Novel 3D printed poly (ethylene glycol) dimethacrylate based photocurable scaffolds for bone regeneration: in vitro and in vivo evaluation Janitha M. Unagolla1, Bipin Gaihre1, A. Champa jayasuriya1,2. 1 Department of Bioengineering, University of Toledo, Toledo, OH; 2 Department of Orthopedic Surgery, University of Toledo, Toledo, OH. Statement of Purpose: The swelling of the scaffolds were negligible and Polyethylene glycol (PEG) hydrogels produced by dimensions of the scaffolds were remained same as the photopolymerization have been investigated extensively printing dimension in both in vitro and in vivo conditions. as biomaterials in applications such as scaffolds for tissue The Compressive modulus of the scaffolds were engineering, and drug delivery. (Sheng L. et al, decreased with the increasing amount of gelatin. The cell Biomacromolecules, 2004; 5, 1280-1287). However, the attachment and cell proliferation were greatly increased use of poly (ethylene glycol dimethacrylate (PEGDMA) with the increasing amount of gelatin. As shown in figure in 3D printing has limited because of the high viscosity 1, rMSC attached and proliferated on both 3% and 6% requirement for extrusion based printing. In this study, D methyl cellulose (MC) was incorporated into the PEGDMA to facilitate the gelation for the first time. Gelatin (GEL), contains integrin with binding affinity for cell surface receptors that initiate cell adhesion, was used to increase the cell affinity of the scaffolds. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was used as the photoinitiator to ensure minimal photoinitiator-induced cytotoxicity. We evaluated the effect of gelatin contain on PEGDMA/MC scaffold concerning mechanical stability, cell viability, cell Figure 1. Live and dead cell images of scaffolds proliferation, and cell differentiation to osteoblast. GEL scaffolds. These results were further clarified by the Further, bone regeneration using PEGDMA/ MC/ GEL DNA assay results, and also, there is no significant scaffolds was evaluated in vivo using a cranial defect difference between the 3% GEL and 6% GEL scaffolds created on Lewis rats. data at both day 7 and day 14. ALP activity of the day 7 Methods: scaffolds was relatively low as shown in figure 2. LAP was dissolved in phosphate buffered saline (PBS) However, scaffolds with gelatin showed significantly high solution, and later MC was added to the PBS solution. ALP activity compared to the PEGDMA only scaffolds. Then PEGDMA (MW-750) was added to the mixture. Similar behavior was observed in the day 14 scaffolds, This mixture was transferred to the ice bath for the but 2-3 fold increase of ALP activity was observed. The gelation of MC. To make GEL contained scaffolds, after surgical lesions of all animals healed without clinical dissolving LAP, GEL was added to make GEL contained signs of inflammation. Since all the animals were scaffolds. Autodesk Fusion 360 software was used to survived after surgery and normal growth was observed, model the 3D pore structure with 500 µm pore size and scaffolds showed good biocompatibility with no toxicity. scaffolds were printed using extrusion based 3D printer The scaffolds were well intergrated in to the surround (ALLEVI2, PA) at room temperature. After printing, tissues according to the visual inspection after scaffolds were subjected to photocuring for 2 minutes euthenization. using blue light (405 nm) radiation. Compressive modulus was determined using ADMET eXpert 2600 series universal testing machine. After proper sterilization, scaffolds were subject to live and dead cell viability assay, and DNA quantification assay to observe the effect of gelatin content on rat mesenchymal stem cell (rMSC) viability and proliferation. Alkaline phosphate assay (ALP) was performed on scaffolds with different gelatin amount by using the ALP assay kit (Biovision, Milpitas, CA). In vivo stability and the osteogenesis of the scaffolds Figure 2. ALP activity of the scaffolds at day 7 and day are evaluated using 8 weeks old inbred Lewis rats 14; * indicates the significance p<0.05 wrt to PEGDMA (ENVIGO). Two circular defects (5 mm) are made on the Conclusions: only scaffolds cranial bone of each rat and rats are euthanized at 6 weeks This novel PEGDMA based scaffolds are bioactive and and 12 weeks. The morphology of the newly formed bone biocompatible. Adding gelatin increases the cell is analyzed using micro-computed tomography (µ-CT). attachment and cell proliferation significanltly. Histological analysis is done using hematoxylin and eosin PEGDMA/MC/gelatin scaffolds showed enhaced (H/E) staining and Masson’s trichrome staining. osteogenic differentiation in vitro. These scaffolds are Results: better suited to non-load bearing bones (eg. cranial bone) All the scaffolds showed good stability after printing. because of the stability and biocompatibility in vivo.