Biphasic calcium phosphate (BCP) with a characteristic needle-shaped submicron surface topography (MagnetOs) has attracted much attention due to its unique bone-forming ability which is essential for repairing critical-size bone defects such as those found in the posterolateral spine. Previous in vitro and ex-vivo data performed by van Dijk LA and Yuan H demonstrated that these specific surface characteristics drive a favorable response from the innate immune system. This study aimed to evaluate and compare the in vivo performance of three commercially-available synthetic bone grafts, (1) i-FACTOR Putty. ®. , (2) OssDsign. ®. Catalyst Putty and (3) FIBERGRAFT. ®. BG Matrix, with that of a novel synthetic bone graft in a clinically-relevant instrumented sheep posterolateral lumbar spine fusion (PLF) model. The novel synthetic bone graft comprised of BCP granules with a needle-shaped submicron surface topography (MagnetOs) embedded in a highly porous and fibrillar collagen matrix (MagnetOs Flex Matrix). Four synthetic bone grafts were implanted as standalone in an instrumented sheep PLF model for 12 weeks (n=3 bilateral levels per group; levels L2/3 & L4/5), after which spinal fusion was determined by manual palpation, radiograph and µCT imaging (based on the Lenke scale), range-of-motion mechanical testing, and histological and histomorphological evaluation. Radiographic fusion assessment determined bilateral robust bone bridging (Lenke scale A) in 3/3 levels for MagnetOs Flex Matrix compared to 1/3 for all other groups. For µCT, bilateral fusion (Lenke scale A) was found in 2/3 levels for MagnetOs Flex Matrix, compared to 0/3 for i-FACTOR Putty. ®. , 1/3 for OssDsign. ®. Catalyst Putty and 0/3 for FIBERGRAFT. ®. BG Matrix. Fusion assessment for MagnetOs Flex Matrix was further substantiated by histology which revealed significant graft resorption complemented by abundant bone tissue and continuous bony bridging between vertebral transverse processes resulting in bilateral spinal fusion in 3/3 implants. These results show that MagnetOs Flex Matrix achieved better fusion rates compared to three commercially-available synthetic bone grafts when used as a standalone in a clinically-relevant instrumented sheep PLF model

Introduction. Low back pain (LBP) is a worldwide leading cause of disability. This preclinical study evaluated the safety of a combined advanced therapy medicinal product developed during the European iPSpine project (#825925) consisting of mesendoderm progenitor cells (MEPC), derived from human induced pluripotent stem cells, in combination with a synthetic poly(N-isopropylacrylamide) hydrogel (NPgel) in an ovine intervertebral disc degeneration (IDD) model. Method. IDD was induced through nucleotomy in 4 adult sheep, 5 lumbar discs each (n=20). After 5 weeks, 3 alternating discs were treated with NPgel (n=6) or NPgel+MEPC (n=6). Before sacrifice, animals were subjected to: MRI of lumbar spines (disc height and Pfirmann grading); blood sampling (hematological, biochemical, metabolic and lymphocyte/monocytes immunological). After 3 months the sheep were sacrificed. The spines were processed for: macroscopic morphology (Thompson grading), microscopic morphology (Histological grading), and glycosaminoglycan content (GAG, DMMB Assay). Furthermore, at sacrifice biodistribution of human MEPC was assessed by Alu-sequences quantification (qPCR) from three tissue samples of heart, liver, spleen, brain, lungs, and kidneys, and PBMCs collected to assess activation of systemic immune cells. To each evaluation, appropriate statistical analysis was applied. Result. Flow cytometry showed no induction of systemic activation of T cells or monocytes. Alu quantification did not give detection of any cells in any organ. Disc height index was slightly increased in discs treated with NPgel+MEPC. Pfirmann's and Thompson's classification showed that treatment with NPgel or NPgel+MEPC gave no adverse reactions. Histological grading showed similar degeneration in vertebrae treated with NPgel+MEPC or with NPgel alone. The amount of GAG was significantly increased in the nucleus pulposus following treatment with NPgel+MEPC compared to NPgel alone, in which a decrease was observed compared to untreated discs in both nucleus pulposus and annulus fibrosus. Conclusion. This study showed the safety of both NPgel+MEPC and NPgel treatments

