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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.
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
2.  The Murine Femoral Allograft Model and a Semi-automated Histomorphometric Analysis Tool 
SUMMARY
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.
doi:10.1007/978-1-62703-989-5_4
PMCID: PMC4003505  PMID: 24482164
VisioPharm; Histomorphometry; Bone Repair; Allograft
3.  PTH promotes allograft integration in a calvarial bone defect 
Molecular pharmaceutics  2013;10(12):4462-4471.
Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones.
Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and micro–computed tomography (μCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes.
Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The μCT scans revealed a significant increase in bone formation in Allograft + PTH–treated mice comparing to Allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the Allograft + PTH–treated animals.
In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.
doi:10.1021/mp400292p
PMCID: PMC3902084  PMID: 24131143
Parathyroid Hormone; endogenous stem cells; osteogenesis; allograft; calvarial bone repair
4.  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.
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1371/journal.pone.0110657
PMCID: PMC4204991  PMID: 25333715
5.  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
6.  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
7.  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
9.  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.
doi:10.3109/03008207.2013.787418
PMCID: PMC3697755  PMID: 23586515
Flexor tendon; Tendoplasty; Autograft; Allograft; Adhesions; Tenocytes; Transforming Growth Factor; Extracellular Matrix; Matrix Metalloproteinase
10.  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.
doi:10.1177/2041731414528736
PMCID: PMC4014079  PMID: 24812579
Flexor tendon; allograft; adhesions; growth and differentiation factor 5; tissue engineering
11.  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.
doi:10.1016/j.biomaterials.2013.12.039
PMCID: PMC3913373  PMID: 24393269
12.  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
13.  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.
doi:10.1016/j.jbiomech.2012.11.039
PMCID: PMC3568260  PMID: 23261243
Fracture Toughness; Raman Spectroscopy; Bone Mineral Density; Bone Quality; Inflammatory Arthritis
14.  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
15.  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
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.  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.
doi:10.1007/s00441-011-1188-4
PMCID: PMC3338097  PMID: 21626290
Parathyroid Hormone (PTH); skeletal repair; fracture insufficiency; allograft
18.  Adeno-Associated Virus–Coated Allografts: A Novel Approach for Cranioplasty 
Bone autografts are considered the gold standard for cranioplasty although they lead to comorbidity. Bone allografts are more easily obtained but have low osteogenic potential and fail to integrate into healthy bone. Previously, we showed that by coating long-bone allografts with freeze-dried recombinant adeno-associated virus (rAAV) vector encoding for an osteogenic gene, enhanced osteogenesis and bone integration were achieved. In this study our aim was to evaluate the bone repair potential of calvarial autografts and allografts coated with either single-stranded rAAV2 vector (SS-rAAV-BMP2) or self-complementary pseudotyped vector (SC-rAAV-BMP2) encoding for bone morphogenetic protein (BMP)–2 in a murine cranioplasty model. The grafts were implanted into critical defects in the calvariae of osteocalcin/luciferase (Oc/Luc) transgenic mice, which allowed longitudinal monitoring of osteogenic activity using bioluminescence imaging (BLI). Our results showed that the bioluminescent signal of the SC-rAAV-BMP2–coated allografts was 40% greater than that of the SS-rAAV-BMP2–coated allografts (p<0.05) and that the bioluminescent signal of the SS-rAAV-BMP2–coated allografts was not significantly different from the signals of the autografts or uncoated allografts. Micro–computed tomography (μCT) confirmed the significant increase in osteogenesis in the SC-rAAV-BMP2 group compared with the SS-rAAV-BMP2 group (p<0.05), indicating a significant difference in bone formation when compared with the other grafts tested. In addition, histological analysis revealed extensive remodeling of the autografts. Collectively, these results demonstrate the feasibility of craniofacial regeneration using SC-rAAV-BMP2–coated allografts, which may be an attractive therapeutic solution for repair of severe craniofacial bone defects.
doi:10.1002/term.1594
PMCID: PMC3652248  PMID: 22941779
gene therapy/therapeutics; craniomaxillofacial surgery; tissue engineering; imaging; bone remodeling/regeneration; bone graft(s)
19.  Mechanisms of bone fragility in a glucocorticoid-treated mouse model of rheumatoid arthritis – implications for insufficiency fracture risk 
Arthritis and rheumatism  2012;64(11):3649-3659.
