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1.  Advances in Regenerative Orthopaedics 
Mayo Clinic proceedings  2013;88(11):1323-1339.
Orthopaedic injuries are very common and a source of much misery and economic stress. Several relevant tissues, such as cartilage, meniscus and intra-articular ligaments, do not heal. And even bone, which normally regenerates spontaneously, can fail to mend. The regeneration of orthopaedic tissues requires four key components: cells, morphogenetic signals, scaffolds and an appropriate mechanical environment. Although differentiated cells from the tissue in question can be used, most cellular research focuses on the use mesenchymal stem cells (MSCs). These can be retrieved from many different tissues, and one unresolved question is the degree to which the origin of the cells matters. Embryonic and induced, pluripotential stem cells are also under investigation. Morphogenetic signals are most frequently supplied by individual, recombinant growth factors or native mixtures provided by, for instance, platelet-rich plasma; MSCs are also a rich source of trophic factors. Obstacles to the sustained delivery of individual growth factors can be addressed by gene transfer or smart scaffolds, but we still lack detailed, necessary information on which delivery profiles are needed. Scaffolds may be based upon natural products, synthetic materials, or devitalized extracellular matrix. Strategies to combine these components to regenerate tissue can follow traditional tissue engineering practices, but these are costly, cumbersome and not well suited to treating large numbers of individuals. More expeditious approaches make full use of intrinsic biological processes in vivo to avoid the need for ex vivo expansion of autologous cells and multiple procedures. Clinical translation remains a bottleneck.
doi:10.1016/j.mayocp.2013.04.027
PMCID: PMC4214280  PMID: 24182709
2.  Preclinical toxicity evaluation of AAV for pain: evidence from human AAV studies and from the pharmacology of analgesic drugs 
Molecular Pain  2014;10:54.
Gene therapy with adeno-associated virus (AAV) has advanced in the last few years from promising results in animal models to >100 clinical trials (reported or under way). While vector availability was a substantial hurdle a decade ago, innovative new production methods now routinely match the scale of AAV doses required for clinical testing. These advances may become relevant to translational research in the chronic pain field. AAV for pain targeting the peripheral nervous system was proven to be efficacious in rodent models several years ago, but has not yet been tested in humans. The present review addresses the steps needed for translation of AAV for pain from the bench to the bedside focusing on pre-clinical toxicology. We break the potential toxicities into three conceptual categories of risk: First, risks related to the delivery procedure used to administer the vector. Second, risks related to AAV biology, i.e., effects of the vector itself that may occur independently of the transgene. Third, risks related to the effects of the therapeutic transgene. To identify potential toxicities, we consulted the existing evidence from AAV gene therapy for other nervous system disorders (animal toxicology and human studies) and from the clinical pharmacology of conventional analgesic drugs. Thereby, we identified required preclinical studies and charted a hypothetical path towards a future phase I/II clinical trial in the oncology-palliative care setting.
doi:10.1186/1744-8069-10-54
PMCID: PMC4237902  PMID: 25183392
Adeno-associated virus; Pain; Gene therapy; Toxicology; Interleukin-10; Beta-endorphin
3.  An overview of underlying causes and animal models for the study of age-related degenerative disorders of the spine and synovial joints 
As human lifespan increases so does the incidence of age-associated degenerative joint diseases, resulting in significant negative socioeconomic consequences. Osteoarthritis (OA) and intervertebral disc degeneration (IDD) are the most common underlying causes of joint-related chronic disability and debilitating pain in the elderly. Current treatment methods are generally not effective and involve either symptomatic relief with non-steroidal anti-inflammatory drugs and physical therapy or surgery when conservative treatments fail. The limitation in treatment options is due to our incomplete knowledge of the molecular mechanism of degeneration of articular cartilage and disc tissue. Basic understanding of the age-related changes in joint tissue is thus needed to combat the adverse effects of aging on joint health. Aging is caused at least in part by time-dependent accumulation of damaged organelles and macromolecules, leading to cell death and senescence and the eventual loss of multipotent stem cells and tissue regenerative capacity. Studies over the past decades have uncovered a number of important molecular and cellular changes in joint tissues with age. However, the precise causes of damage, cellular targets of damage, and cellular responses to damage remain poorly understood. The objectives of this review are (1) to provide an overview of the current knowledge about the sources of endogenous and exogenous damaging agents and how they contribute to age-dependent degenerative joint disease, and (2) highlight animal models of accelerated aging that could potentially be useful for identifying causes of and therapies for degenerative joint diseases.