Chronic rotator cuff tears are a major problem especially in the elderly population. Refixation is associated with high re-rupture rates. Therefore new implants or healing methods are needed. For a control of success biomechanical characteristics of native as well as treated tendons are of particular importance. Currently, tensile tests with static material testing machines are the most common technique for the biomechanical characterization of tendons. Resulting values are the maximum force (Fmax), stiffness and the Young´s modulus. However, no information is given about the allocation of strains over the tendon area. In addition, the determination of Fmax results in tissue destruction thus foreclosing further evaluation like histology. The Digital Image Correlation (DIC) is a contact-free non-destructive optical measuring method which gives information about distribution of strains by tracking the areal shift of an applied speckle pattern. The needed speckle pattern has to have a high contrast, a homogeneous distribution and a good adhesion to the surface. The method is established for the characterization of construction materials [1] to detect e.g. weak points. The present study examined if DIC is applicable for the complementary biomechanical evaluation of the sheep infraspinatus tendon. Fine ground powder extracted from a printer cartridge was chosen as a starting point. Preliminary to the in vitro experiments, the powder was applied on sheets with different methods: brushing, blowing, sieving and stamping. Stamping showed best results and was used for further in vitro tests on cadaveric native tendons (n=5). First, the toner powder was transferred to coarse-grained abrasive paper using a brush and stamped on the tendon surface. Afterwards DIC analysis was performed. For the in vivo tests, the left infraspinatus tendon of two German black-headed Mutton Sheep was detached and then refixed with bone anchors, the right tendon was used as native control (authorization: AZ 33.19-42502-04-17/2739). 12 weeks after surgery the animals were euthanized, the shoulders were explanted and DIC measurement performed. The speckle pattern could be applied adequately on the smooth tendon surfaces of native tendons. All specimens could be analyzed by DIC with sufficient correlation coefficients. The highest displacements were measured in the peripheral areas, whereas the central part of the tendon showed a low displacement. Repaired left tendons showed obvious differences already macroscopically. The tendons were thicker and showed inhomogeneous surfaces. Application of the toner powder by stamping was distinctly more complicated, DIC analysis could not produce sufficient correlation coefficients. In summary, transfer of DIC to native infraspinatus tendons of sheep was successful and can be further transferred to other animal and human tendons. However, irregular surfaces in tendon scar tissues affect the application of an adequate speckle pattern with a stamp technique. Therefore, further modifications are necessary. This research project has been supported by the German Research Foundation “Graded Implants FOR 2180 – tendon- and bone junctions” WE 4262/6-1

Summary. Timing for the application and use of fentanyl patches for pre-emptive analgesia and sedation is crucial to obtain good clinical outcomes. Placement and timing is important to maximise clinical effect and apparent levels of analgesia. Introduction. The use of sheep as preclinical models for the investigation of orthopaedic conditions is gaining momentum, the control of their pain is a significant ethical issue. The daily need for injecting non-steroidal anti-inflammatory drugs (NSAIDs) and/or the shorter acting opioids increases the demand for handling post-operatively which can increase animal distress and risk of human injury. NSAIDs can have a negative effect on bone healing, complicating results. Opioid analgesics have no impact on bone healing. Fentanyl patches have become another option for use in pain management. Pre-emptive analgesia helps reduce the demand on post-operative analgesic use. Fentanyl has the added benefit of producing mild sedation. This study evaluated the pharmacokinetics of fentanyl patches in sheep in an effort to maximise pre and post-surgical analgesia. Methods. Eight sheep were divided into 2 groups of 4. Both groups had a 100µg/kg/hr fentanyl patch (Durogesic – Janssen, Sydney, Australia) applied to the clipped and cleaned skin of the antebrachium and were held in place with a light bandage. (A dose rate range of 1- 1.6µg/kg/hr was achieved). Group 1 had a second patch applied after 72 hours and group 2 had a second patch applied after 24 hours. Blood samples were taken at 0, 3, 6, 12, 24, 36, 48 and 72 hours post patch application. The blood was immediately spun down and the serum drawn off and frozen. Serum levels for fentanyl were measured using commercial ELISA kits and read using a spectrophotometer. Animal behaviour throughout the study was observed and recorded by trained staff (CC, JR). Results. Six hours after the patch application, the sheep were relaxed and easily approachable. They stood calmly while blood was being drawn. This behaviour remained up to the 48 hour time point at which time cornering them in their pen became marginally more difficult, however they still stood calmly for the blood collection. By 72 hours, all sheep co-operation had dissipated. Peak blood levels of Fentanyl were reached by 12 hours post patch application. These levels were maintained with a relatively flat drug plateau for the prescribed 72 hours post application. No difference was found in the peak drug levels post application of the second patch between the two groups. There was no second higher peak in blood levels attained. Discussion. This study quantified the drug absorption and elimination curves of fentanyl using a controlled application method in an effort to better apply and manage post-surgical analgesia in sheep used for orthopaedic studies. The results indicate that the application of fentanyl for pre-emptive surgical analgesia can be applied for the full duration of 72 hours prior to the application of the second patch at the time of surgery. No benefit regarding analgesia appears to be gained from changing the first patch after 24 hours as peak serum levels are not affected. However for peak sedation the second patch can be applied anytime from 6 to 48 hours. This analgesic regime is beneficial to the animal as well as its handling and management

