Publications by authors named "Lara Longobardi"

25 Publications

  • Page 1 of 1

Tibiofemoral Articular Cartilage Composition Differs Based on Serum Biochemical Profiles Following Anterior Cruciate Ligament Reconstruction.

Osteoarthritis Cartilage 2021 Sep 15. Epub 2021 Sep 15.

Department of Exercise and Sports Science, University of North Carolina at Chapel Hill, NC.

Objective: Biochemical joint changes contribute to posttraumatic osteoarthritis (PTOA) development following anterior cruciate ligament reconstruction (ACLR). The purpose of this longitudinal cohort study was to compare tibiofemoral cartilage composition between ACLR patients with different serum biochemical profiles. We hypothesized that profiles of increased inflammation (monocyte chemoattractant protein-1 [MCP-1]), type-II collagen turnover (type-II collagen breakdown [C2C]:synthesis [CPII]), matrix degradation (matrix metalloproteinase-3 [MMP-3] and cartilage oligomeric matrix protein [COMP]) preoperatively to 6-months post-ACLR would be associated with greater tibiofemoral cartilage T1ρ relaxation times 12-months post-ACLR.

Design: Serum was collected from 24 patients (46% female, 22.1±4.2 years old, 24.0±2.6 kg/m body mass index [BMI]) preoperatively (6.4±3.6 days post injury) and 6-months post-ACLR. T1ρ Magnetic Resonance Imaging (MRI) was collected for medial and lateral tibiofemoral articular cartilage at 12-months post-ACLR. A k-means cluster analysis was used to identify profiles based on biomarker changes over time and T1ρ relaxation times were compared between cluster groups controlling for sex, age, BMI, concomitant injury (either meniscal or chondral pathology), and Marx Score.

Results: One cluster exhibited increases in MCP-1 and COMP while the other demonstrated decreases in MCP-1 and COMP preoperatively to 6-months post-ACLR. The cluster group with increases in MCP-1 and COMP demonstrated greater lateral tibial (adjusted mean difference=3.88, 95% confidence intervals [1.97-5.78]) and femoral (adjusted mean difference=12.71, 95% confidence intervals [0.41-23.81]) T1ρ relaxation times.

Conclusion: Profiles of increased serum levels of inflammation and matrix degradation markers preoperatively to 6-months post-ACLR are associated with MRI changes consistent with lesser lateral tibiofemoral cartilage proteoglycan density 12-months post-ACLR.
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http://dx.doi.org/10.1016/j.joca.2021.09.005DOI Listing
September 2021

Association of Increased Serum Lipopolysaccharide but not Microbial Dysbiosis with Obesity-related Osteoarthritis.

Arthritis Rheumatol 2021 Aug 23. Epub 2021 Aug 23.

Division of Rheumatology, Allergy and Immunology and the Thurston Arthritis Research Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina.

Objective: To test the hypothesis that an altered gut microbiota (dysbiosis) plays a role in obesity-associated osteoarthritis (OA).

Methods: Stool and blood samples were collected from 92 participants with BMI ≥ 30 kg/m recruited from the Johnston County Osteoarthritis Project. OA cases (n=50) had hand plus knee OA (Kellgren-Lawrence [KL] grade ≥2 or arthroplasty). Controls (N=42) had no hand OA and KL grade 0-1 knees. Compositional analysis of stool samples was carried out by 16S rRNA amplicon sequencing. Alpha and beta diversity and differences in taxa relative abundances were determined. Blood samples were used for multiplex cytokine analysis and measures of lipopolysaccharide (LPS) and LPS binding protein. Germ-free mice were gavaged with case or control pooled fecal samples and placed on a 40% fat, high sucrose diet for 40 weeks. Knee OA was evaluated histologically.

Results: OA cases were slightly older with more females and higher BMI, WOMAC pain and KL grades than controls. There were no significant differences in alpha or beta diversity or genus level composition between cases and controls. Cases had higher plasma levels of osteopontin (p=0.01) and serum LPS (p<0.0001). Mice transplanted with case or control microbiota exhibited a significant difference in alpha diversity (p=0.02) and beta diversity but no differences in OA severity.

Conclusion: The lack of differences in the gut microbiota yet increased serum LPS levels suggest the possibility that increased intestinal permeability allowing for greater absorption of LPS, rather than a dysbiotic microbiota, may contribute to development of OA associated with obesity.
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http://dx.doi.org/10.1002/art.41955DOI Listing
August 2021

Quality of Life Associates With Moderate to Vigorous Physical Activity Following Anterior Cruciate Ligament Reconstruction.

J Athl Train 2021 Jul 30. Epub 2021 Jul 30.

2Human Movement Science Curriculum, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Context: Higher knee function is linked to psychological readiness to return to sport following anterior cruciate ligament reconstruction (ACLR). Individuals with ACLR participate in less physical activity compared to matched uninjured controls, yet the association between knee function and physical activity following ACLR remains unclear.

