Currently, various methods are employed to investigate the mechanical properties of biological tissues. These methods include techniques such as tensile testing, shear rheometry, and nanoindentation (reviewed by21). While each method has its advantages, nanoindentation offers distinct benefits for distinguishing different tissue components. Nanoindentation provides localized mechanical characterization by indenting the tissue surface with a small probe, allowing for precise measurements of mechanical properties at the microscale22. This makes it particularly suitable for distinguishing different tissue parts. By analyzing parameters like YM, storage modulus (E′), loss modulus (Eʺ), and the E′ − Eʺ difference, nanoindentation enables a comprehensive understanding of the mechanical behavior and relative contributions of elastic and viscous properties within specific tissue regions22. Moreover, it is important to consider that mounting tissue samples for rheological measurements can introduce challenges, particularly for porous tissues.
The mounting process, which involves securing the tissue in place, by using holders or mounting solutions (such as agarose, agar, and commercially available glues) can potentially alter the tissue’s mechanical properties. In the case of porous tissues, mounting solutions can penetrate the pores and introduce inaccuracies in the measurement of properties, like YM, and alter the interpretation of tissue rheology23. Here, a careful optimization of the mounting techniques was performed to minimize these effects and ensure accurate characterization of tissue rheology. In short, tissue pieces were covered with agarose (8 mg mL−1) solution, with the site of indentation exposed. After hardening of agarose around the tissue, the agarose-tissue samples were mounted on a dish using commercial glue, ensuring the stability of the sample. A 10% BSA solution was added in the dish to submerge the sample and prevent adhesion. A schematic representation of the experimental design is illustrated in Fig. 1.
Experimental design: Testicular samples (n = 3 bulls) and female reproductive tracts (n = 6 cows) were collected from a local abattoir. The female reproductive tracts were identified either at follicular or luteal phase (n = 3 per phase), ipsilateral to the dominant follicle (follicular phase) or to the corpus luteum (luteal phase). Samples of endometrium were collected from both horns, medulla and cortex samples from the ovaries, and ampulla and isthmus segments from the oviducts. All the samples were submitted to nanoindentation as native tissues, porosity analysis (n = 3 technical replicate per tissue), characterization (including nuclei staining and DNA levels, n = 1 sample per organ), and decellularization process. The same native samples analyzed in the nanoindenter were submitted to decellularization and analyzed again in the nanoindenter as decellularized tissue. After decellularization, samples were submitted to porosity analysis (n = 3 technical replicate per tissue) and characterization (including nuclei staining and DNA levels, n = 1 sample per organ) to validate the decellularization process (Suppl. Fig. 1).
Elastic and viscoelastic properties of reproductive tissues are location, tissue, and estrous cycle dependent
In the biomechanical analysis of the testis, there were clear differences between the tunica albuginea and the inner testicular tissue in terms of their storage modulus (E′) and loss modulus (Eʺ). Both E′ and Eʺ were found to be significantly higher in the tunica albuginea compared to the inner testis (p < 0.001 for both E′ and Eʺ), indicating that the tunica albuginea is both more elastic and more viscoelastic than the inner testicular tissue. Furthermore, the difference between the storage and loss moduli (E′ − Eʺ) was found to be greater in the tunica albuginea than in the inner testis, suggesting a relative increase in elastic response in the tunica albuginea compared to the inner testicular tissue (Fig. 2a).
Elastic and viscoelastic properties of reproductive tissues. Storage (E′, continuous lines) and Loss (Eʺ, dashed lines) modulus of native inner testis and tunica albuginea (a), endometrium (b), ovarian cortex (c), ovarian medulla (d), oviductal ampulla (e), and oviductal isthmus (f). Female tissues were analyzed for both follicular and luteal phases and ipsi- and contralateral positions.
In the biomechanical analysis of the endometrial tissue, non-distinct differences were observed across the luteal and follicular phases and between the ipsi- and contralateral horns for E′ and Eʺ individually. Nevertheless, a decrease in the difference between the storage modulus and loss modulus (E′ − Eʺ) in the ipsilateral horn during the follicular phase, compared to the other phases and locations, was observed (Fig. 2b).
