Embryonic tendons possess developmental stage-dependent differences in regenerative capacity

Introduction: Tendons are collagenous connective tissues that connect muscle to bone to enable movement. Adult tendons heal poorly as disorganized scar tissue with inferior mechanical properties, decreased functionality, and a high propensity for re-injury. Currently, there are no treatments that promote adult tendon regeneration. Our goal is to develop a tendon treatment strategy that recapitulates aspects of fetal tendon healing to promote adult tendon regeneration. Fetal sheep tendons appear to heal scarlessly[1], but the extent of regeneration and mechanisms of the scarless healing are unknown. The fetal sheep model is challenged by high cost and complications with in utero surgery. Here, we developed a chick embryo calcaneal tendon injury model to investigate mechanisms of fetal tendon healing. The chick embryo model (equivalent to mammalian fetus) is relatively inexpensive and develops fully functional tendons in only 21 days. It also provides direct embryo access through the eggshell, precluding the complications of in utero surgery. We hypothesized that earlier developmental stage chick embryo tendons possess greater regenerative healing capacity, and that the regenerative healing capacity of embryonic tendons diminishes at later developmental stages. We performed partial width calcaneal tendon transections at different embryonic stages and evaluated tendon healing based on collagen matrix organization, nuclear morphology, tensile mechanical properties, and expression of tendon phenotype and pro-inflammatory markers.

Materials and

Methods: Fertilized white leghorn chicken eggs were incubated and then injured at embryonic day (D)13-D14 (younger embryonic tendons; YT) or D16-D17 (older embryonic tendons; OT). For in ovo culture, at D13 and D16, a window was cut in the eggshell at the aircell, a hole was made in the chorioallantoic membrane (CAM), and the right ankle was lifted through the hole in the CAM and held at 90°. Microscissors dipped in India ink were used to transect the middle 25% width of the tendon midsubstance at full thickness. After injury, the egg was incubated at 37.5˚C as the embryo continued to develop. Embryos were sacrificed at D20 and legs were removed, embedded in paraffin, sectioned at 6μm, stained with picrosirius red (PSR) or hematoxylin and eosin (H&E), and imaged with brightfield microscopy. Nuclear morphology was analyzed using ImageJ. For ex ovo culture, D14 and D17 legs were explanted and cultured in standard growth medium. The ankle was held at 90°, and microscissors dipped in India ink were used to transect the middle 25% width of the tendon midsubstance at full thickness. Tendons were harvested after 12h for reverse transcription polymerase chain reaction (RT-PCR) and after 0h and 120h for tensile mechanical testing. For all cultures, contralateral tendons were uninjured controls. Two-tailed Student’s T tests were used to determine significant differences between injured and uninjured controls within each stage, with P< 0.05 considered significant.

Results, Conclusions, and Discussions: Injured YT appeared to have similar collagen matrix morphology as stage-matched uninjured controls, whereas OT collagen matrix appeared less dense and more disorganized than stage-matched uninjured controls (Fig. 1A). Injured YT had similar variability in nuclear area and angle compared to uninjured controls, whereas injured OT had greater variability in nuclear area and angle than uninjured controls (Fig. 1B-D). Immediately after injury (0h), injured YT and OT had lower elastic modulus and peak stress compared to stage-matched uninjured controls. After 120h of healing ex vivo, YT improved elastic modulus and peak stress to levels comparable to that of stage-matched uninjured controls, whereas injured OT did not (Fig. 2). Peak strain was similar between injured and uninjured controls at both timepoints for YT and OT (not shown). At 12h of healing, YT upregulated expression of tenomodulin, collagen type 1, and interleukin 1 receptor 1 (IL-1R1) compared to uninjured controls (Fig. 3). In contrast, expression of these markers was not altered in healing OT relative to uninjured controls (Fig. 3). Our data suggest that injured YT heal scarlessly based on qualitative observations of the collagen matrix and histomorphometric analyses of cell morphology. Additionally, the improvement of healing YT mechanical properties to levels of uninjured controls suggest earlier-stage embryonic tendons possess regenerative capacity. Our data also suggest that later-stage embryonic tendons have lower regenerative potential than earlier-stage embryonic tendons. Healing YT upregulated tendon phenotype and pro-inflammatory markers whereas OT did not, suggesting YT and OT have different cellular responses to injury. Ongoing studies are using histology, immunohistochemistry, and other assays at additional healing timepoints to characterize healing responses and timelines of injured YT and OT. Future studies will identify potential mechanisms for the healing outcomes of YT and OT. Our exciting data highlight stage-dependent differences in the regenerative potential of chick embryo calcaneal tendon injuries. Identifying mechanisms of regenerative embryonic tendon healing could be used to inform regenerative treatment strategies to promote adult tendon regeneration after injury.

Acknowledgements (Optional):

References: [1] Beredjiklian et al. 2003, Annals of biomedical engineering, 31(10); [2] Nguyen et al. 2023, Scientific Reports, 13(1) 9600