Efficacy of bioreactor-activated bone substitute with bone marrow nuclear cells on fusion rate and fusion mass microarchitecture in sheep.

Bioreactors have been used for bone graft engineering in pre-clinical investigations over the past 15 years. The ability of bioreactor-incubated bone marrow nuclear cells (BMNCs) to enhance bone-forming potential varies significantly, and the three-dimensional (3D) distribution of BMNCs within the scaffold is largely unknown. The aims of this study were (1) to investigate the efficacy of a carbonated hydroxyapatite (CHA) with/without BMNCs on spine fusion rate and fusion mass microarchitecture using a highly challenging two-level posterolateral spine fusion without instrumentation; and (2) to evaluate 3D distribution of BMNCs within scaffolds characterized by immunohistochemistry. Fusion rate and fusion mass were quantified by micro-CT, microarchitectural analysis, and histology. While the homogenous 3D distribution of BMNCs was not observed, BMNCs were found to migrate towards a substitute core. In the autograft group, the healing rate was 83.3%, irrespective of the presence of BMNCs. In the CHA group, also 83.3% was fused in the presence of BMNCs, and 66.7% fused without BMNCs. A significant decrease in the fusion mass porosity (p = .001) of the CHA group suggested the deposition of mineralized bone. The autograft group revealed more bone, thicker trabeculae, and better trabecular orientation but less connection compared to the CHA group. Immunohistochemistry confirmed the ability of bioreactors to incubate a large-sized substitute coated with viable BMNCs with the potential for proliferation and differentiation. These findings suggested that a bioreactor-activated substitute is comparable to autograft on spine fusion and that new functional bone regeneration could be achieved by a combination of BMNCs, biomaterials, and bioreactors.

Thymus Extracellular Matrix-Derived Scaffolds Support Graft-Resident Thymopoiesis and Long-Term In Vitro Culture of Adult Thymic Epithelial Cells.

The thymus provides the physiological microenvironment critical for the development of T lymphocytes, the cells that orchestrate the adaptive immune system to generate an antigen-specific response. A diverse population of stroma cells provides surface-bound and soluble molecules that orchestrate the intrathymic maturation and selection of developing T cells. Forming an intricate 3D architecture, thymic epithelial cells (TEC) represent the most abundant and important constituent of the thymic stroma. Effective models for in and ex vivo use of adult TEC are still wanting, limiting the engineering of functional thymic organoids and the understanding of the development of a competent immune system. Here a 3D scaffold is developed based on decellularized thymic tissue capable of supporting in vitro and in vivo thymopoiesis by both fetal and adult TEC. For the first time, direct evidences of feasibility for sustained graft-resident T-cell development using adult TEC as input are provided. Moreover, the scaffold supports prolonged in vitro culture of adult TEC, with a retained expression of the master regulator Foxn1. The success of engineering a thymic scaffold that sustains adult TEC function provides unprecedented opportunities to investigate thymus development and physiology and to design and implement novel strategies for thymus replacement therapies.

Sensing tissue engineered cartilage quality with Raman spectroscopy and statistical learning for the development of advanced characterization assays.

Nasal chondrocyte-derived engineered cartilage has been demonstrated to be safe and feasible for the treatment of focal cartilage lesions with promising preliminary evidences of efficacy. To ensure the quality of the products and processes, and to meet regulatory requirements, quality controls for identity, purity, and potency need to be developed. We investigated the use of Raman spectroscopy, a nondestructive analytical method that measures the chemical composition of samples, and statistical learning methods for the development of quality controls to quantitatively characterize the starting biopsy and final grafts. We provide a proof-of-concept to show how Raman spectroscopy can be used to identify the types of tissues found in a nasal septal biopsy, i.e., hyaline cartilage and perichondrium, for a novel tissue identity assay. The tissues could be classified with a sensitivity of 89% and specificity of 77%. We also show how clinically relevant and mature nasal chondrocyte-derived engineered cartilage can be assessed with Raman spectroscopy for the development of potency assays. The maturity of engineered grafts, based on the quantified ratio of glycosaminoglycans to DNA and histological score, could be accurately assessed (R2 = 0.78 and 0.89, respectively, between predicted and measured values). Our results demonstrate the potential of Raman spectroscopy for the development of characterization assays for regenerative therapies that could be integrated into a good manufacturing practice-compliant process.

Biomarker Signatures of Quality for Engineering Nasal Chondrocyte-Derived Cartilage.

