Osteoarthritis (OA) is the most common type of arthritis and causes a significant deterioration in patients’ quality of life. The high prevalence of OA as well as the current lack of disease-modifying drugs led to a rise in regenerative medicine efforts. The hope is that this will provide a treatment modality with the ability to alter the course of OA via structural modifications of damaged articular cartilage (AC). Regenerative therapy in OA starts with the concept that administered cells may engraft to a lesion site and differentiate into chondrocytes. However, recent studies show that cells, particularly when injected in suspension, rapidly undergo apoptosis after exerting a transient paracrine effect. If the injected stem cells do not lead to structural improvements of a diseased joint, the high cost of cell therapy for OA cannot be justified, particularly when compared with other injection therapeutics such as corticosteroids and hyaluronic acid. Long-term survival of implanted cells that offer prolonged paracrine effects or possible engraftment is essential for a successful cell therapy that will offer durable structural improvements. In this talk, the history and current status of regenerative therapy in OA are summarized along with the conceptual strategy and future directionsfor a successful regenerative therapy that can provide structural modifications in OA.

Adipose-derived stem cells (ADSCs) are an effective alternative for Teno-regeneration. Despite their applications in tendon engineering, the mechanisms promoting tendon healing still need to be understood. Since there is scattered information on ovine ADSCs, this research aims to investigate in vitro their teno-differentiation for potential use in preclinical tendon regeneration models.

Ovine ADSCs were isolated from the tail region according to FAT-STEM laboratories, expanded until passage six (P6), and characterized in terms of stemness, adhesion and MHC markers by Flow Cytometry (FCM) and immunocytochemistry (ICC). Cell proliferation and senescence were evaluated with MTT and Beta-galactosidase assays, respectively. P1 ADSCs’ teno-differentiation was assessed by culturing them with teno-inductive Conditioned Media (CM) or engineering them on tendon-mimetic PLGA scaffolds. ADSCs teno-differentiation was evaluated by morphological, molecular (qRT-PCR), and biochemical (WesternBlot) approaches.

ADSCs exhibited mesenchymal phenotype, positive for stemness (SOX2, NANOG, OCT4), adhesion (CD29, CD44, CD90, CD166) and MHC-I markers, while negative for hematopoietic (CD31, CD45) and MHC-II markers, showing no difference between passages. ICC staining confirmed these results, where ADSCs showed nuclear positivity for SOX2 (≅ 56%) and NANOG (≅ 67%), with high proliferation capacity without senescence until P6. Interestingly, ADSCs cultured with the teno-inductive CM did not express tenomodulin (TNMD) protein or gene. Conversely, ADSCs seeded on scaffolds teno-differentiated, acquiring a spindle shape supported by TNMD protein expression at 48h (p<0.05 vs. ADSCs 48h) with a significant increase at 14 days of culture (p<0.05 vs. ADSCs + fleece 48h).

Ovine ADSCs respond differently upon distinct teno-inductive strategies. While the molecules on the CM could not trigger a teno-differentiation in the cells, the scaffold's topological stimulus did, resulting in the best strategy to apply. More insights are requested to better understand ovine ADSCs’ tenogenic commitment before using them in vivo for tendon regeneration.

Acknowledgements: This research is part of the P4FIT project ESR5, under the H2020MSCA-ITN-EJD-P4 FIT-Grant Agreement ID:955685.

