Publications by authors named "Renny T Franceschi"

74 Publications

Role of Runx2 in prostate development and stem cell function.

Prostate 2021 Mar 7;81(4):231-241. Epub 2021 Jan 7.

Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.

Background: RUNX2, a critical transcription factor in bone development, is also expressed in prostate and breast where it has been linked to cancer progression and cancer stem cells. However, its role in normal prostate biology has not been previously examined.

Methods: Selective growth of murine prostate epithelium under non-adherent conditions was used to enrich for stem cells. Expression of runt domain transcription factors, stem cell and prostate marker messenger RNAs (mRNAs) was determined by quantitative reverse transcription polymerase chain reaction. Effects of Runx2 loss and gain-of-function on prostate epithelial cells were assessed using cells isolated from Runx2 mice transduced with Adeno-Cre or by Adeno-Runx2 transduction of wild type cells. Cellular distribution of RUNX2 and prostate-associated proteins was assessed using immunofluorescence microscopy. In vivo Runx2 knock out was achieved by tamoxifen treatment of Nkx3.1CreERT; Runx2 mice.

Results: Prostate epithelium-derived spheroids, which are enriched in stem cells, were shown to contain elevated levels of Runx2 mRNA. Spheroid formation required Runx2 since adenovirus-Cre mediated knockout of Runx2 in prostatic epithelial cells from Runx2 mice severely reduced spheroid formation and stem cell markers while Runx2 overexpression was stimulatory. In vivo, Runx2 was detected during early prostate development (E16.5) and in adult mice where it was present in basal and luminal cells of ventral and anterior lobes. Prostate-selective deletion of Runx2 in tamoxifen-treated Nkx3.1CreERT; Runx2 mice severely inhibited growth and maturation of tubules in the anterior prostate and reduced expression of stem cell markers and prostate-associated genes.

Conclusion: This study demonstrates an important role for Runx2 in prostate development that may be explained by actions in prostate epithelial stem cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.24099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856111PMC
March 2021

Spatiotemporal control of micromechanics and microstructure in acoustically-responsive scaffolds using acoustic droplet vaporization.

Soft Matter 2020 Jul;16(28):6501-6513

Department of Radiology, University of Michigan, Ann Arbor, MI, USA. and Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA and Applied Physics Program, University of Michigan, Ann Arbor, MI, USA.

Acoustically-responsive scaffolds (ARSs), which are composite fibrin hydrogels, have been used to deliver regenerative molecules. ARSs respond to ultrasound in an on-demand, spatiotemporally-controlled manner via a mechanism termed acoustic droplet vaporization (ADV). Here, we study the ADV-induced, time-dependent micromechanical and microstructural changes to the fibrin matrix in ARSs using confocal fluorescence microscopy as well as atomic force microscopy. ARSs, containing phase-shift double emulsion (PSDE, mean diameter: 6.3 μm), were exposed to focused ultrasound to generate ADV - the phase transitioning of the PSDE into gas bubbles. As a result of ADV-induced mechanical strain, localized restructuring of fibrin occurred at the bubble-fibrin interface, leading to formation of locally denser regions. ADV-generated bubbles significantly reduced fibrin pore size and quantity within the ARS. Two types of ADV-generated bubble responses were observed in ARSs: super-shelled spherical bubbles, with a growth rate of 31 μm per day in diameter, as well as fluid-filled macropores, possibly as a result of acoustically-driven microjetting. Due to the strain stiffening behavior of fibrin, ADV induced a 4-fold increase in stiffness in regions of the ARS proximal to the ADV-generated bubble versus distal regions. These results highlight that the mechanical and structural microenvironment within an ARS can be spatiotemporally modulated using ultrasound, which could be used to control cellular processes and further the understanding of ADV-triggered drug delivery for regenerative applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0sm00753fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377967PMC
July 2020

Spatially-directed cell migration in acoustically-responsive scaffolds through the controlled delivery of basic fibroblast growth factor.

Acta Biomater 2020 09 14;113:217-227. Epub 2020 Jun 14.

Department of Radiology, University of Michigan, Ann Arbor, MI, USA; Applied Physics Program, University of Michigan, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. Electronic address:

Hydrogels are commonly used in regenerative medicine for the delivery of growth factors (GFs). The spatial and temporal presentations of GFs are critical for directing regenerative processes, yet conventional hydrogels do not enable such control. We have developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), where release of a GF is non-invasively and spatiotemporally-controlled using focused ultrasound. The ARS consists of a fibrin matrix doped with a GF-loaded, phase-shift emulsion. The GF is released when the ARS is exposed to suprathreshold ultrasound via a mechanism termed acoustic droplet vaporization. In this study, we investigate how different spatial patterns of suprathreshold ultrasound can impact the biological response upon in vivo implantation of an ARS containing basic fibroblast growth factor (bFGF). ARSs were fabricated with either perfluorohexane (bFGF-C6-ARS) or perflurooctane (bFGF-C8-ARS) within the phase-shift emulsion. Ultrasound generated stable bubbles in bFGF-C6-ARS, which inhibited matrix compaction, whereas transiently stable bubbles were generated in bFGF-C8-ARS, which decreased in height by 44% within one day of implantation. The rate of bFGF release and distance of host cell migration were up to 6.8-fold and 8.1-fold greater, respectively, in bFGF-C8-ARS versus bFGF-C6-ARS. Ultrasound increased the formation of macropores within the fibrin matrix of bFGF-C8-ARS by 2.7-fold. These results demonstrate that spatially patterning suprathreshold ultrasound within bFGF-C8-ARS can be used to elicit a spatially-directed response from the host. Overall, these findings can be used in developing strategies to spatially pattern regenerative processes. STATEMENT OF SIGNIFICANCE: Hydrogels are commonly used in regenerative medicine for the delivery of growth factors (GFs). The spatial and temporal presentations of GFs are critical for directing regenerative processes, yet conventional hydrogels do not enable such control. We have developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), where GF release is non-invasively and spatiotemporally-controlled using focused ultrasound. The ARS consists of a fibrin matrix doped with a phase-shift emulsion loaded with GF, which is released when the ARS is exposed to ultrasound. In this in vivo study, we demonstrate that spatially patterning ultrasound within an ARS containing basic fibroblast growth factor (bFGF) can elicit a spatially-directed response from the host. Overall, these findings can be used in developing strategies to spatially pattern regenerative processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2020.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423759PMC
September 2020

Local delivery of bone morphogenetic protein-2 from near infrared-responsive hydrogels for bone tissue regeneration.

