Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

Hebrew University-Hadassah Faculty of Dental Medicine

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the US alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination intravenous MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (p<0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to intravenous MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.Molecular Therapy (2015); doi:10.1038/mt.2015.211.

PTH induces systemically administered mesenchymal stem cells to migrate to and regenerate spine injuries.

More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of a reporter gene encoding DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging modes to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50μg luciferase plasmid DNA and 5×105 microbubbles followed by ultrasound treatment at 1.4MHz, 200kPa, 100-cycle pulse length, and 540-Hz pulse repetition frequency (PRF) for 2min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p<0.0001, n=5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced sonoporation-induced gene therapies.

Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation.

: Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration; however, their availability and capability of self-renewal are limited. Recent discoveries of somatic cell reprogramming may be used to overcome these challenges. We hypothesized that induced pluripotent stem cells (iPSCs) that were differentiated into MSCs could be used for bone regeneration. Short-term exposure of embryoid bodies to transforming growth factor-β was used to direct iPSCs toward MSC differentiation. During this process, two types of iPSC-derived MSCs (iMSCs) were identified: early (aiMSCs) and late (tiMSCs) outgrowing cells. The transition of iPSCs toward MSCs was documented using MSC marker flow cytometry. Both types of iMSCs differentiated in vitro in response to osteogenic or adipogenic supplements. The results of quantitative assays showed that both cell types retained their multidifferentiation potential, although aiMSCs demonstrated higher osteogenic potential than tiMSCs and bone marrow-derived MSCs (BM-MSCs). Ectopic injections of BMP6-overexpressing tiMSCs produced no or limited bone formation, whereas similar injections of BMP6-overexpressing aiMSCs resulted in substantial bone formation. Upon orthotopic injection into radial defects, all three cell types regenerated bone and contributed to defect repair. In conclusion, MSCs can be derived from iPSCs and exhibit self-renewal without tumorigenic ability. Compared with BM-MSCs, aiMSCs acquire more of a stem cell phenotype, whereas tiMSCs acquire more of a differentiated osteoblast phenotype, which aids bone regeneration but does not allow the cells to induce ectopic bone formation (even when triggered by bone morphogenetic proteins), unless in an orthotopic site of bone fracture.

Human Induced Pluripotent Stem Cells Differentiate Into Functional Mesenchymal Stem Cells and Repair Bone Defects.

Previous studies have associated low pH in intervertebral discs (IVDs) with discogenic back pain. The purpose of this study was to determine whether quantitative CEST (qCEST) MRI can be used to detect pH changes in IVDs in vivo.

Quantitative chemical exchange saturation transfer MRI of intervertebral disc in a porcine model.

A devastating condition that leads to trauma-related morbidity, multiple rib fractures, remain a serious unmet clinical need. Systemic administration of mesenchymal stem cells (MSCs) has been shown to regenerate various tissues. We hypothesized that parathyroid hormone (PTH) therapy would enhance MSC homing and differentiation, ultimately leading to bone formation that would bridge rib fractures.

Systemic administration of mesenchymal stem cells combined with parathyroid hormone therapy synergistically regenerates multiple rib fractures.

More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

Maxim Bez

Skeletal Biotech Laboratory

Hebrew University-Hadassah Faculty of Dental Medicine

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Maxim Bez

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Skeletal Biotech Laboratory

Hebrew University-Hadassah Faculty of Dental Medicine

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Skeletal Biotech Laboratory