Ilan Kallai

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.

Targeted Gene-and-host Progenitor Cell Therapy for Nonunion Bone Fracture Repair

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius bone defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for 10 days before electroporation. Mice were electroporated with either bone morphogenetic protein 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated bone formation bridging the bone gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion bone defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.

Gene-Modified Adult Stem Cells Regenerate Vertebral Bone Defect in a Rat Model

Vertebral compression fractures (VCFs), the most common fragility fractures, account for approximately 700,000 injuries per year. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, a new minimally invasive biological solution to vertebral bone repair is needed. Previously, we showed that adipose-derived stem cells (ASCs) overexpressing a BMP gene are capable of inducing spinal fusion in vivo. We hypothesized that a direct injection of ASCs, designed to transiently overexpress rhBMP6, into a vertebral bone void defect would accelerate bone regeneration. Porcine ASCs were isolated and labeled with lentiviral vectors that encode for the reporter gene luciferase (Luc) under constitutive (ubiquitin) or inductive (osteocalcin) promoters. The ASCs were first labeled with reporter genes and then nucleofected with an rhBMP6-encoding plasmid. Twenty-four hours later, bone void defects were created in the coccygeal vertebrae of nude rats. The ASC-BMP6 cells were suspended in fibrin gel (FG) and injected into the bone void. A control group was injected with FG alone. The regenerative process was monitored in vivo using microCT, and cell survival and differentiation were monitored using tissue specific reporter genes and bioluminescence imaging (BLI). The surgically treated vertebrae were harvested after 12 weeks and subjected to histological and immunohistochemical (against porcine vimentin) analyses. In vivo BLI detected Luc-expressing cells at the implantation site over a 12-week period. Beginning 2 weeks postoperatively, considerable defect repair was observed in the group treated with ASC-BMP6 cells. The rate of bone formation in the stem cell-treated group was two times faster than that in the FG-treated group, and bone volume at the end point was 2-fold compared to the control group. Twelve weeks after cell injection the bone volume within the void reached the volume measured in native vertebrae. Immunostaining against porcine vimentin indicated that the ASC-BMP6 cells contributed to new bone formation. Here we show the potential of injections of BMP-modified ASCs to repair vertebral bone defects in a rat model. Our results could pave the way to a novel approach for the biological treatment of traumatic and osteoporosis-related vertebral bone injuries.

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.

Gene therapy approaches to regenerating bone.

Bone formation and regeneration therapies continue to require optimization and improvement because many skeletal disorders remain undertreated. Clinical solutions to nonunion fractures and osteoporotic vertebral compression fractures, for example, remain suboptimal and better therapeutic approaches must be created. The widespread use of recombinant human bone morphogenetic proteins (rhBMPs) for spine fusion was recently questioned by a series of reports in a special issue of The Spine Journal, which elucidated the side effects and complications of direct rhBMP treatments. Gene therapy - both direct (in vivo) and cell-mediated (ex vivo) - has long been studied extensively to provide much needed improvements in bone regeneration. In this article, we review recent advances in gene therapy research whose aims are in vivo or ex vivo bone regeneration or formation. We examine appropriate vectors, safety issues, and rates of bone formation. The use of animal models and their relevance for translation of research results to the clinical setting are also discussed in order to provide the reader with a critical view. Finally, we elucidate the main challenges and hurdles faced by gene therapy aimed at bone regeneration as well as expected future trends in this field.

Maxillofacial–Derived Stem Cells Regenerate Critical Mandibular Bone Defect

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.

Quantitative microstructural studies of the armor of the marine threespine stickleback (Gasterosteus aculeatus).

In this study, a quantitative investigation of the microstructure and composition of field-caught marine Gasterosteus aculeatus (threespine stickleback) armor is presented, which provides useful phylogenetic information and insights into biomechanical function. Micro-computed tomography (microCT) was employed to create full three-dimensional images of the dorsal spines and basal plate, lateral plates, pelvic girdle and spines and to assess structural and compositional properties such as the spatial distribution of thickness (approximately 100-300 microm), the heterogeneous cross-sectional geometry (centrally thickened), plate-to-plate juncture and overlap (approximately 50% of the plate width), and bone mineral density (634-748 HA/cm(3)). The convolution of plate geometry in conjunction with plate-to-plate overlap allows a relatively constant armor thickness to be maintained throughout the assembly, promoting spatially homogeneous protection and thereby avoiding weakness at the armor unit interconnections. Plate-to-plate junctures act to register and join the plates while permitting compliance in sliding and rotation in selected directions. Mercury porosimetry was used to determine the pore size distribution and volume percent porosity of the lateral plates (20-35 vol.%) and spines (10-15 vol.%). SEM and microCT revealed a porous, sandwich-like cross-section beneficial for bending stiffness and strength at minimum weight. Back-scattered electron microscopy and energy dispersive X-ray analysis were utilized to quantify the weight percent mineral content (58-68%). Scanning electron microscopy and surface profilometry were used to characterize the interior and exterior surface topography (tubercles) of the lateral plates. The results obtained in this study are discussed in the context of mechanical function, performance, fitness, and survivability.

BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells

Bone regeneration achieved using mesenchymal stem cells (MSCs) and nonviral gene therapy holds great promise for patients with fractures seemingly unable to heal. Previously, MSCs overexpressing bone morphogenetic proteins (BMPs) were shown to differentiate into the osteogenic lineage and induce bone formation. In the present study, we evaluated the potential of osteogenic differentiation in porcine adipose tissue- and bone marrow-derived MSCs (ASCs and BMSCs, respectively) in vitro and in vivo when induced by nucleofection with rhBMP-2 or rhBMP-6. Our assessment of the in vivo efficiency of this procedure was made using quantitative micro-computed tomography (micro-CT). Nucleofection efficiency and cell viability were similar in both cell types; however, the micro-CT analyses demonstrated that in both ASCs and BMSCs, nucleofection with rhBMP-6 generated bone tissue faster and of higher volumes than nucleofection with rhBMP-2. RhBMP-6 induced more efficient osteogenic differentiation in vitro in BMSCs, and in fact, greater osteogenic potential was identified in BMSCs both in vitro and in vivo than in ASCs. On the basis of our findings, we conclude that BMSCs nucleofected with rhBMP-6 are superior at inducing bone formation in vivo than all other groups studied.

Microcomputed tomography-based structural analysis of various bone tissue regeneration models

Microcomputed tomography (microCT) analysis is a powerful tool for the evaluation of bone tissue because it provides access to the 3D microarchitecture of the bone. It is invaluable for regenerative medicine as it provides the researcher with the opportunity to explore the skeletal system both in vivo and ex vivo. The quantitative assessment of macrostructural characteristics and microstructural features may improve our ability to estimate the quality of newly formed bone. We have developed a unique procedure for analyzing data from microCT scans to evaluate bone structure and repair. This protocol describes the procedures for microCT analysis of three main types of mouse bone regeneration models (ectopic administration of bone-forming mesenchymal stem cells, and administration of cells after both long bone defects and cranial segmental bone defects) that can be easily adapted for a variety of other models. Precise protocols are crucial because the system is extremely user sensitive and results can be easily biased if standardized methods are not applied. The suggested protocol takes 1.5–3.5 h per sample, depending on bone tissue sample size, the type of equipment used, variables of the scanning protocol and the operators experience.

Three-dimensional structure of the shell plate assembly of the chiton Tonicella marmorea and its biomechanical consequences

This study investigates the three-dimensional structure of the eight plate exoskeletal (shell) assembly of the chiton Tonicella marmorea. X-ray micro-computed tomography and 3D printing elucidate the mechanism of conformational change from a passive (slightly curved, attached to surface) to a defensive (rolled, detached from surface) state of the plate assembly. The passive and defensive conformations exhibited differences in longitudinal curvature index (0.43 vs. 0.70), average plate-to-plate overlap (∼62% vs. ∼48%), cross-sectional overlap heterogeneity (60-82.5% vs. 0-90%, fourth plate), and plate-to-plate separation distance (100% increase in normalized separation distance between plates 4 and 5), respectively. The plate-to-plate interconnections consist of two rigid plates joined by a compliant, actuating muscle, analogous to a geometrically structured shear lap joint. This work provides an understanding of how T. marmorea achieves the balance between mobility and protection. In the passive state, the morphometry of the plates and plate-to-plate interconnections results in an approximately continuous curvature and constant armor thickness, resulting in limited mobility but maximum protection. In the defensive state, the underlying soft tissues gain protection and the chiton gains mobility through tidal flow, but regions of vulnerability open dorsally, due to the increase in plate-to-plate separation and decrease in plate-to-plate overlap. Lastly, experiments using optical and scanning electron microscopy, mercury porosimetry, and Fourier-transform infrared spectroscopy explore the microstructure and spatial distribution of the six layers within the intermediate plates, the role of multilayering in resisting predatory attacks, and the detection of chitin as a major component of the intra-plate organic matrix and girdle.