Lee K, Weir MD, Lippens E, Mehta M, Wang P, Duda GN, Kim WS, Mooney DJ, Xu HHK. Bone regeneration via novel macroporous CPC scaffolds in critical-sized cranial defects in rats. Dent Mater. 2014;30 (7) :e199-207.Abstract
OBJECTIVES: Calcium phosphate cement (CPC) is promising for dental and craniofacial applications due to its ability to be injected or filled into complex-shaped bone defects and molded for esthetics, and its resorbability and replacement by new bone. The objective of this study was to investigate bone regeneration via novel macroporous CPC containing absorbable fibers, hydrogel microbeads and growth factors in critical-sized cranial defects in rats. METHODS: Mannitol porogen and alginate hydrogel microbeads were incorporated into CPC. Absorbable fibers were used to provide mechanical reinforcement to CPC scaffolds. Six CPC groups were tested in rats: (1) control CPC without macropores and microbeads; (2) macroporous CPC+large fiber; (3) macroporous CPC+large fiber+nanofiber; (4) same as (3), but with rhBMP2 in CPC matrix; (5) same as (3), but with rhBMP2 in CPC matrix+rhTGF-β1 in microbeads; (6) same as (3), but with rhBMP2 in CPC matrix+VEGF in microbeads. Rats were sacrificed at 4 and 24 weeks for histological and micro-CT analyses. RESULTS: The macroporous CPC scaffolds containing porogen, absorbable fibers and hydrogel microbeads had mechanical properties similar to cancellous bone. At 4 weeks, the new bone area fraction (mean±sd; n=5) in CPC control group was the lowest at (14.8±3.3)%, and that of group 6 (rhBMP2+VEGF) was (31.0±13.8)% (p<0.05). At 24 weeks, group 4 (rhBMP2) had the most new bone of (38.8±15.6)%, higher than (12.7±5.3)% of CPC control (p<0.05). Micro-CT revealed nearly complete bridging of the critical-sized defects with new bone for several macroporous CPC groups, compared to much less new bone formation for CPC control. SIGNIFICANCE: Macroporous CPC scaffolds containing porogen, fibers and microbeads with growth factors were investigated in rat cranial defects for the first time. Macroporous CPCs had new bone up to 2-fold that of traditional CPC control at 4 weeks, and 3-fold that of traditional CPC at 24 weeks, and hence may be useful for dental, craniofacial and orthopedic applications.
Roche ET, Hastings CL, Lewin SA, Shvartsman D, Brudno Y, Vasilyev NV, O'Brien FJ, Walsh CJ, Duffy GP, Mooney DJ. Comparison of biomaterial delivery vehicles for improving acute retention of stem cells in the infarcted heart. Biomaterials. 2014;35 (25) :6850-6858.Abstract
Cell delivery to the infarcted heart has emerged as a promising therapy, but is limited by very low acute retention and engraftment of cells. The objective of this study was to compare a panel of biomaterials to evaluate if acute retention can be improved with a biomaterial carrier. Cells were quantified post-implantation in a rat myocardial infarct model in five groups (n = 7-8); saline injection (current clinical standard), two injectable hydrogels (alginate, chitosan/β-glycerophosphate (chitosan/ß-GP)) and two epicardial patches (alginate, collagen). Human mesenchymal stem cells (hMSCs) were delivered to the infarct border zone with each biomaterial. At 24 h, retained cells were quantified by fluorescence. All biomaterials produced superior fluorescence to saline control, with approximately 8- and 14-fold increases with alginate and chitosan/β-GP injectables, and 47 and 59-fold increases achieved with collagen and alginate patches, respectively. Immunohistochemical analysis qualitatively confirmed these findings. All four biomaterials retained 50-60% of cells that were present immediately following transplantation, compared to 10% for the saline control. In conclusion, all four biomaterials were demonstrated to more efficiently deliver and retain cells when compared to a saline control. Biomaterial-based delivery approaches show promise for future development of efficient in vivo delivery techniques.
