Relationship between Connective Tissue Morphology and Lower-Limb Stiffness in Endurance Runners. A Prospective Study

lower limb behaves like a spring compressing and decompressing during running, where lower-limb stiffness is one of the most in uential factors. This prospective observational study is aimed at examining the relationship between the connective tissue morphology and lower-limb stiffness and investigating whether the barefoot/shod condition in uences on such relationship. Methods: 14 male amateur runners (10-km time trial <50 0 ) were included. Data were recorded over one session, where participants ran 2 trials (i.e., barefoot and shod conditions) of 3 minutes at 12 km/h, where running spatiotemporal parameters and vertical (Kvert) and leg stiffness (Kleg) were obtained. Prior to testing trials, thickness and cross-sectional area (CSA) were recorded for Achilles (AT) and

amateur runners (10-km time trial <50 0 ) were included. Data were recorded over one session, where participants ran 2 trials (i.e., barefoot and shod conditions) of 3 minutes at 12 km/h, where running spatiotemporal parameters and vertical (Kvert) and leg stiffness (Kleg) were obtained. Prior to testing trials, thickness and cross-sectional area (CSA) were recorded for Achilles (AT) and patellar tendons (PT) and plantar fascia (PF) with ultrasound. Results: Under barefoot condition, a positive correlation was found between Kleg and AT-thickness and CSA and PF-thickness; and between Kvert and AT-thickness and PF thickness. Under shod condition, a positive correlation was found between Kleg and PT-CSA and PT-thickness, and between Kvert and PT-CSA and PT-thickness. Conclusions: The results reveal a speci city of the relationship between the lower-limb stiffness and the morphology of the connective tissue. Greater tendon shows higher lower-limb stiffness when that tendon is specially demanded by the function. Keywords:foot behavior; tendon; stretch-shortening cycle; running 1. Introduction During running, the lower limb behaves like a spring, which compresses and decom- presses over the different gait phases [1,2]. In this spring-like model mechanical energy is stored due to the compression provoked by the runner's body mass during the eccentric phase of stance [3,4]. The release of that energy occurs during the concentric phase of stance facilitating the subsequent movements [3,4]. The behavior of such leg-spring function can be in uenced by several factors such as the foot strike pattern (FSP) [5], footwear condition [6], sex difference [7], velocity [8], fatigue [9] or surface type [10]. It has been shown that two of the most important elements of the spring-like behavior of the leg while running are the stretch-shortening cycle (SSC) [11] and the lower-limb stiffness [12]. In both SSC and lower-limb stiffness the muscle-tendon unit is essential. The prop- erties of the muscle-tendon unit play a vital role for the proper functioning of the SSC (i.e., the proper muscle shortening speed allows the optimization of accumulation and release of energy) [13]. Moreover, the relation between tendon and lower-limb stiffness has Int. J. Environ. Res. Public Health2021,18, 8453.

and lower-limb stiffness the muscle-tendon unit is essential. The prop- erties of the muscle-tendon unit play a vital role for the proper functioning of the SSC (i.e., the proper muscle shortening speed allows the optimization of accumulation and release of energy) [13]. Moreover, the relation between tendon and lower-limb stiffness has Int. J. Environ. Res. Public Health2021,18, 8453.

Int. J. Environ. Res. Public Health2021,18, 8453 2 of 10 been previously demonstrated [14]. Rogers et al. [14] showed an association between AT and lower-limb stiffness in terms of vertical (Kvert) and leg stiffness (Kleg). A greater AT stiffness was linked to improved running performance [14]. Previous studies about lower-limb stiffness used Kvert and Kleg as variables to characterize such neuromuscular mechanism [15]. Kvert refers to the resistance of the center of mass to vertical displacement after being subjected to the reaction force of the ground [16], while Kleg was de ned as the mechanical behavior of the structural components of the leg (i.e., tendons, joints, muscles) is shown by the change in leg length during eccentric phase [17]. It has been demonstrated that both Kvert and Kleg highly contribute to the spatiotemporal running gait characteristics [18]. Lower-limb stiffness operates according to the speci city principle behind a particular task [19]. Thus, changes in the speci c task such as the FSP might alter the behavior of neuromuscular elements (i.e., lower-limb stiffness) in that activity. It is known that the FSP is highly in uenced by the shod/barefoot condition [20]. Generally, runners under shod condition, showed a rearfoot strike pattern, and runners under barefoot condition tended to show midfoot or forefoot strike pattern [21]. During running, the FSP determines that some muscles will be especially demanded. In this way, rearfoot strike pattern, under shod condition, shows higher demand of knee extensors [22] and midfoot or forefoot strike pattern, under barefoot condition, especially demands the ankle plantar exors [23]. As far as the authors know, the relation between morphological characteristics of the main lower-limb connective tissue (i.e., thickness and cross-sectional area (CSA)), and lower-limb stiffness during running remains in debate. Monte et al. [24] found that runners with a greater AT-CSA showed greater Kvert. Similarly, it is unclear whether the shod/barefoot condition, and consequently the FSP, may in uence such relationship. Therefore, the aim of this study is twofold: (i) to examine the likely relation between the morphologic characteristics of the connective tissue and the lower- limb stiffness in terms of

