Young animals are able to grow in a way that allows them to maintain roughly the same shape until they reach their adult size. The growth of embryos is driven by increases in cell size and number, but it is less clear how the body grows after birth. By this point, many of the cells in the body are part of tendons and other fibrous tissues, where they are surrounded by a mesh of fibres made of collagen and other proteins. These fibres provide strength to the tissue, but may also restrict its ability to grow.

Tendons connect muscles to bones. They contain fibres of collagen that run along their length, which enables them to cope with very strong pulling forces. Kalson et al. used electron microscopy to generate highly detailed three-dimensional models of mouse tendons at three stages: in the embryo, at birth and six weeks later.

The experiments identified two stages in tendon development. During the first stage, the number of cells and fibres across the tendon is determined in the embryo. The fibres also slightly expand in diameter and form regular waves called crimps that are important for the structural strength of the tendon. The second stage happens after birth, during which the number of cells and fibres remains constant, but the tendons continue to grow because the fibres increase in diameter and length. The cells also move to form towers of cells running along the tendon.

From these observations, Kalson et al. propose that the numbers and locations of the cells and collagen fibres that determine the shape and size of tendons are established in the embryo. The collagen fibres create a framework for the continued growth of the tendon after birth. Future challenges are to understand how the number and the arrangement of cells in the tendon is determined before the collagen fibres are made, and how these cells control the number of collagen fibres that form.

Tendons transmit tensile forces from muscles to bone and are amongst the heaviest loaded tissues in vertebrates; the forces have been estimated at ∼ 16 kN/Kg of body mass (Harrison et al., 2010). The ability of tendons to transmit such large forces is directly attributable to an extracellular matrix (ECM) comprising collagen fibrils aligned parallel to the tissue long axis. Such forces occur during extended periods of activity with potentially devastating consequences on tissue integrity and cell survival. However, despite the high forces, tendon cells (tenocytes) remain viable and are able to sustain the integrity of the tissue. Our motivation for this study was to understand how tendons increase in size from an anlage of condensed mesenchyme to a mature tissue that is predominately ECM and which can withstand large forces without cell death.

The discovery of transcription factors expressed by tendon progenitors and the identification of signalling pathways in tendon has contributed greatly to our understanding of the earliest stages of tendon specification. However, the absence of a robust method of imaging cell-matrix organisation has precluded a detailed study of how the cell-rich anlage found in embryogenesis grows into a mechanically strong connective tissue in the adult. Advances in serial block face-scanning electron microscopy (SBF-SEM) provide a method to address this problem (Starborg et al., 2013). SBF-SEM is a practical addition to serial section reconstruction and can typically provide data at ∼10 nm resolution in the sectioning plane for ∼100,000 µm³ volume of tissue, which is sufficient to quantify cell number, cell shape, and cell–cell interactions, as well as estimate the number and organisation of individual collagen fibrils. When used in combination with a developmental series, SBF-SEM provides new information on the changes in cell organization and matrix assembly during tissue growth.

The discovery of Scleraxis (Scx), amongst all other transcription factors, transformed studies of tendon development (Brent et al., 2003). The generation of Scx-GFP mice showed the precise location of tendons in the proximal–distal axis of the limb (Schweitzer et al., 2001). Scx specifies important interactions between somatic muscle and cartilage cell lineages leading to tendon development (Brent et al., 2003; Sugimoto et al., 2013a). However, Scx is also expressed in non-tendon tissues including ligaments, intervertebral discs, joints, and cartilage around the chondro-tendinous/ligamentous junction (Sugimoto et al., 2013b). Tenomodulin, a type II transmembrane glycoprotein, is expressed in tendons and ligaments and is a regulator of tenocyte proliferation and is involved in collagen fibril maturation (Docheva et al., 2005). Further studies have shown that Mohawk (Liu et al., 2010), EGR1, and EGR2 are transcription factors that are also involved in tendon formation (Lejard et al., 2011). Furthermore, the ability of EGR1 to promote tendon differentiation is partially mediated by TGFβ2 (Guerquin et al., 2013). Two main signalling pathways, TGFβ and FGF, have been identified in vertebrate tendon development (Maeda et al., 2011, and for review see Tozer and Duprez, 2005; Schweitzer et al., 2010). Collagen type I occurs in virtually all fibrous tissues along with type III, V (Birk and Mayne, 1997), XII, XIV (Ansorge et al., 2009), fibronectin and small proteoglycans such as decorin (Berenson et al., 1996; Zhang et al., 2006). Therefore, the expression of these transcription factors and structural proteins, and the presence of particular signalling pathways, does not, in itself, provide information on the development of tendon structure and function.