Kim J, Li WA, Sands W, Mooney DJ. Effect of pore structure of macroporous poly(lactide-co-glycolide) scaffolds on the in vivo enrichment of dendritic cells. ACS Appl Mater Interfaces. 2014;6 (11) :8505-12.Abstract
The in vivo enrichment of dendritic cells (DCs) in implanted macroporous scaffolds is an emerging strategy to modulate the adaptive immune system. The pore architecture is potentially one of the key factors in controlling enrichment of DCs. However, there have been few studies examining the effects of scaffold pore structure on in vivo DC enrichment. Here we present the effects of surface porosity, pore size, and pore volume of macroporous poly(lactide-co-glycolide) (PLG) scaffolds encapsulating granulocyte macrophage colony-stimulating factor (GM-CSF), an inflammatory chemoattractant, on the in vivo enrichment of DCs. Although in vitro cell seeding studies using PLG scaffolds without GM-CSF showed higher cell infiltration in scaffolds with higher surface porosity, in vivo results revealed higher DC enrichment in GM-CSF loaded PLG scaffolds with lower surface porosity despite a similar level of GM-CSF released. The diminished compressive modulus of high surface porosity scaffolds compared to low surface porosity scaffolds lead to the significant shrinkage of these scaffolds in vivo, suggesting that the mechanical strength of scaffolds was critical to maintain a porous structure in vivo for accumulating DCs. The pore volume was also found to be important in total number of recruited cells and DCs in vivo. Varying the pore size significantly impacted the total number of cells, but similar numbers of DCs were found as long as the pore size was above 10-32 μm. Collectively, these results suggested that one can modulate in vivo enrichment of DCs by altering the pore architecture and mechanical properties of PLG scaffolds.
Chaudhuri O, Koshy ST, Branco da Cunha C, Shin J-W, Verbeke CS, Allison KH, Mooney DJ. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nat Mater. 2014;13 (10) :970-8.Abstract
In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.
Ali OA, Verbeke C, Johnson C, Sands WR, Lewin SA, White D, Doherty E, Dranoff G, Mooney DJ. Identification of immune factors regulating antitumor immunity using polymeric vaccines with multiple adjuvants. Cancer Res. 2014;74 (6) :1670-81.Abstract
The innate cellular and molecular components required to mediate effective vaccination against weak tumor-associated antigens remain unclear. In this study, we used polymeric cancer vaccines incorporating different classes of adjuvants to induce tumor protection, to identify dendritic cell (DC) subsets and cytokines critical to this efficacy. Three-dimensional, porous polymer matrices loaded with tumor lysates and presenting distinct combinations of granulocyte macrophage colony-stimulating factor (GM-CSF) and various Toll-like receptor (TLR) agonists affected 70% to 90% prophylactic tumor protection in B16-F10 melanoma models. In aggressive, therapeutic B16 models, the vaccine systems incorporating GM-CSF in combination with P(I:C) or CpG-ODN induced the complete regression of solid tumors (≤40 mm(2)), resulting in 33% long-term survival. Regression analysis revealed that the numbers of vaccine-resident CD8(+) DCs, plasmacytoid DCs (pDC), along with local interleukin (IL)-12, and granulocyte colony-stimulating factor (G-CSF) concentrations correlated strongly to vaccine efficacy regardless of adjuvant type. Furthermore, vaccine studies in Batf3(-/-) mice revealed that CD8(+) DCs are required to affect tumor protection, as vaccines in these mice were deficient in cytotoxic T lymphocytes priming and IL-12 induction in comparison with wild-type. These studies broadly demonstrate that three-dimensional polymeric vaccines provide a potent platform for prophylactic and therapeutic protection, and can be used as a tool to identify critical components of a desired immune response. Specifically, these results suggest that CD8(+) DCs, pDCs, IL-12, and G-CSF play important roles in priming effective antitumor responses with these vaccines.
