AimsThis study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect.MethodsThis experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis.ResultsWestern blot analysis confirmed the expression of cleaved CASP-1 and melatonin receptor (MT-1A-R) in NPCs. The cultured NPCs were identified by detecting the expression of CD24, collagen type II, and aggrecan. After treatment with hydrogen peroxide, the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3), cleaved CASP-1, N-terminal fragment of gasdermin D (GSDMD-N), interleukin (IL)-18, and IL-1β in NPCs were upregulated, and the number of propidium iodide (PI)-positive cells was also increased, which was able to be alleviated by pretreatment with melatonin. The protective effect of melatonin on pyroptosis was blunted by both the melatonin receptor antagonist luzindole and the nuclear factor erythroid 2–related factor 2 (Nrf2) inhibitor ML385. In addition, the expression of the transcription factor Nrf2 was up- or downregulated when the melatonin receptor was activated or blocked by melatonin or luzindole, respectively.ConclusionMelatonin protects NPCs against reactive oxygen species-induced pyroptosis by upregulating the transcription factor Nrf2 via melatonin receptors.Cite this article: Bone Joint Res 2023;12(3):202–211.

Collagen, commonly used in tissue engineering, is widespread in various tissues. During bone tissue regeneration, collagen can stimulate the cellular response and determine the fate of cells. In this work, we integrated collagen type Ⅱ with procyanidin (PC) onto an implant coating by applying a layer-by-layer technique to demonstrate that collagen and PC can participate in the construction of new biomaterials and serve as multifunctional components. The effects of PC/collagen multilayers on the viability of cocultured bone marrow mesenchymal stem cells (BMSCs) were analyzed by cell counting kit-8 analysis and phalloidin staining. The reactive oxygen species level of BMSCs was revealed through immunofluorescent staining and flow cytometry. Osteogenesis-related genes were detected and in vivo experiment were performed to reveal the effect of newly designed material on the osteogenic differentiation of BMSCs. Our data demonstrated that in BMSCs PC/collagen multilayers accelerated the proliferation and osteogenic differentiation through Wnt/β-catenin signaling pathway and enhanced bone generation around the implant in the bone defect model of rabbit femurs. In summary, combination of collagen and PC provided a new sight for the research and development of implant materials or coatings in the future.

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