Additive manufacturing is a key technology required to realize the production of a personalized bone substitute that exactly meets a patient’s need and fills a patient-specific bone defect. Additive manufacturing can optimize the inner architecture of the scaffold for osteoconduction, allowing fast and reliable defect bridging by promoting rapid growth of new bone tissue into the scaffold. The role of scaffold microporosity/nanoarchitecture in osteoconduction remains elusive. To elucidate this relationship, we produced lithography-based osteoconductive scaffolds from tricalcium phosphate (TCP) with identical macro- and microarchitecture, but varied their nanoarchitecture/microporosity by ranging maximum sintering temperatures from 1000 °C to 1200 °C. After characterization of the different scaffolds’ microporosity, compression strength, and nanoarchitecture, we performed in vivo studies that showed that ingrowth of bone as an indicator of osteoconduction significantly decreased with decreasing microporosity. Moreover, at the 1200 °C peak sinter temperature and lowest microporosity, osteoclastic degradation of the material was inhibited. Thus, even for wide-open porous TCP-based scaffolds, a high degree of microporosity appears to be essential for optimal osteoconduction and creeping substitution, which can prevent non-unions, the major complication during bone regeneration procedures.

Additive manufacturing enables the realization of the macro- and microarchitecture of bone substitutes. The macroarchitecture is determined by the bone defect and its shape makes the implant patient specific. The preset distribution of the 3D-printed material in the macroarchitecture defines the microarchitecture. At the lower scale, the nanoarchitecture of 3D-printed scaffolds is dependent on the post-processing methodology such as the sintering temperature. However, the role of microarchitecture and nanoarchitecture of scaffolds for osteoconduction is still elusive. To address these aspects in more detail, we produced lithography-based osteoconductive scaffolds from hydroxyapatite (HA) of identical macro- and microarchitecture and varied their nanoarchitecture, such as microporosity, by increasing the maximum sintering temperatures from 1100 to 1400 °C. The different scaffold types were characterized for microporosity, compression strength, and nanoarchitecture. The in vivo results, based on a rabbit calvarial defect model showed that bony ingrowth, as a measure of osteoconduction, was independent from scaffold’s microporosity. The same applies to in vitro osteoclastic resorbability, since on all tested scaffold types, osteoclasts formed on their surfaces and resorption pits upon exposure to mature osteoclasts were visible. Thus, for wide-open porous HA-based scaffolds, a low degree of microporosity and high mechanical strength yield optimal osteoconduction and creeping substitution. Based on our study, non-unions, the major complication during demanding bone regeneration procedures, could be prevented.

In regenerative medicine, addressing the complex challenge of bone tissue regeneration demands innovative strategies. Exosomes, nanoscale vesicles rich in bioactive molecules, have shown great promise in tissue repair. This study focuses on exosomes derived from mineralized osteoblasts (MOBs), which play a pivotal role in bone formation. We investigated the therapeutic potential of exosomes isolated from osteoblasts cultured in osteogenic medium for 21 days, delivered via 3D-printed gyroid scaffolds composed of hydroxyapatite (HA) and tricalcium phosphate (TCP). The exosomes were characterized through nanoparticle tracking analysis to determine size, morphology, and concentration, while proteomics revealed their cargo contents. In vitro, rabbit bone marrow mesenchymal stromal cells (rBMSCs) were cultured as monolayers and within ceramic scaffolds, where MOB-derived exosomes were shown to promote osteogenic differentiation. In vivo, their osteoconductive and bone augmentation capabilities were evaluated in two rabbit calvarial models, while the osteoinductive potential was further tested in a heterotopic mouse model. Neo-bone formation was assessed using µCT and histological analysis. Our findings demonstrated that MOB-derived exosomes upregulated bone-related gene expression and promoted mineralization in rBMSCs, even in the absence of osteogenic medium. Proteomics confirmed the presence of bone-associated proteins in these exosomes. In rabbit models, however, exosomes did not significantly enhance bone formation. In contrast, in the heterotopic mouse model, exosomes functionalized onto ceramic scaffolds exhibited strong osteoinductive activity. This study highlights the potential of MOB-derived exosomes to enhance 3D-printed ceramic scaffolds for bone regeneration, offering a promising avenue for bone healing without the need for additional growth factors or stem cells. STATEMENT OF SIGNIFICANCE: The here presented report of our project not only advances our understanding of the role of exosome-functionalized scaffolds in bone regeneration but also proposes a promising alternative to traditional growth factor- or cell-based approaches. We are confident that this study represents a novel and impactful contribution to the field.

Triply periodic minimal surface microarchitectures (TPMS) were developed by mathematicians and evolved in all kingdoms of living organisms. Renowned for their lightweight yet robust attributes, TPMS structures find application in diverse fields, such as the construction of satellites, aircrafts, and electric vehicles. Moreover, these microarchitectures, despite their intricate geometric patterns, demonstrate potential for application as bone substitutes, despite the inherent gothic style of natural bone microarchitecture. Here, we produced three TPMS microarchitectures, D-diamond, G-gyroid, and P-primitive, by 3D printing from hydroxyapatite. We explored their mechanical characterization and, further, implanted them to study their bone augmentation and osteoconduction potential. In terms of strength, the D-diamond and G-gyroid performed significantly better than the P-primitive. In a calvarial defect model and a calvarial bone augmentation model, where osteoconduction is determined as the extent of bony bridging of the defect and bone augmentation as the maximal vertical bone ingrowth, the G-gyroid performed significantly better than the P-primitive. No significant difference in performance was observed between the G-gyroid and D-diamond. Since, in real life, the treatment of bone deficiencies in patients comprises elements of defect bridging and bone augmentation, ceramic scaffolds with D-diamond and G-gyroid microarchitectures appear as the best choice for a TPMS-based scaffold in bone tissue engineering.

The early phase of bone healing is a complex and poorly understood process. With additive manufacturing, we can generate a specific and customizable library of bone substitutes to explore this phase. In this study, we produced tricalcium phosphate-based scaffolds with microarchitectures composed of filaments of 0.50 mm in diameter, named Fil050G, and 1.25 mm named Fil125G, respectively. The implants were removed after only 10 days in vivo followed by RNA sequencing (RNAseq) and histological analysis. RNAseq results revealed upregulation of adaptive immune response, regulation of cell adhesion, and cell migration-related genes in both of our two constructs. However, significant overexpression of genes linked to angiogenesis, regulation of cell differentiation, ossification, and bone development was observed solely in Fil050G scaffolds. Moreover, quantitative immunohistochemistry of structures positive for laminin revealed a significantly higher number of blood vessels in Fil050G samples. Furthermore, µCT detected a higher amount of mineralized tissue in Fil050G samples suggesting a superior osteoconductive potential. Hence, different filament diameters and distances in bone substitutes significantly influence angiogenesis and regulation of cell differentiation involved in the early phase of bone regeneration, which precedes osteoconductivity and bony bridging seen in later phases and as consequence, impacts the overall clinical outcome.