doi:10.1002/jor.22204
PMCID: PMC3628921  PMID: 23483579
Synovial joints; intervertebral disc; DNA repair; aging; oxidative damage
4.  Barriers to the Clinical Translation of Orthopedic Tissue Engineering 
Tissue engineering and regenerative medicine have been the subject of increasingly intensive research for over 20 years, and there is concern in some quarters over the lack of clinically useful products despite the large sums of money invested. This review provides one perspective on orthopedic applications from a biologist working in academia. It is suggested that the delay in clinical application is not atypical of new, biologically based technologies. Some barriers to progress are acknowledged and discussed, but it is also noted that preclinical studies have identified several promising types of cells, scaffolds, and morphogenetic signals, which, although not optimal, are worth advancing toward human trials to establish a bridgehead in the clinic. Although this transitional technology will be replaced by more sophisticated, subsequent systems, it will perform valuable pioneering functions and facilitate the clinical development of the field. Some strategies for achieving this are suggested.
doi:10.1089/ten.teb.2011.0228
PMCID: PMC3223016  PMID: 21682607
5.  Effects of Dexamethasone on Mesenchymal Stromal Cell Chondrogenesis and Aggrecanase Activity: Comparison of Agarose and Self-Assembling Peptide Scaffolds 
Cartilage  2012;4(1):63-74.
Objective
Dexamethasone (Dex) is a synthetic glucocorticoid that has pro-anabolic and anti-catabolic effects in cartilage tissue engineering systems, though the mechanisms by which these effects are mediated are not well understood. We tested the hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine bone marrow stromal cells (BMSCs) cultured in self-assembling peptide and agarose hydrogels.
Design
We cultured young bovine and adult human BMSCs in (RADA)4 self-assembling peptide and agarose hydrogels in medium containing TGF-β1±Dex and analyzed extracellular matrix composition, aggrecan cleavage products, and the effects of the glucocorticoid receptor antagonist RU-486 on proteoglycan content, synthesis, and catabolic processing.
Results
Dex improved proteoglycan synthesis and retention in agarose hydrogels seeded with young bovine cells, but decreased proteoglycan accumulation in peptide scaffolds. These effects were mediated by the glucocorticoid receptor. Adult human BMSCs showed minimal matrix accumulation in agarose, but accumulated ~50% as much proteoglycan and collagen as young bovine BMSCs in peptide hydrogels. Dex reduced aggrecanase activity in (RADA)4 and agarose hydrogels, as measured by anti-NITEGE Western blotting, for both bovine and human BMSC-seeded gels.
Conclusions
The effects of Dex on matrix production are dependent on cell source and hydrogel identity. This is the first report of Dex reducing aggrecanase activity in a tissue engineering culture system.
doi:10.1177/1947603512455196
PMCID: PMC3922645  PMID: 24533173
Chondrogenesis; mesenchymal stem cells; degradative enzymes; articular cartilage; extracellular matrix
6.  EWS-FLI-1-targeted cytotoxic T-cell killing of multiple tumor types belonging to the Ewing Sarcoma Family of Tumors* 
Purpose
The Ewing Sarcoma Family of Tumors (ESFTs) comprises a group of aggressive, malignant bone and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types.
Design
Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of Cytotoxic T-Lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro.
Results
EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing Sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing Sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing Sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing Sarcoma, pPNET, Askin’s Tumor, and Biphenotypic Sarcoma. Stimulation of human Peripheral Blood Mononuclear Cells with YLNPSVDSV peptide resulted in potent CTL-killing.
