JEO. The basic science journal of ESSKA NOTABLE ARTICLES JEO SPECIAL EDITION

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1 JEO The basic science journal of ESSKA NOTABLE ARTICLES JEO SPECIAL EDITION An exclusive collection of important studies and award winning papers from the past two years as selected by the Editor-in-Chief.

2 JEO THE BASIC SCIENCE JOURNAL OF ESSKA JEO The basic science journal of ESSKA Publish in JEO in 2018! Take advantage of 15% discount on submission fee: 15% DISCOUNT for all first authors who are ESSKA members Submit here: editorialmanager.com/jexo For more information contact JEO Editorial Office Centre Médical 76, rue d Eich L-1460 Luxembourg Phone: (+352) Fax: (+352) jeo@esska.org

3 EDITORIAL This collection includes the full versions of the Best Papers 2016/2017 of the Journal of Experimental Orthopaedics and the Journal of Experimental Orthopaedics Young Researcher Award winner. It also highlights very interesting abstracts of articles published since our last ESSKA Congress 2016 in Barcelona selected by the Editor in Chief, some of them from our ESSKA Basic Science Committee and Editorial Board members. The collection aims to bridge the gap between experimental studies and clinical translation, which is the underlying goal of the Journal of Experimental Orthopaedics. We as Journal of Experimental Orthopaedics are already in our fourth year. Especially the past two years have been pivotal for the Journal of Experimental Orthopaedics. The Journal is now listed in PubMed, representing a significant milestone. Other notable successes include a total number of more than 120 published articles with nearly all-time full text downloads to date. The Journal of Experimental Orthopaedics is on an upward slope, and I would like to take this occasion to thank our authors, reviewers and readers for their ongoing support. I also would like to thank our exceptional international Editorial Board members and the ESSKA Board for their input and advocacy which has contributed to the journal`s success and also thank our Editorial Office Team. Being the official basic science journal of the ESSKA is a great privilege, and I am confident that with the ongoing support and your assistance, the Journal of Experimental Orthopaedics will continue to be an important medium for disseminating and discussing new findings in the rapidly evolving area of orthopaedic research. I hope you will enjoy reading and would like to express my hope that this may stimulate you to submit your best scientific work to our Journal of Experimental Orthopaedics! With kind regards, Professor Henning Madry Editor-in-Chief Journal of Experimental Orthopaedics

4 IMPACT OF MECHANICAL STIMULATION ON THE CHONDROGENIC PROCESSES IN HUMAN BONE MARROW ASPIRATES MODIFIED TO OVEREXPRESS SOX9 VIA RAAV VECTORS. 1 JEO. 2017; 4(1):22. JAGADEESH K. VENKATESAN 1 JANINA FRISCH 1 ANA REY-RICO 1 GERTRUD SCHMITT 1 HENNING MADRY 1,2 AND MAGALI CUCCHIARINI 1* JEO AWARD YO U N G RESEARC HER AUTHOR INFORMATION: 1 Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D Homburg/Saar, Germany. 2 Department of Orthopaedic Surgery, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D Homburg/Saar, Germany. * Correspondence: mmcucchiarini@hotmail.com BACKGROUND: Evaluation of gene-based approaches to target human bone marrow aspirates in conditions of mechanical stimulation that aim at reproducing the natural joint environment may allow to develop improved treatments for articular cartilage injuries. In the present study, we investigated the potential of raav-mediated sox9 gene transfer to enhance the chondrogenic differentiation processes in human bone marrow aspirates under established hydrodynamic conditions compared with the more commonly employed static culture conditions. METHODS: Fresh human bone marrow aspirates were transduced with raav-flag-hsox9 (40 μl) and maintained for up to 28 days in chondrogenic medium under mechanically-induced conditions in dynamic flow rotating bioreactors that permit tissue growth and matrix deposition relative to static culture conditions. The samples were then processed to examine the potential effects of sox9 overexpression on the cellular activities (matrix synthesis, proliferation) and on the chondrogenic differentiation potency compared with control treatments (absence of raav vector; reporter raav-lacz, raav-rfp, and raav-luc gene transfer). RESULTS: Prolonged, significant sox9 overexpression via raav was achieved in the aspirates for at least 28 days when applying the raav-flag-hsox9 construct, leading to higher, prolonged levels of matrix biosynthesis and to enhanced chondrogenic activities relative to control treatments especially when maintaining the samples under mechanical stimulation. Administration of sox9 however did not impact the indices of proliferation in the aspirates. Remarkably, sox9 gene transfer also durably delayed hypertrophic and osteogenic differentiation in the samples regardless of the conditions of culture applied versus control treatments. CONCLUSIONS: The current observations show the value of genetically modifying human bone marrow aspirates upon mechanical stimulation by raav sox9 as a promising strategy for future treatments to improve cartilage repair by implantation in lesions where the tissue is submitted to natural mechanical forces. KEYWORDS: Cartilage repair, Bone marrow aspirates, Recombinant adeno-associated virus, Chondrogenic differentiation, Mechanical stimulation. The Editor-in-Chief was not involved in any grading where he had a possible conflict of interest. 4

5 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 2 of 13 Background The articular cartilage is a highly specialized tissue that provides a smooth, low friction weightbearing in diarthrodial joints while protecting bones from damaging loads. The absence of vascularity and lymphatic drainage in the cartilage restricts the ability of this tissue for self-repair in response to injury like after trauma or during degenerative osteoarthritis (Buckwalter & Mankin 1998). While various options are available in the clinics including autologous chondrocyte transplantation and marrow-stimulating procedures, none of them promote the full reproduction of the original hyaline cartilage surface in patients (proteoglycans, type-ii collagen) that can durably withstand mechanical load (Cucchiarini et al. 2014). New, promising avenues of clinical investigation to address such challenges are based on the direct, convenient administration of bone marrow aspirates that carry chondrogenically competent mesenchymal stem cells (MSCs) to sites of articular cartilage lesions (Kim et al. 2014; Slynarski et al. 2006) rather than of isolated MSCs that necessitate more complex steps of preparation and expansion (Cucchiarini et al. 2014). However, the outcomes of such clinically adapted treatments revealed again the production of a firbocartilaginous repair tissue in the lesions that is of lesser structural and mechanical quality than the original hyaline cartilage organization. Therapeutic gene-based modification of bone marrow aspirates prior to re-implantation in sites of injury may enhance the chondroreparative processes to promote the formation of an improved cartilage repair tissue (Cucchiarini et al. 2014; Frisch & Cucchiarini 2016). Among the different factors reported for their chondrogenic properties (transforming growth factor beta - TGF-β, bone morphogenetic proteins - BMPs, basic fibroblast growth factor - FGF-2, insulin-like growth factor I - IGF-I, zinc finger protein ZNF145, Indian hedgehog - Ihh, cartilage oligomeric matrix protein - COMP) (Cucchiarini et al. 2011; Frisch et al. 2015a; Frisch et al. 2016a; Haleem-Smith et al. 2012; Ivkovic et al. 2010; Kawamura et al. 2005; Liu et al. 2011; Neumann et al. 2013; Pagnotto et al. 2007; Steinert et al. 2012), the sex-determining region Y-type high-mobility group box 9 (SOX9) transcription factor might be the best suited candidate to achieve this goal. Remarkably, this transcription factor promotes both chondrocyte differentiation and cartilage formation (Bi et al. 1999) while advantageously restricting terminal differentiation and hypertrophy (Akiyama et al. 2004) and displays superior reparative activities in cartilage lesions in vivo over TGF-β and IGF-I (Frisch et al. 2016b). Interestingly, sox9 gene transfer has been already performed to initiate chondrogenic pathways in isolated human MSCs via nonviral (Babister et al. 2008) and adenoviral vectors (Kupcsik et al. 2010), but the use of such vectors for human gene therapy purposes remains limited by their relatively low and/or short-term efficacy and potential immunogenicity (Frisch et al. 2015b). In contrast, recombinant adeno-associated viral (raav) vectors that allow to achieve higher and prolonged levels of transgene expression may be more adapted for translational approaches (Frisch et al. 2015b). In this regard, we provided evidence of the capability of this vector class to target isolated human bone marrow-derived MSCs (Venkatesan et al. 2012) and most notably of human bone marrow aspirates (Rey-Rico et al. 2015a) maintained in static conditions. As chondrogenic processes in the bone marrow in vivo are subjected to mechanical forces (O Conor et al. 2013; Terraciano et al. 2007), the goal of the present study was thus to further investigate the potential chondrogenic effects mediated by sox9 gene transfer via raav in human bone marrow aspirates upon continuous mechanical stimulation, using dynamic conditions that aim at reproducing a more natural environment (Madry et al. 2013). The present findings demonstrate that effective, prolonged overexpression of sox9 using raav vectors stimulates the chondrogenic differentiation processes in human bone marrow aspirates compared with control treatments, most particularly when applying mechanical forces, with advantageously reduced premature hypertrophy and terminal differentiation, further supporting the concept of providing such a construct to treat articular cartilage defects in patients. Methods Chemicals and reagents All reagents were from Sigma (Munich, Germany) unless otherwise indicated. Recombinant TGF-β was from R&D Systems (Wiesbaden, Germany). The anti-sox9 (C-20) antibody was from Santa Cruz Biotechnology (Heidelberg, Germany), the anti-type-ii collagen (II-II6B3) antibody from the NIH Hybridoma Bank (University of Iowa, Ames, USA), and the anti-type-x collagen (COL-10) antibody from Sigma. Biotinylated secondary antibodies and ABC were from Vector Laboratories (Grünberg, Germany). Luciferase activity was determined with the Luciferase Assay System (Promega GmbH, Mannheim, Germany) and normalized to total cellular proteins using the BCA protein assay kit (Pierce Thermo Scientific, Fisher Scientific GmbH, Schwerte, Germany). The Cell Proliferation Reagent WST-1 was from Roche Applied Science (Mannheim, Germany). The type-ii collagen contents were measured with the native type-ii collagen Arthrogen-CIA Capture ELISA kit (Chondrex, Redmond, WA, USA) and those for type-x collagen using a COL-10 ELISA (Antibodies-Online, Aachen, Germany). Human bone marrow aspirates Bone marrow aspirates (~15 ml; x 10 9 cells/ml) were obtained from the distal femur of patients 5

6 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 3 of 13 undergoing total knee arthroplasty (n = 28, age 73 ± 5 years) (Frisch et al. 2015a; Rey-Rico et al. 2015a). The study was approved by the Ethics Committee of the Saarland Physicians Council (Ärztekammer des Saarlandes, application with reference number Ha06/08). All patients provided informed consent to participate in the study before inclusion in the study. All procedures were in accordance with the Helsinki Declaration. All patients provided informed consent to report individual patient data. Aliquots containing MSCs (Frisch et al. 2015a; Ivkovic et al. 2010; Rey-Rico et al. 2015a) were placed in 96-well plates (150 μl aspirate/well) and maintained in DMEM with 100 U/ml penicillin and 100 μl/ml streptomycin (pen-strep) (basal medium) and 10% FBS (growth medium) at 37 C in a humidified atmosphere with 5% CO 2 for subsequent analyses. Plasmids and raav vectors The constructs were derived from pssv9, an AAV-2 genomic clone (Samulski et al. 1987; Samulski et al. 1989). raav-lacz carries the lacz gene for E. coli β-galactosidase (β-gal), raav-rfp the Discosoma sp. red fluorescent protein (RFP) gene, raav-luc the Firefly luciferase (luc) gene, and raav-flag-hsox9 a 1.7-kb FLAG-tagged human SOX9 sequence, all under the control of the cytomegalovirus immediate-early (CMV-IE) promoter (Cucchiarini et al. 2011; Frisch et al. 2015a; Rey-Rico et al. 2015a; Venkatesan et al. 2012). The vectors were packaged as conventional (not self-complementary) vectors using a helper-free, two-plasmid transfection system in the 293 packaging cell line (an adenovirus-transformed human embryonic kidney cell line) with the packaging plasmid pxx2 and the Adenovirus helper plasmid pxx6 as previously described (Rey-Rico et al. 2015a). The vector preparations were purified by dialysis and titered by real-time PCR (Cucchiarini et al. 2011; Frisch et al. 2015a; Rey-Rico et al. 2015a; Venkatesan et al. 2012), averaging transgene copies/ml (viral particles-tofunctional vectors = 500/1) (Beck et al. 1999). raav-mediated gene transfer Aliquoted aspirates were transduced for 90 min with the raav vectors (40 μl each vector, i.e. 8 x 10 5 functional recombinant viral particles, multiplicity of infection - MOI = 10) or let untreated as previously described (Frisch et al. 2015a; Rey-Rico et al. 2015a). A mixture of fibrinogen (17 mg/ml)/thrombin (5 U/ml) (Baxter, Volketstwil, Switzerland) was then added to the aspirates (70 μl per aspirate) and the samples were evenly distributed in a patient-matched manner in 1.5-ml Eppendorf tubes (one sample per tube, 200 μl medium) (static cultures) and in dynamic flow rotating bioreactors (RCCV-110; Synthecon, Houston, TX) (mechanically-stimulated cultures) that permit tissue growth and matrix deposition under optimal hydrodynamic conditions for chondrogenesis relative to static culture (Madry et al. 2013) for up to 28 days (Madry et al. 2013) using defined chondrogenic medium of DMEM high glucose (4.5 g/l), pen-strep, ITS + Premix (insulin 6.25 μg/ml, transferring 6.25 μg/ml, selenous acid 6.25 μg/ml, linoleic acid 5.35 μg/ml, and bovine serum albumin 1.25 μg/ml), pyruvate (1 mm), ascorbate 2- phosphate (37.5 μg/ml), with dexamethasone (10-7 M) and TGF-β (1 ng/ml) 24 h after application of raav (Frisch et al. 2015a; Rey-Rico et al. 2015a). Transgene expression Transgene expression was determined by detection of live fluorescence, X-Gal staining, analysis of luciferase activity (Luciferase Assay System) with normalization to total cellular proteins, or by immunohistochemical analyses (SOX9) on histological sections using specific primary antibodies, biotinylated secondary antibodies, and the ABC method with diaminobenzidine (DAB) as the chromogen (Cucchiarini et al. 2011; Frisch et al. 2015a; Rey-Rico et al. 2015a; Venkatesan et al. 2012). To control for secondary immunoglobulins, samples were processed with omission of the primary antibody. Samples were examined directly by light microscopy (Olympus BX 45; Hamburg, Germany) or by fluorescent microscopy using an Olympus microscope with a 568-nm filter (CKX41). Histological and immunohistochemical analyses The aspirates were collected, fixed in 4% buffered formalin, and dehydrated in graded alcohols for paraffin embedding (Frisch et al. 2015a; Rey-Rico et al. 2015a; Venkatesan et al. 2012). Paraffin-embedded sections (5 μm) were stained with hematoxylin eosin (H&E) (cellularity), safranin O (matrix proteoglycans) and alizarin red (matrix mineralization) according to routine protocols (Cucchiarini et al. 2011). Fast green was used as a counterstain for the evaluations of transduction efficiencies. Expression of type-ii and -X collagen was detected by immunohistochemistry using specific antibodies (Cucchiarini et al. 2011; Frisch et al. 2015a; Rey-Rico et al. 2015a; Venkatesan et al. 2012). To control for secondary immunoglobulins, samples were processed with omission of the primary antibody. Samples were examined by light microscopy (Olympus BX 45). Histomorphometric analyses The wet weights, the perimeters, the transduction efficiencies (ratio of X-Gal-stained cells to the total number of cells), the % of SOX9-stained cells, the cell densities (cells/mm 2 ), and the histological and immunohistochemical grading scores (safranin O, alizarin red, type-ii and -X collagen) were measured at three standardized sites using replicate samples and ten serial sections per condition 6

