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author:("spelled, Gadi")
1.  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
2.  Smad8/BMP2–Engineered Mesenchymal Stem Cells Induce Accelerated Recovery of the Biomechanical Properties of the Achilles Tendon 
Summary
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), nonengineered 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.
doi:10.1002/jor.22167
PMCID: PMC3479351  PMID: 22696396
Tendon repair; Smad8/BMP2; Tissue engineering; Biomechanics; Achilles tendon
3.  Adeno-Associated Virus–Coated Allografts: A Novel Approach for Cranioplasty 
Bone autografts are considered the gold standard for cranioplasty although they lead to comorbidity. Bone allografts are more easily obtained but have low osteogenic potential and fail to integrate into healthy bone. Previously, we showed that by coating long-bone allografts with freeze-dried recombinant adeno-associated virus (rAAV) vector encoding for an osteogenic gene, enhanced osteogenesis and bone integration were achieved. In this study our aim was to evaluate the bone repair potential of calvarial autografts and allografts coated with either single-stranded rAAV2 vector (SS-rAAV-BMP2) or self-complementary pseudotyped vector (SC-rAAV-BMP2) encoding for bone morphogenetic protein (BMP)–2 in a murine cranioplasty model. The grafts were implanted into critical defects in the calvariae of osteocalcin/luciferase (Oc/Luc) transgenic mice, which allowed longitudinal monitoring of osteogenic activity using bioluminescence imaging (BLI). Our results showed that the bioluminescent signal of the SC-rAAV-BMP2–coated allografts was 40% greater than that of the SS-rAAV-BMP2–coated allografts (p<0.05) and that the bioluminescent signal of the SS-rAAV-BMP2–coated allografts was not significantly different from the signals of the autografts or uncoated allografts. Micro–computed tomography (μCT) confirmed the significant increase in osteogenesis in the SC-rAAV-BMP2 group compared with the SS-rAAV-BMP2 group (p<0.05), indicating a significant difference in bone formation when compared with the other grafts tested. In addition, histological analysis revealed extensive remodeling of the autografts. Collectively, these results demonstrate the feasibility of craniofacial regeneration using SC-rAAV-BMP2–coated allografts, which may be an attractive therapeutic solution for repair of severe craniofacial bone defects.
doi:10.1002/term.1594
PMCID: PMC3652248  PMID: 22941779
gene therapy/therapeutics; craniomaxillofacial surgery; tissue engineering; imaging; bone remodeling/regeneration; bone graft(s)
4.  Gene therapy approaches to regenerating bone 
Advanced drug delivery reviews  2012;64(12):1320-1330.
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.
doi:10.1016/j.addr.2012.03.007
PMCID: PMC3663073  PMID: 22429662
Gene therapy; Bone regeneration; Tissue engineering; Viral vectors; Nonviral vectors
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.  Patterning osteogenesis by inducible gene expression in microfluidic culture systems 
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.
doi:10.1039/c0ib00053a
PMCID: PMC3416042  PMID: 20924519
7.  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
8.  The Effect of Simulated Microgravity on Human Mesenchymal Stem Cells Cultured in an Osteogenic Differentiation System: A Bioinformatics Study 
Tissue Engineering. Part A  2010;16(11):3403-3412.
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.
doi:10.1089/ten.tea.2009.0834
PMCID: PMC2971652  PMID: 20807102
9.  Quantitative, Structural and Image-based Mechanical Analysis of Nonunion Fracture Repaired by Genetically Engineered Mesenchymal Stem Cells 
Journal of biomechanics  2010;43(12):2315-2320.
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 to 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 between in 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.
doi:10.1016/j.jbiomech.2010.04.031
PMCID: PMC2948956  PMID: 20471652
Micro finite element model; Micro computed tomography; Bone tissue regeneration; Mesenchymal stem cells
10.  Direct Gene Therapy for Bone Regeneration: Gene Delivery, Animal Models, and Outcome Measures 
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.
doi:10.1089/ten.teb.2009.0156
PMCID: PMC2865989  PMID: 20143927
11.  Circadian Mechanisms in Murine and Human Bone Marrow Mesenchymal Stem Cells Following Dexamethasone Exposure 
Bone  2008;42(5):861-870.
A core group of transcriptional regulatory factors regulate 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 α, and rev-erb β. The genes displayed a mean oscillatory period of 22.2 to 24.3 hours. The acrophase or peak expression of mRNAs encoding “positive” (bmal1) and “negative” (per3) transcriptional regulatory factors were out of phase with each other by ∼8-12 hours, consistent with in vivo observations. In vivo, glycogen synthase kinase 3β (GSK3β) mediated phosphorylation regulates the turnover of per3 and core circadian transcriptional apparatus. In vitro addition of lithium chloride, a GSK3β inhibitor, significantly shifted the acrophase of all genes by 4.2-4.7 hours 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.
doi:10.1016/j.bone.2007.12.226
PMCID: PMC2423188  PMID: 18302991
Bone Marrow Mesenchymal Stem Cells; Circadian; Dexamethasone; Lithium Chloride; Rev-erb α
12.  Molecular targets for tendon neoformation 
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.
doi:10.1172/JCI33944
PMCID: PMC2214706  PMID: 18246194
13.  Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells 
Journal of Clinical Investigation  2006;116(4):940-952.
Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.
doi:10.1172/JCI22689
PMCID: PMC1421340  PMID: 16585960

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