Biomaterials 2020 05 21;241:119909. Epub 2020 Feb 21.

Hospital Universitario La Paz-IdiPAZ, Paseo de La Castellana 261, Madrid, 28046, Spain; CIBER de Bioingenieria, Biomateriales y Nanomedicina, CIBER-BBN, Spain. Electronic address:

Achievement of spatiotemporal control of growth factors production remains a main goal in tissue engineering. In the present work, we combined inducible transgene expression and near infrared (NIR)-responsive hydrogels technologies to develop a therapeutic platform for bone regeneration. A heat-activated and dimerizer-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in hydrogels based on fibrin and plasmonic gold nanoparticles that transduced incident energy of an NIR laser into heat. In the presence of dimerizer, photoinduced mild hyperthermia induced the release of bioactive BMP-2 from NIR-responsive cell constructs. A critical size bone defect, created in calvaria of immunocompetent mice, was filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the heat-activated and dimerizer-dependent gene circuit. In animals that were treated with dimerizer, NIR irradiation of implants induced BMP-2 production in the bone lesion. Induction of NIR-responsive cell constructs conditionally expressing BMP-2 in bone defects resulted in the formation of new mineralized tissue, thus indicating the therapeutic potential of the technological platform.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2020.119909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263445PMC
May 2020

Controlled delivery of basic fibroblast growth factor (bFGF) using acoustic droplet vaporization stimulates endothelial network formation.

Acta Biomater 2019 10 9;97:409-419. Epub 2019 Aug 9.

Department of Radiology, University of Michigan Health System, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; Applied Physics Program, University of Michigan, Ann Arbor, MI, USA. Electronic address:

The challenge of translating pro-angiogenic growth factors for therapeutic purposes has stimulated a myriad of biomaterials-based, delivery approaches. Many techniques rely on incorporating a growth factor into a hydrogel. The kinetics of release can be tuned based on the physiochemical properties of the growth factor and scaffold. We have developed an acoustically-responsive scaffold (ARS), whereby release of a growth factor is non-invasively and spatiotemporally controlled in an on-demand manner using focused ultrasound. An ARS consists of a fibrin matrix doped with a growth factor-loaded, sonosensitive emulsion. In this study, we used an ARS to investigate the impact of basic fibroblast growth factor (bFGF) release on endothelial tubule formation. The co-culture model of angiogenic sprouting consisted of endothelial cell-coated microbeads and dispersed fibroblasts. bFGF release correlated with the acoustic pressure applied while sprout length correlated with both the volume of bFGF-loaded emulsion in the ARS and acoustic pressure. Minimal bFGF release and sprouting were observed in the absence of ultrasound exposure. Staggering the release of bFGF via multiple ultrasound exposures did not affect sprouting. Additionally, sprouting did not display a dependence on the distance between each microbead and the ARS. Overall, these results highlight the potential of using ultrasound to control regenerative processes via the controlled delivery of a growth factor. STATEMENT OF SIGNIFICANCE: Due to the ineffectiveness of conventional routes of administration, implantable hydrogels are often used as matrices to deliver growth factors (GFs). Spatial control of release is typically realized using anisotropic constructs while temporal control is obtained by modifying matrix properties and GF-scaffold interactions. In this study, we demonstrate how focused ultrasound can be used to non-invasively and spatiotemporally control release of basic fibroblast growth factor (bFGF), in an on-demand manner, from a composite hydrogel. The acoustically-responsive scaffold (ARS) consists of a bFGF-loaded, monodispersed double emulsion embedded within a fibrin matrix. We demonstrate how controlled release of bFGF can stimulate endothelial network formation. These results may be of interest to groups working on controlled release strategies for GFs, especially in the context of stimulating angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2019.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801030PMC
October 2019

Spatiotemporally-controlled transgene expression in hydroxyapatite-fibrin composite scaffolds using high intensity focused ultrasound.

Biomaterials 2019 02 13;194:14-24. Epub 2018 Dec 13.

Applied Physics Program, University of Michigan, Ann Arbor, MI, USA; Department of Radiology, University of Michigan Health System, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. Electronic address:

