Synovial fluid peptidase activity as a biomarker for knee osteoarthritis clinical progression.

AIMS: To analyze the potential role of synovial fluid peptidase activity as a measure of disease burden and predictive biomarker of progression in knee osteoarthritis (KOA).
METHODS: A cross-sectional study of 39 patients (women 71.8%, men 28.2%; mean age of 72.03 years (SD 1.15) with advanced KOA (Ahlbäck grade ≥ 3 and clinical indications for arthrocentesis) recruited through the (Orthopaedic Department at the Complejo Asistencial Universitario de León, Spain (CAULE)), measuring synovial fluid levels of puromycin-sensitive aminopeptidase (PSA), neutral aminopeptidase (NAP), aminopeptidase B (APB), prolyl endopeptidase (PEP), aspartate aminopeptidase (ASP), glutamyl aminopeptidase (GLU) and pyroglutamyl aminopeptidase (PGAP).
RESULTS: Synovial fluid peptidase activity varied significantly as a function of clinical signs, with differences in levels of PEP (p = 0.020), ASP (p < 0.001), and PGAP (p = 0. 003) associated with knee locking, PEP (p = 0.006), ASP (p = 0.001), GLU (p = 0.037), and PGAP (p = 0.000) with knee failure, and PEP (p = 0.006), ASP (p = 0.001), GLU (p = 0.037), and PGAP (p < 0.001) with knee effusion. Further, patients with the greatest functional impairment had significantly higher levels of APB (p = 0.005), PEP (p = 0.005), ASP (p = 0.006), GLU (p = 0.020), and PGAP (p < 0.001) activity, though not of NAP or PSA, indicating local alterations in the renin-angiotensin system. A binary logistic regression model showed that PSA was protective (p = 0.005; Exp (B) 0.949), whereas PEP (p = 0.005) and GLU were risk factors (p = 0.012).
CONCLUSION: These results suggest synovial fluid peptidase activity could play a role as a measure of disease burden and predictive biomarker of progression in KOA. Cite this article: Bone Joint Res 2020;9(11):789-797.

We examined whether synovial fluid peptidase activity has a potential role as a measure of disease burden and predictive biomarker of progression in knee osteoarthritis (KOA).

Synovial fluid peptidase levels varied significantly between patients requiring total knee arthroplasty (TKA) and others with advanced KOA managed conservatively.

To our knowledge, these are the first data to suggest that synovial fluid peptidase activity could play a role as a measure of disease burden and predictive biomarker of progression in KOA.

Limitations of the study include failure to consider potential differences in conservative treatment, and the sample size being relatively small, limiting the reliability and validity of the results.

Introduction

Osteoarthritis (OA) is a chronic inflammatory joint disease that frequently coexists with other comorbidities, reducing joint range of motion and generating pain, functional limitations, and a high economic burden.[1-3] Although there are some partially effective methods,[4] it is generally accepted that treatments do not completely prevent disease progression,[3] which is associated with cartilage erosion and joint inflammation, leading to disability.[1]

Several OA phenotypes have been described,[5] and RNA expression patterns have been found in knee osteoarthritis (KOA) cartilage,[6] indicating the importance of developing new biomarkers to improve the management of this condition. Regarding potential biomarkers, Bauer et al[7] proposed the Burden of Disease, Investigative, Prognostic, Efficacy of Intervention and Diagnostic (BIPED) classification and this has been used in subsequent research;[8] nonetheless, early biomarkers for clinical progression of OA have yet to be established.[9]

Several local renin-angiotensin systems have been described,[10] including one in the synovial fluid and synovium.[11] Animal models indicate its involvement in KOA,[12] and that it promotes periarticular osteopenia by increasing bone resorption and decreasing bone formation.[13] Moreover, research in humans suggests intra-articular renin and angiotensin-converting enzyme may contribute to progression in rheumatoid arthritis (RA).[11] Indeed, aliskiren, a renin inhibitor, has had positive effects in rat models of OA[14] and osteoporosis.[15] Angiotensin II is a known potent proinflammatory mediator,[16] and angiotensin receptor blockers show anti-inflammatory effects in animal models of arthritis.[17] Further, articular chondrocyte angiotensin II type 1 receptor was implicated in KOA progression in a mechanical stress mouse model[18] and activation of the renin-angiotensin system might be involved in the pathogenesis of this condition.[12] Nonetheless, the association between angiotensin-converting enzyme and KOA remains controversial.[19]

