(50g) Initiator System for Encapsulation of Mesenchymal Stem Cells and Analysis of Osteoblastic Differentiation for Orbital Bone Repair

Orbital floor injuries are a devastating form of craniofacial trauma. Current clinical treatments, including implantation of plastics or metals, are often inadequate due to loss of function as well as poor aesthetics. These concerns have led us to investigate tissue engineering approaches for the treatment of orbital bone defects. Engineered bone grafts are often fabricated by encapsulating osteoprogenitor cells within a hydrogel scaffold, however, the components to initiate gel crosslinking may be cytotoxic. This work investigates the effects of a water-soluble radical initiation system, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED), used to crosslink two polymer components, 5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD) and poly(ethylene glycol) diacrylate (PEGDA). Specifically, this work examines the effect of constant culture with the initiators on metabolic activity and viability of osteoprogenitor cells in monolayer. In addition, the ability of the cells to osteodifferentiate when exposed briefly to the initiators is assessed by examining the expression of known osteoblastic markers. Lastly, the cells are encapsulated in the hydrogel system and cultured to assess viability. For all experiments, osteoprogenitor cells were isolated from the bone marrow of young rats. After preculture, the osteoprogenitor cells were plated in monolayer and the experiments were performed. To assess the effect of the initiator system on the metabolic activity, ostoeprogenitor cells were cultured with the initiators at increasing concentrations for 30 minutes, 1, 3, and 6 hours. At the defined time points, the osteoprogenitor cells were analyzed using a toxicology kit based on the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial dehydrogenases to yield formazan crystals. Results indicate that there is no significant difference between control groups and osteoprogenitor cells cultured with 10, 15, and 20 mM concentrations of the initiators for the initial time points. Next, the differentiation of osteoprogenitor cells was examined by exposing them to the initiator system for one minute and then inducing osteodifferentiation. The osteoprogenitor cells were cultured for 1, 4, and 8 days. At the defined time points, the cells were isolated and assayed for expression of alkaline phosphatase and osteocalcin, known to be early and late osteoblastic markers, using quantitative reverse transcription-polymerase chain reaction. Results indicate that brief exposure to the initiation system does not affect the ability of the cell population to osteodifferentiate. Lastly, the cells were encapsulated in PEG-EHD hydrogels using the APS/TEMED initiator system. The cells were encapsulated and cultured in media for one week. Each day the cells were analyzed using a fluorescent live/dead assay. Results qualitatively show that the majority of the osteoprogenitor cell population is able to survive for the examined time points. The results of this work indicate the proposed EHD hydrogel system is a viable approach for the delivery of osteoprogenitor to orbital floor defects.