 Min 6.3 0 0 12.5 Max 43.7 56.3 81.2 75 VAS, Visual Analogue Scale; KOOS PAIN, Pain domain of the KOOS Scale; KOOS SYM, Symptoms domain of the KOOS Scale; KOOS ADL, Activities of Daily Living domain of the KOOS Scale; KOOS SPORT, Sport and Recreational Activities domain of the KOOS Scale; KOOS QOL, Quality of Life domain of the KOOS Scale. Non -co mmerc ial us e o nly Intra-articular injections with CMC in patients affected by knee OA in non-responders to hyaluronic acid Eur J Transl Myol 34 (3) 12413, 2024 doi: 10.4081/ejtm.2024.12413 with bio-mechanical implications that often cause progres- sion of OA in the contralateral knee; arthroplasty is often required at the contralateral knee as well, with all the con- sequent surgical risks and additional biomechanical im- plications.36 Nowadays, non-surgical alternatives for non-responders with advanced OA are however very li- mited.37 Research is ongoing on this topic but there is still little data available. A recent paper involving 9 patients (4 female, 5 male), KL 2-3, showed a reduction of pain and an increase of functional outcomes after intra-articular in- jections in the knee with clodronate plus lidocaine.38 Fin- ding a new treatment option would thus be of paramount importance for two reasons. The first is to give the patient time to think without rushing about the management of their body, having the opportunity to choose the course of care and eventual surgical setting they prefer, considering the possible need for knee replacement surgery. The se- cond, and probably the most important, is to improve pa- tients' QoL even if only for a short period (e.g., up to 4-6 months) by increasing their independence in ADL and em- powering them to carry on their personal passions, hob- bies, and even work activities. The bio-psycho-social approach of the ICF and the holistic view of the person dictate that these aspects must be kept in mind in the re- habilitation setting.39 To the best of our knowledge, at the current time, only two studies have been published regarding the use of CM-C for the treatment of knee OA via injection therapy in human beings, both by Emans et al. One of them40 is the post-hoc analysis of the other.26 The important difference between - 35 - Figure 3. Error-bar of the Pain Domain of KOOS varia- tion during the study timeline. Figure 4. Error-bar of the Sports and Recreational Ac- tivities Domain of KOOS. Figure 5. Error-bar of the ADL Domain of KOOS varia- tion during the study timeline. Figure 6. Error-bar of the QoL Domain of KOOS varia- tion during the study timeline. Non -co mmerc ial us e o nly Intra-articular injections with CMC in patients affected by knee OA in non-responders to hyaluronic acid Eur J Transl Myol 34 (3) 12413, 2024 doi: 10.4081/ejtm.2024.12413 our study and these two is that in the other two, non-respon- ding patients were not recruited. Moreover, despite the small number of participants, this is the first study conducted in Italy aimed to evaluate the ef- ficacy of a single intra-articular CM-C injection for the tre- atment of patients with advanced and symptomatic knee OA unresponsive to HA treatment. In our innovative study, we aimed to evaluate the efficacy of a single intra-articular CM-C injection for the treatment of patients with advanced and symptomatic knee OA unre- sponsive to HA treatment. After data analysis, CM-C seems to show a clear efficacy one month after treatment (T1). Patients reported a signi- ficant reduction in pain and a significant increase in knee function (mobility and swelling), independence in ADL, and general QoL at T1 as evidenced by the changes in the Pain, Symptoms, ADL, and QoL KOOS domains plus VAS. In the following months, though, these indicators showed a trend of gradual worsening: at the following study time-points patients reported ascent of pain and de- scent of KOOS functional outcomes. These results are not in line with the other two available studies on this topic which showed a clear efficacy, although the population dif- ference between the studies should be considered. Emans et al. found clear improvement in pain and functional out- comes up to 6 months post-injection.26,40 In our case, it is important to note that at T2 several scores retained better raw values than T0, albeit without reaching statistical si- gnificance. Even at T3, almost