The missing code in osseointegration: A genome-wide review of RNA sequencing in implant integration.

The molecular mechanisms guiding osseointegration and soft tissue sealing remain poorly understood. Additionally, conventional assays provide limited insight. RNA sequencing (RNA-seq) offers an unbiased, genome-wide profiling of biological events. This review summarizes RNA-seq applications in implant research, highlighting current evidence and future opportunities. A structured search was conducted in PubMed to identify studies that included RNA-seq analyses with titanium surface experiments, including in vitro experiments with osteoblasts/mesenchymal stem cells, fibroblasts, macrophages, soft tissue cells, and in vivo animal models. An overview of the RNA-seq methodology and emerging applications of biomaterials was also included in the search. RNA-seq contributions comprised three categories: (1) confirmation of known responses, such as upregulation of osteogenic differentiation genes on microrough surfaces; (2) discovery of novel functions, including non-canonical Wnt signaling, oxidative stress regulators, and non-coding RNAs mediating osteogenesis and immune modulation; and (3) mechanistic explanations of paradoxes, such as the osteoblast "dilemma" of reduced proliferation but enhanced differentiation. Fibroblast studies linked ultraviolet-activated titanium to proteoglycan/glycosaminoglycan signaling, and macrophage analyses identified zinc-induced metallothioneins as immunomodulatory biomarkers. In vivo, RNA-seq revealed strontium-driven CDH2/β-catenin signaling, age-related suppression of Wnt/angiogenesis, and keratin-mediated soft tissue sealing. Notably, we presented new RNA-seq data demonstrating distinct osteoblast reprogramming on microrough titanium, including the activation of oxidative stress defense, lipid metabolism, and immune-osteoblast crosstalk. RNA-seq is highly useful for establishing implant-tissue interactions, validating clinical observations, and revealing mechanistic explanations. Despite limited studies and protocol heterogeneity, RNA-seq offers a transformative framework for biologically informed and immune-smart implants.