The defining event that signifies the onset of functional tendon development is the appearance of collagen fibrils in the ECM. The distribution of collagen fibril diameters distinguishes two stages in tendon development. In mouse, stage 1 begins at E12.5 when narrow (∼35 nm) diameter collagen fibrils are formed within actin-dependent fibripositors at the cell surface (Canty et al., 2004, 2006; Kalson et al., 2013). The length and width of the tissue doubles in a few days, and is accompanied by two-orders of magnitude increases in elastic modulus and ultimate tensile strength (McBride et al., 1985, 1988). Serial section transmission electron microscopy (ss-TEM) of chick embryonic tendon showed that the tendon matrix contains bundles of collagen fibrils that undergo gradual rotation over several microns (Birk and Trelstad, 1986; Birk et al., 1989). Moreover, the collagen fibril bundles are stabilised by cell–cell connections containing cadherin-11 (Richardson et al., 2007). During stage 1 there is a modest increase in fibril diameter (to ∼40 nm), which has been replicated in vitro by slow stretching of a 3-dimensional (3D) tendon-like construct containing embryonic tenocytes (Kalson et al., 2011). At birth (in the mouse), the unimodal distribution of narrow fibrils is quickly (within a few days) replaced by a bimodal distribution of fibril diameters with a range of 35–400 nm (Goh et al., 2012). The transition from unimodal to bimodal distributions specifies the onset of stage 2.

In addition to increased fibril diameter and changes to cell morphology, the collagen fibrils in tendon develop regular undulations, known as crimp (Diamant et al., 1972). Crimp becomes evident early in embryonic development (Shah et al., 1982) and provides the biomechanically important ‘toe-region’ to the force–extension curve (Shah et al., 1982). Crimp structure has been investigated by plane polarised light microscopy and by SEM (Raspanti et al., 2005; Franchi et al., 2007), but the structure of crimp is still debated; numerous studies describe a planar 2D undulating crimp, but mathematical modelling of crimped collagen fibrils in tendon suggests crimp to be helical in structure (Grytz and Meschke, 2009). Again, technical limitations of techniques used to study crimp have prevented a definitive description; for example, SEM requires chemical treatment of tissue to remove cellular material, precluding analysis of the composite 3D architecture.

Although ss-TEM provided insights into the short-range hierarchical structure of tendon, technical challenges of generating undistorted serial sections and achieving an absolute alignment have been major hurdles in studying hierarchical organisation beyond the level of a few cells. We here describe SBF-SEM studies of tendon development and show 3D reconstructions at three key stages of tendon development: stage 1 (embryonic), the stage 1–2 transition (newborn), and stage 2 (6 week). We interpret our findings as suggesting that the hierarchical structure of tendon, with collagen fibrils organised into fibril bundles with a biomechanically important spiral crimp structure, and the number of collagen fibrils in bundles, is established during embryogenesis. Subsequent postnatal tendon growth is achieved by increase in collagen fibril diameter and fibril length, likely by interface-limited molecular accretion, during which the spatial relationship between cells, fibril bundles and the crimp spiral, is maintained.

The events during tendon development when ECM overtakes cells as the major component of the tissue are poorly understood, largely because of the lack of a suitable technique with which to examine the micro- and macro-scale architecture. At a micro-scale, it was unknown how collagen fibrils increase in diameter from ∼35 nm to ∼400 nm (stage 1 to 2 transition), and curiously, why diameters are independent of the distance from the cell. At a macro-scale it was unknown how tenocytes become aligned in tramlines parallel to the tissue long axis in adult tendon, and how crimp develops. In this study we have shown that structure-based matrix expansion in the presence of stable cell-to-cell junctions is a novel mechanism for driving tissue growth.

The appearance of collagen fibrils and thus the start of tendon development proper, coincides with the formation of extracellular channels whose borders are delineated by cellular extensions (Birk and Trelstad, 1985). As shown in Figure 1, the wrapping of plasma membrane extensions around bundles of collagen fibrils in newborn and 6 week tendon produces a highly convoluted cross-sectional shape of the cell. Contacts between projections of neighbouring cells are stabilised by cell junctions (McNeilly et al., 1996; Ralphs et al., 2002; Richardson et al., 2007). Thus, neighbouring cells define vertical channels into which newly formed collagen fibrils are assembled, thereby generating bundles of collagen fibrils that are corralled into lying parallel to the tendon long axis.

Although there is an increase in the number of cells longitudinally (in cell stacks) between newborn and 6 weeks, the fall in cells per unit volume of tissue due to expansion of the ECM suggest that the major driver of tissue growth is an increase in the ECM in channels between cell stacks. The extended longitudinal cell–cell contacts seen in both newborn and in 6 week tendon tissue, and the maintenance of similar numbers of cell–cell contacts and cell protrusions at birth and 6 weeks, suggests that the cell–cell contacts formed in embryogenesis form the basic pattern for the mature tendon tissue. Positive immunolocalisation of important gap junction components, connexin 32 and 43, at 6 weeks supports this conclusion.

