In the course of evolution, mammals have gone from land to water at several occasions, resulting in separate lineages ranging from Cetacea, which include whales and dolphins, to diverging lines, such as the polar bear (Ursus maritimus) and sea otter (Enhydra lutris) (Uhen, 2007). Polar bears and sea otters have maintained their terrestrial form despite spending a significant portion of their life in water, while other species, in their evolutionary process, have become permanent residents of the aquatic environment, undergoing considerable morphological adaptations to marine life (Uhen, 2007). This long adaptation process that started in the Eocene, is particularly evident in Cetacea (Uhen, 2007; Reidenberg, 2007), where thoracic limbs became flippers, hind limbs regressed and disappeared, and vertebral bones adopted a more uniform morphology (Cozzi et al., 2010; Thewissen et al., 2006).

The main drive for these changes was obviously meeting the physical demands of life in an aquatic environment, where locomotion is largely disentangled from weight-bearing (Turnbull and Cowan, 1999), and thus radically different than as experienced on land. In fact, in water, buoyancy changes the influence of gravity dramatically and water is denser and more viscous than air, influencing resistance (Dejours, 1987). Therefore, locomotion in water happens when the mechanism that propels the animal forward is powerful enough to counter the effects of drag. Different mammalian species specialized in the form of diverse morphologies of their body and limbs (or fins), however, always with the common denominator of the development of propulsive surfaces that oscillate to generate thrust. Swimming of aquatic mammals differs significantly in mechanical efficiency and stress distribution from locomotion in terrestrial mammals relying on limb movement. In particular, pinnipeds, cetaceans and sirenians descend from mammals that evolved to move limbs in a vertical plane, and consequently use a hydrodynamic lift-based momentum exchange to move through water (Farnum, 2007).

In all mammals, regardless whether they live on land or in water, locomotion is effectuated by the musculoskeletal system. Its framework is composed by the skeleton, whose bony components articulate via joints. The function of synovial joints in particular, is to ensure the correct articulation of adjacent bones. In addition, it minimizes friction between the bony components by providing a smooth articulating surface and lubrication to accommodate the biomechanical forces generated by locomotion, transmitting and dampening them (Sophia Fox et al., 2009). The fundamental element that enables physiological function of the synovial joint is the osteochondral unit, composed of articular cartilage and bone. In the front extremities, synovial joints are the principal joint type in terrestrial mammals; in aquatic mammals they have been preserved during evolution at the articulating ends of the humerus, radius, and ulna (Sanchez and Berta, 2010), but have been replaced by fibrous joints in the distal joints of flippers, where bony elements are connected by dense connective tissue (Thewissen, 2009).

Earlier comparative studies on the musculoskeletal system of terrestrial and aquatic mammals have largely focused on bone differences. This is probably because bone is a tissue known to respond extensively and quickly to changes in loading, as described by Wolff’s law and Frost’s mechanostat theory (Wolff, 1893; Frost, 2001). Studies on the macroscopic structures have shown that the bones of the cetacean arm and forearm adopted an hourglass-like form, and that the medullary cavity disappeared to strengthen flipper resistance for steering during swimming (de Buffrénil and Schoevaert, 1988; Felts and Spurrell, 1965). At tissue level, evolutionary adaptations of the bone have been described for both terrestrial (Doube et al., 2010; Mancini et al., 2019) and aquatic mammals (Gray et al., 2007), with a particular focus on how aquatic mammals manage buoyancy by variations in their structural bone density (Gray et al., 2007; Taylor, 2000). However, besides a few studies on some joint diseases (Nganvongpanit et al., 2017; Turnbull and Cowan, 1999), there is very limited knowledge of specific adaptations of the architecture of the cartilage extracellular matrix in aquatic mammals. Moreover, subchondral bone microstructure and related mechanical characteristics, which provide insight in essential aspects of the functionality of the osteochondral unit in joints, have not yet been studied in detail.

