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Supraspinatus tendon overuse results in degenerative changes to tendon insertion region and adjacent humeral cartilage in a rat model.

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Supraspinatus tendon overuse results in degenerative changes to tendon insertion region and adjacent humeral cartilage in a rat model.

J Orthop Res. 2017 09;35(9):1910-1918

Authors: Parks AN, McFaline-Figueroa J, Coogan A, Poe-Yamagata E, Guldberg RE, Platt MO, Temenoff JS

Abstract
The etiology of rotator cuff tendon overuse injuries is still not well understood. Furthermore, how this overuse injury impacts other components of the glenohumeral joint, including nearby articular cartilage, is also unclear. Therefore, this study sought to better understand the time course of tendon protease activity in a rat model of supraspinatus overuse, as well as determine effects of 10 weeks of overuse on humeral head articular cartilage. For these studies, multiplex gelatin zymography was used to characterize protease activity profiles in tendon and cartilage, while histological scoring/mechanical testing and micro-computed tomography (μCT) imaging were used to quantify structural damage in the supraspinatus tendon insertion and humeral articular cartilage, respectively. Histological scoring of supraspinatus tendon insertions revealed tendinopathic cellular and collagen fiber changes after 10 weeks of overuse when compared to controls, while mechanical testing revealed no significant differences between tensile moduli (overuse: 24.5 ± 11.5 MPa; control: 16.3 ± 8.7 MPa). EPIC-μCT imaging on humeral articular cartilage demonstrated significant cartilage thinning (overuse: 119.6 ± 6.34 μm; control: 195.4 ± 13.4μm), decreased proteoglycan content (overuse: 2.1 ± 0.18 cm-1 ; control: 1.65 ± 0.14 cm-1 ), and increased subchondral bone thickness (overuse: 216.2 ± 10.9 μm; control: 192 ± 17.8μm) in the overuse animals. Zymography results showed no significant upregulation of cathepsins or matrix metalloproteinases in tendon or cartilage at 2 or 10 weeks of overuse compared to controls. These results have further elucidated timing of protease activity over 10 weeks and suggest that damage occurs to other tissues in addition to the supraspinatus tendon in this overuse injury model. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1910-1918, 2017.

PMID: 28001327 [PubMed – indexed for MEDLINE]

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Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration.

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Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration.

Tissue Eng Part A. 2017 07;23(13-14):683-695

Authors: Hettiaratchi MH, Chou C, Servies N, Smeekens JM, Cheng A, Esancy C, Wu R, McDevitt TC, Guldberg RE, Krishnan L

Abstract
Tissue engineering strategies involving the in vivo delivery of recombinant growth factors are often limited by the inability of biomaterials to spatially control diffusion of the delivered protein within the site of interest. The poor spatiotemporal control provided by porous collagen sponges, which are used for the clinical delivery of bone morphogenetic protein-2 (BMP-2) for bone regeneration, has necessitated the use of supraphysiological protein doses, leading to inflammation and heterotopic ossification. This study describes a novel tissue engineering strategy to spatially control rapid BMP-2 diffusion from collagen sponges in vivo by creating a high-affinity BMP-2 sink around the collagen sponge. We designed an electrospun poly-ɛ-caprolactone nanofiber mesh containing physically entrapped heparin microparticles, which have been previously demonstrated to bind and retain large amounts of BMP-2. Nanofiber meshes containing 0.05 and 0.10 mg of microparticles/cm2 demonstrated increased BMP-2 binding and decreased BMP-2 release in vitro compared with meshes without microparticles. However, when microparticle-containing meshes were used in vivo to limit the diffusion of BMP-2 delivered by using collagen sponges in a rat femoral defect, no differences in heterotopic ossification or biomechanical properties were observed. Further investigation revealed that, although BMP-2 binding to heparin microparticles was rapid, the presence of serum components attenuated microparticle-BMP-2 binding and increased BMP-2 release in vitro. These observations provide a plausible explanation for the results observed in vivo and suggest that competitive protein binding in vivo may hinder the ability of affinity-based biomaterials to modulate growth factor delivery.