The increase in cell number along the longitudinal axis of the tendon in postnatal growth is compatible with this interpretation. New cells formed by mitosis during tissue growth can be added in cell stacks whilst at the same time maintaining the longitudinal channels containing fibril bundles, which are growing laterally due to a dramatic increase in fibril diameter.

We were able to quantitate the number of fibril profiles, diameter and average length of collagen fibrils during embryonic to postnatal development. As shown in Figure 2, fibril diameters increased during E15.5 to newborn but maintained a unimodal diameter distribution. During embryonic development the number of fibrils per bundle increases (from E15.5 to birth). There was a significant increase in fibril diameter in postnatal tendon, and fibrils developed a bimodal diameter distribution. Also during postnatal growth (between birth and 6 weeks) the number of bundles per cell remained constant and the number of fibril profiles per bundle remained constant. The fact that the number of bundles per cell remained constant was a good indication that the cell–cell junctions between neighbouring cells are stable. SBF-SEM allowed estimation of fibril length, which showed a significant increase in fibril length from E15.5 to newborn. Taken together, these data showed that growth during the period E15.5 to newborn was the result of new fibril formation together with an increase in fibril diameter and length. Recent studies have shown that the collagen fibrils in the extracellular channels, at this early stage of tendon development, are formed in cell-surface fibripositors (Canty et al., 2004; Kalson et al., 2013), and therefore, the cell strictly controls the number of fibrils deposited to the bundles.

SBF-SEM analysis of 6 week postnatal tendon provided explanations for features that have long been a puzzle. As shown in Figure 2, the number of bundles per cell and the number of collagen fibril profiles per bundle remain stable between birth and 6 weeks postnatal. During the same period, fibril diameters increase markedly and the distribution becomes bimodal. These data showed that, in mouse, cells set the number of bundles and number of collagen fibrils per bundle at the time of birth. Local growth in tendon size between newborn and 6 weeks postnatal is driven primarily by increase in collagen fibril diameter and length. Growth models have been proposed for the increase in fibril diameter based on inter-fibrillar fusion and accretion of newly-synthesised collagen molecules (Birk et al., 1995; Kadler et al., 2000; Trotter et al., 2000). Inter-fibrillar fusion can potentially involve tip-to-tip, tip-to-shaft, and shaft-to-shaft fusion. Tip-to-tip fusion results in fibril lengthening. Quantitative mass mapping by scanning transmission electron microscopy and analysis of fibril staining patterns by TEM showed that tip-to-tip fusion occurs during early embryonic tendon morphogenesis and relies on unipolar collagen fibrils, in a process that is regulated by collagen-proteoglycan interactions (Graham et al., 2000). Tip-to-shaft fusion also occurs, which generates branched networks (Kadler et al., 2000). A model for linear and lateral growth of fibrils during tendon development has been proposed in which decorin, and perhaps related small proteoglycans, regulates surface interactions of participating fibrils (Birk et al., 1995). Thus, fibril fusion is regulated by the directionality of collagen molecules in the fibril (Kadler et al., 1990), the availability of tips from unipolar fibrils (Graham et al., 2000), and by the presence of proteoglycans at the fibril surface (Birk et al., 1995; Graham et al., 2000). An important and often overlooked consequence of fibril fusion is the inevitable reduction in fibril numbers as fibrils fuse together, and the decrease in the number of fibrils per cell. It is also to be noted that fibril fusion has no effect on fibril volume fraction as no new fibril volume is created by any type of fusion; we found very few fibril fusion (tip-shaft) events when we tracked fibrils with SBF-SEM. The data presented here show that collagen fibril numbers (in profile) increased during stage 1 of development and remained constant during stage 2, despite increases in fibril diameter (in stage 1 and stage 2). Although we cannot rule out tip-to-tip fusion (which would not be expected to affect fibril diameter) our diameter and fibril number data do not support shaft-to-shaft fusion as a major contributor to increases in fibril diameter during tendon development. We also have not observed irregularly shaped fibrils, which would be expected to occur if lateral fusion was occurring.

