Design and Validation of a Perfusion Bioreactor for Long-term Human Bone Explant Culture

Abstract Ex vivo bone cultures offer a unique platform to study mechanobiology by preserving bone cells and marrow within their native extracellular matrix. We present a 3D-printable modified ZetOS bioreactor for long-term ex vivo culture of human osteoarthritic trabecular bone. Computational fluid dynamics simulations guided design optimization and supported improved average flow velocity and shear stress throughout the central region of the bone construct in the modified bioreactor design. Humeral head osteotomies from four osteoarthritic patients (age: 65-77 years, 1 female, 3 males) were prepared within two hours of surgery. Trabecular bone cores ($\varnothing$9.15 ± 0.14 $\times$ 9.5 ± 2.0 mm) were cultured under static conditions (n = 6) or perfused at 252 μL/min (dynamic I, n = 7) or 752 μL/min (dynamic II, n = 9). Cellular metabolic activity, alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) levels were monitored on 4 time-points. Genomic content (DNA and cell viability were assessed during the culture period, and micro computed tomography (μCT)-based morphometric analysis was performed after completion of the culture. Perfused cores maintained high metabolic activity and showed significantly higher ALP activity compared with static culture, indicating that fluid shear stress alone promotes osteogenic differentiation. LDH release decreased more rapidly in dynamic cultures, suggesting reduced cellular stress under perfusion. DNA content was higher in perfused cores with showed no significant differences. Morphometric analysis from μCT showed no significant differences, likely due to small sample size and inter-donor variability. Power analysis indicated that the current sample sizes were adequate for identifying large changes within groups but were insufficient to reliably detect small between-group differences. The modified ZetOS bioreactor proved biocompatible for long-term ex vivo culture of human trabecular bone tissue, preserving cell viability and enhancing osteogenic activity, while providing a robust platform to evaluate potential biomarkers and guide the development of bone-targeted therapies.