PMID: 28338419 [PubMed – indexed for MEDLINE]

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* Skeletal Myoblast-Seeded Vascularized Tissue Scaffolds in the Treatment of a Large Volumetric Muscle Defect in the Rat Biceps Femoris Muscle.

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* Skeletal Myoblast-Seeded Vascularized Tissue Scaffolds in the Treatment of a Large Volumetric Muscle Defect in the Rat Biceps Femoris Muscle.

Tissue Eng Part A. 2017 09;23(17-18):989-1000

Authors: Li MT, Ruehle MA, Stevens HY, Servies N, Willett NJ, Karthikeyakannan S, Warren GL, Guldberg RE, Krishnan L

Abstract
High velocity impact injuries can often result in loss of large skeletal muscle mass, creating defects devoid of matrix, cells, and vasculature. Functional regeneration within these regions of large volumetric muscle loss (VML) continues to be a significant clinical challenge. Large cell-seeded, space-filling tissue-engineered constructs that may augment regeneration require adequate vascularization to maintain cell viability. However, the long-term effect of improved vascularization and the effect of addition of myoblasts to vascularized constructs have not been determined in large VMLs. Here, our objective was to create a new VML model, consisting of a full-thickness, single muscle defect, in the rat biceps femoris muscle, and evaluate the ability of myoblast-seeded vascularized collagen hydrogel constructs to augment VML regeneration. Adipose-derived microvessels were cultured with or without myoblasts to form vascular networks within collagen constructs. In the animal model, the VML injury was created in the left hind limb, and treated with the harvested autograft itself, constructs with microvessel fragments (MVF) only, constructs with microvessels and myoblasts (MVF+Myoblasts), or left empty. We evaluated the formation of vascular networks in vitro by light microscopy, and the capacity of vascularized constructs to augment early revascularization and muscle regeneration in the VML using perfusion angiography and creatine kinase activity, respectively. Myoblasts (Pax7+) were able to differentiate into myotubes (sarcomeric myosin MF20+) in vitro. The MVF+Myoblast group showed longer and more branched microvascular networks than the MVF group in vitro, but showed similar overall defect site vascular volumes at 2 weeks postimplantation by microcomputed tomography angiography. However, a larger number of small-diameter vessels were observed in the vascularized construct-treated groups. Yet, both vascularized implant groups showed primarily fibrotic tissue with adipose infiltration, poor maintenance of tissue volume within the VML, and little muscle regeneration. These data suggest that while vascularization may play an important supportive role, other factors besides adequate vascularity may determine the fate of regenerating volumetric muscle defects.

PMID: 28372522 [PubMed – indexed for MEDLINE]

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Gender-specific differential expression of exosomal miRNA in synovial fluid of patients with osteoarthritis.

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Gender-specific differential expression of exosomal miRNA in synovial fluid of patients with osteoarthritis.

Sci Rep. 2017 May 17;7(1):2029

Authors: Kolhe R, Hunter M, Liu S, Jadeja RN, Pundkar C, Mondal AK, Mendhe B, Drewry M, Rojiani MV, Liu Y, Isales CM, Guldberg RE, Hamrick MW, Fulzele S

Abstract
The pathogenesis of osteoarthritis (OA) is poorly understood, and therapeutic approaches are limited to preventing progression of the disease. Recent studies have shown that exosomes play a vital role in cell-to-cell communication, and pathogenesis of many age-related diseases. Molecular profiling of synovial fluid derived exosomal miRNAs may increase our understanding of OA progression and may lead to the discovery of novel biomarkers and therapeutic targets. In this article we report the first characterization of exosomes miRNAs from human synovial fluid. The synovial fluid exosomes share similar characteristics (size, surface marker, miRNA content) with previously described exosomes in other body fluids. MiRNA microarray analysis showed OA specific exosomal miRNA of male and female OA. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified gender-specific target genes/signaling pathways. These pathway analyses showed that female OA specific miRNAs are estrogen responsive and target TLR (toll-like receptor) signaling pathways. Furthermore, articular chondrocytes treated with OA derived extracellular vesicles had decreased expression of anabolic genes and elevated expression of catabolic and inflammatory genes. In conclusion, synovial fluid exosomal miRNA content is altered in patients with OA and these changes are gender specific.

