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1.  Lymphatic endothelial cells efferent to inflamed joints produce iNOS and inhibit lymphatic vessel contraction and drainage in TNF-induced arthritis in mice 
Background
In this study, we sought to determine the cellular source of inducible nitric oxide synthase (iNOS) induced in lymphatic endothelial cells (LECs) in response to tumor necrosis factor (TNF), the effects of iNOS on lymphatic smooth muscle cell (LSMC) function and on the development of arthritis in TNF-transgenic (TNF-Tg) mice, and whether iNOS inhibitors improve lymphatic function and reduce joint destruction in inflammatory erosive arthritis.
Methods
We used quantitative polymerase chain reactions, immunohistochemistry, histology, and near-infrared imaging to examine (1) iNOS expression in podoplanin + LECs and lymphatic vessels from wild-type (WT) and TNF-Tg mice, (2) iNOS induction by TNF in WT LECs, (3) the effects of iNOS inhibitors on expression of functional muscle genes in LSMCs, and (4) the effects of iNOS inhibitors on lymphatic vessel contraction and drainage, as well as the severity of arthritis, in TNF-Tg mice.
Results
LECs from TNF-Tg mice had eight fold higher iNOS messenger RNA levels than WT cells, and iNOS expression was confirmed immunohistochemically in podoplanin + LECs in lymphatic vessels from inflamed joints. TNF (0.1 ng/ml) increased iNOS levels 40-fold in LECs. LSMCs cocultured with LECs pretreated with TNF had reduced expression of functional muscle genes. This reduction was prevented by ferulic acid, which blocked nitric oxide production. Local injection of L-N6-(1-iminoethyl)lysine 5-tetrazole-amide into inflamed paws of TNF-Tg mice resulted in recovery of lymphatic vessel contractions and drainage. Treatment of TNF-Tg mice with ferulic acid reduced synovial inflammation as well as cartilage and bone erosion, and it also restored lymphatic contraction and drainage.
Conclusions
iNOS is produced primarily by LECs in lymphatic vessel efferent from inflamed joints of TNF-Tg mice in response to TNF and inhibits LSMC contraction and lymph drainage. Ferulic acid represents a potential new therapy to restore lymphatic function and thus improve inflammatory arthritis by inhibiting local production of nitric oxide by LSMCs.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-016-0963-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13075-016-0963-8
PMCID: PMC4789262  PMID: 26970913
Rheumatoid arthritis; TNF; Lymphatic function; Lymphatic endothelial cells (LECs); Lymphatic smooth muscle cells (LSMC); Inducible nitric oxide synthase (iNOS); Ferulic acid
2.  The Role of the Lymphatic System in Inflammatory-Erosive Arthritis 
Rheumatoid arthritis (RA) is a prevalent inflammatory joint disease with enigmatic flares, which causes swelling, pain, and irreversible connective tissue damage. Recently, it has been demonstrated in murine models of RA that the popliteal lymph node (PLN) is a biomarker of arthritic flare, as it “expands” in size and contrast enhancement during a prolonged asymptomatic phase, prior to when it “collapses” with accelerated synovitis and joint erosion. This PLN collapse is associated with adjacent knee flare, decreases in PLN volume and contrast enhancement, lymphatic pulse and pumping pressure, and an increase in PLN pressure. Currently, it is known that PLN collapse is accompanied by a translocation of B cells from the follicles to the sinuses, effectively clogging the lymphatic sinuses of the PLN, and that B cell depletion therapy ameliorates arthritic flare by eliminating these B cells and restoring passive lymphatic flow from inflamed joints. Here we review the technological advances that have launched this area of research, describe future directions to help elucidate the potential mechanism of PLN collapse, and speculate on clinical translation towards new diagnostics and therapies for RA.
doi:10.1016/j.semcdb.2015.01.001
PMCID: PMC4397133  PMID: 25598390
Rheumatoid arthritis; lymph node; flare; lymphatic vessel
3.  Passive Immunization with Anti-Glucosaminidase Monoclonal Antibodies Protects Mice from Implant-Associated Osteomyelitis by Mediating Opsonophagocytosis of Staphylococcus aureus Megaclusters 
Towards development of a methicillin-resistant S. aureus (MRSA) vaccine we evaluated a neutralizing anti-glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant-associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus placebo (alpha-T2m) and a Gmd-deficient isogenic strain (delta-Gmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro-CT assessment of osteolysis and histomorphometry of abscess numbers (p<0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies delta-Gmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in delta-Gmd cultures or 1C11 treated cultures of a protein A-deficient strain (delta-Spa), suggesting that the combined effects of Gmd inhibition and antibody-mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p<0.01). Collectively, these results suggest that the primary mechanism of anti-Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of SAC and opsonophagocytosis of megaclusters.
doi:10.1002/jor.22672
PMCID: PMC4234088  PMID: 24992290
Osteomyelitis; Methicillin-Resistant Staphylococcus aureus (MRSA); Passive Immunization; Opsonophagocytosis; Electron Microscopy
4.  A Novel Murine Model of Established Staphylococcal Bone Infection in the Presence of a Fracture Fixation Plate to Study Therapies Utilizing Antibiotic-laden Spacers after Revision Surgery 
Bone  2014;0:128-136.