The application of immune regenerative strategies to deal with unsolved pathologies, such as tendinopathies, is getting attention in the field of tissue engineering exploiting the innate immunomodulatory potential of stem cells [1]. In this context, Amniotic Epithelial Cells (AECs) represent an innovative immune regenerative strategy due to their teno-inductive and immunomodulatory properties [2], and because of their high paracrine activity, become a potential stem cell source for a cell-free treatment to overcome the limitations of traditional cell-based therapies. Nevertheless, these immunomodulatory mechanisms on AECs are still not fully known to date. In these studies, we explored standardized protocols [3] to better comprehend the different phenotypic behavior between epithelial AECs (eAECs) and mesenchymal AECs (mAECs), and to further produce an enhanced immunomodulatory AECs-derived secretome by exposing cells to different stimuli. Hence, in order to fulfill these aims, eAECs and mAECs at third passage were silenced for CIITA and Nrf2, respectively, to understand the role of these molecules in an inflammatory response. Furthermore, AECs at first passage were seeded under normal or GO-coated coverslips to study the effect of GO on AECs, and further exposed to LPS and/or IL17 priming to increase the anti-inflammatory paracrine activity. The obtained results demonstrated how CIITA and Nrf2 control the immune response of eAECs and mAECs, respectively, under standard or immune-activated conditions (LPS priming). Additionally, GO exposition led to a faster activation of the Epithelial-Mesenchymal transition (EMT) through the TGFβ/SMAD signaling pathway with a change in the anti-inflammatory properties. Finally, the combinatory inflammatory stimuli of LPS+IL17 enhanced the paracrine activity and immunomodulatory properties of AECs. Therefore, AECs-derived secretome has emerged as a potential treatment option for inflammatory disorders such as tendinopathies.

Acknowledgement: This research is part of the P4FIT project ESR1, funded under the H2020-ITN-EJD-Marie-Skłodowska-Curie grant agreement 955685.

20 years ago, we designed injectable bioactive suspensions in water of calcium phosphate ceramics for bone and periapical regenerations. Because of leakage of these suspensions, we focused on injectable hydrogels before to set in situ by chemical crosslinking to form 3D scaffolds. We set up a platform to develop a series of innovative hydrogels for bone, cartilages and periodontal tissue regeneration. We based our strategy on polysaccharides macromolecules because they are renewable materials, that originate from biological sources and generally are biocompatible, non-toxic and biodegradable. We developed a family of silated macromolecules able to react forming biocompatible hydrogels. The silated polymers are self-setting hydrogels able to covalently crosslink under pH variation, without addition of toxic crosslinking agent. All these macromolecules could be combined in multicomponent hydrogels, representing a strategy for improving mechanical properties of biomaterials or to tailor particular properties to meet specific needs. For mineral scaffolding, we realized composites of calcium phosphates particles or cements with hydrogel, increasing the ductility and creating macroporous scaffold to propose foam bone cements well adapted to bone biomaterials and Bone tissue engineering. Perspectives are 3D printing and bio printing techniques. We will use our hydrogels platform to prepare tunable (bio)inks in skeletal medicine.

The human amniotic membrane (hAM) contains cells of stem cell characteristics with low immunogenicity and anti-inflammatory properties and has for centuries been applied in the clinics especially for ophthalmology and wound care. It has recently been shown to be promising for novel applications such as tissue engineering and regenerative medicine. Towards these novel applications, we have demonstrated the potential of hAM in toto to differentiate towards bone, cartilage, Schwann like cells and recently also a producer of surfactant. We have further investigated the relevance of the location of origin for the therapeutic potential of the membrane. We show that placental and reflected hAM differs distinctly in morphology and functional activity. The placental region has significantly higher mitochondrial activity, however lower levels of reactive oxygen species, which suggests that placental and reflected regions may have different potential for tissue regeneration. We have further investigated the suitability of hAM to support therapeutic cells and have improved its maintenance in vitro towards xeno-free conditions.

Low back pain is thought to relate to intervertebral disc (IVD) degeneration. Although the mechanisms have not been clearly identified, exhaustion of nucleus pulposus cells and their producing matrix is regarded as one cause. The matrix of the IVD is continuously replenished and remodeled by tissue-specialized cells and are crucial in supporting the IVD function. However, due to aging, trauma, and genetic and lifestyle factors, the cells can lose their potency and viability, thereby limiting their collective matrix production capacity.

We have discovered the link between loss of angiopoietin-1 receptor (Tie2)-positive human NP progenitor cells (NPPC) and IVD degeneration. Tie2+ cells were characterized as undifferentiated cells with multipotency and possessing high self-renewal abilities. Thus we and others have proposed Tie2+ NPPC as a potent cell source for regenerative cell therapies against IVD degeneration. However, their utilization is hindered by low Tie2-expressing cell yields from NP tissue, in particular from commonly available older and degenerated tissue sources. Moreover, NPPC show a rapid Tie2 decrease due to cell differentiation as part of standard culture processes. As such, a need exists to optimize or develop new culture methods that enable the maintenance of Tie2-expressing NPPC. Trials to overcome these difficulties will be shared.

