Scaffold-induced compression enhances ligamentization potential of decellularized tendon graft reseeded with ACL-derived cells.

Anterior cruciate ligament (ACL) reconstruction is often performed using a tendon graft. However, the predominant synthesis of fibrotic scar tissue (type III collagen) occurs during the healing process of the tendon graft, resulting in a significantly lower mechanical strength than that of normal ACL tissue. In this study, ACL-derived cells were reseeded to the tendon graft, and scaffold-induced compression was applied to test whether the compressive force results in superior cell survival and integration. Given nanofiber polycaprolactone (PCL) scaffold-induced compression, ACL-derived cells reseeded to a tendon graft demonstrated superior cell survival and integration and resulted in higher gene expression levels of type I collagen compared to non-compressed cell-allograft composites in vitro. Translocation of Yes-associated protein (YAP) into the nucleus was correlated with higher expression of type I collagen in the compression group. These data support the hypothesis of a potential role of mechanotransduction in the ligamentization process.

Comparison of ligamentization potential between anterior cruciate ligament-derived cells and adipose-derived mesenchymal stem cells reseeded to acellularized tendon allograft.

AIMS: To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs). METHODS: Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 106 ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed. RESULTS: In the graft reseeded with ACL-derived cells, a large number of elongated cells that integrated into the matrix were evident at day 3 and day 7. However, in the graft reseeded with ADMSCs, only a small number of elongated cells were found integrated into the matrix. Immunofluorescence for Ki-67 and type I collagen confirmed the pronounced production of type I collagen by Ki-67-positive ACL-derived cells integrated into the ECM. A messenger RNA (mRNA) expression assay demonstrated significantly higher gene expression levels of types I (p = 0.013) and III (p = 0.050) collagen in the composites reseeded with ACL-derived cells than ADMSCs. CONCLUSION: ACL-derived cells, when reseeded to acellularized tendon graft, demonstrated earlier better survival and integration in the tendon ECM and resulted in higher gene expression levels of collagen, which may be essential to the normal ligamentization process compared to ADMSCs.Cite this article: Bone Joint Res 2022;11(11):777-786.

PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue.

PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified as a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.

Inhibition of hydrogen peroxide-induced endothelial apoptosis by 2',4',7-trihydroxyflavanone, a flavonoid form.

Oxidative stress contributes to cellular injury following clinical and experimental ischemia/reperfusion episodes. Oxidative injury can induce cellular and subcellular damage that results in apoptotic cell death. We tested whether 2',4',7-trihydroxyflavanone, a synthetic flavonoid derivative, inhibits hydrogen peroxide (H(2)O(2))-induced toxicity in human umbilical endothelial cells (HUVECs). Cultured HUVECs were incubated for 30 minutes with 0.2 mM H(2)O(2) in the absence and presence of 2',4',7-trihydroxyflavanone at a non-toxic concentration of 50 microM. The effect of 2',4',7-trihydroxyflavanone on apoptosis parameters was compared with that of naringenin and two flavonol derivatives, 2',4',7-trihydroxyflavonol and 2',4',6-trihydroxyflavonol. H(2)O(2) induced cell death within 24 hours over the range of 0.05-1.0 mM, and decreased cell viability by approximately 30% at 0.2 mM. This cytotoxicity was associated with nuclear condensation and DNA fragmentation, indicating induction of apoptosis. 2',4',7-Trihydroxyflavanone, as well as naringenin, was effective as an inhibitor of oxidative stress, protecting cell viability with >/=85% viable cells compared with the control, and no apparent nuclear condensation or DNA fragmentation. In contrast, the flavonol derivatives had no such effect. In addition, immunocytochemical data and Western blot analysis revealed that, unlike flavonol derivatives, the expression of Bcl-2 was markedly up-regulated, and that the expression of Bax and activation of caspase-3 were strongly inhibited by this flavanone derivative, thereby implicating antioxidant activity-related anti-apoptotic mechanisms of 2',4',7-trihydroxyflavanone. These results indicate that the synthetic flavonoid derivative 2',4',7-trihydroxyflavanone is an effective antioxidant, preventing endothelial apoptosis induced by H(2)O(2).

alpha-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production in leukocytes by modulating protein kinase A, p38 kinase, and nuclear factor kappa B signaling pathways.

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in leukocytes via stimulation of alpha-MSH cell surface receptors. However, the signaling mechanism of alpha-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which alpha-MSH inhibits lipopolysaccharide (LPS)-induced TNF-alpha production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor kappa B (NF kappa B) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NF kappa B to induce TNF-alpha production. We found that pretreatment of cells with alpha-MSH blocked LPS-induced p38 kinase and NF kappa B activation as well as TNF-alpha production. This response was proportional to alpha-MSH receptor expression levels, and addition of an alpha-MSH receptor antagonist abolished the inhibitory effects. In addition, alpha-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of alpha-MSH on p38 kinase activation, NF kappa B activation, and TNF-alpha production. Taken together, our results indicate that stimulation of PKA by alpha-MSH causes inhibition of LPS-induced activation of p38 kinase and NF kappa B to block TNF-alpha production.