The definition of quality controls for cell therapy and engineered product manufacturing processes is critical for safe, effective, and standardized clinical implementation. Using the example context of cartilage grafts engineered from autologous nasal chondrocytes, currently used for articular cartilage repair in a phase II clinical trial, we outlined how gene expression patterns and generalized linear models can be introduced to define molecular signatures of identity, purity, and potency. We first verified that cells from the biopsied nasal cartilage can be contaminated by cells from a neighboring tissue, namely perichondrial cells, and discovered that they cannot deposit cartilaginous matrix. Differential analysis of gene expression enabled the definition of identity markers for the two cell populations, which were predictive of purity in mixed cultures. Specific patterns of expression of the same genes were significantly correlated with cell potency, defined as the capacity to generate tissues with histological and biochemical features of hyaline cartilage. The outlined approach can now be considered for implementation in a good manufacturing practice setting, and offers a paradigm for other regenerative cellular therapies.

Entropic elasticity and negative thermal expansion in a simple cubic crystal.

While most solids expand when heated, some materials show the opposite behavior: negative thermal expansion (NTE). In polymers and biomolecules, NTE originates from the entropic elasticity of an ideal, freely jointed chain. The origin of NTE in solids has been widely believed to be different. Our neutron scattering study of a simple cubic NTE material, ScF3, overturns this consensus. We observe that the correlation in the positions of the neighboring fluorine atoms rapidly fades on warming, indicating an uncorrelated thermal motion constrained by the rigid Sc-F bonds. This leads us to a quantitative theory of NTE in terms of entropic elasticity of a floppy network crystal, which is in remarkable agreement with experimental results. We thus reveal the formidable universality of the NTE phenomenon in soft and hard matter.

Bioreactor-manufactured cartilage grafts repair acute and chronic osteochondral defects in large animal studies.

OBJECTIVES: Bioreactor-based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost-effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor-based manufacturing system for the production of cartilage grafts. MATERIALS & METHODS: All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor-based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. RESULTS: The results of these studies demonstrate the safety and feasibility of the bioreactor-based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell-free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor-manufactured grafts may result in a more robust repair in the longer term. CONCLUSION: By demonstrating the safety and efficacy of bioreactor-generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor-based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625.

A perfusion bioreactor-based 3D model of the subarachnoid space based on a meningeal tissue construct.

BACKGROUND: Altered flow of cerebrospinal fluid (CSF) within the subarachnoid space (SAS) is connected to brain, but also optic nerve degenerative diseases. To overcome the lack of suitable in vitro models that faithfully recapitulate the intricate three-dimensional architecture, complex cellular interactions, and fluid dynamics within the SAS, we have developed a perfusion bioreactor-based 3D in vitro model using primary human meningothelial cells (MECs) to generate meningeal tissue constructs. We ultimately employed this model to evaluate the impact of impaired CSF flow as evidenced during optic nerve compartment syndrome on the transcriptomic landscape of MECs. METHODS: Primary human meningothelial cells (phMECs) were seeded and cultured on collagen scaffolds in a perfusion bioreactor to generate engineered meningeal tissue constructs. Engineered constructs were compared to human SAS and assessed for specific cell-cell interaction markers as well as for extracellular matrix proteins found in human meninges. Using the established model, meningeal tissue constructs were exposed to physiological and pathophysiological flow conditions simulating the impaired CSF flow associated with optic nerve compartment syndrome and RNA sequencing was performed. RESULTS: Engineered constructs displayed similar microarchitecture compared to human SAS with regards to pore size, geometry as well as interconnectivity. They stained positively for specific cell-cell interaction markers indicative of a functional meningeal tissue, as well as extracellular matrix proteins found in human meninges. Analysis by RNA sequencing revealed altered expression of genes associated with extracellular matrix remodeling, endo-lysosomal processing, and mitochondrial energy metabolism under pathophysiological flow conditions. CONCLUSIONS: Alterations of these biological processes may not only interfere with critical MEC functions impacting CSF and hence optic nerve homeostasis, but may likely alter SAS structure, thereby further impeding cerebrospinal fluid flow. Future studies based on the established 3D model will lead to new insights into the role of MECs in the pathogenesis of optic nerve but also brain degenerative diseases.

Chondrogenic differentiation of human chondrocytes cultured in the absence of ascorbic acid.