Skeletal sequels of traumatisms, diseases or surgery often lead to bone defects that fail to self-repair. Although the gold standard for bone reconstruction remains the autologous bone graft (ABG), it however exhibits some drawbacks and bone substitutes developed to replace ABG are still far for having its bone regeneration capacity. Herein, we aim to assess a new injectable allogeneic bone substitute (AlloBS) for bone reconstruction. Decellularized and viro-inactivated human femoral heads were crushed then sifted to obtain cortico-spongious powders (CSP). CSP were then partly demineralized and heated, resulting in AlloBS composed of particles consisting in a mineralized core surrounded by demineralized bone matrix, engulfed in a collagen I gelatin. Calvarial defects (5mm in diameter, n=6/condition) in syngeneic Lewis1A rats were filled with CSP, AlloBS±TBM (total bone marrow), BCP (biphasic calcium phosphate)±TBM or left unfilled (control). After 7 weeks, the mineral volume/total volume (MV/TV) ratios were measured by µCT and Movat's pentachrome staining were performed on undemineralized frontal sections. The MV/TV ratios in defects filled with CSP, AlloBS or BCP were equivalent, whereas the MV/TV ratio was higher in AlloBS+TBM compared to CSP, AlloBS or BCP (p<0.01; Mann-Whitney). Histological analyses exhibited a collagen-rich matrix in all the defects, and osteoid at the surface of all implanted biomaterials. Our data indicates that AlloBS is a promising candidate for bone reconstruction, with ease of manipulation, injectability and substantial osteogenic capacity. Further experiments in larger animal models are under consideration to assess whether AlloBS may be a relevant clinical alternative to ABG.

Matrix therapy is a newly coined name emphasizing the importance of the extracellular matrix in regenerative medicine. Heparan sulfates (HS) are key elements of the extracellular matrix (ECM) scaffold which store and protect most growth factors/cytokines controlling the cell migration and differentiation required for healing processes. We have engineered biodegradable nano-polymers (alpha 1–6 polyglucose carboxymethyl sulfate) mimicking (RGTA®) to replace destroyed HS in the damaged ECM scaffolding and to protect cytokines produced by healthy neighbouring cells, thereby restoring the ECM microenvironment and tissue homeostasis and, if needed, provide a homing niche for cell therapy. This matrix therapy approach has considerably improved the quality of healing in various animal models, including muscle and tendon, with reduction or absence of fibrosis resulting in a regeneration process. Over 50 000 patients have been treated in the last years for skin and corneal wounds with dedicated products based on this technology. A randomized controlled trial was performed on 22 racing French Standardbred Trotters (ST) horses to evaluate the efficacy of another polymer, OTR4131 Equitend®, to treat tendinopathies. We evaluated the effect versus placebo on acute superficial digital flexor tendonitis over 4 months by clinical and ultrasonographic measures and their racing performances followed up over the 2 years after treatment. A significant reduction on tendon cross section area was measured in treated animals, racing was 2–3 times more often than placebo, with 3.3 times fewer recurrences and pre-injury performance level was maintained. This study may pave the way for development in humans.

The selection of a proper material to be used as a scaffold or as a hydrogel to support, hold or encapsulate cells is both a critical and a difficult choice that will determine the success of failure of any tissue engineering and regenerative medicine (TERM) strategy. We believe that the use of natural origin polymers, including a wide range of marine origin materials, is the best option for many different approaches that allow for the regeneration of different tissues. In addition to the selection of appropriate material systems it is of outmost importance the development of processing methodologies that allow for the production of adequate scaffolds/matrices, in many cases incorporating bioactive/differentiation agents in their structures. An adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation, and in many cases the selection of a specific sub-population, methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow, mesenchymal stem cells from human adipose tissue, human cells from amniotic fluids and membranes and cells obtained from human umbilical cords. The development of dynamic ways to culture the cells and of distinct ways to stimulate their differentiation in 3D environments, as well as the use of nano-based systems to induce their differentiation and internalization into cells, is also a key part of some of the strategies that are being developed in our research group. The potential of each combination materials/cells, to be used to develop novel useful regeneration therapies will be discussed. The use of different cells and their interactions with different natural origin degradable scaffolds and smart hydrogels will be described. Several examples of TERM strategies to regenerate different types of musculoskeletal tissues will be presented. Relevance to orthopaedics will be highlighted.