Klumpers DD, Mao AS, Smit TH, Mooney DJ. Linear patterning of mesenchymal condensations is modulated by geometric constraints. J R Soc Interface. 2014;11 (95) :20140215.Abstract
The development of the vertebral column starts with the formation of a linear array of mesenchymal condensations, forming the blueprint for the eventual alternating pattern of bone and cartilage. Despite growing insight into the molecular mechanisms of morphogenesis, the impact of the physical aspects of the environment is not well understood. We hypothesized that geometric boundary conditions may play a pivotal role in the linear patterning of condensations, as neighbouring tissues provide physical constraints to the cell population. To study the process of condensation and the patterning thereof under tightly controlled geometric constraints, we developed a novel in vitro model that combines micropatterning with the established micromass assay. The spacing and alignment of condensations changed with the width of the cell adhesive patterns, a phenomenon that could not be explained by cell availability alone. Moreover, the extent of chondrogenic commitment was increased on substrates with tighter geometric constraints. When the in vivo pattern of condensations was investigated in the developing vertebral column of chicken embryos, the measurements closely fit into the quantitative relation between geometric constraints and inter-condensation distance found in vitro. Together, these findings suggest a potential role of geometric constraints in skeletal patterning in a cellular process of self-organization.
Shvartsman D, Storrie-White H, Lee K, Kearney C, Brudno Y, Ho N, Cezar C, McCann C, Anderson E, Koullias J, et al. Sustained delivery of VEGF maintains innervation and promotes reperfusion in ischemic skeletal muscles via NGF/GDNF signaling. Mol Ther. 2014;22 (7) :1243-1253.Abstract
Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both in vitro and in vivo modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.
Huebsch N, Kearney CJ, Zhao X, Kim J, Cezar CA, Suo Z, Mooney DJ. Ultrasound-triggered disruption and self-healing of reversibly cross-linked hydrogels for drug delivery and enhanced chemotherapy. Proc Natl Acad Sci U S A. 2014;111 (27) :9762-7.Abstract
Biological systems are exquisitely sensitive to the location and timing of physiologic cues and drugs. This spatiotemporal sensitivity presents opportunities for developing new therapeutic approaches. Polymer-based delivery systems are used extensively for attaining localized, sustained release of bioactive molecules. However, these devices typically are designed to achieve a constant rate of release. We hypothesized that it would be possible to create digital drug release, which could be accelerated and then switched back off, on demand, by applying ultrasound to disrupt ionically cross-linked hydrogels. We demonstrated that ultrasound does not permanently damage these materials but enables nearly digital release of small molecules, proteins, and condensed oligonucleotides. Parallel in vitro studies demonstrated that the concept of applying temporally short, high-dose "bursts" of drug exposure could be applied to enhance the toxicity of mitoxantrone toward breast cancer cells. We thus used the hydrogel system in vivo to treat xenograft tumors with mitoxantrone, and found that daily ultrasound-stimulated drug release substantially reduced tumor growth compared with sustained drug release alone. This approach of digital drug release likely will be applicable to a broad variety of polymers and bioactive molecules, and is a potentially useful tool for studying how the timing of factor delivery controls cell fate in vivo.
Gu L, Hall DJ, Qin Z, Anglin E, Joo J, Mooney DJ, Howell SB, Sailor MJ. In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles. Nat Commun. 2013;4 :2326.Abstract
Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (<10 ns) emission signals from organic chromophores or tissue autofluorescence. Here using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by >50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.
Chang J, Liu F, Lee M, Wu B, Ting K, Zara JN, Soo C, Al Hezaimi K, Zou W, Chen XH, et al. NF-κB inhibits osteogenic differentiation of mesenchymal stem cells by promoting β-catenin degradation. Proc Natl Acad Sci U S A. 2013;110 (23) :9469-74.Abstract
Mesenchymal stem cell (MSC)-based transplantation is a promising therapeutic approach for bone regeneration and repair. In the realm of therapeutic bone regeneration, the defect or injured tissues are frequently inflamed with an abnormal expression of inflammatory mediators. Growing evidence suggests that proinflammatory cytokines inhibit osteogenic differentiation and bone formation. Thus, for successful MSC-mediated repair, it is important to overcome the inflammation-mediated inhibition of tissue regeneration. In this study, using genetic and chemical approaches, we found that proinflammatory cytokines TNF and IL-17 stimulated IκB kinase (IKK)-NF-κB and impaired osteogenic differentiation of MSCs. In contrast, the inhibition of IKK-NF-κB significantly enhanced MSC-mediated bone formation. Mechanistically, we found that IKK-NF-κB activation promoted β-catenin ubiquitination and degradation through induction of Smurf1 and Smurf2. To translate our basic findings to potential clinic applications, we showed that the IKK small molecule inhibitor, IKKVI, enhanced osteogenic differentiation of MSCs. More importantly, the delivery of IKKVI promoted MSC-mediated craniofacial bone regeneration and repair in vivo. Considering the well established role of NF-κB in inflammation and infection, our results suggest that targeting IKK-NF-κB may have dual benefits in enhancing bone regeneration and repair and inhibiting inflammation, and this concept may also have applicability in many other tissue regeneration situations.
