Publications

2008
Wu X, Yu G, Parks H, Hebert T, Goh BC, Dietrich MA, Pelled G, Izadpanah R, Gazit D, Bunnell BA, Gimble JM. Circadian mechanisms in murine and human bone marrow mesenchymal stem cells following dexamethasone exposure [Internet]. Bone 2008;42(5):861-70. Publisher's VersionAbstract
A core group of regulatory factors control circadian rhythms in mammalian cells. While the suprachiasmatic nucleus in the brain serves as the central core circadian oscillator, circadian clocks also exist within peripheral tissues and cells. A growing body of evidence has demonstrated that >20% of expressed mRNAs in bone and adipose tissues oscillate in a circadian manner. The current manuscript reports evidence of the core circadian transcriptional apparatus within primary cultures of murine and human bone marrow-derived mesenchymal stem cells (BMSCs). Exposure of confluent, quiescent BMSCs to dexamethasone synchronized the oscillating expression of the mRNAs encoding the albumin D binding protein (dbp), brain-muscle arnt-like 1 (bmal1), period 3 (per3), rev-erb alpha (Rev A), and rev-erb beta (Rev B). The genes displayed a mean oscillatory period of 22.2 to 24.3 h. The acrophase or peak expression of mRNAs encoding "positive" (bmal1) and "negative" (per3) components of the circadian regulatory apparatus were out of phase with each other by approximately 8-12 h, consistent with in vivo observations. In vivo, phosphyrylation by glycogen synthase kinase 3beta (GSK3beta) is known to regulate the turnover of per3 and components of the core circadian regulatory apparatus. In vitro addition of lithium chloride, a GSK3beta inhibitor, significantly shifted the acrophase of all genes by 4.2-4.7 h oscillation in BMSCs; however, only the male murine BMSCs displayed a significant increase in the length of the period of oscillation. We conclude that human and murine BMSCs represent a valid in vitro model for the analysis of circadian mechanisms in bone metabolism and stem cell biology.
Steinhardt Y, Aslan H, Regev E, Zilberman Y, Kallai I, Gazit D, Gazit Z. Maxillofacial-derived stem cells regenerate critical mandibular bone defect [Internet]. Tissue Eng Part A 2008;14(11):1763-73. Publisher's VersionAbstract
Stem cell-based bone tissue regeneration in the maxillofacial complex is a clinical necessity. Genetic engineering of mesenchymal stem cells (MSCs) to follow specific differentiation pathways may enhance the ability of these cells to regenerate and increase their clinical relevance. MSCs isolated from maxillofacial bone marrow (BM) are good candidates for tissue regeneration at sites of damage to the maxillofacial complex. In this study, we hypothesized that MSCs isolated from the maxillofacial complex can be engineered to overexpress the bone morphogenetic protein-2 gene and induce bone tissue regeneration in vivo. To demonstrate that the cells isolated from the maxillofacial complex were indeed MSCs, we performed a flow cytometry analysis, which revealed a high expression of mesenchyme-related markers and an absence of non-mesenchyme-related markers. In vitro, the MSCs were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Gene delivery of the osteogenic gene BMP2 via an adenoviral vector revealed high expression levels of BMP2 protein that induced osteogenic differentiation of these cells in vitro and induced bone formation in an ectopic site in vivo. In addition, implantation of genetically engineered maxillofacial BM-derived MSCs into a mandibular defect led to regeneration of tissue at the site of the defect; this was confirmed by performing micro-computed tomography analysis. Histological analysis of the mandibles revealed osteogenic differentiation of implanted cells as well as bone tissue regeneration. We conclude that maxillofacial BM-derived MSCs can be genetically engineered to induce bone tissue regeneration in the maxillofacial complex and that this finding may be clinically relevant.
Aslan H, Kimelman-Bleich N, Pelled G, Gazit D. Molecular targets for tendon neoformation [Internet]. J Clin Invest 2008;118(2):439-44. Publisher's VersionAbstract
Tendons and ligaments are unique forms of connective tissue that are considered an integral part of the musculoskeletal system. The ultimate function of tendon is to connect muscles to bones and to conduct the forces generated by muscle contraction into movements of the joints, whereas ligaments connect bone to bone and provide joint stabilization. Unfortunately, the almost acellular and collagen I-rich structure of tendons and ligaments makes them very poorly regenerating tissues. Injured tendons and ligaments are considered a major clinical challenge in orthopedic and sports medicine. This Review discusses the several factors that might serve as molecular targets that upon activation can enhance or lead to tendon neoformation.
