Publications by authors named "Eric A Lewallen"

30 Publications

  • Page 1 of 1

Surface Roughness of Titanium Orthopedic Implants Alters the Biological Phenotype of Human Mesenchymal Stromal Cells.

Tissue Eng Part A 2021 Aug 16. Epub 2021 Aug 16.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA.

Metal orthopedic implants are largely biocompatible and generally achieve long-term structural fixation. However, some orthopedic implants may loosen over time even in the absence of infection. fixation failure is multifactorial, but the fundamental biological defect is cellular dysfunction at the host-implant interface. Strategies to reduce the risk of short- and long-term loosening include surface modifications, implant metal alloy type, and adjuvant substances such as polymethylmethacrylate cement. Surface modifications (e.g., increased surface rugosity) can increase osseointegration and biological ingrowth of orthopedic implants. However, the localized responses of cells to implant surface modifications need to be better characterized. As an model for investigating cellular responses to metallic orthopedic implants, we cultured mesenchymal stromal/stem cells on clinical-grade titanium disks (Ti6Al4V) that differed in surface roughness as high (porous structured), medium (grit blasted), and low (bead blasted). Topological characterization of clinically relevant titanium (Ti) materials combined with differential mRNA expression analyses (RNA-seq and real-time quantitative polymerase chain reaction) revealed alterations to the biological phenotype of cells cultured on titanium structures that favor early extracellular matrix production and observable responses to oxidative stress and heavy metal stress. These results provide a descriptive model for the interpretation of cellular responses at the interface between native host tissues and three-dimensionally printed modular orthopedic implants, and will guide future studies aimed at increasing the long-term retention of such materials after total joint arthroplasty.
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http://dx.doi.org/10.1089/ten.TEA.2020.0369DOI Listing
August 2021

Molecular pathology of human knee arthrofibrosis defined by RNA sequencing.

Genomics 2020 07 5;112(4):2703-2712. Epub 2020 Mar 5.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States. Electronic address:

Arthrofibrosis is an abnormal histopathologic response, is debilitating for patients, and poses a substantial unsolved clinical challenge. This study characterizes molecular biomarkers and regulatory pathways associated with arthrofibrosis by comparing fibrotic and non-fibrotic human knee tissue. The fibrotic group encompasses 4 patients undergoing a revision total knee arthroplasty (TKA) for arthrofibrosis (RTKA-A) while the non-fibrotic group includes 4 patients undergoing primary TKA for osteoarthritis (PTKA) and 4 patients undergoing revision TKA for non-arthrofibrotic and non-infectious etiologies (RTKA-NA). RNA-sequencing of posterior capsule specimens revealed differences in gene expression between each patient group by hierarchical clustering, principal component analysis, and correlation analyses. Multiple differentially expressed genes (DEGs) were defined in RTKA-A versus PTKA patients (i.e., 2059 up-regulated and 1795 down-regulated genes) and RTKA-A versus RTKA-NA patients (i.e., 3255 up-regulated and 3683 down-regulated genes). Our findings define molecular and pathological markers of arthrofibrosis, as well as novel potential targets for risk profiling, early diagnosis and pharmacological treatment of patients.
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http://dx.doi.org/10.1016/j.ygeno.2020.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217749PMC
July 2020

A multi-chamber tissue culture device for load-dependent parallel evaluation of tendon explants.

BMC Musculoskelet Disord 2019 Nov 18;20(1):549. Epub 2019 Nov 18.

Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA.

Background: Injuries in the musculoskeletal system, such as tendon and ligament ruptures, are challenging to manage and often require surgical reconstructions with limited long-term success. Thus, characterizations of these tissues are urgently needed to better understand cellular mechanisms that regulate tissue homeostasis and healing. Explant culturing systems allow for ex vivo analysis of tissues in an environment that mimics the native microenvironment in vivo.

Methods: Collaborative efforts within our institution facilitated the establishment of a novel explant culturing system. Tissue specimens cultured in single wells, with individual applied loading and/or biological environment, allowed characterization of tissue cultured under a variety of biological loading conditions. Quantitative PCR analysis for selected gene markers was our primary outcome.

Results: Data were stratified for analysis by either culture environment or loading condition. Our gene expression results show that specimens clustered by culture condition may differ in molecular markers related to ECM production (e.g., Col1a1, Adamts4) and/or organization (e.g., Tnc, Dnc). In contrast, loading condition did significantly alter the median gene expression levels of tissues in comparison to unloaded control samples, although gene expression values related to ECM degradation (e.g., Mmp1, Mmp10) were altered in tendons cultured under tension in the device.

Conclusion: Our study demonstrates promising utility of a novel explant culturing system for further characterization of musculoskeletal tissues such as native tendons and ligaments, as well as pathologic fibrotic tissues resulting from arthrofibrosis or Dupuytren's disease.
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http://dx.doi.org/10.1186/s12891-019-2896-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6862789PMC
November 2019

Challenges in the Measurement and Interpretation of Serum Titanium Concentrations.

Biol Trace Elem Res 2020 Jul 6;196(1):20-26. Epub 2019 Nov 6.

Department of Orthopedic Surgery, Mayo Clinic, 200 1st Street, SW, Rochester, MN, 55905, USA.

The measurement of circulating metal ion levels in total hip arthroplasty patients continues to be an area of clinical interest. National regulatory agencies have recommended measurement of circulating cobalt and chromium concentrations in metal-on-metal bearing symptomatic total hip arthroplasty patients. However, the clinical utility of serum titanium (Ti) measurements is less understood due to wide variations in reported values and methodology. Fine-scale instrumentation for detecting in situ Ti levels continues to improve and has transitioned from graphite furnace atomic absorption spectroscopy to inductively coupled plasma optical emission spectrometry or inductively coupled plasma mass spectrometry. Additionally, analytical interferences, variable sample types, and non-standardized sample collection methods complicate Ti measurement and underlie the wide variation in reported levels. Normal reference ranges and pathologic ranges for Ti levels remain to be established quantitatively. However, before these ranges can be recognized and implemented, methodological standardization is necessary. This paper aims to provide background and recommendations regarding the complexities of measurement and interpretation of circulating Ti levels in total hip arthroplasty patients.
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http://dx.doi.org/10.1007/s12011-019-01891-4DOI Listing
July 2020

assessment of high-molecular-weight polyethylene core suture tape for intra-articular ligament reconstruction: an animal study.