Nakaoka R, Hirano Y, Mooney DJ, Tsuchiya T, Matsuoka A. Study on the potential of RGD- and PHSRN-modified alginates as artificial extracellular matrices for engineering bone. J Artif Organs. 2013;16 (3) :284-93.Abstract
Alginate is a polysaccharide that can be crosslinked by divalent cations, such as calcium ions, to form a gel. Chemical modification is typically used to improve its cell adhesive properties for tissue engineering applications. In this study, alginates were modified with peptides containing RGD (arginine-glycine-aspartic acid) or PHSRN (proline-histidine-serine-arginine-asparagine) sequences from fibronectin to study possible additive and synergistic effects on adherent cells. Alginates modified with each peptide were mixed at different ratios to form gels containing various concentrations and spacing between the RGD and PHSRN sequences. When normal human osteoblasts (NHOsts) were cultured on or in the gels, the ratio of RGD to PHSRN was found to influence cell behaviors, especially differentiation. NHOsts cultured on gels composed of RGD- and PHSRN-modified alginates showed enhanced differentiation when the gels contained >33 % RGD-alginate, suggesting the relative distribution of the peptides and the presentation to cells are important parameters in this regulation. NHOsts cultured in gels containing both RGD- and PHSRN-alginates also demonstrated a similar enhancement tendency of calcium deposition that was dependent on the peptide ratio in the gel. However, calcium deposition was greater when cells were cultured in the gels, as compared to on the gels. These results suggest that modifying this biomaterial to more closely mimic the chemistry of natural cell adhesive proteins, (e.g., fibronectin) may be useful in developing scaffolds for bone tissue engineering and provide three-dimensional cell culture systems which more closely mimic the environment of the human body.
Cho A, Haruyama N, Hall B, Danton MJS, Zhang L, Arany P, Mooney DJ, Harichane Y, Goldberg M, Gibson CW, et al. TGF-ß regulates enamel mineralization and maturation through KLK4 expression. PLoS One. 2013;8 (11) :e82267.Abstract
Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.
La Gerche A, Daffy JR, Mooney DJ, Forbes G, Davie AJ. Transit of micro-bubbles through the pulmonary circulation of Thoroughbred horses during exercise. Res Vet Sci. 2013;95 (2) :644-7.Abstract
It has been observed that microbubbles may pass through the pulmonary circulation of dogs and humans during exercise. In humans, this phenomenon has been associated with lower pulmonary artery pressures, enhanced right ventricular function and greater exercise capacity. In the exercising Thoroughbred horse, extraordinarily high cardiac outputs exert significant pulmonary vascular stresses. The aim of this study was to determine, using contrast echocardiography, whether Thoroughbred horses performing strenuous exercise developed pulmonary transit of agitated contrast microbubbles (PTAC). At rest, agitated contrast was observed in the right ventricle, but not in the left ventricle. However, post-exercise microbubbles were observed in the left ventricle, confirming the occurrence of PTAC with exercise but not at rest. Further investigation is warranted to investigate whether this phenomenon may be associated with superior physiology and performance measures as has been implicated in other species.