The osteochondral unit is the pivotal element of the joint, with a fundamental clinical relevance in highly prevalent joint disorders as osteoarthritis (OA), for which no effective, durable treatment exists yet (Lepage et al., 2019). In terrestrial mammals, comparative studies on the osteochondral unit have shown that the biochemical components of the tissues are strongly preserved across a wide range of species (Malda et al., 2013). A negative allometric relationship exists between cartilage thickness and body mass (Malda et al., 2013) and it is the trabecular bone structure that adapts to increasing body mass (Mancini et al., 2019). At present, comparative studies of the osteochondral unit of terrestrial and aquatic mammalian species do not exist, despite the high clinical relevance of the structure and the potential fundamental insights such studies would provide.

The aim of this study was, therefore, to investigate if and how the structure of both the cartilage and the bone component of the osteochondral unit differs between mammals living on land or in water. Given that the biological function of the osteochondral unit is largely structural in nature and determined by the composition and the architecture of the extracellular matrix (ECM) of both the cartilage and the bone part, the present work focused on analyzing the microstructural composition and architectural features of the osteochondral units from the humeral head of six aquatic and nine terrestrial mammalian species.

In the present study, it was investigated how osteochondral tissue has evolved differently in terrestrial and aquatic mammals to accommodate the different demands of life and locomotion on land or in water. Here, the characteristics of articular cartilage of aquatic mammals are described for the first time and they appeared to be widely different from all known terrestrial species in which this has been investigated. In contrast to the arcade-like (Benninghoff, 1925) collagen arrangement in the articular cartilage of terrestrial mammals, articular cartilage collagen fibers did not display this alignment in aquatic mammals. Moreover, the cartilage-bone interface of aquatic mammals entirely lacks the typical calcified layer present in all terrestrial species in which the osteochondral unit has been investigated. Further, it also lacks a dense subchondral plate, featuring an abrupt transition from hyaline cartilage to subchondral trabecular bone. Aquatic mammals lack the intermediate transitional forms of either cartilage (the calcified layer) or bone (the subchondral plate) that are typical of terrestrial species. Interestingly, for semi-aquatic species, such as the harbor seal (Phoca vitulina), there appears still to be some densification of the subchondral bone region, but this needs further investigation. Nevertheless, the trabeculae of the subchondral bone are thinner in aquatics than in terrestrials and they do not increase in size with increasing body mass, in contrast with what has been observed in terrestrial mammals.

Previously, it was shown that cartilage biochemistry is remarkably preserved among species, and that cartilage tissue displays microstructural features that allow functionality in mice as much as in elephants (Malda et al., 2013). One of the important features in the accommodation of different forces generated by body mass is the thickness of the tissue. Our group previously reported that articular cartilage thickness scaled with negative allometry in relationship to body mass (a=0.28), based on the analysis of nearly 60 terrestrial mammalian species (Malda et al., 2013). Interestingly, in the analysis presented here, it was observed that this correlation was similar in aquatic mammals, suggesting that this relationship might be dependent of factors other than loading, such as possibly metabolic limitations (Holliday et al., 2010). In hyaline cartilage, an avascular tissue in which nutrient supply is dependent on diffusion and fluid flow across a poroviscoelastic matrix (Lu and Mow, 2008), metabolite limitation is known to be related to tissue thickness (Malda et al., 2013; White and Seymour, 2003). In the sperm whale, an almost direct transition from hyaline cartilage to trabecular subchondral bone was observed, making diffusion from the rich vascular network of the trabecular bone to the hyaline cartilage probably as easy as diffusion from the synovial fluid at the articular surface of that hyaline cartilage. The increased porosity, and thus the greater potential for transfer of solutes, may permit the great cartilage thickness seen in some aquatic mammals (over 7 mm in the largest whale [Balaenoptera physalus]). As an avascular tissue, cartilage is dependent on diffusion, so critical thickness will be larger if diffusion of nutrients can be provided more efficiently from two sides. The ratio of nutrient exchange is likely to be different in aquatic mammals, as at the few sites where the hyaline cartilage is in direct contact with the subchondral bone in terrestrial animals, solute exchange has been shown to be fivefold higher than through the calcified cartilage (Arkill and Winlove, 2008).