Conventional tissue engineering approaches rely on scaffold-based delivery of exogenous proteins, genes, and/or cells to stimulate regeneration via growth factor signaling. However, scaffold-based approaches do not allow active control of dose, timing, or spatial localization of a delivered growth factor once the scaffold is implanted, yet these are all crucial parameters in promoting tissue regeneration. To address this limitation, we developed a stable cell line containing a heat-activated and rapamycin-dependent gene expression system. In this study, we investigate how high intensity focused ultrasound (HIFU) can spatiotemporally control firefly luciferase (fLuc) transgene activity both in vitro and in vivo by the tightly controlled generation of hyperthermia. Cells were incorporated into composite scaffolds containing fibrin and hydroxyapatite particles, which yielded significant increases in acoustic attenuation and heating in response to HIFU compared to fibrin alone. Using 2.5 MHz HIFU, transgene activation was observed at acoustic intensities of 201 W/cm and higher. Transgene activation was spatially patterned in the scaffolds by rastering HIFU at speeds up to 0.15 mm/s. In an in vivo study, a 67-fold increase in fLuc activity was observed in scaffolds exposed to HIFU and rapamycin versus rapamycin only at 2 days post implantation. Repeated activation of transgene expression was also demonstrated 8 days after implantation. No differences in in vivo scaffold degradation or compaction were observed between +HIFU and -HIFU groups. These results highlight the potential utility of using this heat-activated and rapamycin-dependent gene expression system in combination with HIFU for the controlled stimulation of tissue regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2018.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339574PMC
February 2019

Sequential Payload Release from Acoustically-Responsive Scaffolds Using Focused Ultrasound.

Ultrasound Med Biol 2018 11 2;44(11):2323-2335. Epub 2018 Aug 2.

Applied Physics Program, University of Michigan, Ann Arbor, Michigan, USA; Department of Radiology, University of Michigan Health System, Ann Arbor, Michigan, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA.

Regenerative processes, such as angiogenesis and osteogenesis, often require multiple growth factors with distinct spatiotemporal patterns and expression sequences. Within tissue engineering, hydrogel scaffolds are commonly used for exogenous growth factor delivery. However, direct incorporation of growth factors within conventional hydrogels does not afford spatiotemporally controlled delivery because release is governed by passive mechanisms that cannot be actively controlled after the scaffold is implanted. We have developed acoustically-responsive scaffolds (ARSs), which are fibrin scaffolds doped with payload-containing, sonosensitive emulsions. Payload release from ARSs can be controlled non-invasively and on demand using focused, megahertz-range ultrasound. In the in vitro study described here, we developed and characterized ARSs that enable sequential release of two surrogate, fluorescent payloads using consecutive ultrasound exposures at different acoustic pressures. ARSs were generated with various combinations and volume fractions of perfluoropentane, perfluorohexane, and perfluoroheptane emulsions. Acoustic droplet vaporization and inertial cavitation thresholds correlated with the boiling point/molecular weight of the perfluorocarbon while payload release correlated inversely. Payload release was longitudinally measured and observed to follow a sigmoidal trend versus acoustic pressure. Perfluoropentane and perfluorohexane emulsions were stabilized when incorporated into ARSs with perfluoroheptane emulsion. These results highlight the potential of using ARSs for sequential, dual-payload release for tissue regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ultrasmedbio.2018.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441330PMC
November 2018

Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis.

Arterioscler Thromb Vasc Biol 2018 08;38(8):1878-1889

From the Department of Laboratory Medicine and Pathobiology (M.L., M.H.W., A.S.R., D.N., N.S., G.H., M.P.B.).

Objective- Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. Approach and Results- Ddr1 ; Ldlr (single knock-out) and Ddr1 ; Ldlr (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1 and Ddr1 mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1 vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. Conclusions- These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/ATVBAHA.118.311238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441610PMC
August 2018

Skeletal Stem Cells: Origins, Functions and Uncertainties.

Curr Mol Biol Rep 2017 Dec 19;3(4):236-246. Epub 2017 Oct 19.

Departments of Periodontics and Oral Medicine, University of Michigan School of Medicine, Ann Arbor, MI 48109-0600.

The development and maintenance of the skeleton requires a steady source of skeletal progenitors to provide the osteoblasts and chondrocytes necessary for bone and cartilage growth and development. The current model for skeletal stem cells (SSCs) posits that SSC/progenitor cells are present in bone marrow (BM) and other osteogenic sites such as cranial sutures where they undergo self-renewal and differentiation to give rise to the main skeletal tissues. SSCs hold great promise for understanding skeletal biology and genetic diseases of bone as well as for the advancement of bone tissue engineering and regenerative medicine strategies. In the past few years, a considerable effort has been devoted to identifying and purifying skeletal stem cells and determining their contribution to bone formation and homeostasis. Here, we review recent progress in this area with particular emphasis on the discovery of specific SSC markers, their use in tracking the progression of cell populations along specific lineages and the regulation of SSCs in both the appendicular and cranial skeleton.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40610-017-0075-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802417PMC
December 2017

Genetic inhibition of PPARγ S112 phosphorylation reduces bone formation and stimulates marrow adipogenesis.

Bone 2018 02 26;107:1-9. Epub 2017 Oct 26.

Periodontics & Oral Medicine University of Michigan School of Dentistry, University of Michigan School of Medicine, Ann Arbor, MI, United States; Biological Chemistry, University of Michigan School of Medicine, Ann Arbor, MI, United States. Electronic address:

A common feature of many skeletal diseases is the accumulation of marrow fat. A reciprocal relationship exists between osteogenesis and adipogenesis in bone marrow that is mediated by the relative activity of PPARγ and RUNX2 transcription factors. The ERK/MAPK pathway is an important inducer of MSC differentiation to osteoblasts and an inhibitor of adipogenesis that functions by phosphorylating RUNX2 and PPARγ. To begin to assess the importance of this regulation in vivo, we examined the consequences of blocking one arm of this pathway, PPARγ S112 phosphorylation, by evaluating the bone phenotype of PPARγ S112A mutant mice. This mutation prevents MAPK phosphorylation and inhibition of PPARγ transcriptional activity. Both male and female PPARγ S112A mice had decreased tibial and vertebral BV/TV and decreased trabecular bone relative to wild type littermates. These results were explained by a decrease in bone formation and osteoblast activity in the absence of changes in resorption. In contrast, marrow adipose tissue, adipocyte markers and serum adiponectin were all dramatically increased. Bone marrow stromal cells isolated from PPARγ S112A mice had elevated PPARγ and preferentially differentiated to adipocytes while total and phosphorylated RUNX2 and osteoblastogenesis were inhibited, indicating that the PPARγ S112A mutation affects bone in a cell autonomous manner. Changes in osteoblast/adipocyte lineage allocation in MSC cultures were also seen where CFU-OBs were reduced with a parallel increase in CFU-AD. This study emphasizes the importance of PPARγ phosphorylation in controlling bone mass and marrow adiposity and demonstrates how a regulatory mutation in PPARγ previously associated with peripheral fat metabolism can have broader effects on bone homeostasis that may in turn affect whole body energy metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bone.2017.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743762PMC
February 2018

Control of the Osteoblast Lineage by Mitogen-Activated Protein Kinase Signaling.