Bioreactor systems will likely play a key role in establishing regulatory compliant and cost-effective production systems for manufacturing engineered tissue grafts for clinical applications. However, the automation of bioreactor systems could become considerably more complex and costly due to the requirements for additional storage and liquid handling technologies if unstable supplements are added to the culture medium. Ascorbic acid (AA) is a bioactive supplement that is commonly presumed to be essential for the generation of engineered cartilage tissues. However, AA can be rapidly oxidized and degraded. In this work, we addressed whether human nasal chondrocytes can redifferentiate, undergo chondrogenesis, and generate a cartilaginous extracellular matrix when cultured in the absence of AA. We found that when chondrocytes were cultured in 3D micromass pellets either with or without AA, there were no significant differences in their chondrogenic capacity in terms of gene expression or the amount of glycosaminoglycans. Moreover, 3D pellets cultured without AA contained abundant collagen Types II and I extracellular matrix. Although the amounts of Collagens II and I were significantly lower (34% and 50% lower) than in pellets cultured with AA, collagen fibers had similar thicknesses and distributions for both groups, as shown by scanning electron microscopy imaging. Despite the reduced amounts of collagen, if engineered cartilage grafts can be generated with sufficient properties that meet defined quality criteria without the use of unstable supplements such as AA, bioreactor automation requirements can be greatly simplified, thereby facilitating the development of more compact, user-friendly, and cost-effective bioreactor-based manufacturing systems.

Expansion of Bone Marrow Mesenchymal Stromal Cells in Perfused 3D Ceramic Scaffolds Enhances In Vivo Bone Formation.

Bone marrow-derived mesenchymal stromal cells (BMSC), when expanded directly within 3D ceramic scaffolds in perfusion bioreactors, more reproducibly form bone when implanted in vivo as compared to conventional expansion on 2D polystyrene dishes/flasks. Since the bioreactor-based expansion on 3D ceramic scaffolds encompasses multiple aspects that are inherently different from expansion on 2D polystyrene, we aimed to decouple the effects of specific parameters among these two model systems. We assessed the effects of the: 1) 3D scaffold vs. 2D surface; 2) ceramic vs. polystyrene materials; and 3) BMSC niche established within the ceramic pores during in vitro culture, on subsequent in vivo bone formation. While BMSC expanded on 3D polystyrene scaffolds in the bioreactor could maintain their in vivo osteogenic potential, results were similar as BMSC expanded in monolayer on 2D polystyrene, suggesting little influence of the scaffold 3D environment. Bone formation was most reproducible when BMSC are expanded on 3D ceramic, highlighting the influence of the ceramic substrate. The presence of a pre-formed niche within the scaffold pores had negligible effects on the in vivo bone formation. The results of this study allow a greater understanding of the parameters required for perfusion bioreactor-based manufacturing of osteogenic grafts for clinical applications.

An automated perfusion bioreactor for the streamlined production of engineered osteogenic grafts.

A computer-controlled perfusion bioreactor was developed for the streamlined production of engineered osteogenic grafts. This system automated the required bioprocesses, from the initial filling of the system through the phases of cell seeding and prolonged cell/tissue culture. Flow through chemo-optic micro-sensors allowed to non-invasively monitor the levels of oxygen and pH in the perfused culture medium throughout the culture period. To validate its performance, freshly isolated ovine bone marrow stromal cells were directly seeded on porous scaffold granules (hydroxyapatite/ß-tricalcium-phosphate/poly-lactic acid), bypassing the phase of monolayer cell expansion in flasks. Either 10 or 20 days after culture, engineered cell-granule grafts were implanted in an ectopic mouse model to quantify new bone formation. After four weeks of implantation, histomorphometry showed more bone in bioreactor-generated grafts than cell-free granule controls, while bone formation did not show significant differences between 10 days and 20 days of incubation. The implanted granules without cells had no bone formation. This novel perfusion bioreactor has revealed the capability of activation larger viable bone graft material, even after shorter incubation time of graft material. This study has demonstrated the feasibility of engineering osteogenic grafts in an automated bioreactor system, laying the foundation for a safe, regulatory-compliant, and cost-effective manufacturing process.

Novel Perfused Compression Bioreactor System as an in vitro Model to Investigate Fracture Healing.

Secondary bone fracture healing is a physiological process that leads to functional tissue regeneration via endochondral bone formation. In vivo studies have demonstrated that early mobilization and the application of mechanical loads enhances the process of fracture healing. However, the influence of specific mechanical stimuli and particular effects during specific phases of fracture healing remain to be elucidated. In this work, we have developed and provided proof-of-concept of an in vitro human organotypic model of physiological loading of a cartilage callus, based on a novel perfused compression bioreactor (PCB) system. We then used the fracture callus model to investigate the regulatory role of dynamic mechanical loading. Our findings provide a proof-of-principle that dynamic mechanical loading applied by the PCB can enhance the maturation process of mesenchymal stromal cells toward late hypertrophic chondrocytes and the mineralization of the deposited extracellular matrix. The PCB provides a promising tool to study fracture healing and for the in vitro assessment of alternative fracture treatments based on engineered tissue grafts or pharmaceutical compounds, allowing for the reduction of animal experiments.