et al. [24] found that runners with a greater AT-CSA showed greater Kvert. Similarly, it is unclear whether the shod/barefoot condition, and consequently the FSP, may in uence such relationship. Therefore, the aim of this study is twofold: (i) to examine the likely relation between the morphologic characteristics of the connective tissue and the lower- limb stiffness in terms of Kvert and Kleg, and (ii) to determine whether the shod/barefoot condition in uences the mentioned relation between connective tissue and lower-limb stiffness. We hypothesized that higher values in tendon thickness and CSA would be found alongside greater Kvert and Kleg values, especially when the shod/barefoot condition demands the corresponding connective tissue. 2. Materials and Methods 2.1. Type of Design Prospective observational study. 2.2. Subjects Fourteen recreationally trained male endurance runners (n= 14; age: 27.4 6.3 years; height: 1.75 0.07 m; body mass: 70.9 7.9 kg; BMI: 23.1 2.3) participated in this prospective observational study. All subjects met the inclusion criteria: (i) from 18 to 40 years old, (ii) 3 or more running sessions per week, (iii) 10-km time trial 50 min, and (iv) not suffering from any active known injury. Criteria ii, iii and iv refer to the last 6 months before the data collection. After receiving detailed information on the objectives and procedures of the study, each participant signed an informed consent, which complied with the ethical standards of the World Medical Association's Declaration of Helsinki (2013). It was made clear that the subjects were free to leave the study at any moment. The study was approved by the Institutional Review Board of the San Jorge University (Nº 006-18/19). 2.3. Anthropometric Measurements Body mass (kg) and height (m) were determined using a weighing scale (Tanita BC-601; TANITA Corp., Maeno-Cho, Itabashi-ku, Tokyo, Japan) and a stadiometer (SECA 222; SECA Corp., Hamburg, Germany) for descriptive purposes. The leg length was measured from the great trochanter to the oor in a standing position for lower limb stiffness calculation.

Itabashi-ku, Tokyo, Japan) and a stadiometer (SECA 222; SECA Corp., Hamburg, Germany) for descriptive purposes. The leg length was measured from the great trochanter to the oor in a standing position for lower limb stiffness calculation.

Int. J. Environ. Res. Public Health2021,18, 8453 3 of 10 2.4. Tendon Morphology Characteristics A high-de nition ultrasound images were obtained in B-mode with a linear probe 5–16 MHz (LOGIQ S7 EXPERT, General Electric, Germany, 2013). Longitudinal and transversal views of AT, PT and PF were taken before the running protocol. A recent review suggested that US measures of tendon dimensions are reliable, both in terms of relative and absolute reliability [25]. To assess the AT, subjects were in prone, with both knees extended and the feet outside of the bed keeping the ankle in neutral position [26,27]. A reference of 3 cm proximal to the insertion of the tendon in the calcaneus bone, measured by the ultrasound device, was used to measure the tendon thickness and CSA [26,27]. The PT was measured with subjects in supine, with both knees bent at 30 [26,27]. A reference of 1 cm distal to the lower pole of the patella, identi ed by the ultrasound device, was used to assess the tendon thickness and CSA [26,27]. The PF ultrasound assessment was done with subjects in prone position, with both knees in extension, ankles in neutral position and the ngers extended against the surface of the bed [26,27]. A reference, identi ed by the ultrasound device, located from the anterior edge of the plantar surface of the calcaneus bone vertically to the anterior edge of the PF was used to measure the thickness of the PF [26,27]. For all structures, a frequency of 12 MHz and gain of 100 dB was used. Each measure- ment was recorded twice by a skilled researcher with more than ten years of experience in diagnostic ultrasound imaging. The selected image was the one considered clearest by the examiner for the subsequent calculation of the morphological variables. Before the statistical analysis, thickness and CSA was measured using the software ImageJ (NIH, Baltimore, MD, USA) [28], using the polygon tool for the CSA. 2.5. Procedures The procedure was performed by every participant under the same conditions and researcher control. Before the start of the testing session, the subjects developed a