Simon et al. did not observe a consistent correlation between cartilage thickness and estimated compressive stress on the joint in a study conducted on five species of terrestrial mammals that varied widely in body mass (mouse, rat, dog, sheep, and cow; Simon, 1970). When studying the thicknesses of the different zones that make up articular cartilage, significant differences were observed between aquatic and terrestrial mammals in the deep and middle layers. However, this was not the case for the superficial layer. This supports the notion that the primary role of the superficial layer of cartilage is to protect the deeper layers from shear forces and to distribute impact forces and loads among the tissue (Julkunen et al., 2007; Bevill et al., 2010). Importantly, the load distribution of forces is essential in both aquatic and terrestrial species. Unlike shear forces, compressive loads will, however, be substantially lower in aquatic species, which would be a likely explanation for the relatively thinner deep layer.

A significant difference in the predominance of fiber orientation was found between aquatic and terrestrial mammals in all cartilage layers: collagen fiber orientation in terrestrial species followed the classic Benninghoff arcade model (Benninghoff, 1925). This is known to be conserved in all terrestrial species in which the configuration has been investigated (Kääb et al., 1998) and in which orientation is principally parallel to the surface in the superficial layer, and is changing in the middle zone as the fibers are bending towards an orientation perpendicular to the subchondral bone in the deep layer. The deep layer is known to increase in relative thickness with increasing body mass and therefore increasing compressive loading (Felts and Spurrell, 1965). Conversely, in aquatic mammals, orientation of the collagen fibers throughout the whole thickness of the cartilage was more random. In an aquatic mammal, the total biomechanical loading of the joints will be less because of the absence of gravity and in a relative sense, shear forces, which perhaps are less than in case of terrestrial locomotion, but are still generated by swimming (Dejours, 1987; Williams and Worthy, 2009; Williams, 1999; Williams, 2001), will become more important. This could potentially explain the presence of only a thin superficial layer. Moreover, it is likely that the heterogeneity in the alignment of the collagen fibers in aquatic mammals represents the strongly reduced need for a specifically organized orientation as seen in terrestrial mammals.

The different collagen fiber orientation observed in the aquatic mammals compared to terrestrial mammals is also reflected in the biomechanical behavior of cartilage. To analyze this effect, both peak and equilibrium modulus of cartilage were determined. These parameters are commonly used to describe the mechanical functionality of native and engineered cartilage because they provide a better understanding of how the constituents of cartilage contribute to its overall mechanical function (Mow et al., 1984; Klika et al., 2016). The mechanical responses observed in the aquatic mammals are indicative of an overall less stiff cartilage tissue, compared to that in terrestrial mammals. Furthermore, the absence of a defined collagen network structure in the aquatic mammals is likely to provide less resistance to osmotic swelling, as in terrestrial mammal’s cartilage up to two thirds of the elastic modulus in compression arises from the electrostatic contributions and osmotic swelling that is restricted inside the organized collagen matrix (Lai et al., 1991; Canal Guterl et al., 2010).

The lack of a calcified cartilage layer and a compact subchondral plate, which are typical features of the osteochondral unit in terrestrial animals (Sophia Fox et al., 2009), was highly remarkable. The main function of the calcified layer in the osteochondral unit is to provide a gradual transition in stiffness from hyaline cartilage to subchondral bone and to serve as anchor point for the collagen fibers in the deep layer of the hyaline cartilage, that is to serve as a foundation for the Benninghoff arcades (Sophia Fox et al., 2009; Benninghoff, 1925; Madry et al., 2010). The subchondral plate has a role in distributing the compressive forces acting on the joint. As discussed earlier, in aquatic mammals compressive loading is hardly present and it may well be that firm anchoring of the collagen fibrils is much less important for this reason. Detailed studies using high resolution electron microscopy might shed some light on this issue.

