Engineering Vascularized Bone Grafts With Adipose Derived Stem Cells

Tissue engineered bone grafts could potentially revolutionize treatments for massive bone defects, offering greater tissue quantities and graft customization over the gold standard autograft. However, scale-up of cell-seeded grafts requires rapid vascularization in order to maintain high cell viability and function after implantation. Engineering vasculature inside the graft may greatly accelerate blood perfusion of the whole tissue, rather than relying solely on slow angiogenic ingrowth. Development of a complex tissue graft poses several challenges, including the concurrent stimulation of two lineages, as well as the selection of clinically relevant cell source(s). Therefore, the objective of this thesis was to develop a protocol to engineer vascularized bone grafts with a single, clinically relevant cell source: adipose-derived stem cells (ASCs). ASCs can be harvested autologously via liposuction with very high yield and are unique in that they have both osteogenic and vascular potential, making them a promising cell source to supply key components of a vascularized bone graft. This body of work describes a multistep approach towards the aforementioned objective. First, the potential of ASCs to form functional vasculature networks was explored. By increasing the direct physical interactions amongst cells, it was demonstrated that small lingering sub-populations of vascular cells are capable of spontaneously proliferating and self-assembling into three-dimensional pericyte-stabilized vascular networks. The next two chapters describe the development of a step-wise in vitro protocol to induce vascularized bone by recapitulating the biochemical environment of native bone repair. This study highlighted factors derived from platelets and inflammatory cells that promote vascular growth and stability, as well as osteogenic mineralization. Lastly, the effects of hypoxia on vascular assembly and stability were studied to understand how ASC-seeded grafts may behave in an ischemic bone injury environment. This investigation revealed that hypoxia severely inhibits de novo vascular assembly, but promotes growth and stability of pre-formed vessels. Taken together, these findings have significant implications for how ASCs could be applied clinically for bone regeneration

In Vitro Model of Vascularized Bone: Synergizing Vascular Development and Osteogenesis

Tissue engineering provides unique opportunities for regenerating diseased or damaged tissues using cells obtained from tissue biopsies. Tissue engineered grafts can also be used as high fidelity models to probe cellular and molecular interactions underlying developmental processes. In this study, we co-cultured human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs) under various environmental conditions to elicit synergistic interactions leading to the colocalized development of capillary-like and bone-like tissues. Cells were encapsulated at the 1∶1 ratio in fibrin gel to screen compositions of endothelial growth medium (EGM) and osteogenic medium (OM). It was determined that, to form both tissues, co-cultures should first be supplied with EGM followed by a 1∶1 cocktail of the two media types containing bone morphogenetic protein-2. Subsequent studies of HUVECs and MSCs cultured in decellularized, trabecular bone scaffolds for 6 weeks assessed the effects on tissue construct of both temporal variations in growth-factor availability and addition of fresh cells. The resulting grafts were implanted subcutaneously into nude mice to determine the phenotype stability and functionality of engineered vessels. Two important findings resulted from these studies: (i) vascular development needs to be induced prior to osteogenesis, and (ii) the addition of additional hMSCs at the osteogenic induction stage improves both tissue outcomes, as shown by increased bone volume fraction, osteoid deposition, close proximity of bone proteins to vascular networks, and anastomosis of vascular networks with the host vasculature. Interestingly, these observations compare well with what has been described for native development. We propose that our cultivation system can mimic various aspects of endothelial cell – osteogenic precursor interactions in vivo, and could find utility as a model for studies of heterotypic cellular interactions that couple blood vessel formation with osteogenesis

Engineering Vascularized Bone Grafts With Adipose Derived Stem Cells

Tissue engineered bone grafts could potentially revolutionize treatments for massive bone defects, offering greater tissue quantities and graft customization over the gold standard autograft. However, scale-up of cell-seeded grafts requires rapid vascularization in order to maintain high cell viability and function after implantation. Engineering vasculature inside the graft may greatly accelerate blood perfusion of the whole tissue, rather than relying solely on slow angiogenic ingrowth. Development of a complex tissue graft poses several challenges, including the concurrent stimulation of two lineages, as well as the selection of clinically relevant cell source(s). Therefore, the objective of this thesis was to develop a protocol to engineer vascularized bone grafts with a single, clinically relevant cell source: adipose-derived stem cells (ASCs). ASCs can be harvested autologously via liposuction with very high yield and are unique in that they have both osteogenic and vascular potential, making them a promising cell source to supply key components of a vascularized bone graft. This body of work describes a multistep approach towards the aforementioned objective. First, the potential of ASCs to form functional vasculature networks was explored. By increasing the direct physical interactions amongst cells, it was demonstrated that small lingering sub-populations of vascular cells are capable of spontaneously proliferating and self-assembling into three-dimensional pericyte-stabilized vascular networks. The next two chapters describe the development of a step-wise in vitro protocol to induce vascularized bone by recapitulating the biochemical environment of native bone repair. This study highlighted factors derived from platelets and inflammatory cells that promote vascular growth and stability, as well as osteogenic mineralization. Lastly, the effects of hypoxia on vascular assembly and stability were studied to understand how ASC-seeded grafts may behave in an ischemic bone injury environment. This investigation revealed that hypoxia severely inhibits de novo vascular assembly, but promotes growth and stability of pre-formed vessels. Taken together, these findings have significant implications for how ASCs could be applied clinically for bone regeneration

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

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

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

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

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