Objectives. We sought to determine if a durable bilayer implant composed of trabecular metal with autologous periosteum on top would be suitable to reconstitute large osteochondral defects. This design would allow for secure implant fixation, subsequent integration and remodeling. Materials and Methods. Adult sheep were randomly assigned to one of three groups (n = 8/group): 1. trabecular metal/periosteal graft (TMPG), 2. trabecular metal (TM), 3. empty defect (ED). Cartilage and bone healing were assessed macroscopically, biochemically (type II collagen, sulfated glycosaminoglycan (sGAG) and double-stranded DNA (dsDNA) content) and histologically. Results. At 16 weeks post-operatively, histological scores amongst treatment groups were not statistically different (TMPG: overall 12.7, cartilage 8.6, bone 4.1; TM: overall 14.2, cartilage 9.5, bone 4.9; ED: overall 13.6, cartilage 9.1, bone 4.5). Metal scaffolds were incorporated into the surrounding bone, both in TM and TMPG. The sGAG yield was lower in the neo-cartilage regions compared with the articular cartilage (AC) controls (TMPG 20.8/AC 39.5, TM 25.6/AC 33.3, ED 32.2/AC 40.2 µg sGAG/1 mg respectively), with statistical significance being achieved for the TMPG group (p < 0.05). Hypercellularity of the neo-cartilage was found in TM and ED, as the dsDNA content was significantly higher (p < 0.05) compared with contralateral AC controls (TM 126.7/AC 71.1, ED 99.3/AC 62.8 ng dsDNA/1 mg). The highest type II collagen content was found in neo-cartilage after TM compared with TMPG and ED (TM 60%/TMPG 40%/ED 39%). Inter-treatment differences were not significant. Conclusions. TM is a highly suitable material for the reconstitution of osseous defects. TM enables excellent bony ingrowth and fast integration. However, combined with autologous periosteum, such a biocomposite failed to promote satisfactory neo-cartilage formation. Cite this article: E. H. Mrosek, H-W. Chung, J. S. Fitzsimmons, S. W. O’Driscoll, G. G. Reinholz, J. C. Schagemann. Porous tantalum biocomposites for osteochondral defect repair: A follow-up study in a sheep model. Bone Joint J 2016;5:403–411. DOI: 10.1302/2046-3758.59.BJR-2016-0070.R1

Summary Statement. An autologous thrombin activated 3-fold PRP, mixed with a biphasic calcium phosphate at a 1mL:1cc ratio, is beneficial for early bone healing in older age sheep. Introduction. The management of bone defects continues to present challenges. Upon activation, platelets secrete an array of growth factors that contribute to bone regeneration. Therefore, combining platelet rich plasma (PRP) with bone graft substitutes has the potential to reduce or replace the reliance on autograft. The simple, autologous nature of PRP has encouraged its use. However, this enthusiasm has failed to consistently translate to clinical expediency. Lack of standardisation and improper use may contribute to the conflicting outcomes reported within both pre-clinical and clinical investigations. This study investigates the potential of PRP for bone augmentation in an older age sheep model. Specifically, PRP dose is controlled to provide clearer indications for its clinical use. Methods. Eighty 11mm diameter defects of 20mm in depth were created in the cancellous bone within the epiphyseal region of the medial proximal tibia and distal femur of twenty five-year-old sheep. The defects were treated with three doses of an autologous thrombin activated PRP combined with a biphasic calcium phosphate (BCP). Activated platelet poor plasma (PPP) and the BCP alone provided reference groups, while the autograft and empty defects served as controls. All animals were sacrificed at four weeks post-operatively for radiographic assessment, micro-computed tomography quantification, histological assessment, histomorphometric quantification of new bone area and bone ingrowth, and weekly fluorochrome bone label quantification. TGF-β1 concentrations were quantified using enzyme-linked immunosorbent assays. Results. The PRP had a 2.9-fold (0.4) increase in platelet concentration, a 0.57-fold (0.09) decrease in leukocytes, and a 0.65-fold (0.11) decrease in fibrinogen. After activation, the PRP had an 8.9-fold (1.5) increase in TGF-β1 serum concentration above baseline. Eleven (11) mm diameter cancellous bone defects in the hind legs of five-year-old sheep do not spontaneously heal within four weeks. PRP dose had a significant effect on the radiographic grade. The highest dose of PRP treatment had a significantly greater micro-CT BV/TV over the BCP alone (PRP: 30.6±1.8%; BCP: 24.5±0.1%). All doses of PRP treatment were significantly greater than the BCP alone for both the histomorphometric new bone area (PRP: 14.5±1.3%; BCP: 9.7±1.5%) and bone ingrowth depth (PRP: 2288±210µm; BCP:1151±268µm). From week two onwards, PRP had a significant effect on the weekly bone ingrowth over BCP, however, autograft had the greatest amount of fluorescently labelled bone within the first three weeks. PRP has a significant effect on the shape and density of osteoblasts within the central region of the defect compared to the BCP alone, however, was not significantly different to autograft. TGF-β1 appeared a better predictor of healing outcomes than platelet concentration, however both had relatively weak correlations (r<.324). Conclusion. PRP induces new bone formation with a dose dependant response at four weeks when used with a biphasic calcium phosphate in older age sheep