Objective: To determine the association between patient-reported knee function measured with the Knee Injury and Osteoarthritis Outcomes Score Quality of Life (KOOS-QOL), daily steps, and minutes spent in moderate to vigorous physical activity (MVPA) in individuals with ACLR. Secondarily, we determined associations between KOOS-QOL, daily steps, and MVPA in individuals with ACLR who presented with (symptomatic) and without (asymptomatic) clinically meaningful knee related symptoms.

Design: Cross-sectional study.

Setting: Laboratory, Free-living conditions.

Patients Or Other Participants: Sixty-six individuals with primary unilateral ACLR (55% female, 22±4 years, 28±33 months post-ACLR, BMI: 24.2±2.9 kg/m2).

Outcome Measure(s): We collected KOOS and retrospectively stratified participants into those with (symptomatic [n=30]) or without (asymptomatic [n=36]) clinically meaningful knee related symptoms based on previously defined KOOS cutoffs. We assessed daily steps and MVPA from ActiGraph GT9X Link accelerometers which each participant wore on the right hip for 7 days. We conducted linear regressions to determine associations between KOOS-QOL, daily steps, and MVPA.

Results: No significant associations existed in the entire sample between KOOS-QOL and daily steps (ΔR2=0.01, P=0.50) or MVPA (ΔR2=0.01, P=0.36). In symptomatic individuals, greater KOOS-QOL associated with greater MVPA (ΔR2=0.12, P=0.05,). No significant associations existed between KOOS-QOL, daily steps, and MVPA in the asymptomatic group.

Conclusions: Symptomatic individuals with ACLR who spent more time in MVPA reported higher quality of life.
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http://dx.doi.org/10.4085/1062-6050-0670.20DOI Listing
July 2021

TGF-β type 2 receptor-mediated modulation of the IL-36 family can be therapeutically targeted in osteoarthritis.

Sci Transl Med 2019 05;11(491)

Department of Pediatrics, Rush University Medical Center, Chicago, IL 60612, USA.

Mechanisms that govern the shift from joint homeostasis to osteoarthritis (OA) remain unknown. Here, we identify a pathway used for joint development and homeostasis, and its role in OA. Using a combination of transgenic, pharmacological, and surgical conditions in mouse and human tissues, we found that TGF-β signaling promotes joint homeostasis through regulation of the IL-36 family. We identified IL-36 receptor antagonist (IL-36 in mice and IL-36RN in humans) as a potential disease-modifying OA drug. Specifically, OA development was associated with IL-36α up-regulation and IL-36Ra down-regulation in mice with tissue-specific postnatally induced ablation of , mice treated with a TGF-β signaling inhibitor, mice with posttraumatic OA, and aging mice with naturally occurring OA. In human cartilage, OA severity was associated with decreased TGFBR2 and IL-36RN, whereas IL-36α increased. Functionally, intra-articular treatment with IL-36Ra attenuated OA development in mice, and IL-36RN reduced MMP13 in human OA chondrocytes. These findings highlight the relevance of TGFBR2-IL-36 interplay in joint homeostasis and IL-36RN as a potential therapeutic agent for OA.
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http://dx.doi.org/10.1126/scitranslmed.aan2585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102613PMC
May 2019

Impaired Annulus Fibrosus Development and Vertebral Fusion Cause Severe Scoliosis in Mice with Deficiency of c-Jun NH2-Terminal Kinases 1 and 2.

Am J Pathol 2019 04 19;189(4):868-885. Epub 2019 Jan 19.

Division of Rheumatology, Allergy and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Thurston Arthritis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Electronic address:

Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1Col2-Cre/JNK2) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.
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http://dx.doi.org/10.1016/j.ajpath.2018.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446226PMC
April 2019

A mouse model for chronic pain-induced increase in ethanol consumption.

Pain 2017 03;158(3):457-462

Bowles Center for Alcohol Studies, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Chronic pain conditions are often comorbid with alcohol abuse. "Self-medication" with alcohol introduces a host of problems associated with the abuse of alcohol which over time has the potential of exacerbating the painful condition. Despite the prevalence of chronic pain being associated with alcohol abuse, rodent models which mimic the comorbid conditions are lacking. In this study, we model osteoarthritis (OA) in C57BL/6J mice by surgically destabilizing the medial meniscus (DMM). Sham-operated mice served as controls. Thirteen weeks after surgery, DMM but not sham-operated mice exhibited pronounced incapacitance of the surgically manipulated hind limb compared with the nonsurgically manipulated hind limb. At this time, the mice were exposed to the 2-bottle ethanol choice, beginning with 2.5% with a gradual increasing to 20%. Compared with sham controls, DMM mice consumed more EtOH and preferred EtOH over water at the 20% EtOH concentration. Histological analysis verified that the DMM mice exhibited significant damage to the articular cartilage and osteophyte growth compared with sham controls and these measures of the severity of OA correlated with the amount of ethanol intake. Thus, the combination of the DMM model of OA with the enhanced two-bottle ethanol choice is a potential preclinical approach in mice by which the basis of the comorbid association of alcohol abuse and chronic pain conditions can be explored.
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http://dx.doi.org/10.1097/j.pain.0000000000000780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303149PMC
March 2017

BMP2 Regulation of CXCL12 Cellular, Temporal, and Spatial Expression is Essential During Fracture Repair.

