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1.  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.
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.
PMCID: PMC3787964  PMID: 23775847
Troponin; Knockout Mice; Muscle; Development
2.  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.
PMCID: PMC3540116  PMID: 22991274
recombinant parathyroid hormone (rPTH); angiopoietin; arteriogenesis; vasculogenesis; angiogenesis; allograft healing; fibrosis; mast cell
3.  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.
PMCID: PMC3932510  PMID: 19950010
Inflammatory Arthritis; Lymphangiogenesis; In vivo Imaging; 3D-MRI
4.  Efficacy of B cell Depletion Therapy on Joint Flare is Associated with Increased Lymphatic Flow 
Arthritis and rheumatism  2013;65(1):130-138.
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.
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.
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.
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.
PMCID: PMC3535508  PMID: 23002006
Rheumatoid Arthritis (RA); Flare; Tumor Necrosis Factor (TNF); B cells in Inflamed Lymph Nodes (B-in); Lymphatic Pulse
5.  Elucidating Bone Marrow Edema and Myelopoiesis in Murine Arthritis Using Contrast-Enhanced Magnetic Resonance Imaging 
Arthritis and rheumatism  2008;58(7):2019-2029.
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.
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.
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.
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.
PMCID: PMC2572869  PMID: 18576355
7.  The Murine Femoral Allograft Model and a Semi-automated Histomorphometric Analysis Tool 
Preclinical studies on bone repair remain a high priority due to the unresolved clinical problems associated with treating critical segmental defects and complications of fracture healing. Over the last decade the murine femoral allograft model has gained popularity due to its standardized surgery and potential for examining a vast array of radiographic, biomechanical and histological outcome measures. Here, we describe these methods and a novel semi-automated histomorphometric approach to quantify the amount of bone, cartilage and undifferentiated mesenchymal tissue in demineralized paraffin sections of allografted murine femurs using the VisioPharm Image Analysis Software System.
PMCID: PMC4003505  PMID: 24482164
VisioPharm; Histomorphometry; Bone Repair; Allograft
8.  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.
PMCID: PMC3902084  PMID: 24131143
Parathyroid Hormone; endogenous stem cells; osteogenesis; allograft; calvarial bone repair
9.  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.
PMCID: PMC3530381  PMID: 21490685
10.  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.
PMCID: PMC3334848  PMID: 22172039
Rheumatoid Arthritis; Lymph Node; Flare; Lymphatic Pressure; Lymph Viscosity
11.  Divergent Gene Activation in Peripheral Blood and Tissues of Patients with Rheumatoid Arthritis, Psoriatic Arthritis and Psoriasis following Infliximab Therapy 
PLoS ONE  2014;9(10):e110657.
The immune inflammatory disorders rheumatoid arthritis (RA), psoriatic arthritis (PsA) and psoriasis (Ps) share common pathologic features and show responsiveness to anti-tumor necrosis factor (TNF) agents yet they are phenotypically distinct. The aim of this study was to examine if anti-TNF therapy is associated with divergent gene expression profiles in circulating cells and target tissues of patients with these diseases.
Peripheral blood CD14+ and CD14− cells were isolated from 9 RA, 12 PsA and 10 Ps patients before and after infliximab (IFX) treatment. Paired synovial (n = 3, RA, PsA) and skin biopsies (n = 5, Ps) were also collected. Gene expression was analyzed by microarrays.
26 out of 31 subjects responded to IFX. The transcriptional response of CD14+ cells to IFX was unique for the three diseases, with little overlap (<25%) in significantly changed gene lists (with PsA having the largest number of changed genes). In Ps, altered gene expression was more pronounced in lesional skin (relative to paired, healthy skin) compared to blood (relative to healthy controls). Marked suppression of up-regulated genes in affected skin was noted 2 weeks after therapy but the expression patterns differed from uninvolved skin. Divergent patterns of expression were noted between the blood cells and skin or synovial tissues in individual patients. Functions that promote cell differentiation, proliferation and apoptosis in all three diseases were enriched. RA was enriched in functions in CD14− cells, PsA in CD14+ cells and Ps in both CD14+ and CD14− cells, however, the specific functions showed little overlap in the 3 disorders.
Divergent patterns of altered gene expression are observed in RA, PsA and Ps patients in blood cells and target organs in IFX responders. Differential gene expression profiles in the blood do not correlate with those in target organs.