Bone density deeper under the cartilage tissue (trabecular BV/TV) was relatively higher in terrestrial mammals and increased with body mass, while the opposite was observed in aquatic mammals. For the terrestrial animals the relationship, this is in accordance with the earlier observation in these animals trabecular bone increases with increasing in size (Barak et al., 2013). The second observation, can be attributed to the lack of loading and the fact that in aquatic mammals living in deep waters, in which the absence of gravity obviates the need for such adaptation to increasing body mass, it has been shown that low bone density enables dynamic buoyancy control (Gray et al., 2007), explaining the opposite tendency in terrestrials. According to Dumont et al., bone microanatomy carries an ecological signal that holds information about habitat and locomotion patterns, which is particularly evident in tetrapods that have developed advanced secondary adaptations to life in water (Dumont et al., 2013). In the matter of buoyancy control of aquatic mammals, species recently adapted to aquatic life or living in shallow waters, such as sea cows (sirenians), have bones that are denser than in terrestrial forms to allow static control (ballast), as opposed to a lower bone density typically associated with the dynamic buoyancy control required by animals that exhibit deep diving behavior for longer periods (Gray et al., 2007; Wall, 1983).

Moreover, bone trabecular thickness behaved in a similar manner. This parameter also increased with size in terrestrial mammals, with a trend similar to previous reports in literature (Mancini et al., 2019; Doube et al., 2011), while in aquatic mammals trabecular thickness appeared to scale independently. This independence can possibly be explained by the lack of compressive forces necessary to elicit a proportionate response in the bone for trabecular thickness as suggested by Wolff’s law (Wolff, 1893; Frost, 2001). In fact, Rolvien et al. investigated characteristics of the vertebral bodies of three toothed whale species sperm whale, orca (Orcinus orca) and harbor porpoise (Phocoena phocoena) and found that bone volume fraction (BV/TV) did not scale with body mass, but that trabeculae were thicker and fewer in number in larger whale species (Rolvien et al., 2017). Although research has shown the relationship between bone microanatomical organization, habitats and locomotion patterns in different taxa (Dumont et al., 2013), this was less clear when comparing different species of whales, particularly in view of the variability of for example diving-depth behavior of the different animals (Rolvien et al., 2017).

Interestingly, the histological sections of the aquatic mammals, displayed small islands of cartilaginous tissue embedded within the subchondral bone, and in the larger aquatic species, such as the sperm whale, large islands of cartilage tissue were found deep in the trabecular bone. These appeared as large lacunae upon first inspection with micro-CT, and an initial explanation was that these were a possible areas of osteocyte death due to barotraumas, which have been anecdotally described in deep-diving mammals (Moore and Early, 2004). However, upon histological evaluation, these islands appeared to be composed of healthy cartilage-like tissue naturally embedded in the bone, devoid of evident signs of bone remodeling, not suggesting any form of pathology. The areas may, therefore, represent incomplete osteochondral ossification, well-known in the horse and other species as osteochondrosis (Thomas and Barnes, 2015; McCoy et al., 2013) similarly to what was found and described by Rolvien et al. in the central areas of the vertebrae of sperm whales (Rolvien et al., 2017).

