Orthopedic biomaterial-associated infections remain a major clinical challenge, with Staphylococcus aureus being the most common pathogen. S. aureus biofilm formation enhances immune evasion and antibiotic resistance, resulting in a local, indolent infection that can persist long-term without symptoms before eventual hardware failure, bone non-union, or sepsis. Immune modulation is an emerging strategy to combat host immune evasion by S. aureus. However, most immune modulation strategies are focused on local immune responses at the site of infection, with little emphasis on understanding the infection-induced and orthopedic-related systemic immune responses of the host, and their role in local infection clearance and tissue regeneration. This study utilized a rat bone defect model to investigate how implant-associated infection affects the systemic immune response. Long-term systemic immune dysregulation was observed with a significant systemic decrease in T cells and a concomitant increase in immunosuppressive myeloid-derived suppressor cells (MDSCs) compared to non-infected controls. Further, the control group exhibited a regulated and coordinated systemic cytokine response, which was absent in the infection group. Multivariate analysis revealed high levels of MDSCs to be most correlated with the infection group, while high levels of T cells were most correlated with the control group. Locally, the infection group had attenuated macrophage infiltration and increased levels of MDSCs in the local soft tissue compared to non-infected controls. These data reveal the widespread impacts of an orthopedic infection on both the local and the systemic immune responses, uncovering promising targets for diagnostics and immunotherapies that could optimize treatment strategies and ultimately improve patient outcomes.

BACKGROUND:Our understanding of the biology of ankle arthrodesis is based largely on work in spine and long bone animal models. However, the local soft tissue and vascular anatomy of the foot and ankle is different from that of the spine. Accordingly, the objective of this study was to develop a small animal ankle arthrodesis model.METHODS:A total of 12 Lewis rats successfully underwent ankle arthrodesis with stabilization consisting of a single Kirschner wire across the prepared tibiotalar joint. Based on high nonunion rates with this initial procedure, a modification was made consisting of a second pin crossing the joint. A total of 6 rats underwent the second procedure. Radiographs were taken postoperatively and in 2-week intervals up to 10 weeks. Micro computed tomography (µCT) and histological analysis was conducted at 10 weeks to assess the fusion mass. Osseous bridging of greater than 50% across the tibiotalar joint was deemed a successful fusion.RESULTS:µCT analysis determined that 11 of the 12 rats in the single-pin cohort developed nonunions (8.3% fusion rate). In the dual-pin cohort, all 6 animals successfully fused (100% fusion rate). Histological analysis supported the radiographic imaging conclusions.CONCLUSION:While the initial procedure had a high nonunion rate, enhancing the stability of the fixation greatly increased the union rate.CLINICAL RELEVANCE:The present work demonstrates the first reliable small animal ankle arthrodesis model. We believe that this model can be used in the development of novel therapies aimed at decreasing complications and increasing fusion rates.