Curr Mol Biol Rep 2017 Jun 25;3(2):122-132. Epub 2017 Apr 25.

Departments of Periodontics and Oral Medicine, University of Michigan School of Dentistry and Department of Biological Chemistry, University of Michigan School of Medicine, Ann Arbor, MI 48109-1078.

Purpose Of The Review: This review will provide a timely assessment of MAP kinase actions in bone development and homeostasis with particular emphasis on transcriptional control of the osteoblast lineage.

Recent Findings: ERK and p38 MAP kinases function as transducers of signals initiated by the extracellular matrix, mechanical loading, TGF-β, BMPs and FGF2. MAPK signals may also affect and/or interact with other important pathways such as WNT and HIPPO. ERK and p38 MAP kinase pathways phosphorylate specific osteogenic transcription factors including RUNX2, Osterix, ATF4 and DLX5. For RUNX2, phosphorylation at specific serine residues initiates epigenetic changes in chromatin necessary for decondensation and increased transcription. MAPK also suppresses marrow adipogenesis by phosphorylating and inhibiting PPARγ, which may explain the well-known relationship between reduced skeletal loading and marrow fat accumulation.

Summary: MAPKs transduce signals from the extracellular environment to the nucleus allowing bone cells to respond to changes in hormonal/growth factor signaling and mechanical loading thereby optimizing bone structure to meet physiological and mechanical needs of the body.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40610-017-0059-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647885PMC
June 2017

Controlled release of basic fibroblast growth factor for angiogenesis using acoustically-responsive scaffolds.

Biomaterials 2017 Sep 9;140:26-36. Epub 2017 Jun 9.

Applied Physics Program, University of Michigan, Ann Arbor, MI, USA; Department of Radiology, University of Michigan Health System, Ann Arbor, MI, USA.

The clinical translation of pro-angiogenic growth factors for treatment of vascular disease has remained a challenge due to safety and efficacy concerns. Various approaches have been used to design spatiotemporally-controlled delivery systems for growth factors in order to recapitulate aspects of endogenous signaling and thus assist in translation. We have developed acoustically-responsive scaffolds (ARSs), which are fibrin scaffolds doped with a payload-containing, sonosensitive emulsion. Payload release can be controlled non-invasively and in an on-demand manner using focused, megahertz-range ultrasound (US). In this study, we investigate the in vitro and in vivo release from ARSs containing basic fibroblast growth factor (bFGF) encapsulated in monodispersed emulsions. Emulsions were generated in a two-step process utilizing a microfluidic device with a flow focusing geometry. At 2.5 MHz, controlled release of bFGF was observed for US pressures above 2.2 ± 0.2 MPa peak rarefactional pressure. Superthreshold US yielded a 12.6-fold increase in bFGF release in vitro. The bioactivity of the released bFGF was also characterized. When implanted subcutaneously in mice, ARSs exposed to superthreshold US displayed up to 3.3-fold and 1.7-fold greater perfusion and blood vessel density, respectively, than ARSs without US exposure. Scaffold degradation was not impacted by US. These results highlight the utility of ARSs in both basic and applied studies of therapeutic angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2017.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537721PMC
September 2017

Mesenchymal Stem Cell-Induced DDR2 Mediates Stromal-Breast Cancer Interactions and Metastasis Growth.

Cell Rep 2017 01;18(5):1215-1228

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Increased collagen deposition by breast cancer (BC)-associated mesenchymal stem/multipotent stromal cells (MSC) promotes metastasis, but the mechanisms are unknown. Here, we report that the collagen receptor discoidin domain receptor 2 (DDR2) is essential for stromal-BC communication. In human BC metastasis, DDR2 is concordantly upregulated in metastatic cancer and multipotent mesenchymal stromal cells. In MSCs isolated from human BC metastasis, DDR2 maintains a fibroblastic phenotype with collagen deposition and induces pathological activation of DDR2 signaling in BC cells. Loss of DDR2 in MSCs impairs their ability to promote DDR2 phosphorylation in BC cells, as well as BC cell alignment, migration, and metastasis. Female ddr2-deficient mice homozygous for the slie mutation show inefficient spontaneous BC metastasis. These results point to a role for mesenchymal stem cell DDR2 in metastasis and suggest a therapeutic approach for metastatic BC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.12.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5332146PMC
January 2017

Discoidin Receptor 2 Controls Bone Formation and Marrow Adipogenesis.

J Bone Miner Res 2016 12 31;31(12):2193-2203. Epub 2016 Oct 31.

Departments of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI, USA.

