BACKGROUND CONTEXT: The intervertebral disc (IVD) possesses a minimal capability for self-repair and regeneration. Changes in the differentiation of resident progenitor cells can represent diminished tissue regeneration and a loss of homeostasis. We previously showed that progenitor cells reside in the nucleus pulposus (NP). The effect of the degenerative process on these cells remains unclear. PURPOSE: We sought to explore the effect of IVD degeneration on the abundance of resident progenitor cells in the NP, their differentiation potential, and their ability to give rise to NP-like cells. We hypothesize that disc degeneration affects those properties. STUDY DESIGN: Nucleus pulposus cells derived from healthy and degenerated discs were methodically compared for proliferation, differentiation potential, and ability to generate NP-like cells. METHODS: Intervertebral disc degeneration was induced in 10 skeletally, mature mini pigs using annular injury approach. Degeneration was induced in three target discs, whereas intact adjacent discs served as controls. The disc degeneration was monitored using magnetic resonance imaging for 6 to 8 weeks. After there was a clear evidence of degeneration, we isolated and compared cells from degenerated discs (D-NP cells [NP-derived cells from porcine degenerated discs]) with cells isolated from healthy discs (H-NP cells) obtained from the same animal. RESULTS: The comparison showed that D-NP cells had a significantly higher colony-forming unit rate and a higher proliferation rate in vitro. Our data also indicate that although both cell types are able to differentiate into mesenchymal lineages, H-NP cells exhibit significantly greater differentiation toward the chondrogenic lineage and NP-like cells than D-NP cells, displaying greater production of glycosaminoglycans and higher gene expression of aggrecan and collagen IIa. CONCLUSIONS: Based on these findings, we conclude that IVD degeneration has a meaningful effect on the cells in the NP. D-NP cells clearly go through the regenerative process; however, this process is not powerful enough to facilitate full regeneration of the disc and reverse the degenerative course. These findings facilitate deeper understanding of the IVD degeneration process and trigger further studies that will contribute to development of novel therapies for IVD degeneration.

Osteogenesis of mesenchymal stem cells (MSCs) is highly dependent on oxygen supply. We have shown that perfluorotributylamine (PFTBA), a synthetic oxygen carrier, enhances MSC-based bone formation in vivo. Exploring this phenomenon’s mechanism, we hypothesize that a transient increase in oxygen levels using PFTBA will affect MSC survival, proliferation, and differentiation, thus increasing bone formation. To test this hypothesis, MSCs overexpressing bone morphogenetic protein 2 were encapsulated in alginate beads that had been supplemented with an emulsion of PFTBA or phosphate-buffered saline. Oxygen measurements showed that supplementation of PFTBA significantly increased the available oxygen level during a 96-h period. PFTBA-containing beads displayed an elevation in cell viability, which was preserved throughout 2 weeks, and a significantly lower ratio of dead cells throughout the experiment. Furthermore, the cells from the control group expressed significantly more hypoxia-related genes such as VEGF, DDIT3, and PKG1. Additionally, PFTBA supplementation led to an increase in the osteogenic differentiation and to a decrease in chondrogenic differentiation of MSCs. In conclusion, PFTBA increases the oxygen availability in the vicinity of the MSCs, which suffer oxygen exhaustion shortly after encapsulation in alginate beads. Consequently, cell survival is increased, and hypoxia-related genes are downregulated. In addition, PFTBA promotes osteogenic differentiation over chondrogeneic differentiation, and thereby can accelerate MSC-based bone regeneration.

Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (muCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The muCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.

Real-time bioluminescence functional imaging holds great promise for regenerative medicine because it improves the researcher’s ability to analyze and understand the healing process. Using transgenic mice coupled with gene-modified cells, one can employ this method to monitor host and graft activity in various models of tissue regeneration. We implemented real-time bioluminescence functional imaging to analyze bone formation by following a unique protocol in which the luciferase reporter gene, driven by an osteocalcin promoter, is used to visualize host and graft activity during bone formation. Real-time bioluminescence functional imaging can be used to assess the "host reaction" in transgenic mice models; it can also be used to assess "graft activity" in other animals in which genetically labeled stem cells have been implanted or direct gene delivery has been applied. The suggested imaging protocol requires 25 min per sample. However, special attention must be given to the layout of the experimental design, which determines the specific activity that will be analyzed.

The reduced field-of-view (rFOV) turbo-spin-echo (TSE) technique, which effectively suppresses bowel movement artifacts, is developed for the purpose of chemical exchange saturation transfer (CEST) imaging of the intervertebral disc (IVD) in vivo. Attempts to quantify IVD CEST signals in a clinical setting require high reliability and accuracy, which is often compromised in the conventionally used technique. The proposed rFOV TSE CEST method demonstrated significantly superior reproducibility when compared with the conventional technique on healthy volunteers, implying it is a more reliable measurement. Phantom study revealed a linear relation between CEST signal and glycosaminoglycan (GAG) concentration. The feasibility of detecting IVD degeneration was demonstrated on a healthy volunteer, indicating that the proposed method is a promising tool to quantify disc degeneration.

