Comparison of Osteogenic Capacity and Osteoinduction of Adipose Tissue-Derived Cell Populations.

Stromal vascular fraction (SVF) is the primary isolate obtained after enzymatic digestion of adipose tissue that contains various cell types. Its successful application for cell-based construct preparation in an intra-operative setting for clinical bone augmentation and regeneration has been previously reported. However, the performance of SVF-based constructs compared with traditional ex vivo expanded adipose tissue-derived mesenchymal stromal cells (ATMSCs) remains unclear and direct comparative analyses are scarce. Consequently, we here aimed at comparing the in vitro osteogenic differentiation capacity of donor-matched SVF versus ATMSCs as well as their osteoinductive capacity. Human adipose tissue from nine different donors was used to isolate SVF, which was further purified via plastic-adherence to obtain donor-matched ATMSCs. Both cell populations were immunophenotypically characterized for mesenchymal stromal cell, endothelial, and hematopoietic markers after isolation and immunocytochemical staining was used to identify different cell types during prolonged cell culture. Based on normalization using plastic-adherence fraction determination, SVF and ATMSCs were seeded and cultured in osteogenic differentiation medium for 28 days. Further, SVF and ATMSCs were seeded onto devitalized bovine bone granules and subcutaneously implanted into nude mice. After 42 days of implantation, granules were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) to assess ectopic bone formation. The ATMSCs were shown to be a homogenous cell population during cell culture, whereas SVF cultures consisted of multiple cell types. All donor-matched comparisons showed either accelerated or stronger mineralization for SVF cultures in vitro. However, neither SVF nor ATMSCs loaded on devitalized bone granules induced ectopic bone formation on subcutaneous implantation, as opposed to control granules loaded with bone morphogenetic protein-2 (BMP-2), which triggered ectopic bone formation with 100% incidence. Despite the observed lack of osteoinduction, our findings provide important in vitro evidence on the osteogenic superiority of intra-operatively available SVF as compared with donor-matched ATMSCs. Consequently, further studies should focus on optimizing the efficacy of these cell populations for implementation in orthotopic bone fracture or defect treatment.

Inferring Gene Networks in Bone Marrow Hematopoietic Stem Cell-Supporting Stromal Niche Populations.

The cardinal property of bone marrow (BM) stromal cells is their capacity to contribute to hematopoietic stem cell (HSC) niches by providing mediators assisting HSC functions. In this study we first contrasted transcriptomes of stromal cells at different developmental stages and then included large number of HSC-supportive and non-supportive samples. Application of a combination of algorithms, comprising one identifying reliable paths and potential causative relationships in complex systems, revealed gene networks characteristic of the BM stromal HSC-supportive capacity and of defined niche populations of perivascular cells, osteoblasts, and mesenchymal stromal cells. Inclusion of single-cell transcriptomes enabled establishing for the perivascular cell subset a partially oriented graph of direct gene-to-gene interactions. As proof of concept we showed that R-spondin-2, expressed by the perivascular subset, synergized with Kit ligand to amplify ex vivo hematopoietic precursors. This study by identifying classifiers and hubs constitutes a resource to unravel candidate BM stromal mediators.

Enhanced human bone marrow mesenchymal stromal cell adhesion on scaffolds promotes cell survival and bone formation.

In order to induce an efficient bone formation with human bone marrow mesenchymal stromal cells (hBMSC) associated to a scaffold, it is crucial to determine the key points of the hBMSC action after in vivo transplantation as well as the appropriate features of a scaffold. To this aim we compared the hBMSC behavior when grafted onto two biomaterials allowing different bone potential in vivo. The cancellous devitalized Tutoplast®-processed bone (TPB) and the synthetic hydroxyapatite/ß-tricalcium-phosphate (HA/ßTCP) which give at 6weeks 100% and 50% of bone formation respectively. We first showed that hBMSC adhesion is two times favored on TPB in vitro and in vivo compared to HA/ßTCP. Biomaterial structure analysis indicated that the better cell adhesion on TPB is associated to its higher and smooth open pore architecture as well as its content in collagen. Our 6week time course analysis, showed using qPCR that only adherent cells are able to survive in vivo giving thus an advantage in term of cell number on TPB during the first 4weeks after graft. We then showed that grafted hBMSC survival is crucial as cells participate directly to bone formation and play a paracrine action via the secretion of hIGF1 and hRANKL which are known to regulate the bone formation and resorption pathways respectively. Altogether our results point out the importance of developing a smooth and open pore scaffold to optimize hBMSC adhesion and ensure cell survival in vivo as it is a prerequisite to potentiate their direct and paracrine functions. STATEMENT OF SIGNIFICANCE: Around 10% of skeletal fractures do not heal correctly causing nonunion. An approach involving mesenchymal stromal cells (MSC) associated with biomaterials emerges as an innovative strategy for bone repair. The diversity of scaffolds is a source of heterogeneity for bone formation efficiency. In order to better determine the characteristics of a powerful scaffold it is crucial to understand their relationship with cells after graft. Our results highlight that a biomaterial architecture similar to cancellous bone is important to promote MSC adhesion and ensure cell survival in vivo. Additionally, we demonstrated that the grafted MSC play a direct role coupled to a paracrine effect to enhance bone formation and that both of those roles are governed by the used scaffold.