PMCID: PMC4204991  PMID: 25333715
12.  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.
PMCID: PMC3076794  PMID: 21445993
Modic Changes; CE-MRI; Bone Marrow Edema; Anti-TNF Therapy
13.  Endogenous Tissue Engineering: PTH Therapy for Skeletal Repair 
Cell and Tissue Research  2011;347(3):545-552.
Based on its proven anabolic effects on bone in osteoporosis patients, recombinant parathyroid hormone (PTH1-34) has been evaluated as a potential therapy for skeletal repair. Research in animals has investigated the effect of PTH1-34 in various skeletal repair models such as fractures, allografting, spinal arthrodesis, and distraction osteogenesis. These studies demonstrated that intermittent PTH1-34 treatment enhances and accelerates the skeletal repair process via a number of mechanisms, which includes effects on mesenchymal stem cells (MSC), angiogenesis, chondrogenesis, bone formation and resorption. Furthermore, PTH1-34 was demonstrated to enhance bone repair in challenging animal models of aging, inflammatory arthritis and glucocorticoid-induced bone loss. This pre-clinical success has led to off-label clinical use, and a number of case reports documenting PTH1-34 treatment of delayed-unions and non-unions have been publish. Moreover, a phase 2 clinical trial of PTH1-34 treatment of patient with a radius fracture has now been completed. Although this trial failed to achieve its primary outcome, largely due to effective healing in the placebo group, several secondary outcomes were statistically significant, highlighted several important issues about the appropriate patient population for PTH1-34 therapy for skeletal repair. Here we review our current knowledge of the effects of PTH1-34 therapy for bone healing, enumerate several critical unresolved issues (e.g. appropriate dosing regimen and indications), and discuss this drug’s long term potential as an adjuvant for endogenous tissue engineering.
PMCID: PMC3338097  PMID: 21626290
Parathyroid Hormone (PTH); skeletal repair; fracture insufficiency; allograft
14.  Chronic axial compression of the mouse tail segment induces MRI bone marrow edema changes that correlate with increased marrow vasculature and cellularity 
MRI of bone marrow edema (BME) has been found to be helpful in the diagnosis of back pain attributed to degenerative disk disease (DDD) and spondyloarthropathy (SA), but its interpretation is limited by a lack of knowledge of its nature and natural history. To address this, we assessed effects of compressive forces to mouse tail segments of WT and TNF-Tg mice with SA, via contrast enhanced MRI and histology. Normalized marrow contrast enhancement (NMCE) of uninstrumented WT vertebrae significantly decrease 3-fold (p<0.01) from 8 to 12 weeks of age, consistent with red to yellow marrow conversion, while the NMCE of TNF-Tg vertebrae remained elevated. Chronic compressive loading 6X body weight to WT tails increased NMCE 2-fold (p<0.02) within 2-weeks, which was equal to 6X loaded TNF-Tg tails within 4-weeks. Histology confirmed degenerative changes and that load-induced NMCE corresponded to increased vascular sinus tissue (35± 3% vs. 19± 3%; p<0.01) and cellularity (4,235± 886 vs.1,468± 320 cells/mm2; p<0.01) for the loaded vs. unloaded WT respectively. However, micro-CT analyses failed to detect significant load-induced changes to bone. While the bone marrow of loaded WT and TNF-Tg vertebrae were similar, histology demonstrated mild cellular infiltrate and increased osteoclastic resorption in the WT tails versus severe inflammatory-erosive arthritis in TNF-Tg joints. Significant (p<0.05) decreases in cortical and trabecular bone volume in uninstrumented TNF-Tg vs. WT vertebrae were confirmed by micro-CT. Thus, chronic load-induced DDD causes BME signals in vertebrae similar to those observed from Ankylosing Spondylitis (AS), and both DDD and AS signals correlate with a conversion from yellow to red marrow, with increased vascularity.
PMCID: PMC2891234  PMID: 20187115
Modic Changes; CE-MRI; Bone Marrow Edema; Vertebral Degeneration
15.  Nonerosive arthritis in lupus is mediated by IFN-α stimulated monocyte differentiation that is nonpermissive of osteoclastogenesis 
Arthritis and rheumatism  2010;62(4):1127-1137.
