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OPEN

Experimental study exploring
the factors that promote rib
fragility in the elderly
Christian Liebsch1, Shamila Hübner1, Marco Palanca2, Luca Cristofolini2 &Hans‑Joachim Wilke1*
Rib fractures represent a common injury type due to blunt chest trauma, affecting hospital stayand mortality especially in elderly patients. Factors promoting rib fragility, however, are little
investigated. The purpose of this in vitro study was to explore potential determinants of human rib
fragility in the elderly. 89 ribs from 13 human donors (55–99 years) were loaded in antero-posterior
compression until fracture using a material testing machine, while surface strains were captured
using a digital image correlation system. The effects of age, sex, bone mineral density, rib level andside, four global morphological factors (e.g. rib length), and seven rib cross-sectional morphologicalfactors (e.g. cortical thickness, determined by μCT), on fracture load were statistically examined using
Pearson correlation coefficients, Mann–Whitney U test as well as Kruskal–Wallis test with DunnBonferroni post hoc correction. Fracture load showed significant dependencies (p < 0.05) from bonemineral density, age, antero-posterior rib length, cortical thickness, bone volume/tissue volume ratio,
trabecular number, trabecular separation, and both cross-sectional area moments of inertia and wassignificantly higher at rib levels 7 and 8 compared to level 4 (p = 0.001/0.013), whereas side had no
significant effect (p = 0.989). Cortical thickness exhibited the highest correlation with fracture load(r = 0.722), followed by the high correlation of fracture load with the area moment of inertia aroundthe longitudinal rib cross-sectional axis (r = 0.687). High correlations with maximum external rib
surface strain were detected for bone volume/tissue volume ratio (r = 0.631) and trabecular number(r = 0.648), which both also showed high correlations with the minimum internal rib surface strain(r =  − 0.644/ − 0.559). Together with rib level, the determinants cortical thickness, area moment ofinertia around the longitudinal rib cross-sectional axis, as well as bone mineral density exhibited thelargest effects on human rib fragility with regard to the fracture load. Sex, rib cage side, and global
morphology, in contrast, did not affect rib fragility in this study. When checking elderly patients for rib
fractures due to blunt chest trauma, patients with low bone mineral density and the mid-thoracic area
should be carefully examined.
Clinical treatment of rib fractures has gained growing importance due to the rising number of falls in elderly
people, causing about 75% of all traumatic injuries in patients aged over 60 ­years1. Previous investigations
exhibited increased mortality following rib fractures in advanced age, accounting for more than 20% of patients
aged over 65 years, but only for about 10% in younger p­ atients2–4. While age and number of fractured ribs were
found to be predictive factors for morbidity and mortality after blunt chest ­trauma5, about 20% of all patients
suffer from chronic pain due to rib p­ seudarthrosis6,7.
Despite representing an apparent clinical issue, mechanisms and determinants promoting rib fractures havebeen insufficiently investigated. Recently published findings of an epidemiological study showed that old age,osteoporosis, prior fractures, and falls represent risk factors for rib f­ ractures8. Moreover, several experimental
and numerical studies investigated single factors affecting rib stability and fragility by focusing on few specificparameters such as ­age9–13, ­sex10–13, cortical ­thickness13–15, or rib ­geometry13,16,17, while Agnew et al.13 additionallyanalyzed rib level and bone mineral density as possible promoting factors. However, the effects of all relevant
patient-specific and morphological factors on rib fragility in their entirety have been poorly studied so far.
Knowledge about determinants for rib fragility could help to identify risk factors as part of clinical anamnesis
1

Institute of Orthopaedic Research and Biomechanics, Trauma Research Centre Ulm, University of Ulm,
Helmholtzstraße 14, 89081 Ulm, Germany. 2Department of Industrial Engineering, School of Engineering andArchitecture, Alma Mater Studiorum - Università Di Bologna, Bologna, Italy. *email: hans-joachim.wilke@
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Figure 1.  Scatter diagrams illustrating the effects of age (top) and bone mineral density (bottom) on fractureload. Trend lines are depicted in red.