Kaigler D, Silva EA, Mooney DJ. Guided bone regeneration using injectable vascular endothelial growth factor delivery gel. J Periodontol. 2013;84 (2) :230-8.Abstract
BACKGROUND: Vascularization underlies the success of guided bone regeneration (GBR) procedures. This study evaluates the regenerative potential of GBR in combination with vascular endothelial growth factor (VEGF) delivery via an injectable hydrogel system. METHODS: Critical-sized defects were created in rat calvariae, and GBR procedures were performed with a collagen membrane alone (control), or plus bolus delivery of VEGF, or plus application of VEGF-releasing hydrogels (VEGF-Alg). Four and 8 weeks after treatment, defect sites were evaluated with microcomputed tomographic and histomorphometric analyses for blood vessel and bone formation. RESULTS: At 4 weeks, relative to the control condition, the bolus addition of VEGF did not affect blood vessel density within the defect site, yet the application of VEGF-Alg significantly (P <0.05) increased blood vessel density. Although there was no difference in bone regeneration at 4 weeks, at 8 weeks there was a significant (P <0.05) increase in bone regeneration in the VEGF-Alg-treated defects. CONCLUSIONS: These data demonstrate that the application of VEGF-Alg enhanced early angiogenesis, whereas at a later time point, it enhanced bone regeneration. Controlled delivery approaches of angiogenic growth factors used adjunctively with GBR may be a promising strategy for enhancing outcomes of GBR.
Li WA, Mooney DJ. Materials based tumor immunotherapy vaccines. Curr Opin Immunol. 2013;25 (2) :238-45.Abstract
Immunotherapy is a promising approach for treating cancer. However, there are limitations inherent to current approaches which may be addressed by integrating them with biomaterial-based strategies. Material platforms have been fabricated to interact with immune cells through spatially controlled and temporally controlled delivery of immune modulators and to promote immune cell crosstalk. Particle vaccines have been developed to specifically target and deliver agents to organs, cells and subcellular compartments. These strategies have been shown to generate antigen-specific CTL responses and, in some cases, tumor regression. Therefore, collaboration between immunology and materials engineering is likely to result in the creation of strong vaccines to combat cancer in the future.
Darnell MC, Sun J-Y, Mehta M, Johnson C, Arany PR, Suo Z, Mooney DJ. Performance and biocompatibility of extremely tough alginate/polyacrylamide hydrogels. Biomaterials. 2013;34 (33) :8042-8.Abstract
Although hydrogels now see widespread use in a host of applications, low fracture toughness and brittleness have limited their more broad use. As a recently described interpenetrating network (IPN) of alginate and polyacrylamide demonstrated a fracture toughness of ≈ 9000 J/m(2), we sought to explore the biocompatibility and maintenance of mechanical properties of these hydrogels in cell culture and in vivo conditions. These hydrogels can sustain a compressive strain of over 90% with minimal loss of Young's Modulus as well as minimal swelling for up to 50 days of soaking in culture conditions. Mouse mesenchymal stem cells exposed to the IPN gel-conditioned media maintain high viability, and although cells exposed to conditioned media demonstrate slight reductions in proliferation and metabolic activity (WST assay), these effects are abrogated in a dose-dependent manner. Implantation of these IPN hydrogels into subcutaneous tissue of rats for 8 weeks led to mild fibrotic encapsulation and minimal inflammatory response. These results suggest the further exploration of extremely tough alginate/PAAM IPN hydrogels as biomaterials.
Chan G, Mooney DJ. Ca(2+) released from calcium alginate gels can promote inflammatory responses in vitro and in vivo. Acta Biomater. 2013;9 (12) :9281-91.Abstract
In general, alginate hydrogels are considered to be biologically inert and are commonly used for biomedical purposes that require minimum inflammation. However, Ca(2+), which is commonly used to crosslink alginate, is a critical second messenger in immune cell signaling, and little has been done to understand its effect on immune cell fate when delivered as a component of alginate gels. We found that dendritic cells (DCs) encapsulated in Ca(2+)-crosslinked alginate (calcium alginate) secreted at least fivefold more of the inflammatory cytokine IL-1β when compared to DCs encapsulated in agarose and collagen gels, as well as DCs plated on tissue-culture polystyrene (TCPS). Plating cells on TCPS with the alginate polymer could not reproduce these results, whereas culturing DCs on TCPS with increasing concentrations of Ca(2+) increased IL-1β, MHC class II and CD86 expression in a dose-dependent manner. In agreement with these findings, calcium alginate gels induced greater maturation of encapsulated DCs compared to barium alginate gels. When injected subcutaneously in mice, calcium alginate gels significantly upregulated IL-1β secretion from surrounding tissue relative to barium alginate gels, and similarly, the inflammatory effects of LPS were enhanced when it was delivered from calcium alginate gels rather than barium alginate gels. These results confirm that the Ca(2+) used to crosslink alginate gels can be immunostimulatory and suggest that it is important to take into account Ca(2+)'s bioactive effects on all exposed cells (both immune and non-immune) when using calcium alginate gels for biomedical purposes. This work may strongly impact the way people use alginate gels in the future as well as provide insights into past work utilizing alginate gels.