for the subsequent calculation of the morphological variables. Before the statistical analysis, thickness and CSA was measured using the software ImageJ (NIH, Baltimore, MD, USA) [28], using the polygon tool for the CSA. 2.5. Procedures The procedure was performed by every participant under the same conditions and researcher control. Before the start of the testing session, the subjects developed a struc- tured dynamic 5-min warm-up protocol (squatting, lunging, and hinging) [29]. Just after warming up, an accommodation program over 8 minutes [30] was developed by increasing speed by 1 km/h every minute from 8 to 12 km/h. After that, subjects ran under the rst footwear condition (shod or barefoot) at a speed of 12 km/h for 3 minutes [31], 6 and 8 strides were analyzed to obtain representative data in healthy adults (95% con dence intervals within 5% of error) [32]. Thereafter, subjects ran under the next footwear condi- tion at 12 km/h for another 3 minutes. The order of the shod or barefoot condition was randomized thus half of the sample started the protocol wearing shoes and the other half running barefoot. Both running conditions were completed on a motorized treadmill with a slope of 0% (HP cosmos Pulsar 4 P; HP cosmos Sports & Medical, Gmbh, Nußdorf, Germany) and data were recorded for analysis. The completed protocol was illustrated in Figure. Figure 1.Temporal sequence of the protocol followed in the study.

Int. J. Environ. Res. Public Health2021,18, 8453 4 of 10 2.6. Materials and Testing Data were collected over a 2-trial session in the biomechanics laboratory of the univer- sity during March and April 2019. 2.6.1. Running Spatiotemporal Parameters Contact time (CT; time between touch-down and take-off of the same foot) and ight time (FT; time between take-off of one foot and touch-down of the other) were measured using a photoelectric cell system (Microgate, Bolzano, Italy), which was previously val- idated for the assessment of running gait spatiotemporal parameters [33]. The 2 bars of photoelectric cell systems were xed and stabilized at both sides of the treadmill. 2.6.2. FSP To determine the FSP a high-de nition camera (Imaging Source DFK 33 UX174, The Imaging Source Europe GmbH, Eschborn, Germany) was placed 2 meters lateral to the treadmill, level with the running surface. Videos were sampled at 240 frames per second. Using slow motion video playback, the FSP was determined by one researcher with a wide experience in running biomechanics analysis. This method has been proven valid and reliable previously [34]. 2.6.3. Lower-Limb Stiffness The Kvert and the Kleg were measured to determine lower-limb stiffness [15] using the sine-wave method [24,35]. In order to follow the Morin's method, the collection of information such as body mass, leg length, speed, FT, and CT is required to estimate a runner's Kvert and Kleg. It was shown that Morin's method determines accurately Kvert and Kleg for intra and inter-day designs (ICCs = 0.86–0.99) [36]. Reliability of Kvert and Kleg have been previously reported [16,36]. 2.7. Statistical Analysis Descriptive data are presented as mean and standard deviation (SD). The normal- ity distribution of the data was con rmed by Shapiro-Wilk's test (p> 0.05). To deter- mine the intra-rater reliability of the measures related to the morphology of the connec- tive tissue, intra class correlation coef cients (ICCs) were calculated for each parameter. Additionally, the 95% con dence interval (CI) of the ICC value was provided [37]. To ana- lyze the relationship between the morphology of connective tissue and lower-limb stiffness in endurance runners, a Pearson correlation