Objective
Glucocorticoid (GC) therapy is associated with increased fracture risk in rheumatoid arthritis (RA) patients. To elucidate the cause of this increased risk, we examined the effects of chronic inflammatory-erosive arthritis and GC treatment on bone quality, structure, and biomechanical properties in a murine model.
Methods
Transgenic mice expressing human TNF-α-transgene (TNF-tg) with established arthritis and wild-type (WT) littermates were continually treated with GC (subcutaneous prednisolone controlled-release pellet; 5 mg/kg/day) or placebo for 14, 28 and 42 days. Microstructure, biomechanical properties, chemical composition, and morphology of tibiae and lumbar vertebral bodies were assessed by micro-CT, biomechanical testing, Raman spectroscopy, and histology, respectively. Serum markers of bone turnover were also determined.
Results
TNF-tg and GC treatment additively decreased mechanical strength and stiffness in both tibiae and vertebral bodies. GC treatment in the TNF-tg mice increased the ductility of tibiae under torsional loading. These changes were associated with significant alterations in the biochemical and structural composition of the mineral and organic components of the bone matrix, a decrease in osteoblast activity and bone formation, and an increase in osteoclastic activity.
Conclusions
Our findings indicate that the concomitant decrease in bone strength and increase in ductility associated with chronic inflammation and GC therapy, coupled with the significant changes in the bone quality and structure, may increase the susceptibility of the bone to failure under low energy loading. This may explain the mechanism of symptomatic insufficiency fractures in patients with RA receiving GC therapy without radiographic manifestation of fracture.
doi:10.1002/art.34639
PMCID: PMC3678247  PMID: 22832945
Glucocorticoid; Rheumatoid Arthritis; Bone Quality; Degree of Mineralization
20.  Power Doppler Ultrasound Phenotyping of Expanding versus Collapsed Popliteal Lymph Nodes in Murine Inflammatory Arthritis 
PLoS ONE  2013;8(9):e73766.
Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic “expanding” phase of the disease, and then suddenly “collapse” during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553±0.007 vs. 0.008±0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.
doi:10.1371/journal.pone.0073766
PMCID: PMC3767819  PMID: 24040061
21.  Gene therapy approaches to regenerating bone 
Advanced drug delivery reviews  2012;64(12):1320-1330.
Bone formation and regeneration therapies continue to require optimization and improvement because many skeletal disorders remain undertreated. Clinical solutions to nonunion fractures and osteoporotic vertebral compression fractures, for example, remain suboptimal and better therapeutic approaches must be created. The widespread use of recombinant human bone morphogenetic proteins (rhBMPs) for spine fusion was recently questioned by a series of reports in a special issue of The Spine Journal, which elucidated the side effects and complications of direct rhBMP treatments. Gene therapy—both direct (in vivo) and cell-mediated (ex vivo)—has long been studied extensively to provide much needed improvements in bone regeneration. In this article, we review recent advances in gene therapy research whose aims are in vivo or ex vivo bone regeneration or formation. We examine appropriate vectors, safety issues, and rates of bone formation. The use of animal models and their relevance for translation of research results to the clinical setting are also discussed in order to provide the reader with a critical view. Finally, we elucidate the main challenges and hurdles faced by gene therapy aimed at bone regeneration as well as expected future trends in this field.
doi:10.1016/j.addr.2012.03.007
PMCID: PMC3663073  PMID: 22429662
Gene therapy; Bone regeneration; Tissue engineering; Viral vectors; Nonviral vectors
22.  Quantifying Massive Allograft Healing of the Canine Femur In Vivo and Ex Vivo: A Pilot Study 
Background
Allograft integration in segmental osseous defects is unpredictable. Imaging techniques have not been applied to investigate angiogenesis and bone formation during allograft healing in a large-animal model.
Questions/purposes
We used dynamic contrast-enhanced (DCE)-MRI and cone beam (CB)-CT to quantify vascularity and bone volume in a canine femoral allograft model and determined their relationship with biomechanical testing and histomorphometry.
Methods
Femoral ostectomy was performed in three dogs and reconstructed with a 5-cm allograft and compression plate. At 0.5, 3, and 6 months, we performed DCE-MRI to quantify vascular permeability (Ktrans) and perfused fraction and CB-CT to quantify bone volume. We also performed posteuthanasia torsional testing and dynamic histomorphometry of the grafted and nonoperated femurs.