7 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 4 of 13 using SIS AnalySIS (Olympus), Adobe Photoshop (Adobe Systems, Unterschleissheim, Germany), and Scion Image (Scion Corporation, Frederick, MD, USA) (Cucchiarini et al. 2011; Frisch et al. 2015a; Rey-Rico et al. 2015a; Venkatesan et al. 2012). Safranin O and alizarin red staining and type-ii and -X collagen immunostaining were scored for uniformity and intensity according to a modified Bern Score grading system (Rey-Rico et al. 2015b) as: 0 (no staining), 1 (heterogeneous and/or weak staining), 2 (homogeneous and/or moderate staining), 3 (homogeneous and/ or intense staining), and 4 (very intense staining). Sections were scored blind by two individuals with regard to the conditions. Biochemical assays Cell proliferation in the samples was assessed using the Cell Proliferation Reagent WST-1 with OD 450 nm proportional to the cell numbers according to the manufacturer s recommendations and as previously described (Cucchiarini et al. 2011; Venkatesan et al. 2012). Aspirates were digested in papain solution using previously described protocols (Cucchiarini et al. 2011; Frisch et al. 2015a; Venkatesan et al. 2012). The DNA contents were determined using Hoechst 33258, the proteoglycan contents by binding to the DMMB dye, and those for type- II and -X collagen by ELISA (Cucchiarini et al. 2011; Frisch et al. 2015a; Venkatesan et al. 2012) and data were normalized to total cellular proteins. All measurements were performed with a GENios spectrophotometer/fluorometer (Tecan, Crailsheim, Germany). Total RNA extraction and real-time RT-PCR analyses Total cellular RNA was extracted from the cultures using the RNeasy Protect Mini Kit with an on-column RNase-free DNase treatment (Qiagen, Hilden, Germany) (Cucchiarini et al. 2011; Frisch et al. 2015a; Venkatesan et al. 2012). RNA was eluted in 30 μl RNase-free water. Reverse transcription was carried out with 8 μl of eluate using the 1 st Strand cdna Synthesis kit for RT-PCR (AMV) (Roche Applied Science). Repeated preliminary quantitative evaluations revealed reliable amounts of material in the samples. An aliquot of the cdna product (2 μl) was amplified by real-time PCR using the Brilliant SYBR Green QPCR Master Mix (Stratagene, Agilent Technologies, Waldbronn, Germany) (Cucchiarini et al. 2011; Frisch et al. 2015a; Venkatesan et al. 2012) on a Mx3000P QPCR operator system (Stratagene) as follows: (95 C, 10 min), amplification by 40 cycles (denaturation at 95 C, 30 s; annealing at 55 C, 1 min; extension at 72 C, 30 s), denaturation (95 C, 1 min), and final incubation (55 C, 30 s). The primers (Invitrogen GmbH) used were: SOX9 (chondrogenic marker) (forward 5 -ACACA CAGCTCACTCGACCTTG-3 ; reverse 5 -GGGAATTC TGGTTGGTCCTCT-3 ), type-ii collagen (COL2A1) (chondrogenic marker) (forward 5 -GGACTTTTCTCC CCTCTCT-3 ; reverse 5 -GACCCGAAGGTCTTACA GGA-3 ), type-x collagen (COL10A1) (marker of hypertrophy) (forward 5 -CCCTCTTGTTAGTGCCAACC-3 ; reverse 5 -AGATTCCAGTCCTTGGGTCA-3 ), alkaline phosphatase (ALP) (osteogenic marker) (forward 5 -TGG AGCTTCAGAAGCTCAACACCA-3 ; reverse 5 -ATCT CGTTGTCTGAGTACCAGTCC-3 ), matrix metalloproteinase 13 (MMP13) (marker of terminal differentiation) (forward 5 -AATTTTCACTTTTGGCAATGA-3 ; reverse 5 -CAAATAATTTATGAAAAAGGGATGC-3 ), runtrelated transcription factor 2 (RUNX2) (osteogenic marker) (forward 5 -GCAGTTCCCAAGCATTTCAT-3 ; reverse 5 -CACTCTGGCTTTGGGAAGAG-3 ), β-catenin (mediator of the Wnt signaling pathway for osteoblast lineage differentiation) (forward 5 - CAAGTGGGTGGTA TAGAGG-3 ; reverse 5 -GCGGGACAAAGGGCAAG A-3 ), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (housekeeping gene and internal control) (forward 5 -GAAGGTGAAGGTCGGAGTC-3 ; reverse 5 -GAAGATGGTGATGGGATTTC-3 ) (all 150 nm final concentration) (Cucchiarini et al. 2011; Frisch et al. 2015a; Venkatesan et al. 2012). Control conditions included reactions using water and non-reversetranscribed mrna. Specificity of the products was confirmed by melting curve analysis and agarose gel electrophoresis. The threshold cycle (Ct) value for each gene of interest was measured for each amplified sample using the MxPro QPCR software (Stratagene) and values were normalized to GAPDH expression using the 2 -ΔΔCt method as previously employed (Cucchiarini et al. 2011; Frisch et al. 2015a; Venkatesan et al. 2012). Statistical analysis Each condition was performed in triplicate in three independent experiments. All samples were tested for all the experiments. Data are expressed as mean ± standard deviation (SD) of separate experiments. The t-test and Mann-Whitney Rank Sum Test were employed where appropriate. P values of less than 0.05 were considered statistically significant. Results Maintenance of human bone marrow aspirates in dynamic and static culture conditions upon raav-mediated gene transfer We first examined whether freshly aspirated bone marrow from human donors can be maintained over time in an environment favorable to the formation of a threedimensional composition. To achieve this goal, aspirates were embedded in a fibrin gel and monitored for their stability in dynamic and static culture conditions over a period of 28 days (Madry et al. 2013). 7

8 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 5 of 13 A macroscopic view of aspirates that received no vector treatment revealed that both types of cultivation systems allowed for the formation of a compact structure of the samples, without significant difference in the perimeters of the samples (2.7 ± 0.3 versus 3.4 ± 0.1 mm and 3.0 ± 0.3 versus 3.6 ± 0.2 mm in dynamic and static conditions after 21 and 28 days, respectively, P 0.420; P between similar conditions when comparing days 21 and 28) (Fig. 1a). There was also no significant difference in the wet weight between the samples (0.28 ± 0.01 versus 0.24 ± 0.02 g and 0.30 ± 0.02 versus 0.27 ± 0.02 g in dynamic and static conditions after 21 and 28 days, respectively, P and P between similar conditions when comparing days 21 and 28). When raav-lacz was provided to the aspirates, no noticeable modifications of the overall appearance of the aspirates were observed between systems (perimeters: 2.7 ± 0.2 versus 3.2 ± 0.1 mm and 3.1 ± 0.2 versus 3.3 ± 0.2 mm in dynamic and static conditions after 21 and 28 days, respectively, P and P between similar conditions when comparing days 21 and 28; wet weights: 0.28 ± 0.01 versus 0.23 ± 0.02 g and 0.29 ± 0.02 versus 0.25 ± 0.02 g in dynamic and static conditions after 21 and 28 days, respectively, P and P between similar conditions when comparing days 21 and 28) or when comparing conditions with or without vector treatment (perimeters: P 0.527; wet weights: P 0.293) (Fig. 1a). Efficient raav-mediated transgene expression in human bone marrow aspirates maintained in dynamic and static culture conditions We next assessed whether successful genetic modification of the aspirates could be achieved over time in dynamic and static culture conditions upon raav-mediated gene transfer. Sustained, efficient RFP expression was noted in raav- RFP-transduced aspirates both in dynamic and static culture conditions already after 7 days and for at least 28 days (the longest time point examined) (Madry et al. 2013) Fig. 1 Detection of transgene expression in raav-transduced human bone marrow aspirates in static versus dynamic culture conditions. Aspirates (150 μl) were transduced with raav-rfp, raav-lacz, raav-luc, or raav-flag-hsox9 (40 μl each vector) or let untransduced for maintenance in dynamic or static culture conditions for up to 28 days (n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments) as described in the Methods. a Macroscopic views of the aspirates are presented for each cultivation systems (day 21, scale bars: 1.5 cm for the static cultures and 2 cm for the dynamic cultures; insets: day 28), b Detection of RFP expression by live fluorescence (day 21, scale bars: 50 μm; top insets: day 7; bottom insets: day 28), c detection of lacz expression (day 21) by X-Gal staining (macroscopic views, scale bars: 1.5 cm for the static cultures and 2 cm for the dynamic cultures) and following histological processing of the samples (magnification x20, scale bars: 100 μm, all representative data), d detection of luc expression by analysis of luciferase activity with normalization to total cellular proteins, and e detection of SOX9 (day 28, magnification x40, scale bars: 40 μm; top insets: complete section, magnification x4; bottom insets: H&E staining, magnification x40; all representative data) as described in the Methods. a Statistically significant relative to raav-rfp application 8

9 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 6 of 13 compared with control transduction (absence of vector treatment), showing no visible differences between the two cultivation systems (Fig. 1b). In good agreement, prolonged, effective lacz expression was detected in the aspirates upon administration of raav-lacz both in dynamic and static conditions versus control treatment (absence of vector treatment) (Fig. 1c), with transduction efficiencies reaching ~ 80 85% in either cultivation system (versus below 2% in the controls, P 0.001) as seen on histological sections from aspirates (Fig. 1c). Significant, elevated levels of luciferase activity were also achieved over time in raav-luc-transduced aspirates compared with control (raav-rfp) treatment in the two culture conditions (30.6 ± 0.1 versus 9.9 ± 0.1 and 26.3 ± 0.2 versus 9.8 ± 0.1 RLU/μg total proteins in dynamic environment and static system on day 21, respectively, i.e. an up to 3.1-fold difference, P 0.001) (Fig. 1d). Prolonged, significant expression of the candidate sox9 sequence was also noted in the aspirates following administration of raav-flag-hsox9 compared with control (raav-luc) transduction (85% versus 6% and 84% versus 3% SOX9-stained cells in dynamic environment and static system on day 28, respectively, i.e. an up to 28-fold difference, P 0.001) (Fig. 1e). Effects of raav-mediated gene transfer upon the metabolic activities in human bone marrow aspirates maintained in dynamic and static culture conditions We then tested whether gene transfer via raav altered the proliferative, matrix biosynthetic, and chondrogenic activities in bone marrow aspirates maintained over time in dynamic and static culture conditions while evaluating the possible effects of candidate sox9 gene transfer on these processes using this vector class. Maintenance of the aspirates in dynamic culture conditions led to significant increases in the cell densities in the samples compared with static culture, regardless of the presence or absence of vector (raav-lacz, raav-luc, raav-flag-hsox9) treatment and of the time point evaluated (up to 1.7-fold difference, P 0.001) (Fig. 1c and d; Tables 1 and 2), showing no deleterious effects of raav in either system tested relative to each respective control (untreated) condition (P 0.342). Administration of the sox9 vector did not modify the levels of cell proliferation in the aspirates in any of the systems tested compared with the control treatments at any time point examined (P 0.151). These findings were corroborated by an estimation of the proliferation rates in the aspirates using a WST-1 assay, showing increased proliferative activities in dynamic versus static culture conditions at any time point examined, with or without vector (up to 3.3-fold difference, P 0.001) (Tables 1 and 2), without detrimental effect of raav (P 0.898) while again sox9 treatment did not influence the outcomes (P 0.555). Finally, similar results were obtained when monitoring the DNA contents in the aspirates (up to 1.2-fold increase in dynamic versus static culture conditions independently of the presence or absence of vector treatment and at any time point examined, P 0.001; no deleterious effect of raav: P 0.648; no effect of sox9 application: P 0.342) (Tables 1 and 2). Enhanced levels of cartilage-specific matrix biosynthesis was also achieved in the samples in dynamic Table 1 Biochemical assays in the raav-transduced human bone marrow aspirates (day 21) Assay Static culture Dynamic culture Cell densities (cells/mm 2 ) WST-1 (OD 450 nm ) DNA (μg/mg total proteins) Proteoglycans (mg/mg total proteins) Proteoglycans (mg/μg DNA) Type-II collagen (μg/mg total proteins) Type-II collagen (μg/μg DNA) Type-X collagen (μg/mg total proteins) Type-X collagen (μg/μg DNA) no vector luc sox9 no vector luc sox (94) (0.031) 0.31 (0.12) (0.002) (0.001) (0.002) (0.003) (0.004) (0.003) 5453 (87) (0.046) 0.32 (0.17) (0.003) (0.002) (0.001) (0.002) (0.007) (0.004) 5641 (95) 7044 (104) c 9101 (112) c 9157 (108) c (0.048) (0.118) c (0.107) c (0.122) c (0.17) (0.14) c (0.17) c (0.16) c (0.005) a,b (0.002) c (0.003) c (0.004) a,b,c (0.003) a,b (0.001) c (0.002) c (0.002) a,b,c (0.001) a,b (0.001) c (0.002) c (0.003) a,b,c (0.003) a,b (0.002) c (0.003) c (0.004) a,b,c (0.006) a,b (0.003) (0.004) a,b (0.002) (0.005) (0.004) (0.002) a,b (0.002) a,b Values are given as mean (SD) with n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments. Statistically significant relative to a no vector treatment, b raav-luc application, and c static culture 9

10 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 7 of 13 Table 2 Biochemical assays in the raav-transduced human bone marrow aspirates (day 28) Assay Static culture Dynamic culture Cell densities (cells/mm 2 ) WST-1 (OD 450 nm ) DNA (μg/mg total proteins) Proteoglycans (mg/mg total proteins) Proteoglycans (mg/μg DNA) Type-II collagen (μg/mg total proteins) Type-II collagen (μg/μg DNA) Type-X collagen (μg/mg total proteins) Type-X collagen (μg/μg DNA) no vector luc sox9 no vector luc sox (88) (0.024) 0.29 (0.08) (0.002) (0.003) (0.002) (0.002) (0.003) (0.002) 4987 (91) (0.032) 0.30 (0.11) (0.002) (0.003) (0.002) (0.003) (0.004) (0.003) 5084 (72) 6796 (98) c 7046 (103) c 7892 (76) c (0.037) (0.095) c (0.088) c (0.063) c (0.09) (0.12) c (0.14) c (0.09) c (0.004) a,b (0.002) c (0.003) c (0.003) a,b,c (0.002) a,b (0.002) c (0.002) c (0.004) a,b,c (0.001) a,b (0.001) c (0.002) c (0.002) a,b,c (0.003) a,b (0.002) c (0.002) c (0.003) a,b,c (0.003) a,b (0.002) (0.003) a,b (0.003) (0.003) (0.002) (0.002) a,b (0.002) a,b Values are given as mean (SD) with n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments. Statistically significant relative to a no vector treatment, b raav-luc application, and c static compared with static culture, already starting after 7 days of culture in the presence of raav-flag-hsox9 and for the whole period of evaluation (28 days) and after 21 days in control cultures (no vector treatment, raavluc transduction), as noted on histological sections from aspirates processed of effective chondrogenesis (Madry et al. 2013) for safranin O staining and type-ii collagen immunostaining (Fig. 2a) and scored to grade the staining intensities (Rey-Rico et al. 2015b) (up to 1.3- and 5.7-fold difference for safranin O and type-ii collagen, respectively, P 0.038) (Table 3). No detrimental effect of raav was observed in any of the systems examined at any time point examined (P 0.196). Notably, application of the sox9 vector increased the levels of matrix synthesis in the aspirates in both systems at any time point examined compared with the control treatments (up to 8- and 14-fold difference for safranin O and type- II collagen, respectively, P 0.001). In good agreement, durable increases in the proteoglycan and type-ii collagen contents were noted in dynamic versus static culture conditions at any time point examined, with or without vector (up to 1.8- and 1.7-fold difference for the proteoglycan and type-ii collagen contents, respectively, P 0.001) (Tables 1 and 2). There was no deleterious effect of raav (P 0.195) and again a durable stimulating effect of sox9 compared with the control treatments (up to 1.8- and 1.9-fold difference for the proteoglycan and type-ii collagen contents, respectively, P 0.001). An analysis of the gene expression profiles performed by real-time RT-PCR after 21 days of effective chondrogenesis (Madry et al. 2013) revealed higher levels of SOX9 and COL2A1 expression in the presence of sox9 relative to the control treatments (up to 6.7- and 2.9-fold difference for SOX9 in dynamic and static culture, respectively, and up to 5.6- and 3.1-fold difference for COL2A1 in dynamic and static culture, respectively, P 0.001). The effect that was even more marked in dynamic compared with static culture conditions (up to 1.7-difference for SOX9 and 1.8-fold difference for COL2A1 with sox9, respectively, P 0.001) (Fig. 2b). Effects of raav-mediated gene transfer upon the hypertrophic and terminal differentiation processes in human bone marrow aspirates maintained in dynamic and static culture conditions We finally monitored potential deleterious effects of raav gene transfer on the hypertrophic and terminal differentiation in bone marrow aspirates maintained over time in dynamic and static culture conditions while testing the influence of the candidate sox9 gene transfer on such processes using this vector class. Remarkably, administration of the sox9 vector significantly and durably decreased the levels of matrix mineralization and of hypertrophic/terminal differentiation in the aspirates both in dynamic and static culture conditions compared with the control treatments (no vector, raav-luc) as noted on histological sections from aspirates processed after 21 and 28 days for alizarin red staining and type-x collagen immunostaining, respectively (Fig. 3a). Scores grading the staining intensities (Rey-Rico et al. 2015b) revealed up to 10- and 7.7-fold difference for alizarin red and type-x collagen, respectively) (P 0.012) (Tables 3 and 4). As expected, 10

11 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 8 of 13 Fig. 2 (See legend on next page.) 11