Peptidases can degrade bioactive peptides, modifying their physiological actions, and consequently may regulate cell growth and differentiation, and signal transduction.[20] Specifically, serum peptidases, such as angiotensin-converting enzyme (ACE), angiotensin II-converting enzyme (ACE2), neutral aminopeptidase (NAP), and aminopeptidase A (APA), are important elements of the renin-angiotensin system.[21] For example, ACE is essential for degradation of angiotensin I, to obtain angiotensin II, and regulate blood pressure.[22]

Abnormal peptidase levels have been observed in a range of conditions, from renal cancer (ACE and NAP)[23] to chronic tonsillitis (APA and dipeptidyl-peptidase IV).[24] In OA, proteomic studies have shown inflammatory cytokines and proteases in synovial fluid,[25,26] and evidence suggests the renin-angiotensin system may play a role in the pathophysiology of arthritis.[13,27] Specifically, dipeptidyl-peptidase IV could be involved in OA inflammation.[28] Other synovial peptidases implicated in inflammatory processes (e.g. aminopeptidase N, expressed by fibroblast-like synoviocytes) are present in synovial fluid and could play a role in OA.[29] As de Silveira et al[30] demonstrated, in an animal model of arthritis, joint inflammation was induced by activation of the renin-angiotensin system, but reduced by activation of angiotensin-converting enzyme-2/Ang-(1–7)/Mas receptor pathway.

Further, preoperatory predictive markers of knee joint infection have been proposed,[31-33] but it remains unclear whether the activity of any specific enzymes can be considered a reliable measure of disease burden or predictor of progression in KOA. In this context, we considered that analyzing potential biomarkers could contribute to this line of research. In particular, we explored the potential role of synovial fluid peptidase activity as a measure of disease burden and predictive biomarker of progression in KOA, as this would be useful in clinical practice.

Methods

This cross-sectional study was approved by the Ethics Committee of the University of León, Spain (Agency code: AVPD; ref.: 280310015, 7 October 2011) and conducted in accordance with the Declaration of Helsinki (2013, revised May 5, 2015), ethical regulations and Spanish Laws for Data Protection (15/1999), and Biomedical Research in Human Participants (14/2007). All participants gave written informed consent to arthrocentesis and inclusion in this study.

The study is reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology statement.[34] Patients with advanced KOA were recruited by non-probability sampling through the Orthopaedic Department at CAULE, Spain, between 2011 and 2019.

Inclusion criteria were a diagnosis of KOA (Ahlbäck grade ≥ 3)[35] and clinical indications for arthrocentesis and related treatment, i.e. the extraction of synovial fluid and intra-articular injections for the local treatment of peripheral joint disease. We excluded patients with contraindications to arthrocentesis, biochemical markers of inflammatory activity, or inflammatory comorbidities. A sample size calculation indicated we needed to recruit at least 38 patients. Overall, we included 39 patients (women 71.8%, men 28.2%) with advanced KOA and a mean age of 72.03 years (SD 1.154; 69.69 to 74.36) (see Supplementary table i).

We assessed the activity of the following peptidases: puromycin-sensitive aminopeptidase (PSA) (EC 3.4.11.14, cytosolic form), NAP (EC 3.4.24.11), aminopeptidase B (APB) (EC 3.4.11.6), PEP (EC 3.4.21.26), aspartate aminopeptidase (ASP) (EC 3.4.11.21), glutamyl aminopeptidase (GLU) (EC 3.4.11.7), and pyroglutamyl aminopeptidase (PGAP) (EC 3.4.19.3). At enrolment, synovial fluid samples (blood-free and ≥ 10 ml) were collected from all patients by standard arthrocentesis, centrifuged, and frozen until blind analysis (see Supplementary Material 2). In all cases, samples were collected ≥ six months after the most recent intra-articular injections.

Further, we retrieved data on MRI and laboratory test results (e.g. knee cartilage thickness and blood counts) from health records and collected data on sociodemographic and clinical characteristics through clinical examinations and interviews. Lastly, patient-reported outcome measures ((PROMs) quality of life, physical status, and functional status) were assessed at the most recent visit (see Supplementary Material).