J Bone Miner Res 2015 Nov 15;30(11):2014-27. Epub 2015 Jun 15.

Division of Endocrinology, Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)-dependent fracture healing occurs through a tight control of chemokine C-X-C motif-ligand-12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site-specific response of CXCL12(+)-BMP2(+) endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2(cKO/+)) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2(cKO/+) mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2(cKO/+) mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2(cKO/cKO) endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2-expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12(+)-BMP2(+) perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12(+)-BMP2(+) to osteogenesis while departing their supportive role to angiogenesis. Our findings have far-reaching implications for understanding mechanisms regulating the selective recruitment of distinct cells into the repairing niches and the development of novel pharmacological (by targeting BMP2/CXCL12) and cellular (MSCs, endosteal cells) interventions to promote fracture healing.
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http://dx.doi.org/10.1002/jbmr.2548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970512PMC
November 2015

Synovial joints: from development to homeostasis.

Curr Osteoporos Rep 2015 Feb;13(1):41-51

Department of Pediatrics, University of North Carolina at Chapel Hill, 109 Mason Farm Road, Chapel Hill, NC, 27599-7039, USA,

Synovial joint morphogenesis occurs through the condensation of mesenchymal cells into a non-cartilaginous region known as the interzone and the specification of progenitor cells that commit to the articular fate. Although several signaling molecules are expressed by the interzone, the mechanism is poorly understood. For treatments of cartilage injuries, it is critical to discover the presence of joint progenitor cells in adult tissues and their expression gene pattern. Potential stem cell niches have been found in different joint regions, such as the surface zone of articular cartilage, synovium, and groove of Ranvier. Inherited joint malformations as well as joint-degenerating conditions are often associated with other skeletal defects and may be seen as the failure of morphogenic factors to establish the correct microenvironment in cartilage and bone. Therefore, exploring how joints form can help us understand how cartilage and bone are damaged and develop drugs to reactivate this developing mechanism.
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http://dx.doi.org/10.1007/s11914-014-0247-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306636PMC
February 2015

Expression levels of insulin receptor substrate-1 modulate the osteoblastic differentiation of mesenchymal stem cells and osteosarcoma cells.

Growth Factors 2014 Feb 20;32(1):41-52. Epub 2014 Jan 20.

Laboratory of Experimental Oncology, CRS Development of Molecular Therapies, Orthopaedic Rizzoli Institute , Bologna , Italy .

The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation.
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http://dx.doi.org/10.3109/08977194.2013.870168DOI Listing
February 2014

Stachydrine ameliorates high-glucose induced endothelial cell senescence and SIRT1 downregulation.

J Cell Biochem 2013 Nov;114(11):2522-30

Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy.

Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by accelerating endothelial cell (EC) senescence and limiting the proliferative potential of these cells. Here we aimed to investigate the effect of stachydrine, a proline betaine present in considerable quantities in juices from fruits of the Citrus genus, on EC under high-glucose stimulation, and its underlying mechanism. The senescence model of EC was set up by treating cells with high-glucose (30 mM) for different times. Dose-dependent (0.001-1 mM) evaluation of cell viability revealed that stachydrine does not affect cell proliferation with a similar trend up to 72 h. Noticeable, stachydrine (0.1 mM) significantly attenuated the high-glucose induced EC growth arrest and senescence. Indeed, co-treatment with high-glucose and stachydrine for 48 h kept the percentage of EC in the G0 /G1 cell cycle phase near to control values and significantly reduced cell senescence. Western blot analysis and confocal-laser scanning microscopy revealed that stachydrine also blocked the high-glucose induced upregulation of p16(INK4A) and downregulation of SIRT1 expression and enzyme activity. Taken together, results here presented are the first evidence that stachydrine, a naturally occurring compound abundant in citrus fruit juices, inhibits the deleterious effect of high-glucose on EC and acts through the modulation of SIRT1 pathway. These results may open new prospective in the identification of stachydrine as an important component of healthier eating patterns in prevention of cardiovascular diseases.
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http://dx.doi.org/10.1002/jcb.24598DOI Listing
November 2013

Joint TGF-β type II receptor-expressing cells: ontogeny and characterization as joint progenitors.

Stem Cells Dev 2013 May 15;22(9):1342-59. Epub 2013 Feb 15.

Department of Pediatrics, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599-7039, USA.