The biomechanical properties of the ovarian cortex (Fig. 2c) and medulla (Fig. 2d) were also analyzed, with several significant findings observed. In the luteal phase cortex, the storage modulus from the ipsilateral horn was significantly higher than the contralateral horn (p < 0.001). Same observation was found in the luteal phase medulla for both E′ (p < 0.01) and Eʺ (p = 0.0399). The difference E′ − Eʺ in the luteal contralateral cortex and medulla were reduced when compared to their respective luteal ipsilateral tissues. This decrease in the difference between E′ and Eʺ might suggest an increased viscoelasticity of the contralateral ovarian tissues during the luteal phase.
Regarding the viscoelastic properties of the oviduct, no significant difference on E′ and Eʺ when comparing estrous stage and position were observed in the isthmus (Fig. 2f). Nevertheless, there was a reduction in storage modulus (E′) of the contralateral ampulla when compared to the ipsilateral ampulla in the luteal phase (p < 0.001, Fig. 2e). Moreover, a reduction in the E′ − Eʺ was observed in the luteal ipsilateral ampulla (specially under higher frequencies) when compared to the follicular phase, suggesting an increased viscoelasticity of the ipsilateral ampulla during the luteal phase (Fig. 2e).
Young’s modulus and porosity of testicles are affected by tissue decellularization
Nanoindentation was conducted on pieces of the tunica albuginea and inner testis to evaluate the Young’s modulus of both native and decellularized tissues. Each tissue fragment underwent multiple random indentations as seen in Fig. 3a. The native inner testis exhibited a reduced mean Young’s modulus (YM) of 1.39 ± 0.44 kPa when compared to the decellularized tissue (mean YM of 12.83 ± 7.70 kPa, p < 0.001; Fig. 3b). Differently from the inner testis, no significant changes on Young’s modulus were observed after decellularization for the tunica albuginea (5.88 ± 1.52 vs 12.71 ± 9.83 kPa, native and decellularized, respectively, p = 0.1737; Fig. 3b). Additionally, the native inner testis had a reduced mean Young’s modulus compared to the native tunica albuginea (1.39 ± 0.44 vs 5.88 ± 1.52 kPa, respectively; p = 0.0220). Nevertheless, after decellularization, a lack of difference between inner testis and tunica albuginea YM was observed (p = 1.00).
Young’s modulus and porosity analysis of native and decellularized testicles. In (a) details of sampling for nanoindentation and porosity analysis, testes were cut transversely in half, the tunica vaginalis was removed to allow the isolation of the tunica albuginea and the inner part of the testis, the samples were mounted on a petri dish using 8 mg mL−1 agarose solution. In (b) Young’s modulus of testis (n = 3 bulls). In (c) porosity data (n = 3 bulls). In (d) correlation plots of Porosity and Young’s modulus.
The estimated porosity of the native inner testis was higher than the decellularized inner testis (67.5 ± 15.0 vs 33.4 ± 29.3%, respectively, p = 0.01480; Fig. 3c). Similarly, the decellularization process reduced the porosity of the native tunica albuginea (76.0 ± 14.5% vs 43.1 ± 28.5%, respectively, p = 0.05165; Fig. 3c). Contrary to the YM data, no differences on porosity were observed when comparing inner testis and tunica albuginea, for both native and decellularized tissues (p = 0.8543 and p = 0.8617, respectively). To explore the relationship between porosity and Young’s modulus, a correlation study was conducted. A weak negative correlation between porosity and Young’s modulus was observed only in the native tunica albuginea (R = − 0.59 and p = 0.025, Fig. 3d), while no significant correlations were detected for the other groups.
In summary, the results showed that the decellularized inner testis had a significantly higher YM compared to the native tissue, while no significant changes in YM were observed in the decellularized tunica albuginea. The porosity was higher in the native compared to the decellularized tissues, for both inner testis and tunica albuginea.
Young’s modulus and porosity of female reproductive tissues are estrous cycle dependent and affected by tissue decellularization
Three female reproductive tissues were analyzed from the same animal (endometrium, ovary, and oviduct). In total, six cows were studied, three at follicular phase and three at luteal phase of the estrous cycle. Additionally, samples were analyzed considering the position: ipsi- and contralateral, to the dominant follicle in follicular phase animals or the corpus luteum in luteal phase animals.
