(C-109) Porous 3D Printed Bone Graft Composite For Improved Growth Factor Release and Cell Infiltration

Oral and maxillofacial reconstruction for intra- and extraoral critically sized bone defects occur yearly due to diseases such as osteosarcoma causing maxillofacial maligancies, injuries from high speed impact or projectile blasts, or even congenital conditions such as cleft palate. Currently, a defect is surgically repaired using either xeno- or autograph bone particulate to fill the cavity, which is then encased in a titanium mesh and screwed in place with titanium screws. This approach is limited by its variability, the length of time the patient is exposed to potential infection during surgery, as well as tight packing, often leading to diminished bone regeneration as blood vessels may fail to form. Although the bone particulate signals cells to differentiate along the osteogenic lineage, tight packing of the bone particulate often leads to diminished bone healing because of poor blood vessel formation. To address the fallbacks, we developed a patient specific, osteoinductive 3D printable construct, with a cover core design. The construct was engineered to endure mastication, while also being porous to allow for cell infiltration. The core consists of a hydrogel-tricalcium phosphate composite designed to release growth factors (GFs). This study aimed to optimize the hydrogel-tricalcium phosphate composite for improved cell infiltration and differentiation while withstanding hydrolytic degradation during osteogenesis. We hypothesized that the combination of Methacrylated Alginate (AlgMa) and Methacrylated Gelatin (GelMa) would provide a robust hydrogel to support cell infiltration and that FGF and BMP-2 containing microspheres could be used to develop a GF gradient to induce chemotaxis and osteogenesis.

The GelMa was synthesized by combining gelatin (SigmaAldrich, Massachusettes, USA) and methacrylic anhydride and then dialyzed for a week to remove unreacted Ma. Finally, the GelMa was lyophilized for storage and future use. The AlMa Medium Viscosity 20-40% methcrylation was purchased from SigmaAldrich (Massachusetts, USA).  We used an AR-G2 TA instruments Rheometer to acquire the elastic and storage modulus of AlgMa/GelMa blends. The FGF-2 release was evaluated by preparing 6 mm x 1 mm disks loaded with PLGA microparticles. The disks were placed in 1 mL of 1X PBS buffer and at 1, 2 ,4, 6, 8 hours and 1, 3, 7, 10, 14, 17 and 21 days, the entire sample volume was sampled and then replaced with fresh buffer to maintain sink conditions. The sampled buffer was then stored at -80^oC until it could be analyzed via ELISA (human FGF basic ELISA Kit, Abcam. Massachusetts, USA). Additionally, scanning electron microscopy (SEM) was performed to quantify the porosity differences in hydrogels using a cold field emission high resolution scanning electron microscope (S-4800, Hitachi). Cell migration experiments were performed using Ibidi chemotaxis slides (Gräfelfing, Germany) using GFP expressing NIH 3T3 murine fibroblasts. Immunofluorescent staining was performed using Phalloidin, DAPI and primary and secondary antibodies for fibronectin, collagen and hyaluronic accid to assess for different AlgGelMa hydrogel ratio cell proliferation.

When evaluating the effects of the AlgGelMa ratio we observed that the AlgMa could be used to modulate the matrix stiffness and increase swelling, while the GelMa could be used to reduce swelling and facilitate cell adhesion.We observed that by modulating the AlgGelMa ratio we could lower the pH, swell the hydrogel to ensure cell infiltration, and minimize fibronectin accumulation by increasing GelMa content. The porous cover and core construct will ensure the initial wound clot reaches all the way through to the hydrogel, where the pores are large enough for cells to infiltrate. These findings suggest through the use of this composite, early cell infiltration can be increased and promoted due to FGF release, leading to increased osteointegration.