12 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 9 of 13 (See figure on previous page.) Fig. 2 Evalution of cartilage-specific components in raav-transduced human bone marrow aspirates in static versus dynamic culture conditions. Aspirates (150 μl) were prepared and transduced with raav-luc or raav-flag-hsox9 as described in Fig. 1 or let untransduced for maintenance in dynamic or static culture conditions for up to 28 days (n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments) as described in the Methods. The samples were processed (a) for safranin O staining and to detect the expression of type-ii collagen after 7, 21, and 28 days as described in the Methods (magnification x10, scale bars: 200 μm; insets: complete section, magnification x4; all representative data) and (b) to evaluate the gene expression profiles (SOX9 and COL2A1, with GAPDH serving as a housekeeping gene and internal control) after 21 days by real-time RT-PCR amplification as described in the Methods. Ct values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to untreated aggregates) were measured by using the 2 -ΔΔCt method. Statistically significant relative to a no vector treatment, b raav-luc application, and c static culture hypertrophy and terminal differentiation was not documented early on (day 7) (Fig. 3a and Table 5). No detrimental effect of raav was noted in any of the systems examined at any time point examined (P 0.187) and no difference was reported between dynamic and static culture conditions (P 0.187). These findings were supported by an analysis of the type-x collagen contents in the aspirates, with lower amounts in the presence of sox9 versus control treatments at any time point examined (up to 2.7-fold difference, P 0.001) (Tables 1 and 2) and without effect of the vectors in either system tested nor of the culture conditions (P 0.187). An analysis of the gene expression profiles performed by real-time RT-PCR after 21 days of effective chondrogenesis (Madry et al. 2013) revealed lower levels of COL10A1 expression in the presence of sox9 relative to the control treatments (up to 2.5- and 3.3-fold difference in dynamic and static culture, respectively, P 0.001) but again without significant difference between dynamic and static culture conditions (P 0.141) (Fig. 3b). This was accompanied by decreases in the expression profiles of ALP (up to 2.2- and 2.6-fold difference in dynamic and static culture, respectively), MMP13 (up to 3.7- and 5.3-fold difference in dynamic and static culture, respectively), RUNX2 (up to 5.9- and 4.3-fold difference in dynamic and static culture, respectively), and β-catenin (up to 4- and 6.3-fold difference in dynamic and static culture, respectively) upon raav-flag-hsox9 administration compared with Table 3 Histomorphometric analyses in the raav-transduced human bone marrow aspirates (day 21) Assay Static culture Dynamic culture Safranin O staining Type-II collagen immunostaining Alizarin red staining Type-X collagen immunostaining no vector luc sox9 no vector luc sox9 0.3 (0.2) 2.1 (0.1) 3.3 (0.3) 3.2 (0.2) 0.5 (0.2) 2.2 (0.2) 3.1 (0.4) 3.0 (0.1) (0.3) a,b (0.3) c (0.3) c (0.2) a,b,c (0.1) a,b (0.2) c (0.2) c (0.2) a,b,c (0.4) a,b (0.1) (0.2) a,b (0.4) 3.2 (0.3) 3.0 (0.2) 0.7 (0.2) a,b 0.8 (0.1) a,b Values are given as mean (SD) with n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments. Safranin O and alizarin red staining and type-ii and -X collagen immunostaining were scored as described in Table 5 (Rey-Rico et al. 2015b). Statistically significant relative to a no vector treatment, b raav-luc application, and c static culture the control treatments (always P 0.001). Again, there was no significant difference between dynamic and static culture conditions (P 0.121) (Fig. 3b). Discussion Reproducing an original, biomechanically functional tissue in sites of cartilage damage remains one of the most challenging issue in the clinics as both spontaneous cartilage repair and currently available guided procedures, including those based on the single-step administration of chondrogenically competent bone marrow aspirates (Kim et al. 2014; Slynarski et al. 2006), lead to the formation of a poorly organized tissue of lesser mechanical quality than the hyaline cartilage. In this regard, gene transfer strategies may provide effective tools to enhance the chondroreparative processes in such samples especially when subjected to mechanical stimulation as a means to reproduce the natural environment of the joint. We thus examined the potential benefits of delivering the highly chondrogenic transcription factor sox9 to human bone marrow aspirates using the clinically adapted raav vectors upon continuous maintenance in dynamic culture conditions (flow rotating bioreactors) that permit tissue growth and matrix deposition under optimal hydrodynamic conditions for chondrogenesis relative to static culture (Madry et al. 2013) to extend our previous findings when samples were kept in a static environment (Rey-Rico et al. 2015a). The present findings first reveal that gene transfer via raav allows for a highly effective and durable expression of several reporter genes (RFP, lacz, luc) and of the candidate sox9 sequence in human bone marrow aspirates in a mechanically active environment over an extended period of time (at least 28 days), with transduction efficiencies in the range of those noted when applying static culture conditions (80 85%) (Rey-Rico et al. 2015a). Sustained levels of sox9 expression using raav led to prolonged increases in the production of chondrogenic markers and of cartilage-specific matrix components (SOX9, proteoglycans, type-ii collagen) in the aspirates compared with control treatments (at least 28 days), especially upon mechanical stimulation that may exert direct effects on cell function by hydrodynamic forces and/or indirect flow-induced modifications in mass transfer of nutrients 12

13 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 10 of 13 Fig. 3 (See legend on next page.) 13

14 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 11 of 13 (See figure on previous page.) Fig. 3 Evaluation of hypertrophic and terminal differentiation processes in raav-transduced human bone marrow aspirates in static versus dynamic culture conditions. Aspirates (150 μl) were prepared and transduced with raav-luc or raav-flag-hsox9 as described in Fig. 1 and 2 or let untransduced for maintenance in dynamic or static culture conditions for up to 28 days (n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments) as described in the Methods. The samples were processed (a) for alizarin red staining and to detect the expression of type-x collagen after 7, 21, and 28 days as described in the Methods (magnification x10, scale bars: 200 μm, all representative data) and (b) to evaluate the gene expression profiles (COL10A1, ALP, MMP13, RUNX2, and β-catenin, with GAPDH serving as a housekeeping gene and internal control) after 21 days by real-time RT-PCR amplification as described in the Methods and in Fig. 2. Ct values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to untreated aggregates) were measured by using the 2 -ΔΔCt method. Statistically significant relative to a no vector treatment and b raav-luc application and metabolites. This is in good agreement with the properties of the transcription factor (Bi et al. 1999) and with our preliminary observations in static cultures (Rey-Rico et al. 2015a) or when providing the same construct to isolated human bone marrow-derived MSCs (Venkatesan et al. 2012). In contrast, administration of raav sox9 did not modulate the proliferative processes in the samples over time versus control treatments in any of the systems evaluated (even though mechanical stimulation led to increased activities), again consistent with the known activities of SOX9 (Akiyama et al. 2004) and with our findings in isolated human bone marrow-derived MSCs (Venkatesan et al. 2012). Interestingly, modification of the aspirates via raav sox9 promoted a durable, advantageous reduction of matrix mineralization and hypertrophic/terminal differentiation profiles relative to control treatments, although no difference was noted between samples undergoing mechanical stimulation or kept in static culture conditions. This was probably due to decreased expression levels of the osteogenic transcription factor RUNX2 that controls COL10A1, ALP, and MMP13 expression (Enomoto et al. 2000; Frisch et al. in press) and of the β-catenin signaling mediator that regulates osteoblast lineage differentiation via sox9 gene transfer and overexpression (Akiyama et al. 2004; Topol et al. 2009; Yamashita et al. 2009). These results are concordant with the reported effects of SOX9 on Table 4 Histomorphometric analyses in the raav-transduced human bone marrow aspirates (day 28) Assay Static culture Dynamic culture Safranin O staining Type-II collagen immunostaining Alizarin red staining Type-X collagen immunostaining no vector luc sox9 no vector luc sox9 1.8 (0.2) 2.2 (0.2) 3.2 (0.2) 3.0 (0.2) 1.8 (0.3) 2.2 (0.1) 3.0 (0.3) 3.1 (0.1) 3.3 (0.2) a,b 1.8 (0.2) (0.2) a,b (0.2) (0.3) a,b (0.1) (0.2) a,b (0.2) 2.0 (0.2) 2.5 (0.1) 3.4 (0.2) 3.1 (0.2) 4.0 (0.1) a,b,c 3.6 (0.2) a,b,c 0.7 (0.1) a,b 0.6 (0.1) a,b Values are given as mean (SD) with n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments. Safranin O and alizarin red staining and type-ii and -X collagen immunostaining were scored as described in Table 5 (Rey-Rico et al. 2015b). Statistically significant relative to a no vector treatment, b raav-luc application, and c static culture terminal differentiation and calcification and with our preliminary observations in static culture (Rey-Rico et al. 2015a) and in isolated human bone marrow-derived MSCs (Venkatesan et al. 2012). For comparison, Kupcsik et al. (Kupcsik et al. 2010) reported that sox9 gene transfer in isolated human bone marrow-derived MSCs enhanced the production of matrix components under mechanical stimulation but not when modified cells were maintained in static culture conditions. However, these authors employed a different approach than that tested here, using less effective, highly immunogenic adenoviral vectors that are not adapted for clinical applications to treat non-lethal disorders such as those affecting the articular cartilage and at much higher MOI (100 instead of 10 here with raav, i.e. a 10-fold difference) in conditions of cell seeding in polyurethane scaffolds for maintenance under superimposed compression in a custom-made bioreactor over a shorter period of chondrogenic evaluation (14 instead of 28 days). In addition, the use of bone marrow aspirates here represent a much more convenient, single-step approach and advance relative to the use of MSCs that need to be isolated, thoroughly characterized, and expanded in culture prior to reimplantation in the recipient. Also, our scaffold-free approach is less complex than that employed by these Table 5 Histomorphometric analyses in the raav-transduced human bone marrow aspirates (day 7) Assay Static culture Dynamic culture Safranin O staining Type-II collagen immunostaining Alizarin red staining Type-X collagen immunostaining no vector luc sox9 no vector luc sox9 0.3 (0.2) 0.2 (0.1) 0.2 (0.1) 0.1 (0.1) 0.3 (0.1) 0.3 (0.1) 0.2 (0.2) 0.1 (0.1) 2.0 (0.2) a,b 0.2 (0.1) (0.3) a,b (0.1) 0.1 (0.1) 0.2 (0.1) 0.3 (0.2) 0.2 (0.1) 0.3 (0.1) 0.4 (0.2) 01. (0.1) 0.2 (0.1) 2.6 (0.3) a,b,c 3.3 (0.2) a,b,c 0.2 (0.1) 0.1 (0.1) Values are given as mean (SD) with n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments. Safranin O and alizarin red staining and type-ii and -X collagen immunostaining were scored for uniformity and intensity according to a modified Bern Score grading system (Rey-Rico et al. 2015b) as: 0 (no staining), 1 (heterogeneous and/or weak staining), 2 (homogeneous and/or moderate staining), 3 (homogeneous and/or intense staining), and 4 (very intense staining). Statistically significant relative to a no vector treatment, b raav-luc application, and c static culture 14

15 Venkatesan et al. Journal of Experimental Orthopaedics (2017) 4:22 Page 12 of 13 authors, again allowing for direct, simple strategies for future in vivo applications. Besides, raav might be better suited for translation into the clinics as they appear to be much less immunogenic and more effective over time than vectors based on adenoviruses (Frisch et al. 2015b). Conclusion Overall, the results of the present study using conditions of mechanical stimulation extend our previous work when aspirates were maintained in static culture (Rey-Rico et al. 2015a) and further support the idea of using the current candidate raav sox9 vector for administration in sites of cartilage injury in patients as a means to improve the chondrogenerative processes in a tissue that is naturally submitted to mechanical forces in the joint. Work is ongoing to test whether the therapeutic effects noted in vitro here, including those on hypertrophy and terminal differentiation, may also occur in relevant, orthotopic experimental models in vivo with native mechanical environment (Madry et al. 2013). To achieve this goal, genetically modified bone marrow aspirates may be provided by implantation in focal cartilage lesions to observe the formation of an improved cartilage tissue in a native (cellular, biochemical) environment, a study on itself that also necessitates to translate the current work in human samples in an evaluation of animal bone marrow concentrates. Abbreviations ALP: Alkaline phosphatase; AMV: Avian myeloblastosis virus; BMP: Bone morphogenetic protein; cdna: Complementary deoxyribonucleic acid; CMV-IE: Cytomegalovirus immediate-early; COL10A1: Type-X collagen; COL2A1: Type-II collagen; COMP: Cartilage oligomeric matrix protein; Ct: Threshold cycle; DAB: Diaminobenzidine; DMEM: Dulbecco s modified Eagle s medium; DMMB: Dimethylmethylene blue; DNA: Deoxyribonucleic acid; DNase: Deoxyribonuclease; ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; FGF-2: Basic fibroblast growth factor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IGF-I: Insulin-like growth factor I; Ihh: Indian hedgehog; luc: Firefly luciferase; MMP: Matrix metalloproteinase; MOI: Multiplicity of infection; mrna: Messenger ribonucleic acid; MSCs: Mesenchymal stem cells; OD: Optical density; QPCR: Quantitative polymerase chain reaction; raav: Recombinant adenoassociated virus; RFP: Red fluorescent protein; RNA: Ribonucleic acid; RNase: Ribonuclease; RT-PCR: Reverse-transcriptase polymerase chain reaction; RUNX2: Runt-related transcription factor 2; SD: Standard deviation; SOX: Sex-determining region Y-type high mobility group box; TGF-β: Transformation growth factor beta; Wnt: Wingless/Int; ZNF145: Zinc finger protein 145; β-gal: β-galactosidase Acknowledgements This research was funded by a grant from the Collaborative Research Partner Acute Cartilage Injury Program of AO Foundation, Davos, Switzerland (MC, HM). We thank R. J. Samulski (The Gene Therapy Center, University of North Carolina, Chapel Hill, NC), X. Xiao (The Gene Therapy Center, University of Pittsburgh, Pittsburgh, PA), and E. F. Terwilliger (Division of Experimental Medicine, Harvard Institutes of Medicine and Beth Israel Deaconess Medical Center, Boston, MA) for providing the genomic AAV-2 plasmid clones, the packaging plasmid pxx2 and the Adenovirus helper plasmid pxx6, and the 293 cell line, G. Scherer (Institute for Human Genetics and Anthropology, Albert-Ludwig University, Freiburg, Germany) for the human sox9 cdna, and M. J. Stoddart (AO Research Institute Davos, Switzerland) for sharing fibrinogen and thrombin. Authors contributions JKV carried out most of the experiments, analyzed, interpreted the data, and wrote the manuscript. JF participated in the experiments and in analyzing the data. ARR participated in the experiments and in analyzing the data. GS participated in the experiments. HM offered critical revision and writing. MC was responsible for conception and design of the experiments and critical revision. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D Homburg/Saar, Germany. 2 Department of Orthopaedic Surgery, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D Homburg/Saar, Germany. Received: 25 April 2017 Accepted: 12 June 2017 References Akiyama H, Lyons JP, Mori-Akiyama Y, Yang X, Zhang R, Zhang Z, Deng JM, Taketo MM, Nakamura T, Behringer RR, McCrea PD, de Crombrugghe B (2004) Interactions between Sox9 and beta-catenin control chondrocyte differentiation. 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17 AUGMENTED REPAIR OF RADIAL MENISCUS TEAR WITH BIOMIMETIC ELECTROSPUN SCAFFOLD: AN IN VITRO MECHANICAL ANALYSIS. 2 JEO. 2016;3(1):23. BENJAMIN B. ROTHRAUFF 1,4 PIYA-ON NUMPAISAL 1,5 BRIAN B. LAURO 1,3 PETER G. ALEXANDER 1 RICHARD E. DEBSKI 2,3,4 VOLKER MUSAHL 2,3 ROCKY S. TUAN 1,3,4* JEO AWARD BEST PAPER AUTHOR INFORMATION: 1 Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 450 Technology Drive, Room 221, Pittsburgh, PA 15219, USA. 2 Orthopaedic Robotics Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, 300 Technology Drive, Pittsburgh, PA, USA. 3 Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA. 4 McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA. 5 College of Medicine, National Taiwan University, Taipei, Taiwan. KEYWORDS: Meniscus repair, Radial tear, Scaffold BACKGROUND: Large radial tears that disrupt the circumferential fibers of the meniscus are associated with reduced meniscal function and increased risk of joint degeneration. Electrospun fibrous scaffolds can mimic the topography and mechanics of fibrocartilaginous tissues and simultaneously serve as carriers of cells and growth factors, yet their incorporation into clinically relevant suture repair techniques for radial meniscus tears is unexplored. The purposes of this study were to 1 evaluate the effect of fiber orientation on the tensile properties and suture-retention strength of multilayered electrospun scaffolds and 2 determine the mechanical effects of scaffold inclusion within a surgical repair of a simulated radial meniscal tear. The experimental hypothesis was that augmentation with a multilayered scaffold would not compromise the strength of the repair. METHODS: Three multilayered electrospun scaffolds with different fiber orientations were fabricated aligned, random, and biomimetic. The biomimetic scaffold was comprised of four layers in the following order (deep to superficial) aligned longitudinal, aligned transverse, aligned longitudinal, and random respectively corresponding to circumferential, radial, circumferential, and superficial collagen fibers of the native meniscus. Material properties (i.e., ultimate stress, modulus, etc.) of the scaffolds were determined in the parallel and perpendicular directions, as was suture retention strength. Complete radial tears of lateral bovine meniscus explants were repaired with a double horizontal mattress suture technique, with or without inclusion of the biomimetic scaffold sheath. Both repair groups, as well as native controls, were cyclically loaded between 5 and 20 N for 500 cycles and then loaded to failure. Clamp-to-clamp distance (i.e., residual elongation) was measured following various cycles. Ultimate load, ultimate elongation, and stiffness, were also determined. Group differences were evaluated by one-way ANOVA or Student s t-test where appropriate. RESULTS: Aligned scaffolds possessed the most anisotropic mechanical properties, whereas random scaffolds showed uniform properties in the parallel and perpendicular directions. In comparison, the biomimetic scaffold possessed moduli in the parallel (68.7 ± 14.7 MPa) and perpendicular (39.4 ± 11.6 MPa) directions that respectively approximate the reported circumferential and radial tensile properties of native menisci. The ultimate suture retention load of the biomimetic scaffold in the parallel direction (7.2 ± 1.6 N) was significantly higher than all other conditions (p < 0.001). Biomimetic scaffold augmentation did not compromise mechanical properties when compared against suture repair in terms of residual elongation after 500 cycles (scaffold: 5.05 ± 0.89 mm vs. repair: 4.78 ± 1.24 mm), ultimate failure load (137.1 ± 31.0 N vs ± 21.4 N), ultimate elongation (12.09 ± 5.89 mm vs ± 4.61 mm), and stiffness (20.8 ± 3.6 vs ± 4.7 N/mm). CONCLUSIONS: While multilayered scaffold sheets were successfully fabricated to mimic the ultrastructure and anisotropic tensile properties of native menisci, improvements in suture retention strength or adoption of superior surgical techniques will be needed to further enhance the mechanical strength of repairs of radial meniscal tears. 17