For secondary analysis, the sample was divided into a conservative treatment group (CTG) (n = 18; women 66.7%; mean age 71.33 years (SD 1.926; 67.31 to 75.35)) and knee arthroplasty group (KAG) (n = 21; women 77.8%, mean age 72.83 years (SD 1.135; 70.44 to 75.23)). The CTG contained patients who responded satisfactorily to conservative management, and the KAG those selected for knee arthroplasty following usual criteria: namely, knee locking, failure and/or effusion, pain and joint cartilage damage regardless of the presence of deformity, and others who did not respond satisfactorily to conservative management (see Supplementary Material). Figure 1 shows the flow of patients through the study.

Fig. 1

Flow of patients through the study (Consolidated standards of reporting Trials (CONSORT) flow diagram).

Statistical analysis

All statistical analyses were performed using SPSS Statistics for Windows v25 (IBM, Armonk, New York, USA). The Shapiro-Wilk test was used to check whether quantitative data were normally distributed. Sociodemographic and clinical characteristics of all patients were summarized using descriptive statistics (medians, means, and SDs) and comparisons made using independent-samples t-test or Mann-Whitney U tests for quantitative variables, and chi-squared tests for qualitative variables.

Binary logistic regression models were built to explore the effects of the peptidase activities studied on the dependent binary variable of interest (namely, knee locking, failure, or effusion) with all the samples, and in secondary analysis, the need for arthroplasty (inclusion in the KAG). All enzymes were initially considered candidates and the best model was identified with a backward stepwise procedure, using the Wald statistic. Further, B coefficients were calculated for the predictive equation, as well as Exp(B) values and corresponding 95% confidence intervals for the peptidase activities included. Additionally, having performed Levene’s test to confirm homoscedasticity, independent-samples t-tests were used for between-group comparisons, and standardized means were calculated to assess effect sizes (Cohen’s d).

Results

The synovial fluid peptidase levels studied did not vary significantly with knee cartilage thickness as measured by MRI. Nonetheless, we observed clinically significant differences in NAP as a function of pain and range of motion (Table I). Further, peptidase levels did vary significantly as a function of clinical signs, with differences in levels of PEP, ASP, and PGAP associated with knee locking, PEP, ASP, GLU, and PGAP with knee failure, and PEP, ASP, GLU, and PGAP with knee effusion (Table II). Moreover, the knee locking model showed an influence of PSA, ASP, and GLU, while NAP and PGAP were significant in knee failure and effusion models (Table III). In addition, knee locking, failure, and effusion were all significantly associated with the need for knee arthroplasty (Table IV).

Table I.

Comparison between peptidase activity and patient-reported outcome measures (PROMs): state of the articular cartilage (as assessed by MRI) and pain on movement and range of motion (as assessed with the modified Knee Society Score) (n = 39).[36]

Peptidase activity (U/mg prot)*	Median (IQR)	p-value†	p-value‡	p-value§
NAP	87.8259 (23.42 to 338.79)	0.565	0.051	0.051
PSA	173.0000 (40.56 to 367.44)	0.502	0.154	0.154
ABP	46.8974 (9.00 to 276.54)	0.776	0.103	0.103
PEP	15.3273 (3.00 to 64.15)	0.266	0.103	0.103
ASP	13.7366 (4.93 to 32.99)	0.734	0.205	0.205
GLU	14.0000 (5.00 to 46.88)	0.738	0.564	0.564
PGAP	7.0000 (3.00 to 17.24)	0.426	0.256	0.256

Peptidase activity reported as units of enzyme per milligram of protein; Mann-Whitney U test (p < 0.05).

Comparison between peptidase activity and state of the articular cartilage (assessed by MRI). Mann-Whitney U test.

Comparison between peptidase activity and pain on movement (as assessed with the modified Knee Society Score).

Comparison between peptidase activity and range of motion (as assessed with the modified Knee Society Score).

APB, aminopeptidase B; ASP, aspartate aminopeptidase; GLU, glutamyl aminopeptidase; IQR, interquartile range; NAP, neutral aminopeptidase; PEP, prolyl endopeptidase; PGAP, pyroglutamyl aminopeptidase; PSA, puromycin-sensitive aminopeptidase.

Table II.

Comparison between peptidase activity (reported as units of enzyme per milligram of protein) and clinical signs: knee locking, knee failure, and knee effusion (n = 39).

