Adam Papadimitropoulos

Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering.

Mesenchymal stem/stromal cells (MSC) are typically used to generate bone tissue by a process resembling intramembranous ossification, i.e., by direct osteoblastic differentiation. However, most bones develop by endochondral ossification, i.e., via remodeling of hypertrophic cartilaginous templates. To date, endochondral bone formation has not been reproduced using human, clinically compliant cell sources. Here, we aimed at engineering tissues from bone marrow-derived, adult human MSC with an intrinsic capacity to undergo endochondral ossification. By analogy to embryonic limb development, we hypothesized that successful execution of the endochondral program depends on the initial formation of hypertrophic cartilaginous templates. Human MSC, subcutaneously implanted into nude mice at various stages of chondrogenic differentiation, formed bone trabeculae only when they had developed in vitro hypertrophic tissue structures. Advanced maturation in vitro resulted in accelerated formation of larger bony tissues. The underlying morphogenetic process was structurally and molecularly similar to the temporal and spatial progression of limb bone development in embryos. In particular, Indian hedgehog signaling was activated at early stages and required for the in vitro formation of hypertrophic cartilage. Subsequent development of a bony collar in vivo was followed by vascularization, osteoclastic resorption of the cartilage template, and appearance of hematopoietic foci. This study reveals the capacity of human MSC to generate bone tissue via an endochondral program and provides a valid model to study mechanisms governing bone development. Most importantly, this process could generate advanced grafts for bone regeneration by invoking a “developmental engineering” paradigm.

Engineering of a functional bone organ through endochondral ossification

Embryonic development, lengthening, and repair of most bones proceed by endochondral ossification, namely through formation of a cartilage intermediate. It was previously demonstrated that adult human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) can execute an endochondral program and ectopically generate mature bone. Here we hypothesized that hMSCs pushed through endochondral ossification can engineer a scaled-up ossicle with features of a “bone organ,” including physiologically remodeled bone, mature vasculature, and a fully functional hematopoietic compartment. Engineered hypertrophic cartilage required IL-1β to be efficiently remodeled into bone and bone marrow upon subcutaneous implantation. This model allowed distinguishing, by analogy with bone development and repair, an outer, cortical-like perichondral bone, generated mainly by host cells and laid over a premineralized area, and an inner, trabecular-like, endochondral bone, generated mainly by the human cells and formed over the cartilaginous template. Hypertrophic cartilage remodeling was paralleled by ingrowth of blood vessels, displaying sinusoid-like structures and stabilized by pericytic cells. Marrow cavities of the ossicles contained phenotypically defined hematopoietic stem cells and progenitor cells at similar frequencies as native bones, and marrow from ossicles reconstituted multilineage long-term hematopoiesis in lethally irradiated mice. This study, by invoking a “developmental engineering” paradigm, reports the generation by appropriately instructed hMSC of an ectopic “bone organ” with a size, structure, and functionality comparable to native bones. The work thus provides a model useful for fundamental and translational studies of bone morphogenesis and regeneration, as well as for the controlled manipulation of hematopoietic stem cell niches in physiology and pathology.

Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix

Materials based on synthetic polymers can be extensively tailored in their physical properties but often suffer from limited biological functionality. Here we tested the hypothesis that the biological performance of 3D synthetic polymer-based scaffolds can be enhanced by extracellular matrix (ECM) deposited by cells in vitro and subsequently decellularized. The hypothesis was tested in the context of bone graft substitutes, using polyesterurethane (PEU) foams and mineralized ECM laid by human mesenchymal stromal cells (hMSC). A perfusion-based bioreactor system was critically employed to uniformly seed and culture hMSC in the scaffolds and to efficiently decellularize (94% DNA reduction) the resulting ECM while preserving its main organic and inorganic components. As compared to plain PEU, the decellularized ECM-polymer hybrids supported the osteoblastic differentiation of newly seeded hMSC by up-regulating the mRNA expression of typical osteoblastic genes (6-fold higher bone sialoprotein; 4-fold higher osteocalcin and osteopontin) and increasing calcium deposition (6-fold higher), approaching the performance of ceramic-based materials. After ectopic implantation in nude mice, the decellularized hybrids induced the formation of a mineralized matrix positively immunostained for bone sialoprotein and resembling an immature osteoid tissue. Our findings consolidate the perspective of bioreactor-based production of ECM-decorated polymeric scaffolds as off-the-shelf materials combining tunable physical properties with the physiological presentation of instructive biological signals.

Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion.