and stationary treatment of elderly patients. Moreover, information about rib fragility is essential regarding
future in vitro and in silico studies investigating rib fracture mechanisms, which could help to develop novel
safety devices for elderly people and novel rib fracture treatment strategies.
The purpose of the present in vitro study therefore was to explore determinants of fragility of human ribs fromelderly donors. The significance of the following factors was tested: age, sex, bone mineral density, rib level, rib
cage side, global morphological factors, such as rib length and width, and local, cross-sectional morphologicalfactors, such as the cortical and trabecular thickness.

Results

Fracture types and locations. After fracturing, the vast majority of the 89 investigated ribs exhibited

transverse fractures as defined by Meinberg et al.18, while 54 of the 60 transverse fractures were infractions, i.e.
incomplete fractures. Of the remaining 29 ribs, 25 showed oblique fractures (19 infractions), whereas only 4ribs had multifragment fractures (1 infraction). Fracture locations were detected in the antero-lateral section as
defined by Liebsch et al.19 in 88 of the 89 investigated ribs, while only one rib fractured in the posterior section.

Effects of age, sex, and bone mineral density. Both age and bone mineral density showed medium
correlations with fracture load and absorbed energy, while sex did neither significantly affect fracture load(p = 0.445) nor the other mechanical variables maximum displacement (p = 0.929), rib stiffness (p = 0.981),
absorbed energy (p = 0.981), and maximum external and internal rib surface strains (p = 0.260/0.340). Age exhibited negative correlation coefficients with fracture load (r =  − 0.449, p < 0.001) and absorbed energy (r =  − 0.349,
p = 0.001), meaning that the fracture load tended to decrease with increasing donor age, whereas bone mineraldensity showed positive correlation coefficients with fracture load (r = 0.490, p < 0.001) and absorbed energy(r = 0.382, p = 0.001), entailing a tendency towards increasing fracture load with increasing bone mineral density(Fig. 1).
Effects of rib level and side. Both fracture load and stiffness tended to increase gradually from rib 4 to rib
7, while slightly decreasing for rib 8 compared to rib 7. Fracture load and stiffness were significantly higher for
rib 7 and rib 8 compared to rib 4 (fracture load: p = 0.001, p = 0.013; stiffness: p = 0.002, p = 0.007), respectively(Fig. 2). The rib cage side did neither significantly affect fracture load (p = 0.989) nor the other mechanical variables maximum displacement (p = 0.166), rib stiffness (p = 0.773), absorbed energy (p = 0.354), and maximum
external and internal rib surface strains (p = 0.490/0.338).

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Figure 2.  Boxplot diagram illustrating the effect of rib level on fracture load. Median values are depicted as redlines, the boxes include the 25th to 75th percentile, and the whiskers represent the maximum and minimum
values.