Short Summary. The present study demonstrated the feasibility of culturing a large number of standardised granular MSC-containing constructs in a packed bed/column bioreactor that can produce sheep MSC-containing constructs to repair critical-size bone defects in sheep model. Introduction. Endogenous tissue regeneration mechanisms do not suffice to repair large segmental long-bone defects. Although autologous bone graft remains the gold standard for bone repair, the pertinent surgical technique is limited. Tissue constructs composed of MSCs seeded onto biocompatible scaffolds have been proposed for repairing bone defects and have been established in clinically-relevant animal models. Producing tissue constructs for healing bone defects of clinically-relevant volume requires a large number of cells to heal an approximately 3 cm segmental bone defect. For this reason, a major challenge is to expand cells from a bone marrow aspirate to a much larger, and sufficient, number of MSCs. In this respect, bioreactor systems which provide a reproducible and well-controlled three-dimensional (3D) environment suitable for either production of multiple or large size tissue constructs are attractive approaches to expand MSCs and obtain MSC-containing constructs of clinical grade. In these bioreactor systems, MSCs loaded onto scaffolds are exposed to fluid flow, a condition that provides both enhanced access to oxygen and nutrients as well as fluid-flow-driven mechanical stimulation to cells. The present study was to evaluate bioreactor containing autologous MSCs loaded on coral scaffolds to repair critical-size bone defects in sheep model. Materials and Methods. Animals: 12 two-year-old, female Pre-Alpes sheep were used and reared in accordance with the European Committee for Care. Three-dimensional, porous scaffolds (each 3×3×3 mm) of natural coral exoskeleton were used as substrates for cell attachment. The packed bed/column bioreactor set-up used in the present study was composed of a vertical column filled with MSC-containing constructs. Sheep MSCs were isolated from sheep bone marrow. MSCs were seeded on scaffolds and cultured overnight under standard cell-culture condition. MSC-containing constructs were r placed into the perfusion bioreactor and were either exposed to a perfusion medium flow rate of 10 mL/min for 7 continuous days. Osteoperiosteal segmental (25 mm) defects were made in the left metatarsal bone of 12 sheep. The defect was either filled with coral scaffolds alone (Group 1; five sheep); or filled with coral scaffolds loaded with MSCs (Group 2; five sheep); or filled with autologous bone graft (Group 3; 2 sheep). Results. At 6 month after implantation, radiographs showed resorption of the coral scaffold in all animals but this process was not complete and not the same in all animal. At 6 month radiographs showed more bone formation in group 2 than in group 1. New bone formation volume in each defect was assessed by micro-computed tomography. Volume of bone healing was higher in group 2 than group 1. Discussion. The potential of MSC-containing constructs in a bioreactor for repairing long segmental critical-sized bone defects in sheep was investigated. In one animal of the group 2 the volume of new bone formation was 2066 mm3 and was similar to the bone volume of group 3 (2300 mm3). Our results may have important implications in bone tissue engineering. We observed that the bone tissue regenerationosteogenic ability of bone constructs processed in bioreactor approached the bone autografts

An experimental sheep model was used for impaction allografting of 12 hemiarthroplasty femoral components placed into two equal-sized groups. In group 1, a 50:50 mixture of ApaPore hydroxyapatite bone-graft substitute and allograft was used. In group 2, ApaPore and allograft were mixed in a 90:10 ratio. Both groups were killed at six months. Ground reaction force results demonstrated no significant differences (p > 0.05) between the two groups at 8, 16 and 24 weeks post-operatively, and all animals remained active. The mean bone turnover rates were significantly greater in group 1, at 0.00206 mm/day, compared to group 2 at 0.0013 mm/day (p < 0.05). The results for the area of new bone formation demonstrated no significant differences (p > 0.05) between the two groups. No significant differences were found between the two groups in thickness of the cement mantle (p > 0.05) and percentage ApaPore-bone contact (p > 0.05). The results of this animal study demonstrated that a mixture of ApaPore allograft in a 90:10 ratio was comparable to using a 50:50 mixture

Bone tissue engineering constructs (BTEC) combining natural resorbable osteoconductive scaffolds and mesenchymal stem cells (MSCs) have given promising results to repair critical size bone defect. Yet, results remain inconsistent. Adjonction of an osteoinductive factor to these BTEC, such as rh-BMP-2, to improve bone healing, seems to be a relevant strategy. However, currently supraphysiological dose of this protein are used and can lead to adverse effects such as inflammation, ectopic bone and/or bone cyst formation. Interestingly, in a preliminary study conducted in ectopic site in a murine model, a synergistic effect on bone formation was observed only when a low dose of rh-BMP-2 was associated with MSCs-seeded coral scaffolds but not with a high dose. The objective of the study was then to evaluate a BTEC combining coral scaffold, MSCs and a low dose of rh-BMP-2 in a large animal model of clinical relevance. Sixteen sheep were used for this study. MSCs were isolated from an aspirate of bone marrow harvested from the iliac crest of each sheep receiving BTEC with MSCs, cultivated and seeded on Acroporacoral scaffolds one week before implantation. Rh-BMP-2, used at two different doses (low dose: 68μg/defect and high dose: 680μg/defect), was diluted and absorbed on Acroporacoral scaffold one day before implantation. Metatarsal segmental bone defects (25 mm) were made in the left metatarsal bone of the sheep, stabilized by plate fixation, and filled with Acroporacoral scaffolds loaded with either (i) MSCs and a low dose of rh-BMP-2 (Group 1;n=6), (ii) a low dose of rh-BMP-2 (Group 2;n=5), (iii) a high dose of rh-BMP-2 (Group 3;n=5). Standard radiographs were taken after each surgery and each month until sheep sacrifice, 4 months postoperatively. Bone healing and scaffold resorption were assessed by micro-computed-tomography (μCT) and histomorphometry. Results were compared to a historical control group in which coral scaffolds were loaded with MSCs. Bone volumes (BV) evaluated by μCT and bone surfaces (BS) evaluated by histomorphometry did not differ between groups (BV: 1914±870, 1737±841, 1894±1028 and 1835±1342 mm. 3. ; BS: 25,41±14,25, 19,85±8,31, 25,54±16,98 and 26,08±22,52 %; groups 1, 2, 3 and control respectively); however, an higher bone union was observed in group 1 compared to the others (3, 1, 2 and 2 sheep with bone union in groups 1, 2, 3 and control respectively). No histological abnormalities were observed in any group. Coral resorption was almost complete in all specimens. No significant difference in coral volumes and coral surfaces was observed between groups. A trend towards a higher variability in coral resorption was noted in group 1 compared to the others. There seems to be a benefit to associate low dose of rh-BMP-2 with MSCs-seeded coral scaffolds as this strategy allowed an increase of bone unions in our model. Yet, results remain inconsistent. Although, defective coupling between scaffold resorption and bone formation impaired bone healing in some animals, adjunction of rh-BMP-2 (even at low dose) to CSMs loaded construct is a promising strategy for bone tissue engineering