Peptidase activity (U/mg prot)*	Median (IQR)	p-value†	p-value‡	p-value§
NAP	87.8259 (23.42 to 338.79)	0.251	0.489	0.489
PSA	173.0000 (40.56 to 367.44)	0.392	0.735	0.735
ABP	46.8974 (9.00 to 276.54)	0.041	0.076	0.076
PEP	15.3273 (3.00 to 64.15)	0.020	0.006	0.006
ASP	13.7366 (4.93 to 32.99)	< 0.001	0.001	0.001
GLU	14.0000 (5.00 to 46.88)	0.071	0.037	0.037
PGAP	7.0000 (3.00 to 17.24)	0.003	< 0.001	< 0.001

U/mg prot.: Peptidase activity reported as units of enzyme per milligram of protein; Mann-Whitney U test (p < 0.05).

Comparison between peptidase activity and knee locking.

Comparison between peptidase activity and knee failure.

Comparison between peptidase activity and knee effusion.

APB, aminopeptidase B; ASP, aspartate aminopeptidase; GLU, glutamyl aminopeptidase; IQR, interquartile range; NAP, neutral aminopeptidase; PEP, prolyl endopeptidase; PGAP, pyroglutamyl aminopeptidase; PSA, puromycin-sensitive aminopeptidase.

Table III.

Binary logistic regression models.

Category	B	SD	Wald	Sig.	Exp(B)	95% CI for Exp(B)
Peptidase activity and knee locking *
PSA	−0.016	0.008	4.268	0.039	0.984	0.970 to 0.999
ASP	0.246	0.091	7.268	0.007	1.279	1.069 to 1.529
GLU	0.155	0.086	3.203	0.073	1.167	0.985 to 1.383
Constant	−2.127	1.328	2.565	0.109	0.119	N/A
Peptidase activity and knee failure †
NAP	−0.020	0.010	3.888	0.058	0.980	0.961 to 1.001
PGAP	0.930	0.337	7.643	0.006	2.536	1.311 to 4.904
Constant	−3.273	1.529	4.581	0.032	0.038	N/A
Peptidase activity and knee effusion ‡
NAP	−0.020	0.010	3.888	0.058	0.980	0.961 to 1.001
PGAP	0.930	0.337	7.643	0.006	2.536	1.311 to 4.904
Constant	−3.273	1.529	4.581	0.032	0.038	N/A
Peptidase activity and inclusion in the KAG §
PSA	−0.052	0.019	8.012	0.005	0.949	0.915 to 0.984
PEP	0.414	0.149	7.738	0.005	1.513	1.130 to 2.026
GLU	0.423	0.169	6.250	0.012	1.527	1.096 to 2.128
Constant	−3.625	2.077	3.046	0.081	0.027	N/A

Omnibus p < 0.005; Nagelkerke R2 = 0.577; Hosmer-Lemeshow p = 0.808.

Omnibus p < 0.005; Nagelkerke R2 = 0.520; Hosmer-Lemeshow p = 0.893.

Omnibus p < 0.005; Nagelkerke R2 = 0.520; Hosmer-Lemeshow p = 0.893.

Omnibus p = 0.000; Nagelkerke R2 = 0.812; Hosmer-Lemeshow p = 0.222. This shows the influence of PSA, PEP, and GLU, as independent variables, on a dependent binary variable indicating inclusion in the Knee Arthroplasty Group. PSA was a protective factor, whereas PEP and GLU were risk factors.

ASP, aspartate aminopeptidase; CI, confidence interval; GLU, glutamyl aminopeptidase; KAG, Knee Arthroplasty Group; N/A, not applicable; NAP, neutral aminopeptidase; PEP, prolyl endopeptidase; PGAP, pyroglutamyl aminopeptidase; PSA, puromycin-sensitive aminopeptidase.

Table IV.

Between-group comparison of qualitative clinical variables commonly used in routine clinical practice as criteria for indicating knee arthroplasty (n = 39). All p-values were < 0.05 (chi-squared test).