18 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 2 of 10 (Continued from previous page) Results: Aligned scaffolds possessed the most anisotropic mechanical properties, whereas random scaffolds showed uniform properties in the parallel and perpendicular directions. In comparison, the biomimetic scaffold possessed moduli in the parallel (68.7 ± 14.7 MPa) and perpendicular (39.4 ± 11.6 MPa) directions that respectively approximate the reported circumferential and radial tensile properties of native menisci. The ultimate suture retention load of the biomimetic scaffold in the parallel direction (7.2 ± 1.6 N) was significantly higher than all other conditions (p < 0.001). Biomimetic scaffold augmentation did not compromise mechanical properties when compared against suture repair in terms of residual elongation after 500 cycles (scaffold: 5.05 ± 0.89 mm vs. repair: 4.78 ± 1.24 mm), ultimate failure load (137.1 ± 31.0 N vs ± 21.4 N), ultimate elongation (12.09 ± 5.89 mm vs ± 4.61 mm), and stiffness (20.8 ± 3.6 vs ± 4.7 N/mm). Conclusions: While multilayered scaffold sheets were successfully fabricated to mimic the ultrastructure and anisotropic tensile properties of native menisci, improvements in suture retention strength or adoption of superior surgical techniques will be needed to further enhance the mechanical strength of repairs of radial meniscal tears. Keywords: Meniscus repair, Radial tear, Scaffold Background Meniscus tears involving the central region remain a formidable challenge to orthopaedic surgeons, as the absence of vasculature and a complex loading environment prevent a robust healing response (Abrams et al. 2013; Arnoczky and Warren 1983; Fox et al. 2015). Compared with vertical tears, radial/flap tears, in which there can be disruption of the circumferential fibers, are associated with articular cartilage lesions of increasing severity (Henry et al. 2012). The standard treatment of partial meniscectomy often alleviates pain and mechanical symptoms in the short term but is known to accelerate joint degeneration by increasing contact stresses (Fairbank 1948; Ode et al. 2012). Recent in vitro studies have demonstrated that contact stresses do not differ from native controls until a full-thickness radial tear exceeds 90 % of the meniscus width, prompting renewed efforts to preserve meniscus structure through primary suture repair (Bedi et al. 2012; Mononen et al. 2013; Muriuki et al. 2011; Ode et al. 2012). Clinical studies have reported variable healing rates of repaired meniscal tears involving the avascular region, attributable to differences in tear morphology, tissue quality, and surgical technique (Choi et al. 2010; Henning et al. 1991; Rubman et al. 1998). However, it remains unknown whether successful healing, defined most commonly by neotissue formation as observed by arthroscopy or MRI, restores native meniscus structure and function, thereby maintaining its chondroprotective role in the articular joint. In a canine model, Newman et al. (1989) found that healed radial tear defects consisted of a 3 5 mm gap of fibrovascular scar that failed to restore normal tissue mechanics. As a result, numerous surgical techniques have been explored in an effort to improve repair strength and maintain apposition of the torn edges (Beamer et al. 2015; Branch et al. 2015; Herbort et al. 2010; Lee et al. 2012; Matsubara et al. 2012). Similarly, though largely unexplored in regards to meniscal tears, scaffold sheets can minimize gap formation and augment mechanical properties of surgical repairs of musculoskeletal tissues (McCarron et al. 2010, 2012). To provide mechanical support, scaffolds must possess material properties equivalent to the native tissue, while biological support through the delivery of cells or biological agents must at minimum not compromise the integrity of the surgical repair (Aurora et al. 2012). To that end, tissue engineering strategies including the independent or combinatorial use of cells, scaffolds, and growth factors, have been increasingly investigated as a means of enhancing the healing response (Moran et al. 2015; Yu et al. 2015). In particular, electrospun nanofibers composed of biodegradable polymers can mimic the topographical and mechanical cues of dense fibrocartilaginous tissues, driving fibrochondrogenic differentiation of seeded mesenchymal stem cells (MSCs) (Baker et al. 2010) and enhancing neotissue formation when placed within a vertical tear in vivo (Baker et al. 2012; Qu et al. 2015). Fisher et al. (2015) recently fabricated a multilayered cell-- seeded nanofibrous scaffold capable of mimicking the anisotropic tensile properties of the meniscus that are derived from the complex organization of circumferential and radial tie fibers (Fox et al. 2015). Similarly, our recent report showed that a cell-seeded electrospun nanofibrous scaffold enhanced the mechanical and histological properties of an in vitro repair model of a radial meniscus tear (Shimomura et al. 2015). Because the mechanical effect of incorporating an electrospun scaffold that structurally mimics meniscus fibrous architecture within a surgical repair is unknown, the purposes of this study were to (1) evaluate the effect of fiber orientation on the tensile properties and suture-retention strength of multilayered electrospun scaffolds and (2) determine the mechanical effects of scaffold inclusion within a 18

19 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 3 of 10 surgical repair of a simulated radial meniscal tear. The experimental hypothesis was that augmentation with a multilayered scaffold would not compromise the strength of the repair. Methods Study design Individual sheets of aligned or randomly oriented electrospun nano/microfibers were combined to form biomimetic multilayered scaffolds. In addition to fiber diameter characterization, the tensile and sutureretention properties of the scaffolds were determined. The biomimetic scaffold, modeling the fibrous structure of the native meniscus, was incorporated as a sheath enveloping the tibia and femoral surfaces of a radially transected lateral bovine meniscus. The mechanical properties of the suture repair, with or without inclusion of the biomimetic scaffold, were determined following cyclic loading and subsequent load to failure. Fabrication of multilayered nanofibrous scaffold Multilayered nanofibrous scaffolds were fabricated through electrospinning, as shown in Fig. 1. The electrospinning apparatus is shown in Fig. 1A C. Three orientations of fibers aligned longitudinal (Fig. 1D), aligned transverse (Fig. 1E), and random (Fig. 1F) were utilized to create three designs of multilayered scaffolds (1) aligned (Fig. 1G), (2) random (Fig. 1H), or (3) biomimetic (Fig. 1I). The biomimetic scaffold was comprised of four layers in the following order (deep to superficial): aligned longitudinal, aligned transverse, aligned longitudinal, and random. This design was inspired by the fibrous structure of the native meniscus, in which the circumferential collagen fibers resist hoop stresses (aligned longitudinal) while the tie fibers resist radial stresses (aligned transverse) and random fibers constitute the meniscal surfaces (random) (Fox et al. 2015; Makris et al. 2011). Each layer was fabricated from a solution of poly-ε-caprolactone (PCL, MW = 70 k-90 kd, Sigma-Aldrich, St. Louis, MO) prepared at 15 % w/v in 1:1 (v/v) tetrahydrofuran (THF, Sigma- Aldrich):dimethylformamide (DMF, Sigma-Aldrich). The PCL solution was loaded into a 10 ml syringe and extruded through an 18-gauge blunt tip needle at 3.0 ml/h using a syringe pump (PY ; Harvard Apparatus, Holliston, MA). The needle tip was placed 10 cm from a custom-designed cylindrical mandrel, which rotated at a surface velocity of 10 m/s for aligned fibers or Fig. 1 Fabrication of multilayered electrospun scaffolds. (A) Electrospinning apparatus consisting of (a) syringe with polymer solution, (b) syringe pump, (c) 18-gauge blunt tip needle, (d) rotating mandrel, and (e) aluminum shield. (B) Taylor cone (arrow) with emerging polymer fiber creates (C) nanofibrous sheet. (D F) SEM images of fiber orientations comprising individual layers. Scale bar, 10 μm. (G I) Individual layers are combined to form three types of multilayered scaffolds, (G) aligned, (H) random, and (I) biomimetic (consisting of alternating layers of aligned and random layers) 19

20 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 4 of m/s for random fibers kv DC potential (Gamma High Voltage, Ormand Beach, FL) was applied to the polymer solution while an 8 kv potential was applied to two aluminum shields placed perpendicular to the mandrel axis but parallel to the needle axis (Fig. 1A). For the biomimetic scaffolds, a given layer was removed, reoriented, and reattached to the mandrel such that the fibers of the subsequent layer were electrospun directly onto the former. Characterization of multilayered nanofibrous scaffold Average fiber diameter in each layer was determined by scanning electron microscopy (SEM). Briefly, scaffold layers were dried under vacuum and mounted on aluminum stubs, sputter-coated with 4.5 nm of gold, and imaged by SEM (field emission, JEOL JSM6335F, Peabody, MA) operated at 3 kv accelerating voltage and 8 mm working distance. Images were morphometrically analyzed using Image J (National Institutes of Health, Bethesda, MD). Each multilayered scaffold was cut into dumbbellshaped constructs with a central rectangular area measuring 25 mm by 5 mm. Construct thickness was measured with digital calipers at three sites and averaged, from which the cross-sectional area (CSA) was calculated. Constructs were then clamped into a materials testing machine (Model 4502; Instron, Norwood, MA) and loaded under tension in a direction either parallel or perpendicular to fiber alignment. This distinction was arbitrary for random scaffolds. For the biomimetic scaffold, the scaffold was oriented with the two aligned longitudinal layers defining parallel. After preloading to 0.5 N, constructs were preconditioned from 0 to 2 % strain (estimated from clamp-to-clamp distance) for 15 cycles at 20 mm/min before undergoing load to failure at the same elongation rate. A custom digital motion tracking system (Spica Technology, Kihei, Maui, HI; 0.01 mm accuracy) was used to track the vertical displacement of the strain markers (black pen) using a single video camera aligned perpendicular to the plane. These data were inputted into ABAQUS software (ABA- QUS/CAE Student Version 6.4; Simulia, Providence, RI) to determine strain. Both structural and material properties were determined. Of note, stiffness (modulus) was determined from the slope of the linear region of the load-elongation (stress-strain) curve while yield load (stress) and yield elongation (strain) were found at the intersection of the data curve and the tangent line with a 0.2 % positive offset along the x-axis, as described previously (Czaplewski et al. 2014). To determine suture retention strength, scaffolds were clamped on both ends, as in the tensile testing protocol described above, and evenly transected. A single loop of 3-0 prolene suture was passed through the midline of each construct at a distance of 5 mm from the cut edge and secured to a immovable cylindrical rod mounted on the material testing machine. A preload of 0.5 N was applied before loading to failure at 20 mm/min. The maximum load was recorded as the suture retention strength. Suture repair of radial tear of lateral meniscus Twenty-four fresh-frozen lateral menisci of adult cows (2 3 years old, JW Treuth & Sons Inc., Catonsville, MD) were used to simulate repair of a radial tear. Menisci were radially transected in the midbody, beginning in the central region and extending to a width of 90 %, corresponding to a length of ~27 mm (out of 30 mm). A single horizontal stitch of 2 0 braided polyester suture (TiCron, Covidien, Dublin, Ireland) was placed 7 mm from the tear edges and in the center of the tear width to reduce the transected edges (Fig. 2a). The remaining 10 % width was then transected to complete the tear. In the scaffold-augmented group, an hour-glass shaped biomimetic scaffold was wrapped around the tear site so as to cover the femoral and tibial surfaces of the Fig. 2 Suture repair of meniscal tears and mechanical testing set-up. a Suture repair of fully transected meniscus. Inset shows dimensions of suture placement. b Scaffold-augmented repair. c Suture repaired meniscus clamped in materials testing machine prior to tensile loading protocol 20

21 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 5 of 10 meniscus. The scaffold was oriented such that the two layers of aligned longitudinal nanofibers were parallel to the circumferential fibers of the meniscus. For both the suture repair and scaffold-augmented groups (n = 8 per group), a double horizontal mattress suture technique was subsequently performed in which the sutures were positioned 5 mm medial and lateral to the reducing suture and 5 mm from the tear margin (Fig. 2a, b). The double horizontal sutures passed through, and laid superficial to, the scaffold sheath so as to ensure its stable incorporation into the repair. Native menisci served as intact controls against which both repair groups were compared. Mechanical testing of suture repair Three groups of menisci were tested (1) intact controls, (2) suture repairs, and (3) scaffold-augmented repairs, as described above. For each, the anterior and posterior portions of the menisci were trimmed to provide relatively flat surfaces for clamping (Fig. 2). Menisci were clamped in a materials testing machine such that the axis of tension was perpendicular to the simulated tear (Fig. 2c). After preloading to 5 N, the construct was cyclically loaded from 5 to 20 N for 500 cycles at a rate of 20 mm/min, pausing after 250 cycles to tighten the clamps. Thereafter, the construct was loaded to failure at the same rate. Residual elongation (indicative of gap formation) was determined after 1, 10, 50, 100, 250, and 500 cycles. Ultimate load, ultimate elongation, and stiffness were determined from the load to failure. Statistical analysis A priori power calculations utilizing pilot data and values from relevant literature revealed a minimum sample size of seven was required to detect a 15 % difference in ultimate load when comparing scaffold-augmented repairs against suture repair alone (power = 0.80, α = 0.05). As a result, eight menisci were allocated to each group for mechanical testing. In analyzing scaffold properties, a two-way ANOVA with fixed factors scaffold type (3) and direction (2) was employed to determine main and interactive effects. Subsequent one-way ANOVA with Tukey post-hoc tests or Student s t-tests were performed to determine statistical differences between conditions. One-way ANOVA with post-hoc Tukey s test was performed to evaluate differences in native meniscus, suture repairs, and scaffold-augmented repairs, after undergoing both cyclic loading and load to failure. p < 0.05 was considered statistically significant. Results Scaffold properties Three orientations of PCL fibers (1) aligned longitudinal (fiber diameter: 811 ± 388 nm, Fig. 1D), (2) aligned transverse (diameter: 772 ± 408 nm, Fig. 1E), and (3) random (diameter: 1562 ± 524 nm, Fig. 1F) were successfully prepared by electrospinning. From these orientations, three multilayered scaffolds were fabricated (1) aligned (thickness: 0.60 ± 0.04 mm, Fig. 1G), (2) random (thickness: 0.37 ± 0.08 mm, Fig. 1H), and (3) biomimetic (thickness: 0.56 ± 0.05 mm, Fig. 1I). Both structural and material tensile properties were determined, with the latter presented in Table 1. For all parameters tested, the aligned scaffolds possessed the greatest degree of anisotropy, i.e., a difference when comparing values in the parallel and perpendicular direction (p < 0.001). Conversely, random scaffolds demonstrated isotropic properties, with no significant differences between the two directions. The biomimetic scaffold, much like the native meniscus, exhibited higher values for ultimate stress (p < 0.001), yield stress (p = 0.002), and modulus (p < 0.001, Fig. 3a), in the parallel (i.e., circumferential) direction, as compared to the perpendicular (i.e., radial). The biomimetic scaffold possessed lower material properties than the aligned scaffolds in the parallel direction, although these differences did not consistently reach statistical significance. Conversely, the biomimetic scaffold was superior to both the aligned and random scaffolds when considering the perpendicular direction, as found for ultimate stress (p < vs. aligned, p = vs. random), yield stress (p < vs. aligned, p = vs. random), and modulus (p < Table 1 Material Properties of Scaffold Designs a Ultimate Stress (MPa) Aligned Random Biomimetic Parallel 12.9 ± 4.3 *, *** 3.4 ± 1.1 *** *, *** 8.5 ± 1.9 Perpendicular 1.2 ± 0.3 **** 3.8 ± 1.0 *** 5.1 ± 1.0 *** Ultimate Strain (mm/mm) Parallel 0.40 ± 0.03 * 3.28 ± 1.49 **** 0.34 ± 0.08 Perpendicular 3.69 ± ± ± 0.07 *** Modulus (MPa) Parallel 93.6 ± 33.9 *, ***** *, ***** 16.9 ± ± 14.7 Perpendicular 2.7 ± 0.5 *** 18.5 ± 5.4 *** 39.4 ± 11.6 **** Yield Stress (MPa) Parallel 4.9 ± 1.5 *, ***** **, ****** 1.7 ± ± 1.3 Perpendicular 0.4 ± 0.1 **** 1.7 ± 0.4 *** 2.2 ± 0.4 *** Yield Strain (mm/mm) Parallel 0.06 ± 0.01 ***** 0.11 ± ± 0.01 ***** Perpendicular 0.12 ± ± ± 0.02 a For a given scaffold type, significant difference when comparing parallel vs. perpendicular direction, * p < 0.001, ** p < 0.05; Significantly different from both scaffolds, *** p < 0.05, **** p < 0.001; Significantly different from random scaffold, ***** p < 0.01, ****** p <