PMID: 28515465 [PubMed – in process]

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Enhanced in vivo retention of low dose BMP-2 via heparin microparticle delivery does not accelerate bone healing in a critically sized femoral defect.

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Enhanced in vivo retention of low dose BMP-2 via heparin microparticle delivery does not accelerate bone healing in a critically sized femoral defect.

Acta Biomater. 2017 09 01;59:21-32

Authors: Hettiaratchi MH, Rouse T, Chou C, Krishnan L, Stevens HY, Li MA, McDevitt TC, Guldberg RE

Abstract
Bone morphogenetic protein-2 (BMP-2) is an osteoinductive growth factor used clinically to induce bone regeneration and fusion. Some complications associated with BMP-2 treatment have been attributed to rapid release of BMP-2 from conventional collagen scaffolds, motivating the development of tunable sustained-release strategies. We incorporated BMP-2-binding heparin microparticles (HMPs) into a hydrogel scaffold to improve spatiotemporal control of BMP-2 delivery to large bone defects. HMPs pre-loaded with BMP-2 were mixed into alginate hydrogels and compared to hydrogels containing BMP-2 alone. BMP-2 release from scaffolds in vitro, BMP-2 retention within injury sites in vivo, and bone regeneration in a critically sized femoral defect were evaluated. Compared to hydrogel delivery alone, BMP-2-loaded HMPs reduced BMP-2 release in vitro and increased early BMP-2 retention in the bone defect. BMP-2-loaded HMPs induced bone formation at both ectopic and orthotopic sites; however, the volume of induced bone was lower for defects treated with BMP-2-loaded HMPs compared to hydrogel delivery. To better understand the effect of HMPs on BMP-2 release kinetics, a computational model was developed to predict BMP-2 release from constructs in vivo. The model suggested that HMPs limited BMP-2 release into surrounding tissues, and that changing the HMP density could modulate BMP-2 release. Taken together, these experimental and computational results suggest the importance of achieving a balance of BMP-2 retention within the bone defect and BMP-2 release into surrounding soft tissues. HMP delivery of BMP-2 may provide a method of tuning BMP-2 release in vivo that can be further investigated to improve current methods of bone regeneration.
STATEMENT OF SIGNIFICANCE: The development of effective biomaterials for sustained protein delivery is a crucial component of tissue engineering strategies. However, in most applications, including bone repair, the optimal balance between protein presentation in the injury site and protein release into the surrounding tissues is unknown. Herein, we introduced heparin microparticles (HMPs) into a tissue engineered construct to increase in vivo retention of bone morphogenetic protein-2 (BMP-2) and enhance healing in femoral defects. Although HMPs induced bone regeneration, no increase in bone volume was observed, leading to further experimental and computational analysis of the effect of HMP-BMP-2 interactions on protein retention and release. Ultimately, this work provides insight into designing tunable protein-material interactions and their implications for controlling BMP-2 delivery.

PMID: 28645809 [PubMed – indexed for MEDLINE]

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Deformation and fatigue of tough 3D printed elastomer scaffolds processed by fused deposition modeling and continuous liquid interface production.

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Deformation and fatigue of tough 3D printed elastomer scaffolds processed by fused deposition modeling and continuous liquid interface production.