Qualitative clinical variable	Conservative treatment group (n = 18)	Knee arthroplasty group (n = 21)
Knee locking, n (%)	4 (16)	21 (84)
Knee failure, n (%)	5 (19.2)	21 (80.8)
Knee effusion, n (%)	5 (19.2)	21 (80.8)

Regarding the secondary analysis by need for arthroplasty (CTG vs KAG), the groups were similar, with no significant differences in sociodemographic or the majority of clinical variables (Tables V and VI and Supplementary table iii). Nonetheless, as expected, there were significant differences in variables commonly used as criteria for indicating knee arthroplasty (Table VII). Specifically, KAG patients had not substantially benefited from previous treatment, such as hyaluronic acid injections, and had highly inflamed knee joints, with recurrent severe distention of the joint capsule, intense pain, and joint effusion, locking, and failure in all cases.

Table VII.

Between-group comparison of qualitative clinical variables.

Clinical variable	Conservative treatment group (n = 18)	Knee arthroplasty group (n = 21)	p-value*
Degree of OA (Ahlbäck grade), n (%)†
3	3 (16.7)	4 (19.0)	0.006
4	8 (44.4)	17 (81.0)
5	7	0
Change in quality of life over 12 months (EuroQol EQ-5D)†	18 (100)	21 (100)	< 0.001
BMI, kg/m2‡
18.5 to 24.9 (normal weight)	4 (22.2)	5 (23.8)	0.981
25.0 to 29.9 (grade I and II overweight)	10 (55.6)	11 (52.4)
30.0 to 39.9 (grade I and II obesity)	4 (22.2)	5 (23.8)
History of surgery for OA in contralateral knee, n (%)			0.052
No	15 (83.3)	21 (100)
Yes	3 (16.7)	0 (0)
History of surgical interventions in the knee, n (%)			0.002
No	13 (72.2)	5 (23.8)
Yes	5 (27.8)	16 (76.2)
Knee locking, n (%)†			< 0.001
No	14 (77.8)	0 (0)
Yes	4 (22.2)	21 (100)
Knee failure, n (%)†			< 0.001
No	13 (72.2)	0 (0)
Yes	5 (27.8)	21 (100)
Knee effusion, n (%)†			< 0.001
No	13 (72.2)	0 (0)
Yes	5 (27.8)	21 (100)
History of treatment
Only oral analgesia	11 (61.1)	9 (42.9)	< 0.001
Oral analgesia + physical therapy + intra-articular injections	7 (38.9)	12 (57.1)

Chi-squared test.

Commonly used in routine clinical practice as criteria for indicating knee arthroplasty.

Classification as recommended by Spanish Society for the Study of Obesity[37] and Federation of European Nutrition Societies (FENS),[38] 2020.

BMI, body mass index; OA, osteoarthritis.

All peptidases studied were detected in all patients, and several between-group differences were found. Specifically, KAG patients showed significantly higher activity of APB, PEP, ASP, GLU, and PGLU, but not PNA or PSA (Table VIII).

Table VIII.

Between-group comparison of levels of activity of synovial fluid peptidases. Each column of p-values signify comparisons between groups.

Peptidase activity (U/mg prot)*	Conservative treatment group (n = 18)			Knee arthroplasty group (n = 21)			p-value‡
	Average (median or mean (SD))	IQR or 95% CI	p-value†	Average (median or mean (SD))	IQR or 95% CI	p-value†
NAP	106.500	41.00 to 266.00	0.041	85.5089	23.42 to 338.79	0.001	0.223
PSA	202.2193 (87.06746)	95% CI 158.9217 to 245.516	0.222	152.8595	40.56 to 367.44	0.013	0.183
ABP	17.500	9.00 to 242.00	0.000	55.5021	28.11 to 276.54	0.000	0.005
PEP	12.8358 (5.55793)	95% CI 10.0719 to 15.5997	0.859	16.9785	9.06 to 64.15	0.000	0.006
ASP	8.500	5.00 to 22.40	0.023	19.0425	4.93 to 32.99	0.045	< 0.001
GLU	11.000	5.00 to 30.88	0.037	16.3267	8.79 to 46.88	0.003	0.020
PGAP	6.000	3.00 to 13.00	0.012	9.4819 (3.21187)	95% CI 8.0199 to 10.9439	0.080	< 0.001

Peptidase activity is reported as units of enzyme per milligram of protein (U/mg prot).

Shapiro-Wilk test.

Mann-Whitney U test.