Summary. MSCs could promote bone regeneration in sheep when loaded on natural fully-resorbable scaffolds, but results are highly variable. Improving the ultimate performance of cell-containing constructs cannot be limited to the decreased rate of scaffold resorption. Introduction. Tissue constructs containing mesenchymal stem cells (MSCs) are an appealing strategy for repairing massive segmental bone defects. However, their therapeutic effectiveness does not match that of autologous bone grafts; among the failure reasons the scaffold resorbability has been identified as a critical feature for achieving bone regeneration. In the present study, the osteogenic potential of 2 constructs obtained by expanding in a bioreactor autologous MSCs onto granules of Acropora or Porites coral, natural fully-resorbable scaffolds, was compared. Materials and methods. 15 sheep underwent a 25 mm long metatarsal ostectomy stabilised with a 3.5 DCP plate. Bone defects were replaced with (i) MSCs-Acropora constructs (n=7), (ii) MSCs-Porites constructs (n=6), (iii) autograft (n=2). Animals were sacrificed 4 months later and bone healing and coral resorption was documented by radiographic, histologic and microCT studies. Results. Results were highly variable in both scaffold groups. Bone formation. Non-union occurred in half cases of each group. In the other half, abundant new bone formation within the defect was observed. This permitted full bone regeneration in 2 animals from the Acropora group and 1 from the Porites group. MicroCT and histomorphometric analysis confirmed great variations as regard of the amount of newly formed bone in defects. Two Acropora-filled defects showed greater amount of newly formed bone than all the Porites-filled defects and were equivalent to the autograft-filled defects, however the difference between the 2 groups wasn't significant. In all groups, the amount of newly formed bone was similar in the proximal, central, and distal thirds of the defects. Coral resorption. The quantitative analysis provided evidence that the Acropora scaffold resorption rate was slower than the Porites one. Bone formation was not statistically associated with coral resorption. However, the 2 Acropora-filled defects with the highest rate of resorption showed a less extend bone formation. Discussion and conclusions. Interestingly, osteogenesis within the 2 constructs was not only found continuous with the bony stumps, but also at the core of the implants. Moreover, bone was observed inside the residual coral fragments. Scaffold resorption was almost complete at 4 months, leading to full bone regeneration in 3 animals. These results provided evidence that MSCs could promote bone regeneration in sheep when loaded on a natural fully-resorbable scaffold. The capacity of the 2 scaffolds to repair defects is statistically similar, despite their different resorption rates and kinetics. This finding suggests that improving the ultimate performance of cell-containing constructs cannot be limited to the decreased rate of scaffold resorption

Abstract. Objectives. A promising therapy for early osteoarthritis (OA) is the transplantation of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). The synovial fluid (SF) from a pre-clinical ovine model treated with hUC-MSCs has been profiled using proteomics and bioinformatics to elucidate potential mechanisms of therapeutic effect. Methods. Four weeks after a medial meniscus transection surgery, sheep were injected with 10. 7. hUC-MSCs in Phosphate Buffered Saline (PBS) or PBS only (n=7) and sacrificed at 12 weeks. SF was normalised for protein abundance (ProteoMiner. TM. ) and analysed using label-free quantitation proteomics. Bioinformatics analyses (Ingenuity Pathway Analysis (IPA) and STRING) were used to assess differentially regulated functions from the proteomic data. Human orthologues were identified for the ovine proteins using UniProt and DAVID resources and proteins that were ≥±1.3 fold differentially abundant between treatment groups, were included in the bioinformatics analyses. Results. hUC-MSC treated animals demonstrated significantly less joint space narrowing. Nineteen SF proteins were differentially abundant in treated cf. control sheep (FC±2.0; p<0.05). Biglycan (a small leucine-rich proteoglycan of the cartilage extracellular matrix) abundance was increased by 2.1 fold in treated compared to untreated sheep (p=0.024). IPA indicated that lipid synthesis (z-score=1.772; p=0.00267) and immune cell migration pathways (cell movement of mononuclear leukocytes: z-score=1.761; p=0.00259), amongst others, were likely to be activated in the treated sheep. Conversely, tissue damage (z-score=−2; p=0.00019), senescence (z-score=−1.981; p=0.00007) and necrosis (z-score=−1.728; p=0.00829) associated pathways as well as inflammation (z-score=−1.718; p=0.00057) and vascular permeability (z-score=−1.698; p=0.00002) were likely to be inhibited in treated cf. untreated sheep. Conclusions. hUC-MSC treatment prevented/delayed OA progression, demonstrated via a reduction in joint space narrowing. SF proteome bioinformatics revealed potential mechanisms of therapeutic action related to immunomodulation and the inhibition of multiple cell death, and tissue damage associated pathways. Further, a potential predicted upregulation in lipid synthesis in treated sheep represents a novel mechanism warranting further investigation. Additional work is required to validate these discovery phase proteomic findings in studies which specifically target and manipulate the proposed mechanisms highlighted. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Low back pain (LBP) is a worldwide leading cause of disability. Treatment of intervertebral disc (IVD) with stem cells has been used on degenerate discs (IDD), cause of around 40% of LBP cases. Despite pain reduction, clinical studies' follow-up have not shown a structural IVD improvement. A valid alternative may be the use of notocordal cells (NC) or their precursors. Mesendoderm progenitor cells (MEPC) have the ability to replicate and differentiate toward NC. In this preliminary study we evaluated in a preclinical IDD model the viability and NC differentiation of MEPC derived from induced pluripotent stem cells (iPSC). MEPC derived from iPSC were developed during the iPSpine project (# 825925), thawed, plated for 24h on laminin and labeled with PKH26. Two adult sheep were subjected to nucleotomy of five lumbar discs for the induction of IDD. After 5 weeks, 3 degenerated discs were treated with MEPC at 3 different doses (low, medium and high). One sheep was sacrificed after 7 days and one after 30 days. Clinical parameters were collected to evaluate the safety of treatment. Discs were analysed using histological techniques. Survival (PKH26), proliferation (PCNA), notocordal cell differentiation (Brachyury, Cytokeratin 8/18/19, Sox9, Foxa2) and endodermal differentiation (Sox17) were evaluated. At 7 days from treatment, both sheep lost about 20% of body weight. Only in discs treated with the highest dose PKH26 stained cells were alive up to 30 days. These cells turn out to be: proliferating (PCNA); positive for Brachyury, cytokeratin 8/18/19 and Foxa2; positive for SOX17 in a small percentage. This preliminary study shows that MEPC, derived from iPSC and injected into ovine discs degenerated by nucleotomy, are able to survive up to 30 days and differentiate within the disc predominantly towards the notocordal phenotype