In contrast to rheumatoid arthritis (RA), Jaccoud arthritis (JA) joint inflammation in systemic lupus erythematosus (SLE) is nonerosive. Although the mechanism responsible is unknown, the anti-osteoclastogenic cytokine interferon-alpha (IFN-α), whose transcriptome is present in SLE monocytes, may be responsible. To test this, we examined effects of IFN-α versus lupus disease on osteoclasts and erosion in the NZBxNZW F1 SLE mouse model with K/BxN serum-induced arthritis (SIA).
Elevated systemic IFN-α levels were obtained by administration of an adenoviral vector expressing IFN-α (Ad-IFN-α). SLE disease was marked by anti-dsDNA antibody titer and proteinuria, and Ifi202 and Mx1 expression represented the IFN-α transcriptome. Micro-CT was used to evaluate bone erosions. Flow cytometry for CD11b and CD11c was used to evaluate the frequency of circulating osteoclast precursors (OCP) and myeloid dendritic cells (mDC) in blood.
Administration of Ad-IFN-α to NZBxNZW F1 mice induced osteopetrosis. Pre-autoimmune NZBxNZW F1 mice are fully susceptible to focal erosions in the setting of SIA. However, NZBxNZW F1 mice with high anti-dsDNA antibody titers and the IFN-α transcriptome were protected against bone erosions. Ad-IFN-α pre-treatment of NZW mice before K/BxN serum administration also resulted in protection against bone erosion (r2=0.4720, p<0.01), which was associated with a decrease in circulating CD11b+CD11c− OCP, and a concomitant increase in CD11b+CD11c+ cells (r2=0.6330, p<0.05) that are phenotypic of mDC.
These findings suggest that IFN-α in SLE shifts monocyte development toward mDC at the expense of osteoclastogenesis thereby resulting in decreased bone erosion.
PMCID: PMC2854832  PMID: 20131244
Jaccoud arthritis (JA); Lupus; Osteoclast; Interferon-alpha (IFN-α)
16.  Effects of anti-resorptive agents on osteomyelitis: Novel insights on osteonecrosis of the jaw (ONJ) pathogenesis 
The effects of anti-resorptive agents (i.e. alendronate (Aln), osteoprotegerin (OPG)) on bone infection are unknown. Thus, their effects on implant-associated osteomyelitis (OM) were investigated in mice using PBS (placebo), gentamycin and etanercept (TNFR:Fc) controls. None of the drugs affected humoral immunity, angiogenesis, or chronic infection. However, the significant (p<0.05 vs. PBS) inhibition of cortical osteolysis and decreased draining lymph node size in Aln and OPG treated mice was associated with a significant (p<0.05) increase in the incidence of high-grade infections during the establishment of OM. In contrast, the high-grade infections in TNFR:Fc treated mice were associated with immunosuppression, as evidenced by the absence of granulomas and presence of Gram+ biofilm in the bone marrow. Collectively, these findings indicate that while anti-resorptive agents do not exacerbate chronic OM, they can increase the bacterial load during early infection by decreasing lymphatic drainage and preventing the removal of necrotic bone that harbors the bacteria.
PMCID: PMC2947350  PMID: 20392222
17.  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.
PMCID: PMC2865989  PMID: 20143927
18.  Cellular and Molecular Factors in Flexor Tendon Repair and Adhesions: A Histological and Gene Expression Analysis 
Connective tissue research  2013;54(3):218-226.
Flexor tendon healing is mediated by cell proliferation, migration, and ECM synthesis that contribute to the formation of scar tissue and adhesion. The biological mechanisms of flexor tendon adhesion formation has been linked to TGF-β. To elucidate the cellular and molecular events in this pathology, we implanted live FDL grafts from the reporter mouse Rosa26LacZ/+ in WT recipients, and used histological β-galactosidase (β-gal) staining to evaluate the intrinsic versus extrinsic cellular origins of scar, and RT-PCR to measure gene expression of TGF-β and its receptors, extracellular matrix (ECM) proteins, and MMPs and their regulators. Over the course of healing, graft cellularity and β-gal activity progressively increased, and β-gal-positive cells migrated out of the Rosa26LacZ/+ graft. In addition, there was evidence of influx of host cells (β-gal-negative) into the gliding space and the graft, suggesting that both graft and host cells contribute to adhesions. Interestingly, we observed a biphasic pattern in which Tgfb1 expression was highest in the early phases of healing and gradually decreased thereafter, whereas Tgfb3 increased and remained upregulated later. The expression of TGF-β receptors was also upregulated throughout the healing phases. In addition, type III collagen and fibronectin were upregulated during the proliferative phase of healing, confirming that murine flexor tendon heals by scar tissue. Furthermore, gene expression of MMPs showed a differential pattern in which inflammatory MMPs were highest early and matrix MMPs increased over time. These findings offer important insights into the complex cellular and molecular factors during flexor tendon healing.