APB, aminopeptidase B; ASP, aspartate aminopeptidase; CI, confidence interval; GLU, glutamyl aminopeptidase; IQR, interquartile range; NAP, neutral aminopeptidase; PEP, prolyl endopeptidase; PGAP, pyroglutamyl aminopeptidase; PSA, puromycin-sensitive aminopeptidase.

A binary logistic regression model showed effects of PSA, PEP, and GLU on the need for arthroplasty. PSA was a protective factor, whereas PEP and GLU were risk factors (Table III) (Nagelkerke R2 p = 0.812; Hosmer-Lemeshow p = 0.222; Omnibus p < 0.001).

There was a significant difference between groups (Levene’s p = 0.138; independent-samples t-test p = 0.011); specifically, the difference between means was −8.22763 (95% confidence interval (CI) −1.98798 to −14.46727), with higher levels in the KAG than the CTG. The effect sizes obtained were 0.36, 0.79, and 0.64 for PSA, PEP, and GLU, respectively.

Discussion

To our knowledge, this study provides the first evidence for the role of synovial fluid peptidase activity as a measure of disease burden and predictive biomarker of progression in KOA. Specifically, we found significant differences in biomarker levels as a function of pain and range of motion, with somewhat different patterns in each case.

To date, few studies have investigated synovial fluid aminopeptidase activity in intra-articular inflammatory processes in humans. Some years ago, human articular chondrocytes obtained from patients with OA undergoing arthroplasty were found to express angiotensin II receptors.[39] In line with this, losartan showed anti-inflammatory action in human arthritis.[40] Further, Cobankara et al[11] have suggested that the local articular renin-angiotensin system is involved in joint destruction in RA, and recent studies support the view that this system plays a role in the pathophysiology of arthritis.[13,27]

We have found significant differences in synovial fluid levels of some biomarkers as a function of clinical signs: knee locking, failure, and effusion, although not as a function of PROMs (visual analogue scale, modified Knee Society Score, or EQ-5D score). Moreover, in patients with advanced KOA, synovial fluid peptidase analysis revealed significant differences between patients requiring TKA and those managed conservatively, the former (those with the greatest functional impairment) having significantly higher peptidase activities, indicating alterations in the local renin-angiotensin system in the cases of APB, PEP, ASP, GLU, and PGAP, but not NAP or PSA. This is consistent with the higher APB activity in synovial fluid from swollen knees in a rat model of RA[41] and synovial fluid NAP activity inducing T-cell chemotaxis in a similar animal model.[42] Further, NAP seems to be involved in the pathogenesis of RA[43] and possibly also OA.[29]

The predictive model for knee locking showed the influence of PSA, ASP, and GLU, while APN and PGLU were significant in knee failure and effusion models (Table III). Further, the model for the need for arthroplasty (Table III) showed the influence of PSA, PEP, and GLU. Specifically, this model indicated that PSA activity was a protective factor, whereas PEP and GLU activities were risk factors. We could have studied other inflammatory cytokines and matrix metalloproteinases, but opted to focus on synovial soluble peptidases involved in the articular renin-angiotensin system, which has been associated with clinical progression in KOA.

Although between-group differences were not significant for NAP or PSA, both were considered candidates for inclusion in this model, given their clinical relevance, and the results indicated a role as a predictive factor for PSA, but not for NAP. The potential importance of NAP is related to its expression by fibroblast-like synoviocytes in inflamed synovial tissue in humans, suggesting a role in acute inflammatory arthritis.[29] Regarding PSA, while there is a paucity of data, spinorphin, an endogenous enzyme inhibitor, has been reported to break down enkephalin and play a role in pain and inflammation.[44] The possible role of PSA in pain strengthens its clinical relevance, in that, as underlined by scientific societies, pain is an important clinical feature in deciding whether to indicate arthroplasty, and this is what motivated us to include it in our study. Notably, patients with chronic pain associated with fibromyalgia have enzyme activity characterized by abnormally low serum enkephalin-degrading enzyme activity,[45] suggesting that it may influence musculoskeletal pain and that PSA might be involved in pain neuromodulatory mechanisms in OA. More studies are needed to clarify these issues.