Tai K, Pelled G, Sheyn D, Bershteyn A, Han L, Kallai I, Zilberman Y, Ortiz C, Gazit D. Nanobiomechanics of repair bone regenerated by genetically modified mesenchymal stem cells [Internet]. Tissue Eng Part A 2008;14(10):1709-20. Publisher's VersionAbstract
Genetically modified mesenchymal stem cells (MSCs), overexpressing a BMP gene, have been previously shown to be potent inducers of bone regeneration. However, little was known of the chemical and intrinsic nanomechanical properties of this engineered bone. A previous study utilizing microcomputed tomography, back-scattered electron microscopy, energy-dispersive X-ray, nanoindentation, and atomic force microscopy showed that engineered ectopic bone, although similar in chemical composition and topography, demonstrated an elastic modulus range (14.6-22.1 GPa) that was less than that of the native bone (16.6-38.5 GPa). We hypothesized that these results were obtained due to the specific conditions that exist in an intramuscular ectopic implantation site. Here, we implanted MSCs overexpressing BMP-2 gene in an orthotopic site, a nonunion radial bone defect, in mice. The regenerated bone tissue was analyzed using the same methods previously utilized. The samples revealed high similarity between the engineered and native radii in chemical structure and elemental composition. In contrast to the previous study, nanoindentation data showed that, in general, the native bone exhibited a statistically similar elastic modulus values compared to that of the engineered bone, while the hardness was found to be marginally statistically different at 1000 muN and statistically similar at 7000 muN. We hypothesize that external loading, osteogenic cytokines and osteoprogenitors that exist in a fracture site could enhance the maturation of engineered bone derived from BMP-modified MSCs. Further studies should determine whether longer duration periods postimplantation would lead to increased bone adaptation.
Sheyn D, Pelled G, Zilberman Y, Talasazan F, Frank JM, Gazit D, Gazit Z. Nonvirally engineered porcine adipose tissue-derived stem cells: use in posterior spinal fusion [Internet]. Stem Cells 2008;26(4):1056-64. Publisher's VersionAbstract
Multiple factors alter intervertebral disc volume, structure, shape, composition, and biomechanical properties, often leading to low back pain. Spinal fusion is frequently performed to treat this problem. We recently published results of our investigation of a novel system of in vivo bone formation, in which we used nonvirally nucleofected human mesenchymal stem cells that overexpress a bone morphogenetic protein gene. We hypothesized that primary porcine adipose tissue-derived stem cells (ASCs) nucleofected with plasmid containing recombinant human bone morphogenetic protein-6 (rhBMP-6) could induce bone formation and achieve spinal fusion in vivo. Primary ASCs were isolated from freshly harvested porcine adipose tissue. Overexpression of rhBMP-6 was achieved ex vivo by using a nucleofection technique. Transfection efficiency was monitored by assessing a parallel transfection involving an enhanced green fluorescent protein reporter gene and flow cytometry analysis. rhBMP-6 protein secreted by the cells was measured by performing an enzyme-linked immunosorbent assay. Genetically engineered cells were injected into the lumbar paravertebral muscle in immunodeficient mice. In vivo bone formation was monitored by a quantitative microcomputed tomography (muCT). The animals were euthanized 5 weeks postinjection, and spinal fusion was evaluated using in vitro muCT and histological analysis. We found formation of a large bone mass adjacent to the lumbar area, which produced posterior spinal fusion of two to four vertebrae. Our data demonstrate that efficient bone formation and spinal fusion can be achieved using ex vivo, nonvirally transfected primary ASCs. These results could pave the way to a novel biological solution for spine treatment.