J Mech Behav Biomed Mater. 2017 11;75:1-13

Authors: Miller AT, Safranski DL, Wood C, Guldberg RE, Gall K

Abstract
Polyurethane (PU) based elastomers continue to gain popularity in a variety of biomedical applications as compliant implant materials. In parallel, advancements in additive manufacturing continue to provide new opportunities for biomedical applications by enabling the creation of more complex architectures for tissue scaffolding and patient specific implants. The purpose of this study was to examine the effects of printed architecture on the monotonic and cyclic mechanical behavior of elastomeric PUs and to compare the structure-property relationship across two different printing approaches. We examined the tensile fatigue of notched specimens, 3D crosshatch scaffolds, and two 3D spherical pore architectures in a physically crosslinked polycarbonate urethane (PCU) printed via fused deposition modeling (FDM) as well as a photo-cured, chemically-crosslinked, elastomeric PU printed via continuous liquid interface production (CLIP). Both elastomers were relatively tolerant of 3D geometrical features as compared to stiffer synthetic implant materials such as PEEK and titanium. PCU and crosslinked PU samples with 3D porous structures demonstrated a reduced tensile failure stress as expected without a significant effect on tensile failure strain. PCU crosshatch samples demonstrated similar performance in strain-based tensile fatigue as solid controls; however, when plotted against stress amplitude and adjusted by porosity, it was clear that the architecture had an impact on performance. Square shaped notches or pores in crosslinked PU appeared to have a modest effect on strain-based tensile fatigue while circular shaped notches and pores had little impact relative to smooth samples. When plotted against stress amplitude, any differences in fatigue performance were small or not statistically significant for crosslinked PU samples. Despite the slight difference in local architecture and tolerances, crosslinked PU solid samples were found to perform on par with PCU solid samples in tensile fatigue, when appropriately adjusted for material hardness. Finally, tests of samples with printed architecture localized to the gage section revealed an effect in which fatigue performance appeared to drastically improve despite the localization of strain.

PMID: 28689135 [PubMed – indexed for MEDLINE]

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Wireless Implantable Sensor for Noninvasive, Longitudinal Quantification of Axial Strain Across Rodent Long Bone Defects.

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Wireless Implantable Sensor for Noninvasive, Longitudinal Quantification of Axial Strain Across Rodent Long Bone Defects.

J Biomech Eng. 2017 Nov 01;139(11):

Authors: Klosterhoff BS, Ghee Ong K, Krishnan L, Hetzendorfer KM, Chang YH, Allen MG, Guldberg RE, Willett NJ

Abstract
Bone development, maintenance, and regeneration are remarkably sensitive to mechanical cues. Consequently, mechanical stimulation has long been sought as a putative target to promote endogenous healing after fracture. Given the transient nature of bone repair, tissue-level mechanical cues evolve rapidly over time after injury and are challenging to measure noninvasively. The objective of this work was to develop and characterize an implantable strain sensor for noninvasive monitoring of axial strain across a rodent femoral defect during functional activity. Herein, we present the design, characterization, and in vivo demonstration of the device’s capabilities for quantitatively interrogating physiological dynamic strains during bone regeneration. Ex vivo experimental characterization of the device showed that it possessed promising sensitivity, signal resolution, and electromechanical stability for in vivo applications. The digital telemetry minimized power consumption, enabling extended intermittent data collection. Devices were implanted in a rat 6 mm femoral segmental defect model, and after three days, data were acquired wirelessly during ambulation and synchronized to corresponding radiographic videos, validating the ability of the sensor to noninvasively measure strain in real-time. Together, these data indicate the sensor is a promising technology to quantify tissue mechanics in a specimen specific manner, facilitating more detailed investigations into the role of the mechanical environment in dynamic bone healing and remodeling processes.

PMID: 28975256 [PubMed – indexed for MEDLINE]

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Contrast enhanced μCT imaging of early articular changes in a pre-clinical model of osteoarthritis.

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Contrast enhanced μCT imaging of early articular changes in a pre-clinical model of osteoarthritis.

