A fundamental understanding of normal tendon development is needed for any future biologically based treatments of tendon injuries. The current studies are aimed at producing information that will be useful in the identification of novel strategies for potential biological therapies. In particular, we have identified spatial and temporal landmarks of mouse patellar tendon proliferation and differentiation, which can be used to assess the degree of tendon healing, and some of the signaling pathways that may control these.
Tendons contain highly abundant ECM glycoproteins and proteoglycans, which are essential for both organization of the tendon and its healing. The timing and position of appearance of these in the tendon can be used as molecular markers for healing, or for attempts to develop tendons from stem cells in culture. In this work, we found that COL1, FMOD, and TNMD were present throughout the developing mouse patella tendon; whereas COMP, TNC, and BGN were concentrated at the insertion site. COL1 is the most abundant protein in the tendon and is important for its strength.24
Therefore, it is logical to see the dramatic increase in COL1 during the first 2 weeks after birth, when the patellar tendon becomes functional. FMOD and TNMD are thought to contribute to the distribution and patterning of the tendon collagen.25,26
Null mutations in Fmod
mice have shown irregular fiber bundles and abnormal morphology in Achilles and tail tendons.27
Mice in which Tnmd
had been targeted also developed abnormally organized collagen fibrils.25
These findings indicated that FMOD and TNMD regulate the composition of fibril bundles in tendons, and correlate well with the increase in both of these proteins at the same period as the increase in COL1. Although several studies showed that type II collagen (COL2) is present at the insertion site, we did not observe COL2 expression by immunocytochemistry during the period studied (data not shown). COL2 was expressed at high levels by adjacent articular cartilage cells, thus indicating that the anti-COL2 antibody used in the current study was able to detect COL2 protein. It may be that COL2 appears later in the differentiation of the patellar tendon.
The localization of BGN at the insertion site of the patella tendon was consistent with the previous findings using the Achilles tendon.28
COMP expression has been observed in the tendons/ligaments of both humans and different animal models29
, whereas TNC is known to be expressed in many tissues during normal embryogenesis as well as in some cases after tendon rupture.30,31
However, to our knowledge, the current study is the first to show that the COMP and TNC are concentrated in the insertion site during normal tendon development at early postnatal stages. Interestingly, it has been shown that the expression of COMP and TNC are elevated during the healing process in the tendon-to-bone insertion site.30,32
This correlation between normal tendon development and healing suggests that COMP and TNC might play roles during the formation of tendon-to-bone insertion sites. In addition, ECM proteins such as FMOD, bigylcan, and tenasin-c have been suggested to be involved in the maintenance of stem cell niches.33,34
Therefore, COMP, BGN, and TNC might also play roles in regulating niches of tendon progenitor cells at insertion sites. However, how these might function in tendon cell generation is still unclear. It will be important to further investigate their functional roles in the formation of the insertion site. Further, it will be important to identify ECM components that have different expression patterns in the normal developing patellar tendon in culture models of tendon differentiation, because they may serve as markers for differentiation of mid-substance vs. insertion sites.
In the current study, we have identified the spatial and temporal expression patterns of activated cell signaling pathways during mouse patellar tendon development. Among these, Hh signaling is particular interesting. We observed that Gli1-LacZ -positive cells appeared in the tibial insertion site, thus indicating that the Hh signaling pathway was active in the insertion site. We also observed that the Gli1-LacZ positive cells were present at the cruciate ligament insertion sites (data not shown), thus suggesting the general importance of Hh signaling at tendon/ligament-to-cartilage insertion sites. The maturation of tendon-to-cartilage insertion sites requires contributions from both the tenocyte and chondrocyte.35
In addition, it is known that the differentiation and proliferation of chondrocytes is regulated by Hh signaling.36,37
Therefore, the localization of Hh-responding cells in the insertion site suggests that the Hh signaling pathway might control the chondrification of the tenocytes at the insertion site. There are also spatial and temporal correlations between the expression of BGN and TNC and Hh responding cells at the insertion site, thus suggesting that the Hh signaling pathway might regulate the expression of these ECM proteins in the developing insertion site of mouse patella tendon. Alternatively, the ECM glycoproteins and proteoglycans may actually control the function of the Hh signaling pathway in the tendon. It has been shown that proteoglycans such as BGN can bind to signaling ligands and control their range of action.38
It will be important to learn the relationship between these ECM components and the Hh signaling pathway in the insertion site. In addition, we also found that there were fewer Gli1-LacZ positive cells at P7 and P14 than there were at P1, thus suggesting that the activity of Hh signaling was decreased at the insertion site after P7. It is well known that the tendon-to-bone insertion is a common region for overuse injuries and is hard to repair. However, why exactly it is so difficult to reconstruct an injured insertion site is still unclear. We suggest two reasons: first, the loss of Hh signaling at the insertion site at later postnatal stages, and second, the ossification of the target cartilage to bone at later postnatal stages. It will be important to further explore the functional roles of Hh signaling pathway in the differentiation of the insertion site as well as its role in tissue repair. Two Hh ligands; sonic Hh and Indian Hh, have been shown to regulate the development of the musculoskeletal system.39
It will be important to learn which ligand is functional at the insertion site.
Many studies have shown that BMP, FGF, and TGFβ signaling are important during tendon development, including the initiation of tendon cell fate.40
However, how these signals regulate tendon differentiation is not fully understood. Here, we show that TGFβ and BMP signaling were active throughout the period studied and throughout the whole tendon. FGF signaling is responsible for the initiation of tendon development during embryogenesis.13,15,41
In the current study, we found that FGF signaling was decreased at P14 (), thus suggesting that it may not be essential after the initial period of tendon differentiation is over. Several studies have shown that the healing process of tendons and ligaments can be enhanced by FGF,42–44
which might therefore re-awaken a developmental pathway in the tendon. Although the chief mechanism among these is not clear, it will be important to compare activation of FGF signaling during tendon healing with its timing of activation during the normal development of tendon.
Although we did not find any evidence to support the presence of canonical Wnt signaling in the developing mouse patellar tendon, our results could not exclude the involvement of noncanonical WNT signaling in regulation of tendon growth. Further analysis of noncanonical WNT signaling in tendon development is needed.
This study shows that Scx-expressing cells are still in the cell cycle at E17.5 and P1. However, by P7 and P14, most of the cycling cells were not ScxGFP positive, thus suggesting that most tenocytes have exited the cell cycle by this time. Occasional Scx-positive cells still in the cycle can be identified at the two later time points. It is not yet clear how this data might fit with the finding that cycling tenocytes are generated from tendon cell cultures, or with the suggestion that cells from the epitenon and endotenon might contribute to the healing process of tendon.45
It is not yet known whether cells in the connective tissues of the tendon can activate Scx expression and become tenocytes, It is also not clear whether the repair process of an injured tendon generates a fibrotic scar,46,47
or a tissue containing genuine tenocytes. It is most likely that healing cells in the tendon require signals that are present during normal development of the tendon for the generation of genuine tendon at the wound site. The markers identified in this study and the signaling pathways identified as active during normal development should be useful in gaining the answers to these important questions.