TGF-β type II receptor (Tgfbr2) signaling plays an essential role in joint-element development. The Tgfbr2(PRX-1KO) mouse, in which the Tgfbr2 is conditionally inactivated in developing limbs, lacks interphalangeal joints and tendons. In this study, we used the Tgfbr2-β-Gal-GFP-BAC mouse as a LacZ/green fluorescent protein (GFP)-based read-out to determine: the spatial and temporally regulated expression pattern of Tgfbr2-expressing cells within joint elements; their expression profile; and their slow-cycling labeling with bromodeoxyuridine (BrdU). Tgfbr2-β-Gal activity was first detected at embryonic day (E) 13.5 within the interphalangeal joint interzone. By E16.5, and throughout adulthood, Tgfbr2-expressing cells clustered in a contiguous niche that comprises the groove of Ranvier and the synovio-entheseal complex including part of the perichondrium, the synovium, the articular cartilage superficial layer, and the tendon's entheses. Tgfbr2-expressing cells were found in the synovio-entheseal complex niche with similar temporal pattern in the knee, where they were also detected in meniscal surface, ligaments, and the synovial lining of the infrapatellar fat pad. Tgfbr2-β-Gal-positive cells were positive for phospho-Smad2, signifying that the Tgfbr2 reporter was accurate. Developmental-stage studies showed that Tgfbr2 expression was in synchrony with expression of joint-morphogenic genes such as Noggin, GDF5, Notch1, and Jagged1. Prenatal and postnatal BrdU-incorporation studies showed that within this synovio-entheseal-articular-cartilage niche most of the Tgfbr2-expressing cells labeled as slow-proliferating cells, namely, stem/progenitor cells. Tgfbr2-positive cells, isolated from embryonic limb mesenchyme, expressed joint progenitor markers in a time- and TGF-β-dependent manner. Our studies provide evidence that joint Tgfbr2-expressing cells have anatomical, ontogenic, slow-cycling trait and in-vivo and ex-vivo expression profiles of progenitor joint cells.
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http://dx.doi.org/10.1089/scd.2012.0207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629851PMC
May 2013

TGF-β type II receptor/MCP-5 axis: at the crossroad between joint and growth plate development.

Dev Cell 2012 Jul;23(1):71-81

Department of Pediatrics, University of North Carolina at Chapel Hill, NC 27599, USA.

Despite its clinical significance, the mechanisms of joint morphogenesis are elusive. By combining laser-capture microdissection for RNA sampling with microarrays, we show that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by downregulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in vivo, ex vivo, and in vitro approaches, we show that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-β-type-II-receptor (TβRII) in their limbs (Tgfbr2(Prx1KO)), which lack joint development and fail chondrocyte hypertrophy, show upregulation of interzone-MCP-5. In vivo and ex vivo blockade of the sole MCP-5 receptor, CCR2, led to the rescue of joint formation and growth plate maturation in Tgfbr2(Prx1KO) but an acceleration of growth plate mineralization in control mice. Our study characterized the TβRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth.
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http://dx.doi.org/10.1016/j.devcel.2012.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401383PMC
July 2012

Comparison of microCT and an inverse finite element approach for biomechanical analysis: results in a mesenchymal stem cell therapeutic system for fracture healing.

J Biomech 2012 Aug 4;45(12):2164-70. Epub 2012 Jul 4.

Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232, USA.

An important concern in the study of fracture healing is the ability to assess mechanical integrity in response to candidate therapeutics in small-animal systems. In recent reports, it has been proposed that microCT image-derived densitometric parameters could be used as a surrogate for mechanical property assessment. Recently, we have proposed an inverse methodology that iteratively reconstructs the modulus of elasticity of the lumped soft callus/hard callus region by integrating both intrinsic mechanical property (from biomechanical testing) and geometrical information (from microCT) within an inverse finite element analysis (FEA) to define a callus quality measure. In this paper, data from a therapeutic system involving mesenchymal stem cells is analyzed within the context of comparing traditional microCT densitometric and mechanical property metrics. In addition, a novel multi-parameter regression microCT parameter is analyzed as well as our inverse FEA metric. The results demonstrate that the inverse FEA approach was the only metric to successfully detect both longitudinal and therapeutic responses. While the most promising microCT-based metrics were adequate at early healing states, they failed to track late-stage mechanical integrity. In addition, our analysis added insight to the role of MSCs by demonstrating accelerated healing and was the only metric to demonstrate therapeutic benefits at late-stage healing. In conclusion, the work presented here indicates that microCT densitometric parameters are an incomplete surrogate for mechanical integrity. Additionally, our inverse FEA approach is shown to be very sensitive and may provide a first-step towards normalizing the often challenging process of assessing mechanical integrity of healing fractures.
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http://dx.doi.org/10.1016/j.jbiomech.2012.05.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782612PMC
August 2012

Systemically delivered insulin-like growth factor-I enhances mesenchymal stem cell-dependent fracture healing.

Growth Factors 2012 Aug 4;30(4):230-41. Epub 2012 May 4.

Department of Pediatrics, Division of Endocrinology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7039, USA.