Low back pain (LBP) is the leading cause of disability worldwide. Recently, treatment of the intervertebral disc (IVD) with stem cells has been used for the treatment of degenerate discs (IDD) which cause at least the 40% of LBP cases. Despite pain reduction, follow-up in clinical studies have not shown an improvement in the structural integrity of IVD. A valid alternative could be the use of progenitor disc cells (notocordal cells, NC) or of their precursors. Mesendoderm progenitor cells (MEPC) have the ability to replicate and differentiate toward NC. In this preliminary study we evaluated in a preclinical large animal IDD model the viability and NC differentiation of MEPC derived from induced pluripotent stem cells (iPSC). MEPC, derived from iPSC and developed during the iPSpine project (# 825925), were thawed and plated on laminin for 24h and labeled with PKH26. Two adult sheep were subjected to nucleotomy of five lumbar discs for the induction of IDD. After 5 weeks, 3 of the 5 degenerate discs were treated with MEPC at 3 different doses (low, medium and high). One sheep was sacrificed after 7 days and the other after 30 days from the treatment injection procedure. Clinical parameters were collected to evaluate the safety of treatment. Discs were paraffin embedded and analysed using histological techniques. Survival (PKH26), proliferation (PCNA), notocordal cell differentiation (Brachyury, Cytokeratin 8/18/19, Sox9, Foxa2) and endodermal differentiation (Sox17) were evaluated. After the injection of the cells, both sheep lost about 20% of body weight. The analysis showed that only in discs treated with the highest dose the PKH26 stained cells resulted alive after 30 days from the procedure. These cells turn out to be:. -. in proliferation (PCNA). -. positive for Brachyury, cytokeratin 8/18/19 and Foxa2. -. a small percentage positive for SOX17. This preliminary study shows that MEPC, derived from iPSC and injected into ovine discs degenerated by nucleotomy, are able to survive 30 days from treatment and differentiate within the disc predominantly towards the notocordal phenotype

Tryfonidou leads the Horizon 2020 consortium (iPSpine; 2019–2023) bringing a transdisciplinary team of 21 partners together to address the challenges and bottlenecks of iPS-based advanced therapies towards their transition to the clinic. Here, chronic back pain due to intervertebral disc degeneration is employed as a show case. The project develops the iPS-technology and designed smart biomaterials to carry, protect and instruct the iPS cells within the degenerate disc environment. This work will be presented including ongoing activities focus on translating the developed methodology and tools towards clinically relevant animal models. The consortium optimized the protocol for the differentiated iPS-notochordal-like cells (iPS-NLCs) and shortlisted two biomaterials shortlisted based on their physicochemical, cytotoxicity, biomechanical and biocompatibility testing. Both were shown to be safe and have been tested with the progenitors of iPS-NLCs. An advanced platform (e.g., the dynamic loading bioreactor for disc tissue) was used to evaluate their performance: the biomaterials supported the iPS-NLC progenitors after injection into the degenerate disc and seem to also support their maturation towards NLCs. Furthermore, we confirmed the capacity of these cells to survive inside degenerated discs at 30 days upon injection in sheep, whereafter we continued with their evaluation at 3 months post-injection. We achieved full evaluation of the sheep spines, including biomechanical analysis using the portable spine biomechanics tester prior analysis at the macro- and microscopic, and biochemical level