Sheyn D, Kimelman-Bleich N, Pelled G, Zilberman Y, Gazit D, Gazit Z. Ultrasound-based nonviral gene delivery induces bone formation in vivo [Internet]. Gene Ther 2008;15(4):257-66. Publisher's VersionAbstract
Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to bone tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human bone morphogenetic protein-9 (rhBMP-9) would induce bone formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day 10. Bone tissue was formed in the site of rhBMP-9 delivery, as was shown by micro-computerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring bone tissue regeneration after further development that will increase the transfection efficiency.
2007
Zvonic S, Ptitsyn AA, Kilroy G, Wu X, Conrad SA, Scott LK, Guilak F, Pelled G, Gazit D, Gimble JM. Circadian oscillation of gene expression in murine calvarial bone [Internet]. J Bone Miner Res 2007;22(3):357-65. Publisher's VersionAbstract
UNLABELLED: The genes encoding the core circadian transcription factors display an oscillating expression profile in murine calvarial bone. More than 26% of the calvarial bone transcriptome exhibits a circadian rhythm, comparable with that observed in brown and white adipose tissues and liver. Thus, circadian mechanisms may directly modulate oxidative phosphorylation and multiple metabolic pathways in bone homeostasis. INTRODUCTION: Although circadian rhythms have been associated historically with central regulatory mechanisms, there is emerging evidence that the circadian transcriptional apparatus exists in peripheral tissues. The aim of this study was to determine the presence and extent of circadian oscillation in the transcriptome of murine calvarial bone. MATERIALS AND METHODS: Cohorts of 8-week-old male AKR/J mice were maintained in a controlled 12-h light:12-h dark cycle on an ad libitum diet for 2 weeks. Groups of three mice were killed every 4 h over a 48-h period. The level of gene expression at successive times-points was determined by quantitative RT-PCR and Affymetrix microarray. Data were analyzed using multiple statistical time series algorithms, including Cosinor, Fisher g-test, and the permutation time test. RESULTS: Both the positive (Bmal1, Npas2) and negative (Cry1, Cry2, Per1, Per2, Per3) elements of the circadian transcriptional apparatus and their immediate downstream targets and mediators (Dbp, Rev-erbalpha, Rev-erbbeta) exhibited oscillatory expression profiles. Consistent with findings in other tissues, the positive and negative elements were in antiphase relative to each other. More than 26% of the genes present on the microarray displayed an oscillatory profile in calvarial bone, comparable with the levels observed in brown and white adipose tissues and liver; however, only a subset of 174 oscillating genes were shared among all four tissues. CONCLUSIONS: Our findings show that the components of the circadian transcriptional apparatus are represented in calvarial bone and display coordinated oscillatory behavior. However, these are not the only genes to display an oscillatory expression profile, which is seen in multiple pathways involving oxidative phosphorylation and lipid, protein, and carbohydrate metabolism.
Risbud MV, Guttapalli A, Tsai TT, Lee JY, Danielson KG, Vaccaro AR, Albert TJ, Gazit Z, Gazit D, Shapiro IM. Evidence for skeletal progenitor cells in the degenerate human intervertebral disc [Internet]. Spine (Phila Pa 1976) 2007;32(23):2537-44. Publisher's VersionAbstract
STUDY DESIGN: To identify and characterize endogenous progenitor cell population from intervertebral disc. OBJECTIVE: To determine if progenitor cells exist in degenerate human discs. SUMMARY OF BACKGROUND DATA: Back pain, a significant source of morbidity in our society, is directly linked to the pathology of the intervertebral disc. Because disc disease is accompanied by a loss of cellularity, there is considerable interest in regeneration of cells of both the anulus fibrosus (AF) and nucleus pulposus (NP). METHODS: To determine if skeletal progenitor cells are present in the disc, samples were obtained from the degenerate AF and NP of 5 patients (Thompson grade 2 and 3, mean age 34 +/- 7.6 years) undergoing anterior cervical discectomy and fusion procedures as well as adult rat lumbar spine. RESULTS: Cells isolated from degenerate human tissues expressed CD105, CD166, CD63, CD49a, CD90, CD73, p75 low affinity nerve growth factor receptor, and CD133/1, proteins that are characteristic of marrow mesenchymal stem cells. In osteogenic media, there was an induction of alkaline phosphatase activity and expression of alkaline phosphatase, osteocalcin, and Runx-2 mRNA. When maintained in adipogenic media, a small percentage of cells displayed evidence of adipogenic differentiation: accumulation of cytosolic lipid droplets and increased expression of peroxisome proliferator-activated receptor-gamma2 and lipoprotein lipase mRNA. AF- and NP-derived cells also evidenced chondrogenic differentiation. CD133 (+) cells in the AF were able to commit to either the chondrogenic or adipogenic lineages. The results of the human disc studies were confirmed using cell derived from the NP and AF tissue of the mature rat disc. CONCLUSION: The analytical data indicated that the pathologically degenerate human disc contained populations of skeletal progenitor cells. These findings suggest that these endogenous progenitors may be used to orchestrate the repair of the intervertebral disc.
