The objective of this work was to investigate the effects of macroporous hydrogel architecture on the osteogenic signal expression and differentiation of human mesenchymal stem cells (hMSCs). In particular, we have proposed a tissue engineering approach for orbital bone repair based on a cyclic acetal biomaterial formed from 5-ethyl-5-(hydroxymethyl)-beta,beta-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD) and poly(ethylene glycol) diacrylate (PEGDA). The EHD monomer and PEGDA polymer may be fabricated into macroporous EH-PEG hydrogels by radical polymerization and subsequent porogen leaching, a novel technique for hydrophilic gels. We hypothesized that EH-PEG hydrogel macroporosity facilitates intercellular signaling among hMSCs. To investigate this phenomenon, hMSCs were loaded into EH-PEG hydrogels with varying pore size and porosity. The viability of hMSCs, the expression of bone morphogenetic protein-2 (BMP-2), BMP receptor type 1A, and BMP receptor type 2 by hMSCs, and the differentiation of hMSCs were then assessed. Results demonstrate that macroporous EH-PEG hydrogels support hMSCs and that this macroporous environment promotes a dramatic increase in BMP-2 expression by hMSCs. This upregulation of BMP-2 expression is associated by a more rapid hMSC differentiation, as measured by alkaline phosphatase expression. Altering hMSC interactions with the EH-PEG hydrogel surface, by the addition of fibronectin, did not appear to augment BMP-2 expression. We therefore speculate that EH-PEG hydrogel macroporosity facilitates autocrine and paracrine signaling by localizing endogenously expressed factors within the hydrogel's pores and thus promotes hMSC osteoblastic differentiation and bone regeneration.

Many systems have been proposed for the encapsulation of bone marrow stromal cells (BMSCs) within degradable hydrogels. Here, we use a novel cyclic acetal-based biomaterial formed from 5-ethyl-5-(hydroxymethyl)-beta,beta-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD) and poly(ethylene glycol) diacrylate (PEGDA). A cyclic acetal-based hydrogel may be preferred as cyclic acetals hydrolytically degraded into diols and carbonyls as primary degradation products, which may not affect local acidity, unlike other widely investigated polymers. The EHD monomer and PEGDA polymer may be fabricated into a EH-PEG hydrogel by radical polymerization initiated by the ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) system. The objective of this work is to determine whether the components utilized in the fabrication of EH-PEG hydrogels as well as the EH-PEG hydrogels permit BMSC viability, metabolic activity, and osteodifferentiation. Cell viability and metabolic activity were assessed after 30 min, 1 h, and 3 h of exposure to pertinent concentrations of the initiator system (10-20 mM). Osteodifferentiation was assessed by alkaline phosphatase and osteocalcin expression after a short exposure to the initiator system to simulate the encapsulation process. Lastly, cell viability was assessed immediately after encapsulation and after 7 days of culture within the EH-PEG hydrogels. Results indicate that the metabolic activity and viability of BMSCs are minimally affected, and that osteodifferentiation is not significantly affected by the APS-TEMED initiator system. Also, encapsulated BMSCs maintained viability within EH-PEG hydrogels for 7 days. This work demonstrates that the EH-PEG hydrogel is a viable option for the encapsulation and osteodifferentiation of BMSCs.

A simple porogen-leaching method to fabricate macroporous cyclic acetal hydrogel cell scaffolds is presented. Optical coherence tomography (OCT) was applied for nondestructive imaging and quantitative characterization of the scaffold structures. High-resolution OCT reveals the microstructures of the engineered tissue scaffolds in three dimensions. It also enables subsequent image processing to investigate quantitatively several key morphological design parameters for macroporous scaffolds, including the volume porosity, pore interconnectivity, and pore size. Two image-processing algorithms were adapted: three-dimensional labeling was applied to assess the interconnectivity, and erosion was applied to assess the pore size. Scaffolds with different design parameters were imaged, characterized, and compared. OCT imaging and image processing successfully discriminated scaffolds made from different formulations in terms of volume porosity, interconnectivity, and pore size. The cell viability and their spread across the scaffolds were confirmed by the fluorescence microscopy co-registered with OCT.

We examined the distribution of silver in pregnant mice and embryos/fetuses following intravenous injections of 10 nm silver nanoparticles (AgNPs) or soluble silver nitrate (AgNO3) at dose levels of 0 (citrate buffer control) or 66 µg Ag/mouse to pregnant mice on gestation days (GDs) 7, 8 and 9. Selected maternal tissues and all embryos/fetuses from control, AgNP- and AgNO3-treated groups on GD10 and control and AgNP-treated groups on GD16 were processed for the measurement of silver concentrations, intracellular AgNP localization, histopathology and gross examination of tissue morphology. Inductively-coupled plasma mass spectrometry revealed silver in all examined tissues following either AgNP or AgNO3 treatment, with highest concentrations of silver in maternal liver, spleen and visceral yolk sac (VYS), and lowest concentrations in embryos/fetuses. For VYS, mean silver concentration following AgNO3 treatment (4.87 ng Ag/mg tissue) was approximately two-fold that following AgNP treatment (2.31 ng Ag/mg tissue); for all other tissues examined, mean silver concentrations following either AgNP or AgNO3 treatment were not significantly different from each other (e.g. 2.57 or 2.84 ng Ag/mg tissue in maternal liver and 1.61 or 2.50 ng Ag/mg tissue in maternal spleen following AgNP or AgNO3 treatment, respectively). Hyperspectral imaging revealed AgNP aggregates in maternal liver, kidney, spleen and VYS from AgNP-treated mice, but not AgNO3-treated mice. Additionally, one or more embryos collected on GD10 from eight of ten AgNP-treated mice appeared small for their age (i.e. Theiler stage 13 [GD8.5] or younger). In the control group (N = 11), this effect was seen in embryos from only one mouse. In conclusion, intravenous injection of 10 nm AgNPs to pregnant mice resulted in notable silver accumulation in maternal liver, spleen and VYS, and may have affected embryonic growth. Silver accumulation in embryos/fetuses was negligible.