PMCID: PMC3697755  PMID: 23586515
Flexor tendon; Tendoplasty; Autograft; Allograft; Adhesions; Tenocytes; Transforming Growth Factor; Extracellular Matrix; Matrix Metalloproteinase
19.  Freeze-dried allograft-mediated gene or protein delivery of growth and differentiation factor 5 reduces reconstructed murine flexor tendon adhesions 
Journal of Tissue Engineering  2014;5:2041731414528736.
Advances in allograft processing have opened new horizons for clinical adaptation of flexor tendon allografts as delivery scaffolds for antifibrotic therapeutics. Recombinant adeno-associated-virus (rAAV) gene delivery of the growth and differentiation factor 5 (GDF-5) has been previously associated with antifibrotic effects in a mouse model of flexor tendoplasty. In this study, we compared the effects of loading freeze-dried allografts with different doses of GDF-5 protein or rAAV-Gdf5 on flexor tendon healing and adhesions. We first optimized the protein and viral loading parameters using reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and in vivo bioluminescent imaging. We then reconstructed flexor digitorum longus (FDL) tendons of the mouse hindlimb with allografts loaded with low and high doses of recombinant GDF-5 protein and rAAV-Gdf5 and evaluated joint flexion and biomechanical properties of the reconstructed tendon. In vitro optimization studies determined that both the loading time and concentration of the growth factor and viral vector had dose-dependent effects on their retention on the freeze-dried allograft. In vivo data suggest that protein and gene delivery of GDF-5 had equivalent effects on improving joint flexion function, in the range of doses used. Within the doses tested, the lower doses of GDF-5 had more potent effects on suppressing adhesions without adversely affecting the strength of the repair. These findings indicate equivalent antifibrotic effects of Gdf5 gene and protein delivery, but suggest that localized delivery of this potent factor should also carefully consider the dosage used to eliminate untoward effects, regardless of the delivery mode.
PMCID: PMC4014079  PMID: 24812579
Flexor tendon; allograft; adhesions; growth and differentiation factor 5; tissue engineering
20.  The Emerging Field of Osteoimmunology 
Immunologic research  2009;10.1007/s12026-009-8093-x.
Recent studies have elucidated unanticipated connections between the immune and skeletal systems, and this relationship has led to the development of a new field known as osteoimmunology. The goal of research in this field is to: 1) further understand how the bone microenvironment influences immune cell ontogeny and subsequent effector functions, and 2) translate basic science findings in bone biology to clinical applications for autoimmune diseases that target the skeleton such as rheumatoid arthritis. In this review, we will examine the recent findings of the interplay between the immune and skeletal systems. This discussion will focus on the cells and signaling pathways in osteoimmune interactions and how innate and adaptive immune effector cells as well as cytokines and chemokines play a role in the maintenance and dysregulation of skeletal-immune homeostasis. We will also discuss how immunomodulatory biologic drugs, which specifically target these cells and effector molecules, have transformed the treatment of autoimmune mediated inflammatory diseases (IMIDs) and metabolic bone diseases such as osteoporosis.