22 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 6 of 10 Meniscus repair properties Residual elongation values following cyclic loading are shown in Table 2. All repairs survived the cyclic loading protocol and exhibited significantly larger displacement at a given cycle when compared against native controls (p < 0.001). There was no statistical difference between the repair groups. Similarly, both repair groups demonstrated significantly lower ultimate failure load (p < 0.001) and stiffness (p < 0.001) than the native control, yet no differences between repair groups were found (Table 3). All repaired constructs failed by suture breakage, whereas the native meniscus specimens experienced clamp slippage. Fig. 3 Moduli and suture retention strength of multilayered scaffolds. a Tensile modulus of three scaffold designs in parallel (i.e., circumferential) and perpendicular (i.e., radial) direction. b Ultimate suture retention load by scaffold design. * (p <0.05) and # (p <0.001) indicate significant difference across scaffold types for a given direction. Horizontal lines above columns indicate a significant difference (p < 0.001) between directions for a given scaffold type vs. both). Nevertheless, no scaffold, regardless of direction, possessed a high resistance to suture pull-through (Fig. 3b), even though the biomimetic scaffold was significantly stronger than either scaffold when oriented in the parallel direction (p < 0.001). This finding, coupled with its anisotropic material properties reminiscent of native menisci, supported the surgical incorporation of the biomimetic scaffold positioned such that the two layers of aligned longitudinal fibers (i.e., parallel in Table 1) were parallel to the circumferential fibers of the underlying meniscus (Fig. 2b). Discussion In this study, an electrospun scaffold that mimicked the fibrous architecture and anisotropic mechanical properties of the meniscus was fabricated. The biomimetic scaffold was incorporated within a double horizontal mattress suture repair of a complete radial tear without diminishing the mechanical properties of the repair. The biomimetic scaffold contained two layers of aligned nanofibers, imparting the greatest tensile strength in a direction parallel to these fibers, while a layer of aligned fibers oriented transversely resisted tension applied in the perpendicular direction. When used as an augmentation to meniscal repair, these fibers are intended to mimic the circumferential and radial tie fibers of the native meniscus, respectively (Fithian et al. 1990). Similarly, the layer of randomly oriented fibers mimics the surface of the native meniscus (Fithian et al. 1990; Fox et al. 2015). The anisotropic tensile properties of the biomimetic scaffold grossly matched those of native meniscus as well. Namely, the average modulus of the scaffold in the parallel (i.e. circumferential) direction was 67.8 ± 14.7 MPa, which falls within the range of reported modulus values (~59 to 294 MPa) of circumferentially oriented specimens obtained from both bovine and human menisci (Fithian et al. 1990; Lechner et al. 2000; Proctor et al. 1989; Tissakht and Ahmed 1995). Similarly, the scaffold modulus in the perpendicular direction (39.4 ± 11.6 MPa) was within the range of native meniscus specimens (~3 to 60 MPa) (Fithian et al. 1990; Tissakht Table 2 Residual Elongation (mm) During 500 Cycles Between 5 and 20 N Cycle Native Suture Repair Scaffold-Augmented ± 0.16 a 1.14 ± ± ± 0.23 a 1.75 ± ± ± 0.33 a 2.57 ± ± ± 0.39 a 3.15 ± ± ± 0.51 a 4.29 ± ± ± 0.49 a 4.78 ± ± 0.89 a Native control significantly less (p < 0.001) than either repair group at given cycle 22

23 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 7 of 10 Table 3 Mechanical Properties of Native and Repaired Menisci Pulled to Failure Native Suture Repair Scaffold-Augmented Ultimate Load (N) a ± ± ± 31.0 Ultimate Elongation 5.12 ± ± ± 5.89 (mm) b Stiffness (N/mm) ± 42.4 a 18.4 ± ± 3.6 a Native control significantly greater (p < 0.001) than either repair group b Scaffold-augmented group significantly greater (p = 0.022) than native control and Ahmed 1995). As the range of values suggests, the material properties of native menisci vary broadly by region (Fithian et al. 1990; Proctor et al. 1989; Tissakht and Ahmed 1995) and are further affected by the crosssectional area of the specimen undergoing tensile testing (Lechner et al. 2000). The scaffold fabrication parameters utilized in this study could be modified to provide increased stiffness in the circumferential direction with corresponding reductions in the radial direction, as exaggerated in the aligned scaffold. The resulting moduli would then more closely match the average reported moduli of native menisci. However, the superficial regions of the menisci exhibit more isotropic properties than the deeper regions, a finding worthy of consideration when implementing a scaffold as a sheath, such as in this study (Fithian et al. 1990). While the biomimetic scaffold mimicked the topography and tensile properties of native menisci, it did not improve the mechanical properties of a simulated radial meniscal tear repaired with suture. Both the ultimate failure load (~125 N) and stiffness (~19 N/mm) of the repair and scaffold-augmented groups are comparable to reported values using similar suture techniques in cadaveric models (Beamer et al. 2015; Bhatia et al. 2015; Branch et al. 2015; Herbort et al. 2010). In testing different suture techniques, Herbort et al. (2010) and Branch et al. (2015) confirmed that multiple sutures are superior to a single suture loop, providing in vitro support to the clinical standard of using an inside-out double horizontal suture technique for repair of radial tears. In this study, a single horizontal stitch was placed equidistant from the central and peripheral rims to reduce the torn edges before performing a double horizontal mattress suture repair, with or without inclusion of the biomimetic scaffold serving as a sheath. As visualization of tear apposition was impossible with the opaque scaffold in place, the reducing stitch was necessary. This third stitch likely accounts for the elevated ultimate load found in this study, as compared against those reported by Herbort et al. (2010) (109 N) and Bhatia et al. (2015) (106 N) when a double horizontal suture technique was evaluated. Unfortunately, the scaffold also prevented direct visualization of markers that might otherwise be used to track tissue strain and gap formation, as utilized in related studies (Beamer et al. 2015; Bhatia et al. 2015). Consequently, residual elongation (i.e. clamp-to-clamp distance) was used to indicate gap formation, although the ~1 mm elongation found after 500 cycles in the native controls suggests that clamp slippage and/or viscoelastic creep partially contributed to this measurement of gap formation. The biomimetic scaffold of this study enveloped the tear site and was secured as part of the double horizontal suture repair. This approach, inspired by the application of scaffold sheets to augment rotator cuff repairs (Ratcliffe et al. 2015), stands in contrast to a previous clinical study in which autologous fascia was used to wrap surgical repairs of complex meniscal tears (Henning et al. 1991). Namely, the fascia sheath was not intended to provide any mechanical support to the suture repair and therefore was attached to the meniscus only along the peripheral rim. In this study, suture breakage was the mode of failure for all repairs, obviating any possible benefit of including a mechanically robust scaffold. If improvements in surgical materials or techniques were sufficient to alter the mechanism of failure to suture pull-through (as seen with rotator cuff repairs), augmentation with mechanically robust scaffolds could further enhance repair strength. In order to provide mechanical support to the surgical repair, the scaffold should possess (1) material properties similar to that of the native tissue and (2) suture retention strength equal to, or greater than, the surgical repair (Aurora et al. 2012). While the biomimetic scaffold met the former criterion, it exhibited a poor ability to hold suture with an ultimate pullout load of 7.2 ± 1.6 N. Categorically, nonwoven electrospun nanofibers do not adequately hold suture. However, the implementation of textile patterns such as braiding or weaving could provide added resistance to suture pull-through (Hakimi et al. 2015; McCarron et al. 2010). To that end, future studies will explore fabrication methods capable of enhancing the suture retention strength of the biomimetic scaffold. Similarly, in vitro culture of cells on the biomimetic scaffold may increase mechanical properties through deposition of extracellular matrix proteins (Fisher et al. 2015) while also contributing to neotissue formation if localized to the lesion (Shimomura et al. 2015). Baek et al. (2015) and Fisher et al. (2015) independently developed cell-seeded multilayered electrospun scaffolds that mimicked the anisotropic fibrous ultrastructure of the meniscus. When implanted within a vertical longitudinal tear created within an explant (Baek et al. 2016) and animal (Qu et al. 2015) model, these electrospun scaffolds promoted neotissue formation within the tear site. In contrast, the biomimetic scaffold of the present study was applied as a sheath 23

24 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 8 of 10 enveloping the tear site. Application of the scaffold as a sheath is not biomimetic in the same sense as the former electrospun scaffolds placed within the body of the meniscus, whereby through the process of contact guidance the scaffolds may direct cells to deposit collagen fibers in the same orientation as the circumferential and radial fibers of the surrounding native tissue (Baek et al. 2016; Baker et al. 2010). On the other hand, radial tears are less amenable to the stable localization of scaffolds within the tear site as compared to longitudinal or horizontal tears, given the greater propensity for gapping when the meniscus is loaded. Therefore, the application of the scaffold as a sheath, if drawing lessons from augmented rotator cuff repairs (Aurora et al. 2012), could theoretically off-load the surgical repair while also protecting a cell-seeded construct placed within the defect site. Furthermore, we recently demonstrated in an explant model of a radial meniscus tear that a cell-seeded scaffold sheath could augment neotissue formation and associated mechanical properties through cell migration into the lesion and/or paracrine-mediated effects on the fibrochondrocytes in the native tissue (Shimomura et al. 2015). Nevertheless, the ex vivo culture conditions of our recent study did not replicate the mechanical demands or inflammatory mediators expected within an injured joint environment, tempering the generalization of these promising in vitro results to a clinical scenario. Stable integration of a scaffold sheath, mediated in part through enhanced suture retention strength, will be needed for translational success. Similarly, broad clinical adoption would likely require timely and secure fixation through an arthroscopic surgical approach. Related clinical studies have reported secure fixation of fascia sheaths (Henning et al. 1991) and collagen matrix membranes (Piontek et al. 2016) enveloping meniscus repairs, suggesting the feasibility of applying the scaffold sheet through an arthroscopic approach. Beyond these opportunities to improve the scaffold design and implementation, there were several limitations inherent in this study. While the scaffold was fabricated to mimic the tensile properties of native menisci, its compressive and shear properties were not evaluated. In particular, a high coefficient of friction of the scaffold sheet could potentially abrade the articulating hyaline cartilage or enveloped meniscus. However, discrepancies between the friction coefficients of meniscus devices and native articular cartilage do not inevitably lead to joint degeneration, depending upon the ability of the device to support neotissue formation, including surface lubrication (Bonnevie et al. 2014; Lee et al. 2014). The immune response to the device is of similar concern. As PCL is a biodegradable, biocompatible biomaterial with a record of clinical safety (Sell et al. 2010), the scaffold sheet should not promote an adverse inflammatory response when implanted in vivo. Nevertheless, secure fixation of the scaffold to the meniscal lesion would be required to prevent dislodgement, with possible disruption of normal joint articulation. At present, these concerns can only be sufficiently evaluated in a large animal model. An additional limitation to this study was that the mechanical properties of the surgical repair were only evaluated under tension. Although commonly employed in similar studies to evaluate suture techniques (Bhatia et al. 2015; Branch et al. 2015; Herbort et al. 2010), this protocol does not replicate how the meniscus functions in vivo. Instead, the native meniscus encounters a complex loading environment consisting of tensile, compressive, and shear forces, exerted dynamically in the context of other joint tissues such as cartilage, ligaments, and synovium. Analysis of surface contact stresses, as well as joint kinematics via robotic systems, would provide further insight into how novel repair techniques and biomaterials affect time-zero mechanics (Maher et al. 2011). Similarly, dynamic loading protocols simulating gait could provide information on the stability of biomaterials under more physiological conditions. To that end, such investigations should ideally be performed with human cadaveric samples, although homologous structure-function relationships of the meniscus exist across species (Proffen et al. 2012). Ultimately, long-term preclinical and clinical studies will be required to determine the potential benefit of promising results in vitro. Conclusions This study showed that a novel biomimetic scaffold fabricated by electrospinning could be incorporated into the repair of a radial meniscus tear without compromising the tensile properties of the repair. Future research will explore methods to enhance suture retention strength. Additionally, the effect of seeding the scaffolds with adult stem cells to further improve long-term durability and integration will be examined. With further modification, the scaffold presented in this study may provide a potential approach to enhance healing of meniscus tears in patients. Abbreviations CSA: Cross-sectional area; DMF: Dimethylformamide; MSC: Mesenchymal stem cell; PCL: Poly-ε-caprolactone; SEM: Scanning electron microscopy; THF: Tetrahydrofuran Acknowledgments The authors thank Matthew Miller and Gerald Ferrer for their technical assistance in mechanical testing and Dr. Bryson Lesniak for his recommendations on surgical technique. Funding This work was supported by the U.S. Department of Defense (W81XWH ). Benjamin B. Rothrauff is a pre-doctoral trainee supported by an NIH Training Grant (5 T32 EB001026). 24

25 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 9 of 10 Authors contributions All authors have read and approved the final submitted manuscript. Contributions are as follows: Research design, or the acquisition, analysis or interpretation of data: BBR, PN, BBL, PGA, RED, VM, RST. Drafting the paper or revising it critically: BBR, VM, RST. Approval of submitted and final versions: BBR, PN, BBL, PGA, RED, VM, RST. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. 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26 Rothrauff et al. Journal of Experimental Orthopaedics (2016) 3:23 Page 10 of 10 Matrix Wrapping and Bone Marrow Blood Injection: A 2-Year Clinical Follow- Up. Cartilage 7: Proctor CS, Schmidt MB, Whipple RR, Kelly MA, Mow VC (1989) Material properties of the normal medial bovine meniscus. J Orthop Res 7: Proffen BL, McElfresh M, Fleming BC, Murray MM (2012) A comparative anatomical study of the human knee and six animal species. Knee 19: Qu F, Pintauro MP, Haughan JE, Henning EA, Esterhai JL, Schaer TP, Mauck RL, Fisher MB (2015) Repair of dense connective tissues via biomaterial-mediated matrix reprogramming of the wound interface. Biomaterials 39:85 94 Ratcliffe A, Butler DL, Dyment NA, Cagle PJ Jr, Proctor CS, Ratcliffe SS, Flatow EL (2015) Scaffolds for Tendon and Ligament Repair and Regeneration. Ann Biomed Eng 43: Rubman MH, Noyes FR, Barber-Westin SD (1998) Arthroscopic repair of meniscal tears that extend into the avascular zone A review of 198 single and complex tears. Am J Sports Med 26:87 95 Sell SA, Wolfe PS, Garg K, McCool JM, Rodriguez IA, Bowlin GL (2010) The Use of Natural Polymers in Tissue Engineering: A Focus on Electrospun Extracellular Matrix Analogues. Polymers 2: Shimomura K, Bean AC, Lin H, Nakamura N, Tuan RS (2015) In Vitro Repair of Meniscal Radial Tear Using Aligned Electrospun Nanofibrous Scaffold. Tissue Eng A 21: Tissakht M, Ahmed AM (1995) Tensile stress-strain characteristics of the human meniscal material. J Biomech 28: Yu H, Adesida AB, Jomha NM (2015) Meniscus repair using mesenchymal stem cells a comprehensive review. Stem Cell Res Ther 6:86 Submit your manuscript to a journal and benefit from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the field 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com 26

27 SCAFFOLD-FREE TISSUE ENGINEERING FOR INJURED JOINT SURFACE RESTORATION. 3 JEO. 2018; 5(1):2. SHIMOMURA K 1,2 ANDO W 2 FUJIE H 3 HART DA 4 YOSHIKAWA H 2 NAKAMURA N 5,6 AUTHOR INFORMATION: 1 Medicine for Sports and Performing Arts, Department of Health and Sport Sciences, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka, , Japan. 2 Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka, , Japan. 3 Division of Human Mechatronics Systems, Faculty of System Design, Tokyo Metropolitan University, 6-6 Asahigaoka, Hino City, Tokyo, , Japan. 4 McCaig Institute for Bone & Joint Health, University of Calgary, 3330 Hospital Drive Northwest, Calgary, AB, T2N 4N1, Canada. 5 Institute for Medical Science in Sports, Osaka Health Science University, Tenma, Kita-ku, Osaka City, Osaka, , Japan. norimasa.nakamura@ohsu.ac.jp. 6 Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita City, Osaka, , Japan. norimasa.nakamura@ohsu.ac.jp. Articular cartilage does not heal spontaneously due to its limited healing capacity, and thus effective treatments for cartilage injuries has remained challenging. Since the first report by Brittberg et al. in 1994, autologous chondrocyte implantation (ACI) has been introduced into the clinic. Recently, as an alternative for chondrocyte-based therapy, mesenchymal stem cell (MSC)-based therapy has received considerable research attention because of the relative ease in handling for tissue harvest, and subsequent cell expansion and differentiation. In this review, we discuss the latest developments regarding stem cell-based therapies for cartilage repair, with special focus on recent scaffold-free approaches. 27