In this study, we examined the effectiveness of systemic subcutaneous delivery of recombinant Insulin-like growth factor (IGF)-I concurrently with primary cultured bone marrow-derived mesenchymal stem cell (MSC) transplant on fracture repair. We found that the fracture callus volume increased in mice with a stabilized tibia fracture that received IGF-I+MSC when compared with that in either untreated or MSC alone treated mice. In evaluating the callus tissue components, we found that the soft and new bone tissue volumes were significantly increased in IGF-I+MSC recipients. Histological and in-situ hybridization analyses confirmed a characteristic increase of newly forming bone in IGF-I+MSC recipients and that healing progressed mostly through endochondral ossification. The increase in soft and new bone tissue volumes correlated with increased force and toughness as determined by biomechanical testing. In conclusion, MSC transplant concurrent with systemic delivery of IGF-I improves fracture repair suggesting that IGF-I+MSC could be a novel therapeutic approach in patients who have inadequate fracture repair.
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http://dx.doi.org/10.3109/08977194.2012.683188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752908PMC
August 2012

Mesenchymal stem cells expressing insulin-like growth factor-I (MSCIGF) promote fracture healing and restore new bone formation in Irs1 knockout mice: analyses of MSCIGF autocrine and paracrine regenerative effects.

Stem Cells 2011 Oct;29(10):1537-48

Department of Pediatrics, Division of Pediatric Endocrinology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Failures of fracture repair (nonunions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe, and feasible. The contributions of MSC to the reparative process can occur through autocrine and paracrine effects. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow-derived MSCs expressing insulin-like growth factor-I (MSC(IGF)), to promote these seed-and-soil actions of MSC to fully implement their regenerative abilities in fracture repair and nonunions. MSC(IGF) or traceable MSC(IGF)-Lac-Z were transplanted into wild-type or insulin-receptor-substrate knockout (Irs1(-/-)) mice with a stabilized tibia fracture. Healing was assessed using biomechanical testing, microcomputed tomography (μCT), and histological analyses. We found that systemically transplanted MSC(IGF) through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF-I adapted the response of transplanted MSC(IGF) to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF-I-induced osteoglastogenesis in MSCs was dependent of an intact IRS1-PI3K signaling. Furthermore, using Irs1(-/-) mice as a nonunion fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF-I on MSC restored the fracture new bone formation and promoted the occurrence of a well-organized callus that bridged the gap. A callus that was basically absent in Irs1(-/-) left untransplanted or transplanted with MSCs. We provided evidence of effects and mechanisms for transplanted MSC(IGF) in fracture repair and potentially to treat nonunions.
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http://dx.doi.org/10.1002/stem.697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622704PMC
October 2011

Mesenchymal stem cells at the intersection of cell and gene therapy.

Expert Opin Biol Ther 2010 Dec;10(12):1663-79

University of North Carolina at Chapel Hill, Department of Pediatrics, Chapel Hill, NC 27599-7239, USA.

Importance Of The Field: Mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. Along with differentiation, MSCs can modulate inflammation, home to damaged tissues and secrete bioactive molecules. These properties can be enhanced through genetic-modification that would combine the best of both cell and gene therapy fields to treat monogenic and multigenic diseases.

Areas Covered In This Review: Findings demonstrating the immunomodulation, homing and paracrine activities of MSCs followed by a summary of the current research utilizing MSCs as a vector for gene therapy, focusing on skeletal disorders, but also cardiovascular disease, ischemic damage and cancer.

What The Reader Will Gain: MSCs are a possible therapy for many diseases, especially those related to the musculoskeletal system, as a standalone treatment, or in combination with factors that enhance the abilities of these cells to migrate, survive or promote healing through anti-inflammatory and immunomodulatory effects, differentiation, angiogenesis or delivery of cytolytic or anabolic agents.

Take Home Message: Genetically-modified MSCs are a promising area of research that would be improved by focusing on the biology of MSCs that could lead to identification of the natural and engrafting MSC-niche and a consensus on how to isolate and expand MSCs for therapeutic purposes.
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http://dx.doi.org/10.1517/14712598.2010.531257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057936PMC
December 2010

Use of glycol chitosan modified by 5beta-cholanic acid nanoparticles for the sustained release of proteins during murine embryonic limb skeletogenesis.

J Control Release 2010 May 28;144(1):101-8. Epub 2010 Jan 28.

Department of Pediatrics, Division of Pediatric Endocrinology, University of North Carolina at Chapel Hill, Mason Farm Road, Campus Box: 7039, Chapel Hill, NC 27599-7039, USA.

Murine embryonic limb cultures have invaluable roles in studying skeletogenesis. Substance delivery is an underdeveloped area in developmental biology that has primarily relied on Affi-Gel-Blue-agarose-beads. However, the lack of information about the efficiency of agarose-bead loading and release and difficulties for a single-bead implantation represent significant limitations. We optimized the use of glycol chitosan-5beta-cholanic acid conjugates (HGC) as a novel protein delivery system in mouse embryonic limbs. To this purpose, we loaded HGC either with recombinant Noggin, or bovine serum albumin (BSA). The size, morphology and stability of the protein-loaded-HGC were determined by transmission electron microscopy and dynamic-light-scattering. HGC-BSA and HGC-Noggin loading efficiencies were 80-90%. Time-course study revealed that Noggin and BSA were 80-90% released after 48 h. We developed several techniques to implant protein-loaded-HGC into murine embryonic joints from embryonic age E13.5 to E15.5, including a micro-injection system dispensing nanoliters. HGC did not interfere with skeletogenesis. Using CBR-3BA staining, we detected HGC nanoparticles within implanted tissues. Furthermore, a sustained release of BSA and Noggin was demonstrated in HGC-BSA and HGC-Noggin injected regions. HGC-released Noggin was biologically active in blocking the BMP signaling in in vitro mesenchyme limb micromasses as well as in ex-vivo limb cultures. Results reveal that HGC is a valuable protein-delivery system in developmental biology.
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http://dx.doi.org/10.1016/j.jconrel.2010.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3363971PMC
May 2010

