Scaffold materials for bone regeneration are crucial for supporting endogenous healing after accidents, infections, or tumor resection. Although beneficial impacts of microtopological or nanotopological cues in scaffold topography are commonly acknowledged, less consideration is given to the interplay between the microscale and nanoscale. Here, micropores with a 60.66 ± 24.48 µm diameter ordered by closely packed collagen fibers are identified in pre-wetted Spongostan, a clinically-approved collagen sponge. On a nanoscale level, a corrugated surface of the collagen sponge is observable, leading to the presence of 32.97 ± 1.41 nm pores. This distinct micro-and nanotopography is shown to be solely sufficient for guiding osteogenic differentiation of human stem cells in vitro. Transplantation of Spongostan into a critical-size calvarial rat bone defect further leads to fast regeneration of the lesion. However, masking the micro-and nanotopographical cues using SiO 2 nanoparticles prevents bone regeneration in vivo. Therefore, we demonstrate that the identified micropores allow migration of stem cells, which are further driven towards osteogenic differentiation by scaffold nanotopography. The present findings emphasize the necessity of considering both microand nanotopographical cues to guide intramembranous ossification, and might provide an optimal cell-and growth-factor-free scaffold for bone regeneration in clinical settings. Cells 2020, 9, 654 2 of 17 Cells 2020, 9, 654 3 of 17 Materials and Methods Study DesignThe study design is depicted in Figure 1. Briefly, micropores and nanopores were identified in Spongostan, followed by assessment of their osteoinductive capacity in vitro. For investigation of bone regeneration in vivo, Spongostan was transplanted into critical-size calvarial defects. Next to an empty control, we applied sole collagen fibers (control lacking the microtopography of Spongostan) and Spongostan masked with nanoparticles (control lacking nano-and microtopography).Cells 2020, 9, x 3 of 18 Study DesignThe study design is depicted in Figure 1. Briefly, micropores and nanopores were identified in Spongostan, followed by assessment of their osteoinductive capacity in vitro. For investigation of bone regeneration in vivo, Spongostan was transplanted into critical-size calvarial defects. Next to an empty control, we applied sole collagen fibers (control lacking the microtopography of Spongostan) and Spongostan masked with nanoparticles (control lacking nano-and microtopography).

Bone substitute materials are becoming increasingly important in oral and maxillofacial surgery. Reconstruction of critical size bone defects is still challenging for surgeons. Here, we compared the clinically applied organic bone substitute materials NanoBone® (nanocrystalline hydroxyapatite and nanostructured silica gel; n = 5) and Actifuse (calcium phosphate with silicate substitution; n = 5) with natural collagen-based Spongostan™ (hardened pork gelatin containing formalin and lauryl alcohol; n = 5) in bilateral rat critical-size defects (5 mm diameter). On topological level, NanoBone is known to harbour nanopores of about 20 nm diameter, while Actifuse comprises micropores of 200–500 µm. Spongostan™, which is clinically applied as a haemostatic agent, combines in its wet form both nano- and microporous topological features by comprising 60.66 ± 24.48 μm micropores accompanied by nanopores of 32.97 ± 1.41 nm diameter. Micro-computed tomography (µCT) used for evaluation 30 days after surgery revealed a significant increase in bone volume by all three bone substitute materials in comparison to the untreated controls. Clearly visual was the closure of trepanation in all treated groups, but granular appearance of NanoBone® and Actifuse with less closure at the margins of the burr holes. In contrast, transplantion of Spongostan™ lead to complete filling of the burr hole with the highest bone volume of 7.98 ccm and the highest bone mineral density compared to all other groups. In summary, transplantation of Spongostan™ resulted in increased regeneration of a rat calvarial critical size defect compared to NanoBone and Actifuse, suggesting the distinct nano- and microtopography of wet Spongostan™ to account for this superior regenerative capacity. Since Spongostan™ is a clinically approved product used primarily for haemostasis, it may represent an interesting alternative in the reconstruction of defects in the maxillary region.