Cartilage graft engineering by co-culturing primary human articular chondrocytes with human bone marrow stromal cells.

Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM-MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair.

Rapid prototyped porous nickel-titanium scaffolds as bone substitutes.

While calcium phosphate-based ceramics are currently the most widely used materials in bone repair, they generally lack tensile strength for initial load bearing. Bulk titanium is the gold standard of metallic implant materials, but does not match the mechanical properties of the surrounding bone, potentially leading to problems of fixation and bone resorption. As an alternative, nickel-titanium alloys possess a unique combination of mechanical properties including a relatively low elastic modulus, pseudoelasticity, and high damping capacity, matching the properties of bone better than any other metallic material. With the ultimate goal of fabricating porous implants for spinal, orthopedic and dental applications, nickel-titanium substrates were fabricated by means of selective laser melting. The response of human mesenchymal stromal cells to the nickel-titanium substrates was compared to mesenchymal stromal cells cultured on clinically used titanium. Selective laser melted titanium as well as surface-treated nickel-titanium and titanium served as controls. Mesenchymal stromal cells had similar proliferation rates when cultured on selective laser melted nickel-titanium, clinically used titanium, or controls. Osteogenic differentiation was similar for mesenchymal stromal cells cultured on the selected materials, as indicated by similar gene expression levels of bone sialoprotein and osteocalcin. Mesenchymal stromal cells seeded and cultured on porous three-dimensional selective laser melted nickel-titanium scaffolds homogeneously colonized the scaffold, and following osteogenic induction, filled the scaffold's pore volume with extracellular matrix. The combination of bone-related mechanical properties of selective laser melted nickel-titanium with its cytocompatibility and support of osteogenic differentiation of mesenchymal stromal cells highlights its potential as a superior bone substitute as compared to clinically used titanium.

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.

Atomic force microscopy to investigate spatial patterns of response to interleukin-1beta in engineered cartilage tissue elasticity.

Atomic force microscopy (AFM) has been proposed as a tool to evaluate the structural and mechanical properties of cartilage tissue. Here, we aimed at assessing whether AFM can be employed to quantify spatially resolved elastic response of tissue engineered cartilage (TEC) to short exposure to IL-1ß, thus mimicking the initially inflammatory implantation site. TEC generated by 14 days of pellet-culture of expanded human chondrocytes was left untreated (ctr) or exposed to IL-1ß for 3 days. TEC pellets were then cut in halves that were glued on a Petri dish. Profiles of elasticity were obtained by sampling with a nanometer sized, pyramidal indenting tip, with 200µm step resolution, the freshly exposed surfaces along selected directions. Replicate TECs were analyzed biochemically and histologically. GAG contents and elasticity of pellets decreased (1.4- and 2.6-fold, respectively, p<0.05) following IL-1ß stimulation. Tissue quality was evaluated by scoring histological pictures taken at 200µm intervals, using the Bern-score grading system. At each distance, scores of ctr TEC were higher than those IL-1ß treated, with the largest differences between the two groups observed in the central regions. Consistent with the histological results, elasticity of IL-1ß-treated TEC was lower than in ctr pellets (up to 3.4-fold at 200µm from the center). IL-1ß treated but not ctr TEC was intensely stained for MMP-13 and DIPEN (cryptic fragment of aggrecan) especially in the central regions. The findings indicate the potential of AFM to investigate structure/function relationships in TEC and to perform tests aimed at predicting the functionality of TEC upon implantation.

Interleukin-1ß modulates endochondral ossification by human adult bone marrow stromal cells.

Inflammatory cytokines present in the milieu of the fracture site are important modulators of bone healing. Here we investigated the effects of interleukin-1ß (IL-1ß) on the main events of endochondral bone formation by human bone marrow mesenchymal stromal cells (BM-MSC), namely cell proliferation, differentiation and maturation/remodelling of the resulting hypertrophic cartilage. Low doses of IL-1ß (50 pg/mL) enhanced colony-forming units-fibroblastic (CFU-f) and -osteoblastic (CFU-o) number (up to 1.5-fold) and size (1.2-fold) in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold) upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1ß enhanced calcium deposition (62.2-fold) and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1ß-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold) in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas), resulting in the formation of mature bone and bone marrow after 12 weeks. In conclusion, IL-1ß finely modulates early and late events of the endochondral bone formation by BM-MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC and as well as improve the engineering of implantable tissues.