Pelled G, Tai K, Sheyn D, Zilberman Y, Kumbar S, Nair LS, Laurencin CT, Gazit D, Ortiz C. Structural and nanoindentation studies of stem cell-based tissue-engineered bone [Internet]. J Biomech 2007;40(2):399-411. Publisher's VersionAbstract
Stem cell-based gene therapy and tissue engineering have been shown to be an efficient method for the regeneration of critical-sized bone defects. Despite being an area of active research over the last decade, no knowledge of the intrinsic ultrastructural and nanomechanical properties of such bone tissue exists. In this study, we report the nanomechanical properties of engineered bone tissue derived from genetically modified mesenchymal stem cells (MSCs) overexpressing the rhBMP2 gene, grown in vivo in the thigh muscle of immunocompetent mice for 4 weeks, compared to femoral bone adjacent to the transplantation site. The two types of bone had similar mineral contents (61 and 65 wt% for engineered and femoral bone, respectively), overall microstructures showing lacunae and canaliculi (both measured by back-scattered electron microscopy), chemical compositions (measured by energy dispersive X-ray analysis), and nanoscale topographical morphologies (measured by tapping-mode atomic force microscopy imaging or TMAFM). Nanoindentation experiments revealed that the small length scale mechanical properties were statistically different with the femoral bone (indented parallel to the bone long axis) being stiffer and harder (apparent elastic modulus, E approximately 27.3+/-10.5 GPa and hardness, H approximately 1.0+/-0.7G Pa) than the genetically engineered bone (E approximately 19.8+/-5.6 GPa, H approximately 0.9+/-0.4G Pa). TMAFM imaging showed clear residual indents characteristic of viscoelastic plastic deformation for both types of bone. However, fine differences in the residual indent area (smaller for the engineered bone), pile up (smaller for the engineered bone), and fracture mechanisms (microcracks for the engineered bone) were observed with the genetically engineered bone behaving more brittle than the femoral control.
Xie C, Reynolds D, Awad H, Rubery PT, Pelled G, Gazit D, Guldberg RE, Schwarz EM, O'Keefe RJ, Zhang X. Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering [Internet]. Tissue Eng 2007;13(3):435-45. Publisher's VersionAbstract
The presence of live periosteal progenitor cells on the surface of bone autografts confers better healing than devitalized allograft. We have previously demonstrated in a murine 4 mm segmental femoral bone-grafting model that live periosteum produces robust endochondral and intramembraneous bone formation that is essential for effective healing and neovascularization of structural bone grafts. To the end of engineering a live pseudo-periosteum that could induce a similar response onto devitalized bone allograft, we seeded a mesenchymal stem cell line stably transfected with human bone morphogenic protein-2/beta-galactosidase (C9) onto devitalized bone allografts or onto a membranous small intestinal submucosa scaffold that was wrapped around the allograft. Histology showed that C9-coated allografts displayed early cartilaginous tissue formation at day 7. By 6 and 9 weeks, a new cortical shell was found bridging the segmental defect that united the host bones. Biomechanical testing showed that C9-coated allografts displayed torsional strength and stiffness equivalent to intact femurs at 6 weeks and superior to live isografts at 9 weeks. Volumetric and histomorphometric micro-computed tomography analyses demonstrated a 2-fold increase in new bone formation around C9-coated allografts, which resulted in a substantial increase in polar moment of inertia (pMOI) due to the formation of new cortical shell around the allografts. Positive correlations between biomechanics and new bone volume and pMOI were found, suggesting that the biomechanical function of the grafted femur relates to both morphological parameters. C9-coated allograft also exhibited slower resorption of the graft cortex at 9 weeks than live isograft. Both new bone formation and the persistent allograft likely contributed to the improved biomechanics of C9-coated allograft. Taken together, we propose a novel strategy to combine structural bone allograft with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering.