Effect of global rib morphology. Antero-posterior rib length exhibitedlow positive correlation with fracture load (r = 0.287, p = 0.006), whereas rib width, rib length/width ratio, and external rib edge length were notsignificantly correlated with fracture load. Medium correlation coefficients were found for stiffness with both
rib width (r =  − 0.446, p < 0.001) and rib length/width ratio (r = 0.409, p < 0.001), while external rib edge length
presented solely low correlation coefficients with maximum displacement (r = 0.248, p = 0.020).
Effect of rib cross‑sectional morphology. Rib cross-sectional variables overall showed higher correla-tion coefficients with the mechanical variables compared to global rib morphological variables. Cortical thickness (r = 0.722, p < 0.001) and area moment of inertia around the longitudinal rib cross-sectional axis (r = 0.687,
p < 0.001) exhibited high positive correlation with fracture load (Fig. 3). These two variables further showed highcorrelation coefficients with absorbed energy (cortical thickness: r = 0.617, p < 0.001; area moment of inertia
around the longitudinal rib cross-sectional axis: r = 0.640, p < 0.001). Medium correlation coefficients with fracture load were found for bone volume/tissue volume ratio (r = 0.448, p < 0.001), trabecular number (r = 0.479,
p < 0.001), and trabecular separation (r =  − 0.367, p < 0.001). The area moment of inertia around the transverse
rib cross-sectional axis (r = 0.227, p = 0.033) exhibited a low correlation coefficient, while trabecular thickness(r = 0.201, p = 0.058) did not exhibit substantial correlation. High correlation coefficients were also detected
for both bone volume/tissue volume ratio and trabecular number together with absorbed energy (r = 0.542,
p < 0.001; r = 0.539, p < 0.001), maximum external rib surface strain (r = 0.631, p < 0.001; r = 0.648, p < 0.001), andminimum internal rib surface strain (r =  − 0.644, p < 0.001; r =  − 0.559, p = 0.001).
All results including the experimental raw data and the statistical variables are retrievable from the supplementary material files attached to the online version of this publication.

Discussion

Rib fractures in elderly people represent a major clinical issue due to both increased risk of falling and higher
mortality compared to the younger population, necessitating the exploration of mechanisms and promoting factors for rib fracture emergence. This study aimed to explore determinants of rib fragility using an experimental
test design.
The outcomes of the present study indicated a combined, however hierarchically organized effect of theinvestigated continuous variables on human rib fragility. Based on the correlation analysis, solely considering
statistically significant findings and arranging them in descending order according to their correlation coefficients, the following ranking order can be derived for increased rib fragility, defined as reduction in fractureload, which was considered as the primary indicator: (1) Low cortical thickness, (2) low area moment of inertia
around the longitudinal rib cross-sectional axis, (3) low bone mineral density, (4) low trabecular number of therib cross-section, (5) high age, (6) low bone volume/tissue volume ratio, (7) high trabecular separation of the ribcross-section, (8) shorter antero-posterior rib length, and (9) lower area moment of inertia around the transverse
rib cross-sectional axis. Rib width, rib length/width ratio, external rib edge length, and trabecular thickness of
the rib cross-section, in contrast, were not found to substantially affect rib fragility when defined as reduction
in fracture load. Overall, correlation coefficients were relatively low, potentially indicating mutual dependency
between single variables. Therefore, it can be assumed that there is no definite determinant of rib fragility, but
a combination of several, differently weighted factors. Out of the discrete variables, rib level was found to be an
important parameter that significantly affected rib fragility, in contrast to sex and rib cage side. While highest
fracture resistance was found for rib levels 7 and 8, it gradually decreased in superior direction up to level 4. Thismight be explained by the effects of cortical thickness, which was significantly higher in rib 8 compared to level 4(p = 0.022), and area moment of inertia around the longitudinal rib cross-sectional axis, which was significantly

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Figure 3.  Scatter diagrams illustrating the effects of cortical thickness and area moment of inertia around thelongitudinal axis of the rib cross-sectional area (xx) on fracture load. Trend lines are depicted in red.