Mice are the small animal model of choice in biomedical research due to the low cost and availability of genetically engineered lines. However, the devices utilized in current mouse models of implant-associated bone infection have been limited to intramedullary or trans-cortical pins, which are not amenable to treatments involving extensive debridement of a full-thickness bone loss and placement of a segmental antibiotic spacer. To overcome these limitations, we developed a clinically faithful model that utilizes a locking fracture fixation plate to enable debridement of an infected segmental bone defect (full-thickness osteotomy) during a revision surgery, and investigated the therapeutic effects of placing an antibiotic-laden spacer in the segmental bone defect.
To first determine the ideal time point for revision following infection, a 0.7 mm osteotomy in the femoral mid-shaft was stabilized with a radiolucent PEEK fixation plate. The defect was inoculated with bioluminescent Staphylococcus aureus, and the infection was monitored over 14 days by bioluminescent imaging (BLI). Osteolysis and reactive bone formation were assessed by X-ray and micro-computed tomography (micro-CT). The active bacterial infection peaked by 5 days post-inoculation, however the stability of the implant fixation became compromised by 10–14 days post-inoculation due to osteolysis around the screws. Thus, day 7 was defined as the ideal time point to perform the revision surgery.
During the revision surgery, the infected tissue was debrided and the osteotomy was widened to 3 mm to place a poly-methyl methacrylate spacer, with or without vancomycin. Half of the groups also received systemic vancomycin for the remaining 21 days of the study. The viable bacteria remaining at the end of the study were measured using colony forming unit assays. Volumetric bone changes (osteolysis and reactive bone formation) were directly measured using micro-CT image analysis. Mice that were treated with local or systemic vancomycin did not display gross pathology at the end of the study. While localized vancomycin delivery alone tended to decrease the bacterial burden and osteolysis, these effects were only significant when combined with systemic antibiotic therapy.
This novel mouse model replicates key features of implant-associated osteomyelitis that make treatment extremely difficult, such as biofilm formation and osteolysis, and imitates the clinical practice of placing an antibiotic-laden spacer after infected tissue debridement. In addition, the model demonstrates the limitations of current PMMA spacers and could be an invaluable tool for evaluating alternative antimicrobial treatments for implant-associated bone infection.
doi:10.1016/j.bone.2014.11.019
PMCID: PMC4282971  PMID: 25459073
Bone infection; osteomyelitis; mouse; revision; antibiotics; PMMA
5.  Loss of the PGE2 Receptor EP1 enhances bone acquisition, which protects against age and ovariectomy-induced impairments in bone strength 
Bone  2014;72:92-100.
PGE2 exerts anabolic and catabolic effects on bone through the discrete actions of four prostanoid receptors (EP1-4). We have previously demonstrated that loss EP1 accelerates fracture repair by enhancing bone formation. In the present study we defined the role of EP1 in bone maintenance and homeostasis during aging and in response to ovariectomy. The femur and L4 vertebrae of wild type (WT) and EP1−/− mice were examined at 2-months, 6-months, and 1-year of age, and in WT and EP1−/− mice following ovariectomy (OVX) or sham surgery. Bone volume fraction, trabecular architecture and mechanical properties were maintained during aging in EP1−/− mice to a greater degree than age-matched WT mice. Moreover, significant increases in bone formation rate (BFR) (+60%) and mineral apposition rate (MAR) (+50%) were observed in EP1−/−, relative to WT, while no change in osteoclast number and osteoclast surface were observed. Following OVX, loss of EP1 was protective against bone loss in both femur and L4 vertebrae, with increased bone volume/total volume (BV/TV) (+32% in femur) and max load at failure (+10% in femur) relative to WT OVX, likely resulting from the increased bone formation rate that was observed in these mice. Taken together these studies identify inhibition of EP1 as a potential therapeutic approach to suppress bone loss in aged or post-menopausal patients.
doi:10.1016/j.bone.2014.11.018
PMCID: PMC4437532  PMID: 25446888
EP1; PGE2; ovariectomy; age-related bone loss; mouse model; bone homeostasis
6.  TGF-beta1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair 
Journal of cellular physiology  2015;230(2):318-326.