The number of individuals and species from which samples could be obtained was based on species availability and practical (such as the possibility of tissue harvest shortly after death from aquatic mammals) and does not compare to the numbers of earlier multispecies investigations in terrestrial animals (Mancini et al., 2019; Malda et al., 2013), which could be considered as a major limitation of this study. However, the included aquatic species covered a wide range of body masses (from 54 to 48,000 kg) and can, given the high consistency of the findings and the clear and unambiguous landslide differences with terrestrial species, be considered representative, especially in view of the very limited and subtle differences observed between a large number of different terrestrial species in the earlier studies (Mancini et al., 2019; Malda et al., 2013). Taken together, the findings of the current study add significantly to our knowledge of the basic aspects of the biology of the osteochondral unit and clearly reflect the crucial influence of loading. We know from evolution that life started to develop in the water and that later species came on land to develop further (Colbert and Morales, 2001). We also know that all mammals have initially evolved on land and that some of them returned to the water to become what we now call aquatic mammals (Colbert and Morales, 2001). Where we do not have histological data from the joints of the land-borne species from which our current aquatic mammalian species have evolved, it is reasonable to assume that their osteochondral units were similar to the common format of all currently existing terrestrial mammalian species. The huge structural differences between the osteochondral units of the currently living aquatic and terrestrial mammals make hence clear that accommodating loading is the most important evolutionary pressure on the osteochondral unit of terrestrial mammals. Further, it highlights that this challenge is dealt with by the architecture and structural composition of the osteochondral unit and not by molecular composition or cellular diversity. The last two elements of the osteochondral unit are similar in aquatics and terrestrials. It is the specific tissue architecture and structural organization of especially the cartilage-bone interface that got lost in aquatic mammals, once the loading-based evolutionary pressure disappeared. This increased insight in the fundamental biology of the structural components of the osteochondral unit, obtained by observing Nature, is of great importance from a tissue repair perspective, as it is a natural guide for which considerations should be prioritized in the quest for recapitulation of the native template for regenerative purposes. In this sense, the study presents strong support for a recent call to rethink the current paradigms in articular cartilage regeneration (Malda et al., 2019), through focusing on solutions with a proper architecture that allow restoration of (biomechanical) function, rather than on mere cellular activity in terms of production of extracellular matrix components.

Data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figure 1.

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Author details

1. Irina AD Mancini

   1. Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
   2. Regenerative Medicine Utrecht, Utrecht University, Utrecht, Netherlands

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Riccardo Levato

   1. Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
   2. Regenerative Medicine Utrecht, Utrecht University, Utrecht, Netherlands
   3. Department of Orthopedics, University Medical Centre Utrecht, Utrecht, Netherlands

   Contribution

   Data curation, Formal analysis, Supervision, Methodology, Writing – review and editing

   Contributed equally with

   Marlena M Ksiezarczyk

   Competing interests

   No competing interests declared

3. Marlena M Ksiezarczyk

   1. Regenerative Medicine Utrecht, Utrecht University, Utrecht, Netherlands
   2. Department of Orthopedics, University Medical Centre Utrecht, Utrecht, Netherlands

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Contributed equally with

   Riccardo Levato

   Competing interests

   No competing interests declared

4. Miguel Dias Castilho

   1. Regenerative Medicine Utrecht, Utrecht University, Utrecht, Netherlands
   2. Department of Orthopedics, University Medical Centre Utrecht, Utrecht, Netherlands
   3. Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands

   Contribution

   Data curation, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

5. Michael Chen

   Department of Mathematical Sciences, University of Adelaide, Adelaide, Australia

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Mattie HP van Rijen

   1. Regenerative Medicine Utrecht, Utrecht University, Utrecht, Netherlands
   2. Department of Orthopedics, University Medical Centre Utrecht, Utrecht, Netherlands

   Contribution

   Data curation, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

7. Lonneke L IJsseldijk

   Division of Pathology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

   Contribution

   Resources, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7288-9118

8. Marja Kik

   Division of Pathology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

   Contribution

   Funding acquisition, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

9. P René van Weeren

   1. Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
   2. Regenerative Medicine Utrecht, Utrecht University, Utrecht, Netherlands

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6654-1817

10. Jos Malda

    1. Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
    2. Regenerative Medicine Utrecht, Utrecht University, Utrecht, Netherlands
    3. Department of Orthopedics, University Medical Centre Utrecht, Utrecht, Netherlands

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    j.malda@umcutrecht.nl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9241-7676

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