Bone Joint J 2019 10;101-B(10):1238-1247

Department of Orthopaedic Surgery, Mayo Clinic, Rochester, Minnesota, USA.

Aims: Options for the treatment of intra-articular ligament injuries are limited, and insufficient ligament reconstruction can cause painful joint instability, loss of function, and progressive development of degenerative arthritis. This study aimed to assess the capability of a biologically enhanced matrix material for ligament reconstruction to withstand tensile forces within the joint and enhance ligament regeneration needed to regain joint function.

Materials And Methods: A total of 18 New Zealand rabbits underwent bilateral anterior cruciate ligament reconstruction by autograft, FiberTape, or FiberTape-augmented autograft. Primary outcomes were biomechanical assessment (n = 17), microCT (µCT) assessment (n = 12), histological evaluation (n = 12), and quantitative polymerase chain reaction (qPCR) analysis (n = 6).

Results: At eight weeks, FiberTape alone or FiberTape-augmented autograft demonstrated increased biomechanical stability compared with autograft regarding ultimate load to failure (p = 0.035), elongation (p = 0.006), and energy absorption (p = 0.022). FiberTape-grafted samples also demonstrated increased bone mineral density in the bone tunnel (p = 0.039). Histological evaluation showed integration of all grafts in the bone tunnels by new bone formation, and limited signs of inflammation overall. A lack of prolonged inflammation in all samples was confirmed by quantification of inflammation biomarkers. However, no regeneration of ligament-like tissue was observed along the suture tape materials. Except for one autograft failure, no adverse events were detected.

Conclusion: Our results indicate that FiberTape increases the biomechanical performance of intra-articular ligament reconstructions in a verified rabbit model at eight weeks. Within this period, FiberTape did not adversely affect bone tunnel healing or invoke a prolonged elevation in inflammation. Cite this article: 2019;101-B:1238-1247.
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http://dx.doi.org/10.1302/0301-620X.101B10.BJJ-2018-1282.R2DOI Listing
October 2019

Inhibition of COX-2 Pathway as a Potential Prophylaxis Against Arthrofibrogenesis in a Rabbit Model of Joint Contracture.

J Orthop Res 2019 12 26;37(12):2609-2620. Epub 2019 Aug 26.

Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, Minnesota.

Arthrofibrosis is a common complication following total knee arthroplasty caused by pathologic fibroblast activation and excessive collagen deposition around a synovial joint leading to debilitating loss of motion. Treatment options are limited because the pathologic mechanisms remain to be characterized. Dysregulation of the inflammatory cascade may lead to communication between myofibroblasts and immune cells triggering tissue metaplasia, and excessive collagen deposition described clinically as arthrofibrosis. We explored the novel use of celecoxib (selective cyclooxygenase-2 [COX-2] inhibitor) to disrupt the downstream effects of the post-traumatic inflammatory cascade and inhibit scar tissue formation in a validated rabbit model of arthrofibrosis combined with new parameters for quantifying the stiffness of the posterior capsule. Biomechanical and molecular analyses, of contracted rabbit knee posterior capsule tissue after COX-2 inhibition revealed increased maximal passive extension and down-regulation of collagen messenger RNA compared with controls. Histopathologic examination suggested a trend of decreased quantities of dense fibrous connective tissue with COX-2 inhibition. These data may suggest that inhibiting the inflammatory cascade could potentially reduce pathologic myofibroblast activation, thereby reducing scar tissue formation and increasing the range of motion in arthrofibrotic joints. Implementing a multi-modal pharmacologic approach may simultaneously target numerous cellular components contributing to the complex process of arthrofibrogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2609-2620, 2019.
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http://dx.doi.org/10.1002/jor.24441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848758PMC
December 2019

Seeding decellularized nerve allografts with adipose-derived mesenchymal stromal cells: An in vitro analysis of the gene expression and growth factors produced.

J Plast Reconstr Aesthet Surg 2019 Aug 9;72(8):1316-1325. Epub 2019 May 9.

Department of Orthopedic Surgery, Division of Hand and Microvascular Surgery, Mayo Clinic, 200 First Street S.W., Rochester, MN 55905, USA. Electronic address:

Mesenchymal stromal cells (MSCs) secrete many soluble growth factors and have previously been shown to stimulate nerve regeneration. MSC-seeded processed nerve allografts could potentially be a promising method for large segmental motor nerve injuries. Further progress in our understanding of how the functions of MSCs can be leveraged for peripheral nerve repair is required before making clinical translation. The present study, therefore, investigated whether interactions of adipose-derived MSCs with decellularized nerve allografts can improve gene and protein expression of growth factors that may support nerve regeneration. Human nerve allografts (n = 30) were decellularized and seeded with undifferentiated human adipose-derived MSCs. Subsequently, the MSCs and MSC-seeded grafts were isolated on days 3, 7, 14, and 21 in culture for RNA expression analysis by qRT-PCR. Evaluated genes included NGF, BDNF, PTN, GAP43, MBP, PMP22, VEGF, and CD31. Growth factor production was evaluated and quantified using enzyme-linked immunosorbent assay (ELISA). On day 21, semi-quantitative RT-PCR analysis showed that adherence of MSCs to nerve allografts significantly enhances mRNA expression of neurotrophic, angiogenic, endothelial, and myelination markers (e.g., BDNF, VEGF, CD31, and MBP). ELISA results revealed an upregulation of BDNF and reduction of both VEGF and NGF protein levels. This study demonstrates that seeding of undifferentiated adipose-derived MSCs onto processed nerve allografts permits the secretion of neurotrophic and angiogenic factors that can stimulate nerve regeneration. These favorable molecular changes suggest that MSC supplementation of nerve allografts may have potential in improving nerve regeneration.
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http://dx.doi.org/10.1016/j.bjps.2019.04.014DOI Listing
August 2019

Flyingfish (Exocoetidae) species diversity and habitats in the eastern tropical Pacific Ocean.