Degeneration of intervertebral disc (IVD) Nucleus Pulposus (NP) is a major cause of low back pain (LBP). Healthy NP contains two cell types: notochordal cells (NTC) and nucleopulpocytes (NPCytes). While NTC are embryonic notochord derived cells that are regarded as the resident stem cells of NP, NPCytes are considered the mature NP cells responsible for extracellular matrix (ECM) synthesis. During IVD aging, some still unknown cues drive NTC disappearance. This loss of NTC alters their dialog with NPCytes thereby jeopardizing cell viability and ECM homeostasis, which in turn drives NP degeneration. In this context, NP regeneration by re-establishing this NTC/NPCytes dialog has been contemplated with clinical interest. We will first share our view of the mesenchymal stem cells (MSC)-based therapies that have been preclinically and clinically assessed in LBP. We will then comment on the biomaterial-assisted MSC therapies that recently enter the scene of IVD regeneration. Finally, we will present our REMEDIV project that aims at developing a NP substitute containing stem cells-derived NPCytes and NTC within an injectable hydrogel. We will share our results regarding the generation of NPCytes from adipose-derived MSC and our recent unpublished evidences that human induced-pluripotent stem cells can be differentiated into NTC. Finally, we will consider our ability to transplant these regenerative cells using hydrogels in various animal models. Whether this concept could open new therapeutic windows in the management of discogenic low back pain will finally be discussed.

Summary

Mesenchymal stem cells from human semitendinosus and gracilis tendons (TSPCs) could be a promising MSCs resource for tissue-engineering application. In comparison to adipose-derived stem cells, TSPCs possess similar stem-cells properties and a higher chondrogenic differentiation potential.

Regenerative medicine (RM) promises to restore both the mechanical functionality and the biological composition of tissues after damage. Three-dimensional scaffolds are used in RM to host cells and let them produce proteins that are the building blocks of the native tissues. While regenerating tissues evolve over time through dynamic biomechanical and biochemical changes, current scaffolds’ generation are passive causing mechanical mismatch, suboptimal growth, and pain. Furthermore, current scaffolds ignore the complexity of the reciprocal bio-mechanics regulation, hindering the design of the next-gen scaffolds. To regenerate tissues and organs, biofabrication strategies that impart spatiotemporal control over cell-cell and cell-extracellular matrix communication, often through control over cell and material deposition and placement, are being developed. To achieve these targets, the spatiotemporal control over biological signals at the interface between cells and materials is often aimed for. Alternatively, biological activity can be triggered through the control of mechanical cues, harnessing more fundamental know-how in mechanobiology that could be combined with biofabrication strategies. Here, I present some of our most recent advancements in merging mechanobiology with biofabrication that enabled the control of cell activity, moving towards enhanced tissue regeneration as well as the possibility to create more complex 3D in vitro models to study biological processes

Extensive bone defects, caused by severe trauma or resection of large bone tumors, are difficult to treat. Regenerative medicine, including stem cell transplantation, may provide a novel solution for these intractable problems and improve the quality of life in affected patients. Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine due to their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. However, the osteogenic potential of ASCs is lower than that of bone marrow-derived stromal/stem cells. To address this disadvantage, our group has employed various methods to enhance osteogenic differentiation of ASCs, including factors such as bone morphogenetic protein or Vitamin D, coculture with bone marrow stem cells, VEGF transfection, and gene transfer of Runx-2 and osterix. Recently, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation while GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species and increased GSH/GSSG ratios were also detected in GSTT1- transfected ASCs. GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones

Mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) have great promise in the field of orthopaedic nanomedicine due to their regenerative, as well as immunomodulatory and anti-inflammatory properties. Researchers are interested in harnessing these biologically sourced nanovesicles as powerful therapeutic tools with intrinsic bioactivity to help treat various orthopaedic diseases and defects. Recently, a new class of EV mimetics has emerged known as nanoghosts (NGs). These vesicles are derived from the plasma membrane of ghost cells, thus inheriting the surface functionalities and characteristics of the parent cell while at the same time allowing for a more standardized and reproducible production and significantly greater yield when compared to EVs. This study aims to investigate and compare the osteoinductive potential of MSC-EVs and MSC-NGs in vitro as novel tools in the field of bone tissue engineering and nanomedicine. To carry out this investigation, MSC-EVs were isolated from serum-free MSC conditioned media through differential ultracentrifugation. The remaining cells were treated with hypotonic buffer to produce MSC-ghosts that were then homogenized and serially extruded through 400 and 200 nm polycarbonate membranes to form the MSC-NGs. The concentration, size distribution, zeta potential, and protein content of the isolated nanoparticles were assessed. Afterwards, MSCs were treated with either MSC-EVs or MSC-NGs under osteogenic conditions, and their differentiation was assessed through secreted ALP assay, qPCR, and Alizarin Red mineralization staining. Isolation of MSC-EVs and MSC-NGs was successful, with relatively similar mean diameter size and colloidal stability. No effect on MSC viability and metabolic activity was observed with either treatment. Both MSC-EV and MSC-NG groups had enhanced osteogenic outcomes compared to the control; however, a trend was observed that suggests MSC-NGs as better osteoinductive mediators compared to MSC-EVs. Acknowledgements: The authors would like to acknowledge Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, CHRP, and McGill's Faculty of Dental Medicine and Oral Health Sciences for their financial support

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2. Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining. Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation. Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S

The treatment of osteochondral lesions and osteoarthritis remains an ongoing clinical challenge in orthopaedics. This review examines the current research in the fields of cartilage regeneration, osteochondral defect treatment, and biological joint resurfacing, and reports on the results of clinical and pre-clinical studies. We also report on novel treatment strategies and discuss their potential promise or pitfalls. Current focus involves the use of a scaffold providing mechanical support with the addition of chondrocytes or mesenchymal stem cells (MSCs), or the use of cell homing to differentiate the organism’s own endogenous cell sources into cartilage. This method is usually performed with scaffolds that have been coated with a chemotactic agent or with structures that support the sustained release of growth factors or other chondroinductive agents. We also discuss unique methods and designs for cell homing and scaffold production, and improvements in biological joint resurfacing. There have been a number of exciting new studies and techniques developed that aim to repair or restore osteochondral lesions and to treat larger defects or the entire articular surface. The concept of a biological total joint replacement appears to have much potential.

Cite this article: Bone Joint Res 2013;2:193–9.

Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs). Results. The supernatant contained several GFs/CKs, with especially high levels of basic fibroblast growth factor, and CD34+ cells as the stem/progenitor cell fraction. With regard to biological potential, we confirmed that cell proliferation, osteoinduction, and angiogenesis in hMSCs and HUVECs were enhanced by the supernatant. Conclusions. The current study demonstrates the potential of a new point-of-care strategy for regenerative medicine using skeletal muscle supernatant. This attractive approach and readily-available material could be a promising option for tissue repair/regeneration in the clinical setting. Cite this article: M. Yoshikawa, T. Nakasa, M. Ishikawa, N. Adachi, M. Ochi. Evaluation of autologous skeletal muscle-derived factors for regenerative medicine applications. Bone Joint Res 2017;6:277–283. DOI: 10.1302/2046-3758.65.BJR-2016-0187.R1