Poly(para-phenylene) (PPP) is a novel aromatic polymer with higher strength and stiffness than polyetheretherketone (PEEK), the gold standard material for polymeric load-bearing orthopaedic implants. The amorphous structure of PPP makes it relatively straightforward to manufacture different architectures, while maintaining mechanical properties. PPP is promising as a potential orthopaedic material; however, the biocompatibility and osseointegration have not been well investigated. The objective of this study was to evaluate biological and mechanical behavior of PPP, with or without porosity, in comparison to PEEK. We examined four specific constructs: 1) solid PPP, 2) solid PEEK, 3) porous PPP and 4) porous PEEK. Pre-osteoblasts (MC3T3) exhibited similar cell proliferation among the materials. Osteogenic potential was significantly increased in the porous PPP scaffold as assessed by ALP activity and calcium mineralization. In vivo osseointegration was assessed by implanting the cylindrical materials into a defect in the metaphysis region of rat tibiae. Significantly more mineral ingrowth was observed in both porous scaffolds compared to the solid scaffolds, and porous PPP had a further increase compared to porous PEEK. Additionally, porous PPP implants showed bone formation throughout the porous structure when observed via histology. A computational simulation of mechanical push-out strength showed approximately 50% higher interfacial strength in the porous PPP implants compared to the porous PEEK implants and similar stress dissipation. These data demonstrate the potential utility of PPP for orthopaedic applications and show improved osseointegration when compared to the currently available polymeric material. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The strategy of vascular tissue engineering is to create a vascular substitute by combining autologous vascular cells with a tubular-shaped biodegradable scaffold. We have previously developed a novel electrospun bilayered vascular scaffold that provides proper biological and biomechanical properties as well as structural configuration. In this study, we investigated the clinical feasibility of a cellularized vascular scaffold in a preclinical large animal model. We fabricated the cellularized vascular construct with autologous endothelial progenitor cell (EPC)-derived endothelial cells (ECs) and smooth muscle cells (SMCs) followed by a pulsatile bioreactor preconditioning. This fully cellularized vascular construct was tested in a sheep carotid arterial interposition model. After preconditioning, confluent and mature EC and SMC layers in the scaffold were achieved. The cellularized constructs sustained the structural integrity with a high degree of graft patency without eliciting an inflammatory response over the course of the 6-month period in sheep. Moreover, the matured EC coverage on the lumen and a thick smooth muscle layer were formed at 6 months after transplantation. We demonstrated that electrospun bilayered vascular scaffolds in conjunction with autologous vascular cells may be a clinically applicable alternative to traditional prosthetic vascular graft substitutes.Statement of SignificanceThis study demonstrates the utility of tissue engineering to provide platform technologies for rehabilitation of patients recovering from severe, devastating cardiovascular diseases. The long-term goal is to provide alternatives to vascular grafting using bioengineered blood vessels derived from an autologous cell source with a functionalized vascular scaffold. This novel bilayered vascular construct for engineering blood vessels is designed to offer "off-the-shelf" availability for clinical translation. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Bone marrow derived mesenchymal stem cells (MSCs) are regularly utilized for translational therapeutic strategies including cell therapy, tissue engineering, and regenerative medicine and are frequently used in preclinical mouse models for both mechanistic studies and screening of new cell based therapies. Current methods to culture murine MSCs (mMSCs) select for rapidly dividing colonies and require long-term expansion. These methods thus require months of culture to generate sufficient cell numbers for feasibility studies in a lab setting and the cell populations often have reduced proliferation and differentiation potential, or have become immortalized cells. Here we describe a simple and reproducible method to generate mMSCs by utilizing hypoxia and basic fibroblast growth factor supplementation. Cells produced using these conditions were generated 2.8 times faster than under traditional methods and the mMSCs showed decreased senescence and maintained their multipotency and differentiation potential until passage 11 and beyond. Our method for mMSC isolation and expansion will significantly improve the utility of this critical cell source in pre-clinical studies for the investigation of MSC mechanisms, therapies, and cell manufacturing strategies.

Lee, Sang Jin; Ahn, Hyunhee PhD; Ju, Young Min PhD; Takahashi, Hironobu; Okano, Teruo; Jackson, John PhD; Yoo, James MD, PhD; Atala, Anthony MD, FACS Author Information

Tissue engineering offers an attractive approach to creating functional small-diameter (<5mm) blood vessels by combining autologous cells with a natural and/or synthetic scaffold under suitable culture conditions, which results in a tubular construct that can be implanted in vivo. We have previously developed a vascular scaffold fabricated by electrospinning poly(ε-caprolactone) (PCL) and type I collagen that mimics the structural and biomechanical properties of native vessels. In this study, we investigated whether a smooth muscle cell (SMC) sheet could be combined with the electrospun vascular scaffolds to produce a more mature smooth muscle layer as compared to the conventional cell seeding method. The pre-fabricated SMC sheet, wrapped around the vascular scaffold, provided high cell seeding efficiency (approx. 100%) and a mature smooth muscle layer that expressed strong cell-to-cell junction, connexin 43 (CX43), and contractile proteins, α smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK). Moreover, bioreactor-associated preconditioning of the SMC sheet-combined vascular scaffold maintained high cell viability (95.9±2.7%) and phenotypes and improved cellular infiltration and mechanical properties (35.7% of tensile strength, 47.5% of elasticity, and 113.2% of elongation at break).