Mar Biodivers 2018 Dec 9;48(4):1755-1765. Epub 2017 Mar 9.

Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada.

Flyingfishes are large enough to eat zooplankton, small enough to be consumed by top predators, and therefore form a central mid-trophic component of tropical epipelagic marine food webs. Characterizing patterns of flyingfish abundance, distribution, and habitat preference have important implications for understanding both localized and generalized functions of marine ecosystems. The eastern tropical Pacific Ocean (ETP) supports many flyingfish species and their predators, yet no studies to date have identified oceanographic factors that define flyingfish habitats or estimate species richness and diversity at broad taxonomic and geographic scales. In this study, we analyzed 11,125 flyingfish representing 25 species and all 7 named genera, collected from the ETP over a 21-year period. We applied spatially-explicit analysis methods (ARCGIS, DIVA-GIS, MAXENT) and compared specimen locality data to remotely-sensed oceanographic data, and previously described oceanographic partitions. Our results show that is the most abundant genus (49%), and the most abundant species (32%) of flyingfishes in the ETP. Mean sea surface temperature was most important for defining flyingfish habitats (19.2-41.7%) and species richness (highest in the North Equatorial Current). Additionally, flyingfish species diversity was found to be highest in coastal regions of the study area (Shannon indices > 1.5). Together, these results provide unprecedented characterizations of a mid-trophic epipelagic community in an economically valuable region during a time when sea surface temperatures are predicted to increase as a result of global climate change.
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http://dx.doi.org/10.1007/s12526-017-0666-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260952PMC
December 2018

Molecular pathology of adverse local tissue reaction caused by metal-on-metal implants defined by RNA-seq.

Genomics 2019 12 21;111(6):1404-1411. Epub 2018 Sep 21.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States. Electronic address:

Total hip arthroplasty (THA) alleviates hip pain and improves joint function. Current implant design permits long-term survivorship of THAs, but certain metal-on-metal (MoM) articulations can portend catastrophic failure due to adverse local tissue reactions (ALTR). Here, we identified biological and molecular differences between periacetabular synovial tissues of patients with MoM THA failure undergoing revision THA compared to patients undergoing primary THA for routine osteoarthritis (OA). Analysis of tissue biopsies by RNA-sequencing (RNA-seq) revealed that MoM patient samples exhibit significantly increased expression of immune response genes but decreased expression of genes related to extracellular matrix (ECM) remodeling. Thus, interplay between local tissue inflammation and ECM degradation may account for the pathology and compromised clinical outcomes in select patients with MoM implants. We conclude that adverse responses of host tissues to implant materials result in transcriptomic modifications in patients with MoM implants that permit consideration of strategies that could mitigate ECM damage.
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http://dx.doi.org/10.1016/j.ygeno.2018.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428632PMC
December 2019

A Drug Eluting Scaffold for the Treatment of Arthrofibrosis.

Tissue Eng Part C Methods 2018 09;24(9):514-523

1 Department of Orthopedic Surgery, Mayo Clinic , Rochester, Minnesota.

Introduction: The inflammatory cascade and production of prostaglandins may play a role in the pathogenesis of arthrofibrosis, a debilitating condition after joint replacement and other orthopedic procedures. Cyclooxygenase 2 (COX-2) inhibitors may mitigate the inflammatory response and formation of arthrofibrosis, but oral delivery is associated with risk of systemic side effects in many patients. The nonsteroidal anti-inflammatory drug, celecoxib, may have therapeutic benefits for arthrofibrosis, but current methods for its local delivery (e.g., biologically derived microspheres) are not translatable to immediate clinical use. Therefore, we investigated the use of a drug scaffold for sustainable intra-articular delivery of therapeutic doses of celecoxib.

Materials And Methods: Celecoxib was eluted from clinically approved biodegradable collagen membranes over 7 days as measured by UV spectroscopy and high-performance liquid chromatography/mass spectroscopy. Eluted concentrations of celecoxib were examined for toxicity (live/dead staining) and profibrotic gene expression (real-time-quantitative polymerase chain reaction) in rabbit knee capsular fibroblasts in vitro.

Results: Sustained concentrations of celecoxib eluted from the membrane over 7 days from both a wet and dry scaffold, with a burst release (30-45%) of celecoxib in the first 2 h. Rabbit cells treated with eluted concentrations experienced a toxic response to the burst release doses, and inhibitory effects on profibrotic genes were seen in response to the sustained doses eluted from the scaffold.

Conclusions: This study characterized the novel use of collagen scaffolds for intra-articular drug delivery to treat arthrofibrosis. Scaffolds exhibit celecoxib release through an initial burst release followed by sustained release of antifibrotic doses over 7 days. Thus, collagen scaffolds are promising for clinician-directed treatment of arthrofibrosis.
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http://dx.doi.org/10.1089/ten.TEC.2018.0136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426273PMC
September 2018

Joint contracture is reduced by intra-articular implantation of rosiglitazone-loaded hydrogels in a rabbit model of arthrofibrosis.

J Orthop Res 2018 11 13;36(11):2949-2955. Epub 2018 Jul 13.

Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905.