the intra-rater reliability of the measures related to the morphology of the connec- tive tissue, intra class correlation coef cients (ICCs) were calculated for each parameter. Additionally, the 95% con dence interval (CI) of the ICC value was provided [37]. To ana- lyze the relationship between the morphology of connective tissue and lower-limb stiffness in endurance runners, a Pearson correlation analysis was conducted for the whole group. The following criteria were adopted to interpret the magnitude of correlations between measurement variables: <0.1 (trivial), 0.1–0.3 (small), 0.3–0.5 (moderate), 0.5–0.7 (large), 0.7–0.9 (very large), and 0.9–1.0 (almost perfect) [38]. A cluster k-means analysis matched the whole group into 2 sub-groups regarding lower-limb stiffness, in terms of Kvert and Kleg, for each running condition (i.e., barefoot vs. shod). An analysis of variance (ANOVA) was conducted between the created sub-groups during each running condition (i.e., barefoot vs. shod) for each dependent variable (i.e., connective tissue morphology). The magnitude of the differences between values was also interpreted using the Cohen's d effect size (ES) (between-group differences) [39]. Effect sizes are reported as: trivial (<0.19), small (0.2–0.49), medium (0.5–0.79), and large ( 0.8) (Cohen, 1988). All statistical analyses were performed using SPSS software version 25.0 (SPSS Inc., Chicago, IL, USA) and statistical signi cance was accepted at an alpha level of 0.05. 3. Results The mean values for the AT-CSA were 55.43 10.91 mm 2 and for the AT-thickness 6.28 0.68 mm. For the PT the mean values of PT-CSA were 99.25 22.07 mm 2 and for the PT-thickness 3.54 0.51 mm. For PF-thickness, the mean value was 2.84 0.36 mm. An excellent intra-rater reliability was reported for the measures related to the mor- phology of the connective tissue (ICC > 0.989, 95% CI: 0.913–0.996).

Int. J. Environ. Res. Public Health2021,18, 8453 5 of 10 For the FSP, 79% (n= 11) of the subjects showed a rearfoot strike pattern and a 21% (n= 3) showed a midfoot or forefoot strike pattern, under shod condition. Under barefoot condition, 86% (n= 12) of the subjects showed a midfoot or forefoot strike pattern and a 14% (n= 2) showed a rearfoot strike pattern. The Pearson correlation analysis (Table) reported signi cant relationships ( p< 0.05) between Kvert_shod with AT-thickness (r = 0.577) and PF-thickness (r = 0.513), and between Kleg_barefoot and PF-thickness (r = 0.516). Table 1. Relationship (Pearson coef cient) between connective tissue morphology and lower- limb stiffness. Kvert Shod Kleg Shod Kvert Barefoot Kleg Barefoot PT-thickness 0.276 0.194 0.250 0.470 PT-CSA 0.234 0.125 0.201 0.379 AT- thickness 0.577 * 0.421 0.041 0.495 AT-CSA 0.306 0.311 0.202 0.051 PF-thickness 0.513 * 0.395 0.206 0.516 * *p< 0.05; AT: Achilles' tendon; CSA: cross-sectional area; Kleg: leg stiffness; Kvert: vertical stiffness; PT: patellar tendon; PF: plantar fascia. The cluster k-means analysis matched the whole group into 2 sub-groups for each running condition (i.e., barefoot vs. shod) regarding lower limb stiffness (Table). Table 2.Leg and Vertical lower-limb stiffness in barefoot and shod conditions (ANOVA). Barefoot p-Value ES (d) Shod p-Value ES (d) HSG (n= 6) LSG (n= 8) HSG ( n= 5) LSG (n= 9) Kvert (kN/m) 31.05 (3.21) 26.30 (2.41) 0.05 1.85 36.50 (2.26) 22.99 (2.27) 0.001 6.44 Kleg (kN/m) 14.72 (1.92) 10.48 (1.37) 0.001 2.82 10.34 (0.83) 8.10 (0.77) 0.003 3.04 HSG: higher-stiffness group; Kleg: leg stiffness; Kvert: vertical stiffness; LSG: lower-stiffness group; ES: effect size. Table those sub-groups (higher stiffness group [HSG] vs. lower stiffness group [LSG]) in both barefoot and shod running conditions. In the barefoot condition, no signi cant differ- ences in the morphology of connective tissue were found between the HSG and the LSG (p 0.05). In the shod condition, the LSG reported higher values in AT-thickness (p= 0.023) with large ES (ES = 2.00), whereas the rest of parameters showed no between- group differences.

condition, no signi cant differ- ences in the morphology of connective tissue were found between the HSG and the LSG (p 0.05). In the shod condition, the LSG reported higher values in AT-thickness (p= 0.023) with large ES (ES = 2.00), whereas the rest of parameters showed no between- group differences.

The study investigates how connective tissue affects lower-limb stiffness in runners.