higher in levels 6 and 7 compared to level 4 (p = 0.015/0.036). Indirectly, rib level therefore might be a further
essential determinant for rib fragility together with the above stated continuous variables.
Regarding clinically determinable parameters, rib level, bone mineral density, and age might be the most
crucial determinants when assessing rib fragility of elderly patients, whereas sex and rib cage side might benegligible in clinical practice. While age and bone mineral density might be closely related to each other as wellas to most of the analyzed morphological factors, rib level represents a remarkable determinant for rib fragility,
which should be further investigated in future studies to explore its role regarding fracture resistance of the entire
rib cage complex. Bone mineral density, on the other hand, represents a variable, which is affected by several
influential factors due to its systemic character, such as diseases or nutritional and hormonal effects. The fact thatsex was not affecting rib fragility in the present study, however, might indicate that bone mineral density can beseen as an independent determinant for rib fragility. In the present study, bone mineral density was evaluatedfrom vertebral bodies, since there was no established procedure for ribs, which was seen as justifiable due to thesystemic feature of bone mineral ­density20.
Rib fragility was predominantly affected by local, cross-sectional morphological factors rather than by global
morphology in this study. Indeed, previous studies showed that both cortical thickness and cross-sectional
area moment of inertia were significantly higher in lateral and posterior rib sections compared to the anterior
rib section and increased with rib l­evel21,22, corresponding to the findings of the present study, where fractures
occurred primarily in the anterior and antero-lateral rib sections and fragility gradually decreased from rib
levels 4–7. While the determinants affecting the cross-sectional rib shape are not known, it can be assumed
that rib cross-sectional geometry is not optimally adapted to high antero-posterior rib cage compression as
in case of frontal impacts, since the inner cortical bone is about 40% thicker compared to the outer cortical­bone22. Antero-posterior rib length exhibited low positive correlation with fracture load, stiffness, and absorbed
energy. Since the antero-posterior rib length was significantly increased at rib levels 6–8 compared to level 4(p = 0.002/ < 0.001/ < 0.001) as well as at level 7 compared to level 5 (p = 0.002), corresponding to previous findingsthat the antero-posterior rib length approximately doubled from level 1 to level 7 and then slightly d­ ecreased23,
increased antero-posterior rib length may therefore most probably reduce rib fragility indirectly via the rib
level, which itself might be influenced by local, cross-sectional morphological determinants, such as the cortical
thickness or the area moment of inertia.
In a previous in vitro study, investigating the fracture mode after antero-posterior compression in eight ribs,
Daegling et al. detected five transverse, two oblique, and one multifragmentary fracture t­ ypes24, corresponding
to the findings of the present study, where of the 89 tested ribs, 60 exhibited transverse, 25 oblique, and four multifragmentary fracture types. However, the study of Daegling et al. solely showed four incomplete fractures inScientific Reports |Vol:.(1234567890)(2021) 11:9307 |https://doi.org/10.1038/s41598-021-88800-94

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Donor ID

Sex

Age in years

Rib level and side (l = left / r = right)

1

Female

55

8 l/r

2

Male

62

4 l/r, 5 l/r, 6 l/r, 7 l/r, 8 l/r

3

Male

63

4 l/r, 5 l/r, 7 l/r, 8 l/r

4Female64

6 l, 7 l/r, 8 l/r

5Female65

5 l, 6 l, 8 l

6

Male

68

4 l/r, 5 l/r, 6 l/r, 7 l/r, 8 l/r

7Female71

4 l, 5 l/r, 7 l/r, 8 l/r

8

Male

80

4 l/r, 5 l/r, 6 l/r, 7 l/r, 8 l/r

9

Male

81

4 l/r, 5 l/r, 7 l/r, 8 l/r

10Female84

4 l/r, 5 l/r, 6 l/r, 7 l/r, 8 r

11

Male

91

4 l/r, 5 l/r, 7 r, 8 r

12

Male

96

4 l/r, 5 l/r, 7 l/r, 8 l/r

13Female99

4 r, 7 l/r

Table 1.  Data on specimens used for this in vitro study.