Trauma, surgery, and other inflammatory conditions can lead to debilitating joint contractures. Adjunct pharmacologic modalities may permit clinical prevention and treatment of recalcitrant joint contractures. We investigated the therapeutic potential of rosiglitazone by intra-articular delivery via oligo[poly(ethylene glycol)fumarate] (OPF) hydrogels in an established rabbit model of arthrofibrosis. OPF hydrogels loaded with rosiglitazone were characterized for drug elution properties upon soaking in minimum essential media (MEM) with 10% fetal bovine serum and measurements of drug concentrations via High Performance Liquid Chromatography (HPLC). Drug-loaded scaffolds were surgically implanted into 24 skeletally mature female New Zealand White rabbits that were divided into equal groups receiving OPF hydrogels loaded with rosiglitazone (1.67 mg), or vehicle control (10 µl DMSO). After 8 weeks of joint immobilization, rabbits were allowed unrestricted cage activity for 16 weeks. Contracture angles of rabbit limbs treated with rosiglitazone showed statistically significant improvements in flexion compared to control animals (mean angles, respectively, 64.4° vs. 53.3°, p < 0.03). At time of sacrifice (week 24), animals in the rosiglitazone group continued to exhibit less joint contracture than controls (119.0° vs. 99.5°, p = 0.014). The intra-articular delivery of rosiglitazone using implanted OPF hydrogels decreases flexion contractures in a rabbit model of arthrofibrosis without causing adverse effects (e.g., gross inflammation or arthritis). Statement of Clinical Significance: Post-traumatic joint contractures are common and debilitating, with limited available treatment options. Pharmacologic interventions can potentially prevent and treat such contractures. This study is translational in that a commercially approved medication has been repurposed through a novel delivery device. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2949-2955, 2018.
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http://dx.doi.org/10.1002/jor.24068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347955PMC
November 2018

Fibrin glue mediated delivery of bone anabolic reagents to enhance healing of tendon to bone.

J Cell Biochem 2018 07 25;119(7):5715-5724. Epub 2018 Mar 25.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.

Tendon graft healing in bone tunnels for the fixation of intra-articular ligament reconstructions may limit clinical outcome by delaying healing. This study assesses the effects of hydrogel-mediated delivery of bone anabolic growth factors in a validated model of tendon-to-bone tunnel healing. Forty-five Wistar rats were randomly allocated into three groups (BMP2-treated, GSK126-treated, and placebo). All animals underwent a tendon-to-bone tunnel reconstruction. Healing was evaluated at 4 weeks by biomechanical assessment, micro-computed tomography (bone mineral density, bone volume, cross sectional area of bone tunnels), and traditional histology. Adverse events associated with the hydrogel-mediated delivery of drugs were not observed. Results of our biomechanical assessment demonstrated favorable trends in animals treated with bone anabolic factors for energy absorption (P = 0.116) and elongation (P = 0.054), while results for force to failure (P = 0.691) and stiffness (P = 0.404) did not show discernible differences. Cross sectional areas for BMP2-treated animals were reduced, but neither BMP2 nor GSK126 administration altered bone mineral density (P = 0.492) or bone volume in the bone tunnel. These results suggest a novel and positive effect of bone anabolic factors on tendon-to-bone tunnel healing. Histological evaluation confirmed absence of collagen fibers crossing the soft tissue-bone interface indicating immature graft integration as expected at this time point. Our study indicates that hydrogel-mediated delivery of BMP2 and GSK126 appears to be safe and has the potential to enhance tendon-to-bone tunnel healing in ligament reconstructions.
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http://dx.doi.org/10.1002/jcb.26755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993592PMC
July 2018

Molecular pathology of total knee arthroplasty instability defined by RNA-seq.

Genomics 2018 09 22;110(5):247-256. Epub 2017 Nov 22.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States. Electronic address:

Total knee arthroplasty (TKA) is a durable and reliable procedure to alleviate pain and improve joint function. However, failures related to flexion instability sometimes occur. The goal of this study was to define biological differences between tissues from patients with and without flexion instability of the knee after TKA. Human knee joint capsule tissues were collected at the time of primary or revision TKAs and analyzed by RT-qPCR and RNA-seq, revealing novel patterns of differential gene expression between the two groups. Interestingly, genes related to collagen production and extracellular matrix (ECM) degradation were higher in samples from patients with flexion instability. Partitioned clustering analyses further emphasized differential gene expression patterns between sample types that may help guide clinical interpretations of this complication. Future efforts to disentangle the effects of physical and biological (e.g., transcriptomic modifications) risk factors will aid in further characterizing and avoiding flexion instability after TKA.
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http://dx.doi.org/10.1016/j.ygeno.2017.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963991PMC
September 2018

Local Cellular Responses to Titanium Dioxide from Orthopedic Implants.

Biores Open Access 2017 1;6(1):94-103. Epub 2017 Jul 1.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.

We evaluated recently published articles relevant to the biological effects of titanium dioxide (TiO) particles on local endogenous cells required for normal bone homeostasis, repair, and implant osseointegration. Structural characteristics, size, stability, and agglomeration of TiO particles alter the viability and behavior of multiple bone-related cell types. Resulting shifts in bone homeostasis may increase bone resorption and lead to clinical incidents of osteolysis, implant loosening, and joint pain. TiO particles that enter cells (through endocytosis or Trojan horse mechanism) may further disrupt implant retention. We propose that cellular responses to titanium-based nanoparticles contribute to pathological mechanisms underlying the aseptic loosening of titanium-based metal implants.
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http://dx.doi.org/10.1089/biores.2017.0017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627672PMC
July 2017

Intra-articular injection of a substance P inhibitor affects gene expression in a joint contracture model.

J Cell Biochem 2018 02 20;119(2):1326-1336. Epub 2017 Nov 20.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.