28 AUTOLOGOUS AND MICRO-FRAGMENTED ADIPOSE TISSUE FOR THE TREATMENT OF DIFFUSE DEGENERATIVE KNEE OSTEOARTHRITIS. 4 JEO. 2017; 4(1):33. RUSSO A 1 CONDELLO V 2 MADONNA V 2 GUERRIERO M 3 ZORZI C 2 AUTHOR INFORMATION: 1 Orthopaedic Department, Sacro Cuore - Don Calabria Hospital, Via Don A. Sempreboni, 5, 37024, Negrar, VR, Italy. arcangelorusso@yahoo.it. 2 Orthopaedic Department, Sacro Cuore - Don Calabria Hospital, Via Don A. Sempreboni, 5, 37024, Negrar, VR, Italy. 3 Computer Science Department, University of Verona, Verona, VR, Italy. BACKGROUND: Chondral lesions of the knee represent a challenge for the orthopaedic surgeon. Several treatments have been proposed with variable success rate. Recently, new therapeutic approaches, such as the use of mesenchymal stem cells, have shown promising results. The adipose tissue is a good source of these naturally occurring regenerative cells, due to its abundance and easy access. In addition, it can be used to provide cushioning and filling of structural defects. The 1-year safety and outcome of a single intra-articular injection of autologous and micro-fragmented adipose tissue in 30 patients affected by diffuse degenerative chondral lesions was evaluated. METHODS: Micro-fragmented adipose tissue was obtained using a minimal manipulation technique in a closed system. The safety of the procedure was evaluated by recording type and incidence of any adverse event. The clinical outcomes were determined using the KOOS, IKDC-subjective, Tegner Lysholm Knee, and VAS pain scales taken pre-operatively and at 12 months follow-up. A level of at least 10 points of improvement in the scores has been selected as cut-off representing a clinically significant difference. RESULTS: No relevant complications nor clinical worsening were recorded. A total median improvement of 20 points has been observed in IKDC-subjective and total KOOS, and a higher percentage of success was found in VAS pain and Tegner Lysholm Knee, where the total median improvement was 24 and 31 points, respectively. CONCLUSION: The results of this study show the safety and feasibility of using autologous and micro-fragmented adipose tissue in patients affected by diffuse degenerative chondral lesions. The technique is safe, minimally invasive, simple, one-step, with low percentage of complications, and compliant with the regulatory panorama. 28

29 CURRENT TRENDS IN TENDINOPATHY: CONSENSUS OF THE ESSKA BASIC SCIENCE COMMITTEE. PART I: BIOLOGY, BIOMECHANICS, ANATOMY AND AN EXERCISE-BASED APPROACH. 5 JEO. 2017; 4(1):18. ABAT F 1 ALFREDSON H 2,3,4 CUCCHIARINI M 5 MADRY H 6 MARMOTTI A 7 MOUTON C 8 OLIVEIRA JM 9,10 PEREIRA H 11,12,13 PERETTI GM 14 ROMERO-RODRIGUEZ D 15,16 SPANG C 17 STEPHEN J 18,19 VAN BERGEN CJA 2 DE GIROLAMO L 21 AUTHOR INFORMATION: 1 Department of Orthopaedic Sports Medicine, ReSport Clinic, Passeig Fabra i Puig 47, 08030, Barcelona, Spain. abat@resportclinic.com. 2 Sports Medicine Unit, University of Umeå, Umeå, Sweden. 3 Alfredson Tendon Clinic Inc, Umeå, Sweden. 4 Pure Sports Medicine Clinic, ISEH, UCLH, London, UK. 5 Molecular Biology, Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr Bldg 37, 66421, Homburg/Saar, Germany. 6 Lehrstuhl für Experimentelle Orthopädie und Arthroseforschung, Universität des Saarlandes, Gebäude 37, Kirrbergerstr 1, 66421, Homburg, Germany. 7 Department of Orthopaedics and Traumatology, San Luigi Gonzaga Hospital, Orbassano, University of Turin, Turin, Italy. 8 Department of Orthopedic Surgery, Clinique d Eich-Centre Hospitalier de Luxembourg, 76, rue d Eich, L-1460, Luxembourg, Luxembourg. 9 3B s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, , Barco, GMR, Portugal. 10 ICVS/3B s - PT Government Associate Laboratory, Braga, Guimarães, Portugal. 11 3B s Research Group University of Minho, ICVS/3B s-pt Government Associate Laboratory, Braga, Guimarães, Portugal. 12 Orthopedic Department Centro Hospitalar Póvoa de Varzim, Vila do Conde, Portugal. 13 Ripoll y De Prado Sports Clinic - FIFA Medical Centre of Excellence, Murcia, Madrid, Spain. 14 IRCCS Istituto Ortopedico Galeazzi, Department of Biomedical Sciences for Health, University of Milan, Milan, Italy. 15 Department of Physical Therapy and Sports Rehabilitation, ReSport Clinic Barcelona, Barcelona, Spain. 16 EUSES Sports Science, University of Girona, Girona, Spain. 17 Department of Integrative Medical Biology, Anatomy Section, Umeå University, Umeå, Sweden. 18 Fortius Clinic, 17 Fitzhardinge St, London, W1H 6EQ, UK. 19 The Biomechanics Group, Department of Mechanical Engineering, Imperial College, London, UK. 20 Department of Orthopedic Surgery, Amphia Hospital Breda, Breda, The Netherlands. 21 Orthopaedic Biotechnology Laboratory, Galeazzi Orthopaedic Institute, Milan, Italy. Chronic tendinopathies represent a major problem in the clinical practice of sports orthopaedic surgeons, sports doctors and other health professionals involved in the treatment of athletes and patients that perform repetitive actions. The lack of consensus relative to the diagnostic tools and treatment modalities represents a management dilemma for these professionals. With this review, the purpose of the ESSKA Basic Science Committee is to establish guidelines for understanding, diagnosing and treating this complex pathology. 29

30 DOES ANATOMIC SINGLE-BUNDLE ACL RECONSTRUCTION USING HAMSTRING AUTOGRAFT PRODUCE ANTEROLATERAL MENISCAL ROOT TEARING? 6 JEO. 2017; 4(1):17. IRARRÁZAVAL S 1 MASFERRER-PINO A 2 IBAÑEZ M 2 SHEHATA TMA 3 NAHARRO M 4 MONLLAU JC 2,5 AUTHOR INFORMATION: 1 Department of Orthopaedic Surgery, School of Medicine, Pontificia Universidad Católica de Chile, Diagonal Paraguay 362, Santiago, Chile. sirarraz@med.puc.cl. 2 ICATME, Hospital Universitari Dexeus, Barcelona, Spain. 3 Tadawi General Hospital, Dammam, Saudi Arabia. 4 Complejo Hospitalario Universitario de Pontevedra, Galicia, Spain. 5 Department of Orthopaedic Surgery and Traumatology, Hospital del Mar, Universitat Autònoma de Barcelona, Barcelona, Spain. BACKGROUND: To determine if tibial tunnel reaming during anatomic single-bundle anterior cruciate ligament (ACL) reconstruction using hamstring autograft can result in anterolateral meniscal root injury, as diagnosed by magnetic resonance imaging (MRI). METHODS: A case series of 104 primary anatomic single-bundle ACL reconstructions using hamstring autograft was retrospectively reviewed. Pre- and post-operative (>1 year) MRIs were radiologically evaluated for each patient, with a lateral meniscus extrusion > 3 mm at the level of the medial collateral ligament midportion on a coronal MRI, to establish anterolateral meniscal root injury. RESULTS: No patients presented radiological findings of anterolateral meniscalroot injury in this case series. CONCLUSIONS: Examining a single-bundle ACL reconstruction technique using hamstring autograft that considered tibial tunnel positioning in the center of the tibial footprint, this case series found no evidence of anterolateral meniscal root injury in patient MRIs, even more than 1-year post-operation. 30

31 THREE-DIMENSIONAL GEOMETRIC MORPHOMETRIC ANALYSIS REVEALS ETHNIC DIMORPHISM IN THE SHAPE OF THE FEMUR. 7 JEO. 2017; 4(1):13. CAVAIGNAC E 1,2 LI K 3 FARUCH M 4 SAVALL F 4 CHIRON P 5 HUANG W 3 TELMON N 4 AUTHOR INFORMATION: 1 Laboratoire AMIS, UMR 5288 CNRS, Université Paul Sabatier, 37 allée Jules Guesdes, 31000, Toulouse, France. cavaignac.etienne@gmail.com. 2 Institut de l appareil locomoteur, Hôpital Pierre-Paul Riquet, Toulouse, CHU, France. cavaignac.etienne@gmail.com. 3 Department of Orthopedic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China. 4 Laboratoire AMIS, UMR 5288 CNRS, Université Paul Sabatier, 37 allée Jules Guesdes, 31000, Toulouse, France. 5 Institut de l appareil locomoteur, Hôpital Pierre-Paul Riquet, Toulouse, CHU, France. BACKGROUND: Ethnic dimorphism in the distal femur has never been studied in a three-dimensional analysis focused on shape instead of size. Yet, this dimorphism has direct implications in orthopedic surgery and in anthropology. The goal of this study was to show that differences in distal femur shape related to ethnic dimorphism could be identified, visualized, and quantified using 3D geometric morphometric analysis. METHODS: CT scans of the distal femur were taken from 482 patients who were free of any bone-related pathology: 240 patients were European (E) and 242 were Asian (A). Ten osteometric landmarks based on standard bone landmarks used in anthropometry were placed on these scans. Geometric morphometric analysis, principal component analysis (PCA), canonical variates analysis (CVA), and other discriminant analyses (Goodall s F-test and Mahalanobis distance) were performed. A cross-validation analysis was carried out to determine the percentage of cases in which the ethnicity was correctly estimated. RESULTS: The shape of the E and A distal femur differed significantly (Goodall s F = 94.43, P < and Mahalanobis D2 distance = 1.85, P < 0.001). PCA identified a difference in distal femur shape between A and E. The CVA revealed that correct ethnicity was assigned in 82% of cases and the cross-validation revealed a 75% rate of correct ethnic group estimation. CONCLUSION: The distal femur exhibits ethnic dimorphism. 3D geometric morphometric analysis made it possible to demonstrate these differences. The large number of subjects studied has helped modernize the references for certain bone measurements, with direct implication for orthopedic surgery and anthropology. 31

32 THE CYTOKINE EXPRESSION IN SYNOVIAL MEMBRANE AND THE RELATIONSHIP WITH PAIN AND PATHOLOGICAL FINDINGS AT HIP ARTHROSCOPY. 8 JEO. 2017; 4(1):12. FUKUSHIMA K 1 INOUE G 2 FUJIMAKI H 1 UCHIDA K 1 MIYAGI M 1 NAGURA N 1 UCHIYAMA K 1 TAKAHIRA N 1 TAKASO M 1 AUTHOR INFORMATION: 1 Department of Orthopaedic Surgery, Kitasato University School of Medicine, , Kitazato, Minami-ku, Sagamihara, , Kanagawa, Japan. 2 Department of Orthopaedic Surgery, Kitasato University School of Medicine, , Kitazato, Minami-ku, Sagamihara, , Kanagawa, Japan. ginoue@kitasato-u.ac.jp. BACKGROUND: Synovial membrane inflammation is the most common finding presenting during hip arthroscopy, and may play a role in hip pain. We sought to determinethe relationships between synovial cytokine levels, hip pain, and arthroscopicfindings of the hip joint. METHODS: We prospectively included 33 patients who underwent arthroscopic hip surgery (34 hips). For all patients, radiographs and severity of pain were evaluated preoperatively. During arthroscopy, we classified the chondral injury and synovitis, noted the incidence of labral tear and its instability, and a sample of the synovial membrane was harvested for quantitative PCR to determine levels of TNFα, IL1β, IL6, ADAMTS4, MMP1, and MMP3. The relationships between the levels of these cytokines, severity of hip pain, and the pathological findings during arthroscopy were examined. RESULTS: Pain intensity and cytokine levels were not significantly different between patients with labral tear or instability and those without. By contrast, the expression of TNFα, IL1β, IL6, and MMP1 mrna was significantly higher in patients with diffuse synovitis than in patients with focal synovitis. VAS score during rest showed significant positive correlation with IL6 (r = 0.45,p < 0.01), while VAS score on walking showed a positive correlation with TNFα (r = 0.47, p < 0.01), and ADAMTS4 (r = 0.51, p < 0.01). The modified Harris Hip pain score showed a negative correlation with TNFα (r = -0.38, p = 0.04) and IL6 (r = -0.58, p < 0.01). CONCLUSIONS: The severity of synovitis and chondral injury are considered to be more important in the pathology of hip pain than labral tear or instability. Inflammatory cytokines, especially TNFα and IL6 might play an important role in the pathogenesis of pain in patients indicated for hip arthroscopy, possibly depending on the severity of synovitis. 32

33 THE HAEMATOMA AND ITS ROLE IN BONE HEALING. 9 JEO. 2017; 4(1):5. SCHELL H 1 DUDA GN 1,2 PETERS A 1 TSITSILONIS S 1,2,3 JOHNSON KA 4, SCHMIDT-BLEEK K 5,6 AUTHOR INFORMATION: 1 Julius Wolff Institut and Center for Musculoskeletal Surgery Charité - Universitätsmedizin Berlin, Berlin, Germany. 2 Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany. 3 Center for Musculoskeletal Surgery, Charité - Universitätsmedizin Berlin, Berlin, Germany. 4 Faculty of Veterinary Science, University of Sydney, Sydney, Australia. 5 Julius Wolff Institut and Center for Musculoskeletal Surgery Charité - Universitätsmedizin Berlin, Berlin, Germany. katharina.schmidt-bleek@charite.de. 6 Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Berlin, Germany. katharina.schmidt-bleek@charite.de. Fracture treatment is an old endeavour intended to promote bone healing and to also enable early loading and regain of function in the injured limb. However, in today s clinical routine the healing potential of the initial fracture haematoma is still not fully recognized. The Arbeitsgemeinschaft für Osteosynthesefragen (AO) formed in Switzerland in 1956 formulated four AO principles of fracture treatment which are still valid today. Fracture treatment strategies have continued to evolve further, as for example the relatively new concept of minimally invasive plate osteosynthesis (MIPO). This MIPO treatment strategy harbours the benefit of an undisturbed original fracture haematoma that supports the healing process. The extent of the supportive effect of this haematoma for the bone healing process has not been considered in clinical practice so far. The rising importance of osteoimmunological aspects in bone healing supports the essential role of the initial haematoma as a source for inflammatory cells that release the cytokine pattern that directs cell recruitment towards the injured tissue. In reviewing the potential benefits of the fracture haematoma, the early development of angiogenic and osteogenic potentials within the haematoma are striking. Removing the haematoma during surgery could negatively influence the fracture healing process. In an ovine open tibial fracture model the haematoma was removed 4 or 7 days after injury and the bone that formed during the first two weeks of healing was significantly reduced in comparison with an undisturbed control. These findings indicate that whenever possible the original haematoma formed upon injury should be conserved during clinical fracture treatment to benefit from the inherent healing potential. 33

34 QUANTITATIVE COMPUTED TOMOGRAPHY (QCT) DERIVED BONE MINERAL DENSITY (BMD) IN FINITE ELEMENT STUDIES: A REVIEW OF THE LITERATURE. 10 KNOWLES NK 1,2,3 REEVES JM 4,5,6 FERREIRA LM 7,4,5 JEO. 2016; 3(1):36. AUTHOR INFORMATION: 1 Graduate Program in Biomedical Engineering, The University of Western Ontario, 1151 Richmond St, London, ON, Canada. nknowle@uwo.ca. 2 Roth McFarlane Hand and Upper Limb Centre, Surgical Mechatronics Laboratory, St. Josephs Health Care, 268 Grosvenor St, London, ON, Canada. nknowle@uwo.ca. 3 Collaborative Training Program in Musculoskeletal Health Research, and Bone and Joint Institute, The University of Western Ontario, 1151 Richmond St, London, ON, Canada. nknowle@uwo.ca. 4 Roth McFarlane Hand and Upper Limb Centre, Surgical Mechatronics Laboratory, St. Josephs Health Care, 268 Grosvenor St, London, ON, Canada. 5 Collaborative Training Program in Musculoskeletal Health Research, and Bone and Joint Institute, The University of Western Ontario, 1151 Richmond St, London, ON, Canada. 6 Department of Mechanical and Materials Engineering, The University of Western Ontario, 1151 Richmond St, London, ON, Canada. 7 Graduate Program in Biomedical Engineering, The University of Western Ontario, 1151 Richmond St, London, ON, Canada. BACKGROUND: Finite element modeling of human bone provides a powerful tool to evaluate a wide variety of outcomes in a highly repeatable and parametric manner. These models are most often derived from computed tomography data, with mechanical properties related to bone mineral density (BMD) from the x-ray energy attenuation provided from this data. To increase accuracy, many researchers report the use of quantitative computed tomography (QCT), in which a calibration phantom is used during image acquisition to improve the estimation of BMD. Since model accuracy is dependent on the methods used in the calculation of BMD and density-mechanical property relationships, it is important to use relationships developed for the same anatomical location and using the same scanner settings, as these may impact model accuracy. The purpose of this literature review is to report the relationships used in the conversion of QCT equivalent density measures to ash, apparent, and/or tissue densities in recent finite element (FE) studies used in common density-modulus relationships. For studies reporting experimental validation, the validation metrics and results are presented. RESULTS: Of the studies reviewed, 29% reported the use of a dipotassium phosphate (K2HPO4) phantom, 47% a hydroxyapatite (HA) phantom, 13% did not report phantom type, 7% reported use of both K2HPO4 and HA phantoms, and 4% alternate phantom types. Scanner type and/or settings were omitted or partially reported in 31% of studies. The majority of studies used densitometric and/or density-modulus relationships derived from different anatomical locations scanned in different scanners with different scanner settings. The methods used to derive various densitometric relationships are reported and recommendations are provided toward the standardization of reporting metrics. CONCLUSIONS: This review assessed the current state of QCT-based FE modeling with use of clinical scanners. It was found that previously developed densitometric relationships vary by anatomical location, scanner type and settings. Reporting of all parameters used when referring to previously developed relationships, or in the development of new relationships, may increase the accuracy and repeatability of future FE models. 34

