The effect of simulated microgravity on human mesenchymal stem cells cultured in an osteogenic differentiation system: a bioinformatics study.

One proposed strategy for bone regeneration involves ex vivo tissue engineering, accomplished using bone-forming cells, biodegradable scaffolds, and dynamic culture systems, with the goal of three-dimensional tissue formation. Rotating wall vessel bioreactors generate simulated microgravity conditions ex vivo, which lead to cell aggregation. Human mesenchymal stem cells (hMSCs) have been extensively investigated and shown to possess the potential to differentiate into several cell lineages. The goal of the present study was to evaluate the effect of simulated microgravity on all genes expressed in hMSCs, with the underlying hypothesis that many important pathways are affected during culture within a rotating wall vessel system. Gene expression was analyzed using a whole genome microarray and clustering with the aid of the National Institutes of Health's Database for Annotation, Visualization and Integrated Discovery database and gene ontology analysis. Our analysis showed 882 genes that were downregulated and 505 genes that were upregulated after exposure to simulated microgravity. Gene ontology clustering revealed a wide variety of affected genes with respect to cell compartment, biological process, and signaling pathway clusters. The data sets showed significant decreases in osteogenic and chondrogenic gene expression and an increase in adipogenic gene expression, indicating that ex vivo adipose tissue engineering may benefit from simulated microgravity. This finding was supported by an adipogenic differentiation assay. These data are essential for further understanding of ex vivo tissue engineering using hMSCs.

Genetically modified mesenchymal stem cells induce mechanically stable posterior spine fusion.

Most spine fusion procedures involve the use of prosthetic fixation devices combined with autologous bone grafts rather than biological treatment. We had shown that spine fusion could be achieved by injection of bone morphogenetic protein-2 (BMP-2)-expressing mesenchymal stem cells (MSCs) into the paraspinal muscle. In this study, we hypothesized that posterior spinal fusion achieved using genetically modified MSCs would be mechanically comparable to that realized using a mechanical fixation. BMP-2-expressing MSCs were injected bilaterally into paravertebral muscles of the mouse lumbar spine. In one control group BMP-2 expression was inhibited. Microcomputed tomography and histological analyses were used to evaluate bone formation. For comparison, a group of mouse spines were bilaterally fused with stainless steel pins. The harvested spines were later tested using a custom four-point bending apparatus and structural bending stiffness was estimated. To assess the degree to which MSC vertebral fusion was targeted and to quantify the effects of fusion on adjacent spinal segments, images of the loaded spine curvature were analyzed to extract rigidity of the individual spinal segments. Bone bridging of the targeted vertebrae was observed in the BMP-2-expressing MSC group, whereas no bone formation was noted in any control group. The biomechanical tests showed that MSC-mediated spinal fusion was as effective as stainless steel pin-based fusion and significantly more rigid than the control groups. Local analysis showed that the distribution of stiffness in the MSC-based fusion group was similar to that in the steel pin fusion group, with the majority of spinal stiffness contributed by the targeted fusion at L3-L5. Our findings demonstrate that MSC-induced spinal fusion can convey biomechanical rigidity to a targeted segment that is comparable to that achieved using an instrumental fixation.

Genetically modified cells in regenerative medicine and tissue engineering.

Regenerative medicine appears to take as its patron, the Titan Prometheus, whose liver was able to regenerate daily, as the field attempts to restore lost, damaged, or aging cells and tissues. The tremendous technological progress achieved during the last decade in gene transfer methods and imaging techniques, as well as recent increases in our knowledge of cell biology, have opened new horizons in the field of regenerative medicine. Genetically engineered cells are a tool for tissue engineering and regenerative medicine, albeit a tool whose development is fraught with difficulties. Gene-and-cell therapy offers solutions to severe problems faced by modern medicine, but several impediments obstruct the path of such treatments as they move from the laboratory toward the clinical setting. In this review we provide an overview of recent advances in the gene-and-cell therapy approach and discuss the main hurdles and bottlenecks of this approach on its path to clinical trials and prospective clinical practice.

The use of a synthetic oxygen carrier-enriched hydrogel to enhance mesenchymal stem cell-based bone formation in vivo.

A major hurdle to surmount in bone-tissue engineering is ensuring a sufficient oxygen supply to newly forming tissue to avoid cell death or delayed development of osteogenic features. We hypothesized that an oxygen-enriched hydrogel scaffold would enhance tissue-engineered bone formation in vivo. To test this, we used a well-characterized mesenchymal stem cell (MSC) line, Tet-off BMP2 MSC, whose cells were engineered to express recombinant human bone morphogenetic protein-2. Cells were suspended in hydrogel supplemented with perfluorotributylamine (PFTBA) and implanted subcutaneously in an ectopic site, a radial bone defect, or a lumbar paravertebral muscle (mouse model of spinal fusion) in C3H/HeN mice. For controls, we used cells suspended in the same gel without PFTBA. In the ectopic site, there were significant increases in bone formation (2.5-fold increase), cell survival, and osteocalcin activity in the PFTBA-supplemented groups. PFTBA supplementation significantly increased structural parameters of bone in radial bone defects and triggered a significant 1.4-fold increase in bone volume in the spinal fusion model. We conclude that synthetic oxygen carrier supplementation of tissue-engineered implants enhances ectopic bone formation and yields better bone quality and volume in bone-repair and spinal fusion models, probably due to increased cell survival.

Genetically engineered mesenchymal stem cells: applications in spine therapy.

Spine disorders and intervertebral disc degeneration are considered the main causes for the clinical condition commonly known as back pain. Spinal fusion by implanting autologous bone to produce bony bridging between the two vertebrae flanking the degenerated-intervertebral disc is currently the most efficient treatment for relieving the symptoms of back pain. However, donor-site morbidity, complications and the long healing time limit the success of this approach. Novel developments undertaken by regenerative medicine might bring more efficient and available treatments. Here we discuss the pros and cons of utilizing genetically engineered mesenchymal stem cells for inducing spinal fusion. The combination of the stem cells, gene and carrier are crucial elements for achieving optimal spinal fusion in both small and large animal models, which hopefully will lead to the development of clinical applications.

Nanobiomechanics of repair bone regenerated by genetically modified mesenchymal stem cells.

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.

Nonvirally engineered porcine adipose tissue-derived stem cells: use in posterior spinal fusion.

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.

Structural and nanoindentation studies of stem cell-based tissue-engineered bone.

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.

Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration.

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.