METHODS: Invasive penile cancer cases from 2010-2012 were identified from the NCDB.Pathologic tumor stage was recorded including spongiosal versus carvernosal involvement.Differences in demographic (age, race, comorbid status) and pathologic features (size of tumor, grade, nodal status, LVI, histology, and extranodal extension) between T2 and T3 tumors were compared using c 2 and t-tests.Univariate and multivariate logistic regression was performed to determine the odds of positive lymph nodes (pN+) at inguinal lymph node dissection (ILND) relative to T-stage.RESULTS: There were 367 T2 and 507 T3 patients with penile cancer.The proportion of cases with pN+ disease was 15%, 32%, 46% and 58% for T1, T2, T3 and T4 cases, respectively.Compared to T2 tumors, T3 tumors were larger (mean size 5.8 cm vs. 4.3 cm), more often treated with radical penectomy (36% vs 17%), had higher positive surgical margin rates (12% vs 9%), more aggressive pathology (32% vs 27% poorly differentiated), and were more likely to have lymphovascular invasion (42% vs 31%) (all p < 0.05).In univariate analysis, compared to T1 tumors, T2 (OR 2.8, 95% CI 1.9-4.2) and T3 (OR 4.7, 95% CI 3.3-6.8)were both associated with an increased risk of positive lymph nodes.Although in multivariate analysis, both T2 (OR 2.0, 95% CI 1.2-3.3)and T3 (OR 2.3, 95% CI 1.4-3.6)remained significantly associated with risk of positive lymph nodes compared to T1 disease, there was no increase in risk between T2 and T3 disease (OR 1.1, 95% CI 0.7-1.8,p ¼ 0.56).CONCLUSIONS: The proposed new AJCC staging system for the penile cancer distinguishes spongiosal (T2) from cavernosal (T3) involvement and identifies significant differences in pathologic features of the tumors (grade, LVI and size).There does not appear to be a difference in positive lymph node status between the two grades when other clinical and pathological variables are considered.Further study is required to confirm these findings and the prognostic implications of the proposed new staging system.

You have accessJournal of UrologyTransplantation & Vascular Surgery II1 Apr 2014MP72-08 CELLULARIZED ELECTROSPUN VASCULAR GRAFTS FOR RENAL ARTERY RECONSTRUCTION Hyunhee Ahn, Young Miin Ju, Cheil Kim, John D. Jackson, James J. Yoo, Sang Jin Lee, and Anthony Atala Hyunhee AhnHyunhee Ahn More articles by this author , Young Miin JuYoung Miin Ju More articles by this author , Cheil KimCheil Kim More articles by this author , John D. JacksonJohn D. Jackson More articles by this author , James J. YooJames J. Yoo More articles by this author , Sang Jin LeeSang Jin Lee More articles by this author , and Anthony AtalaAnthony Atala More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2247AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Abnormal vascular conditions, such as renal artery stenosis due to fibrous dysplasia or aneurysms may require the use of vascular bypass grafts. However, the use of autologous vascular grafts in these patients is often limited by the availability and size mismatch. We previously have shown that an electrospun synthetic vascular scaffold is able to withstand physiological vascular conditions. In this study we examined whether fully cellularized vascular grafts could be created for use in renal artery reconstruction. METHODS Bilayered vascular scaffolds were electrospun using collagen type I mixed with poly(å-caprolactone) (PCL) in a 1:1 ratio. To demonstrate the applicability, autologous smooth muscle and endothelial cells were seeded on the exterior and luminal surface of the vascular scaffold, respectively, followed by preconditioning in a bioreactor system. Preconditioned cellularized scaffolds were implanted as an arterial interposition in the left carotid artery of sheep. Grafts without cells served as controls. The animals were evaluated at 4, 12, and 24 weeks after surgery using doppler ultrasound, CT angiography and histomorphological analyses. RESULTS The electrospun vascular grafts provided adequate surgical handling. Dual cell seeded scaffolds withstood physiologic hemodynamic conditions and the vessel constructs remained patent for 6 month in vivo. Grafts without cells resulted in stenosis and occlusion. The implanted grafts maintained a stable midgraft diameter and confirmed the absence of aneurysmal degeneration. The explanted grafts showed normal vascular tissue configuration without inflammation. CONCLUSIONS Dual cell seeded PCL/collagen electrospun grafts maintained a high degree of patency and structural integrity without eliciting a histologic inflammatory response over the course of a 6-month period in a sheep arterial interposition model. This study indicates that cellularized electrospun grafts may provide an alternative option for patients with renal artery stenosis. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e826 Advertisement Copyright & Permissions© 2014Metrics Author Information Hyunhee Ahn More articles by this author Young Miin Ju More articles by this author Cheil Kim More articles by this author John D. Jackson More articles by this author James J. Yoo More articles by this author Sang Jin Lee More articles by this author Anthony Atala More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Appropriate surface wettability and roughness of biomaterials is an important factor in cell attachment and proliferation. In this study, we investigated the correlation between surface wettability and roughness, and biological response in human adipose-derived stem cells (hADSCs). We prepared wettable and rough gradient polyethylene (PE) surfaces by increasing the power of a radio frequency corona discharge apparatus with knife-type electrodes over a moving sample bed. The PE changed gradually from hydrophobic and smooth surfaces to hydrophilic (water contact angle, 90° to ~50°) and rough (80 to ~120 nm) surfaces as the power increased. We found that hADSCs adhered better to highly hydrophilic and rough surfaces and showed broadly stretched morphology compared with that on hydrophobic and smooth surfaces. The proliferation of hADSCs on hydrophilic and rough surfaces was also higher than that on hydrophobic and smooth surfaces. Furthermore, integrin beta 1 gene expression, an indicator of attachment, and heat shock protein 70 gene expression were high on hydrophobic and smooth surfaces. These results indicate that the cellular behavior of hADSCs on gradient surface depends on surface properties, wettability and roughness.