Young’s modulus and porosity of endometrium
Tissue sections from contra- and ipsilateral horns of the endometrium in the luteal and follicular phases of the estrous cycle were evaluated (Fig. 4a). An increase in the YM was observed in decellularized compared to native tissues only at luteal phase (Fig. 4b), in both contralateral (9.24 ± 5.22 vs 2.75 ± 3.6 kPa, p = 0.00837) and ipsilateral horns (16.25 ± 9.69 vs 2.51 ± 1.55 kPa, p < 0.0001), but not at follicular phase, neither in contralateral (7.51 ± 6.40 vs 2.75 ± 3.6 kPa, decellularized and native, respectively, p > 0.05) nor ipsilateral (4.22 ± 1.65 vs 2.13 ± 1.62, decellularized and native, respectively, p > 0.05). An increase in YM in the luteal compared to the follicular phase was observed for both native and decellularized ipsilateral horns (p = 0.0208 and p < 0.0001, respectively), while no significant differences were observed for the contralateral horns. Nevertheless, no differences on YM were observed between native contralateral and ipsilateral horns (for a full comparison of groups see Suppl. Data 1).
Young’s modulus and porosity analysis of native and decellularized endometrium. In (a) details of sampling for nanoindentation and porosity analysis, uteri were opened and a transversal sample was collected of each horn (ipsi- and contralateral), the endometrium was collected with the myometrium to stabilize the positioning of the tissue (myometrium was not evaluated) and the sample was mounted on a petri dish using 8 mg mL−1 agarose solution. In (b) Young’s modulus of endometrial tissue from native and decellularized tissue (n = 3 cows in follicular phase; n = 3 cows in luteal phase). In (c) porosity data from endometrial tissue from native and decellularized tissue (n = 3 cows in follicular phase; n = 3 cows in luteal phase).
There was a reduction of estimated porosity values for contralateral native tissues compared to the decellularized tissues in the follicular phase (77.7 ± 5.1 vs 45.8 ± 9.0%, p < 0.0001), which was not significant in the luteal phase (70.9 ± 13.7 vs 60.1 ± 13.7%, p = 0.28177, Fig. 4c). Same reduction was observed in the ipsilateral samples between native and decellularized tissues during follicular (70.4 ± 10.8 vs 42.5 ± 15.2%, p < 0.0001) and luteal (79.2 ± 9.2 vs 56.8 ± 9.7%, p = 0.00196) phases. No significant differences were observed when contralateral and ipsilateral horns were compared (for a full comparison of groups see Suppl. Data 1). Similarly to the testis, a correlation study between YM and porosity was performed, weak negative correlations for contralateral decellularized luteal and ipsilateral native luteal endometrium were observed (R = − 0.59 and p = 0.02; R = − 0.1 and p = 0.03, respectively; Suppl. Fig. 2), while all other groups had no significant correlations.
In summary, the native endometrium of the ipsilateral horn showed lower YM in the follicular phase compared to the luteal phase. After decellularization, the stiffness increased only in horns collected at luteal phase, in both ipsi- and contralateral horns. The porosity of the decellularized tissues was lower than that of the native tissues in follicular-contralateral and both phases in ipsilateral horns.
Young’s modulus and porosity of ovary
Ipsi- and contralateral ovaries from luteal and follicular phases of the estrous cycle were evaluated. For nanoindentation analysis, thin slices were cut transversely to analyze all layers of the ovary. Multiple indentations were performed with a 150 μm interval in the x-axis starting with a distance of 1500 μm (600 μm estimated cortex and tunica layers and 900 μm of medulla layer) to accurately measure all layers of the ovary (Tunica albuginea, cortex, and medulla, as shown in Fig. 5a). Because a non-precise distinction between Tunica albuginea and cortex was possible, data was separated and presented as cortex and medulla. For porosity measurements, the cortex and the medulla were separately dissected as described in the methods.
Young’s modulus and porosity analysis of native and decellularized ovaries. In (a) details of sampling for nanoindentation, ipsi- and contralateral ovaries to the dominant follicle in follicular phase animals (n = 3 cows) or to the corpus luteum in luteal animals (n = 3 cows) were collected for each measurement and were cut transversely in half to evaluate the different layers of the ovary separately (cortex and medulla). For porosity measurements, the cortex and the medulla were separately dissected as described in the methods. In (b) boxplot analysis of Young’s modulus measurements of ipsi- and contralateral ovaries of follicular and luteal phases of native (n = 6 ovaries) and decellularized (n = 6 ovaries) tissues. In (c) boxplot analysis of porosity data of ipsi- and contralateral ovaries of follicular and luteal phases of native (n = 6 ovaries) and decellularized tissues (n = 6 ovaries).
