Osteogenic differentiation and mineralization of bone marrow stromal (BMS) cells depends on the cells' interactions with bioactive peptides associated with the matrix proteins. The RGD peptides of ECM proteins interact with BMS cells through integrin surface receptors to facilitate cell spreading and adhesion. The BMP peptide corresponding to residues 73-92 of bone morphogenetic protein-2 promotes differentiation and mineralization of BMS cells. The objective of this work was to investigate the effects of RGD and BMP peptides, grafted to a hydrogel substrate, on osteogenic differentiation and mineralization of BMS cells. RGD peptide was acrylamide-terminated by reacting acrylic acid with the N-terminal amine group of the peptide to produce the functionalized Ac-GRGD peptide. The PEGylated BMP peptide was reacted with 4-carboxybenzenesulfonazide to produce an azide functionalized Az-mPEG-BMP peptide. Poly (lactide-co-ethylene oxide- co-fumarate) (PLEOF) macromer was cross-linked with Ac-GRGD peptide and propargyl acrylate to produce an RGD conjugated hydrogel. Az-mPEG-BMP peptide was grafted to the hydrogel by "click chemistry". The RGD and BMP peptide density on the hydrogel surface was 1.62+/-0.37 and 5.2+/-0.6 pmol/cm2, respectively. BMS cells were seeded on the hydrogels and the effect of RGD and BMP peptides on osteogenesis was evaluated by measuring ALPase activity and calcium content with incubation time. BMS cells cultured on RGD conjugated, BMP peptide grafted, and RGD+BMP peptide modified hydrogels showed 3, 2.5, and 5-fold increase in ALPase activity after 14 days incubation. BMS cells seeded on RGD+BMP peptides modified hydrogel showed 4.9- and 11.8-fold increase in calcium content after 14 and 21 days, respectively, which was significantly higher than RGD conjugated or BMP grafted hydrogels. These results demonstrate that RGD and BMP peptides, grafted to a hydrogel substrate, act synergistically to enhance osteogenic differentiation and mineralization of BMS cells. These findings are potentially useful in developing engineered scaffolds for bone regeneration.

Reconstruction of large bone defects is limited by insufficient vascularization and slow bone regeneration. The objective of this work was to investigate the effect of spatial and temporal release of recombinant human bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) on the extent of osteogenic and vasculogenic differentiation of human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) encapsulated in a patterned hydrogel. Nanogels (NGs) based on polyethylene glycol (PEG) macromers chain-extended with short lactide (L) and glycolide (G) segments were used for grafting and timed-release of BMP2 and VEGF. NGs with 12 kDa PEG molecular weight (MW), 24 LG segment length, and 60/40 L/G ratio (P12-II, NG(10)) released the grafted VEGF in 10 days. NGs with 8 kDa PEG MW, 26 LG segment length, and 60/40 L/G ratio (P8-I, NG(21)) released the grafted BMP2 in 21 days. hMSCs and NG-BMP2 were encapsulated in a patterned matrix based on acrylate-functionalized lactide-chain-extended star polyethylene glycol (SPELA) hydrogel and microchannel patterns filled with a suspension of hMSCs+ECFCs and NG-VEGF in a crosslinked gelatin methacryloyl (GelMA) hydrogel. Groups included patterned constructs without BMP2/VEGF (None), with directly added BMP2/VEGF, and NG-BMP2/NG-VEGF. Based on the results, timed-release of VEGF in the microchannels in 10 days from NG(10) and BMP2 in the matrix in 21 days from NG(21) resulted in highest extent of osteogenic and vasculogenic differentiation of the encapsulated hMSCs and ECFCs compared to direct addition of VEGF and BMP2. Further, timed-release of VEGF from NG(10) in hMSC+ECFC encapsulating microchannels and BMP2 from NG(21) in hMSC encapsulating matrix sharply increased bFGF expression in the patterned constructs. The results suggest that mineralization and vascularization are coupled by localized secretion of paracrine signaling factors by the differentiating hMSCs and ECFCs.

Bone marrow stromal cells (BMSCs) possess multi-lineage differentiation potential and can be induced to undergo differentiation into various cell types with the correct combination of chemical and environmental factors. Although, they have shown great prospects in therapeutic and medical applications, less is known about their behavior on nanosurfaces mimicking the extra cellular matrix (ECM). In this report we have employed 2D substrates coated with tobacco mosaic virus (TMV) nanorods to study the differentiation process of BMSCs into osteoblast-like cells. TMV is a rod-shaped plant virus with an average length of 300 nm and diameter of 18 nm. The osteogenic differentiation of BMSCs on TMV was studied over time points of 7, 14 and 21 days. We examined the temporal gene expression changes during these time points by real time quantitative PCR (RT-qPCR) analysis. As expected, osteo-specific genes (osteocalcin, osteopontin and osteonectin) were upregulated and showed a maximum change in expression on TMV at 14 days which was 7 days earlier than on tissue culture plastic (TCP). Based on the genes expression profile generated by RT-qPCR experiments, we proposed that the early interaction of cells with TMV triggers on signaling pathways which regulate speedy expression of osteocalcin in turn, resulting in early mineralization of the cells. To further investigate these regulating factors we studied global changes in gene expression (DNA microarray analyses) during osteogenic differentiation on the nanosubstrate. Multitudes of genes were affected by culturing cells on nanorod substrate, which corroborated our initial PCR findings. Microarray analysis further revealed additional targets influenced by the presence of nanorods on the surface, of which, the expression of bone morphogenetic protein 2 (BMP2) was of particular interests. Further investigation into the temporal change of BMP2, revealed that it acts as a major promoter in signaling the early regulation of osteocalcin on TMV coated substrates.

A novel polymer, oligo(poly(ethylene glycol) fumarate) (OPF), cross-linked with a thermal radical initiation system has recently been developed in our laboratory as an injectable, biodegradable cell carrier for regeneration of orthopaedic tissues. The cross-linking, swelling, and degradative properties of hydrogels prepared from OPF with poly(ethylene glycol) of two different chain lengths were assessed. The two OPF types had similar gelation onset times ( approximately 3.6 min) but, when cross-linked for 8 min at 37 degrees C, exhibited significantly different swelling characteristics (fold swelling: 17.5 +/- 0.2 vs 13.4 +/- 0.4). Rat marrow stromal cells (MSCs) were then directly combined with the hydrogel precursors and encapsulated in a model OPF formulation at approximately 14 million cells/mL, cultured in vitro in the presence of osteogenic supplements (dexamethasone), and monitored over 28 days via histology. MSC differentiation in these samples (6 mm diameter x 0.5 mm thick before swelling), as determined by Von Kossa staining for calcified matrix, was apparent by day 21. At day 28, mineralized matrix could be seen throughout the samples, many microns away from the cells. These experiments strongly support the usefulness of thermally cross-linked OPF hydrogels as injectable cell carriers for bone regeneration.

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