Arenas, Juan E. MD; Ahn, Hyunhee PhD; Hill, Tanner K. BS; Young, Ju M. PhD; Chang, Hwang PhD; Yoo, James MD, PhD; Lee, Sang J. PhD Author Information

336 ©2013 Society For Biomaterials

Using a complete spinal cord transection model, the present study employed a combinatorial strategy comprising rat bone marrow stem cells (rBMSCs) and polymer scaffolds to regenerate neurological function after spinal cord injury (SCI) of different lengths. SCI models with completely transected lesions were prepared by surgical removal of 1 mm (SC1) or 3 mm (SC3) lengths of spinal cord in the eighth-to-ninth spinal vertebrae, a procedure that resulted in bilateral hindlimb paralysis. A cylindrical poly(D,L-lactide-co-glycolide)/small intestinal submucosa scaffold 1 or 3 mm in length with or without rBMSCs was fitted into the completely transected lesion. Rats in SC1 and SC3 groups implanted with rBMSC-containing scaffolds received Basso-Beattie-Bresnahan scores for hindlimb locomotion of 15 and 8, respectively, compared with ∼3 for control rats in SC1-C and SC3-C groups implanted with scaffolds lacking rBMSCs. The amplitude of motor-evoked potentials recorded in the hindlimb area of the sensorimotor cortex after stimulation of the injured spinal cord averaged ∼100 μV in SC1-C and 10-50 μV in SC3-C groups at 4 weeks, and then declined to nearly zero at 8 weeks. In contrast, the amplitude of motor-evoked potentials increased from ∼300 to 350 μV between 4 and 8 weeks in SC1 rats and from ∼200 to ∼250 μV in SC3 rats. These results demonstrate functional recovery in rBMSC-transplanted rats, especially those with smaller defects. Immunohistochemically stained sections of the injury site showed clear evidence for axonal regeneration only in rBMSC-transplanted SC1 and SC3 models. In addition, rBMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating cell survival in SCI. Collectively, our results indicate that therapeutic rBMSCs in a poly(D,L-lactide-co-glycolide)/small intestinal submucosa scaffold induced nerve regeneration in a complete spinal cord transection model and showed that functional recovery further depended on defect length.

The sol-to-gel transition occurring at around body temperature makes the MPEG-PCL diblock copolymer an ideal candidate material for use as an injectable in situ-forming gel containing human adipose tissue-derived stem cells (hADSCs). The sol can be prepared at room temperature, and the gel forms at body temperature. Solutions of the copolymer containing hADSCs and osteogenic factors injected into rats formed gel scaffolds at the injection sites. The gels thus formed showed the interconnective pore structure required to support growth, proliferation, and differentiation of hADSCs. Bromodeoxyuridine-labeled hADSCs were confirmed to be present in gels formed in vivo. Bone formation was observed only in gel implants containing both hADSCs and osteogenic factors. Subcutaneous implantation of the in situ-forming gel scaffold demonstrated that hADSCs embedded in the gel stimulated much lower host tissue responses than did the gel alone, probably because of the unique immunomodulatory properties of hADSCs. In conclusion, our data on hADSCs embedded in an in situ gel scaffold suggest that this formulation may provide numerous benefits as a noninvasive alternative for tissue-engineered bone formation.