PMCID: PMC2934875  PMID: 19184540
Osteoimmunology; receptor-activator of nuclear factor kappa B (RANK); RANK-ligand; osteoprotegerin (OPG); arthritis; osteoporosis
21.  Efficacy of Colistin Impregnated Beads to Prevent Multi-drug Resistant A. baumannii Implant-Associated Osteomyelitis 
Osteomyelitis (OM) from multidrug-resistant (MDR) Acinetobacter has emerged in >30% of combat-related injuries in Iraq and Afghanistan. While most of these strains are sensitive to colistin, the drug is not availible in bone void fillers for local high-dose delivery. To address this we developed a mouse model with MDR strains isolated from wounded military personnel. In contrast to S. aureus OM, which is osteolytic and characterized by biofilm in necrotic bone, A. baumannii OM results in blastic lesions that do not contain apparent biofilm. We also found that mice mount a specific IgG response against 3 proteins (40, 47 & 56KDa) regardless of the strain used, suggesting that these may be immunuo-dominant antigens. PCR for the A. baumannii specific parC gene confirmed a 100% infection rate with 75% of the MDR strains, and in vitro testing confirmed that all strains were sensitive to colistin. We also developed a real-time quantitative PCR (RTQ-PCR) assay that could detect as few as 10 copies of parC in a sample. To demonstrate the efficacy of colistin prophylaxis in this model, mice were treated with either parenteral colistin (0.2mg colistinmethate i.m. for 7 days), local colistin (PMMA bead impregnated with 1.0mg colistin sulfate), or an unloaded PMMA bead control. While the parenteral colistin failed to demonstrate any significant effects vs. the placebo, the colistin PMMA bead significantly reduced the infection rate such that only 29.2% of the mice had detectable levels of parC at 19 days (p<0.05 vs. i.m. colistin and placebo).
PMCID: PMC2810872  PMID: 19173261
Multi-Drug Resistant; Acinetobacter baumannii; Osteomyelitis; Colistin
22.  The effect of mesenchymal stem cell sheets on structural allograft healing of critical-sized femoral defects in mice 
Biomaterials  2014;35(9):2752-2759.
Structural bone allografts are widely used in the clinic to treat critical sized bone defects, despite lacking the osteoinductive characteristics of live autografts. To address this, we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets, which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates, as a tissue engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets, suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo, allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of x-ray plain radiography, 3D microCT, histology, and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation, as well as graft-host osteointegration. Moreover, a large periosteal callus was observed spanning the allografts with MSC sheets, which partially mimics live autograft healing. Finally, biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together, MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair, demonstrating the feasibility of this tissue engineering solution for massive allograft healing.
PMCID: PMC3913373  PMID: 24393269
23.  Bone Fragility Beyond Strength and Mineral Density: Raman Spectroscopy Predicts Femoral Fracture Toughness in a Murine Model of Rheumatoid Arthritis 
Journal of biomechanics  2012;46(4):723-730.
Clinical prediction of bone fracture risk primarily relies on measures of bone mineral density (BMD). BMD is strongly correlated with bone strength, but strength is independent of fracture toughness, which refers to the bone’s resistance to crack initiation and propagation. In that sense, fracture toughness is more relevant to assessing fragility-related fracture risk, independent of trauma. We hypothesized that bone biochemistry, determined by Raman spectroscopy, predicts bone fracture toughness better than BMD. This hypothesis was tested in tumor necrosis factor-transgenic mice (TNF-tg), which develop inflammatory-erosive arthritis and osteoporosis. The left femurs of TNF-tg and wild type (WT) littermates were measured with Raman spectroscopy and micro-computed tomography. Fracture toughness was assessed by cutting a sharp notch into the anterior surface of the femoral mid-diaphysis and propagating the crack under 3 point bending. Femoral fracture toughness of TNF-tg mice was significantly reduced compared to WT controls (p=0.04). A Raman spectrum-based prediction model of fracture toughness was generated by partial least squares regression (PLSR). Raman spectrum PLSR analysis produced strong predictions of fracture toughness, while BMD was not significantly correlated and produced very weak predictions. Raman spectral components associated with mineralization quality and bone collagen were strongly leveraged in predicting fracture toughness, reiterating the limitations of mineralization density alone.