all variables returned to raw values that still showed a small improvement though being roughly similar to those observed before treatment. Pa- tients thus on average reported a positive trend in the first few months after treatment, and probably the small sample size prevented greater statistical evidence. Limitations This study has several limitations such as a small sample size, a short follow-up period, the absence of a control group and its retrospective design. These limitations did not allow a more in-depth statistical analysis. Conclusions This is the first study conducted in Italy to evaluate the ef- fects of a single intra-articular CM-C injection for the tre- atment of patients with knee OA unresponsive to HA. This retrospective study suggests a short-lasting overall ef- ficacy of CM-C for the treatment of patients with advanced knee OA (KL≥3) non-responders to intra-articular HA in- jection. Reduction in pain and increase in functional outco- mes were observed clearly at one month after CM-C injections but lasted only as a small improvement in the next study time-points up to 6 months. CM-C could then appear as a treatment option for this po- pulation to extend the time to surgery and make the decision more informed. The albeit small improvement in QoL in people who have few or no alternatives for treatment of a disabling condition such as advanced knee OA should not be neglected. List of acronyms ADL: Activities of Daily Living. CM-C: Carboxymethyl-chitosan. HA: hyaluronic acid. KL: Kellgren-Lawrence. KOOS: Knee Injury and Osteoarthritis Outcome Score. NSAIDs: Non-Steroidal Anti-Inflammatories Drugs. OA: Osteoarthritis. QoL: Quality of Life. SD: Standard Deviation. VAS: Visual Analogue Scale. Contributions NM, investigation, writing - original draft, review and edi- ting; CL, methodology, writing – review and editing; NP, GV: investigation, writing – original draft; RS, formal ana- lysis; CF, conceptualization, supervision, writing - review and editing. Conflict of interest All authors have read and approved this manuscript. The authors declare no conflicts of interest. Funding No funding was needed or requested. Ethics approval The Territorial Ethics Committee “Lazio Area 2” approved this study (173.24). The study conforms with the Helsinki Declaration of 1964, as revised in 2013, concerning human and animal rights. Informed consent All patients participating in this study signed a written in- formed consent form for participating in this study. Patient consent for publication Written informed consent was obtained from a legally au- thorized representative(s) for anonymized patient informa- tion to be published in this article. Availability of data and materials All data generated or analyzed during this study are inclu- ded in this published article. Corresponding author Calogero Foti, Physical and Rehabilitation Medicine, Cli- nical Sciences and Translational Medicine Department, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy. Tel.: +39.0620.900594. - 36 - Non -co mmerc ial us e o nly Intra-articular injections with CMC in patients affected by knee OA in non-responders to hyaluronic acid Eur J Transl Myol 34 (3) 12413, 2024 doi: 10.4081/ejtm.2024.12413 ORCID ID: 0000-0003-2246-348X E-mail: foti@med.uniroma2.it Nicola Manocchio ORCID ID: 0009-0009-9900-4725 E-mail: nicola.manocchio@uniroma2.it Concetta Ljoka ORCID ID: 0000-0001-6260-8474 E-mail: concetta.ljoka@ptvonline.it Nicolò Piacentini ORCID ID: 0009-0004-3773-3934 E-mail: nicolo.piacentini@students.uniroma2.eu Roberto Sorge ORCID ID: 0000-0002-4513-151X E-mail: sorge@uniroma2.it Giulia Vita ORCID ID: 0009-0009-3586-171X E-mail: giulia.vita@students.uniroma2.eu References 1. Allen KD, Thoma LM, Golightly YM. Epidemiology of osteoarthritis. Osteoarthr Cartilage 2022;30:184–95. 2. Jang S, Lee K, Ju JH. Recent updates of diagnosis, pa- thophysiology, and treatment on osteoarthritis of the knee. IJMS 2021;22:2619. 3. Dantas LO, Salvini TDF, McAlindon TE. 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Disclaimer All claims expressed in this article are solely those of the authors and do not necessarily represent those of their af- filiated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Submitted: 21 February 2024. Accepted: 2 May 2024. Early access: 22 August 2024. - 38 - Non -co mmerc ial us e o nly