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.
Micro-electroporation is an electroporation technology in which the electrical field that induces cell membrane poration is focused onto a single cell contained in a micro-electromechanical structure. Micro-electroporation has many unique attributes including that it facilitates real time control over the process of electroporation at the single cell level. Flow-through micro-electroporation expands on this principle and was developed to facilitate electroporation of a large numbers of cells with control over the electroporation of every single cell. However, our studies show that when electroporation employs conventional direct current (DC) electrical pulses the micro-electroporation system fails, because of electrolysis induced gas bubble formation. We report in this study that when certain alternating currents (AC) electrical pulses are used for micro-electroporation it becomes possible to avoid electrolytic gas bubble formation in a micro-electroporation flow-through system. The effect of AC micro-electroporation on electrolysis was found to depend on the AC frequency used. This concept was tested with mesenchymal stem cells and preliminary results show successful electroporation using this system.
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.
The human body displays central circadian rhythms of activity. Recent findings suggest that peripheral tissues, such as bone, possess their own circadian clocks. Studies have shown that osteocalcin protein levels oscillate over a 24-hour period, yet the specific skeletal sites involved and its transcriptional profile remain unknown. The current study aimed to test the hypothesis that peripheral circadian mechanisms regulate transcription driven by the osteocalcin promoter. Transgenic mice harboring the human osteocalcin promoter linked to a luciferase reporter gene were used. Mice of both genders and various ages were analyzed non-invasively at sequential times throughout 24-hour periods. Statistical analyses of luminescent signal intensity of osteogenic activity from multiple skeletal sites indicated a periodicity of 24 hrs. The maxillomandibular complex displayed the most robust oscillatory pattern. These findings have implications for dental treatments in orthodontics and maxillofacial surgery, as well as for the mechanisms underlying bone remodeling in the maxillomandibular complex.
Our objectives were to determine the chondrogenic potential of a murine Brachyury-transformed mesenchymal progenitor cell line in the presence of rheumatoid arthritis-activated synovial fibroblasts (RASFs). Brachyury-transformed mesenchymal progenitor cells were implanted alone or combined with RASFs isolated from diseased human joints in each of six immunodeficient SCID mice. De novo tissue formation was analysed by histology and immunohistochemistry after 60 days. Spheroid nodules resembling cartilage morphologically and by the expression of proteoglycans and collagen II developed in four of six implants in the absence and in five of six implants in the presence of RASFs. No evidence for hypertrophic differentiation could be observed. Mesenchymal progenitor cells transformed with Brachyury are able to produce a cartilage like tissue in vivo over an extended period of time that is resistant to the destructive effect of RASF. This observation may provide opportunities for a cell-based reconstructive treatment in joint disease.
This work advances fibered confocal microscopy (FCM) as a functional imaging platform for in vivo assessment of tissue mechanics. Building on our earlier studies demonstrating proof of principle and introducing an analytical framework for FCM image processing, here we present data that improve and validate several critical aspects of FCM. Specifically, we have considerably reduced the invasiveness of the imaging procedure, and verified that endoscopic imaging through a transcutaneous access point does not induce functional changes in passive ankle joint biomechanics. We have also verified that periodic (weekly) measurements on uninjured tendons are reproducible. Importantly, we have further proven that the method can sensitively detect and quantify compromised tendon mechanics in injured tendons. These incremental but essential developments further push FCM measurement of tissue mechanics from a novel concept to a usable tool that fills an important niche by functionally imaging living tissue at the highest available spatial resolution of any currently available in vivo imaging method. It is expected that functional FCM imaging will eventually enable accelerated screening of preclinical therapies, and allow researchers to quantifiably relate implanted cell behavior with resulting changes in tissue structure and function.
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.