Given the high specificity in the clinical characterization of these patients, and in line with the BIPED classification,[7,8] our results suggest a role for synovial fluid peptidase activity as a measure of disease burden and predictive biomarker of progression in KOA, which would be useful in clinical practice. Generally, patients with advanced KOA are seen by trauma and orthopaedic specialists every six months at outpatient appointments, in which arthrocentesis may be performed to drain synovial fluid, and intra-articular injections given. During such appointments, a small quantity of synovial fluid could be collected easily, inexpensively, and without causing bleeding, and sent for peptidase analysis, which is straightforward and quick. The use of PSA, PEP, and GLU activities as indicators of disease burden and predictors of progression could help clinicians decide whether to indicate knee arthroplasty. This might not only reduce delays, avoiding patients waiting a further year and potentially experiencing functional deterioration, but also the risk of complications (such as infection) should surgery become necessary,[31-33] and the need for prosthesis arthroplasty, reducing surgical reintervention and failure rates.

This study has limitations, including failure to consider differences in conservative treatment, or other proteins and enzymes likely involved in KOA. Other limitations are the small sample size, and that we only analyzed samples from patients undergoing arthrocentesis. All the participants had a marked inflammatory component, and our results may not apply to less inflamed joints. Future studies are needed to explore whether enzyme activity is influenced by treatment type (anti-inflammatory corticosteroids, chondroprotective agents, or platelet-rich plasma), and confirm whether peptidase levels are indeed reliable bioindicators of KOA progression.

Table V.

Between-group comparison of quantitative sociodemographic and clinical variables.

Variable	Conservative treatment group (n = 18)			Knee arthroplasty group (n = 21)
	Average (median or mean (SD))	IQR or 95% CI	p-value*	Average (median or mean (SD))	IQR or 95% CI	p-value*	p-value†	p-value‡
Age, yrs	72.83 (4.817)	95% CI 70.44 to 75.23	0.518	71.33 (8.828)	95% CI 67.31 to 75.35	0.317	0. 507	0.922
Duration of pain, yrs	9.50	2 to 30	0.007	9.00	6 to 22	0.005	0.563	0.282
Pain VAS (1 to 10)	8.00	4 to 10	0.003	8.00	6 to 10	0.029	0.205	0.174
mKSS ROM (0 to 100)	26.67 (13.284)	95% CI 20.06 to 33.27	0.096	28.33 (15.838)	95% CI 21.12 to 35.54	0.077	0.726	0.707
mKSS pain on movement (0 to 100)	22.17 (16.525)	95% CI 13.95 to 30.38)	0.091	13.00	0 to 69	0.018	0.919	0.967

Shapiro-Wilk test.

Independent-samples t-test.

Mann-Whitney U test.

CI, confidence interval; IQR, interquartile range; mKSS, Insall’s modified Knee Society Score; ROM, range of motion; VAS, visual analogue scale.

Table VI.

Between-group comparison of qualitative sociodemographic and clinical variables.

Variable	Conservative treatment group (n = 18)	Knee arthroplasty group (n = 21)	p-value*
Sex, n (%)			0.442
Female	14 (77.8)	14 (66.7)
Male	4 (22.4)	7 (33.3)
Surgical risk based on physical status (ASA class), n (%)
No systemic conditions	6 (33.3)	5 (23.8)	0.700
Mild systemic disease	10 (55.6)	12 (57.1)
Severe systemic disease	2 (11.1)	4 (19.0)
Laterality, n (%)			0.882
Right	9 (50)	10 (47.6)
Left	9 (50)	11 (52.4)
Contralateral pain, n (%)			0.159
No	6 (33.3)	3 (14.3)
Yes	12 (66.6)	18 (85.7)
mKSS pain on movement (Insall’s modified Knee Society Score), n (%)
Acceptable (60 to 69)	0 (0)	1 (4.8)	0.348
Poor (< 60)	18 (100)	20 (95.2)
mKSS ROM, n (%)
Acceptable (60 to 69)	0 (0)	1 (4.8)	0.348
Poor (< 60)	18 (100)	20 (95.2)
State of the articular cartilage (assessed by MRI), n (%)
Ulceration (depth < 50%)	2 (11.1)	0 (0)	0.267
Ulceration (depth ≥ 50%)	8 (44.4)	12 (57.1)
Exposure of subchondral bone	8 (44.4)	9 (42.9)

Chi-squared test.

ASA, American Society of Anesthesiologists; mKSS, Insall’s modified Knee Society Score;ROM, range of motion.