The only significant difference on YM was observed when comparing the native versus decellularized tissues in the contralateral medulla from the follicular phase (6.02 ± 4.84 vs 2.06 ± 1.12 kPa, p < 0.01, Fig. 5b). All the other analysis were similar when comparing native versus decellularized, respectively, 7.37 ± 4.44 vs 5.07 ± 3.19 kPa (contralateral cortex at follicular phase), 2.51 ± 1.64 vs 4.38 ± 5.00 kPa (contralateral cortex at luteal phase), 6.03 ± 3.54 vs 3.47 ± 2.94 kPa (ipsilateral cortex at follicular phase), 5.43 ± 3.01 vs 2.91 ± 1.58 kPa (ipsilateral cortex at luteal phase), 2.18 ± 0.91 vs 2.78 ± 3.81 kPa (contralateral medulla at luteal phase), 3.41 ± 1.55 vs 1.54 ± 1.11 kPa (ipsilateral medulla at follicular phase), and 4.45 ± 3.22 vs 2.51 ± 1.33 kPa (ipsilateral medulla at luteal phase). No effects of estrous cycle or contra-ipsilateral positions on YM were observed for native tissues (for a full comparison of groups see Suppl. Data 1).
Although not statistically different, native cortex mean YM were higher than native medulla YM for both follicular ipsilateral (6.0 vs 3.4 kPa), follicular contralateral (7.4 vs 6.0 kPa), luteal ipsilateral (5.4 vs 4.4 kPa), and luteal contralateral (2.5 vs 2.2 kPa). Interestingly, a reduction in Young’s modulus was observed in all analyzed native tissues, when transitioning from medulla to cortex (900–1050 μm distance; Suppl. Fig. 3).
Porosity was significantly higher in native than decellularized medulla during luteal phase for both contralateral (83.8 ± 9.7 vs 61.3 ± 12.8%) and ipsilateral (79.7 ± 7.4 vs 56.7 ± 6.9%) tissues (p < 0.01 for both, Fig. 5c), but not during follicular phase (77.2 ± 6.4 vs 63.3 ± 12.8% in contralateral medulla and 75.7 ± 7.0 vs 62.5 ± 8.3% in ipsilateral medulla). Similarly, no differences were observed in cortex samples when comparing native versus decellularized, respectively, 69.5 ± 9.2 vs 57.4 ± 7.9% (contralateral-follicular), 65.6.5 ± 14.3 vs 65.6 ± 5.4% (contralateral-luteal), 71.8 ± 17.3 vs 57.9 ± 18.1% (ipsilateral-follicular), and 68.6 ± 10.8 vs 59.1 ± 7.8% (ipsilateral-luteal). No effects of estrous cycle or contra-ipsilateral positions on porosity were observed for native tissues (for a full comparison of groups see Suppl. Data 1).
A correlation study between YM and porosity was performed for cortex and medulla separately, a weak negative correlation for native cortex contralateral luteal (R = − 0.59, p = 0.034) and a strong positive correlation for decellularized cortex ipsilateral luteal (R = 0.77, p = 0.0021) were observed (Suppl. Fig. 4), while all other groups had no significant correlations. Similarly, weak correlations for native ipsi- (R = − 0.49, p = 0.033) and contralateral (R = 0.48, p = 0.014) follicular medulla samples were observed (Suppl. Fig. 5), while all other groups had no significant correlations.
In summary, the results showed that the decellularized tissues generally had lower YM values compared to the native tissues, but the differences were not statistically significant except for the contralateral medulla in the follicular phase. No significant effects of the estrous cycle or contra-ipsilateral positions were observed on YM or porosity in the native tissues, except for porosity differences between native and decellularized medulla during the luteal phase. Additionally, a reduction in YM was observed when transitioning from the medulla to the cortex in all analyzed native tissues, but no statistical differences were observed.
Young’s modulus and porosity of oviduct
For the analysis of oviductal tissue, ipsi- and contralateral oviducts from follicular and luteal phases of the estrous cycle were evaluated. Both nanoindentation and porosity were performed separately in the ampulla and isthmus segments. Multiple nanoindentation measurements were conducted with 200 μm increments for the analysis of luminal epithelium, stroma, muscular, and tunica layers as shown in Fig. 6a and Suppl. Fig. 6. Adherence was a significant challenge in our nanoindentation measurements, particularly in the analysis of the oviductal tissue. Despite employing adherence mode, a technique aimed at reducing adherence effects by retracting the probe and holding it for a brief period, and using BSA to prevent adherence, the decellularized oviductal tissues exhibited heightened stickiness and stronger adhesion compared to the native tissues. Due to this adherence to the nanoindentation probe and the difficulties of precisely measuring the thin oviductal samples, it was not possible to obtain sufficient measurements to analyze the tissue’s layers as first planned (lumen-stroma surface vs muscle-tunica surface, Suppl. Fig. 7). Therefore, results are presented only separated by ampulla vs isthmus. For porosity measurements, all the tissue layers were analyzed as one, for both ampulla and isthmus segments.