35 BONE MARROW ASPIRATE CONCENTRATE FOR THE TREATMENT OF OSTEOCHONDRAL LESIONS OF THE TALUS: A SYSTEMATIC REVIEW OF OUTCOMES. 11 JEO. 2016; 3(1):38. CHAHLA J 1 CINQUE ME 1 SHON JM 1 LIECHTI DJ 1 MATHENY LM 1 LAPRADE RF 2,3 CLANTON TO 4,1 AUTHOR INFORMATION: 1 Steadman Philippon Research Institute, 181 West Meadow Drive, Suite 400, Vail, CO, 81657, USA. 2 The Steadman Clinic, 181 West Meadow Drive, Suite 400, Vail, CO, 81657, USA. drlaprade@sprivail.org. 3 Steadman Philippon Research Institute, 181 West Meadow Drive, Suite 400, Vail, CO, 81657, USA. drlaprade@sprivail.org. 4 The Steadman Clinic, 181 West Meadow Drive, Suite 400, Vail, CO, 81657, USA. BACKGROUND: The goal of this perform a systematic review on the outcomes of bone marrow aspirate concentrate (BMAC) for the treatment of chondral defects and osteoarthritis (OA) of the talus. RESULTS: The systematic search performed identified 47 studies after duplicates were removed. After inclusion criteria were applied four studies were considered for insightful analysis for the treatment of focal chondral defects in the foot and ankle with the use of BMAC. Three studies were retrospective and one study was prospective in nature. One study was a comparative cohort study and three studies were case series. CONCLUSIONS: This review denotes that there exists an overwhelming paucity of long-term data and high-level evidence supporting BMAC for the treatment of chondral defects. Nonetheless, the evidence available showed varying degrees of beneficial results of BMAC for the treatment of ankle cartilage defects. The limited literature presented in this review demonstrates the need for more advanced, comparative studies to further investigate the efficacy, safety and techniques for BMAC in the treatment of OLTs. The authors recommend that BMAC therapy should be performed with careful consideration until the application and target population for this treatment are established. 35

36 KNEE DONOR-SITE MORBIDITY AFTER MOSAICPLASTY - A SYSTEMATIC REVIEW. 12 JEO. 2016; 3(1):31. ANDRADE R 1,2,3 VASTA S 4 PEREIRA R 1,2,3,5 PEREIRA H 3,6,7,8,9 PAPALIA R 4 KARAHAN M 10 OLIVEIRA JM 2,7,8 REIS RL 7,8, ESPREGUEIRA-MENDES J 2,3,7,8,11,* AUTHOR INFORMATION: 1 Faculty of Sports, University of Porto, Porto, Portugal. 2 Clínica do Dragão, Espregueira-Mendes Sports Centre - FIFA Medical Centre of Excellence, Porto, Portugal. 3 Dom Henrique Research Centre, Porto, Portugal. 4 Orthopaedic and Trauma Department, Campus Biomedico University of Rome, Rome, Italy. 5 Faculty of Health Sciences, University of Fernando Pessoa, Porto, Portugal. 6 Orthopaedic Department, Centro Hospitalar Póvoa de Varzim, Vila do Conde, Portugal. 7 3B s Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, Barco, , Guimarães, Portugal. 8 ICVS/3B s-pt Government Associate Laboratory, Braga/Guimarães, Portugal. 9 Ripoll y De Prado Sports Clinic FIFA Medical Centre of Excellence, Murcia-Madrid, Spain. 10 Department of Orthopaedic Surgery, Acibadem University, Istanbul, Turkey. 11 Orthopaedics Department of Minho University, Minho, Portugal. * espregueira@dhresearchcentre.com. BACKGROUND: Mosaicplasty has been associated with good short- to long-term results. Nevertheless, the osteochondral harvesting is restricted to the donor-site area available and it may lead to significant donor-site morbidity. PURPOSE: Provide an overview of donor-site morbidity associated with harvesting of osteochondral plugs from the knee joint in mosaicplasty procedure. METHODS: Comprehensive search using Pubmed, Cochrane Library, SPORTDiscus and CINAHL databases was carried out through 10th October of As inclusion criteria, all English-language studies that assessed the knee donor-site morbidity after mosaicplasty were accepted. The outcomes were the description and rate of knee donor-site morbidity, sample s and cartilage defect s characterization and mosaicplasty-related features. Correlation between mosaicplasty features and rate of morbidity was performed. The methodological and reporting quality were assessed according to Coleman s methodology score. RESULTS: Twenty-one studies were included, comprising a total of 1726 patients, with 1473 and 268 knee and ankle cartilage defects were included. The defect size ranged from 0.85 cm2 to 4.9 cm2 and most commonly 3 or less plugs (averaging 2.9 to 9.4 mm) were used. Donor-site for osteochondral harvesting included margins of the femoral trochlea (condyles), intercondylar notch, patellofemoral joint and upper tibio-fibular joint. Mean donor-site morbidity was 5.9 % and 19.6 % for knee and ankle mosaicplasty procedures, respectively. Concerning knee-to-knee mosaicplasty procedures, the most common donor-site morbidity complaints were patellofemoral disturbances (22 %) and crepitation (31 %), and in knee-to-ankle procedures there was a clear tendency for pain or instability during daily living or sports activities (44 %), followed by patellofemoral disturbances, knee stiffness and persistent pain (13 % each). There was no significant correlation between rate of donor-site morbidity and size of the defect, number and size of the plugs (p > 0.05). CONCLUSIONS: Osteochondral harvesting in mosaicplasty often results in considerable donor-site morbidity. The donor-site morbidity for knee-to-ankle (16.9 %) was greater than knee-to-knee (5.9 %) mosaicplasty procedures, without any significant correlation between rate of donor-site morbidity and size of the defect, number and size of the plugs. Lack or imcomplete of donor-site morbidity reporting within the mosaicplasty studies is a concern that should be addressed in future studies. LEVEL OF EVIDENCE: Level IV, systematic review of Level I-IV studies. 36

37 THREE-DIMENSIONAL KINEMATIC AND KINETIC ANALYSIS OF KNEE ROTATIONAL STABILITY IN ACL-DEFICIENT PATIENTS DURING WALKING, RUNNING AND PIVOTING. 13 JEO. 2016; 3(1):27. BOHN MB 1 PETERSEN AK 2 NIELSEN DB 3 SØRENSEN H 3 LIND M 4 AUTHOR INFORMATION: 1 Division of Sportstrauma, Department of Orthopedics, Aarhus University Hospital, Tage Hansens Gade 2, 8000, Aarhus C, Denmark. marie.bagger.bohn@clin.au.dk. 2 Department of Physiotherapy and Occupational Therapy, Aarhus University. Hospital, Palle Juul-Jensens Boulevard 99, 8200, Aarhus N, Denmark. 3 Department of Public Health - Sport, Aarhus University, Dalgas Avenue 4, 8000, Aarhus C, Denmark. 4 Division of Sportstrauma, Department of Orthopedics, Aarhus University.Hospital, Tage Hansens Gade 2, 8000, Aarhus C, Denmark. BACKGROUND: Anterior cruciate ligament (ACL) deficiency leads to altered stability of the knee. The purpose of this study was to compare the dynamic, rotational stability of the knee, expressed as rotational stiffness, between anterior cruciate ligament-deficient (ACLD) knees, their contralateral intact knees (ACLI) and a knee healthy control group during walking, running and 90 pivoting. We hypothesized a larger tibial internal rotation, a smaller knee joint external moment and a lower rotational stiffness in the ACLD group compared to the ACLI and the control group. METHODS: Kinematic and kinetic data were collected from both legs of 44 ACLD patients and 16 healthy controls during walking, running and a pivoting maneuver (descending a staircase and immediately pivoting 90 on the landing leg). Motion data were captured using 8 high-speed cameras and a force-plate. Reflective markers were attached to bony landmarks of the lower limb and rigid clusters on the shank and thigh (CASH model). Maximum internal tibial rotation and the corresponding rotational moment were identified for all tasks and groups and used to calculate rotational stiffness (= Δmoment /Δrotation) of the knee. RESULTS: The tibial internal rotation of the ACLD knee was not significantly different from the ACLI knee during all three tasks. During walking and running, the tibial rotation of the control group was significantly different from both legs of the ACL-injured patient. For pivoting, no difference in tibial rotation between knees of the ACLD, ACLI and the control group was found. Knee joint external moments were not significantly different between the three groups during walking and pivoting. During running, the moments of the ACLI group were significantly higher than both the knees of the ACLD and the control group. Rotational stiffness did not differ significantly between groups in any of the three tasks. CONCLUSION: A high-intensity activity combining stair descent and pivoting produces similar angular rotations, knee joint external moments and rotational stiffness in ACLD knees compared to ACLI knees and the control group. During running, the ACLI knee displayed a higher external moment than the ACLD and the healthy control group. This could indicate some type of protective strategy or muscular adaptation in the ACL-injured patients. 37

38 BASIC SCIENCE OF OSTEOARTHRITIS. 14 JEO. 2016; 3(1):22. CUCCHIARINI M 1 DE GIROLAMO L 2 FILARDO G 3 OLIVEIRA JM 4,5 ORTHP 6,7 PAPE D 8,9 REBOUL P 10 AUTHOR INFORMATION: 1 Center of Experimental Orthopaedics, Saarland University Medical Center and Saarland University, Kirrbergerstr. Bldg 37, D-66421, Homburg, Germany. mmcucchiarini@hotmail.com. 2 Orthopaedic Biotechnology Laboratory, Galeazzi Orthopaedic Institute, Milan, Italy. 3 Orthopaedic and Traumatologic I Clinic, Biomechanics Laboratory, Rizzoli Orthopaedic Institute, University of Bologna, Bologna, Italy. 4 3B s Research Group - Biomaterials, Biodegradables and Biomimetics, Univ. Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark - Parque de Ciência e Tecnologia, Zona Industrial da Gandra, Barco GMR, Barco, Guimarães, Portugal. 5 ICVS/3B s - PT Government Associated Laboratory, Barco, Guimarães, Portugal. 6 Center of Experimental Orthopaedics, Saarland University Medical Center and Saarland University, Kirrbergerstr. Bldg 37, D-66421, Homburg, Germany. 7 Department of Orthopaedic Surgery, Saarland University Medical Center and Saarland University, Homburg, Saar, Germany. 8 Department of Orthopaedic Surgery, Centre Hospitalier de Luxembourg, Luxembourg ville, Luxembourg. 9 Sports Medicine Research Laboratory, Public Research Centre for Health, Luxembourg, Centre Médical de la Fondation Norbert Metz, Luxembourg ville, Luxembourg. 10 UMR 7365 CNRS-Université de Lorraine, IMoPA, Biopôle de l Université de Lorraine, Campus Biologie-Santé, Vandoeuvre-lès-Nancy, France. Osteoarthritis (OA) is a prevalent, disabling disorder of the joints that affects a large population worldwide and for which there is no definitive cure. This review provides critical insights into the basic knowledge on OA that may lead to innovative end efficient new therapeutic regimens. While degradation of the articular cartilage is the hallmark of OA, with altered interactions between chondrocytes and compounds of the extracellular matrix, the subchondral bone has been also described as a key component of the disease, involving specific pathomechanisms controlling its initiation and progression. The identification of such events (and thus of possible targets for therapy) has been made possible by the availability of a number of animal models that aim at reproducing the human pathology, in particular large models of high tibial osteotomy (HTO). From a therapeutic point of view, mesenchymal stem cells (MSCs) represent a promising option for the treatment of OA and may be used concomitantly with functional substitutes integrating scaffolds and drugs/growth factors in tissue engineering setups. Altogether, these advances in the fundamental and experimental knowledge on OA may allow for the generation of improved, adapted therapeutic regimens to treat human OA. 38

39 FUNCTIONAL OUTCOME MEASURES IN A SURGICAL MODEL OF HIP OSTEOARTHRITIS IN DOGS. 15 JEO. 2016; 3(1):17. LITTLE D 1,2 JOHNSON S 3 HASH J 4 OLSON SA 3 ESTES BT 3,5 MOUTOS FT 3,5 LASCELLES BD 4 GUILAK F 5,6 AUTHOR INFORMATION: 1 Department of Orthopaedic Surgery, Duke University Medical Center, 375 MSRB 1, BOX 3093 DUMC, Durham, NC, 27710, USA. dianne.little@duke.edu. 2 Department of Basic Medical Sciences, Purdue University College of Veterinary Medicine, 625 Harrison St West Lafayette, IN, USA. dianne.little@duke.edu. 3 Department of Orthopaedic Surgery, Duke University Medical Center, 375 MSRB 1, BOX 3093 DUMC, Durham, NC, 27710, USA. 4 Comparative Pain Research Laboratory and Comparative Medicine Institute, Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC, USA. 5 Cytex Therapeutics Inc, Durham, NC, 27705, USA. 6 Department of Orthopaedic Surgery, Washington University and Shriners Hospitals for Children - St. Louis, St. Louis, MO, 63110, USA. BACKGROUND: The hip is one of the most common sites of osteoarthritis in the body, second only to the knee in prevalence. However, current animal models of hip osteoarthritis have not been assessed using many of the functional outcome measures used in orthopaedics, a characteristic that could increase their utility in the evaluation of therapeutic interventions. The canine hip shares similarities with the human hip, and functional outcome measures are well documented in veterinary medicine, providing a baseline for pre-clinical evaluation of therapeutic strategies for the treatment of hip osteoarthritis. The purpose of this study was to evaluate a surgical model of hip osteoarthritis in a large laboratory animal model and to evaluate functional and end-point outcome measures. METHODS: Seven dogs were subjected to partial surgical debridement of cartilage from one femoral head. Pre- and postoperative pain and functional scores, gait analysis, radiographs, accelerometry, goniometry and limb circumference were evaluated through a 20-week recovery period, followed by histological evaluation of cartilage and synovium. RESULTS: Animals developed histological and radiographic evidence of osteoarthritis, which was correlated with measurable functional impairment. For example, Mankin scores in operated limbs were positively correlated to radiographic scores but negatively correlated to range of motion, limb circumference and 20-week peak vertical force. CONCLUSIONS: This study demonstrates that multiple relevant functional outcome measures can be used successfully in a large laboratory animal model of hip osteoarthritis. These measures could be used to evaluate relative efficacy of therapeutic interventions relevant to human clinical care. 39

40 MUSCLE INJURIES AND STRATEGIES FOR IMPROVING THEIR REPAIR. 16 LAUMONIER T 1 MENETREY J 2 JEO. 2016; 3(1):15. AUTHOR INFORMATION: 1 Department of Orthopaedic Surgery, Geneva University Hospitals & Faculty of Medicine, 4, Rue Gabrielle Perret-Gentil, 1211, Geneva 14, Switzerland. thomas.laumonier@unige.ch. 2 Department of Orthopaedic Surgery, Geneva University Hospitals & Faculty of Medicine, 4, Rue Gabrielle Perret-Gentil, 1211, Geneva 14, Switzerland. Satellite cells are tissue resident muscle stem cells required for postnatal skeletal muscle growth and repair through replacement of damaged myofibers. Muscle regeneration is coordinated through different mechanisms, which imply cell-cell and cell-matrix interactions as well as extracellular secreted factors. Cellular dynamics during muscle regeneration are highly complex. Immune, fibrotic, vascular and myogenic cells appear with distinct temporal and spatial kinetics after muscle injury. Three main phases have been identified in the process of muscle regeneration; a destruction phase with the initial inflammatory response, a regeneration phase with activation and proliferation of satellite cells and a remodeling phase with maturation of the regenerated myofibers. Whereas relatively minor muscle injuries, such as strains, heal spontaneously, severe muscle injuries form fibrotic tissue that impairs muscle function and lead to muscle contracture and chronic pain. Current therapeutic approaches have limited effectiveness and optimal strategies for such lesions are not known yet. Various strategies, including growth factors injections, transplantation of muscle stem cells in combination or not with biological scaffolds, anti-fibrotic therapies and mechanical stimulation, may become therapeutic alternatives to improve functional muscle recovery. 40