Kimelman N, Pelled G, Helm GA, Huard J, Schwarz EM, Gazit D. Review: gene- and stem cell-based therapeutics for bone regeneration and repair [Internet]. Tissue Eng 2007;13(6):1135-50. Publisher's VersionAbstract
Many clinical conditions require regeneration or implantation of bone. This is one focus shared by neurosurgery and orthopedics. Current therapeutic options (bone grafting and protein-based therapy) do not provide satisfying solutions to the problem of massive bone defects. In the past few years, gene- and stem cell-based therapy has been extensively studied to achieve a viable alternative to current solutions offered by modern medicine for bone-loss repair. The use of adult stem cells for bone regeneration has gained much focus. This unique population of multipotential cells has been isolated from various sources, including bone marrow, adipose, and muscle tissues. Genetic engineering of adult stem cells with potent osteogenic genes has led to fracture repair and rapid bone formation in vivo. It is hypothesized that these genetically modified cells exert both an autocrine and a paracrine effects on host stem cells, leading to an enhanced osteogenic effect. The use of direct gene delivery has also shown much promise for in vivo bone repair. Several viral and nonviral methods have been used to achieve substantial bone tissue formation in various sites in animal models. To advance these platforms to the clinical setting, it will be mandatory to overcome specific hurdles, such as control over transgene expression, viral vector toxicity, and prolonged culture periods of therapeutic stem cells. This review covers a prospect of cell and gene therapy for bone repair as well as some very recent advancements in stem cell isolation, genetic engineering, and exogenous control of transgene expression.
2006
Aslan H, Ravid-Amir O, Clancy BM, Rezvankhah S, Pittman D, Pelled G, Turgeman G, Zilberman Y, Gazit Z, Hoffmann A, Gross G, Domany E, Gazit D. Advanced molecular profiling in vivo detects novel function of dickkopf-3 in the regulation of bone formation [Internet]. J Bone Miner Res 2006;21(12):1935-45. Publisher's VersionAbstract
UNLABELLED: A bioinformatics-based analysis of endochondral bone formation model detected several genes upregulated in this process. Among these genes the dickkopf homolog 3 (Dkk3) was upregulated and further studies showed that its expression affects in vitro and in vivo osteogenesis. This study indicates a possible role of Dkk3 in regulating bone formation. INTRODUCTION: Endochondral bone formation is a complex biological process involving numerous chondrogenic, osteogenic, and angiogenic proteins, only some of which have been well studied. Additional key genes may have important roles as well. We hypothesized that to identify key genes and signaling pathways crucial for bone formation, a comprehensive gene discovery strategy should be applied to an established in vivo model of osteogenesis. MATERIALS AND METHODS: We used in vivo implanted C3H10T1/2 cells that had been genetically engineered to express human bone morphogenetic protein-2 (BMP2) in a tetracycline-regulated system that controls osteogenic differentiation. Oligonucleotide microarray data from the implants (n = 4 repeats) was analyzed using coupled two-way clustering (CTWC) and statistical methods. For studying the effects of dickkopf homolog 3 (Dkk3) in chondrogenesis and osteogenesis, C3H10T1/2 mesenchymal progenitors were used. RESULTS: The CTWC revealed temporal expression of Dkk3 with other chondrogenesis-, osteogenesis-, and Wnt-related genes. Quantitative RT-PCR confirmed the expression of Dkk3 in the implants. C3H10T1/2 cells that expressed Dkk3 in the presence of BMP2 displayed lower levels of alkaline phosphatase and collagen I mRNA expression than control C3H10T1/2 cells that did not express Dkk3. Interestingly, the levels of collagen II mRNA expression, Alcian blue staining, and glucose aminoglycans (GAGs) production were not influenced by Dkk3 expression. In vivo microCT and bioluminescence imaging revealed that co-expression of Dkk3 and BMP2 by implanted C3H10T1/2 cells induced the formation of significantly lower quantities of bone than cells expressing only BMP2. CONCLUSIONS: A bioinformatics analysis enabled the identification of Dkk3 as a pivotal gene with a novel function in endochondral bone formation. Our results showed that Dkk3 might have inhibitory effects on osteogenesis, but no effect on chondrogenesis, indicating that Dkk3 plays a regulatory role in endochondral bone formation. Further mechanistic studies are required to reveal the mechanism of action of Dkk3 in endochondral bone formation.
