Fabrication and analysis of in vivo bioengineered GelMA tooth bud constructs. (a) Cultured dental cells are harvested, pelleted, and resuspended in GelMA hydrogel (a1). The GelMA/cell solution is then pipetted into PDMS ring molds that are placed within a 24-well plate (a2). UV exposure is used to photocrosslink the GelMA/ cell solution (a3). The PDMS molds are then removed and media is added for in vitro culture (a4). (b) Four incisions are made on the backs of immunocompromised rats (b1) to make subcutaneous pockets. The GelMA tooth bud constructs are carefully placed within the pockets (b2). To harvest, the skin and subcutaneous tissue are dissected to reveal the tooth bud constructs (b3). Formalin-fixed paraffin-embedded sections can be stained with various histological stains such as H&E (c). IF and IHC can be used to investigate the expression of various proteins including amelogenin (AM, d) and dentin sialophosphoprotein (DSPP, e) which will be detected by brown staining in contrast to a negative control (f). Scale bar: c-d 200 μm, insets 50 μm