BACKGROUND: Brown adipose tissue plays a pivotal role in mammal metabolism and thermogenesis. It has a great therapeutic potential in several metabolic disorders such as obesity and diabetes. Mesenchymal stem cells (MSCs) are suitable candidates for brown adipose tissue formation de novo. Ppargamma2 and C/ebpalpha are nucleic receptors known to mediate adipogenic differentiation. We hypothesized that overexpression of the Ppargamma2 and C/ebpalpha genes in MSCs would lead to the formation of adipose tissue. MATERIALS & METHODS: MSCs bearing the Luc reporter gene were transfected to overexpress Ppargamma2 and C/ebpalpha. Differentiation of nucleofected cells was evaluated in vitro and in vivo following ectopic implantation of the cells in C3H/HeN mice. RESULTS: After implantation, the engineered cells survived for 5 weeks and brown adipose-like tissue was observed in histological samples. Immunostaining and bioluminescent imaging showed new adipocytes expressing Luc and the brown adipose tissue marker, UCP1, in vitro and in vivo. CONCLUSION: We show that gene delivery of transcription factors into MSCs generates brown adipose tissue in vitro and in vivo.

Tendon tissue regeneration is an important goal for orthopedic medicine. We hypothesized that implantation of Smad8/BMP2-engineered MSCs in a full-thickness defect of the Achilles tendon (AT) would induce regeneration of tissue with improved biomechanical properties. A 2 mm defect was created in the distal region of murine ATs. The injured tendons were then sutured together or given implants of genetically engineered MSCs (GE group), non-engineered MSCs (CH3 group), or fibrin gel containing no cells (FG group). Three weeks later the mice were killed, and their healing tendons were excised and processed for histological or biomechanical analysis. A biomechanical analysis showed that tendons that received implants of genetically engineered MSCs had the highest effective stiffness (>70% greater than natural healing, p < 0.001) and elastic modulus. There were no significant differences in either ultimate load or maximum stress among the treatment groups. Histological analysis revealed a tendon-like structure with elongated cells mainly in the GE group. ATs that had been implanted with Smad8/BMP2-engineered stem cells displayed a better material distribution and functional recovery than control groups. While additional study is required to determine long-term effects of GE MSCs on tendon healing, we conclude that genetically engineered MSCs may be a promising therapeutic tool for accelerating short-term functional recovery in the treatment of tendon injuries.

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 operator’s experience.

SUMMARY: This study investigated the influence of ovarian hormone deficiency on core circadian regulatory protein (CCRP) in the context of bone loss. Our data suggest that ovarian hormone deficiency disrupts diurnal rhythmicity and CCRP expression in bone. Further studies should determine if chronobiology provides a novel therapeutic target for osteoporosis intervention. INTRODUCTION: CCRP synchronize metabolic activities and display an oscillatory expression profile in murine bone. In vitro studies using bone marrow mesenchymal stromal/stem cells have demonstrated that the CCRP is present and can be regulated within osteoblast progenitors. In vivo studies have shown that the CCRP regulates bone mass via leptin/neuroendocrine pathways. The current study used an ovariectomized murine model to test the hypothesis that ovarian hormone deficiency is associated with either an attenuation and/or temporal phase shift of the CCRP oscillatory expression in bone and that these changes are correlated with the onset of osteoporosis. METHODS: Sham-operated controls and ovariectomized female C57BL/6 mice were euthanized at 4-h intervals 2 weeks post-operatively. RESULTS: Ovariectomy attenuated the oscillatory expression of CCRP mRNAs in the femur and vertebra relative to the controls and reduced the wheel-running activity profile. CONCLUSION: Ovarian hormone deficiency modulates the expression profile of the CCRP with potential impact on bone marrow mesenchymal stem cell lineage commitment.

The development of transitional interfacial zones between adjacent tissues remains a significant challenge for developing tissue engineering and regenerative medicine strategies. Using osteogenic differentiation as a model, we describe a novel approach to spatially regulate expression and secretion of the bone morphogenetic protein (BMP-2) in a two-dimensional field of cultured cells, by flow patterning the modulators of inducible BMP-2 gene expression. We first demonstrate control of gene expression, and of osteogenic differentiation of the cell line with inducible expression of BMP-2. Then we design laminar flow systems, with patterned delivery of Doxycycline (Dox), the expression modulator of BMP-2. The patterned concentration profiles were verified by computational simulation and dye separation experiments. Patterned differentiation experiments conducted in the flow systems for a period of three weeks showed the Dox concentration dependent osteogenic differentiation, as evidenced by mineral deposition. In summary, by combining inducible gene expression with laminar flow technologies, this study provided an innovative way to engineer tissue interfaces.

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 (microCT) 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.