Conclusions
These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development.
doi:10.1158/1078-0432.CCR-12-1985
PMCID: PMC3463738  PMID: 22879388
Ewing Sarcoma Family of Tumors; Ewing Sarcoma; pPNET; Askin’s Tumor; Biphenotypic sarcoma; EWS-FLI-1; Immunotherapy; vaccine; cancer; HLA-A2.1; HLA-A*0201
7.  Gene therapy of the rheumatic diseases: 1998 to 2008 
During the decade since the launch of Arthritis Research, the application of gene therapy to the rheumatic diseases has experienced the same vicissitudes as the field of gene therapy as a whole. There have been conceptual and technological advances and an increase in the number of clinical trials. However, funding has been unreliable and a small number of high-profile deaths in human trials, including one in an arthritis gene therapy trial, have provided ammunition to skeptics. Nevertheless, steady progress has been made in a number of applications, including rheumatoid arthritis and osteoarthritis, Sjögren syndrome, and lupus. Clinical trials in rheumatoid arthritis have progressed to phase II and have provided the first glimpses of possible efficacy. Two phase I protocols for osteoarthritis are under way. Proof of principle has been demonstrated in animal models of Sjögren syndrome and lupus. For certain indications, the major technological barriers to the development of genetic therapies seem to have been largely overcome. The translational research necessary to turn these advances into effective genetic medicines requires sustained funding and continuity of effort.
doi:10.1186/ar2563
PMCID: PMC2688220  PMID: 19232068
8.  Evaluation of BMP-2 gene-activated muscle grafts for cranial defect repair 
Journal of Orthopaedic Research  2011;30(7):1095-1102.
Large, osseous, segmental defects heal poorly. Muscle has a propensity to form bone when exposed to an osteogenic stimulus such as that provided by transfer and expression of cDNA encoding bone morphogenetic protein-2 (BMP-2). The present study evaluated the ability of genetically modified, autologous muscle to heal large cranial defects in rats. Autologous grafts (8mm × 2mm) were punched from the biceps femoris muscle and transduced intraoperatively with recombinant adenovirus vector containing human BMP-2 or green fluorescent protein cDNA. While the muscle biopsies were incubating with the vector, a central parietal 8mm defect was surgically created in the calvarium of the same animal. The gene-activated muscle graft was then implanted into the cranial defect. After 8 weeks, crania were examined radiographically, histologically, and by micro-computed tomography and dual energy X-ray absorptiometry. Although none of the defects were completely healed in this time, muscle grafts expressing BMP-2 deposited more than twice as much new bone as controls. Histology confirmed the anatomical integrity of the newly formed bone, which was comparable in thickness and mineral density to the original cranial bone. This study confirms the in vivo osteogenic properties of genetically modified muscle and suggests novel strategies for healing bone.
doi:10.1002/jor.22038
PMCID: PMC3349003  PMID: 22213093
Gene Therapy; Bone Healing; Muscle; BMP-2; Osteogenesis
9.  Synovial fibroblasts spread rheumatoid arthritis to unaffected joints 
Nature medicine  2009;15(12):1414-1420.
Active rheumatoid arthritis is characterized by originating from few but affecting subsequently the majority of joints. Thus far, the pathways of the progression of the disease are largely unknown. As rheumatoid arthritis synovial fibroblasts (RASFs) are key players in joint destruction and migrate in vitro, the current study evaluated the potential of RASFs to spread the disease in vivo. To simulate the primary joint of origin, healthy human cartilage was co-implanted subcutaneously into SCID mice together with RASFs. At the contralateral flank, healthy cartilage was implanted without cells. RASFs showed an active movement to the naïve cartilage via the vasculature independent of the site of application of RASFs into the SCID mouse, leading to a strong destruction of the target cartilage. These findings support the hypothesis that the characteristic clinical phenomenon of destructive arthritis spreading between joints is mediated, at least in part, by the transmigration of activated RASFs.
doi:10.1038/nm.2050
PMCID: PMC3678354  PMID: 19898488
10.  BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells 
Cytotherapy  2010;12(4):505-513.
Background aims
To date there are only very few data available on the ligamentogenic differentiation capacity of mesenchymal stromal/progenitor cells (MSC) and anterior cruciate ligament (ACL) fibroblasts.