the eight tested ribs, whereas in the present study, 74 of the 89 fracture types were infractions, which might be
explained by differences in testing conditions and the use of rehydrated rib specimens, since it was shown thatmoisture level can affect the fragility of cortical b­ one25. Compared to data of a retrospective clinical study, however, rib fracture locations of the present study showed similar patterns as ribs of cardiopulmonary resuscitated­patients19, which might be explained by equivalent loading conditions in both cases. Therefore, it can be assumed
that pure antero-posterior rib cage compression predominantly results in anterior or antero-lateral rib fractures.
As in the present study, another large experimental study by Agnew et al.13 also detected bone mineral density,
global rib geometry, and cross-sectional geometry as predictors of the structural properties of human ribs. However, cortical thickness did not appear to be a useful predictor in this study, which is in contrast to the findingsof the present study and the numerical study of Li et al.14, whereas increasing age as a factor for increased rib
fragility was also detected in most previous ­studies9,11,13. In contrast, sex exhibited major effects on the structuralproperties of human ribs in other s­ tudies11,13, which, however, could not be determined in the present study.
The present study entailed several limitations due to its experimental design. First, the boundary conditionsof the test setup were simplified and did not exactly replicate the loading conditions during blunt chest trauma interms of loading direction and rate. For instance, it was not feasible to align the rib ends reproducibly in anteroposterior loading direction without creating primary constraint forces due to the complex three-dimensional
morphology of the individual ribs. Beyond that, a low loading rate was chosen in order to enable surface strain
measurement, which requires low deformation velocities. Moreover, potential damping effects of the costal
cartilage were neglected in order to ensure more reproducible testing conditions. Apart from that, the anterior
PMMA embedding could have accelerated the fracture process by creating stress concentrations at the transition
between rib and PMMA, which could not be prevented due to the experimental nature of this study. Furthermore,measurement inaccuracies of the load cell, the digital image correlation system, and the micro-CT might haveaffected the outcome variables and should be considered when interpreting the results of the present study. While
inaccuracies are overall estimated low with regard to the accuracy ranges in case of the load cell and the digital
image correlation system, potential inaccuracy in case of the micro-CT is also dependent from the evaluatorand therefore difficult to estimate despite standardized evaluation techniques. Moreover, samples for micro-CT
analysis were taken from the fracture site instead of a defined anatomical landmark in order to compare themorphological values of the weakest points of the respective ribs. While these were in the anterolateral part of
the ribs in almost all cases, this might have affected the comparability of the morphological values nonetheless.
One substantial limitation is the usage of fine sandpaper for the preparation of the rib surface in order to assure
valid results with regard to surface strain measurements. However, the sandpaper was carefully used to remove
remaining tendon tissue and the periosteum, which were assumed to have no distinct effect on the mechanicalstability of the ribs, while care was taken not to remove bone tissue. Therefore, effects of this approach on the
results were considered as low by the authors, but have, nevertheless, to be taken into account. Finally, regarding
the statistical evaluation, the inclusion of multiple ribs from same donors also represents a limitation of this study.
In conclusion, reduced cortical thickness, area moment of inertia around the longitudinal axis of the rib crosssection, and bone mineral density predominantly promote rib fragility, whereas sex, global morphology, andrib cage side do not represent essential fragility determinants. When examining elderly patients for rib fracturesdue to blunt chest trauma, for instance following falls from low height, especially patients with low bone mineraldensity and the mid-thoracic area of the patients should therefore be carefully checked.

Methods

Specimens. A total of 89 human ribs from 13 donors were prepared for experimental testing, while solely
ribs from levels 4 to 8 were included since highest fracture rates were detected at these levels in a recent retrospective ­study19. Donor age ranged from 55 to 99 years with a mean of 75 years, including seven male and six
female donors and a total of 45 left and 44 right ribs (Table 1). The use of the specimens was approved by the
ethics committee board of the University of Ulm (vote no. 92/20). The specimens were acquired from body
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Figure 4.  Illustration of the test setup.