Cell-extracellular matrix (ECM) interactions play major roles in controlling progenitor cell fate and differentiation. The receptor tyrosine kinase, discoidin domain receptor 2 (DDR2), is an important mediator of interactions between cells and fibrillar collagens. DDR2 signals through both ERK1/2 and p38 MAP kinase, which stimulate osteoblast differentiation and bone formation. Here we show that DDR2 is critical for skeletal development and differentiation of marrow progenitor cells to osteoblasts while suppressing marrow adipogenesis. Smallie mice (Ddr2 ), which contain a nonfunctional Ddr2 allele, have multiple skeletal defects. A progressive decrease in tibial trabecular bone volume/total volume (BV/TV) was observed when wild-type (WT), Ddr2 , and Ddr2 mice were compared. These changes were associated with reduced trabecular number (Tb.N) and trabecular thickness (Tb.Th) and increased trabecular spacing (Tb.Sp) in both males and females, but reduced cortical thickness only in Ddr2 females. Bone changes were attributed to decreased bone formation rather than increased osteoclast activity. Significantly, marrow fat and adipocyte-specific mRNA expression were significantly elevated in Ddr2 animals. Additional skeletal defects include widened calvarial sutures and reduced vertebral trabecular bone. To examine the role of DDR2 signaling in cell differentiation, bone marrow stromal cells (BMSCs) were grown under osteogenic and adipogenic conditions. Ddr2 cells exhibited defective osteoblast differentiation and accelerated adipogenesis. Changes in differentiation were related to activity of runt-related transcription factor 2 (RUNX2) and PPARγ, transcription factors that are both controlled by MAPK-dependent phosphorylation. Specifically, the defective osteoblast differentiation in calvarial cells from Ddr2 mice was associated with reduced ERK/MAP kinase and RUNX2-S319 phosphorylation and could be rescued with a constitutively active phosphomimetic RUNX2 mutant. Also, DDR2 was shown to increase RUNX2-S319 phosphorylation and transcriptional activity while also increasing PPARγ-S112 phosphorylation, but reducing its activity. DDR2 is, therefore, important for maintenance of osteoblast activity and suppression of marrow adipogenesis in vivo and these actions are related to changes in MAPK-dependent RUNX2 and PPARγ phosphorylation. © 2016 American Society for Bone and Mineral Research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.2893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135576PMC
December 2016

Protein Phosphatase PP5 Controls Bone Mass and the Negative Effects of Rosiglitazone on Bone through Reciprocal Regulation of PPARγ (Peroxisome Proliferator-activated Receptor γ) and RUNX2 (Runt-related Transcription Factor 2).

J Biol Chem 2016 Nov 29;291(47):24475-24486. Epub 2016 Sep 29.

From the Department of Orthopaedic Surgery,; the Center for Diabetes and Endocrine Research, University of Toledo Health Science Campus, Toledo, Ohio 43614 and; Physiology and Pharmacology, and. Electronic address:

Peroxisome proliferator-activated receptor γ (PPARγ) and runt-related transcription factor 2 (RUNX2) are key regulators of mesenchymal stem cell (MSC) differentiation toward adipocytes and osteoblasts, respectively. Post-translational modifications of these factors determine their activities. Dephosphorylation of PPARγ at Ser-112 is required for its adipocytic activity, whereas phosphorylation of RUNX2 at serine 319 (Ser-319) promotes its osteoblastic activity. Here we show that protein phosphatase 5 (PP5) reciprocally regulates each receptor by targeting each serine. Mice deficient in PP5 phosphatase have increased osteoblast numbers and high bone formation, which results in high bone mass in the appendicular and axial skeleton. This is associated with a substantial decrease in lipid-containing marrow adipocytes. Indeed, in the absence of PP5 the MSC lineage allocation is skewed toward osteoblasts and away from lipid accumulating adipocytes, although an increase in beige adipocyte gene expression is observed. In the presence of rosiglitazone, PP5 translocates to the nucleus, binds to PPARγ and RUNX2, and dephosphorylates both factors, resulting in activation of PPARγ adipocytic and suppression of RUNX2 osteoblastic activities. Moreover, shRNA knockdown of PP5 results in cells refractory to rosiglitazone treatment. Lastly, mice deficient in PP5 are resistant to the negative effects of rosiglitazone on bone, which in wild type animals causes a 50% decrease in trabecular bone mass. In conclusion, PP5 is a unique phosphatase reciprocally regulating PPARγ and RUNX2 activities in marrow MSC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.752493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114402PMC
November 2016

In vitro and in vivo assessment of controlled release and degradation of acoustically responsive scaffolds.

Acta Biomater 2016 12 27;46:221-233. Epub 2016 Sep 27.

Applied Physics Program, University of Michigan, Ann Arbor, MI, USA; Department of Radiology, University of Michigan Health System, Ann Arbor, MI, USA.

Spatiotemporally controlled release of growth factors (GFs) is critical for regenerative processes such as angiogenesis. A common strategy is to encapsulate the GF within hydrogels, with release being controlled via diffusion and/or gel degradation (i.e., hydrolysis and/or proteolysis). However, simple encapsulation strategies do not provide spatial or temporal control of GF delivery, especially non-invasive, on-demand controlled release post implantation. We previously demonstrated that fibrin hydrogels, which are widely used in tissue engineering and GF delivery applications, can be doped with perfluorocarbon emulsion, thus yielding an acoustically responsive scaffold (ARS) that can be modulated with focused ultrasound, specifically via a mechanism termed acoustic droplet vaporization. This study investigates the impact of ARS and ultrasound properties on controlled release of a surrogate payload (i.e., fluorescently-labeled dextran) and fibrin degradation in vitro and in vivo. Ultrasound exposure (2.5MHz, peak rarefactional pressure: 8MPa, spatial peak time average intensity: 86.4mW/cm), generated up to 7.7 and 21.7-fold increases in dextran release from the ARSs in vitro and in vivo, respectively. Ultrasound also induced morphological changes in the ARS. Surprisingly, up to 2.9-fold greater blood vessel density was observed in ARSs compared to fibrin when implanted subcutaneously, even without delivery of pro-angiogenic GFs. The results demonstrate the potential utility of ARSs in generating controlled release for tissue regeneration.