Mesenchymal stromal/stem cell (MSC) expansion in conventional monolayer culture on plastic dishes (2D) leads to progressive loss of functionality and thus challenges fundamental studies on the physiology of skeletal progenitors, as well as translational applications for cellular therapy and molecular medicine. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow nucleated cells within 3D porous scaffolds in a perfusion-based bioreactor system. The 3D-perfusion system generated a stromal tissue that could be enzymatically treated to yield CD45- MSC. As compared to 2D-expanded MSC (control), those derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7–8 doublings) better maintained their progenitor properties, as assessed by a 4.3-fold higher clonogenicity and the superior differentiation capacity towards all typical mesenchymal lineages. Transcriptomic analysis of MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability and a significant upregulation of multipotency-related gene clusters following 3D-perfusion- as compared to 2D-expansion. Interestingly, the differences in functionality and transcriptomics between MSC expanded in 2D or under 3D-perfusion were only partially captured by cytofluorimetric analysis using conventional surface markers. The described system offers a multidisciplinary approach to study how factors of a 3D engineered niche regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems.

Fibroblast growth factor-2 maintains a niche-dependent population of self-renewing highly potent non-adherent mesenchymal progenitors through FGFR2c.

Bone marrow (BM) mesenchymal stem/stromal cells (MSC) are a heterogeneous population of multipotent progenitors currently under investigation for a variety of applications in regenerative medicine. While self‐renewal of stem cells in different tissues has been demonstrated to be regulated by specialized microenvironments called niches, it is still unclear whether a self‐renewing niche also exists for MSC. Here, we show that primary human BM cultures contain a population of intrinsically non‐adherent mesenchymal progenitors (NAMP) with features of more primitive progenitors than the initially adhering colony‐forming units‐fibroblast (CFU‐f). In fact, NAMP could generate an adherent progeny: (a) enriched with early mesenchymal populations (CD146+, SSEA‐1+, and SSEA‐4+); (b) with significantly greater proliferation and multilineage differentiation potential in vitro; and (c) capable of threefold greater bone formation in vivo than the corresponding CFU‐f. Upon serial replating, NAMP were able to regenerate and expand in suspension as non‐adherent clonogenic progenitors, while also giving rise to an adherent progeny. This took place at the cost of a gradual loss of proliferative potential, shown by a reduction in colony size, which could be completely prevented when NAMP were expanded on the initially adhering BM fraction. Mechanistically, we found that NAMP crucially depend on fibroblast growth factor (FGF)‐2 signaling through FGFR2c for their survival and expansion. Furthermore, NAMP maintenance depends at least in part on humoral signals distinct from FGF‐2. In conclusion, our data show a niche/progenitor organization in vitro, in which the BM adherent fraction provides a self‐renewing microenvironment for primitive NAMP. Stem Cells2012;30:1455–1464

A collagen network phase improves cell seeding of open‐pore structure scaffolds under perfusion

Scaffolds with open‐pore morphologies offer several advantages in cell‐based tissue engineering, but their use is limited by a low cell‐seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open‐pore architecture of polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds increases human bone marrow stromal cells (hBMSCs) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL–TCP scaffolds, rapid prototyped with a honeycomb‐like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen‐free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSCs directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSCs under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5‐fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non‐crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell‐based therapies, collagen‐filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion‐based bioreactor devices to streamline graft manufacture. Copyright

Effect of bone sialoprotein coating of ceramic and synthetic polymer materials on in vitro osteogenic cell differentiation and in vivo bone formation

In this study, we addressed whether Bone Sialoprotein (BSP) coating of various substrates could enhance the in vitro osteogenic differentiation and in vivo bone formation capacity of human Bone Marrow Stromal Cells (BMSC). Moreover, we tested whether synthetic polymer-based porous scaffolds, despite the absence of a mineral component, could support ectopic bone formation by human BMSC if coated with BSP. Adsorption of recombinant human BSP on tissue culture-treated polystyrene (TCTP), beta-tricalcium phosphate (Osteologic) or synthetic polymer (Polyactive) substrates was dose dependent, but did not consistently accelerate or enhance in vitro BMSC osteogenic differentiation, as assessed by the mRNA expression of osteoblast-related genes. Similarly, BSP coating of porous beta-tricalcium phosphate scaffolds (Skelite) did not improve the efficiency of bone tissue formation following loading with BMSC and ectopic implantation in nude mice. Finally, Polyactive foams seeded with BMSC did not form bone tissue in the same ectopic assay, even if coated with BSP. We conclude that BSP coating of a variety of substrates is not directly associated with an enhancement of osteoprogenitor cell differentiation in vitro or in vivo, and that presentation of BSP on polymeric materials is not sufficient to prime BMSC functional osteoblastic differentiation in vivo.