Klumpers DD, Zhao X, Mooney DJ, Smit TH. Cell mediated contraction in 3D cell-matrix constructs leads to spatially regulated osteogenic differentiation. Integr Biol (Camb). 2013;5 (9) :1174-83.Abstract
During embryonic development, morphogenetic processes give rise to a variety of shapes and patterns that lead to functional tissues and organs. While the impact of chemical signals on these processes is widely studied, the role of physical cues is less understood. The aim of this study was to test the hypothesis that the interplay of cell mediated contraction and mechanical boundary conditions alone can result in spatially regulated differentiation in simple 3D constructs. An experimental model consisting of a 3D cell-gel construct and a finite element (FE) model were used to study the effect of cellular traction exerted by mesenchymal stem cells (MSCs) on an initially homogeneous matrix under inhomogeneous boundary conditions. A robust shape change is observed due to contraction under time-varying mechanical boundary conditions, which is explained by the finite element model. Furthermore, distinct local differences in osteogenic differentiation are observed, with a spatial pattern independent of osteogenic factors in the culture medium. Regions that are predicted to have experienced relatively high shear stress at any time during contraction correlate with the regions of distinct osteogenesis. Taken together, these results support the underlying hypothesis that cellular contractility and mechanical boundary conditions alone can result in spatially regulated differentiation. These results will have important implications for tissue engineering and regeneration.
Lui KO, Zangi L, Silva EA, Bu L, Sahara M, Li RA, Mooney DJ, Chien KR. Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA. Cell Res. 2013;23 (10) :1172-86.Abstract
Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.
Brudno Y, Ennett-Shepard AB, Chen RR, Aizenberg M, Mooney DJ. Enhancing microvascular formation and vessel maturation through temporal control over multiple pro-angiogenic and pro-maturation factors. Biomaterials. 2013;34 (36) :9201-9.Abstract
Therapeutic stimulation of angiogenesis to re-establish blood flow in ischemic tissues offers great promise as a treatment for patients suffering from cardiovascular disease or trauma. Since angiogenesis is a complex, multi-step process, different signals may need to be delivered at appropriate times in order to promote a robust and mature vasculature. The effects of temporally regulated presentation of pro-angiogenic and pro-maturation factors were investigated in vitro and in vivo in this study. Pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) cooperatively promoted endothelial sprouting and pericyte detachment in a three-dimensional in vitro EC-pericyte co-culture model. Pro-maturation factors platelet-derived growth factor B (PDGF) and angiopoietin 1 (Ang1) inhibited the early stages of VEGF- and Ang2-mediated angiogenesis if present simultaneously with VEGF and Ang2, but promoted these behaviors if added subsequently to the pro-angiogenesis factors. VEGF and Ang2 were also found to additively enhance microvessel density in a subcutaneous model of blood vessel formation, while simultaneously administered PDGF/Ang1 inhibited microvessel formation. However, a temporally controlled scaffold that released PDGF and Ang1 at a delay relative to VEGF/Ang2 promoted both vessel maturation and vascular remodeling without inhibiting sprouting angiogenesis. Our results demonstrate the importance of temporal control over signaling in promoting vascular growth, vessel maturation and vascular remodeling. Delivering multiple growth factors in combination and sequence could aid in creating tissue engineered constructs and therapies aimed at promoting healing after acute wounds and in chronic conditions such as diabetic ulcers and peripheral artery disease.