Introduction. Degenerative meniscal tears are the most common meniscal lesions, representing huge clinical and socio-economic burdens. Their role in knee osteoarthritis (OA) onset and progression is well established and demonstrated by several retrospective studies. Effective preventive measures and non-surgical treatments for degenerative meniscal lesions are still lacking, also because of the lack of specific and accurate animal models in which test them. Thus, we aim to develop and validate an accurate animal model of meniscus degeneration. Method. Three different surgical techniques to induce medial meniscus degenerative changes in ovine model were performed and compared. A total of 32 sheep (stifle joints) were subjected to either one of the following surgical procedures: a) direct arthroscopic mechanical meniscal injury; b) peripheral devascularization and denervation of medial meniscus; c) full thickness medial femoral condyle cartilage lesion. In all the 3 groups, the contralateral joint served as a control. Result. From a visual examination of the knee joint emerged a clear difference between control and operated groups, in the menisci but also in the cartilage, indicating the onset of OA-related cartilage degeneration. The meniscal and cartilaginous lesions were characterized by different severity and location in the different groups. For instance, a direct meniscal injury caused cartilaginous lesions especially in the medial part of the condyles, and the other approaches presented specific signature. Evaluation of scoring scales (e.g. ICRS score) allowed the quantification of the damage and the identification of differences among the four groups. Conclusion. We were effectively able to develop and validate a sheep model of meniscal degeneration which led to the onset of OA. This innovative model will allow to test in a pre-clinical relevant setting innovative approaches to prevent meniscal-related OA. Funding. Project PNRR-MAD-2022-12375978 funded by Italian Ministry of Health

In the course of uneventful secondary bone healing, a fracture gap is progressively overgrown by callus which subsequently calcifies and remodels into new bone. It is widely accepted that callus formation is promoted by mechanical stimulation of the tissue in the fracture gap. However, the optimal levels of the interfragmentary motion's amplitude, frequency and timing remain unknown. The aim of this study was to develop an active fixation system capable of installing a well-controlled mechanical environment in the fracture gap with continuous monitoring of the bone healing progression. The experimental model was adapted from Tufekci et al. 2018 and required creation of a critical size defect and an osteotomy in a sheep tibia. They were separated by a mobile bone fragment. The distal and proximal parts of the tibia were fixed with an external fixator, whereas the mobile fragment was connected to the proximal part with an active fixator equipped with a linear actuator to move it axially for mechanical stimulation of the tissue in the fracture gap. This configuration installed well-controlled mechanical conditions in the osteotomy, dependent only on the motion of the active fixator and shielded from the influence of the sheep's functional weightbearing. A load sensor was integrated to measure the force acting in the fracture gap during mechanical stimulation. The motion of the bone fragment was controlled by means of a custom-made controller allowing to program stimulation protocols of various profiles, amplitudes and frequencies of loading events. Following in vitro testing, the system was tested in two Swiss White Alpine Sheep. It was configured to simulate immediate weightbearing for one of the animals and delayed weightbearing for the other. The applied loading protocol consisted of 1000 loading events evenly distributed over 12 hours resulting in in a single loading event every 44 seconds. Bench testing confirmed the ability of the system to operate effectively with frequencies up to 1Hz over a range of stimulation amplitudes from 0.1 to 1.5 mm. Continuous measurements of in vivo callus stiffness revealed progressive fracture consolidation in the course of each experiment. A delayed onset of fracture healing was observed in the sheep with simulated delayed weightbearing. The conducted preclinical experiments demonstrated its robustness and reliability. The system can be applied for further preclinical research and comprehensive in-depth investigation of fracture healing

The course of secondary fracture healing typically consists of four major phases including inflammation, soft and hard callus formation, and bone remodeling. Callus formation is promoted by mechanical stimulation, yet little is known about the healing tissue response to strain stimuli over shorter timeframes on hourly and daily basis. The aim of this study was to explore the hourly, daily and weekly variations in bone healing progression and to analyze the short-term response of the repair tissue to well-controlled mechanical stimulation. A system for continuous monitoring of fracture healing was designed for implantation in sheep tibia. The experimental model was adapted from Tufekci et al. 2018 and consisted of 3 mm transverse osteotomy and 30 mm bone defect resulting in an intermediate mobile bone fragment in the tibial shaft. Whereas the distal and proximal parts of the tibia were fixed with external fixator, the mobile fragment was connected to the proximal part via a second, active fixator. A linear actuator embedded in the active fixator moved the mobile fragment axially, thus stimulating mechanically the tissue in the osteotomy gap via well-controlled displacement being independent from the sheep's functional weightbearing. A load sensor was integrated in the active fixation to measure the force acting in the osteotomy gap. During each stimulation cycle the displacement and force magnitudes were recorded to determine in vivo fracture stiffness. Following approval of the local ethics committee, experiments were conducted on four skeletally mature sheep. Starting from the first day after surgery, the daily stimulation protocols consisted of 1000 loading events equally distributed over 12 hours from 9:00 to 21:00 resulting in a single loading event every 44 seconds. No stimulation was performed overnight. One animal had to be excluded due to inconsistencies in the load sensor data. The onset of tissue stiffening was detected around the eleventh day post-op. However, on a daily basis, the stiffness was not steadily increasing, but instead, an abrupt drop was observed in the beginning of the daily stimulations. Following this initial drop, the stiffness increased until the last stimulation cycle of the day. The continuous measurements enabled resolving the tissue response to strain stimuli over hours and days. The presented data contributes to the understanding of the influence of patient activity on daily variations in tissue stiffness and can serve to optimize rehabilitation protocols post fractures