Substance P (SP), a neurotransmitter released after injury, has been linked to deregulated tissue repair and fibrosis in musculoskeletal tissues and other organs. Although SP inhibition is an effective treatment for nausea, it has not been previously considered as an anti-fibrotic therapy. Although there are extensive medical records of individuals who have used SP antagonists, our analysis of human registry data revealed that patients receiving these antagonists and arthroplasty are exceedingly rare, thus precluding a clinical evaluation of their potential effects in the context of arthrofibrosis. Therefore, we pursued in vivo studies to assess the effect of SP inhibition early after injury on pro-fibrotic gene expression and contractures in an animal model of post-traumatic joint stiffening. Skeletally mature rabbits (n = 24) underwent surgically induced severe joint contracture, while injected with either fosaprepitant (a selective SP antagonist) or saline (control) early after surgery (3, 6, 12, and 24 h). Biomechanical testing revealed that differences in mean contracture angles between the groups were not statistically significant (P = 0.27), suggesting that the drug neither mitigates nor exacerbates joint contracture. However, microarray gene expression analysis revealed that mRNA levels for proteins related to cell signaling, pro-angiogenic, pro-inflammatory, and collagen matrix production were significantly different between control and fosaprepitant treated rabbits (P < 0.05). Hence, our study demonstrates that inhibition of SP alters expression of pro-fibrotic genes in vivo. This finding will motivate future studies to optimize interventions that target SP to reduce the formation of post-traumatic joint contractures.
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http://dx.doi.org/10.1002/jcb.26256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388635PMC
February 2018

Diabetes Mellitus and Hyperglycemia and the Risk of Aseptic Loosening in Total Joint Arthroplasty.

J Arthroplasty 2017 09 2;32(9S):S251-S253. Epub 2017 Mar 2.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.

Background: It is unknown to what extent diabetes mellitus modifies the long-term risk of aseptic loosening in total hip arthroplasty (THA) and total knee arthroplasty (TKA). We examined the association between diabetes mellitus, perioperative hyperglycemia, and the likelihood of revisions for aseptic loosening.

Methods: We studied 16,085 primary THA and TKA procedures performed at a large tertiary care hospital between 2002 and 2009. All blood glucose values around the time of surgery (within 1 week) were retrieved. Subsequent revision surgeries and the reasons for revision were ascertained through the institutional joint registry. Multivariate Cox models were used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) for aseptic loosening associated with diabetes mellitus and hyperglycemia adjusting for age, gender, body mass index, and surgery type.

Results: A total of 2911 (18%) surgeries had a diagnosis of diabetes mellitus at the time of surgery. Glucose testing was performed at least once in 7055 (44%) procedures within ±1 week of surgery. Although diabetic patients did not experience a higher risk of revision for aseptic loosening (HR, 0.87; 95% CI, 0.55-1.38), higher preoperative glucose values on the day before surgery were significantly associated with both the overall risk of revisions (HR, 2.80; 95% CI, 1.00-7.85) and revisions for aseptic loosening (HR, 4.95; 95% CI, 1.26-19.54).

Conclusion: High preoperative hyperglycemia is a potential risk factor for aseptic loosening in THA and TKA.
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http://dx.doi.org/10.1016/j.arth.2017.02.056DOI Listing
September 2017

Phylogenetics and biogeography of the two-wing flyingfish (Exocoetidae: ).

Ecol Evol 2017 03 12;7(6):1751-1761. Epub 2017 Feb 12.

Department of Biological Sciences University of Toronto Scarborough Toronto ON Canada.

Two-wing flyingfish (.) are widely distributed, epipelagic, mid-trophic organisms that feed on zooplankton and are preyed upon by numerous predators (e.g., tunas, dolphinfish, tropical seabirds), yet an understanding of their speciation and systematics is lacking. As a model of epipelagic fish speciation and to investigate mechanisms that increase biodiversity, we studied the phylogeny and biogeography of , a highly abundant holoepipelagic fish taxon of the tropical open ocean. Morphological and molecular data were used to evaluate the phylogenetic relationships, species boundaries, and biogeographic patterns of the five putative species. We show that the most widespread species () is sister to all other species, and we find no evidence for cryptic species in this taxon. Sister relationship between (Indo-Pacific) and (Atlantic) indicates the Isthmus of Panama and/or Benguela Barrier may have played a role in their divergence via allopatric speciation. The sister species and are found in different regions of the Pacific Ocean; however, our molecular results do not show a clear distinction between these species, indicating recent divergence or ongoing gene flow. Overall, our phylogeny reveals that the most spatially restricted species are more recently derived, suggesting that allopatric barriers may drive speciation, but subsequent dispersal and range expansion may affect the distributions of species.
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http://dx.doi.org/10.1002/ece3.2786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355192PMC
March 2017

Safety Studies for Use of Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells in a Rabbit Model for Osteoarthritis to Support a Phase I Clinical Trial.

Stem Cells Transl Med 2017 03 26;6(3):910-922. Epub 2016 Oct 26.

Department of Orthopedic Surgery, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, Minnesota, USA.

Adipose-derived mesenchymal stem cells (AMSCs) offer potential as a therapeutic option for clinical applications in musculoskeletal regenerative medicine because of their immunomodulatory functions and capacity for trilineage differentiation. In preparation for a phase I clinical trial using AMSCs to treat patients with osteoarthritis, we carried out preclinical studies to assess the safety of human AMSCs within the intra-articular joint space. Culture-expanded human AMSCs grown in human platelet-lysate were delivered via intra-articular injections into normal healthy rabbit knees and knees at risk for the development of osteoarthritis after bilateral medial anterior hemimeniscectomy. Treatment outcomes and safety were evaluated by assessing the general health, function, and behavior of the animals. Joint tissues were analyzed by x-ray, magnetic resonance imaging, and histopathology. Intra-articular AMSC therapy was well tolerated in this study. We did not observe adverse systemic reactions, nor did we find evidence of damage to intra-articular joint tissues. Thus, the data generated in this study show a favorable safety profile for AMSCs within the joint space in support of a phase I clinical trial evaluating the clinical utility of AMSCs to treat osteoarthritis. Stem Cells Translational Medicine 2017;6:910-922.
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http://dx.doi.org/10.5966/sctm.2016-0097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442773PMC
March 2017

Clinical Factors, Disease Parameters, and Molecular Therapies Affecting Osseointegration of Orthopedic Implants.

