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1.  BMP6-Engineered MSCs Induce Vertebral Bone Repair in a Pig Model: A Pilot Study 
Stem Cells International  2015;2016:6530624.
Osteoporotic patients, incapacitated due to vertebral compression fractures (VCF), suffer grave financial and clinical burden. Current clinical treatments focus on symptoms' management but do not combat the issue at the source. In this pilot study, allogeneic, porcine mesenchymal stem cells, overexpressing the BMP6 gene (MSC-BMP6), were suspended in fibrin gel and implanted into a vertebral defect to investigate their effect on bone regeneration in a clinically relevant, large animal pig model. To check the effect of the BMP6-modified cells on bone regeneration, a fibrin gel only construct was used for comparison. Bone healing was evaluated in vivo at 6 and 12 weeks and ex vivo at 6 months. In vivo CT showed bone regeneration within 6 weeks of implantation in the MSC-BMP6 group while only minor bone formation was seen in the defect site of the control group. After 6 months, ex vivo analysis demonstrated enhanced bone regeneration in the BMP6-MSC group, as compared to control. This preclinical study presents an innovative, potentially minimally invasive, technique that can be used to induce bone regeneration using allogeneic gene modified MSCs and therefore revolutionize current treatment of challenging conditions, such as osteoporosis-related VCFs.
doi:10.1155/2016/6530624
PMCID: PMC4685143  PMID: 26770211
2.  Detection of Low Back Pain Using pH Level-Dependent Imaging of the Intervertebral Disc Using the Ratio of R1ρ Dispersion and –OH Chemical Exchange Saturation Transfer (RROC) 
Magnetic resonance in medicine  2014;73(3):1196-1205.
Purpose
Low pH is associated with intervertebral disc (IVD)-generated low back pain (LBP). The purpose of this work was to develop an in vivo pH level-dependent magnetic resonance imaging (MRI) method for detecting discogenic LBP, without using exogenous contrast agents.
Methods
The ratio of R1ρ dispersion and chemical exchange saturation transfer (CEST) (RROC) was used for pH-level dependent imaging of the IVD while eliminating the effect of labile proton concentration. The technique was validated by numerical simulations and studies on phantoms and ex vivo porcine spines. Four male (ages 42.8 ± 18.3) and two female patients (ages 55.5 ± 2.1) with LBP and scheduled for discography were examined with the method on a 3.0 Tesla MR scanner. RROC measurements were compared with discography outcomes using paired t-test.
Results
Simulation and phantom results indicated RROC is a concentration independent and pH level-dependent technique. Porcine spine study results found higher RROC value was related to lower pH level. Painful discs based on discography had significant higher RROC values than those with negative diagnosis (P < 0.05).
Conclusion
RROC imaging is a promising pH level dependent MRI technique that has the potential to be a noninvasive imaging tool to detect painful IVDs in vivo.
doi:10.1002/mrm.25186
PMCID: PMC4511600  PMID: 24700573
Intervertebral disc; pH imaging; RROC; R1ρ dispersion; CEST
3.  PTH promotes allograft integration in a calvarial bone defect 
Molecular pharmaceutics  2013;10(12):4462-4471.
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 micro–computed tomography (μCT) 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 μCT 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.
doi:10.1021/mp400292p
PMCID: PMC3902084  PMID: 24131143
Parathyroid Hormone; endogenous stem cells; osteogenesis; allograft; calvarial bone repair
5.  Gene-Modified Adult Stem Cells Regenerate Vertebral Bone Defect in a Rat Model 
Molecular pharmaceutics  2011;8(5):1592-1601.
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, 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 endpoint was twofold 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.
doi:10.1021/mp200226c
PMCID: PMC3220930  PMID: 21834548
vertebral fracture; bone regeneration; gene-and-cell therapy; stem cell tracking
6.  Genetically Modified Mesenchymal Stem Cells Induce Mechanically Stable Posterior Spine Fusion 
Tissue Engineering. Part A  2010;16(12):3679-3686.
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.
doi:10.1089/ten.tea.2009.0786
PMCID: PMC2991214  PMID: 20618082

Results 1-6 (6)