Tumor Necrosis Factor Improves Vascularization in Osteogenic Grafts Engineered with Human Adipose-Derived Stem/Stromal Cells

<div><p>The innate immune response following bone injury plays an important role in promoting cellular recruitment, revascularization, and other repair mechanisms. Tumor necrosis factor-α (TNF) is a prominent pro-inflammatory cytokine in this cascade, and has been previously shown to improve bone formation and angiogenesis in a dose- and timing-dependent manner. This ability to positively impact both osteogenesis and vascular growth may benefit bone tissue engineering, as vasculature is essential to maintaining cell viability in large grafts after implantation. Here, we investigated the effects of exogenous TNF on the induction of adipose-derived stem/stromal cells (ASCs) to engineer pre-vascularized osteogenic tissue <i>in vitro</i> with respect to dose, timing, and co-stimulation with other inflammatory mediators. We found that acute (2-day), low-dose exposure to TNF promoted vascularization, whereas higher doses and continuous exposure inhibited vascular growth. Co-stimulation with platelet-derived growth factor (PDGF), another key factor released following bone injury, increased vascular network formation synergistically with TNF. ASC-seeded grafts were then cultured within polycaprolactone-fibrin composite scaffolds and implanted in nude rats for 2 weeks, resulting in further tissue maturation and increased angiogenic ingrowth in TNF-treated grafts. VEGF-A expression levels were significantly higher in TNF-treated grafts immediately prior to implantation, indicating a long-term pro-angiogenic effect. These findings demonstrate that TNF has the potential to promote vasculogenesis in engineered osteogenic grafts both <i>in vitro</i> and <i>in vivo</i>. Thus, modulation and/or recapitulation of the immune response following bone injury may be a beneficial strategy for bone tissue engineering.</p></div

In vivo integration of vascularized osteogenic grafts.

<p><i>In vitro</i>-induced grafts were implanted subcutaneously in athymic nude rats for 14 days. Staining of cryosections (10 µm thick): (<b>A, B</b>) H&E, (<b>C, D</b>) human-specific Lamin A/C (LMNA) to label implanted cells, (<b>E, F</b>) osteogenic markers, (<b>G, H</b>) vascular markers (human-specific CD31 and laminin (LAM) for rat/human vessels). Scale bars  = 500 µm. (<b>I</b>) Quantification of immunostaining. Values shown as mean ± SEM. Significance indicated as **<i>p</i><0.01.</p

Combined effects of TNF and PDGF-BB on vascular and osteogenic induction within fibrin gels.

<p>(<b>A</b>) Fibrin-encapsulated ASC aggregates underwent dual induction with the addition of TNF and/or PDGF-BB. (<b>B</b>) Whole-mount immunostaining for CD31 (green) and OCN (blue). Scale bar  = 500 µm. Quantification of immunostains: (<b>C</b>) vascular network length, (<b>D</b>) vascular network interconnectivity, and (<b>E</b>) mean intensity of OCN deposits. (<b>F</b>) Total calcium content. (<b>G</b>) Calcium content normalized to DNA content. Values shown as mean ± SEM. Significance indicated as **<i>p</i><0.01 or ***<i>p</i><0.001.</p

Schematic of experimental approaches.

<p>(<b>A</b>) Osteogenic (monolayer) and vascular (aggregates in fibrin gel) cultures were studied separately in this experiment to study the effects of TNF dose (0 to 100 ng/ml for the first 2 days only) on each lineage. (<b>B</b>) Fibrin-encapsulated ASC aggregates underwent dual (vascular and osteogenic) induction using a step-wise approach <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107199#pone.0107199-Hutton1" target="_blank">[1]</a>. The cells were treated with TNF and/or PDGF-BB to study their individual and combined effects. (<b>C</b>) ASC aggregates were seeded with fibrin gel into the pores of 3D-printed PCL scaffolds and induced to form vessels and mineral in order to study the spatial organization of tissue assembly within grafts. (<b>D</b>) These cultured grafts were implanted subcutaneously in athymic nude rats for 2 weeks to assess <i>in vivo</i> integration and maturation of the grafts.</p

Effects of acute TNF exposure on independent lineage induction.

<p>ASCs were induced towards either osteogenic differentiation (2D monolayer) or vascular morphogenesis (spheroids in 3D fibrin gel) and treated with varying doses of exogenous TNF for the first 48 hours. Osteogenic cultures were assessed via Alizarin Red S stain for calcium deposits (<b>A</b>), as well as quantification of total calcium content (<b>C</b>) and calcium normalized to DNA content (<b>D</b>) (dotted line: non-osteogenic control). Vascular cultures were assessed via whole-mount immunostaining for CD31 (green) and αSMA (red) (<b>B</b>), as well as quantification of vascular network length (<b>E</b>) and interconnectivity (<b>F</b>). Scale bars  = 500 µm. Values shown as mean ± SEM. *<i>p</i><0.05, **<i>p</i><0.01, or ***<i>p</i><0.001 versus 0 ng/ml TNF.</p

Surface marker characterization of cell population.

<p>Flow cytometry histograms from a representative population of passage 2 human ASCs for mesenchymal markers CD73 and CD105, pericyte marker PDGFR-β, and endothelial markers CD31 and CD34. Grey histogram: cells labeled with isotype control antibody; black outline histogram: cells labeled with antigen-specific antibody.</p