Subcellular localization of IRS-1 in IGF-I-mediated chondrogenic proliferation, differentiation and hypertrophy of bone marrow mesenchymal stem cells.

Growth Factors 2009 Oct;27(5):309-20

Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7039, USA.

Bone marrow derived mesenchymal stem cells (BM-MSC) can differentiate into chondrocytes. Understanding the mechanisms and growth factors that control the MSC stemness is critical to fully implement their therapeutic use in cartilage diseases. The activated type 1 insulin-like growth factor receptor (IGF-IR), interacting with the insulin receptor substrate-1 (IRS-1), can induce cancer cell proliferation and transformation. In cancer or transformed cells, IRS-1 has been shown to localize in the cytoplasm where it activates the canonical Akt pathway, as well as in the nucleus where it binds to nuclear proteins. We have previously demonstrated that IGF-I has distinct time-dependent effect on primary BM-MSC chondrogenic pellets: initially (2-day culture), IGF-I induces proliferation; subsequently, IGF-I promotes chondrocytic differentiation (7-day culture). In the present study, by using MSC from the BM of IRS-1(- / - ) mice we show that IRS-1 mediates almost 50% of the IGF-I mitogenic response and the MAPK-MEK/ERK signalling accounts for the other 50%. After stimulation with IGF-I, we found that in 2-day old human and mouse derived BM-MSC pellets, IRS-1 (total and phosphorylated) is nuclearly localized and that proliferation prevails over differentiation. The IGF-I mitogenic effect is Akt-independent. In 7-day MSC pellets, IGF-I stimulates the chondrogenic differentiation of MSC into chondrocytes, pre-hypertrophic and hypertrophic chondrocytes and IRS-1 accumulates in the cytoplasm. IGF-I-dependent differentiation is exclusively Akt-dependent. Our data indicate that in the physiologically relevant model of primary cultured MSC, IGF-I induces a temporally regulated nuclear or cytoplasmic localization of IRS-1 that correlate with the transition from proliferation to chondrogenic differentiation.
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http://dx.doi.org/10.1080/08977190903138874DOI Listing
October 2009

Regenerative effects of transplanted mesenchymal stem cells in fracture healing.

Stem Cells 2009 Aug;27(8):1887-98

Departments of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat nonunions. The use of MSC to treat fractures is attractive for several reasons. First, MSCs would be implementing conventional reparative process that seems to be defective or protracted. Secondly, the effects of MSCs treatment would be needed only for relatively brief duration of reparation. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation, and their role in modulating the injury-related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time- and dose-dependent and, it is exclusively CXCR4-dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro-computed tomography and histological studies. Transplanting CMV-Cre-R26R-Lac Z-MSC, we found that MSCs engrafted within the callus endosteal niche. Using MSCs from BMP-2-Lac Z mice genetically modified using a bacterial artificial chromosome system to be beta-gal reporters for bone morphogenic protein 2 (BMP-2) expression, we found that MSCs contributed to the callus initiation by expressing BMP-2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow design of novel MSC-based therapies to treat fractures.
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http://dx.doi.org/10.1002/stem.103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426453PMC
August 2009

Role of mesenchymal stem cells in regenerative medicine: application to bone and cartilage repair.

Expert Opin Biol Ther 2008 Mar;8(3):255-68

University of North Carolina at Chapel Hill, Division of Endocrinology, Department of Pediatrics, 3341 Medical Biomolecular Research Building, 103 Mason Farm Road Campus Box: 7039, Chapel Hill North Carolina 27599-7239, USA.

Background: Mesenchymal stem cells (MSC) are multipotent cells with the ability to differentiate into mesenchyme-derived cells including osteoblasts and chondrocytes.

Objective: To provide an overview and expert opinion on the in vivo ability of MSC to home into tissues, their regenerative properties and potential applications for cell-based therapies to treat bone and cartilage disorders.

Methods: Data sources including the PubMed database, abstract booklets and conference proceedings were searched for publications pertinent to MSC and their properties with emphasis on the in vivo studies and clinical use in cartilage and bone regeneration and repair. The search included the most current information possible.