While various problems with bone healing remain, the greatest clinical change is the absence of an effective approach to manage large segmental defects in limbs and craniofacial bones caused by trauma or cancer. Thus, nontraditional forms of medicine, such as gene therapy, have been investigated as a potential solution. The use of osteogenic genes has shown great potential in bone regeneration and fracture healing. Several methods for gene delivery to the fracture site have been described. The majority of them include a cellular component as the carrying vector, an approach known as cell-mediated gene therapy. Yet, the complexity involved with cell isolation and culture emphasizes the advantages of direct gene delivery as an alternative strategy. Here we review the various approaches of direct gene delivery for bone repair, the choice of animal models, and the various outcome measures required to evaluate the efficiency and safety of each technique. Special emphasis is given to noninvasive, quantitative, in vivo monitoring of gene expression and biodistribution in live animals. Research efforts should aim at inducing a transient, localized osteogenic gene expression within a fracture site to generate an effective therapeutic approach that would eventually lead to clinical use.

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.

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.

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.

In order to investigate intervertebral disc (IVD) degeneration and repair, a quantitative non-invasive tool is needed. Various MRI methods including qCPMG, which yields dipolar echo relaxation time (T(DE)), magnetization transfer contrast (MTC), and (1)H and (2)H double quantum filtered (DQF) MRI were used in the present work to monitor changes in rat IVD after ablation of the nucleus pulposus (NP), serving as a model of severe IVD degeneration. In the intact IVD, a clear distinction between the annulus fibrosus (AF) and the NP is obtained on T(2) and T(DE) weighted images as well as on MTC maps, reflecting the high concentration of ordered collagen fibers in the AF. After ablation of the NP, the distinction between the compartments is lost. T(2) and T(DE) relaxation times are short throughout the disc and MTC is high. (1)H and (2)H DQF signal, which in intact discs is obtained only for the AF, is now observable throughout the tissue. These results indicate that after ablation, there is an ingression of collagen fibers from the AF into the area that was previously occupied by the NP, as was confirmed by histology.

Stimuli responsive or "smart" hydrogels are of interest for tissue-engineering applications, featuring the advantages of minimally invasive application. Currently, these materials have yet to be used as a biological replacement in restoring the function of damaged tissues or organs. The aim of this study was to demonstrate the advantages of thermoresponsive, peptide-containing hydrogels as a supportive matrix for genetically engineered stem cells. We used injectable hydrogels, enabling cell delivery to the desired site and providing adequate scaffolding postimplantation. Thermoresponsive hydrogels were developed based on amphiphilic block copolymers of polyethylene-oxide and polypropylene-oxide end-capped with methacrylate or maleimide entities and further reacted with RGD-containing peptides. Cell metabolic activity and survival within those hydrogels was studied, illustrating that the stable peptide-polymer conjugate is required for prolonged cell support. The unique polymer characteristics, combined with its enhanced cell interactions, suggest the potential use of these biomaterials in various tissue engineering applications.

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.

Stem cell-mediated gene therapy for fracture repair, utilizes genetically engineered mesenchymal stem cells (MSCs) for the induction of bone growth and is considered a promising approach in skeletal tissue regeneration. Previous studies have shown that murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). Nanoindentation studies of bone tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine bone, eight weeks post-treatment. In the present study we sought to investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius bones, following the implantation of MSCs. High-resolution micro-computed tomography (micro-CT) was performed 10 and 35 weeks post MSC implantation, followed by micro-finite element (micro-FE) analysis. The results have shown that the regenerated bone tissue remodels over time, as indicated by a significant decrease in bone volume, total volume, and connectivity density combined with an increase in mineral density. In addition, the axial stiffness of limbs repaired with MSCs was 2-1.5 times higher compared to the contralateral intact limbs, at 10 and 35 weeks post-treatment. These results could be attributed to the fusion that occurred in between the ulna and radius bones. In conclusion, although MSCs induce bone formation, which exceeds the fracture site, significant remodeling of the repair callus occurs over time. In addition, limbs treated with an MSC graft demonstrated superior biomechanical properties, which could indicate the clinical benefit of future MSC application in nonunion fracture repair.

Fibered confocal laser scanning microscopes have given us the ability to image fluorescently labeled biological structures in vivo and at exceptionally high spatial resolutions. By coupling this powerful imaging modality with classic optical elastography methods, we have developed novel techniques that allow us to assess functional mechanical integrity of soft biological tissues by measuring the movements of cells in response to externally applied mechanical loads. Using these methods we can identify minute structural defects, monitor the progression of certain skeletal tissue disease states, and track subsequent healing following therapeutic intervention in the living animal. Development of these methods using a murine Achilles tendon model has revealed that the hierarchical and composite anatomical structure of the tendon presents various technical challenges that can confound a mechanical analysis of local material properties. Specifically, interfascicle gliding can yield complex cellular motions that must be interpreted within the context of an appropriate anatomical model. In this study, we explore the various classes of cellular images that may result from fibered confocal microscopy of the murine Achilles tendon, and introduce a simple two-fascicle model to interpret the images in terms of mechanical strains within the fascicles, as well as the relative gliding between fascicles.