Kimelman N, Pelled G, Gazit Z, Gazit D. Applications of gene therapy and adult stem cells in bone bioengineering [Internet]. Regen Med 2006;1(4):549-61. Publisher's VersionAbstract
Bone tissue engineering is an emerging field, that could become a main therapeutic strategy in orthopedics in coming years. While bone has regenerative abilities that enable the self repair and regeneration of fractures, there are extreme situations in which the extent of bone loss is too large for complete regeneration to occur. In order to achieve bone regeneration, osteogenic genes (mainly from the bone morphogenetic protein family) can be delivered either directly into the target tissue, or by using adult stem cells, which are later implanted into the target site. Engineered adult stem cells combined with biodegradable polymeric scaffolds can be implanted into target sites, with or without ex vivo culture period. Several important factors influence the success of bone engineering approaches including: choice of cell and scaffold, the vector used in order to deliver the osteogenic gene, and the osteogenic gene itself. Cutting-edge imaging technologies, bioinformatics-based analysis of gene expression and exogenous regulation of transgene expression are among the tools that are being used to optimize and control bone formation in vivo. In this review we have attempted to provide an overview of the main factors that should be considered when utilizing adult stem cells and gene therapy strategies to regenerate bone defects or to promote new bone formation in vivo.
Gafni Y, Zilberman Y, Ophir Z, Abramovitch R, Jaffe M, Gazit Z, Domb A, Gazit D. Design of a filamentous polymeric scaffold for in vivo guided angiogenesis [Internet]. Tissue Eng 2006;12(11):3021-34. Publisher's VersionAbstract
Angiogenesis is mandatory for reperfusion of viable tissues, and lack of vascularization may cause ischemia. The increasing disparity between the demand and availability of adequate substitutes for small-diameter human blood vessels has prompted an intensive search for artificial materials or biological allograft tissues, both of which usually fail in the long term. The objective of this study was to pioneer a novel model for in vivo guided angiogenesis based on a specific design process of a filamentous polymeric scaffold with endothelial cells in a 3-dimensional culture system. To our knowledge, this is the first report of an in vivo guided angiogenesis approach based on a 2-step model, composed of endothelial cells and a filamentous polymeric scaffold framework. Endothelial cells that had been cultured on a specifically designed filamentous polymeric scaffold within a regulated dynamic tissue culture system were shown in vivo to induce guided angiogenesis. Cells seeded on a biodegradable polymeric scaffold were implanted into mice. On day 28 after implantation, analysis revealed a guided angiogenic process along the path of the implanted polymeric scaffold as well as initial evidence for early maturation of engineered vessels, allowing red blood cells to flow through the forming lumina of new vessels as the polymer degraded. The authors conclude that in vivo guided angiogenesis can be achieved by combining endothelial cells with biodegradable filamentous polymeric scaffolds and that this model can lay the cornerstone for vascular engineering and future development of clinically available protocols aimed to treat life-threatening cardiovascular conditions.
Snedeker JG, Pelled G, Zilberman Y, Gerhard F, Muller R, Gazit D. Endoscopic cellular microscopy for in vivo biomechanical assessment of tendon function [Internet]. J Biomed Opt 2006;11(6):064010. Publisher's VersionAbstract
This study explores a novel method to quantify in vivo soft tissue biomechanics from endoscopic confocal fluorescence microscope images of externally loaded biological tissues. A custom algorithm based on normalized cross-correlation is used to track fluorescently labeled cells within soft tissue structures as they deform. Cellular displacements are subsequently reduced to tissue strains by deriving the spatial gradient of the spline smoothed cellular displacement field. The relative performance of the tracking method is verified using a synthetic dataset with known underlying deformation. In biological application of the method, tissue strains are measured in the Achilles tendon of an anesthetized mouse. Over repeated trials, structural strain in the tendon (i.e., the relative change in distance between cells located at view field extremes) is 20.3+/-3.1%, thus establishing the reproducibility of the loading protocol. Analysis of local tendon tissue strains reveal primary engineering strains in the tissue to range from 5 to 55%, signifying a highly inhomogeneous strain state, with complex relative motions of neighboring tendon substructures. In summary, the current work establishes a baseline for a promising experimental method, and demonstrates its technical feasibility.