Flexor tendon injuries caused by deep lacerations to the hands are a challenging problem as they often result in debilitating adhesions that prevent the movement of the afflicted fingers. Evidence exists that tendon adhesions as well as scarring throughout the body are largely precipitated by the pleiotropic growth factor, TGF-β1, but the effects of TGF-β1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-β1 causes gene expression changes in tenocytes that are consistent with scar tissue and adhesion formation, including upregulation of the anti-fibrinolytic protein, PAI-1. Therefore, we hypothesized that TGF-β1 contributes to scarring and adhesions by reducing the activity of proteases responsible for ECM degradation and remodeling, such as plasmin and MMPs, via upregulation of PAI-1. To test our hypothesis, we examined the effects of TGF-β1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-β1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-β1 on protease activity were completely abolished in tendon cells from homozygous PAI-1 KO mice, which are unable to express PAI-1. Our findings support the hypothesis that TGF-β1 induces PAI-1, which suppresses plasmin and plasmin-mediated MMP activity, and provide evidence that PAI-1 may be a novel therapeutic target for preventing adhesions and promoting a scarless, regenerative repair of flexor tendon injuries.
doi:10.1002/jcp.24707
PMCID: PMC4276543  PMID: 24962629
TGF-β1; PAI-1; Tendon; Adhesions; MMP; Tenocyte; Plasmin
7.  Validation of power Doppler Versus Contrast Enhanced Magnetic Resonance Imaging Quantification of Joint Inflammation in Murine Inflammatory Arthritis 
Contrast enhancement magnetic resonance imaging (CE-MRI) of synovial volume is the radiographic gold standard to quantify joint inflammation but cost limits use. Therefore, we examined if power Doppler-ultrasound (PD-US) outcomes of synovitis in tumor necrosis factor transgenic (TNF-Tg) mice correlate with CE-MRI. TNF-Tg mice underwent PD-US of their knees to measure the joint space volume (JSV) and PD volume (PDV), and the results were correlated with synovial volume determined by CE-MRI. Immunohistochemistry for CD31 was performed to corroborate the PD signal. Synovial volume strongly correlated with both JSV and PDV (p<0.01). CD31+ blood vessels were observed in inflamed synovium proximal to the joint surface, which corresponded to areas of intense PD signals. JSV and PDV are valid measures of joint inflammation that correlate with synovial volume determined by CE-MRI and are associated with vascularity. Given the emergence of PD-US as a non-quantitative outcome of joint inflammation, we find JSV and PDV to be feasible and highly cost-effective for longitudinal studies in animal models. Furthermore, given the increasing utilization of PD-US in standard clinical practice, JSV and PDV could be translated to better quantify joint flare and response to therapy in RA patients.
doi:10.1002/jbmr.2392
PMCID: PMC4376603  PMID: 25359523
Bone ultrasound; Rheumatoid Arthritis (RA); Magnetic Resonance Imaging (MRI)
8.  Analysis of new bone, cartilage, and fibrosis tissue in healing murine allografts using whole slide imaging and a new automated histomorphometric algorithm 
Bone Research  2016;4:15037-.
Histomorphometric analysis of histologic sections of normal and diseased bone samples, such as healing allografts and fractures, is widely used in bone research. However, the utility of traditional semi-automated methods is limited because they are labor-intensive and can have high interobserver variability depending upon the parameters being assessed, and primary data cannot be re-analyzed automatically. Automated histomorphometry has long been recognized as a solution for these issues, and recently has become more feasible with the development of digital whole slide imaging and computerized image analysis systems that can interact with digital slides. Here, we describe the development and validation of an automated application (algorithm) using Visiopharm’s image analysis system to quantify newly formed bone, cartilage, and fibrous tissue in healing murine femoral allografts in high-quality digital images of H&E/alcian blue-stained decalcified histologic sections. To validate this algorithm, we compared the results obtained independently using OsteoMeasureTM and Visiopharm image analysis systems. The intraclass correlation coefficient between Visiopharm and OsteoMeasure was very close to one for all tissue elements tested, indicating nearly perfect reproducibility across methods. This new algorithm represents an accurate and labor-efficient method to quantify bone, cartilage, and fibrous tissue in healing mouse allografts.
doi:10.1038/boneres.2015.37
PMCID: PMC4717440  PMID: 26816658
9.  Are Biologic Treatments a Potential Approach to Wear- and Corrosion-related Problems? 
Where Are We Now?
Biological treatments, defined as any nonsurgical intervention whose primary mechanism of action is reducing the host response to wear and/or corrosion products, have long been postulated as solutions for osteolysis and aseptic loosening of total joint arthroplasties. Despite extensive research on drugs that target the inflammatory, osteoclastic, and osteogenic responses to wear debris, no biological treatment has emerged as an approved therapy. We review the extensive preclinical research and modest clinical research to date, which has led to the central conclusion that the osteoclast is the primary target. We also allude to the significant changes in health care, unabated safety concerns about chronic immunosuppressive/antiinflammatory therapies, industry’s complete lack of interest in developing an intervention for this condition, and the practical issues that have narrowly focused the possibilities for a biologic treatment for wear debris-induced osteolysis.
Where Do We Need to Go?
Based on the conclusions from research, and the economic, regulatory, and practical issues that limit the future directions toward the development of a biologic treatment, there are a few rational approaches that warrant investigation. These largely focus on FDA-approved osteoporosis therapies that target the osteoclast (bisphosphonates and anti-RANK ligand) and recombinant parathyroid hormone (teriparatide) prophylactic treatment to increase osseous integration of the prosthesis to overcome high-risk susceptibility to aseptic loosening. The other roadblock that must be overcome if there is to be an approved biologic therapy to prevent the progression of periprosthetic osteolysis and aseptic loosening is the development of radiological measures that can quantify a significant drug effect in a randomized, placebo-controlled clinical trial. We review the progress of volumetric quantification of osteolysis in animal studies and clinical pilots.
