Publications

2014
Duda GN, Grainger DW, Frisk ML, Bruckner-Tuderman L, Carr A, Dirnagl U, Einhäupl KM, Gottschalk S, Gruskin E, Huber C, et al. Changing the mindset in life sciences toward translation: a consensus. Sci Transl Med. 2014;6 (264) :264cm12.Abstract
Participants at the recent Translate! 2014 meeting in Berlin, Germany, reached a consensus on the rate-limiting factor for advancing translational medicine.
Brudno Y, Silva EA, Kearney CJ, Lewin SA, Miller A, Martinick KD, Aizenberg M, Mooney DJ. Refilling drug delivery depots through the blood. Proc Natl Acad Sci U S A. 2014;111 (35) :12722-7.Abstract
Local drug delivery depots have significant clinical utility, but there is currently no noninvasive technique to refill these systems once their payload is exhausted. Inspired by the ability of nanotherapeutics to target specific tissues, we hypothesized that blood-borne drug payloads could be modified to home to and refill hydrogel drug delivery systems. To address this possibility, hydrogels were modified with oligodeoxynucleotides (ODNs) that provide a target for drug payloads in the form of free alginate strands carrying complementary ODNs. Coupling ODNs to alginate strands led to specific binding to complementary-ODN-carrying alginate gels in vitro and to injected gels in vivo. When coupled to a drug payload, sequence-targeted refilling of a delivery depot consisting of intratumor hydrogels completely abrogated tumor growth. These results suggest a new paradigm for nanotherapeutic drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.
Roche ET, Wohlfarth R, Overvelde JTB, Vasilyev NV, Pigula FA, Mooney DJ, Bertoldi K, Walsh CJ. A bioinspired soft actuated material. Adv Mater. 2014;26 (8) :1200-6.Abstract
A class of soft actuated materials that can achieve lifelike motion is presented. By embedding pneumatic actuators in a soft material inspired by a biological muscle fibril architecture, and developing a simple finite element simulation of the same, tunable biomimetic motion can be achieved with fully soft structures, exemplified here by an active left ventricle simulator.
Silva EA, Eseonu C, Mooney DJ. Endothelial cells expressing low levels of CD143 (ACE) exhibit enhanced sprouting and potency in relieving tissue ischemia. Angiogenesis. 2014;17 (3) :617-30.Abstract
The sprouting of endothelial cells from pre-existing blood vessels represents a critical event in the angiogenesis cascade. However, only a fraction of cultured or transplanted endothelial cells form new vessels. Moreover, it is unclear whether this results from a stochastic process or instead relates to certain endothelial cells having a greater angiogenic potential. This study investigated whether there exists a sub-population of cultured endothelial cells with enhanced angiogenic potency in vitro and in vivo. First, endothelial cells that participated in sprouting, and non-sprouting cells, were separately isolated from a 3D fibrin gel sprouting assay. Interestingly, the sprouting cells, when placed back into the same assay, displayed a sevenfold increase in the number of sprouts, as compared to control cells. Angiotensin-converting enzyme (CD143) was significantly down regulated on sprouting cells, as compared to regular endothelial cells. A subset of endothelial cells with low CD143 expression was then prospectively isolated from an endothelial cell culture. Finally, these cells were found to have greater potency in alleviating local ischemia, and restoring regional blood perfusion when transplanted into ischemic hindlimbs, as compared to unsorted endothelial cells. In summary, this study indicates that low expression of CD143 can be used as a biomarker to identify an endothelial cell sub-population that is more capable to drive neovascularization.
Koshy ST, Ferrante TC, Lewin SA, Mooney DJ. Injectable, porous, and cell-responsive gelatin cryogels. Biomaterials. 2014;35 (8) :2477-87.Abstract
The performance of biomaterials-based therapies can be hindered by complications associated with surgical implant, motivating the development of materials systems that allow minimally invasive introduction into the host. In this study, we created cell-adhesive and degradable gelatin scaffolds that could be injected through a conventional needle while maintaining a predefined geometry and architecture. These scaffolds supported attachment, proliferation, and survival of cells in vitro and could be degraded by recombinant matrix metalloproteinase-2 and -9. Prefabricated gelatin cryogels rapidly resumed their original shape when injected subcutaneously into mice and elicited only a minor host response following injection. Controlled release of granulocyte-macrophage colony-stimulating factor from gelatin cryogels resulted in complete infiltration of the scaffold by immune cells and promoted matrix metalloproteinase production leading to cell-mediated degradation of the cryogel matrix. These findings suggest that gelatin cryogels could serve as a cell-responsive platform for biomaterial-based therapy.
Madl CM, Mehta M, Duda GN, Heilshorn SC, Mooney DJ. Presentation of BMP-2 mimicking peptides in 3D hydrogels directs cell fate commitment in osteoblasts and mesenchymal stem cells. Biomacromolecules. 2014;15 (2) :445-55.Abstract
Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by (1)H NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.
Cezar CA, Kennedy SM, Mehta M, Weaver JC, Gu L, Vandenburgh H, Mooney DJ. Biphasic ferrogels for triggered drug and cell delivery. Adv Healthc Mater. 2014;3 (11) :1869-76.Abstract
Ferrogels are an attractive material for many biomedical applications due to their ability to deliver a wide variety of therapeutic drugs on-demand. However, typical ferrogels have yet to be optimized for use in cell-based therapies, as they possess limited ability to harbor and release viable cells. Previously, an active porous scaffold that exhibits large deformations and enhanced biological agent release under moderate magnetic fields has been demonstrated. Unfortunately, at small device sizes optimal for implantation (e.g., 2 mm thickness), these monophasic ferrogels no longer achieve significant deformation due to a reduced body force. A new biphasic ferrogel, containing an iron oxide gradient, capable of large deformations and triggered release even at small gel dimensions, is presented in this study. Biphasic ferrogels demonstrate increased porosity, enhanced mechanical properties, and potentially increased biocompatibility due to their reduced iron oxide content. With their ability to deliver drugs and cells on-demand, it is expected that these ferrogels will have wide utility in the fields of tissue engineering and regenerative medicine.
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.
2013
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.

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