Methods
We describe the in vitro potential of MSC and ACL cells to undergo ligamentogenic differentiation upon transduction with adenoviral vectors encoding the human cDNA for bone morphogenetic protein (BMP) 12 and BMP13, also known as growth and differentiation factors (GDF) 6 and 7, respectively.
Results
Transgene expression for at least 14 days was confirmed by Western blot analyzes. After 21 days of cell culture within collagen type I hydrogels, histochemical (hematoxylin/eosin (H&E), Azan and van Gieson), immunohistochemical and polymerase chain reaction (PCR) analyzes of the genetically modified constructs of both cell types revealed elongated, viable fibroblast-like cells embedded in a ligament-like matrix rich in collagens, vimentin, fibronectin, decorin, elastin, scleraxis, tenascin, and tenomodulin.
Conclusions
It appears that both MSC and ACL fibroblasts are capable of ligamentogenic differentiation with these factors. This information may aid in the development of biologic approaches to repair and restore ACL after injury.
doi:10.3109/14653241003709652
PMCID: PMC3580941  PMID: 20334610
adenovirus; anterior cruciate ligament; BMP12; BMP13; collagen hydrogel; fibroblasts; gene transfer; mesenchymal stromal cells
11.  Arginase Treatment Prevents the Recovery of Canine Lymphoma and Osteosarcoma Cells Resistant to the Toxic Effects of Prolonged Arginine Deprivation 
PLoS ONE  2013;8(1):e54464.
Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6–8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.
doi:10.1371/journal.pone.0054464
PMCID: PMC3554772  PMID: 23365669
12.  Getting arthritis gene therapy into the clinic 
Nature reviews. Rheumatology  2010;7(4):244-249.
Gene transfer technologies enable the controlled, targeted and sustained expression of gene products at precise anatomical locations, such as the joint. In this way, they offer the potential for more-effective, less-expensive treatments of joint diseases with fewer extra-articular adverse effects. A large body of preclinical data confirms the utility of intra-articular gene therapy in animal models of rheumatoid arthritis and osteoarthritis. However, relatively few clinical trials have been conducted, only one of which has completed phase II. This article summarizes the status in 2010 of the clinical development of gene therapy for arthritis, identifies certain constraints to progress and suggests possible solutions.
doi:10.1038/nrrheum.2010.193
PMCID: PMC3460537  PMID: 21135882
13.  Genetic mismatch affects the immunosuppressive properties of mesenchymal stem cells in vitro and their ability to influence the course of collagen-induced arthritis 
Arthritis Research & Therapy  2012;14(4):R167.
Introduction
The immunological and homing properties of mesenchymal stem cells (MSCs) provide a potentially attractive treatment for arthritis. The objective of this study was to determine effects of genetic disparity on the immunosuppressive potential of MSCs in vitro and in vivo within collagen induced arthritis (CIA).
Methods
The ability of DBA/1, FVB and BALB/c MSC preparations to impact the cytokine release profile of CD3/CD28 stimulated DBA/1 T cells was assessed in vitro. The effect of systemically delivered MSCs on the progression of CIA and cytokine production was assessed in vivo.
Results
All MSC preparations suppressed the release of TNFα and augmented the secretion of IL-4 and IL-10 by stimulated DBA/1 T-cells. However, assessment of the ratio of IFNγ to IL-4 production indicated that the more genetically distant BALB/c MSCs had significantly less immunosuppressive capacity. Systemic delivery of BALB/c MSC resulted in an exacerbation of CIA disease score in vivo and a higher erosive disease burden. This was not seen after treatment with syngeneic or partially mismatched MSCs. An increase in serum levels of IL-1β was observed up to 20 days post treatment with allogeneic MSCs. An initial elevation of IL-17 in these treatment groups persisted in those treated with fully mismatched BALB/c MSCs. Over the course of the study, there was a significant suppression of serum IL-17 levels in groups treated with syngeneic MSCs.
