Injuries to ligaments are common, painful and debilitating, causing joint instability and impaired protective proprioception sensation around the joint. Healing of torn ligaments usually fails to take place, and surgical replacement or reconstruction is required. Previously, we showed that in vivo application of the recombinant human amelogenin protein (rHAM +) resulted in enhanced healing of the tooth‐supporting tissues. The aim of this study was to evaluate whether amelogenin might also enhance repair of skeletal ligaments. The rat knee medial collateral ligament (MCL) was chosen to prove the concept. Full thickness tear was created and various concentrations of rHAM +, dissolved in propylene glycol alginate (PGA) carrier, were applied to the transected MCL. 12 weeks after transection, the mechanical properties, structure and composition of transected ligaments treated with 0.5 μg/μl rHAM + were similar to the normal un‐transected ligaments, and were much stronger, stiffer and organized than control ligaments, treated with PGA only. Furthermore, the proprioceptive free nerve endings, in the 0.5 μg/μl rHAM + treated group, were parallel to the collagen fibres similar to their arrangement in normal ligament, while in the control ligaments the free nerve endings were entrapped in the scar tissue at different directions, not parallel to the axis of the force. Four days after transection, treatment with 0.5 μg/μl rHAM + increased the amount of cells expressing mesenchymal stem cell markers at the injured site. In conclusion application of rHAM + dose dependently induced mechanical, structural and sensory healing of torn skeletal ligament. Initially the process involved recruitment and proliferation of cells expressing mesenchymal stem cell markers.
Lateral ligament tears, also known as high‐grade ankle sprains, are common, debilitating, and usually heal slowly. Ten to thirty percent of patients continue to suffer from chronic pain and ankle instability even after 3 to 9 months. Previously, we showed that the recombinant human amelogenin (rHAM+) induced regeneration of fully transected rat medial collateral ligament, a common proof‐of‐concept model. Our aim was to evaluate whether rHAM+ can regenerate torn ankle calcaneofibular ligament (CFL), an important component of the lateral ankle stabilizers. Right CFLs of Sabra rats were transected and treated with 0, 0.5, or 1 µg/µL rHAM+ dissolved in propylene glycol alginate (PGA). Results were compared with the normal group, without surgery. Healing was evaluated 12 weeks after treatment by mechanical testing (ratio between the right and left, untransected ligaments of the same rat), and histology including immunohistochemical staining of collagen I and S100. The mechanical properties, structure, and composition of transected ligaments treated with 0.5 μg/μL rHAM+ (experimental) were similar to untransected ligaments. PGA (control) treated ligaments were much weaker, lax, and unorganized compared with untransected ligaments. Treatment with 1 μg/μL rHAM+ was not as efficient as 0.5 μg/μL rHAM+. Normal arrangement of collagen I fibers and of proprioceptive nerve endings, parallel to the direction of the force, was detected in ligaments treated with 0.5 μg/μL rHAM+, and scattered arrangement, resembling scar tissue, in control ligaments. In conclusion, we showed that rHAM+ induced significant mechanical and structural regeneration of torn rat CFLs, which might be translated into treatment for grades 2 and 3 ankle sprain injuries.
Little is known about tuftelin expression in the developing embryo, previously it was thought to play a role in tooth enamel mineralization. In this study we show tuftelinʼs spatio-temporal expression in mineralizing and nonmineralizing tissues of the craniofacial complex in the developing mouse embryo. Embryos aged E10.5−E18.5 and newborns aged P3 were used in this study. Polymerase chain reaction (PCR), Real-time PCR, sequencing, and in-situ hybridization were used to detect and quantify messenger RNA (mRNA) expression in different developmental stages. We applied indirect immunohistochemistry and western-blot analyses to investigate protein expression. Two tuftelin mRNA transcripts and a single 64KDa protein were detected throughout embryonic development. Tuftelin was detected in tissues which develop from different embryonic origins; ectoderm, ectomesenchyme, and mesoderm. Tuftelin mRNA and protein were expressed already at E10.5, before the initiation of tooth formation and earlier than previously described. The expression pattern of tuftelin mRNA and protein exhibits dynamic spatio-temporal changes in various tissues. Tuftelin is expressed in neuronal tissues, thus fitting with its described correlation to nerve growth factor. A shift between cytoplasmatic and perinuclear/nuclear expression implies a possible role in regulation of transcription. Recent studies showed tuftelin is induced under hypoxic conditions in-vitro and in-vivo, through the hypoxia-inducible factor 1-α pathway. These results led to the hypothesis that tuftelin is involved in adaptation to hypoxic conditions. The fact that much of mammalian embryogenesis occurs at O 2 concentrations of 1-5%, raises the possibility that tuftelin expression throughout development is due to its role in the adaptive mechanisms in response to hypoxia.
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