Conclusion: MSC can migrate to injured tissues and some of their reparative properties are mediated by paracrine mechanisms including their immunomodulatory actions. MSC possess a critical potential in regenerative medicine for the treatment of skeletal diseases, such as osteoarthritis or fracture healing failure, where treatments are partially effective or palliative.
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http://dx.doi.org/10.1517/14712598.8.3.255DOI Listing
March 2008

TGF-beta signaling is essential for joint morphogenesis.

J Cell Biol 2007 Jun;177(6):1105-17

Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

Despite its clinical significance, joint morphogenesis is still an obscure process. In this study, we determine the role of transforming growth factor beta (TGF-beta) signaling in mice lacking the TGF-beta type II receptor gene (Tgfbr2) in their limbs (Tgfbr2(PRX-1KO)). In Tgfbr2(PRX-1KO) mice, the loss of TGF-beta responsiveness resulted in the absence of interphalangeal joints. The Tgfbr2(Prx1KO) joint phenotype is similar to that in patients with symphalangism (SYM1-OMIM185800). By generating a Tgfbr2-green fluorescent protein-beta-GEO-bacterial artificial chromosome beta-galactosidase reporter transgenic mouse and by in situ hybridization and immunofluorescence, we determined that Tgfbr2 is highly and specifically expressed in developing joints. We demonstrated that in Tgfbr2(PRX-1KO) mice, the failure of joint interzone development resulted from an aberrant persistence of differentiated chondrocytes and failure of Jagged-1 expression. We found that TGF-beta receptor II signaling regulates Noggin, Wnt9a, and growth and differentiation factor-5 joint morphogenic gene expressions. In Tgfbr2(PRX-1KO) growth plates adjacent to interphalangeal joints, Indian hedgehog expression is increased, whereas Collagen 10 expression decreased. We propose a model for joint development in which TGF-beta signaling represents a means of entry to initiate the process.
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http://dx.doi.org/10.1083/jcb.200611031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064369PMC
June 2007

Effect of IGF-I in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling.

J Bone Miner Res 2006 Apr 5;21(4):626-36. Epub 2006 Apr 5.

Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2579, USA.

Unlabelled: A novel role for IGF-I in MSC chondrogenesis was determined. IGF-I effects were evaluated in the presence or absence of TGF-beta signaling by conditionally inactivating the TGF-beta type II receptor. We found that IGF-I had potent chondroinductive actions on MSCs. IGF-I effects were independent from and additive to TGF-beta.

Introduction: Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow (BM), expanded, and differentiated into several cell types, including chondrocytes. The role of IGF-I in the chondrogenic potential of MSCs is poorly understood. TGF-beta induces MSC chondrogenic differentiation, although its actions are not well defined. The aim of our study was to define the biological role of IGF-I on proliferation, chondrogenic condensation, apoptosis, and differentiation of MSCs into chondrocytes, alone or in combination with TGF-beta and in the presence or absence of TGF-beta signaling.

Materials And Methods: Mononuclear adherent stem cells were isolated from mouse BM. Chondrogenic differentiation was induced by culturing high-density MSC pellets in serum- and insulin-free defined medium up to 7 days, with or without IGF-I and/or TGF-beta. We measured thymidine incorporation and stained 2-day-old pellets with TUNEL, cleaved caspase-3, peanut-agglutinin, and N-cadherin. Seven-day-old pellets were measured in size, stained for proteoglycan synthesis, and analyzed for the expression of collagen II and Sox-9 by quantitative real time PCR. We obtained MSCs from mice in which green fluorescent protein (GFP) was under the Collagen2 promoter and determined GFP expression by confocal microscopy. We conditionally inactivated the TGF-beta type II receptor (TbetaRII) in MSCs using a cre-lox system, generating TbetaRII knockout MSCs (RIIKO-MSCs).

Results And Conclusions: IGF-I modulated MSC chondrogenesis by stimulating proliferation, regulating cell apoptosis, and inducing expression of chondrocyte markers. IGF-I chondroinductive actions were equally potent to TGF-beta1, and the two growth factors had additive effects. Using RIIKO-MSCs, we showed that IGF-I chondrogenic actions are independent from the TGF-beta signaling. We found that the extracellular signal-related kinase 1/2 mitogen-activated protein kinase (Erk1/2 MAPK) pathway mediated the TGF-beta1 mitogenic response and in part the IGF-I proliferative action. Our data, by showing the role of IGF-I and TGF-beta1 in the critical steps of MSC chondrogenesis, provide critical information to optimize the therapeutic use of MSCs in cartilage disorders.
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http://dx.doi.org/10.1359/jbmr.051213DOI Listing
April 2006

Cartilage disorders: potential therapeutic use of mesenchymal stem cells.

Endocr Dev 2005 ;9:17-30

Departments of Pediatrics and Cancer Biology, Vanderbilt University Medical Center, T-0107 Medical Center North, Nashville, Tenn., USA.