PMCID: PMC3568260  PMID: 23261243
Fracture Toughness; Raman Spectroscopy; Bone Mineral Density; Bone Quality; Inflammatory Arthritis
24.  Differential effects of biologic vs bisphosphonate inhibition of wear debris-induced osteolysis assessed by longitudinal micro-CT 
Aseptic loosening secondary to periprosthetic osteolysis remains a serious orthopaedic problem and the greatest limitation of total joint replacement. This process is caused by wear debris-induced osteoclastic bone resorption, for which effective small molecule (bisphosphonates, BPs) and biologic (RANK antagonists) drugs have been developed. While BPs have proven to be effective in preventing metabolic bone loss in non-inflammatory conditions such as osteoporosis, they do not have the same efficacy in the setting of inflammatory bone loss such as that observed in periprosthetic osteolysis. Since this difference has been attributed to the anti-apoptotic inflammatory signals that protect osteoclasts from BP-induce apoptosis, but not RANK antagonists, we tested the hypothesis that osteoprotegerin (OPG) is more effective in preventing wear debris-induced osteolysis than zoledronic acid (ZA) or alendronate (Aln) in the murine calvaria model. Based on the shortcomings of previous animal studies that focused on 2D imaging and histology endpoints, and the emergence of quantitative 3D-CT, we developed in vivo micro-CT methods for the murine calvaria model to more rigorously test our hypothesis and correlate the osteolysis results with traditional histology. Although this approach proved to be incompatible with titanium (Ti) particles, due to metal artifact, we were able to demonstrate a 3.2-fold increase in osteolytic volume over 10 days induced by ultra high molecular weight polyethylene (PE) particles vs. sham controls (0.49 +/− 0.23mm3 vs. 0.15 +/− 0.067mm3; p<0.01). While OPG and high dose ZA completely inhibited this PE-induced osteolysis (p<0.001), pharmacological doses of ZA and Aln were less effective but still reached statistical significance (p<0.05). Traditional histomorphometry of the sagital suture area of calvaria from both Ti and PE treated mice confirmed the remarkable suppression of resorption by OPG (p<0.001) vs. the lack of effect by physiological BPs. The differences in drug effects on osteolysis were largely explained by the significant difference in osteoclast numbers observed between OPG vs. BPs in both Ti and PE treated calvaria; and linear regression analyses that demonstrated a highly significant correlation between osteolysis volume and sagittal suture area vs. osteoclast numbers (p<0.001). Taken together our results demonstrate the sensitivity and utility of in vivo 3D-CT to detect the effects of BPs on wear debris-induced osteolysis that could not be observed by histology alone; and that the greater suppression of bone resorption observed with OPG treatment vs. BPs is due to its ability to dramatically reduce osteoclast numbers in the presence of inflammatory signals.
PMCID: PMC2742224  PMID: 18404739
Aseptic Loosening; wear debris; osteolysis; osteoprotegerin (OPG); bisphosphonate (BP); 3D-micro-CT
25.  The effect of surface demineralization of cortical bone allograft on the properties of recombinant adeno-associated virus coatings 
Biomaterials  2008;29(28):3882-3887.
Freeze-dried recombinant adeno-associated virus (rAAV) coated structural allografts have emerged as an approach to engender necrotic cortical bone with host factors that will persist for weeks following surgery to facilitate revascularization, osteointegration, and remodeling. However, one major limitation is the nonporous cortical surface that prohibits uniform distribution of the rAAV coating prior to freeze-drying. To overcome this we have developed a demineralization method to increase surface absorbance while retaining the structural integrity of the allograft. Demineralized bone wafers (DBW) made from human femoral allograft rings demonstrated a significant 21.1 % (73.6 ± 3.9 % vs. 52.5 ± 2.6 %; p<0.001) increase in percent surface area coating versus mineralized controls. Co-incubation of rAAV-luciferase (rAAV-Luc) coated DBW with a monolayer of C3H10T1/2 cells in culture led to peak luciferase levels that were not significantly different from soluble rAAV-Luc controls (p>0.05), although the peaks occurred at 60hrs and 12hrs, respectively. To assess the transduction efficiency of rAAV-Luc coated DBW in vivo, we first performed a dose response with allografts containing 107, 109 or 1010 particles that were surgically implanted into the quadriceps of mice, and assayed by in vivo bioluminescence imaging (BLI) on days 1, 3, 5, 7, 10, 14, and 21. The results demonstrated a dose response in which the DBW coated with 1010 rAAV-Luc particles achieved peak gene expression levels on day 3, which persisted until day 21, and was significantly greater than the 107 dose throughout this time period (p<0.01). A direct comparison of mineralized versus DBW coated with 1010 rAAV-Luc particles failed to demonstrate any significant differences in transduction kinetics or efficiency in vivo. Thus, surface demineralization of human cortical bone allograft increase its absorbance for uniform rAAV coating, without affecting vector transduction efficiency.
PMCID: PMC2570090  PMID: 18590929
Allograft; Bone; Recombinant Adeno-Associated Virus (rAAV); Freeze-dried; Bioluminescence Imaging (BLI)

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