Young’s modulus and porosity analysis of native and decellularized oviducts. In (a) details of sampling for nanoindentation, ipsilateral (n = 3 cows) and contralateral (n = 3 cows) thin transversal slices of the ampulla and isthmus segments of the oviducts (ampullary-isthmic junction was discarded). Boxplots depicting Young’s modulus (b) and Porosity (c) measurements of ipsi- and contralateral ampulla and isthmus of follicular and luteal phases, for both native (n = 6 oviducts) and decellularized (n = 6 oviducts) tissues are shown.
The YM was only significantly different in the contralateral oviducts, either when considering the estrous cycle phase, in which the isthmus was stiffer than the ampulla at follicular phase (6.55 ± 2.69 vs 5.36 ± 4.37 kPa, p < 0.01, Fig. 6b), or considering the decellularization process, in which the decellularized isthmus was stiffer than the native one at luteal phase (16.80 ± 7.56 vs 6.74 ± 3.82 kPa, p < 0.01, Fig. 6b). All the other analysis were similar when comparing native versus decellularized, respectively, 5.36 ± 4.37 vs 3.92 ± 2.19 kPa (contralateral ampulla at follicular phase), 6.55 ± 2.69 vs 14.12 ± 7.73 kPa (contralateral isthmus at follicular phase), 5.46 ± 4.57 vs 15.93 ± 13.63 kPa (ipsilateral ampulla at follicular phase), 7.06 ± 5.38 vs 3.69 ± 1.98 kPa (ipsilateral isthmus at follicular phase), 4.27 ± 4.00 vs 7.38 ± 6.58 kPa (contralateral ampulla at luteal phase), 7.35 ± 6.65 vs 12.64 ± 14.08 kPa (ipsilateral ampulla at luteal phase), and 6.88 ± 4.74 vs 13.32 ± 9.60 kPa (ipsilateral isthmus at luteal phase). No other effects of estrous stage nor position were observed in native tissues (for a full comparison of groups see Suppl. Data 1). Nevertheless, although significance could not be determined, higher YM were observed in muscle-tunica surfaces than in lumen-stroma surfaces for native ampulla contralateral follicular (8.6 vs 2.1 kPa), native ampulla contralateral luteal (5.9 vs 1.7 kPa), native ampulla ipsilateral follicular (9.2 vs 1.0 kPa), native ampulla ipsilateral luteal (9.9 vs 2.6 kPa), which was less evident in native isthmus contralateral follicular (6.7 vs 5.3 kPa), native isthmus contralateral luteal (7.7 vs 4.9 kPa), native isthmus ipsilateral follicular (8.3 vs 4.0 kPa), and native isthmus ipsilateral luteal (7.7 vs 4.8 kPa; Suppl. Fig. 7b). Regarding the porosity, no effects of estrous cycle or contra- ipsilateral positions on porosity were observed for all tissues (Fig. 6c; for a full comparison of groups see Suppl. Data 1).
A correlation study between YM and porosity was also performed for ampulla and isthmus separately. In the ampulla, a weak negative correlation for decellularized ampulla contralateral follicular (R = − 0.56, p = 0.045) and a weak positive correlation for native ampulla ipsilateral follicular (R = 0.61, p = 0.049) were observed (Suppl. Fig. 8), while all other groups had no significant correlations. In isthmus, a positive correlation for native ipsi- (R = 0.63, p = 0.017) and contralateral (R = 0.71, p = 0.031) follicular isthmus and a negative correlation for decellularized ipsilateral luteal (R = − 0.65, p = 0.0089) were observed (Suppl. Fig. 9), while all other groups showed no significant correlations.
In summary, a significant YM increase was observed in the isthmus than in the ampulla at follicular phase in contralateral native tissues, with decellularization leading to increased YM in contralateral luteal isthmus. Ipsilateral native ampulla tended to be stiffer than contralateral in both phases.
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- Source: https://www.nature.com/articles/s41598-024-57867-5