Bone marrow mesenchymal stem cell-derived exosomal lactate dehydrogenase A promotes tendon-bone healing via histone lactylation-mediated cartilage regeneration.

Background: Anterior cruciate ligament reconstruction (ACLR) is the dominant clinical modality for the treatment of anterior cruciate ligament injuries. The success of ACLR is largely dependent on tendon-bone healing, and stem cell biotherapies are often used to facilitate this process. Histone lactylation modifications are involved in the regulation of various diseases. Lactate dehydrogenase A (LDHA) has been shown to play an important role in exosomes.

Aim: To explore the regulation of tendon-bone healing after ACLR by LDHA in exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos).

Methods: BMSC-Exos and LDHA were characterized and analyzed by transmission electron microscopy, qNano, immunofluorescence and western blotting assay. The corresponding low expression cell lines were obtained using RNA interference transfection; LDHA expression in rat bone tissues after ACLR was analyzed by western blotting. The volume of newborn bone tissues was monitored by micro-computed tomography imaging. Tendon and fibrocartilage regeneration were further analyzed and calculated by histological analysis, including hematoxylin and eosin and Safranin O-Fast green staining, respectively; LDHA levels of chondrocyte stem cells (CSPCs) after co-incubation with BMSC-Exos were analyzed by western blotting. Extracellularly secreted lactic acid content was determined by lactate assay kit. Cell viability was assessed by cell counting kit 8 assay, and the proliferation and differentiation ability of cells was further examined by the expression of collagen II, SOX9 and aggrecan. Histone H3K18 lactylation modification was analyzed by western blotting. H3K18 La binding on bone morphogenetic protein 7 (BMP7) promoter was analyzed by chromatin immunoprecipitation-quantitative polymerase chain reaction; BMP7 promoter activity was analyzed by dual luciferase reporter gene; BMP7 protein expression was analyzed using quantitative polymerase chain reaction and western blotting. Then, the proliferation of CSPCs promoted by BMSC-Exos LDHA was analyzed by protein expression levels of LDHA, BMP7, collagen II, SOX9, aggrecan, extracellular lactate content, and cell counting kit 8 assay.

Results: The spherical nanosized BMSC-Exos could be uptaken by CSPCs. LDHA was highly expressed in BMSC-Exos, which could infiltrate into the bone tissue of ACLR rats and promoted the generation of new bone tissue, as well as significantly increased the regeneration of tendon and fibrocartilage. Co-incubation of CSPCs with high-expressing LDHA BMSC-Exos increased the secretion of lactate content from CSPCs, cell viability, and the expression of markers related to cell proliferation and differentiation, including collagen II, SOX9, and aggrecan; LDHA in BMSC-Exos upregulated BMP7 through histone H3K18 lactate modification; high LDHA expression reversed the knockdown of BMP7, further increasing the proliferation and differentiation of CSPCs, thereby inducing cartilage formation.

Conclusion: LDHA in BMSC-Exos promotes BMP7 expression via H3K18 lactylation modification, which further promotes tendon-bone healing after ACLR.