First works focuses on the characterization (physical and biological) of this biomaterial. Current work had studied osteoinductive and osteoconductive capacity of these hydrogels. In vivoresults highlight a significant bone reconstruction two months after implantations on bone lesions in mice. Bone is a dynamic and vascularized tissue that has the ability of naturally healing upon damage. Nevertheless, in the case of critical size defects this potential is impaired. Present approaches mainly consider autografts and allografts, which presents several limitations. Bone Tissue Engineering (BTE) is based on the use of 3D matrices to guide both cellular growth, differentiation to promote bone regeneration. Hence, matrices can contain biological materials such as cells and growth factors. Our project aims to design a hydrogel for BTE, particularly for bone lesion filling. We previously showed that a porous 3D hydrogel, Glycosyl-Nucleoside-Fluorinated (GNF) is: 1) non-cytotoxic to clustered human Adipose Mesenchymal Stem Cells (hASCs), 2) bioinjectable and 3) biodegradable. Therefore, this novel class of hydrogels show promise for the development of therapeutic solutions for BTE [1]. The hypothesis of this research was that improving the capacity to promote the adhesion of cells by adding collagen gel matrices and bone morphogenic protein 2 (BMP-2) to improve the bone regenerative potential of this gel. Collagen is a protein matrix well known for its cytocompatibility [2]. BMP-2, have been shown ability to induce bone formation in combination with an adequate matrix [3]. Thereby, the overall aim of this work was to design, develop and validate a new composite hydrogel for BTE. GNF was prepared as previously described in detail[1], at a concentration of 3% (w/v). Type I-collagen gel was prepared from rat-tail tendons at a concentration of 4 g/L [2]. hASCs were isolated from human adipose tissue in our laboratory. To establish a suitable microenvironment for cell proliferation and differentiation cells were seeded in collagen and then GNF gel was added and the resulting mixture was blended, BMP-2 (InductOs ® Kit) is added to this preparation (5µm BMP-2/ml). Fluorometry was used to follow BMP2 release in vitro andin vivo(NOG mices;n=6), orthotopic calvariumbone critical defect (3.3 mm) has been selected to challenge the bone repair. Adding collagen hydrogel improve cell adhesion, survivals and proliferation rather than simple GNF hydrogel. This novel gel composite has the ability to sustain hASCs adhesion and differentiation towards the osteoblastic lineage (positive ALP cells). Fluorometry showed the ability of our hydrogel to prolong the residence of BMP-2 (in vitro and in vivo) compared to collagen hydrogel sponges. Implantation of hydrogel containing hASC and BMP-2 has shown encouraging results in bone reconstruction: 2 months after implantation of biomaterials a significant bone reconstruction can be observed using X-Ray imaging. Adding collagen to GNF allowed to obtain gels showing satisfactory cell-behaviour. In parallel, the presence of GNF hydrogel helps to improve mechanical properties of the biomaterial (hydrogel stability and controlled release of BMP-2). The first in vivostudies have shown encouraging bone regeneration capacity of these hydrogels. The implantation performed on a larger number of animals and quantitative microCT analysis will enable us to judge the effectiveness of this hydrogel as a new injectable biomaterial for BTE. This work was partially supported by NSERC-Canada, FRQ-NT-Quebec, FRQ-S- Quebec, and CFI-Canada. Mathieu Maisani was awarded of a NSERC CREATE Program in Regenerative Medicine (www.ncprm.ulaval.ca)

Ovine articular chondrocytes were isolated from cartilage biopsy and culture expanded in vitro. Approximately 30 million cells per ml of cultured chondrocytes were incorporated with autologous plasma-derived fibrin to form a three-dimensional construct. Full-thickness punch hole defects were created in the lateral and medial femoral condyles. The defects were implanted with either an autologous ‘chondrocyte-fibrin’ construct (ACFC), autologous chondrocytes (ACI) or fibrin blanks (AF) as controls. Animals were killed after 12 weeks. The gross appearance of the treated defects was inspected and photographed. The repaired tissues were studied histologically and by scanning electron microscopy analysis.

All defects were assessed using the International Cartilage Repair Society (ICRS) classification. Those treated with ACFC, ACI and AF exhibited median scores which correspond to a nearly-normal appearance. On the basis of the modified O’Driscoll histological scoring scale, ACFC implantation significantly enhanced cartilage repair compared to ACI and AF. Using scanning electron microscopy, ACFC and ACI showed characteristic organisation of chondrocytes and matrices, which were relatively similar to the surrounding adjacent cartilage.

Implantation of ACFC resulted in superior hyaline-like cartilage regeneration when compared with ACI. If this result is applicable to humans, a better outcome would be obtained than by using conventional ACI.