Exosome-functionalized collagen-coated 3D-printed PCL scaffold enhancing osteogenesis and bone regeneration

The critical role of bone within the skeletal system becomes particularly evident in the context of injuries resulting from trauma, congenital anomalies, tumors, or age-related degeneration [1]. Such conditions often lead to the loss of bone tissue, compromised load-bearing capacity, and diminished protection of internal organs [2].The reconstruction of these defects remains one of the most challenging, costly, and painful procedures in orthopedic surgery. Autologous bone grafts (e.g., iliac crest) and allogeneic transplants have been previously used clinically [3,4,5]. However, both approaches suffer from significant drawbacks. Autologous grafting is constrained by limited donor availability and carries the risk of additional morbidity at the harvest site due to secondary surgery [6]. Allogeneic grafts are also associated with considerable immunological rejection and high failure rates [7]. To address these limitations, bone tissue engineering (BTE) strategies have been developed, integrating scaffolds, cells, and bioactive cues to enhance osteogenesis, vascularization, and matrix deposition [8,9,10]. Scaffolds used in BTE with porosity levels exceeding 50% and pore sizes ranging between 200 and 500 micrometers play a pivotal role not only in promoting osteoinduction and osteoconduction but also in serving as effective 3D matrices that facilitate cell adhesion, proliferation, and extracellular matrix (ECM) deposition at the site of injury [6, 11,12,13,14]. In recent years, extrusion-based additive manufacturing, known as fused deposition modeling, has become an innovative method for fabricating scaffolds with precise architectural control. This technique enables controlled material deposition, geometric customization, and surface modification, addressing limitations related to tissue heterogeneity and complex design needs. The structural and biological performance of biomimetic scaffolds in bone tissue engineering applications has been significantly enhanced [15,16,17].

PCL, a linear aliphatic thermoplastic polyester, has garnered significant interest in tissue engineering scaffold fabrication due to its favorable properties, such as biocompatibility, low immunogenicity, cost-effectiveness, ease of processing, and slow degradation rate. PCL is particularly suitable for long-term applications in BTE [18, 19]. PCL’s low melting temperature enhances its compatibility with extrusion-based printing [20]. Bone scaffolds tailored to individual patients, constructed from PCL, provide mechanical support and gradually degrade in vivo, thus obviating the necessity for secondary surgical interventions [21, 22]. The inherent hydrophobicity of native PCL limits cellular adhesion and proliferation. Surface modification strategies, including plasma treatment and subsequent collagen or ECM-protein coating, have been utilized to enhance the hydrophilicity of PCL and improve cell adhesion and bioactivity, thereby supporting BTE [22,23,24]. Despite advances in bioactive and mechanically supporting scaffolds that mimic the native bone microenvironment, bone regeneration also relies on biological signals derived from cells and their secreted factors for new tissue production.

Among these, mesenchymal stromal cells (MSCs) are non-hematopoietic, multipotent cells with self-renewal capacity, essential for bone regeneration via differentiation into osteogenic lineages and paracrine modulation of the surrounding microenvironment [25,26,27]. Of the various MSC sources, hEnMSCs have emerged as a significant candidate for BTE platforms. hEnMSCs not only display classical MSC characteristics, including clonogenicity, multipotency, and paracrine activity, but also provide the notable benefit of minimally invasive harvesting. Due to their inherent osteogenic potential and responsiveness to stimuli, hEnMSCs are increasingly utilized as a model for assessing new osteoinductive strategies [28,29,30,31].

Beyond the cellular component, bone regeneration is strongly governed by the signaling molecules released into the extracellular environment. Among these, exosomes (Exo) are nanosized extracellular vesicles secreted by various cell types that play a crucial role in mediating intercellular communication [32]. Despite their small size (30–180 nm), they transport a wide range of bioactive molecules, such as proteins, nucleic acids, lipids, and metabolites, which significantly affect intercellular communication and alter the behavior and function of recipient cells [25, 33]. Due to their low immunogenicity, efficient cellular uptake, and extended systemic circulation, exosomes have emerged as promising agents for bone regeneration [34]. These exosomes demonstrate osteoinductive and immunomodulatory properties, making them appropriate for applications as bone tissue engineering scaffolds for repairing critical-sized bone defects [35,36,37].

While substantial research has focused on the effects of MSC-derived exosomes in bone regeneration, the role of exosomes derived from alternative osteogenic cellular models in promoting osteogenesis has not been fully elucidated. In this study, the ability of osteoblast-derived exosomes to induce osteogenesis within the context of BTE has been investigated. We have developed a 3D-printed porous PCL scaffold coated with collagen (PCL/Col), which, upon incorporation of these vesicles, significantly enhanced the osteogenic differentiation of hEnMSCs in vitro, even in the absence of standard osteogenic medium. Furthermore, in vivo assessments demonstrated improved calvarial bone regeneration. These findings highlight the potential of exosomes-functionalized, cell-free scaffolds as an effective strategy for promoting bone repair in critical-sized defects in a rat model.