How Do We Get There?
Accepting the aforementioned rigid boundaries, we describe the emergence of repurposing FDA-approved drugs for new indications and public (National Institutes of Health, FDA, Centers for Disease Control and Prevention) and private (universities and drug and device manufactures) partnerships as the future roadmap for clinical translation. In the case of biologic treatments for wear debris-induced osteolysis, this will involve combined federal and industry funding of multicenter clinical trials that will be run by thought leaders at large medical centers.
Electronic supplementary material
The online version of this article (doi:10.1007/s11999-014-3765-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s11999-014-3765-9
PMCID: PMC4397762  PMID: 24993143
10.  Troponin T3 expression in skeletal and smooth muscle is required for growth and postnatal survival: Characterization of Tnnt3tm2a(KOMP)Wtsi mice 
Genesis (New York, N.Y. : 2000)  2013;51(9):667-675.
Summary
The troponin complex, which consists of three regulatory proteins (troponin C, troponin I and troponin T), is known to regulate muscle contraction in skeletal and cardiac muscle, but its role in smooth muscle remains controversial. Troponin T3 (TnnT3) is a fast skeletal muscle troponin believed to be expressed only in skeletal muscle cells. To determine the in vivo function and tissue specific expression of Tnnt3, we obtained the heterozygous Tnnt3+/flox/lacZ mice from Knockout Mouse Project (KOMP) Repository. Tnnt3lacZ/+ mice are smaller than their WT littermates throughout development, but do not display any gross phenotypes. Tnnt3lacZ/lacZ embryos are smaller than heterozygotes, and die shortly after birth. Histology revealed hemorrhagic tissue in Tnnt3lacZ/lacZ liver and kidney, which was not present in Tnnt3lacZ/+ or WT, but no other gross tissue abnormalities. X-gal staining for Tnnt3 promoter-driven lacZ transgene expression revealed positive staining in skeletal muscle and diapharam, and smooth muscle cells located in the aorta, bladder, and bronchus. Collectively, these findings suggest that troponins are expressed in smooth muscle, and are required for normal growth and breathing for postnatal survival. Moreover, future studies with this mouse model can explore TnnT3 function in adult muscle function using the conditional-inducible gene deletion approach.
doi:10.1002/dvg.22407
PMCID: PMC3787964  PMID: 23775847
Troponin; Knockout Mice; Muscle; Development
11.  Increased Insulin mRNA Binding Protein-3 Expression Correlates with Vascular Enhancement of Renal Cell Carcinoma by Intravenous Contrast-CT and is Associated with Bone Metastasis 
Journal of bone oncology  2015;4(3):69-76.
Purpose
To: 1) assess the correlation between CT vascularity and a candidate molecular marker of RCC metastasis (insulin-like mRNA binding protein-3 (IMP3)); and 2) demonstrate the differential expression of IMP3 in high vs. low vascular tumors.
Experimental Design
Retrospectively obtained contrast CT from 72 patients with primary RCC were used to establish threshold values for Low, Intermediate and High tumor vascularity. Paired histopathology specimens from 33 of these patients were used for immunohistochemistry (IHC) to correlate CT with IMP-3 expression. IMP-3 gene expression studies were performed on RCC and poorly vascular prostate cancer (PC) human bone metastases samples to confirm presence of IMP3 in metastatic samples from RCC. Gene expression studies were performed on RCC 786-O and PC3 cell lines to confirm the presence of high expression of IMP3 in the RCC cell line.
Results
IMP-3 expression positively correlated with CT vascular enhancement (p<0.01). IMP3 expression by IHC was strongly positive in all RCC, but weak in PC bone metastases. Real time RT-PCR demonstrated a significant 4-fold increase in imp-3 expression in RCC 786-O vs. PC3 cells in vitro (p<0.001).
Conclusion
Quantitation of pre-operative CT is a feasible method to phenotype primary RCC vascularity, which correlates with IMP-3 expression. In situ and cell line studies demonstrate an association between high IMP-3 expression and RCC bone metastasis. Studies aimed at defining the diagnostic potential of biomarkers for RCC bone metastasis, and functional significance of IMP-3 in RCC vascularity and tumor progression are warranted.