Results
DCE-MRI confirmed the avascular nature of allograft healing (perfused fraction, 2.08%–3.25%). CB-CT demonstrated new bone formation at 3 months (26.2, 3.7, and 2.2 cm3) at the graft-host junctions, which remodeled down at 6 months (14.0, 2.2, and 2.0 cm3). The increased bone volume in one subject was confirmed with elevated Ktrans (0.22) at 3 months. CB-CT-identified remodeled bone at 6 months was corroborated by histomorphometry. Allografted femurs recovered only 40% of their strength at 6 months.
Conclusions
CB-CT and DCE-MRI can discriminate differences in angiogenesis and bone formation in the canine allograft model, which has potential to detect a small (32%) drug or device effect on biomechanical healing with only five animals per group.
Clinical Relevance
These radiographic tools may have the potential to assess adjuvant effects on vascular invasion and new bone formation after segmental allograft transplantation.
doi:10.1007/s11999-012-2349-9
PMCID: PMC3830092  PMID: 22528382
23.  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.
doi:10.1002/jor.21103
PMCID: PMC2891234  PMID: 20187115
Modic Changes; CE-MRI; Bone Marrow Edema; Vertebral Degeneration
24.  Dual Differentiation-Exogenous Mesenchymal Stem Cell Therapy for Traumatic Spinal Cord Injury Repair in a Murine Hemisection Model 
Stem Cells International  2013;2013:928982.
Mesenchymal stem cell (MSC) transplantation has shown tremendous promise as a therapy for repair of various tissues of the musculoskeletal, vascular, and central nervous systems. Based on this success, recent research in this field has focused on complex tissue damage, such as that which occurs from traumatic spinal cord injury (TSCI). As the critical event for successful exogenous, MSC therapy is their migration to the injury site, which allows for their anti-inflammatory and morphogenic effects on fracture healing, neuronal regeneration, and functional recover. Thus, there is a need for a cost-effective in vivo model that can faithfully recapitulate the salient features of the injury, therapy, and recovery. To address this, we review the recent advances in exogenous MSC therapy for TSCI and traumatic vertebral fracture repair and the existing challenges regarding their translational applications. We also describe a novel murine model designed to take advantage of multidisciplinary collaborations between musculoskeletal and neuroscience researchers, which is needed to establish an efficacious MSC therapy for TSCI.
doi:10.1155/2013/928982
PMCID: PMC3762188  PMID: 24027587
25.  Conditional Activation of β-Catenin Signaling Leads to Severe Defects in Intervertebral Disc Tissue 
Arthritis and rheumatism  2012;64(8):2611-2623.
Objective
The incidence of low back pain is extremely high and is often linked to intervertebral disc (IVD) degeneration. The mechanism of this disease is currently unknown. In this study, we have investigated the role of β-catenin signaling in IVD tissue function.
Methods
β-catenin protein levels were measured in disc samples derived from patients with disc degeneration and normal subjects by immunohistochemistry (IHC). To generate β-catenin conditional activation (cAct) mice, Col2a1-CreERT2 transgenic mice were bred with β-cateninfx(Ex3)/fx(Ex3) mice. Changes in disc tissue morphology and function were analyzed by micro-CT, histology and real-time PCR assays.
Results
We found that β-catenin protein was up-regulated in disc tissues from patients with disc degeneration. To assess the effects of increased β-catenin on disc tissue we generated β-catenin cAct mice. Overexpression of β-catenin in disc cells led to extensive osteophyte formation in 3- and 6-month-old β-catenin cAct mice which were associated with significant changes in the cells and extracellular matrix of disc tissues and growth plate. Gene expression analysis demonstrated that activation of β-catenin could enhance Runx2-dependent Mmp13 and Adamts5 expression. Moreover, genetic ablation of the Mmp13 or Adamts5 under β-catenin cAct background, or treatment of β-catenin cAct mice with a specific MMP13 inhibitor, ameliorated the mutant phenotype.
Conclusions
β-catenin signaling pathway plays a critical role in disc tissue function.
doi:10.1002/art.34469
PMCID: PMC3632450  PMID: 22422036

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