Hoffmann A, Pelled G, Turgeman G, Eberle P, Zilberman Y, Shinar H, Keinan-Adamsky K, Winkel A, Shahab S, Navon G, Gross G, Gazit D. Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells [Internet]. J Clin Invest 2006;116(4):940-52. Publisher's VersionAbstract
Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.
Aslan H, Zilberman Y, Arbeli V, Sheyn D, Matan Y, Liebergall M, Li JZ, Helm GA, Gazit D, Gazit Z. Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration [Internet]. Tissue Eng 2006;12(4):877-89. Publisher's VersionAbstract
There are several gene therapy approaches to tissue regeneration. Although usually efficient, virusbased approaches may elicit an immune response against the viral proteins. An alternative approach, nonviral transfer, is safer, and can be controlled and reproduced. We hypothesized that in vivo bone formation could be achieved using human mesenchymal stem cells (hMSCs) nonvirally transfected with the human bone morphogenetic protein-2 (hBMP-2) or -9 (hBMP-9) gene. Human MSCs were transfected using nucleofection, a unique electropermeabilization-based technique. Postnucleofection, cell viability was 53.6 +/- 2.5% and gene delivery efficiency was 51% to 88% (mean 68.2 +/- 4.1%), as demonstrated by flow cytometry in enhanced green fluorescent protein (EGFP)-nucleofected hMSCs. Transgene expression lasted longer than 14 days and was very low 21 days postnucleofection. Both hBMP-2- and hBMP-9-nucleofected hMSCs in culture demonstrated a significant increase in calcium deposition compared with EGFP-nucleofected hMSCs. Human BMP-2- and hBMP-9-nucleofected hMSCs transplanted in ectopic sites in NOD/SCID mice induced bone formation 4 weeks postinjection. We conclude that in vivo bone formation can be achieved by using nonvirally nucleofected hMSCs. This could lead to a breakthrough in the field of regenerative medicine, in which safer, nonviral therapeutic strategies present a very attractive alternative.
Aslan H, Zilberman Y, Kandel L, Liebergall M, Oskouian RJ, Gazit D, Gazit Z. Osteogenic differentiation of noncultured immunoisolated bone marrow-derived CD105+ cells [Internet]. Stem Cells 2006;24(7):1728-37. Publisher's VersionAbstract
The culture expansion of human mesenchymal stem cells (hMSCs) may alter their characteristics and is a costly and time-consuming stage. This study demonstrates for the first time that immunoisolated noncultured CD105-positive (CD105(+)) hMSCs are multipotent in vitro and exhibit the capacity to form bone in vivo. hMSCs are recognized as promising tools for bone regeneration. However, the culture stage is a limiting step in the clinical setting. To establish a simple, efficient, and fast method for applying these cells for bone formation, a distinct population of CD105(+) hMSCs was isolated from bone marrow (BM) by using positive selection based on the expression of CD105 (endoglin). The immunoisolated CD105(+) cell fraction represented 2.3% +/- 0.45% of the mononuclear cells (MNCs). Flow cytometry analysis of freshly immunoisolated CD105(+) cells revealed a purity of 79.7% +/- 3.2%. In vitro, the CD105(+) cell fraction displayed significantly more colony-forming units-fibroblasts (CFU-Fs; 6.3 +/- 1.4) than unseparated MNCs (1.1 +/- 0.3; p < .05). Culture-expanded CD105(+) cells expressed CD105, CD44, CD29, CD90, and CD106 but not CD14, CD34, CD45, or CD31 surface antigens, and these cells were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. In addition, freshly immunoisolated CD105(+) cells responded in vivo to recombinant bone morphogenetic protein-2 by differentiating into chondrocytes and osteoblasts. Genetic engineering of freshly immunoisolated CD105(+) cells was accomplished using either adenoviral or lentiviral vectors. Based on these findings, it is proposed that noncultured BM-derived CD105(+) hMSCs are osteogenic cells that can be genetically engineered to induce tissue generation in vivo.