Figure 5.  Image sequence of a typical loading test.
donation programs which require legally valid informed consents from a parent or legal guardian (Science Care
Inc., Phoenix, USA; Anatomy Gifts Registry program, Hanover, USA). All methods were performed in accordance with relevant guidelines and regulations and following the Declaration of Helsinki. Rib cage specimens
were gathered fresh frozen and stored at − 20 °C. Prior to preparation, bone quality was assessed by CT scans(Somatom Definition AS, Siemens Healthcare, Erlangen, Germany) with a resolution of 512 × 512x804 pixels
and a voxel size of 0.87 × 0.87x0.6 mm in terms of bone mineral density of the respective spines. The bone mineral density values were evaluated from the trabecular bones of the lumbar vertebral bodies L2, L3, and L4 using
standard clinical density-reference phantoms (Osteo Phantom, Siemens Healthineers, Erlangen, Germany),
which were included in the patient table of the CT device and averaged for every specimen, while the calibration curves were automatically fitted using a standardized software protocol for clinical evaluation, comparable
to the procedure of a previous ­investigation26. After thawing the specimens at 5 °C for about 12 h, the ribs wereseparated from the spines by cutting the connecting ligaments. All muscular and ligamentous tissues were carefully removed and the costal cartilage was severed close to the cartilage-bone junction. Ribs showing any signs ofbony defects, previous fractures, or degeneration were excluded from the study. Rib heads and shafts were coaxially embedded in a polymethylmethacrylate cylinder (Technovit 3040, Heraeus Kulzer, Wehrheim, Germany)for a length of about 40 mm, while care was taken that the rib was uniformly surrounded by PMMA to ensure
rigid fixation in the testing device. After preparation, the ribs were frozen again and stored at − 20 °C in sealed
polyethylene bags in order to keep the exposure to room temperature to the same time period for all ribs. Prior
to testing, every rib was thawed for about 12 h in 0.9% saline solution. Immediately before testing, remaining
tendon tissue and the periosteum were carefully removed using fine sandpaper while taking care not to damage
bone substance. In the next step, the ribs were primed with a thin layer of white water-based paint and sprayedwith a black speckle pattern for surface strain measurement with digital image correlation (Fig. 4). This speckle
technique was verified in a preliminary study not to affect the biomechanical properties of biological t­ issue27.

Biomechanical testing. The specimens were loaded in antero-posterior compression until fracture using

a material testing machine (Zwick Roell Z010, Zwick Roell AG, Ulm, Germany) equipped with a 200 N load cell
(Hottinger Baldwin Messtechnik GmbH, Darmstadt, Germany) (Fig. 4), having a measuring error of < 0.3%.
Load and displacement data was acquired using the software testXpert II (Zwick Roell AG, Ulm, Germany)with a sampling frequency of 100 Hz. To create loading conditions that were as reproducible as possible, theposterior end of the rib was rigidly fixed in a ball joint, which was adjusted in a way that the rib head and theanterior end of the rib shaft were preferably aligned in loading direction without creating constraint forces on
the rib, while the anterior rib end was clamped in a cardan joint allowing quasi-physiological rotations. Loading
was performed in antero-posterior direction with a constant velocity of 1 mm/s after applying a preload of 5 N
to remove any effect of mechanical play between the components of the loading system (Fig. 5). Fracture occurrence was determined from the decline in the load–displacement curve after combined audible cracking of thebone and appearance of a clearly visible fracture line. Simultaneously with load application, full-field analysis
was performed using a 3D digital image correlation system (Q400, Dantec Dynamics, Denmark) consisting of
two cameras in order to measure the surface strain (Fig. 4), exhibiting a relative displacement error of < 1/3 pixels and a strain error of < 180 μStrain. This technique has been validated and successfully applied to the spine in

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Figure 6.  Illustrative summary of all evaluated cross-sectional morphological parameters.

previous in vitro s­ tudies28–31. To detect maximum and minimum surface strains during rib deformation, about
half of the ribs were examined from a lateral view and the other half from a medial view, while care was taken toevenly distribute the specimens with respect to age, sex, bone mineral density, rib cage side, and rib level.