41 ON THE HETEROGENEITY OF THE FEMORAL ENTHESIS OF THE HUMAN ACL: MICROSCOPIC ANATOMY AND CLINICAL IMPLICATIONS. 17 JEO. 2016; 3(1):14. BEAULIEU ML 1,2 CAREY GE 3,4 SCHLECHT SH 5 WOJTYS EM 5,6 ASHTON-MILLER JA 3,4 AUTHOR INFORMATION: 1 School of Kinesiology, University of Michigan, 1402 Washington Heights, Ann Arbor, MI, 48109, USA. mbeaulie@umich.edu. 2 Biomechanics Research Laboratory, Department of Mechanical Engineering, University of Michigan, 2350 Hayward Street, GG Brown Building, Ann Arbor, MI, 48109, USA. mbeaulie@umich.edu. 3 School of Kinesiology, University of Michigan, 1402 Washington Heights, Ann Arbor, MI, 48109, USA. 4 Biomechanics Research Laboratory, Department of Mechanical Engineering, University of Michigan, 2350 Hayward Street, GG Brown Building, Ann Arbor, MI, 48109, USA. 5 Orthopaedic Research Laboratories, Department of Orthopaedic Surgery, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI, 48109, USA. 6 MedSport, University of Michigan, Domino s Farms, 24 Frank Lloyd Wright Drive, Lobby A, P.O. Box 391, Ann Arbor, MI, 48106, USA. BACKGROUND: Most ruptures of the native anterior cruciate ligament (ACL) and ACL graft occur at, or near, the femoral enthesis, with the posterolateral fibers of the native ligament being especially vulnerable during pivot landings. Characterizing the anatomy of the ACL femoral enthesis may help us explain injury patterns which, in turn, could help guide injury prevention efforts. It may also lead to improved anatomic reconstruction techniques given that the goal of such techniques is to replicate the knee s normal anatomy. Hence, the aim of this study was to investigate the microscopic anatomy of the ACL femoral enthesis and determine whether regional differences exist. METHODS: Fifteen human ACL femoral entheses were histochemically processed and sectioned along the longitudinal axis of the ACL at 20, 40, 60, and 80 % of the width of the enthesis. Four thick sections (100 μm) per enthesis were prepared, stained, and digitized. From these sections, regional variations in the quantity of calcified and uncalcified fibrocartilage, the angle at which the ligament originates from the bone, and the shape profile of the tidemark were quantified. RESULTS: At least 33 % more calcified fibrocartilage and 143 % more uncalcified fibrocartilage were found in the antero-inferior region, which corresponds to the inferior margin of the origin of the anteromedial ACL fibers, than all other regions (Ps < 0.05). In addition, the anteromedial fibers of the ACL originated from the femur at an angle six times greater than did its posterolateral fibers (P = 0.032). Finally, average entheseal tidemark profiles correlated bilaterally (Pearson s r = 0.79; P = 0.036), the most common profile being convex with a single re-entrant. CONCLUSIONS: Systematic regional differences were found in fibrocartilage quantity and collagen fiber attachment angles. The marked differences may reflect differences in the loading history of the various regions of the ACL femoral enthesis. These differences, which could affect the potential for injury, should also be considered when developing new ACL reconstruction approaches. 41

42 THE EFFECT OF FOOT LANDING POSITION ON BIOMECHANICAL RISK FACTORS ASSOCIATED WITH ANTERIOR CRUCIATE LIGAMENT INJURY. 18 JEO. 2016; 3(1):13. TRAN AA 1,2,3 GATEWOOD C 2,3 HARRIS AH 2 THOMPSON JA 2,4,3 DRAGOO JL 5,6,7 AUTHOR INFORMATION: 1 Stanford University School of Medicine, Stanford University, Stanford, CA, USA. 2 Department of Orthopaedic Surgery, Stanford University, Stanford, CA, USA. 3 Human Performance Lab, Stanford Sports Medicine Center, 341 Galvez Street, Stanford, CA, 94305, USA. 4 Department of Bioengineering, Stanford University, Stanford, CA, USA. 5 Stanford University School of Medicine, Stanford University, Stanford, CA, USA. jdragoo@stanford.edu. 6 Department of Orthopaedic Surgery, Stanford University, Stanford, CA, USA. jdragoo@stanford.edu. 7 Human Performance Lab, Stanford Sports Medicine Center, 341 Galvez Street, 7 Stanford, CA, 94305, USA. jdragoo@stanford.edu. BACKGROUND: Identification of biomechanical risk factors associated with anterior cruciate ligament (ACL) injury can facilitate injury prevention. The purpose of this study is to investigate the effects of three foot landing positions, toe-in, toe-out and neutral, on biomechanical risk factors for ACL injury in males and females. The authors hypothesize that 1) relative to neutral, the toe-in position increases the biomechanical risk factors for ACL injury, 2) the toe-out position decreases these biomechanical risk factors, and 3) compared to males, females demonstrate greater changes in lower extremity biomechanics with changes in foot landing position. METHODS: Motion capture data on ten male and ten female volunteers aged years (26.4 ± 2.50) were collected during double-leg jump landing activities. Subjects were asked to land on force plates and target one of three pre-templated foot landing positions: 0 ( neutral ), 30 internal rotation ( toe-in ), and 30 external rotation ( toe-out ) along the axis of the anatomical sagittal plane. A mixed-effects ANOVA and pairwise Tukey post-hoc comparison were used to detect differences in kinematic and kinetic variables associated with biomechanical risk factors of ACL injury between the three foot landing positions. RESULTS: Relative to neutral, landing in the toe-in position increased peak hip adduction, knee internal rotation angles and moments (p < 0.01), and peak knee abduction angle (p < 0.001). Landing in the toe-in position also decreased peak hip flexion angle (p < 0.001) and knee flexion angle (p = 0.023). Landing in the toe-out position decreased peak hip adduction, knee abduction, and knee internal rotation angles (all p < 0.001). Male sex was associated with a smaller increase in hip adduction moment (p = 0.043) and knee internal rotation moment (p = 0.032) with toe-in landing position compared with female sex. CONCLUSIONS: Toe-in landing position exacerbates biomechanical risk factors associated with ACL injury, while toe-out landing position decreases these factors. 42

43 QUALITATIVE AND QUANTITATIVE MEASUREMENT OF THE ANTERIOR AND POSTERIOR MENISCAL ROOT ATTACHMENTS OF THE NEW ZEALAND WHITE RABBIT. 19 JEO. 2016; 3(1):10. CIVITARESE D 1 DONAHUE TL 2 LAPRADE CM 3 SAROKI AJ 4 MOULTON SG 5 SCHON JM 6 LAPRADE RF 7,8 AUTHOR INFORMATION: 1 Department of Biomedical Engineering, Steadman Philippon Research Institute, 181 West Meadow Drive Suite 1000, Vail, CO, 81657, USA. davidcivitarese11@gmail.com. 2 Department of Mechanical Engineering, Colorado State University, Building A106. Engineering, Fort Collins, CO, 80523, USA. Tammy.Donahue@colostate.edu. 3 Department of Biomedical Engineering, Steadman Philippon Research Institute, 181 West Meadow Drive Suite 1000, Vail, CO, 81657, USA. claprade@mcw.edu. 4 Department of Biomedical Engineering, Steadman Philippon Research Institute, 181 West Meadow Drive Suite 1000, Vail, CO, 81657, USA. ajsaroki@umich.edu. 5 Department of Biomedical Engineering, Steadman Philippon Research Institute, 181 West Meadow Drive Suite 1000, Vail, CO, 81657, USA. sam.g. moulton@gmail.com. 6 Department of Biomedical Engineering, Steadman Philippon Research Institute, 181 West Meadow Drive Suite 1000, Vail, CO, 81657, USA. jschon@sprivail.org. 7 Department of Biomedical Engineering, Steadman Philippon Research Institute, 181 West Meadow Drive Suite 1000, Vail, CO, 81657, USA. drlaprade@sprivail.org. 8 The Steadman Clinic, 181 West Meadow Drive Suite 400, Vail, CO, 81657, USA. drlaprade@sprivail.org. BACKGROUND: The purpose of this study was to quantify the meniscal root anatomy of the New Zealand white rabbit to better understand this animal model for future in vitro and in vivo joint degeneration studies. METHODS: Ten non-paired fresh frozen New Zealand white rabbit knee stifle joints were carefully disarticulated for this study. Measurements were made for all bony landmarks and ligamentous structure attachment sites on the tibial plateau. The following soft tissue structures were consistently identified in the rabbit stifle joint: the anterior root attachment of the lateral meniscus, the anterior root attachment of the medial meniscus, the anterior cruciate ligament, the posterior root attachment of the medial meniscus, the ligament of Wrisberg, the posterior cruciate ligament, and the posterior meniscotibial ligament. The following bony landmarks were consistently identified: the extensor digitorum longus groove, the medial tibial eminence, the center of the tibial tuberosity, and the lateral tibial eminence. RESULTS: The center of the anterior cruciate ligament and the medial tibial eminence apex were found to be 3.4 ± 0.3 mm ( ) and 6.1 ± 0.6 mm ( ) respectively from the center of the medical anterior root attachment. The center of the anterior cruciate ligament and the lateral tibial eminence apex were found to be 2.1 ± 0.5 mm ( ) and 7.0 ± 0.6 mm ( ) respectively from the center of the lateral anterior root attachment. The center of the posterior cruciate ligament and the medial tibial eminence apex were found to be 2.0 ± 0.7 mm ( ) and 1.8 ± 0.4 mm ( ) respectively from the center of the medial posterior root attachment. CONCLUSIONS: This study augments our understanding of the comparative anatomy of the rabbit stifle joint. This information will be useful for future biomechanical, surgical, and in vitro studies utilizing the rabbit stifle as a model for human knee joint degenerative diseases. 43

44 JOINT SPARING TREATMENTS IN EARLY ANKLE OSTEOARTHRITIS: CURRENT PROCEDURES AND FUTURE PERSPECTIVES. 20 JEO. 2016; 3(1):3. CASTAGNINI F 1 PELLEGRINI C 2 PERAZZO L 2 VANNINI F 2 BUDA R 3 AUTHOR INFORMATION: 1 Clinic of Orthopaedics and Traumatology, Rizzoli Orthopaedic Institute, Bologna, Italy. francescocastagnini@hotmail.it. 2 Clinic of Orthopaedics and Traumatology, Rizzoli Orthopaedic Institute, Bologna, Italy. 3 Orthopaedics and Traumatology, I Clinic, Rizzoli Orthopaedic Institute, University of Bologna, Bologna, Italy. Ankle osteoarthritis (AOA) is a severe pathology, mostly affecting a post-traumatic young population. Arthroscopic debridement, arthrodiastasis, osteotomy are the current joint sparing procedures, but, in the available studies, controversial results were achieved, with better outcomes in case of limited degeneration. Only osteotomy in case of malalignment is universally accepted as a joint sparing procedure in case of partial AOA. Recently, the biological mechanism of osteoarthritis has been intensively studied: it is a whole joint pathology, affecting cartilage, bone and synovial membrane. In particular, the first stage is characterized by a reversible catabolic activity with a state of chondropenia. Thus, biological procedures for early AOA were proposed in order to delay or to avoid end stage procedures. Mesenchymal stem cells (MSCs) may be a good solution to prevent or reverse degeneration, due to their immunomodulatory features (able to control the catabolic joint environment) and their regenerative osteochondral capabilities (able to treat the chondral defects). In fact, MSCs may regulate the cytokine cascade and the metalloproteinases release, restoring the osteochondral tissue as well. After interesting reports of mesenchymal stem cells seeded on scaffold and applied to cartilage defects in non-degenerated joints, bone marrow derived cells transplantation appears to be a promising technique in order to control the degenerative pathway and restore the osteochondral defects. 44

45 OPEN VERSUS ARTHROSCOPIC REPAIR OF THE TRIANGULAR FIBROCARTILAGE COMPLEX: A SYSTEMATIC REVIEW. 21 ANDERSSON JK 1,2 ÅHLÉN M 3,4 ANDERNORD D 4,5,6 JEO. 2018; 5(1):6. AUTHOR INFORMATION: 1 Department of Hand Surgery, Sahlgrenska University Hospital, SE , Göteborg, Sweden. jonny_a@telia.com. 2 Department of Orthopaedics, Institute of Clinical Sciences, The Sahlgrenska Academy, University of Gothenburg, Göteborg, Sweden. jonny_a@telia.com. 3 Department of Hand Surgery, Sahlgrenska University Hospital, SE , Göteborg, Sweden. 4 Department of Orthopaedics, Institute of Clinical Sciences, The Sahlgrenska Academy, University of Gothenburg, Göteborg, Sweden. 5 Vårdcentralen Gripen, Karlstad, Sweden. 6 Centre for Clinical Research, County Council of Värmland, Karlstad, Sweden. BACKGROUND AND PURPOSE: To investigate the outcome of open versus arthroscopic repair of injuries of the triangular fibrocartilage complex (TFCC). METHODS: An electronic literature search of articles published between January 1, 1985, and May 26, 2016, in PubMed, Embase, and the Cochrane Library was carried out in May 2016 and updated in March and December Studies comparing open and arthroscopic repair of TFCC injury with a mean follow up of more than 1 year were eligible for inclusion. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist guided the extraction and reporting of data. The methodological quality of the included articles was assessed with the Cochrane Collaboration s tool for assessing risk of bias. The primary outcome measure was the rate of postoperative distal radioulnar joint (DRUJ) re-instability. Secondary outcome measures were range of motion (ROM), grip strength, residual pain, functional wrist scores and the rates of complications and re-operations. RESULTS: A total of 868 articles were identified by the electronic search. After duplicate removal and subsequent study selection, a total of two studies were included in this systematic review. The methodological quality of the included articles displayed risks of bias. There was no difference in DRUJ re-instability between open and arthroscopic repair of the TFCC. There were no differences in obtained postoperative ROM, grip strength or values in functional outcome scores, between open and arthroscopic TFCC repair in the two included studies, except for the Disability of the Arm Shoulder and Hand (DASH) questionnaire - in favor of arthroscopic surgery - in one of the included studies. CONCLUSIONS: This systematic review shows comparable results between open and arthroscopic repair of the TFCC, in terms of DRUJ re-instability and functional outcome scores. There is insufficient evidence to recommend one technique over the other in clinical practice. There is an immense lack of comparison studies with high level of evidence in the area of wrist ligament repair and reconstruction, including TFCC-injuries and DRUJ-instability. KEYWORDS: Arthroscopic repair, Systematic review; Open repair; Triangular fibrocartilage complex 45

46 TRANSLATIONAL MOVEMENT WITHIN THE GLENOHUMERAL JOINT AT DIFFERENT ROTATION VELOCITIES AS SEEN BY CINE MRI. 22 JEO. 2018; 5(1):7. MATSUI K 1,2 TACHIBANA T 3 NOBUHARA K 4 UCHIYAMA Y 5 AUTHOR INFORMATION: 1 Department of Physical Therapy, Graduate School of Medicine, School of Health Sciences, Nagoya University, Daiko-Minami, Higashi-ku, Nagoya-shi, Aichi-ken, Nagoya City, , Japan. 2 Department of Rehabilitation, Gifu Junior College of Health Science, 2-92 Higashi-Uzura, Gifu, , Japan. 3 Department of Physiotherapy, Nobuhara Hospital, 720 Haze, Issai, Tatsuno, Hyogo, , Japan. 4 Institute of Biomechanics, Nobuhara Hospital, 720 Haze, Issai, Tatsuno, Hyogo, , Japan. 5 Department of Physical Therapy, Graduate School of Medicine, School of Health Sciences, Nagoya University, Daiko-Minami, Higashi-ku, Nagoya-shi, Aichi-ken, Nagoya City, , Japan. uchiyama@met.nagoya-u.ac.jp. BACKGROUND: The glenohumeral joint is subjected to opposing forces when the direction of shoulder motion is changed, accelerating and decelerating to make the movements. The influence of motion velocity or acceleration on translation of the humeral head has not been evaluated although direction and distance of humeral head translation has been analyzed in real time in normal shoulders. We hypothesized that, in a normal shoulder, the humeral head does not deviate significantly or suddenly during active shoulder rotation regardless of motion velocity. The purpose of this study was to clarify normal intraarticular kinematics of humeral head position and translation during axial shoulder rotation with the arm by the side of the body at different rotational velocities using cine magnetic resonance imaging (MRI). METHODS: Both shoulders of ten healthy adults (mean age group between ± 6.05 years) were used in this study. Prior to MRI scan, dynamic glenohumeral stability was confirmed by physical examination. The glenohumeral joint was scanned during active shoulder rotation at three angular velocities (low, medium and high velocities), with the arm by the side of the body by real-time cine MRI while recording with the help of a video camera. Translation of the humeral head and rotation angles on MR imaging and video camera were measured to match shoulder rotational positions. RESULTS: There were no statistical differences of the humeral head position and translation among three rotation velocities (p > 0.05). Translation of the humeral head was distributed from 1.44 ± 2.45 mm anteriorly to 0.65 ± 1.84 mm posteriorly at low velocity, from 0.74 ± 1.92 mm anteriorly to 0.75 ± 2.17 mm posteriorly at medium velocity, and from 2.62 ± 2.19 anteriorly to 1.17 ± 1.44 mm posteriorly at high velocity. CONCLUSIONS: Translation of the humeral head was shown to undergo no significant change throughout the ranges of internal and external rotation, or among different rotational velocities in dynamic stability of the glenohumeral joint. KEYWORDS: Cine MRI; Dynamic glenohumeral stability; Intraarticular movement; Rotation velocity 46

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