2005
Hasharoni A, Zilberman Y, Turgeman G, Helm GA, Liebergall M, Gazit D. Murine spinal fusion induced by engineered mesenchymal stem cells that conditionally express bone morphogenetic protein-2 [Internet]. J Neurosurg Spine 2005;3(1):47-52. Publisher's VersionAbstract
OBJECT: The authors hypothesized that spinal fusion can be achieved and monitored by using cell-mediated gene therapy. Mesenchymal stem cells (MSCs) genetically engineered to express recombinant human bone morphogenetic protein-2 (rhBMP-2) conditionally, were implanted into the paraspinal muscles of mice to establish spinal fusion. The goal was to demonstrate an MSC-based gene therapy platform in which controlled gene expression is used to obtain spinal fusion in a murine model. METHODS: Mesenchymal stem cells expressing the rhBMP-2 gene were injected into the paravertebral muscle in mice. Bone formation in the paraspinal region was longitudinally followed by performing micro-computerized tomography scanning, histological studies, and an analysis of osteocalcin expression to demonstrate the presence of engrafted engineered MSCs. The minimal period of rhBMP-2 expression by the engineered MSCs required to induce fusion was determined. The results of this study demonstrate that genetically engineered MSCs induce bone formation in areas adjacent to and touching the posterior elements of the spine. This newly formed bone fuses the spine, as demonstrated by radiological and histological studies. The authors demonstrate that injected cells induce active osteogenesis at the site of implantation for up to 4 weeks postinjection. They found that a 7-day induction of rhBMP-2 expression in genetically engineered MSCs was sufficient to form new bone tissue, although the quantity of this bone increased as longer expression periods were implemented. CONCLUSIONS: After their injection genetically engineered MSCs can efficiently form new bone in the paraspinal muscle of the mouse to obtain spinal fusion. The extent and quantity of this newly formed bone can be monitored by controlling the duration of rhBMP-2 gene expression.
Eliyahu H, Makovitzki A, Azzam T, Zlotkin A, Joseph A, Gazit D, Barenholz Y, Domb AJ. Novel dextran-spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers [Internet]. Gene Ther 2005;12(6):494-503. Publisher's VersionAbstract
Recently, a novel cationic polymer, dextran-spermine (D-SPM) was developed for gene delivery. An efficient transfection was obtained using this polycation for a variety of genes and cell lines in serum-free or serum-poor medium. However, transfection using the water-soluble D-SPM-based polyplexes decreased with increasing serum concentration in cell culture in a concentration-dependent manner, reaching 95% inhibition at 50% serum in the cell growth medium. In order to overcome this obstacle, oleyl derivatives of D-SPM (which form micelles in aqueous phase) were synthesized at 1, 10, and 20 mol% of oleyl moiety to polymer epsilon-NH2 to form N-oleyl-D-SPM (ODS). Polyplexes based on ODS transfected well in medium containing 50% serum. Comparison with polyplexes based on well-established polymers (branched and linear polyethyleneimine) and with DOTAP/Cholesterol lipoplexes showed that regarding beta-galactosidase transgene expression level and cytotoxicity in tissue culture, the D-SPM and ODS compare well with the above polyplexes and lipoplexes. Intracellular trafficking using FITC-labeled ODS and Rhodamine-labeled pGeneGrip plasmid cloned with hBMP2 monitored by confocal microscopy revealed that during the transfection process the fluorescent-labeled polymer concentrates in the Golgi apparatus and around the nucleus, while the cell cytoplasm was free of fluorescent particles, suggesting that the polyplexes move in the cell toward the nucleus by vesicular transport through the cytoplasm and not by a random diffusion. We found that the plasmids penetrate the cell nucleus without the polymer. Preliminary results in zebra fish and mice demonstrate the potential of ODS to serve as an efficient nonviral vector for in vivo transfection.

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