Effects of a perfusion bioreactor activated novel bone substitute in spine fusion in sheep.

PURPOSE: To evaluate the effect of a large perfusion-bioreactor cell-activated bone substitute, on a two-level large posterolateral spine fusion sheep model. METHODS: A 50 mm long porous biphasic-calcium-phosphate bone substitute reinforced with poly(D,L-lactide) and, activated with bone marrow derived mononuclear-cells (BMNC) was used. Eighteen sheep were divided into two groups and one group (n = 9) had BMNC-activated bone substitutes and cell-free substitutes implanted. The second group (n = 9) had autograft supplemented with BMNC and regular autograft implanted. The implant material was alternated between spine level L2-L3 and L4-L5 in both groups. MicroCT was used to compare the spine fusion efficacy and bone structure of the two groups as well as the implanted bone substitutes and non-implanted substitutes. RESULTS: After 4½ months six sheep survived in both groups and we found five spine levels were fused when using activated bone substitute compared to three levels with cell-free bone substitute (p = 0.25). Five sheep fused at both levels in the autograft group. A significant increased bone density (p < 0.05) and anisotropy (p < 0.05) was found in the group of activated bone substitutes compared to cell-free bone substitute and no difference existed on the other parameters. The implanted bone substitutes had a significant higher bone density and trabecular thickness than non-implanted bone substitutes, thus indicating that the PLA reinforced BCP had osteoconductive properties (p < 0.05). No effect of the supplemented BMNC to autograft was observed. The autograft group had a significant higher bone density, trabecular thickness and degree of anisotropy than the implanted bone substitutes (p < 0.05), but a lower connectivity density existed (p < 0.05). This indicates that though the activated substitute might have a similar fusion efficacy to autograft, the fusion bridge is not of equal substance. CONCLUSION: We found that bioreactor-generated cell-based bone substitutes seemed superior in fusion ability when compared to cell-free bone substitute and comparable to autograft in fusion ability, but not in bone structure. This combined with the favorable biocompatible abilities and strength comparable to human cancellous bone indicates that it might be a suitable bone substitute in spine fusion procedures.

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.

The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding.

In natural tissues, the extracellular matrix composition, cell density and physiological properties are often non-homogeneous. Here we describe a model system, in which the distribution of cells throughout tissue engineering scaffolds after perfusion seeding can be influenced by the pore architecture of the scaffold. Two scaffold types, both with gyroid pore architectures, were designed and built by stereolithography: one with isotropic pore size (412 ± 13 µm) and porosity (62 ± 1%), and another with a gradient in pore size (250-500 µm) and porosity (35%-85%). Computational fluid flow modelling showed a uniform distribution of flow velocities and wall shear rates (15-24 s(-1)) for the isotropic architecture, and a gradient in the distribution of flow velocities and wall shear rates (12-38 s(-1)) for the other architecture. The distribution of cells throughout perfusion-seeded scaffolds was visualised by confocal microscopy. The highest densities of cells correlated with regions of the scaffolds where the pores were larger, and the fluid velocities and wall shear rates were the highest. Under the applied perfusion conditions, cell deposition is mainly determined by local wall shear stress, which, in turn, is strongly influenced by the architecture of the pore network of the scaffold.

Toward modeling the bone marrow niche using scaffold-based 3D culture systems.

In the bone marrow, specialized microenvironments, called niches, regulate hematopoietic stem cell (HSC) maintenance and function through a complex crosstalk between different cell types. Although in vivo studies have been instrumental to elucidate some of the mechanisms by which niches exert their function, the establishment of an in vitro model that recapitulates the fundamental interactions of the niche components in a controlled setting would be of great benefit. We have previously shown that freshly harvested bone marrow- or adipose tissue-derived cells can be cultured under perfusion within porous scaffolds, allowing the formation of an organized 3D stromal tissue, composed by mesenchymal and endothelial progenitors and able to support hematopoiesis. Here we describe 3D scaffold-based perfusion systems as potential models to reconstruct ex vivo the bone marrow stem cell niche. We discuss how several culture parameters, including scaffold properties, cellular makeup and molecular signals, can be varied and controlled to investigate the role of specific cues in affecting HSC fate. We then provide a perspective of how the system could be exploited to improve stem cell-based therapies and how the model can be extended toward the engineering of other specialized stromal niches.