doi:10.1016/j.jbo.2015.07.001
PMCID: PMC4607090  PMID: 26478857
12.  PTH Enhanced Structural Allograft Healing is Associated with Decreased Angiopoietin-2 Mediated Arteriogenesis, Mast Cell Accumulation and Fibrosis 
Recombinant parathyroid hormone (rPTH) therapy has been evaluated for skeletal repair in animal studies and clinical trials based on its known anabolic effects, but its effects on angiogenesis and fibrosis remain poorly understood. We examined the effects of rPTH therapy on blood vessel formation and osseous integration in a murine femoral allograft model, which caused a significant increase in small vessel numbers, and decreased large vessel formation (p < 0.05). Histology showed that rPTH also reduced fibrosis around the allografts to similar levels observed in live autografts, and decreased mast cells at the graft-host junction. Similar effects on vasculogenesis and fibrosis were observed in femoral allografts from Col1caPTHR transgenic mice. Gene expression profiling revealed rPTH induced angiopoietin-1 (8-fold), while decreasing angiopoietin-2 (70-fold) at day 7 of allograft healing. Finally, we demonstrate anti-angiopoietin-2 peptibody(L1-10) treatment mimics rPTH effects on angiogenesis and fibrosis. Collectively, these findings demonstrate that intermittent rPTH treatment enhances structural allograft healing by two processes: 1) anabolic effects on new bone formation via small vessel angiogenesis, and 2) inhibition of angiopoietin-2 mediated arteriogenesis. The latter effect may function as a vascular sieve to limit mast cell access to the site of tissue repair, which decreases fibrosis around and between the fractured ends of bone. Thus, rPTH therapy may be generalizable to all forms of tissue repair that suffer from limited biointegration and excessive fibrosis.
doi:10.1002/jbmr.1765
PMCID: PMC3540116  PMID: 22991274
recombinant parathyroid hormone (rPTH); angiopoietin; arteriogenesis; vasculogenesis; angiogenesis; allograft healing; fibrosis; mast cell
13.  The Role of Bone Marrow Edema and Lymphangiogenesis in Inflammatory-Erosive Arthritis 
A common feature of autoimmune diseases is perpetual production of macrophage, dendritic and/or osteoclast effector cells, which mediate parenchymal tissue destruction in end organs. In support of this, we have demonstrated previously that patients and mice with inflammatory-erosive arthritis have a marked increase in circulating CD11b+ precursor cells, which are primed for osteoclastogenesis, and that this increase in osteoclast precursors (OCPs) is due to systemically increased TNF production. From these data, we proposed a unifying hypothesis to explain these osteoimmunologic findings during the pathogenesis of inflammatory-erosive arthritis, which has three postulates: 1) myelopoiesis chronically induce by TNF has profound effects on the bone marrow and joint tissues that should be evident from longitudinal MRI; 2) TNF alters the chemokine/chemokine receptor axis in the bone marrow to stimulate OCP release into the blood, and 3) OCP-mediated lymphangiogenesis occurs in the end organ as a compensatory mechanism to drain the inflammation and remove by-products of joint catabolism. Here, we describe our recent experimental findings that support these hypotheses and speculate on how this information can be used as diagnostic biomarkers and tools to discover novel therapies to treat patients with inflammatory-erosive arthritis.
doi:10.1007/978-1-4419-1050-9_1
PMCID: PMC3932510  PMID: 19950010
Inflammatory Arthritis; Lymphangiogenesis; In vivo Imaging; 3D-MRI
14.  Periosteal Progenitor Cell Fate in Segmental Cortical Bone Graft Transplantations: Implications for Functional Tissue Engineering 
A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ~70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP-2–producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene-enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.
Introduction
The loss of cellular activity in a structural bone allograft markedly reduces its healing potential compared with a live autograft. To further understand the cellular mechanisms for structural bone graft healing and repair and to devise a therapeutic strategy aimed at enhancing the performance of allograft, we established a segmental femoral structural bone graft model in mice that permits qualitative and quantitative analyses of graft healing and neovascularization.
Materials and Methods
Using this segmental femoral bone graft model, we transplanted live isografts harvested from Rosa 26A mice that constitutively express β-galactosidase into their wildtype control mice. In an attempt to emulate the osteogenic and angiogenic properties of periosteum, we applied a cell-based, adenovirus-mediated gene therapy approach to engraft BMP-2–producing bone marrow stromal cells onto devitalized allografts.
Results
X-gal staining for donor cells allowed monitoring the progression of periosteal progenitor cell fate and showed that 70% of osteogenesis was attributed to cellular proliferation and differentiation of donor progenitor cells on the surface of the live bone graft. Quantitative µCT analyses showed a 3-fold increase in new bone callus formation and a 6.8-fold increase in neovascularization for BMP-2/stromal cell–treated allograft compared with control acellular allografts. Histologic analyses showed the key features of autograft healing in the BMP-2/stromal cell–treated allografts, including the formation of a mineralized bone callus completely bridging the segmental defects, abundant neovascularization, and extensive resorption of bone graft.
Conclusions
The marked improvement of healing in these cellularized allografts suggests a clinical strategy for engineering a functional periosteum to improve the osteogenic and angiogenic properties of processed allografts.
doi:10.1359/JBMR.050806
PMCID: PMC4527562  PMID: 16294266
allografts; isografts; autografts; periosteum; functional tissue engineering
15.  Efficacy of B cell Depletion Therapy on Joint Flare is Associated with Increased Lymphatic Flow 
Arthritis and rheumatism  2013;65(1):130-138.