Conclusions
These data demonstrate a significant difference in the immunosuppressive properties of syngeneic and allogeneic MSCs in vitro and in vivo, which needs to be appreciated when developing MSC based therapies for inflammatory arthritis.
doi:10.1186/ar3916
PMCID: PMC3580561  PMID: 22812502
14.  Mesenchymal Stem Cell Characteristics of Human Anterior Cruciate Ligament Outgrowth Cells 
Tissue Engineering. Part A  2011;17(9-10):1375-1388.
When ruptured, the anterior cruciate ligament (ACL) of the human knee has limited regenerative potential. However, the goal of this report was to show that the cells that migrate out of the human ACL constitute a rich population of progenitor cells and we hypothesize that they display mesenchymal stem cell (MSC) characteristics when compared with adherent cells derived from bone marrow or collagenase digests from ACL. We show that ACL outgrowth cells are adherent, fibroblastic cells with a surface immunophenotype strongly positive for cluster of differentiation (CD)29, CD44, CD49c, CD73, CD90, CD97, CD105, CD146, and CD166, weakly positive for CD106 and CD14, but negative for CD11c, CD31, CD34, CD40, CD45, CD53, CD74, CD133, CD144, and CD163. Staining for STRO-1 was seen by immunohistochemistry but not flow cytometry. Under suitable culture conditions, the ACL outgrowth-derived MSCs differentiated into chondrocytes, osteoblasts, and adipocytes and showed capacity to self-renew in an in vitro assay of ligamentogenesis. MSCs derived from collagenase digests of ACL tissue and human bone marrow were analyzed in parallel and displayed similar, but not identical, properties. In situ staining of the ACL suggests that the MSCs reside both aligned with the collagenous matrix of the ligament and adjacent to small blood vessels. We conclude that the cells that emigrate from damaged ACLs are MSCs and that they have the potential to provide the basis for a superior, biological repair of this ligament.
doi:10.1089/ten.tea.2010.0413
PMCID: PMC3079172  PMID: 21247268
15.  Autologous bone grafting on steroids: preliminary clinical results. A novel treatment for nonunions and segmental bone defects 
International Orthopaedics  2010;35(4):599-605.
Clinical management of delayed healing or nonunion of long bone fractures and segmental bone defects poses a substantial orthopaedic challenge. Surgical advances and bone tissue engineering are providing new avenues to stimulate bone growth in cases of bone loss and nonunion. The reamer-irrigator-aspirator (RIA) device allows surgeons to aspirate the medullary contents of long bones and use the progenitor-rich “flow-through” fraction in autologous bone grafting. Dexamethasone (DEX) is a synthetic steroid that has been shown to induce osteoblastic differentiation. A series of 13 patients treated with RIA bone grafting enhanced with DEX for nonunion or segmental defect was examined retrospectively to assess the quality of bony union and clinical outcomes. Despite the initial poor prognoses, promising results were achieved using this technique; and given the complexity of these cases the observed success is of great value and warrants controlled study into both standardisation of the procedure and concentration of the grafting material.
doi:10.1007/s00264-010-1013-9
PMCID: PMC3066321  PMID: 20414656
16.  Effects of short-term glucocorticoid treatment on changes in cartilage matrix degradation and chondrocyte gene expression induced by mechanical injury and inflammatory cytokines 
Arthritis Research & Therapy  2011;13(5):R142.
Introduction
Traumatic joint injury damages cartilage and causes adjacent joint tissues to release inflammatory cytokines, increasing the risk of developing osteoarthritis. The main objective of this study was to determine whether the combined catabolic effects of mechanical injury, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) on cartilage could be abolished by short-term treatment with glucocorticoids such as dexamethasone.
Methods
In an initial dexamethasone-dose-response study, bovine cartilage explants were treated with TNFα and increasing concentrations of dexamethasone. Bovine and human cartilage explants were then subjected to individual and combined treatments with TNFα, IL-6/sIL-6R and injury in the presence or absence of dexamethasone. Treatment effects were assessed by measuring glycosaminoglycans (GAG) release to the medium and synthesis of proteoglycans. Additional experiments tested whether pre-exposure of cartilage to dexamethasone could prevent GAG loss and inhibition of biosynthesis induced by cytokines, and whether post-treatment with dexamethasone could diminish the effects of pre-established cytokine insult. Messenger ribonucleic acid (mRNA) levels for genes involved in cartilage homeostasis (proteases, matrix molecules, cytokines, growth and transcription factors) were measured in explants subjected to combined treatments with injury, TNFα and dexamethasone. To investigate mechanisms associated with dexamethasone regulation of chondrocyte metabolic response, glucocorticoid receptor (GR) antagonist (RU486) and proprotein convertase inhibitor (RVKR-CMK) were used.