Statement Of Significance: Simple encapsulation of a molecular payload within a conventional hydrogel scaffold does not provide spatial or temporal control of payload release. Yet, spatiotemporally controlled release of bioactive payloads is critical for tissue regeneration, which often utilizes hydrogel scaffolds to facilitate processes such as angiogenesis. This work investigates the design and performance (both in vitro and in vivo) of hydrogel scaffolds where release of a fluorescent payload is non-invasively and spatiotemporally-controlled using focused ultrasound. We also quantitatively characterize the degradation and vascularization of the scaffolds. Our results may be of interest to groups working on controlled release strategies for implants, especially within the field of tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2016.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097683PMC
December 2016

DNMT1 Regulates Epithelial-Mesenchymal Transition and Cancer Stem Cells, Which Promotes Prostate Cancer Metastasis.

Neoplasia 2016 09;18(9):553-66

Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI 48109, USA. Electronic address:

Cancer metastasis is a multistep process associated with the induction of an epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs). Although significant progress has been made in understanding the molecular mechanisms regulating EMT and the CSC phenotype, little is known of how these processes are regulated by epigenetics. Here we demonstrate that reduced expression of DNA methyltransferase 1 (DNMT1) plays an important role in the induction of EMT and the CSC phenotype by prostate cancer (PCa) cells, with enhanced tumorigenesis and metastasis. First, we observed that reduction of DNMT1 by 5-azacitidine (5-Aza) promotes EMT induction as well as CSCs and sphere formation in vitro. Reduced expression of DNMT1 significantly increased PCa migratory potential. We showed that the increase of EMT and CSC activities by reduction of DNMT1 is associated with the increase of protein kinase C. Furthermore, we confirmed that silencing DNMT1 is correlated with enhancement of the induction of EMT and the CSC phenotype in PCa cells. Additionally, chromatin immunoprecipitation assay reveals that reduction of DNMT1 promotes the suppression of H3K9me3 and H3K27me3 on the Zeb2 and KLF4 promoter region in PCa cells. Critically, we found in an animal model that significant tumor growth and more disseminated tumor cells in most osseous tissues were observed following injection of 5-Aza pretreated-PCa cells compared with vehicle-pretreated PCa cells. Our results suggest that epigenetic alteration of histone demethylation regulated by reduction of DNMT1 may control induction of EMT and the CSC phenotype, which facilitates tumorigenesis in PCa cells and has important therapeutic implications in targeting epigenetic regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neo.2016.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031902PMC
September 2016

MAP Kinase-Dependent RUNX2 Phosphorylation Is Necessary for Epigenetic Modification of Chromatin During Osteoblast Differentiation.

J Cell Physiol 2017 Sep 10;232(9):2427-2435. Epub 2017 Apr 10.

Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan.

RUNX2, an essential transcription factor for osteoblast differentiation and bone formation is activated by ERK/MAP kinase-dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2-binding regions of Bglap2 and Ibsp. Growth of MC3T3-E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin-bound P-ERK, P-RUNX2, p300, and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di-methylation, another gene activation-associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono-, di-, and tri-methylation in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation-dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2-deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non-phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin-associated P-ERK, p300, and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P-RUNX2-dependent process involving epigenetic modifications of specific promoter regions. J. Cell. Physiol. 232: 2427-2435, 2017. © 2016 Wiley Periodicals, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.25517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303561PMC
September 2017

In Situ Transfection by Controlled Release of Lipoplexes Using Acoustic Droplet Vaporization.

Adv Healthc Mater 2016 07 18;5(14):1764-74. Epub 2016 May 18.

Department of Radiology, University of Michigan Health System, Ann Arbor, MI, 48109, USA.

Localized delivery of nucleic acids to target sites (e.g., diseased tissue) is critical for safe and efficacious gene therapy. An ultrasound-based technique termed acoustic droplet vaporization (ADV) has been used to spatiotemporally control the release of therapeutic small molecules and proteins contained within sonosensitive emulsions. Here, ADV is used to control the release of lipoplex-containing plasmid DNA encoding an enhanced green fluorescent protein reporter-from a sonosensitive emulsion. Focused ultrasound (3.5 MHz, mechanical index (MI) ≥ 1.5) generates robust release of fluorescein (i.e., surrogate payload) and lipoplex from the emulsion. In situ release of the lipoplex from the emulsion using ADV (MI = 1.5, 30 cycles) yields a 55% release efficiency, resulting in 43% transfection efficiency and 95% viability with C3H/10T1/2 cells. Without exposure to ultrasound, the release and transfection efficiencies are 5% and 7%, respectively, with 99% viability. Lipoplex released by ADV retains its bioactivity while the ADV process does not yield any measureable sonoporative enhancement of transfection. Co-encapsulation of Ficoll PM 400 within the lipoplex-loaded emulsion, and its subsequent release using ADV, yield higher transfection efficiency than the lipoplex alone. The results demonstrate that ADV can have utility in the spatiotemporal control of gene delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adhm.201600008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956527PMC
July 2016

Use of Hydroxyapatite Doping to Enhance Responsiveness of Heat-Inducible Gene Switches to Focused Ultrasound.

Ultrasound Med Biol 2016 Mar 19;42(3):824-30. Epub 2015 Dec 19.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; School of Dentistry, University of Michigan, Ann Arbor, MI, USA; Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA.

