Effect of knee flexion angle on infrapatellar fat pad hemoglobin during isometric quadriceps exercise

Abstract

[Purpose] This study aimed to elucidate the optimal knee flexion angles and contraction intensities for isometric quadriceps exercises to enhance the hemoglobin concentration in the infrapatellar fat pad. [Participants and Methods] This observational, cross-sectional study included 13 healthy young adults. Oxygenated hemoglobin in the infrapatellar fat pad was measured using near-infrared spectroscopy, and the change was calculated by subtracting the value after isometric quadriceps exercise from the value during exercise. Infrapatellar fat pad hardness was measured using shear wave elastography with ultrasonography, and the change was calculated by subtracting the value at rest from that during isometric quadriceps exercises. The changes were assessed under six combinations of knee flexion angle and isometric quadriceps exercise intensity based on maximum voluntary contraction (5°–10%, 5°–50%, 5°–70%, 30°–10%, 30°–50%, and 30°–70%), and were analyzed using a linear mixed model for angles and contraction intensities. [Results] Changes in oxygenated hemoglobin concentration and infrapatellar fat pad hardness at 50% and 70% intensities were significantly higher at 5° knee flexion than at 30°. At 5° of knee flexion, the change in infrapatellar fat pad hardness increased significantly with exercise intensity. [Conclusion] Isometric quadriceps exercise at 5° of knee flexion significantly increased the oxygenated hemoglobin concentration in the infrapatellar fat pad.

INTRODUCTION

Knee osteoarthritis (KOA) is a highly prevalent condition that causes knee pain^{1, 2)}. Recent studies have highlighted the role of inflammation and fibrosis of the infrapatellar fat pad (IFP) in the pathogenesis of KOA^{3, 4)}. The IFP is located deep to the patellar ligament and disperses mechanical stress during knee movements^{5, 6)}. When the IFP becomes fibrotic, it alters the even distribution of mechanical stress during knee movement. This compromises its ability to absorb shock, which may in turn worsen the damage to the knee cartilage^{7, 8)}. Therefore, mitigating IFP fibrosis is crucial to prevent the progression of KOA.

Research using a KOA rat model has demonstrated that hypoxia within the IFP induces fibrosis⁹⁾. Isometric quadriceps exercise (IQE) was reported as a method to improve IFP hypoxia¹⁰⁾. This cross-sectional study included healthy young participants who performed 10° knee flexion and 10% IQE of maximal muscle strength, which resulted in changes in hemoglobin concentration and promoted IFP oxygenation¹⁰⁾. This suggests that improving hypoxia and enhancing hemoglobin concentration changes could prevent IFP fibrosis. However, the optimal knee flexion angles and contraction intensities causing maximum changes in hemoglobin concentration are unknown. Furthermore, whether improvement in hypoxia requires improvements in knee joint angle and strength in patients with KOA remains unclear. The anatomical positioning of the IFP, surrounded by the femur, tibia, and patellar tendon^{11, 12)}, suggests that variations in knee flexion angles and contraction intensities might influence IFP hemoglobin concentration during IQE.

This study aimed to examine the optimal conditions for IQE to enhance hemoglobin concentration in the IFP, focusing on knee flexion angles and contraction intensities, in healthy individuals. We hypothesized that IQE performed with 5° knee flexion and 50% intensity would most effectively promote hemoglobin concentration changes in the IFP.

PARTICIPANTS AND METHODS

To investigate the effects of different knee flexion angles and contraction intensities on hemodynamics in the IFP, we used a cross-sectional study design, which is appropriate for assessing the relationships between variables at a single point in time¹³⁾. This study evaluated data collected from healthy volunteers between July 2023 and April 2024. Participants were recruited via convenience sampling¹⁴⁾ using word-of-mouth within Morinomiya University of Medical Sciences. This inclusion criteria required participants to be healthy young men who reported no knee pain and had no previous history of surgical procedures on the knee, traumatic knee injuries, or any neurological disorders. Women were excluded because of the influence of menstruation on blood pressure¹⁵⁾. Those with autonomic nervous system disorders, intake of drugs acting on the autonomic nervous system, systolic blood pressure at rest >140 mmHg, and diastolic blood pressure >90 mmHg were excluded.