Objective
B cell depletion therapy (BCDT) ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritic flare in tumor necrosis factor transgenic (TNF-Tg) mice is associated with efferent lymph node (LN) “collapse,” triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. We examined whether BCDT efficacy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells.
Methods
We developed contrast-enhancement (CE) MRI imaging, near-infrared indocyanine green (NIR-ICG) imaging, and intravital immunofluorescent imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-Tg mice. We tested to see if BCDT efficacy is associated with restoration of lymphatic draining and cell egress from arthritic joints.
Results
Unlike active lymphatics to normal and pre-arthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescent imaging demonstrated that CD11b+ monocytes/macrophages in lymphatic vessels afferent to expanding LN travel at high velocity (186 ± 37 micrometer/sec), while these cells are stationary in lymphatic vessels afferent to collapsed PLN. BCDT of flaring TNF-Tg mice significantly decreased knee synovial volume by 50% from the baseline level, and significantly increased lymphatic clearance versus placebo (p<0.05). This increased lymphatic drainage restored macrophages egress from inflamed joints without recovery of the lymphatic pulse.
Conclusion
These results support a novel mechanism in which BCDT of flaring joints lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.
doi:10.1002/art.37709
PMCID: PMC3535508  PMID: 23002006
Rheumatoid Arthritis (RA); Flare; Tumor Necrosis Factor (TNF); B cells in Inflamed Lymph Nodes (B-in); Lymphatic Pulse
16.  Elucidating Bone Marrow Edema and Myelopoiesis in Murine Arthritis Using Contrast-Enhanced Magnetic Resonance Imaging 
Arthritis and rheumatism  2008;58(7):2019-2029.
Objective
While bone marrow edema (BME) detected by magnetic resonance imaging (MRI) is a biomarker of arthritis, its nature remains poorly understood due to the limitations of clinical studies. In this study, MRI of murine arthritis was used to elucidate its cellular composition and vascular involvement.
Methods
BME was quantified using normalized bone marrow intensity (NBMI) from precontrast MRI and normalized marrow contrast enhancement (NMCE) following intravenous administration of gadopentate dimeglumine. Wild-type (WT) and tumor necrosis factor (TNF)-transgenic mice were scanned from 2 to 5 months of age, followed by histologic or fluorescence-activated cell sorting (FACS) analysis of marrow. In efficacy studies, TNF-transgenic mice were treated with anti-TNF or placebo for 8 weeks, and then were studied using bimonthly MRI and histologic analysis.
Results
NBMI values were similar in WT and TNF-transgenic mice at 2 months. The values in WT mice steadily decreased thereafter, with mean values becoming significantly different from those of TNF-transgenic mice at 3.5 months (mean ± SD 0.29 ± 0.08 versus 0.46 ± 0.13; P < 0.05). Red to yellow marrow transformation occurred in WT but not TNF-transgenic mice, as observed histologically at 5 months. The marrow of TNF-transgenic mice that received anti-TNF therapy converted to yellow marrow, with lower NBMI values versus placebo at 6 weeks (mean ± SD 0.26 ± 0.07 versus 0.61 ± 0.22; P < 0.05). FACS analysis of bone marrow revealed a significant correlation between NBMI values and CD11b+ monocytes (R2 = 0.91, P = 0.0028). Thresholds for “normal” red marrow versus pathologic BME were established, and it was also found that inflammatory marrow is highly permeable to contrast agent.
Conclusion
BME signals in TNF-transgenic mice are caused by yellow to red marrow conversion, with increased myelopoiesis and increased marrow permeability. The factors that mediate these changes warrant further investigation.
doi:10.1002/art.23546
PMCID: PMC2572869  PMID: 18576355
17.  Remodeling of Murine Intrasynovial Tendon Adhesions following Injury: MMP and Neotendon Gene Expression 
Tendon injury frequently results in the formation of adhesions that reduce joint range of motion. To study the cellular, molecular, and biomechanical events involved in intrasynovial tendon healing and adhesion formation, we developed a murine flexor tendon healing model in which the flexor digitorum longus (FDL) tendon of C57BL/6 mice was transected and repaired using suture. This model was used to test the hypothesis that murine flexor tendons heal with differential expression of matrix metalloproteases (MMPs), resulting in the formation of scar tissue as well as the subsequent remodeling of scar and adhesions. Healing tendons were evaluated by histology, gene expression via real-time RT-PCR, and in situ hybridization, as well as biomechanical testing to assess the metatarsophalangeal (MTP) joint flexion range of motion (ROM) and the tensile failure properties. Tendons healed with a highly disorganized fibroblastic tissue response that was progressively remodeled through day 35 resulting in a more organized pattern of collagen fibers. Initial repair involved elevated levels of Mmp-9 at day 7, which is associated with catabolism of damaged collagen fibers. High levels of Col3 are consistent with scar tissue, and gradually transition to the expression of Col1. Scleraxis expression peaked at day 7, but the expression was limited to the original tendon adjacent to the injury site, and no expression was present in granulation tissue involved in the repair response. The MTP joint ROM with standardized force on the tendon was decreased on days 14 and 21 compared to day 0, indicating the presence of adhesions. Peak expressions of Mmp-2 and Mmp-14 were observed at day 21, associated with tendon remodeling. At day 28, two genes associated with neotendon formation, Smad8 and Gdf-5, were elevated and an improvement in MTP ROM occurred. Tensile strength of the tendon progressively increased, but by 63 days the repaired tendons had not reached the tensile strength of normal tendon. The murine model of primary tendon repair, described here, provides a novel mechanism to study the tendon healing process, and further enhances the understanding of this process at the molecular, cellular, and biomechanical level.