OBJECTIVES: Major risk factors of oral squamous cell carcinoma (OSCC) are environmental and can lead to DNA mutagenesis. Mismatch repair (MMR) system functions to repair small DNA lesions, which can be targeted for promoter hypermethylation. We therefore wanted to test whether hypermethylation of MMR genes (hMLH1, hMSH2) could contribute to oral carcinogenesis by correlating the information to patient clinical data. METHODS: Genomic DNA was extracted from 28 OSCC and six normal oral epithelium samples. The methylation status of the two MMR genes was assessed using Methylation Specific PCR after DNA modification with sodium bisulfite. Serial sections of the same tissues were immunostained with antibodies against hMLH1 and hMSH2 protein. RESULTS: Promoter hypermethylation was observed in 14/28 OSCC cases. Remarkably, 100% of patients with multiple oral malignancies showed hypermethylation in hMLH1 or hMSH2 compared with 31.5% of single tumor patients. In 10 cancer cases, expression of the hMLH1 and hMSH2 genes by immunostaining showed reduced or absence of expression of one of the genes, although some did not reflect the methylation status. CONCLUSIONS: Hypermethylation of hMLH1 and hMSH2 might play a role in oral carcinogenesis and may be correlated with a tendency to develop multiple oral malignancies.
Osteoarthritis (OA) affects both cartilage and bone tissues, and the subsequent breakdown of the two tissues appears to be interrelated. The interest in the role of subchondral bone changes with OA is growing, and one suggestion is that a simple inverse correlation exists between the cartilage loss and increased bone mineral density. In this work the STR/ort mouse is used as a model for human OA, in order to investigate disease progression. The aim of the work is to elucidate the tempero-spatial relationships between bone and cartilage architecture and determine whether a simple inverse correlation is satisfactory. We employ 3D whole joint quantitative imaging techniques for assessment of subchondral bone and articular cartilage. The knee joints of mice aged 3, 4, 7 and 10 months are scanned with muCT and then the tibial plateaus are scanned with CLSM. The results show that depending on site (medial and lateral), compartment (epiphyseal, metaphyseal, cortical), and age (3, 4, 7, 10 months), the subchondral bone undergoes changes that lead to an altered architecture. This is primarily seen as densification of the cortex and epiphysis in the STR/ort mice, with a significant change occurring between 7 and 10 months, while the medial cartilage thickness is significantly reduced after 7 months. Using a novel multimodal imaging approach, morphometric changes in the murine osteoarthritic knee joint are elucidated. It is seen that a complex interplay of events - both spatially and temporally - is involved in OA onset and progression. The initial measured differences between the two strains suggest a possible morphological phenotype involved in OA resistance/vulnerability. Temporally the changes have a strong strain:age dependence, although no separate timeline of events between the two tissues could be discerned. Spatially, the changes to medial and lateral morphometry across the cartilage and bone, indicate a relationship to altered joint mechanics.
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.
Monitoring gene expression in vitro and in vivo, is crucial when analyzing osteogenesis and developing effective bone gene therapy protocols. Until recently, molecular analytical tools were only able to detect protein expression either in vitro or in vivo. These systems include histology and immunohistochemistry, fluorescent imaging, PET (micro-PET), CT (micro-CT), and bioluminescent imaging. The last is the only system to date that can enable efficient quantitative monitoring of gene expression both in vitro and in vivo. Effective bioluminescent imaging in bone can be achieved by using transgenic mice harboring the luciferase reporter gene, downstream of an osteogenesis specific promoter. The aim of this chapter is to comprehensively describe the various protocols needed for the detection of bioluminescence in bone development and repair.
Fluorescence molecular tomography (FMT) is a novel tomographic near-infrared (NIR) imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals at any depth. This study demonstrates a noninvasive, quantitative method of monitoring engineered bone remodeling via FMT. Murine mesenchymal stem cells overexpressing the osteogenic gene BMP2 (mMSCs-BMP2) were implanted into the thigh muscle and into a radial nonunion bone defect model in C3H/HeN mice. Real-time imaging of bone formation was performed following systemic administration of the fluorescent bisphosphonate imaging agent OsteoSense, an hydroxyapatite-directed bone-imaging probe. The mice underwent imaging on days 7, 14, and 21 postimplantation. New bone formation at the implantation sites was quantified using micro-computed tomography (micro-CT) imaging. A higher fluorescent signal occurred at the site of the mMSC-BMP2 implants than that found in controls. Micro-CT imaging revealed a mass of mature bone formed in the implantation sites on day 21, a finding also confirmed by histology. These findings highlight the effectiveness of FMT as a functional platform for molecular imaging in the field of bone regeneration and tissue engineering.
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.
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.
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.