Curr Mol Biol Rep 2016 Sep 29;2(3):123-132. Epub 2016 Jun 29.

Department of Orthopedic Surgery, Mayo Clinic, 200 First St SW, Rochester, MN 55905.

Total hip and knee arthroplasty are effective interventions for management of end-stage arthritis. Indeed, about 7 million Americans are currently living with artificial hip and knee joints. The majority of these individuals, however, will outlive their implants and require revision surgeries, mostly due to poor implant osseointegration and aseptic loosening. Revisions are potentially avoidable with better management of patient-related risk factors that affect the osseointegration of orthopedic implants. In this review, we summarize the published clinical literature on the role of demographics, biologic factors, comorbidities, medications and aseptic loosening risk. We focus on several systemic and local factors that are particularly relevant to implant osseointegration. Examples include physiological and molecular processes that are linked to hyperglycemia, oxidative stress, metabolic syndrome and dyslipidemia. We discuss how orthopedic implant osseointegration can be affected by a number of molecular therapies that are antiresorptive or bone anabolic (i.e. calcium, vitamin D, bisphosphonates, calcitonin, strontium, hormone replacement therapy, selective estrogen-receptor modulators).
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http://dx.doi.org/10.1007/s40610-016-0042-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5166702PMC
September 2016

Population Genetic Structure of the Tropical Two-Wing Flyingfish (Exocoetus volitans).

PLoS One 2016 13;11(10):e0163198. Epub 2016 Oct 13.

Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada.

Delineating populations of pantropical marine fish is a difficult process, due to widespread geographic ranges and complex life history traits in most species. Exocoetus volitans, a species of two-winged flyingfish, is a good model for understanding large-scale patterns of epipelagic fish population structure because it has a circumtropical geographic range and completes its entire life cycle in the epipelagic zone. Buoyant pelagic eggs should dictate high local dispersal capacity in this species, although a brief larval phase, small body size, and short lifespan may limit the dispersal of individuals over large spatial scales. Based on these biological features, we hypothesized that E. volitans would exhibit statistically and biologically significant population structure defined by recognized oceanographic barriers. We tested this hypothesis by analyzing cytochrome b mtDNA sequence data (1106 bps) from specimens collected in the Pacific, Atlantic and Indian oceans (n = 266). AMOVA, Bayesian, and coalescent analytical approaches were used to assess and interpret population-level genetic variability. A parsimony-based haplotype network did not reveal population subdivision among ocean basins, but AMOVA revealed limited, statistically significant population structure between the Pacific and Atlantic Oceans (ΦST = 0.035, p<0.001). A spatially-unbiased Bayesian approach identified two circumtropical population clusters north and south of the Equator (ΦST = 0.026, p<0.001), a previously unknown dispersal barrier for an epipelagic fish. Bayesian demographic modeling suggested the effective population size of this species increased by at least an order of magnitude ~150,000 years ago, to more than 1 billion individuals currently. Thus, high levels of genetic similarity observed in E. volitans can be explained by high rates of gene flow, a dramatic and recent population expansion, as well as extensive and consistent dispersal throughout the geographic range of the species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163198PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063402PMC
May 2017

Safety of Intra-Articular Implantation of Oligo[Poly(ethylene glycol) Fumarate] Scaffolds into the Rabbit Knee.

Tissue Eng Part C Methods 2016 10;22(10):991-998

3 Department of Orthopedic Surgery, Mayo Clinic , Rochester, Minnesota.

Implantable biomaterials supporting extended release of pharmacologic agents may permit localized intra-articular delivery of drugs that modulate the fibrotic response to injuries and surgery. Oligo[poly (ethylene glycol)] fumarate (OPF) is an attractive organic carrier, but its safety profile within synovial joints remains unclear. Here, we assessed the safety of OPF sponges using a validated in vivo model of knee arthrofibrosis. A cohort of 102 rabbits was divided into five groups: arthrotomy only (24), arthrotomy with OPF scaffold placement (24), surgically induced contracture (24), surgically induced contracture with OPF scaffold placement (24), and control without any surgical intervention (6). Six rabbits per surgical group were sacrificed at 72 h, 2, 8, and 24 weeks. Outcomes included biomechanical testing of range of motion, histologic analysis of synovial and cartilage tissues, and scaffold degradation. Cartilage histology and biomechanical measurements were comparable between groups with and without OPF. Synovial inflammation scores were similar among most groups with a minimally elevated score in the rabbits with arthrotomy and OPF versus those with arthrotomy alone. Scores for synovial tissues in rabbits with contracture and OPF were clinically equivalent to those with contractures alone. Most animals (92%) retained scaffold fragments at 24 weeks. Thus, OPF scaffolds implanted into native or arthrofibrotic rabbit knees neither induce nor aggravate cartilage damage, synovial inflammation, or contractures. The apparent safety of OPF scaffolds suggests that they are suitable carriers for the controlled delivery of reagents into the intra-articular joint space to treat arthrofibrosis.
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http://dx.doi.org/10.1089/ten.TEC.2016.0209DOI Listing
October 2016

The synovial microenvironment of osteoarthritic joints alters RNA-seq expression profiles of human primary articular chondrocytes.