Results
Dexamethasone dose-dependently inhibited GAG loss and the reduction in biosynthesis caused by TNFα. The combination of mechanical injury, TNFα and IL-6/sIL-6R caused the most severe GAG loss; dexamethasone reduced this GAG loss to control levels in bovine and human cartilage. Additionally, dexamethasone pre-treatment or post-treatment of bovine explants lowered GAG loss and increased proteoglycan synthesis in cartilage explants exposed to TNFα. Dexamethasone did not down-regulate aggrecanase mRNA levels. Post-transcriptional regulation by dexamethasone of other genes associated with responses to injury and cytokines was noted. GR antagonist reversed the effect of dexamethasone on sulfate incorporation. RVKR-CMK significantly reduced GAG loss caused by TNFα + IL-6 + injury.
Conclusions
Short-term glucocorticoid treatment effectively abolished the catabolic effects exerted by the combination of pro-inflammatory cytokines and mechanical injury: dexamethasone prevented proteoglycan degradation and restored biosynthesis. Dexamethasone appears to regulate the catabolic response of chondrocytes post-transcriptionally, since the abundance of transcripts encoding aggrecanases was still elevated in the presence of dexamethasone.
doi:10.1186/ar3456
PMCID: PMC3308070  PMID: 21888631
17.  Intra-articular gene delivery and expression of interleukin-1Ra mediated by self-complementary adeno-associated virus 
The journal of gene medicine  2009;11(7):605-614.
Background
The adeno-associated virus (AAV) has many safety features that favor its use in the treatment of arthritic conditions; however, the conventional, single-stranded vector is inefficient for gene delivery to fibroblastic cells that primarily populate articular tissues. This has been attributed to the inability of these cells to convert the vector to a double-stranded form. To overcome this, we evaluated double-stranded self-complementary (sc) AAV as a vehicle for intra-articular gene delivery.
Methods
Conventional and scAAV vectors were used to infect lapine articular fibroblasts in culture to determine transduction efficiency, transgene expression levels, and nuclear trafficking. scAAV containing the cDNA for interleukin (IL)-1 receptor antagonist (Ra) was delivered to the joints of naïve rabbits and those with IL-1β-induced arthritis. From lavage of the joint space, levels of transgenic expression and persistence were measured by enzyme-linked immunosorbent assay. Infiltrating leukocytes were quantified using a hemocytometer.
Results
Transgene expression from scAAV had an earlier onset and was approximately 25-fold greater than conventional AAV despite the presence of similar numbers of viral genomes in the nuclei of infected cells. Fibroblasts transduced with scAAV produced amounts of IL1-Ra comparable to those transduced with adenoviral and lentiviral vectors. IL1-Ra was present in lavage fluid of most animals for 2 weeks in sufficient quantities to inhibit inflammation of the IL-1β-driven model. Once lost, neither subsequent inflammatory events, nor re-administration of the virus could re-establish transgene expression.
Conclusions
scAAV-mediated intra-articular gene transfer is robust and similarly efficient in both normal and inflamed joints; the resulting transgenic expression is sufficient to achieve biological relevance in joints of human proportion.
doi:10.1002/jgm.1334
PMCID: PMC2876984  PMID: 19384892
adeno-associated virus; arthritis; gene therapy; interleukin-1; interleukin-1 receptor antagonist
18.  Enhanced In Vitro Chondrogenesis of Primary Mesenchymal Stem Cells by Combined Gene Transfer 
Tissue Engineering. Part A  2008;15(5):1127-1139.