An alternative mechanism of fibril growth is the surface-nucleation-and-propagation (SNP) model in which collagen molecules accrete onto the growing tips of fibrils (Holmes et al., 1992, 1998) and to the molecular reversal region in bipolar fibrils (Trotter et al., 1998, 2000). The SNP model predicts that collagen fibrils have specific binding sites for collagen molecules, leading to growth in diameter and length. Thus, fibril growth is determined by local structure at the fibril surface and is an example of interface-controlled growth as opposed to diffusion-limited accretion onto the fibril surface. The occurrence of an abrupt limitation in fibril diameter at the growing tips of vertebrate collagen fibrils also suggests a structure-based mechanism for fibril growth (Holmes et al., 1998). Evidence from collagen fibril diameter measurements in vivo also supports a structure-based assembly mechanism. If collagen molecules could assemble onto non-specific binding sites on the fibril in an uncoordinated manner, the axial mass profiles would be expected to show pronounced random fluctuations. In the case of diffusion-limited growth, collagen fibrils closer to the cell might be expected to have larger diameters because of the ready availability of collagen molecules emerging from the cell. However, as fibril diameters are independent of distance from the cell, it seems more likely that fibril growth (especially in postnatal tendon) is strongly influenced by the local structure of the fibril surface. Thus, the establishment of fibril number during stage 1 of development and subsequent growth in fibril diameter during stage 2 by the SNP mechanism (in the absence of overt shaft-to-shaft fibril fusion) would explain the observed increase in fibril diameter in stage 2 and the lateral growth of the tissue.

The ability of SBF-SEM to examine relatively large volumes of tissue at good resolution enabled us to assess cell shape with precision. As shown in Figure 4, cells at E15.5 and newborn had convoluted profiles, were slender cylinders with overlapping ends, and were ∼50–60 µm in length. In contrast, cells in 6 weeks postnatal had prominent lateral cellular extensions that reached deep into the ECM, were short (∼25 µm) and stacked in rows on their blunt ends. Importantly, cells at 6 weeks postnatal tendon remained in contact with each other via their cellular extensions regardless of the distance between the cell bodies where the nucleus was located. The number of collagen bundles at the cell periphery (and therefore the number of cell–cell contacts) was stable between newborn and 6 weeks postnatal. The data show that the main cell bodies move laterally apart because of the expansion of the ECM but maintain their longitudinal relationship, thereby generating the tramline organisation seen in mature tendon.

An unexpected result was visualisation of a spiral 3D organisation of collagen bundles to form the tendon crimp. In relaxed tendons, the bundles of collagen fibrils are buckled into a wavy pattern, called crimp, which is readily visible using plane polarized light (Kapacee et al., 2008). Unbuckling of the crimp during longitudinal loading functions as a natural shock-absorber on initial loading as well as being important in elastic recoil (reviewed by [Benjamin et al., 2008]). Crimp first appears during embryonic tendon development (Shah et al., 1982) but the mechanism of crimp formation remains unknown, largely because of the impracticalities of studying its formation in vivo. In our studies we were careful to preserve the tension in the tendon, avoiding major changes in crimp structure (Shah et al., 1977), by fixing whole tails without disturbing the axial attachment to the skeleton. Crimp 3D structure has been debated over a prolonged period. We found no kinks as previously described (Raspanti et al., 2005) but instead observed a left-handed spiral running the length of our 3D reconstructions. A spring/spiral nature of the tendon crimp has been predicted in a mathematical model (Grytz and Meschke, 2009). It is important to note that fibril bundles are continuous over the distances we have measured here, forming parallel spirals in axial register to provide uniform biomechanical properties when loaded, and there is a close relationship between the cell and the fibril bundle. The close association of cells with bundles suggests that the cell shape strongly influences the configuration of the crimp structure. This is consistent with studies suggesting that cell force generated by the actin-myosin system can create a crimp structure (Herchenhan et al., 2012). As illustrated in Figure 7, we showed that the crimp wavelength lengthens from ∼14 µm at E15.5 to ∼99 µm at 6 weeks postnatal. The length increase of the mouse-tail from newborn to 6 weeks by a factor of ∼6.2× closely matches the increase in crimp wavelength of ∼7.1×. This suggests a simple crimp elongation process in synchrony with bundle and tissue elongation.

In conclusion, we have shown that tendon development occurs in a two-stage process. In stage 1 the organisation of fibroblasts and the cellular channels for fibril deposition and growth are established, and cell-mediated control of collagen fibril deposition occurs. This sets the developmental template for structure-based matrix expansion of the existing matrix in stage 2, which drives the increase in the size of the matrix that underpins tissue growth. Important features include the cell-regulated number of cell processes that define the boundaries of collagen fibril bundles and help to maintain the tramline organization of cells in the mature tissue. These mechanisms could be a paradigm for the growth of other matrix-rich tissues that are abundant in collagen fibrils.

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Author details

1. Nicholas S Kalson

   Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

   Contribution

   NSK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   nickkalson@gmail.com

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-8394-3060

2. Yinhui Lu

   Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

   Contribution

   YL, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Susan H Taylor

   Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

   Contribution

   SHT, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Tobias Starborg

   Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

   Contribution

   TS, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. David F Holmes

   Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

   Contribution

   DFH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Karl E Kadler

   Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

   Contribution

   KEK, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   karl.kadler@manchester.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

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