Muscle acellular scaffold as a biomaterial: effects on C2C12 cell differentiation and interaction with the murine host environment.

The extracellular matrix (ECM) of decellularized organs possesses the characteristics of the ideal tissue-engineering scaffold (i.e., histocompatibility, porosity, degradability, non-toxicity). We previously observed that the muscle acellular scaffold (MAS) is a pro-myogenic environment in vivo. In order to determine whether MAS, which is basically muscle ECM, behaves as a myogenic environment, regardless of its location, we analyzed MAS interaction with both muscle and non-muscle cells and tissues, to assess the effects of MAS on cell differentiation. Bone morphogenetic protein treatment of C2C12 cells cultured within MAS induced osteogenic differentiation in vitro, thus suggesting that MAS does not irreversibly commit cells to myogenesis. In vivo MAS supported formation of nascent muscle fibers when replacing a muscle (orthotopic position). However, heterotopically grafted MAS did not give rise to muscle fibers when transplanted within the renal capsule. Also, no muscle formation was observed when MAS was transplanted under the xiphoid process, in spite of the abundant presence of cells migrating along the laminin-based MAS structure. Taken together, our results suggest that MAS itself is not sufficient to induce myogenic differentiation. It is likely that the pro-myogenic environment of MAS is not strictly related to the intrinsic properties of the muscle scaffold (e.g., specific muscle ECM proteins). Indeed, it is more likely that myogenic stem cells colonizing MAS recognize a muscle environment that ultimately allows terminal myogenic differentiation. In conclusion, MAS may represent a suitable environment for muscle and non-muscle 3D constructs characterized by a highly organized structure whose relative stability promotes integration with the surrounding tissues. Our work highlights the plasticity of MAS, suggesting that it may be possible to consider MAS for a wider range of tissue engineering applications than the mere replacement of volumetric muscle loss.

Platelet lysate coating on scaffolds directly and indirectly enhances cell migration, improving bone and blood vessel formation.

Suitable colonization and vascularization of tissue-engineered constructs after transplantation represent critical steps for the success of bone repair. Human platelet lysate (hPL) is composed of numerous growth factors known for their proliferative, differentiative and chemo-attractant effects on various cells involved in wound healing and bone growth. The aim of this study was to determine whether the delivery of human mesenchymal stromal cells (hMSC) seeded on hPL-coated hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) scaffolds could enhance vascularization and bone formation, as well as to investigate the mechanisms by which hMSC participate in tissue regeneration. Our study demonstrates that hPL can be coated on HA/ß-TCP scaffolds, which play direct and indirect effects on implanted and/or resident stem cells. Effectively, we show that hPL coating directly increases chemo-attraction to and adhesion of hMSC and endothelial cells on the scaffold. Moreover, we show that hPL coating induces hMSC to produce and secrete pro-angiogenic proteins (placental growth factor and vascular endothelial growth factor) which allow the proliferation and specific chemo-attraction of endothelial cells in vitro, thus improving in vivo neovascularization and new bone formation. This study highlights the potential of functionalizing biomaterials with hPL and shows that this growth factor combination can have synergistic effects leading to enhanced bone and blood vessel formation.

In vivo and in vitro comparison of three different allografts vitalized with human mesenchymal stromal cells.

Bone allografts are commonly used by orthopedists to provide a mechanical support and template for cellular colonization and tissue repair. There is an increasing demand for bone graft substitutes that are safe and easy to store but which are equally effective in supporting new bone growth. In this study, we compared three different human bone allografts: (1) the cryopreserved allograft (frozen), (2) the gamma-irradiated and cryopreserved allograft (γ-irradiated), and (3) the solvent dehydrated and γ-irradiated-processed bone allograft (Tutoplast(®) Process Bone [TPB]). Human mesenchymal stromal cells (hMSCs) have the potential to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Our results showed that hMSC seeding efficiency was equivalent among the three bone allografts. However, differences were observed in terms of cell metabolism (viability), osteoblastic gene expression, and in vivo bone formation. Frozen allografts had the higher frequency of new bone formation in vivo (89%). Compared with frozen allografts, we demonstrated that TPB allografts allowed optimal hMSC viability, osteoblastic differentiation, and bone formation to occur in vivo (72%). Further, the frequency of successful bone formation was higher than that obtained with the γ-irradiated allograft (55%). Moreover, after hMSC osteoinduction, 100% of the TPB and frozen allografts formed bone in vivo whereas only 61% of the γ-irradiated allografts did. As healthcare teams around the world require bone-grafting scaffolds that are safe and easy to store, the TPB allograft appears to be a good compromise between efficient bone formation in vivo and convenient storage at room temperature.