Morphological analysis. After each trial, fracture type and location were visually assessed and docu-

mented. Cross-sectional rib morphology was acquired using micro-CT scans (Skyscan 1172 Micro-CT, Skyscan,
Kontich, Belgium) of about 10 mm long samples, which were cut posteriorly from the fracture position and as
closely as possible to the fracture site, respectively. The scans were performed using a tube voltage of 100 kV, atube current of 100 µA, an Al-Cu filter for beam hardening reduction, and a scan period of 38 min, obtaining an
isotropic voxel size of 5 µm. The following parameters were examined using a standard threshold technique forgreyscale values within the software CTAn 1.17.7.2 (Skyscan, Kontich, Belgium) in combination with a calibration phantom (Bruker MicroCT, Kontich, Belgium): Bone volume/tissue volume ratio, average thickness, number, and separation of the trabeculae, average cortical thickness, and area moments of inertia Ixx and Iyy aroundthe longitudinal (xx) and transverse (yy) cross-sectional axes (Fig. 6). All values were automatically calculatedby the software for every cross-section and finally averaged for a determined region of interest over 50 adjacentsample slices. Thickness values were defined as the minimum thickness at different locations of the respective
area. Ixx and Iyy were defined as the integral of the quadratic distance of all bony elements to the axes xx andyy over the cross-sectional bony area, reflecting potential resistance against bending deformation around theseaxes. Global morphology of the ribs was identified regarding rib length and width prior to embedding using atape measure. Rib length was measured as both the external edge length, characterizing the length of the external
rib side from rib head to anterior tip of the rib shaft, and the antero-posterior length, describing the direct distance between rib head and anterior tip of the rib shaft (Fig. 4). Rib width was defined as the maximum distance
between the rib shaft and the connecting line between rib head and anterior tip of the rib shaft.

Data processing and statistics. Load–displacement data were post-processed using Matlab 2018 (MathWorks Inc., Natick, USA) in order to calculate fracture load, crosshead displacement at fracture, apparent stiffness and absorbed energy, while the displacement was first set manually to zero for the starting point of loadincrease. Stiffness was defined as the gradient of the load–displacement curve within the interval starting from
5 N and ending at the point where 20% of the peak load were reached, while absorbed energy was specified as thearea under the load–displacement curve up to fracture. All biomechanical, morphological, and donor-specificdata were collected and post-processed using Excel 2016 (Microsoft Corp., Redmond, USA). Statistical analyses
were performed in SPSS 24 (IBM Corp., Armonk, USA). For all continuous variables, linear relationships ofcontinuous variables were investigated using the Pearson correlation coefficient r defining |r|≤ 0.1 as no linear
correlation, 0.1 <|r|≤ 0.3 as low linear correlation, 0.3 <|r|≤ 0.5 as medium linear correlation, and |r|> 0.5 as highlinear correlation according to the recommendation of C
­ ohen32. For all statistical analyses, the significance level
was set to 0.05. For comparisons between two groups, the Mann–Whitney U test was used, whereas for comparisons between multiple groups, a Kruskal–Wallis test with Dunn-Bonferroni post hoc correction was performed.
Information on which variables were treated as independent and which as dependent is given in Table 3 andTable 4 of the supplementary material file.
Approval for human experiments. The use of the specimens was approved by the ethics committeeboard of the University of Ulm (92/20).

Data availability

All data including the experimental raw data and the statistical variables are retrievable from the supplementarymaterial file attached to the online version of this publication.
Received: 1 February 2021; Accepted: 7 April 2021

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Acknowledgements

The authors gratefully acknowledge Daniele Marras for assisting in the experimental testings.

Author contributions

Study conception by C.L. and H.J.W., funding acquisition by H.J.W., data acquisition by S.H., C.L., M.P., andL.C., data analysis by C.L. and S.H., data interpretation by S.H. and C.L., manuscript writing and illustrations
by C.L., critical revision of the manuscript by S.H., M.P., L.C., and H.J.W.

Funding

Open Access funding enabled and organized by Projekt DEAL. This study was funded by the German Research
Foundation (DFG, Project WI 1352/20-2). The funding agency had no influence on study design, data collection,
analysis, or interpretation, manuscript preparation, or the decision to submit the manuscript for publication.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​021-​88800-9.
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