Recently, we demonstrated that ultrasound-based hyperthermia can activate cells containing a heat-activated and ligand-inducible gene switch in a spatio-temporally controlled manner. These engineered cells can be incorporated into hydrogel scaffolds (e.g., fibrin) for in vivo implantation, where ultrasound can be used to non-invasively pattern transgene expression. Due to their high water content, the acoustic attenuation of fibrin scaffolds is low. Thus, long ultrasound exposures and high acoustic intensities are needed to generate sufficient hyperthermia for gene activation. Here, we demonstrate that the attenuation of fibrin scaffolds and the resulting hyperthermia achievable with ultrasound can be increased significantly by doping the fibrin with hydroxyapatite (HA) nanopowder. The attenuation of a 1% (w/v) fibrin scaffold with 5% (w/v) HA was similar to soft tissue. Transgene activation of cells harboring the gene switch occurred at lower acoustic intensities and shorter exposures when the cells were encapsulated in HA-doped fibrin scaffolds versus undoped scaffolds. Inclusion of HA in the fibrin scaffold did not affect the viability of the encapsulated cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ultrasmedbio.2015.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744111PMC
March 2016

Design and Characterization of Fibrin-Based Acoustically Responsive Scaffolds for Tissue Engineering Applications.

Ultrasound Med Biol 2016 Jan 30;42(1):257-71. Epub 2015 Oct 30.

Applied Physics Program, University of Michigan, Ann Arbor, Michigan, USA; Department of Radiology, University of Michigan Health System, Ann Arbor, Michigan, USA.

Hydrogel scaffolds are used in tissue engineering as a delivery vehicle for regenerative growth factors. Spatiotemporal patterns of growth factor signaling are critical for tissue regeneration, yet most scaffolds afford limited control of growth factor release, especially after implantation. We previously found that acoustic droplet vaporization can control growth factor release from a fibrin scaffold doped with a perfluorocarbon emulsion. This study investigates properties of the acoustically responsive scaffold (ARS) critical for further translation. At 2.5 MHz, acoustic droplet vaporization and inertial cavitation thresholds ranged from 1.5 to 3.0 MPa and from 2.0 to 7.0 MPa peak rarefactional pressure, respectively, for ARSs of varying composition. Viability of C3H/10T1/2 cells, encapsulated in the ARS, did not decrease significantly for pressures below 4 MPa. ARSs with perfluorohexane emulsions displayed higher stability versus those with perfluoropentane emulsions, while surrogate payload release was minimal without ultrasound. These results enable the selection of ARS compositions and acoustic parameters needed for optimized spatiotemporally controlled release.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ultrasmedbio.2015.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666743PMC
January 2016

Reciprocal Control of Osteogenic and Adipogenic Differentiation by ERK/MAP Kinase Phosphorylation of Runx2 and PPARγ Transcription Factors.

J Cell Physiol 2016 Mar;231(3):587-96

Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, Michigan.

In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPARγ, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPARγ. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPARγ phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPARγ-dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant-negative suppression of activity had the opposite effect. In rescue experiments using Runx2(-/-) mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation-deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation-resistant S112A mutant PPARγ strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg(-/-) MEFs, while S112E mutant PPARγ was less active. Competition between RUNX2 and PPARγ was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPARγ phosphorylation in the control of osteoblast and adipocyte lineages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.25102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745986PMC
March 2016

Temporal and spatial patterning of transgene expression by near-infrared irradiation.

Biomaterials 2014 Sep 21;35(28):8134-8143. Epub 2014 Jun 21.

Hospital Universitario La Paz-IdiPAZ, 28046 Madrid, Spain.

We investigated whether near-infrared (NIR) light could be employed for patterning transgene expression in plasmonic cell constructs. Hollow gold nanoparticles with a plasmon surface band absorption peaking at ∼750 nm, a wavelength within the so called "tissue optical window", were used as fillers in fibrin-based hydrogels. These composites, which efficiently transduce NIR photon energy into heat, were loaded with genetically-modified cells that harbor a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation in the presence of ligand triggered 3-dimensional patterns of transgene expression faithfully matching the illuminated areas of plasmonic cell constructs. This non-invasive technology was proven useful for remotely controlling in vivo the spatiotemporal bioavailability of transgenic vascular endothelial growth factor. The combination of spatial control by means of NIR irradiation along with safe and timed transgene induction presents a high application potential for engineering tissues in regenerative medicine scenarios.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2014.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4101380PMC
September 2014

Patterning expression of regenerative growth factors using high intensity focused ultrasound.

Tissue Eng Part C Methods 2014 Oct 11;20(10):769-79. Epub 2014 Mar 11.

1 Department of Periodontics and Oral Medicine, Center for Craniofacial Regeneration, University of Michigan School of Dentistry , Ann Arbor, Michigan.

Temporal and spatial control of growth factor gradients is critical for tissue patterning and differentiation. Reinitiation of this developmental program is also required for regeneration of tissues during wound healing and tissue regeneration. Devising methods for reconstituting growth factor gradients remains a central challenge in regenerative medicine. In the current study we develop a novel gene therapy approach for temporal and spatial control of two important growth factors in bone regeneration, vascular endothelial growth factor, and bone morphogenetic protein 2, which involves application of high intensity focused ultrasound to cells engineered with a heat-activated- and ligand-inducible gene switch. Induction of transgene expression was tightly localized within cell-scaffold constructs to subvolumes of ∼30 mm³, and the amplitude and projected area of transgene expression was tuned by the intensity and duration of ultrasound exposure. Conditions for ultrasound-activated transgene expression resulted in minimal cytotoxicity and scaffold damage. Localized regions of growth factor expression also established gradients in signaling activity, suggesting that patterns of growth factor expression generated by this method will have utility in basic and applied studies on tissue development and regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEC.2013.0518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4186636PMC
October 2014

Crystallinity and compositional changes in carbonated apatites: Evidence from P solid-state NMR, Raman, and AFM analysis.