Chondrogenesis is a well-orchestrated process driven by chondroprogenitors that undergo to condensation, proliferation and chondrocyte differentiation. Because cartilage lacks regenerative ability, treatments for cartilage diseases are primarily palliative. Adult bone marrow contains a reservoir of mesenchymal stem cells (MSC) with in vitro and in vivo potential of becoming cartilage. To optimize the potential therapeutic use of MSC in cartilage disorders, we need to understand the mechanisms by which growth factors determine their chondrogenic potential. Insulin-like growth factors (IGFs) play a central role in chondrogenesis as indicated by the severe growth failure observed in animals carrying null mutations of Igfs and Igf1R genes. We have found that IGF-I has potent chondrogenic effects in MSC. Effects are similar to transforming growth factor-Beta (TGF-Beta). Insulin-like growth factor binding protein-3 (IGFBP-3), well characterized as IGF carrier, has intrinsic bioactivities that are independent of IGF binding. IGFBP-3 levels are increased in degenerative cartilage diseases such as osteoarthritis. We have demonstrated that IGFBP-3 has IGF-independent growth inhibitory effects in chondroprogenitors. We now show that IGFBP-3 induces MSC apoptosis and antagonizes TGF-Beta chondroinductive effects in chondroprogenitors. Understanding IGF-I chondroinductive and IGFBP-3 chondroinhibitory effects would provide critical information to optimize the therapeutic use of MSC in cartilage disorders.
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http://dx.doi.org/10.1159/000085719DOI Listing
July 2005

RUNX3 inhibits cell proliferation and induces apoptosis by reinstating transforming growth factor beta responsiveness in esophageal adenocarcinoma cells.

Surgery 2004 Aug;136(2):310-6

Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

Background: SEG-1, a Barrett's-derived esophageal adenocarcinoma cell line, is not responsive to transforming growth factor beta (TGF-beta) growth effects. We hypothesize that SEG-1 cells lack the tumor-suppressor gene Runt domain transcription factor 3 (RUNX3) and that its reinstatement can restore the antiproliferative and apoptotic effects of TGF-beta.

Methods: RUNX3 expression was assessed by immunoblotting. SEG-1 cells were transfected with RUNX3 and treated with TGF-beta. The effects of RUNX3 transfection on cell proliferation and apoptosis were determined. Smad-mediated TGF-beta transcriptional activity was evaluated with the use of dual-luciferase assay.

Results: SEG-1 cells are not responsive to TGF-beta. SEG-1 cells lack RUNX3 protein expression, while RUNX3 is highly expressed in normal human gastric and esophageal epithelium. Although the Smad-2 signaling is activated by TGF-beta, SEG-1 cells lack Smad-mediated TGF-beta transcriptional activity. In cells transfected with RUNX3, TGF-beta acquired an antiproliferative effect and induced apoptosis (P = .001). RUNX3 transfection, in the absence of TGF-beta, had no effect on proliferation and apoptosis of SEG-1 cells. RUNX3 expression dramatically increases SMAD-mediated TGF-beta-induced transcriptional activity when compared with controls (P = .0001).

Conclusions: RUNX3 is not expressed in SEG-1 cells, while it is present in normal esophageal mucosa. RUNX3 is essential for the antiproliferative and apoptotic effects of TGF-beta in SEG-1 cells and for the Smad-mediated transcriptional activity of TGF-beta.
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http://dx.doi.org/10.1016/j.surg.2004.05.005DOI Listing
August 2004

A novel insulin-like growth factor (IGF)-independent role for IGF binding protein-3 in mesenchymal chondroprogenitor cell apoptosis.

Endocrinology 2003 May;144(5):1695-702

Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2579, USA.

Chondrogenesis results from the condensation of mesenchymal chondroprogenitor cells (MCC) that proliferate and differentiate into chondrocytes. We have previously shown that IGF binding protein (IGFBP)-3 has an IGF-independent antiproliferative effect in MCC. The current study evaluates the IGF-independent apoptotic effect of IGFBP-3 on MCC to modulate chondrocyte differentiation. We employed the RCJ3.1C5.18 chondrogenic cell line, which in culture progresses from MCC to differentiated chondrocytes; cells do not express IGFs or IGFBP-3. We also used IGFBP-3 mutants with decreased (I56 substituted to G56; L80 and L81 to G80G81) or abolished binding for IGFs (I56, L80, and L81 to G56G80G81). MCC transfected with IGFBP-3 detached, changed their phenotype, and underwent apoptosis. A maximal IGFBP-3 apoptotic effect was observed 24 h after transfection (463 +/- 73% of controls; P < 0.001). Remarkably, IGFBP-3 mutants had similar effects, demonstrating that the IGFBP-3 apoptotic action was clearly IGF independent. In addition, treatment with IGFBP-3 in serum-free conditions resulted in a significant increase of apoptosis (173 +/- 23% of controls; P < 0.05). Moreover, this apoptotic effect was selective for MCC, resulting in a selective reduction of chondrocytic nodules and a significant decrease in type II collagen expression and proteoglycan synthesis. In summary, we have identified a novel IGF-independent role for IGFBP-3 in the modulation of chondrocyte differentiation.
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http://dx.doi.org/10.1210/en.2002-220959DOI Listing
May 2003
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