Osteoarthritis Cartilage. 2018 01;26(1):118-127

Authors: Reece DS, Thote T, Lin ASP, Willett NJ, Guldberg RE

Abstract
OBJECTIVE: The objective of this study was to characterize early osteoarthritis (OA) development in cartilage and bone tissues in the rat medial meniscus transection (MMT) model using non-destructive equilibrium partitioning of an ionic contrast agent micro-computed tomography (EPIC-μCT) imaging. Cartilage fibrillation, one of the first physiological developments in OA, was quantified in the rat tibial plateau as three-dimensional (3D) cartilage surface roughness using a custom surface-rendering algorithm.
METHODS: Male Lewis rats underwent MMT or sham-operation in the left leg. At 1- and 3-weeks post-surgery, the animals (n = 7-8 per group) were euthanized and the left legs were scanned using EPIC-μCT imaging to quantify cartilage and bone parameters. In addition, a custom algorithm was developed to measure the roughness of 3D surfaces. This algorithm was validated and used to quantify cartilage surface roughness changes as a function of time post-surgery.
RESULTS: MMT surgery resulted in significantly greater cartilage damage and subchondral bone sclerosis with the damage increasing in both severity and area from 1- to 3-weeks post-surgery. Analysis of rendered 3D surfaces could accurately distinguish early changes in joints developing OA, detecting significant increases of 45% and 124% in surface roughness at 1- and 3-weeks post-surgery respectively.
CONCLUSION: Disease progression in the MMT model progresses sequentially through changes in the cartilage articular surface, extracellular matrix composition, and then osteophyte mineralization and subchondral bone sclerosis. Cartilage surface roughness is a quantitative, early indicator of degenerative joint disease in small animal OA models and can potentially be used to evaluate therapeutic strategies.

PMID: 29107695 [PubMed – indexed for MEDLINE]

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Getting PEEK to Stick to Bone: The Development of Porous PEEK for Interbody Fusion Devices.

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Getting PEEK to Stick to Bone: The Development of Porous PEEK for Interbody Fusion Devices.

Tech Orthop. 2017 Sep;32(3):158-166

Authors: Torstrick FB, Safranski DL, Burkus JK, Chappuis JL, Lee CSD, Guldberg RE, Gall K, Smith KE

Abstract
Interbody fusion cages are routinely implanted during spinal fusion procedures to facilitate arthrodesis of a degenerated or unstable vertebral segment. Current cages are most commonly made from polyether-ether-ketone (PEEK) due to its favorable mechanical properties and imaging characteristics. However, the smooth surface of current PEEK cages may limit implant osseointegration and may inhibit successful fusion. We present the development and clinical application of the first commercially available porous PEEK fusion cage (COHERE®, Vertera, Inc., Atlanta, GA) that aims to enhance PEEK osseointegration and spinal fusion outcomes. The porous PEEK structure is extruded directly from the underlying solid and mimics the structural and mechanical properties of trabecular bone to support bone ingrowth and implant fixation. Biomechanical testing of the COHERE® device has demonstrated greater expulsion resistance versus smooth PEEK cages with ridges and greater adhesion strength of porous PEEK versus plasma-sprayed titanium coated PEEK surfaces. In vitro experiments have shown favorable cell attachment to porous PEEK and greater proliferation and mineralization of cell cultures grown on porous PEEK versus smooth PEEK and smooth titanium surfaces, suggesting that the porous structure enhances bone formation at the cellular level. At the implant level, preclinical animal studies have found comparable bone ingrowth into porous PEEK as those previously reported for porous titanium, leading to twice the fixation strength of smooth PEEK implants. Finally, two clinical case studies are presented demonstrating the effectiveness of the COHERE® device in cervical spinal fusion.

PMID: 29225416 [PubMed – in process]

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Design and Structure-Function Characterization of 3D Printed Synthetic Porous Biomaterials for Tissue Engineering.

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Design and Structure-Function Characterization of 3D Printed Synthetic Porous Biomaterials for Tissue Engineering.

Adv Healthc Mater. 2018 Apr;7(7):e1701095

Authors: Kelly CN, Miller AT, Hollister SJ, Guldberg RE, Gall K

Abstract
3D printing is now adopted for use in a variety of industries and functions. In biomedical engineering, 3D printing has prevailed over more traditional manufacturing methods in tissue engineering due to its high degree of control over both macro- and microarchitecture of porous tissue scaffolds. However, with the improved flexibility in design come new challenges in characterizing the structure-function relationships between various architectures and both mechanical and biological properties in an assortment of clinical applications. Presently, the field of tissue engineering lacks a comprehensive body of literature that is capable of drawing meaningful relationships between the designed structure and resulting function of 3D printed porous biomaterial scaffolds. This work first discusses the role of design on 3D printed porous scaffold function and then reviews characterization of these structure-function relationships for 3D printed synthetic metallic, polymeric, and ceramic biomaterials.

PMID: 29280325 [PubMed – in process]

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