This study aims to investigate the mechanical properties of a rotator cuff tear repaired with a polypropylene interposition graft in an ovine infraspinatus ex-vivo model. Twenty fresh shoulders from skeletally mature sheep were used in this study. A tear size of 20 mm from the tendon joint was created in the infraspinatus tendon to simulate a large tear in fifteen specimens. This was repaired with a polypropylene mesh used as an interposition graft between the ends of the tendon. Eight specimens were secured with mattress stitches while seven were secured to the remnant tendon on the greater tuberosity side by continuous stitching. Remaining five specimens with an intact tendon served as a control group. All specimens underwent cyclic loading with a universal testing machine to determine the ultimate failure load and gap distance. Gap distance increased with progressive cyclic loading through 3000 cycles for all repaired specimens. Mean gap distance after 3000 cycles for both continuous and mattress groups are 1.7 mm and 4.2 mm respectively (P = .001). Significantly higher mean ultimate failure load was also observed with 549.2 N in the continuous group, 426.6 N in the mattress group and 370 N in the intact group. The use of a polypropylene mesh as an interposition graft for large irreparable rotator cuff tears is biomechanically suitable and results in a robust repair that is comparable to an intact rotator cuff tendon. When paired with a continuous suturing technique, it demonstrates significantly resultant superior biomechanical properties that may potentially reduce re-tear rates after repairing large or massive rotator cuff tears

Introduction and Objective. It is widely accepted that interfragmentary strain stimulus promotes callus formation during secondary bone healing. However, the impact of the temporal variation of mechanical stimulation on fracture healing is still not well understood. Moreover, the minimum strain value that initiates callus formation is unknown. The goal of this study was to develop an active fixation system that allows for in vivo testing of varying temporal distribution of mechanical stimulation and that enables detection of the strain limit that initiates callus formation. Materials and Methods. We employed a previously established wedge defect model at the sheep tibia. The model incorporates two partial osteotomies directed perpendicularly to each other, thus creating a bone fragment in the shape of a wedge. The defect was instrumented with an active fixator that tilts the wedge around its apex to create a gradient of interfragmentary strain along the cutting line. The active fixator was equipped with a force and displacement sensors to measure the stiffness of the repair tissue during the course of healing. We developed a controller that enabled programming of different stimulation protocols and their autonomous execution during the in vivo experiment. The system was implanted in two sheep for a period of five weeks. The device was configured to execute immediate stimulation for one animal (stimulation from Day 1), and delayed stimulation for the other (stimulation from Day 22). The daily stimulation protocol consisted of 1’000 loading events evenly distributed over 12 hours from 9:00 am to 9:00 pm. The healing progression was monitored by the in vivo stiffness measurements provided by the fixator and by weekly radiographs. The impact of the local strain magnitude on bone formation was qualitatively evaluated on a post-mortem high-resolution CT scan of the animal with immediate stimulation. Results. The animals tolerated the fixator system well. Both devices operated seamlessly throughout the entire experiment. Callus formation was initiated earlier for the immediately stimulated animal which was also confirmed by a faster stiffness increase. In this pilot feasibility experiment, the initiation of callus formation was observed between 0% and 4% local interfragmentary strain. Conclusions. We developed an autonomous stimulation system for large animal research that enables systematic investigation of fracture healing processes. The in vivo pilot study demonstrated the feasibility of the system and delivered first interesting insides on temporal stimulation impact and callus induction strain limit. These observations, however, require further validation

Secondary bone healing is impacted by the extent of interfragmentary motion at the fracture site. It provides mechanical stimulus that is required for the formation of fracture callus. In clinical settings, interfragmentary motion is induced by physiological loading of the broken bone – for example, by weight-bearing. However, there is no consensus about when mechanical stimuli should be applied to achieve fast and robust healing response. Therefore, this study aims to identify the effect of the immediate and delayed application of mechanical stimuli on secondary bone healing. A partial tibial osteotomy was created in twelve Swiss White Alpine sheep and stabilized using an active external fixator that induced well-controlled interfragmentary motion in form of a strain gradient. Animals were randomly assigned into two groups which mimicked early (immediate group) and late (delayed group) weight-bearing. The immediate group received daily stimulation (1000 cycles/day) from the first day post-op and the delayed group from the 22nd day post-op. Healing progression was evaluated by measurements of the stiffness of the repair tissue during mechanical stimulation and by quantifying callus area on weekly radiographs. At the end of the five weeks period, callus volume was measured on the post-mortem high-resolution computer tomography (HRCT) scan. Stiffness of the repair tissue (p<0.05) and callus progression (p<0.01) on weekly radiographs were significantly larger for the immediate group compared to the delayed group. The callus volume measured on the HRCT was nearly 3.2 times larger for the immediate group than for the delayed group (p<0.01). This study demonstrates that the absence of immediate mechanical stimuli delays callus formation, and that mechanical stimulation already applied in the early post-op phase promotes bone healing