Comparative study of desktop- and synchrotron radiation-based micro computed tomography analyzing cell-seeded scaffolds in tissue engineering of bone

In the field of tissue engineering, micro computed tomography (μCT) should allow non-destructively assessing the extra-cellular matrix deposited by cells within porous scaffolds in-vitro. While synchrotron radiation-based μCT combines micrometer resolution with a high signal-to-noise ratio (contrast), recent advances in desktop μCT devices have achieved comparable results with benefits in availability and user-friendliness. In this study we compare the performance of the commercially available, entry-level desktop device 1174 (SkyScan, Belgium) with the μCT at HASYLAB (DESY, Hamburg, Germany) by characterizing porous interconnected 3D scaffolds and monitoring the development of engineered human bone constructs upon culture in such an environment. Expansion of human osteogenic cells has been performed with the use of perfusion bioreactors and 3D scaffolds, serving as cell carriers. Constructs based on low X-ray absorbing, rapid-prototyped fibrous scaffolds were analyzed with a nominal spatial resolution of better than 5 μm. Direct 3D image analysis allowed for the accurate quantification of the scaffold morphometry parameters, where both μCT techniques yielded comparable results. However, due to the monochromatic nature of X-rays available at the synchrotron radiation source, drastically reduced beam hardening effects and higher density resolution (higher dynamic range) has been obtained at HASYLAB. Studies in this direction could be useful to highlight the mechanisms that are involved in bone-like tissue growth and to further understand how it can be affected by the choice of cell type, 3D culture environment and scaffold type and architecture.

Engineering Small-Scale and Scaffold-Based Bone Organs via Endochondral Ossification Using Adult Progenitor Cells

Bone development, growth, and repair predominantly occur through the process of endochondral ossification, characterized by remodelling of cartilaginous templates. The same route efficiently supports engineering of bone marrow as a niche for hematopoietic stem cells (HSC). Here we describe a combined in vitro/in vivo system based on bone marrow-derived Mesenchymal Stem/Stromal Cells (MSC) that duplicates the hallmark cellular and molecular events of endochondral ossification during development. The model requires MSC culture with instructive molecules to generate hypertrophic cartilage tissues. The resulting constructs complete the endochondral route upon in vivo implantation, in the timeframe of up to 12 weeks. The described protocol is clearly distinct from the direct ossification approach typically used to drive MSC towards osteogenesis. Recapitulation of endochondral ossification allows modelling of stromal-HSC interactions in physiology and pathology and allows engineering processes underlying bone regeneration.

Abstract 328: Original microenvironment of different cancer types is maintained upon culture of primary tissues in perfused bioreactors

The development of novel three-dimensional (3D) culture systems allowing the survival/expansion of primary tumors in vitro may help to bridge the gap between conventional bi-dimensional (2D) cultures and animal models, poorly predictive of therapeutic responses. We have investigated the suitability of a previously developed perfused bioreactor system for the in vitro culture of human primary tumor tissues. Surgical specimens of colorectal cancer (CRC, n = 15), glioblastoma (n = 3), breast cancer (n = 3), sarcoma (n = 2), and melanoma (n = 1) were used. Tumor fragments, obtained upon mechanical mincing and enzymatic digestion, were cultured on collagen scaffolds, in medium supplemented with human serum, under alternate perfusion up to 20 days. Characterization of expanded tissues was performed by histo-morphological analysis, immunofluorescence and gene expression profiling. CRC tissues were effectively expanded in perfused 3D cultures in 10 out of 15 cases, whereas no expansion was observed under static culture conditions. Gene profiles of expanded tumor tissues suggested a heterogeneous tissue composition, as indicated by the expression of EpCAM, CD90, CD8, CD16 and Foxp3 genes. Phenotypic analysis confirmed that expanded tissues included epithelial and stromal cells, as assessed by EpCAM and vimentin staining, respectively. Evidence of tumor cell proliferation was provided by Ki67 staining. Furthermore, infiltrating CD4+ and CD8+ lymphocytes were consistently identified within cultured tumor fragments. The established protocol could easily be adapted to other tumor types, including breast-cancer, glioblastoma, sarcoma and melanoma, with highly effective tissue formation. Taken together, our results indicate that culture of primary tumor fragments within perfused bioreactors can be successfully achieved over a short-time period in a reproducible manner, and results in the expansion of epithelial and interstitial cells. These ex-vivo generated tissues might mirror features of the original tumor more effectively than 2D or 3D static cultures, and of patient-derived xenografts, thus possibly representing useful tools for the evaluation of sensitivity to chemotherapies or new targeted treatments. Citation Format: Christian Hirt, Manuele G. Muraro, Valentina Mele, Francesca Amicarella, Celeste Manfredonia, Savas D. Soysal, Simone Muenst, Luigi Mariani, Christoph Kettelhack, Michael Heberer, Giulio C. Spagnoli, Ivan Martin, Giandomenica Iezzi, Adam Papadimitropoulos. Original microenvironment of different cancer types is maintained upon culture of primary tissues in perfused bioreactors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 328. doi:10.1158/1538-7445.AM2015-328