Because articular cartilage has a poor regeneration capacity, numerous cell-based approaches to therapy are currently being explored. The present study involved the use of gene transfer as a means to provide sustained delivery of chondrogenic proteins to primary mesenchymal stem cells (MSCs). In previous work, we found that adenoviral-mediated gene transfer of transforming growth factor-beta1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2), but not insulin-like growth factor 1 (IGF-1), could be used to induce chondrogenic differentiation of MSCs in an aggregate culture system. In the present study, we examined the effects on chondrogenesis of these transgenes when delivered in combination. Cultures of bone marrow–derived MSCs were infected with 2.5 × 102 or 2.5 × 103 viral particles/cell of each adenoviral vector individually, or in combination, seeded into aggregates, and cultured for 3 weeks in a defined serum-free medium. Levels of transgene product in the medium were initially high, approximately 100 ng/mL TGF-β1, 120 ng/mL BMP-2, and 80 ng/mL IGF-1 at day 3, and declined thereafter. We found that co-expression of IGF-1 and TGF-β1, BMP-2, or both at low doses resulted in larger aggregates, higher levels of glycosaminoglycan synthesis, stronger staining for proteoglycans and collagen type II and X, and greater expression of cartilage-specific marker genes than with either transgene alone. Gene-induced chondrogenesis of MSCs using multiple genes that act synergistically may enable the administration of reduced viral doses in vivo and could be of considerable benefit for the development of cell-based therapies for cartilage repair.
doi:10.1089/ten.tea.2007.0252
PMCID: PMC2810414  PMID: 18826340
19.  Clinical Responses to Gene Therapy in Joints of Two Subjects with Rheumatoid Arthritis 
Human Gene Therapy  2009;20(2):97-101.
Abstract
This paper provides the first evidence of a clinical response to gene therapy in human arthritis. Two subjects with rheumatoid arthritis received ex vivo, intraarticular delivery of human interleukin-1 receptor antagonist (IL-1Ra) cDNA. To achieve this, autologous synovial fibroblasts were transduced with a retrovirus, MFG-IRAP, carrying IL-1Ra as the transgene, or remained as untransduced controls. Symptomatic metacarpophalangeal (MCP) joints were injected with control or transduced cells. Joints were clinically evaluated on the basis of pain; the circumference of MCP joint 1 was also measured. After 4 weeks, joints underwent surgical synovectomy. There were no adverse events in either subject. The first subject responded dramatically to gene transfer, with a marked and rapid reduction in pain and swelling that lasted for the entire 4 weeks of the study. Remarkably, joints receiving IL-1Ra cDNA were protected from flares that occurred during the study period. Analysis of RNA recovered after synovectomy revealed enhanced expression of IL-1Ra and reduced expression of matrix metalloproteinase-3 and IL-1β. The second subject also responded with reduced pain and swelling. Thus, gene transfer to human, rheumatoid joints can be accomplished safely to produce clinical benefit, at least in the short term. Using this ex vivo procedure, the transgene persisted within the joint for at least 1 month. Further clinical studies are warranted.
doi:10.1089/hum.2008.075
PMCID: PMC2855248  PMID: 18986219
20.  Regional and cellular localisation of BMPs and their inhibitors’ expression in human fractures 
International Orthopaedics  2008;33(1):281-288.
The objective of this study was to determine whether BMP-2 and -14, noggin, and chordin could be detected in human fractures and to assess their regional and cellular distribution. The expression of these proteins was detected by immunohistochemistry in an archive of human fractures. BMP-2 and BMP-14 expression was strongest in areas of cartilage formation and, to a lesser extent, in areas of bone formation. Within areas of cartilage formation, both BMP-2 and BMP-14 were expressed more strongly by the non-hypertrophic chondrocytes. The BMP inhibitors noggin and chordin were also expressed most intensely in areas of cartilage formation and there was no difference in their expression between the non-hypertrophic and hypertrophic chondrocytes. Our study demonstrates the expression of BMP-14 and the BMP inhibitors in human fractures for the first time, and our findings will contribute to an improved understanding of the physiological processes in bone repair.
