In vivo skeletal regeneration using a nanocomposite silicate-based bioink

Introduction: An ideal hydrogel for skeletal biofabrication should aim to preserve cell viability during printing and sustain proliferation right after. Here we have developed a nanocomposite bioink to allow high printing fidelity of human bone marrow stromal cells (HBMSCs) with elevated cell viability in vitro and functionality in vivo. Objectives: (i) To fabricate and characterize HBMSCs laden constructs in vitro; (ii) to evaluate integration and vascular infiltration in an ex vivo model; (iii) to implant and investigate ectopic bone formation of BMP 2 loaded 3D clay based scaffolds. Methods: 5 × 106 HBMSCs ml−1 were encapsulated in nanoclay (Laponite), alginate, and methylcellulose bioink, printed, and evaluated for viability after live/dead confocal analysis. 3D printed scaffolds were implanted in a vascularized chorioallantoic membrane (CAM) of a developing chick egg and vascular infiltration assessed by Chalkley score and histology. MF 1 mice were anesthetized, three constructs were implanted on each side, and computed tomography (CT) was carried out using Bruker Skyscan 1176 after 28 days. Results: Cell viability was investigated at days 1 (81.1 % ± 13.3), 7 (88.0 % ± 6.7), 14 (88.3 % ± 9.2), and 21 (88.8 % ± 8.0). Confocal analysis demonstrated HBMSCs proliferation over time. Alkaline phosphatase staining at day 1 confirmed HBMSCs differentiation. Investigation of clay based 3D printed scaffolds in an ex vivo CAM model showed integration and vasculature infiltration after 7 days. BMP 2 loaded and −free biofabricated scaffolds implanted within an in vivo subcutaneous mouse model showed significantly higher mineralized tissue volume when compared with bulk alginate BMP 2 loaded and −free hydrogel (15.17 ± 1.98 mm3 [p < 0.0001] and 13.75 ± 3.98 mm3 [p < 0.0001], respectively). Micro CT analysis of BMP 2 loaded 3D printed clay scaffolds demonstrated a significant fivefold increase in bone formation and bone mineral density compared with alginate BMP 2 control (p < 0.0001). Conclusion: We have successfully demonstrated viability and functionality of biofabricated clay based scaffolds. Implantation in CAM and subcutaneous models showed chick vessels infiltration and new mineralized tissue formation, respectively, proving clinical relevance of this new nanoclay biofabrication 3D platform with implications for orthopedic reparative strategies.