The study protocol was approved by the Ethics Committee of Morinomiya University of Medical Sciences (approval number: 2023-124) and conducted in accordance with the principles of the Declaration of Helsinki. All participants provided written informed consent before their inclusion in the study.

The maximum voluntary isometric contraction (MVC) torque of the knee extensors was measured during IQE at 5° and 30° of knee flexion using a Biodex System 3 isokinetic dynamometer (Biodex Medical Systems, Shirley, NY, USA). Participants were seated with their trunk and thighs secured by straps, and the dynamometer’s lever arm was attached to the distal lower leg. Following a warm-up, participants performed three 5-second maximal contractions at each angle, with a 60-second rest period between trials. The highest torque value was recorded as the MVC. Target torques for the experimental tasks were set at 10%, 50%, and 70% of the MVC¹⁶⁾ for each respective angle.

Changes in tissue hemodynamics within the IFP were monitored using a NIRS system (NIRO-200NX; Hamamatsu Photonics Inc., Shizuoka, Japan), as previously described^{10, 17)}. The system used three laser diodes (775, 810, and 850 nm) and a photodiode receiver, with data sampled at 0.5 Hz. Two NIRS probes were placed medially and laterally to the patellar tendon with an inter-optode distance of 3.0 cm, providing a signal penetration depth of approximately 1.5 cm to target the IFP¹⁸⁾. Relative changes in the concentrations of oxygenated hemoglobin (O₂Hb), deoxygenated hemoglobin (HHb), and total hemoglobin (cHb) were calculated using the modified Beer–Lambert law. The protocol began with a pre-task resting period to establish a stable hemodynamic baseline, which was subsequently normalized to zero. Participants then performed 10 sets of IQE, each consisting of a 10-second contraction followed by a 3-second rest. A 5-minute recovery period was recorded post-exercise. Surface electromyography (COMETA; ArchiveTips Inc., Tokyo, Japan) of the vastus medialis was recorded to confirm appropriate muscle contraction and relaxation during the IQE. For statistical analysis, representative values were extracted from the continuous time-series data for three phases: Baseline (mean of the final 60 seconds of pre-task rest), IQE-task (mean of the 10 nadirs during contractions), and Post (mean of a 60-second window starting 3 min into recovery), as illustrated in Fig. 1. All extracted values were expressed as deviations from the Baseline. The magnitude of the post-exercise hemodynamic response was calculated as the difference between the Post and IQE-task values.

Fig. 1.

Typical time-series waveform data of changes in hemoglobin concentrations in the infrapatellar fat pad during isometric quadriceps exercise performed with 5° knee flexion and 10% intensity.

Typical changes in oxygenated hemoglobin (O₂Hb, red line), deoxygenated hemoglobin (HHb, blue line), and total hemoglobin (cHb, gray line) concentrations are illustrated as time-series waveform data. The data for the statistical analysis before (Baseline), during (IQE-task), and after (Post) isometric quadriceps exercise (IQE) are defined as the mean value at rest 1 min before the task, the mean of the 10 lower limits during the task, and the mean value at 1 min after 3 min of rest after the task, respectively. The change in each hemoglobin concentration is defined as the IQE-task value minus the Post value.

IFP hardness was quantified using shear wave elastography with an Aplio 300 ultrasound system and a 14-MHz linear transducer (PLT-1005BT; Canon Medical Systems Inc., Tochigi, Japan), following a previously described protocol¹⁰⁾. The transducer was positioned longitudinally over the patellar tendon, with imaging settings of 80 for gain, 70 for dynamic range, and a frame rate of 30 frames per minute. A representative hardness value was obtained by averaging the measurements from three distinct regions of interest within the IFP. The change in IFP hardness was defined as the difference between hardness during IQE and hardness at rest.

All dependent variables (hemoglobin concentrations and IFP hardness) were measured under six experimental conditions, comprising two knee angles (5° and 30°) and three IQE intensities (10%, 50%, and 70% MVC). The order of these six conditions was randomized for each participant.