doi:10.1007/s00264-008-0691-z
PMCID: PMC2899214  PMID: 19023570
21.  Osteogenic Potential of Reamer Irrigator Aspirator (RIA) Aspirate Collected from Patients Undergoing Hip Arthroplasty 
Intramedullary nailing preceded by canal reaming is the current standard of treatment for long-bone fractures requiring stabilization. However, conventional reaming methods can elevate intramedullary temperature and pressure, potentially resulting in necrotic bone, systemic embolism, and pulmonary complications. To address this problem, a reamer irrigator aspirator (RIA) has been developed that combines irrigation and suction for reduced-pressure reaming with temperature modulation. Osseous particles aspirated by the RIA can be recovered by filtration for use as an autograft, but the flow-through is typically discarded. The purpose of this study was to assess whether this discarded filtrate has osteogenic properties that could be used to enhance the total repair potential of aspirate. RIA aspirate was collected from five patients (ages 71–78) undergoing hip hemiarthroplasty. Osseous particles were removed using an open-pore filter, and the resulting filtrate (230 ± 200 mL) was processed by Ficoll-gradient centrifugation to isolate mononuclear cells (6.2 ± 5.2 × 106 cells/mL). The aqueous supernatant contained FGF-2, IGF-I, and latent TGF-β1, but BMP-2 was below the limit of detection. The cell fraction included culture plastic-adherent, fibroblastic cells that displayed a surface marker profile indicative of mesenchymal stem cells and that could be induced along the osteogenic, adipogenic, and chondrogenic lineages in vitro. When compared to outgrowth cells from the culture of osseous particles, filtrate cells were more sensitive to seeding density during osteogenic culture but had similar capacity for chondrogenesis. These results suggest using RIA aspirate to develop improved, clinically expeditious, cost-effective technologies for accelerating the healing of bone and other musculoskeletal tissues.
doi:10.1002/jor.20715
PMCID: PMC2648608  PMID: 18655129
intramedullary reaming; reamer irrigator aspirator; hemiarthroplasty; bone regeneration; mesenchymal stem cells
22.  Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer 
Arthritis Research & Therapy  2009;11(5):R148.
Introduction
The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.
Methods
Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 × 102 viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.
Results
Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size.
Conclusions
BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.
doi:10.1186/ar2822
PMCID: PMC2787261  PMID: 19799789
23.  Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells 
Introduction
Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.
Methods
Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.
Results
We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.
Conclusion
We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.
doi:10.1186/ar2436
PMCID: PMC2483456  PMID: 18533030
24.  Arthritis gene therapy's first death 
In July 2007 a subject died while enrolled in an arthritis gene therapy trial. The study was placed on clinical hold while the circumstances surrounding this tragedy were investigated. Early in December 2007 the Food and Drug Administration removed the clinical hold, allowing the study to resume with minor changes to the protocol. In the present article we collate the information we were able to obtain about this clinical trial and discuss it in the wider context of arthritis gene therapy.
doi:10.1186/ar2411
PMCID: PMC2483436  PMID: 18510784
25.  Major biological obstacles for persistent cell-based regeneration of articular cartilage 
Hyaline articular cartilage, the load-bearing tissue of the joint, has very limited repair and regeneration capacities. The lack of efficient treatment modalities for large chondral defects has motivated attempts to engineer cartilage constructs in vitro by combining cells, scaffold materials and environmental factors, including growth factors, signaling molecules, and physical influences. Despite promising experimental approaches, however, none of the current cartilage repair strategies has generated long lasting hyaline cartilage replacement tissue that meets the functional demands placed upon this tissue in vivo. The reasons for this are diverse and can ultimately result in matrix degradation, differentiation or integration insufficiencies, or loss of the transplanted cells and tissues. This article aims to systematically review the different causes that lead to these impairments, including the lack of appropriate differentiation factors, hypertrophy, senescence, apoptosis, necrosis, inflammation, and mechanical stress. The current conceptual basis of the major biological obstacles for persistent cell-based regeneration of articular cartilage is discussed, as well as future trends to overcome these limitations.
doi:10.1186/ar2195
PMCID: PMC2206353  PMID: 17561986

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