Gene 2016 Oct 1;591(2):456-64. Epub 2016 Jul 1.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States; Department of Biochemistry & Molecular Biology, Mayo Clinic, Rochester, MN, United States; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, United States. Electronic address:

Osteoarthritis (OA) is a disabling degenerative joint disease that prompts pain and has limited treatment options. To permit early diagnosis and treatment of OA, a high resolution mechanistic understanding of human chondrocytes in normal and diseased states is necessary. In this study, we assessed the biological effects of OA-related changes in the synovial microenvironment on chondrocytes embedded within anatomically intact cartilage from joints with different pathological grades by next generation RNA-sequencing (RNA-seq). We determined the transcriptome of primary articular chondrocytes derived from anatomically unaffected knees and ankles, as well as from joints affected by OA. The GALAXY bioinformatics platform was used to facilitate biological interpretations. Comparisons of patient samples by k-means, hierarchical clustering and principal component analyses together reveal that primary chondrocytes exhibit OA grade-related differences in gene expression, including genes involved in cell-adhesion, ECM production and immune response. We conclude that diseased synovial microenvironments in joints with different histopathological OA grades directly alter gene expression in chondrocytes. One ramification of this finding is that anatomically intact cartilage from OA joints is not an ideal source of healthy chondrocytes, nor should these specimens be used to generate a normal baseline for the molecular characterization of diseased joints.
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http://dx.doi.org/10.1016/j.gene.2016.06.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989726PMC
October 2016

RNA-seq analysis of clinical-grade osteochondral allografts reveals activation of early response genes.

J Orthop Res 2016 11 3;34(11):1950-1959. Epub 2016 Mar 3.

Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, Minnesota, 55905.

Preservation of osteochondral allografts used for transplantation is critical to ensure favorable outcomes for patients after surgical treatment of cartilage defects. To study the biological effects of protocols currently used for cartilage storage, we investigated differences in gene expression between stored allograft cartilage and fresh cartilage from living donors using high throughput molecular screening strategies. We applied next generation RNA sequencing (RNA-seq) and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) to assess genome-wide differences in mRNA expression between stored allograft cartilage and fresh cartilage tissue from living donors. Gene ontology analysis was used to characterize biological pathways associated with differentially expressed genes. Our studies establish reduced levels of mRNAs encoding cartilage related extracellular matrix (ECM) proteins (i.e., COL1A1, COL2A1, COL10A1, ACAN, DCN, HAPLN1, TNC, and COMP) in stored cartilage. These changes occur concomitantly with increased expression of "early response genes" that encode transcription factors mediating stress/cytoprotective responses (i.e., EGR1, EGR2, EGR3, MYC, FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND). The elevated expression of "early response genes" and reduced levels of ECM-related mRNAs in stored cartilage allografts suggests that tissue viability may be maintained by a cytoprotective program that reduces cell metabolic activity. These findings have potential implications for future studies focused on quality assessment and clinical optimization of osteochondral allografts used for cartilage transplantation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1950-1959, 2016.
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http://dx.doi.org/10.1002/jor.23209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993686PMC
November 2016

Human Adipose-Derived Mesenchymal Stromal/Stem Cells Remain Viable and Metabolically Active Following Needle Passage.

PM R 2016 09 28;8(9):844-54. Epub 2016 Jan 28.

Department of Physical Medicine & Rehabilitation, W14, Mayo Building, Mayo Clinic, 200 1st St, SW, Rochester, MN 55905; Department of Radiology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN; Department of Anatomy, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN(‡‡). Electronic address:

Objective: To assess the biological effects of passage through clinically relevant needles on the viability and metabolic activity of culture-expanded, human adipose tissue-derived mesenchymal stromal/stem cells (AMSCs).

Design: Prospective observational pilot study.

Setting: Academic medical center.

Participants: Patient-derived clinical-grade culture expanded AMSCs.

Interventions: AMSCs were passed through syringes without a needle attached (control), with an 18-gauge (25.4-mm) needle attached and with a 30-gauge (19-mm) needle attached at a constant injection flow rate and constant cell concentrations. Each injection condition was completed in triplicate.

Main Outcome Measures: Cell number and viability, proliferative capacity, metabolic activity, and acute gene expression as measured by cell counts, mitochondrial activity, and quantitative real time reverse-transcription polymerase chain reaction on day 0 (immediately), day 1, and day 4 after injection.

Results: AMSC viability was not significantly affected by injection, and cells proliferated normally regardless of study group. Postinjection, AMSCs robustly expressed both proliferation markers and extracellular matrix proteins. Stress-response mRNAs were markedly but transiently increased independently of needle size within the first day in culture postinjection.

Conclusions: Human, culture-expanded AMSCs maintain their viability, proliferative capacity, and metabolic function following passage through needles as small as 30-gauge at constant flow rates of 4 mL/min, despite an early, nonspecific stress/cytoprotective response. These initial findings suggest that culture-expanded AMSCs should tolerate the injection process during most cell-based therapeutic interventions.
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http://dx.doi.org/10.1016/j.pmrj.2016.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724701PMC
September 2016

Osteogenic potential of human adipose-tissue-derived mesenchymal stromal cells cultured on 3D-printed porous structured titanium.

Gene 2016 May 13;581(2):95-106. Epub 2016 Jan 13.

Department of Orthopedic Surgery, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA; Department of Biomedical Engineering and Physiology, Mayo Graduate School, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA. Electronic address:

Integration of porous metal prosthetics, which restore form and function of irreversibly damaged joints, into remaining healthy bone is critical for implant success. We investigated the biological properties of adipose-tissue-derived mesenchymal stromal/stem cells (AMSCs) and addressed their potential to alter the in vitro microenvironment of implants. We employed human AMSCs as a practical source for musculoskeletal applications because these cells can be obtained in large quantities, are multipotent, and have trophic paracrine functions. AMSCs were cultured on surgical-grade porous titanium disks as a model for orthopedic implants. We monitored cell/substrate attachment, cell proliferation, multipotency, and differentiation phenotypes of AMSCs upon osteogenic induction. High-resolution scanning electron microscopy and histology revealed that AMSCs adhere to the porous metallic surface. Compared to standard tissue culture plastic, AMSCs grown in the porous titanium microenvironment showed differences in temporal expression for genes involved in cell cycle progression (CCNB2, HIST2H4), extracellular matrix production (COL1A1, COL3A1), mesenchymal lineage identity (ACTA2, CD248, CD44), osteoblastic transcription factors (DLX3, DLX5, ID3), and epigenetic regulators (EZH1, EZH2). We conclude that metal orthopedic implants can be effectively seeded with clinical-grade stem/stromal cells to create a pre-conditioned implant.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054723PMC
http://dx.doi.org/10.1016/j.gene.2016.01.015DOI Listing
May 2016

Identification of differentially methylated regions in new genes associated with knee osteoarthritis.

