OC 69.2 - Lymphatic platelet thrombosis limits bone repair by precluding lymphatic transporting DAMPs

Background: Lymphatic vessels, interdigitated with blood vessels, travel and form an extensive transport network in the musculoskeletal system. Our previous work demonstrated that sufficient lymphatic drainage improves traumatic fracture healing and its early complications. However, the molecular mechanism of lymphatic drainage regulated bone repair is unclear.

Aims: To reveal of the molecular mechanism of lymphatic drainage regulated bone repair.

Methods: (1) For lymphatic drainage function, near infrared indocyanine green lymphatic imaging and ultrasound were employed. (2) For lymphatic platelet thrombosis, immunofluorescence staining by CD41 and Podoplanin and transmission electron microscopy were utilized. (3) For evaluating bone repair, Micro-CT, biomechanical testing and ABOG staining were employed. (4) For observing the phenotype of osteoblast and BMSCs, microscopic observation, cell counting and CCK8 were utilized. (5) For identification of neutrophil and marcophage, Wright-Giemsa staining and flow cytometry were used. (6) For measuring the concentrations of DAMPs, 4D label-free quantitative proteome analysis of rat lymph, ELISA ( lymph,venous blood and hematoma tissue) and mIHC of PLN are employed.

Results: (1) We for first time identified that a high incidence of lymphatic platelet thrombosis.
(2) Lymphatic platelet thrombosis at fracture site, leads to the accumulation of DAMPs and excessive immune and inflammatory response that negatively affect osteoblast survival and BMSC proliferation.
(3) Elimination of lymphatic platelet thrombosis using a low dose of blood thinner could improve bone repair by unblocking lymphatic draining function.
(4) Unblocked lymphatic drainage support osteoblasts and BMSCs via significantly decreasing neutrophiles and increasing M2-like macrophages at hematoma niche.
(5) Facture-associated DAMPs with high molecular weight are transported mainly via the lymphatic capillaries rather than blood capillaries.

Conclusion(s): These findings demonstrate that (1) lymphatic platelet thrombosis is the crucial pathological mechanism of trauma induced lymphatic drainage insufficiency. (2) lymphatic platelet thrombosis limits bone regeneration by blocking lymphatic drainage from transporting DAMPs.