All statistical analyses were performed using SPSS version 27 (IBM Corp., Armonk, NY, USA). Linear mixed models were used to assess the effects of knee angle (5° and 30°) and contraction intensity (10%, 50%, and 70%) on the changes in O₂Hb, HHb, cHb, and IFP hardness. The models included angle, intensity, and their interaction as fixed effects, with participant ID as a random effect. Age, height, weight, and MVC were included as covariates. The assumptions of normality and homoscedasticity of the residuals were confirmed by visual inspection of residual plots. When significant main effects or interactions were detected, pairwise comparisons of the estimated marginal means were conducted with a Bonferroni correction for multiple comparisons. The significance level was set at p<0.05.

RESULTS

Fifteen individuals met the inclusion criteria, of whom two were excluded based on the exclusion criteria. Thus, data from 13 male participants were analyzed (age: 22.1 ± 2.9 years, height: 169.8 ± 5.4 cm, weight: 62.5 ± 8.2 kg).

Regarding O₂Hb concentration changes, in the linear mixed model, the main effect between angles was significant (p=0.002), whereas the main effect between intensities was not significant (p=0.845), with no significant interaction effect (p=0.075) (Table 1). A post hoc test revealed that the change in O₂Hb concentration was significantly higher at 5° than at 30° (mean difference [MD], 2.870; 95% confidence interval [CI], 1.122–4.618; p=0.002).

Table 1. Data value of each variable for angle and intensity, and results of linear mixed models for the infrapatellar fat pad hardness and each hemoglobin concentration.

	Intensity (%)	Angle		Fixed effects		Interaction	Covariate
		5°	30°	Angle	Intensity
Hardness (cm/s)	10	0.51 ± 0.24	0.15 ± 0.22	<0.001	<0.001	<0.001	Age Height Weight MVC
	50	1.05 ± 0.44	0.19 ± 0.18
	70	1.55 ± 0.56	0.27 ± 0.38
O2Hb (µmol/dL)	10	8.75 ± 3.63	7.19 ± 3.79	0.002	0.845	0.075
	50	11.18 ± 5.03	5.38 ± 4.34
	70	9.23 ± 4.93	7.98 ± 3.59
HHb (µmol/dL)	10	4.24 ± 3.38	2.38 ± 1.48	0.071	0.345	0.292
	50	4.73 ± 2.34	3.70 ± 2.00
	70	3.62 ± 2.28	3.72 ± 2.31
cHb (µmol/dL)	10	12.99 ± 5.68	9.57 ± 4.45	0.002	0.668	0.147
	50	15.91 ± 6.66	9.08 ± 5.03
	70	12.86 ± 6.55	11.70 ± 5.09

Data are presented as means ± standard deviation. Fixed effects and interaction values indicate p-values. O₂Hb: oxygenated hemoglobin; HHb: deoxygenated hemoglobin; cHb: total hemoglobin; MVC: maximum voluntary contraction.

Regarding HHb concentration changes, in the linear mixed model, the main effects between the angles (p=0.071) and between the intensities (p=0.345) were not significant, with no significant interaction effect (p=0.292) (Table 1).

Regarding cHb concentration changes, in the linear mixed model, the main effect between angles was significant (p=0.002), whereas the main effect between the intensities was not significant (p=0.668), with no significant interaction effect (p=0.147) (Table 1). A post hoc test revealed that cHb concentration change was significantly higher at 5° than at 30° (MD, 3.800; 95% CI, 1.507–6.093; p=0.002).

Regarding IFP hardness changes, in the linear mixed model, the main effects between the angles (5° and 30°; p<0.001) and between the intensities (10%, 50%, and 70%; p<0.001) were significant, with a significant interaction effect (p<0.001) (Table 1). A post hoc test revealed that the changes in IFP hardness at 50% and 70% intensities were significantly higher at 5° than at 30° (50%: MD, 0.859; 95% CI, 0.614–1.104; p<0.001 vs. 70%: MD, 1.285; 95% CI, 1.040–1.530; p<0.001). At 5°, the change in IFP hardness was significantly higher with increasing intensity (50% vs. 10%: MD, 0.544; 95% CI, 0.299–0.789; p<0.001 and 70% vs. 50%: MD, 0.498; 95% CI, 0.253–0.743; p=0.003).