J Solid State Chem 2013 Oct;206

Department of Chemistry and University of Michigan, Ann Arbor, MI 48109-1055.

Solid-state (magic-angle spinning) NMR spectroscopy is a useful tool for obtaining structural information on bone organic and mineral components and synthetic model minerals at the atomic-level. Raman and P NMR spectral parameters were investigated in a series of synthetic B-type carbonated apatites (CAps). Inverse P NMR linewidth and inverse Raman PO ν bandwidth were both correlated with powder XRD crystallinity over the 0.3-10.3 wt% CO range investigated. Comparison with bone powder crystallinities showed agreement with values predicted by NMR and Raman calibration curves. Carbonate content was divided into two domains by the P NMR chemical shift frequency and the Raman phosphate ν band position. These parameters remain stable except for an abrupt transition at 6.5 wt% carbonate, a composition which corresponds to an average of one carbonate per unit cell. This near-binary distribution of spectroscopic properties was also found in AFM-measured particle sizes and Ca/P molar ratios by elemental analysis. We propose that this transition differentiates between two charge-balancing ion-loss mechanisms as measured by Ca/P ratios. These results define a criterion for spectroscopic characterization of B-type carbonate substitution in apatitic minerals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jssc.2013.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835554PMC
October 2013

Acoustic droplet-hydrogel composites for spatial and temporal control of growth factor delivery and scaffold stiffness.

Acta Biomater 2013 Jul 25;9(7):7399-409. Epub 2013 Mar 25.

Department of Radiology, University of Michigan Health System, Ann Arbor, MI, USA.

Wound healing is regulated by temporally and spatially restricted patterns of growth factor signaling, but there are few delivery vehicles capable of the "on-demand" release necessary for recapitulating these patterns. Recently we described a perfluorocarbon double emulsion that selectively releases a protein payload upon exposure to ultrasound through a process known as acoustic droplet vaporization (ADV). In this study, we describe a delivery system composed of fibrin hydrogels doped with growth factor-loaded double emulsion for applications in tissue regeneration. Release of immunoreactive basic fibroblast growth factor (bFGF) from the composites increased up to 5-fold following ADV and delayed release was achieved by delaying exposure to ultrasound. Releasates of ultrasound-treated materials significantly increased the proliferation of endothelial cells compared to sham controls, indicating that the released bFGF was bioactive. ADV also triggered changes in the ultrastructure and mechanical properties of the fibrin as bubble formation and consolidation of the fibrin in ultrasound-treated composites were accompanied by up to a 22-fold increase in shear stiffness. ADV did not reduce the viability of cells suspended in composite scaffolds. These results demonstrate that an acoustic droplet-hydrogel composite could have broad utility in promoting wound healing through on-demand control of growth factor release and/or scaffold architecture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2013.03.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746980PMC
July 2013

Spatiotemporal control of vascular endothelial growth factor expression using a heat-shock-activated, rapamycin-dependent gene switch.

Hum Gene Ther Methods 2013 Jun 6;24(3):160-70. Epub 2013 May 6.

Hospital Universitario La Paz-IdiPAZ, Madrid 28046, Spain.

A major challenge in regenerative medicine is to develop methods for delivering growth and differentiation factors in specific spatial and temporal patterns, thereby mimicking the natural processes of development and tissue repair. Heat shock (HS)-inducible gene expression systems can respond to spatial information provided by localized heating, but are by themselves incapable of sustained expression. Conversely, gene switches activated by small molecules provide tight temporal control and sustained expression, but lack mechanisms for spatial targeting. Here we combine the advantages of HS and ligand-activated systems by developing a novel rapamycin-regulated, HS-inducible gene switch that provides spatial and temporal control and sustained expression of transgenes such as firefly luciferase and vascular endothelial growth factor (VEGF). This gene circuit exhibits very low background in the uninduced state and can be repeatedly activated up to 1 month. Furthermore, dual regulation of VEGF induction in vivo is shown to stimulate localized vascularization, thereby providing a route for temporal and spatial control of angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hgtb.2013.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732131PMC
June 2013

Tracking circadian rhythms of bone mineral deposition in murine calvarial organ cultures.

J Bone Miner Res 2013 Aug;28(8):1846-54

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

Osteoblasts, which orchestrate the deposition of small apatite crystals through the expression of nucleating proteins, have been shown to also express clock genes associated with the circadian signaling pathway. We hypothesized that protein-mediated bone mineralization may be linked to circadian oscillator mechanisms functioning in peripheral bone tissue. In this study, Per1 expression in ex vivo neonatal murine calvaria organ cultures was monitored for 6 days using a Per1-luciferase transgene as a bioluminescent indicator of clock function. Fluctuations in Per1 expression had a period of 25 ± 4 hours (n = 14) with early expression at CT09:59 ± 03:37 (CT = circadian time). We also established the kinetics of mineral deposition in developing bone by using noninvasive Raman microscopy to track mineral accumulation in calvarial tissue. The content and quality of newly deposited mineral was continually examined at the interparietal bone/fontanel boundary for a period of 6 days with 1-hour temporal resolution. Using this approach, mineralization over time exhibited bursts of mineral deposition followed by little or no deposition, which was recurrent with a periodicity of 26.8 ± 9.6 hours. As many as six near-daily mineralization events were observed in the calvaria before deposition ceased. Earliest mineralization events occurred at CT16:51 ± 03:45, which is 6 hours behind Per1 expression. These findings are consistent with the hypothesis that mineralization in developing bone tissue is regulated by a local circadian oscillator mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.1924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720727PMC
August 2013