Abstract: SmartBone® (SB) is a biohybrid bone substitute advantageously proposed as a class III
medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic,
and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed.
SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell
(MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB
scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs
(38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly
with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro
tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB
or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with
respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other
hand, the process was still present, but tissue formation was less organized at 60 days. On both
scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless,
SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed
trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and
the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively.
This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as
fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β