doi:10.1002/jor.20769
PMCID: PMC4472455  PMID: 19051246
flexor tendon healing; matrix metalloproteases; adhesions; mouse model; biomechanics
18.  Validation of GAITRite and PROMIS as High-Throughput Physical Function Outcome Measures Following ACL Reconstruction 
New healthcare demands for quality measures of elective procedures, such as anterior cruciate ligament (ACL) reconstructive surgery, warrant the establishment of high through-put outcomes for high volume clinics. To this end we evaluated the PROMIS and GAITRite as physical function outcome measures to quantify early healing and post-operative complications in 106 patients at pre-op and 3, 10, 20 and 52 weeks post-ACL reconstruction with bone-tendon-bone autograft, and compared the results to the current IKDC validated outcome measure. The results showed that both PROMIS and GAITRite were significantly quicker to administer versus IKDC (p < 0.0001). Additional advantages were that PROMIS and GAITRite detected a significant decrease in physical function at 3 weeks post-op, and a significant improvement at 10 weeks post-op, versus pre-op (p<0.001), which were not detected with IKDC. GAITRite was limited by a low ceiling that could not detect improvement of physical function beyond 20 weeks, while both PROMIS and IKDC detected significant improvement out to 52 weeks postop (p<0.001). Linear regressions demonstrated a significant relationship between IKDC and PROMIS, with a combined correlation value of 0.8954 (p<.001) for all time points. Finally, ROC curve analysis demonstrated that PROMIS is a diagnostic test for poor outcomes.
doi:10.1002/jor.22591
PMCID: PMC3976703  PMID: 24532421
ACL reconstruction; PROMIS; Physical Function; Gait
20.  Aging Periosteal Progenitor Cells have Reduced Regenerative Responsiveness to Bone Injury and to the Anabolic Actions of PTH 1-34 Treatment 
Bone  2014;62:79-89.
A stabilized tibia fracture model was used in young (8-week old) and aged (1-year old) mice to define the relative bone regenerative potential and the relative responsiveness of the periosteal progenitor population with aging and PTH 1-34 (PTH) systemic therapy. Bone regeneration was assessed through gene expressions, radiographic imaging, histology/histomorphometry, and biomechanical testing. Radiographs and microCT showed increased calcified callus tissue and enhanced bone healing in young compared to aged mice. A key mechanism involved reduced proliferation, expansion, and differentiation of periosteal progenitor cell populations in aged mice. The experiments showed that PTH increased calcified callus tissue and torsional strength with a greater response in young mice. Histology and quantitative histomorphometry confirmed that PTH increased callus tissue area due primarily to an increase in bone formation, since minimal changes in cartilage and mesenchyme tissue area occurred. Periosteum examined at 3, 5, and 7 days showed that PTH increased cyclin D1 expression, the total number of cells in the periosteum, and width of the periosteal regenerative tissue. Gene expression showed that aging delayed differentiation of both bone and cartilage tissues during fracture healing. PTH resulted in sustained Col10a1 expression consistent with delayed chondrocyte maturation, but otherwise minimally altered cartilage gene expression. In contrast, PTH 1-34 stimulated expression of Runx2 and Osterix, but resulted in reduced Osteocalcin. β-catenin staining was present in mesenchymal chondroprogenitors and chondrocytes in early fracture healing, but was most intense in osteoblastic cells at later times. PTH increased active β-catenin staining in the osteoblast populations of both young and aged mice, but had a lesser effect in cartilage. Altogether the findings show that reduced fracture healing in aging involves decreased proliferation and differentiation of stem cells lining the bone surface. While PTH 1-34 enhances the proliferation and expansion of the periosteal stem cell population and accelerates bone formation and fracture healing, the effects are proportionately reduced in aged mice compared to young mice. β-catenin is induced by PTH in early and late fracture healing and is a potential target of PTH 1-34 effects.
doi:10.1016/j.bone.2014.02.002
PMCID: PMC4085793  PMID: 24530870
Fracture healing; Parathyroid hormone; Periosteum; Wnt-β-catenin signaling; Mesenchymal stem cell
21.  3D Printing of Composite Calcium Phosphate and Collagen Scaffolds for Bone Regeneration 
Biomaterials  2014;35(13):4026-4034.
Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1–2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.
doi:10.1016/j.biomaterials.2014.01.064
PMCID: PMC4065717  PMID: 24529628
22.  Investigation of the peak action wavelength of light-activated gene transduction (LAGT) 
Gene therapy  2011;18(11):1043-1051.
Light-activated gene transduction (LAGT) is an approach to localize gene therapy via preactivation of cells with UV light, which facilitates transduction by recombinant adeno-associated virus vectors. Prior studies demonstrated that UVC induces LAGT secondary to pyrimidine dimer formation, while UVA induces LAGT secondary to reactive oxygen species (ROS) generation. However, the empirical UVB boundary of these UV effects is unknown. Thus, we aimed to define the action spectra for UV-induced LAGT independent of DNA damage, and determine an optimal wavelength to maximize safety and efficacy. Results: UV at 288, 311 and 320nm produced significant dose-dependent LAGT effects, of which the maximum (800-fold) was observed with 4kJ/m2 at 311nm. Consistent with its robust cytotoxicity, 288nm produced significantly high levels of DNA damage at all doses tested, while 311, 320 and 330nm did not generate pyrimidine dimers and produced low levels of DNA damage detected by comet assay. While 288nm failed to induce ROS, the other wavelengths were effective, with the maximum (10-fold) effect observed with 30 kJ/m2 at 311nm. An in vivo pilot study assessing 311nm-induced LAGT of rabbit articular chondrocytes demonstrated a significant 6.6-fold (p<0.05) increase in transduction with insignificant cytotoxicity. Conclusion: 311nm was found to be the optimal wavelength for LAGT based on its superior efficacy at the peak dose, and its broad safety range that is remarkably wider than the other UV wavelengths tested.
doi:10.1038/gt.2011.47
PMCID: PMC3530381  PMID: 21490685
23.  Measuring intranodal pressure and lymph viscosity to elucidate mechanisms of arthritic flare and therapeutic outcomes 
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints, whose etiology is largely unknown. Recent studies in mice demonstrated alterations in lymphatics from affected joints precede flares. Thus, we aimed to develop novel methods for measuring lymph node pressure and lymph viscosity in limbs of mice. Pressure measurements were performed by inserting a glass micropipette connected to a pressure transducer into popliteal lymph nodes (PLN) or axillary lymph nodes (ALN) of mice and determined that the lymphatic pressures were 9 and 12 cm of water, respectively. We are also developing methods for measuring lymph viscosity in lymphatic vessels afferent to PLN, which can be measured by multi-photon fluorescence recovery after photobleaching (MP-FRAP) of FITC-BSA injected into the hind footpad. These results demonstrate the potential of lymph node pressure and lymph viscosity measurements, and warrant future studies to test these outcomes as biomarkers of arthritic flare.
doi:10.1111/j.1749-6632.2011.06237.x
PMCID: PMC3334848  PMID: 22172039
Rheumatoid Arthritis; Lymph Node; Flare; Lymphatic Pressure; Lymph Viscosity
24.  Cartilage Transplants Hold Promise for Challenging Bone Defects 
Nature reviews. Rheumatology  2014;10(3):129-130.
The challenges of healing have led investigators to question existing paradigms in the hopes of uncovering overlooked solutions. Such is the case in a recent study showing that introduction of a cartilage construct into a mouse tibial defect induces remarkable healing owing to the transformation of donor chondrocytes into new bone.
doi:10.1038/nrrheum.2013.216
PMCID: PMC4312000  PMID: 24418762
25.  TNF is required for the induction but not the maintenance of compression-induced BME signals in murine tail vertebrae: limitations of anti-TNF therapy for degenerative disc disease 
While bone marrow edema (BME) is diagnostic of spondyloarthropathy, its nature remains poorly understood. In contrast, BME in ankylosing spondylitis is caused by TNF-induced vascular and cellular changes. To investigate the relationship between chronic compression and TNF signaling in compression induced BME we utilized a tail vertebrae compression model with WT, TNF-Tg and TNFR1&2−/− mice to evaluate: 1) healing following release of chronic compression, 2) induction of BME in the absence of TNFR, and 3) efficacy of anti-TNF therapy. Compression-induced normalized marrow contrast enhancement (NMCE) in WT was significantly decreased 3-fold (p<0.01) within 2 weeks of release, while the NMCE values in TNF-Tg vertebrae remained elevated, but had a significant decrease (p<0.05) by 6 weeks after the release of compression. TNFR1&2−/− mice were resistant to compression-induced BME. Anti-TNF therapy did not affect NMCE vs. placebo. Histological examination revealed that NMCE values significantly correlated with marrow vascularity and cellularity (p<0.05), which account for 76% of the variability of NMCE. Collectively, these data demonstrate a critical role for TNF in the induction of chronic compression-induced BME, but not in its maintenance. Amelioration of BME is achieved through biomechanical stability, but is not affected by anti-TNF therapy.
doi:10.1002/jor.21370
PMCID: PMC3076794  PMID: 21445993
Modic Changes; CE-MRI; Bone Marrow Edema; Anti-TNF Therapy

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