DISCUSSION

This study investigated the optimal conditions for IQE to enhance hemoglobin concentration in the IFP, focusing on knee flexion angles and contraction intensities. The O₂Hb and cHb concentration changes were significantly higher during 5° knee flexion than during 30° knee flexion. For IQE with 50% and 70% intensities, the changes in IFP hardness were significantly higher during 5° knee flexion than during 30° knee flexion. For 5° knee flexion, the change in IFP hardness was significantly high with increasing intensity. Thus, the change in IFP hardness was affected by knee flexion angles and contraction intensities, and O₂Hb and cHb concentrations were affected by knee flexion angles.

In a previous study, changes in hemoglobin concentration in the IFP occurred during 10° knee flexion and 10% of maximal strength¹⁰⁾. Changes in hemoglobin concentration during contraction at different intensities have been reported in muscles, tendons, and other tissues¹⁶⁾. Contraction intensity has been shown to influence changes in hemoglobin concentration in muscles and tendons. The present study revealed that changes in hemoglobin concentration in the IFP were influenced by knee flexion angles but not contraction intensities.

Changes in O₂Hb and cHb concentrations and IFP hardness were significantly higher during 5° knee flexion than during 30° knee flexion. During IQE with 5° knee flexion, anterior tibial sliding causes the IFP to be compressed by the patellar tendon, femur, and tibia, increasing IFP hardness^{17, 18)}. However, during IQE with 30° knee flexion, the IFP may be less affected by this mechanical stress. In the case of 5° knee flexion, repeated mechanical stress causes compression and release of capillaries within the IFP, thus increasing blood flow and inducing reactive hyperemia^{10, 17, 19)}. Thus, the oxygenation of the IFP might have improved. In contrast, contraction intensities did not affect the changes in hemoglobin concentration during IQE; however, IFP hardness increased significantly with increasing intensity during 5° knee flexion. IFP capillaries may also close in response to minor pressures similar to capillaries in skeletal muscles²⁰⁾; therefore, differences in contraction intensities might not have caused changes in hemoglobin concentration. This may be due to the structural and circulatory features of the IFP.

Fibrosis of the IFP affects the pathophysiology of KOA, and no exercise therapy that can prevent IFP fibrosis has been reported. Fibrosis of the IFP is associated with hypoxia⁹⁾, and IQE performed with 5° knee flexion, during which O₂Hb concentration improves considerably, may be an exercise therapy that can prevent IFP fibrosis. Patients with KOA often have knee joint extension limitation^21,22,23), which indicates that improving extension limitation by performing IQE in knee joint extension rather than knee joint flexion is beneficial. Future interventional studies should be conducted to clarify whether IQE with 5° knee flexion leads to improved oxygenation of the IFP in these patients.

This study has several limitations. First, as the study was conducted on young, healthy individuals, whether similar results would be achieved in patients with KOA is unclear. Second, because the participants were men, we could not elucidate the influence of sex-related differences.

In conclusion, the most critical factor for improving O₂Hb concentration in the IFP during IQE is a near-extension knee angle (5°), rather than the contraction intensity. Our findings indicate that this position-dependent mechanical stress is sufficient to induce a hemodynamic response, likely through a mechanism of reactive hyperemia following vascular compression. This represents a key physiological pathway for preventing or mitigating the IFP fibrosis implicated in the pathogenesis of KOA. This study provides foundational evidence for exercise prescription that could be beneficial for patients, particularly those with knee extension limitations. Further longitudinal clinical trials are necessary to confirm if this exercise approach can effectively prevent IFP fibrosis and improve patient symptoms such as pain, stiffness, and physical function in the long term.

Data availability

All summary data and statistics supporting the findings of this study are available within the manuscript. The underlying raw dataset contains personal information and are therefore not publicly available. However, the anonymized raw data may be made available to qualified researchers by the corresponding author upon reasonable request that includes a clear research plan and execution of a data use agreement.

Author contributions

Syoya Nakanishi: Conceptualization, Methodology, Validation, Formal analysis, Writing—Original draft, Visualization. Naoya Uemura: Validation, Formal analysis, Investigation, Resources. Masahiro Tsutsumi: Writing—Review and editing. Takashi Kitagawa: Writing—Review and editing. Toshinori Miyashita: Writing—Review and editing. Shintarou Kudo: Conceptualization, Methodology, Writing—Review and editing, Supervision, Project administration, Funding acquisition.

Funding

This work was supported by KAKENHI grant number JP24K02805.

Conflict of interest

There are no conflicts of interest.