Gene 2016 Jan 17;576(1 Pt 2):312-8. Epub 2015 Oct 17.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States; Department of Biochemistry & Molecular Biology, Mayo Clinic, Rochester, MN, United States. Electronic address:

Epigenetic changes in articular chondrocytes are associated with osteoarthritis (OA) disease progression. Numerous studies have identified differentially methylated cytosines in OA tissues; however, the consequences of altered CpG methylation at single nucleotides on gene expression and phenotypes are difficult to predict. With the objective of detecting novel genes relevant to OA, we conducted a genome-wide assessment of differentially methylated sites (DMSs) and differentially methylated regions (DMRs). DNA was extracted from visually damaged and normal appearing, non-damaged human knee articular cartilage from the same joint and then subjected to reduced representation bisulfite sequencing. DMRs were identified using a genome-wide systematic bioinformatics approach. A sliding-window of 500 bp was used for screening the genome for regions with clusters of DMSs. Gene expression levels were assessed and cell culture demethylation experiments were performed to further examine top candidate genes associated with damaged articular cartilage. More than 1000 DMRs were detected in damaged osteoarthritic cartilage. Nineteen of these contained five or more DMSs and were located in gene promoters or first introns and exons. Gene expression assessment revealed that hypermethylated DMRs in damaged samples were more consistently associated with gene repression than hypomethylated DMRs were with gene activation. Accordingly, a demethylation agent induced expression of most hypermethylated genes in chondrocytes. Our study revealed the utility of a systematic DMR search as an alternative to focusing on single nucleotide data. In particular, this approach uncovered promising candidates for functional studies such as the hypermethylated protein-coding genes FOXP4 and SHROOM1, which appear to be linked to OA pathology in humans and warrant further investigation.
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http://dx.doi.org/10.1016/j.gene.2015.10.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679536PMC
January 2016

Multi-disciplinary antimicrobial strategies for improving orthopaedic implants to prevent prosthetic joint infections in hip and knee.

J Orthop Res 2016 Feb 29;34(2):177-86. Epub 2015 Dec 29.

Department of Orthopedic Surgery, Mayo Clinic, 200 1st St SW, Rochester, Minnesota 55905.

Like any foreign object, orthopaedic implants are susceptible to infection when introduced into the human body. Without additional preventative measures, the absolute number of annual prosthetic joint infections will continue to rise, and may exceed the capacity of health care systems in the near future. Bacteria are difficult to eradicate from synovial joints due to their exceptionally diverse taxonomy, complex mechanistic attachment capabilities, and tendency to evolve antibiotic resistance. When a primary orthopaedic implant fails from prosthetic joint infection, surgeons are generally challenged by limited options for intervention. In this review, we highlight the etiology and taxonomic groupings of bacteria known to cause prosthetic joint infections, and examine their key mechanisms of attachment. We propose that antimicrobial strategies should focus on the most harmful bacteria taxa within the context of occurrence, taxonomic diversity, adhesion mechanisms, and implant design. Patient-specific identification of organisms that cause prosthetic joint infections will permit assessment of their biological vulnerabilities. The latter can be targeted using a range of antimicrobial techniques that exploit different colonization mechanisms including implant surface attachment, biofilm formation, and/or hematogenous recruitment. We anticipate that customized strategies for each patient, joint, and prosthetic component will be most effective at reducing prosthetic joint infections, including those caused by antibiotic-resistant and polymicrobial bacteria.
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http://dx.doi.org/10.1002/jor.23068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824296PMC
February 2016

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2.

J Biol Chem 2015 Nov 30;290(46):27604-17. Epub 2015 Sep 30.

From the Departments of Orthopedic Surgery, Biochemistry & Molecular Biology,

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.
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http://dx.doi.org/10.1074/jbc.M115.672345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646012PMC
November 2015

Histone deacetylase inhibition destabilizes the multi-potent state of uncommitted adipose-derived mesenchymal stromal cells.

J Cell Physiol 2015 Jan;230(1):52-62

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.

Human adipose-derived mesenchymal stromal cells (AMSCs) grown in platelet lysate are promising agents for therapeutic tissue regeneration. Here, we investigated whether manipulation of epigenetic events by the clinically relevant histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) alters differentiation of AMSCs. The multipotency of AMSCs was validated by their ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. High-throughput RNA sequencing and RT-qPCR established that human histone deacetylases (HDAC1 to HDAC11, and SIRT1 to SIRT7) are differentially expressed in AMSCs. SAHA induces hyper-acetylation of histone H3 and H4, stimulates protein expression of the HDAC-responsive gene SLC9A3R1/NHERF1 and modulates the AKT/FOXO1 pathway. Biologically, SAHA interferes with osteogenic, chondrogenic and adipogenic lineage commitment of multipotent AMSCs. Mechanistically, SAHA-induced loss of differentiation potential of uncommitted AMSCs correlates with multiple changes in the expression of principal transcription factors that control mesenchymal or pluripotent states. We propose that SAHA destabilizes the multi-potent epigenetic state of uncommitted human AMSCs by hyper-acetylation and perturbation of key transcription factor pathways. Furthermore, AMSCs grown in platelet lysate may provide a useful biological model for screening of new HDAC inhibitors that control the biological fate of human mesenchymal stromal cells.
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http://dx.doi.org/10.1002/jcp.24680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225068PMC
January 2015

High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells.

J Cell Biochem 2014 Oct;115(10):1816-28

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.
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http://dx.doi.org/10.1002/jcb.24852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225070PMC
October 2014
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