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.

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.

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.

An RGD-restricted substrate interface is sufficient for the adhesion, growth and cartilage forming capacity of human chondrocytes.

This study aimed at testing whether an RGD-restricted substrate interface is sufficient for adhesion and growth of human articular chondrocytes (HAC), and whether it enhances their post expansion chondrogenic capacity. HAC/substrate interaction was restricted to RGD by modifying tissue culture polystyrene (TCPS) with a poly(ethylene glycol) (PEG) based copolymer system that renders the surface resistant to protein adsorption while at the same time presenting the bioactive RGD-containing peptide GCRGYGRGDSPG (RGD). As compared to TCPS, HAC cultured on RGD spread faster (1.9-fold), maintained higher type II collagen mRNA expression (4.9-fold) and displayed a 19% lower spreading area. On RGD, HAC attachment efficiency (66±10%) and proliferation rate (0.56±0.04 doublings/day), as well as type II collagen mRNA expression in the subsequent chondrogenic differentiation phase, were similar to those of cells cultured on TCPS. In contrast, cartilaginous matrix deposition by HAC expanded on RGD was slightly but consistently higher (15% higher glycosaminoglycan-to-DNA ratio). RDG (bioinactive peptide) and PEG (no peptide ligand) controls yielded drastically reduced attachment efficiency (lower than 11%) and proliferation (lower than 0.20 doublings/day). Collectively, these data indicate that restriction of HAC interaction with a substrate through RGD peptides is sufficient to support their adhesion, growth and maintenance of cartilage forming capacity. The concept could thus be implemented in materials for cartilage repair, whereby in situ recruited/infiltrated chondroprogenitor cells would proliferate while maintaining their ability to differentiate and generate cartilage tissue.

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.

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.

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.

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.

Osteochondral tissue engineering.

Osteochondral defects (i.e., defects which affect both the articular cartilage and underlying subchondral bone) are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. Current surgical limits in the treatment of complex joint lesions could be overcome by grafting osteochondral composite tissues, engineered by combining the patient's own cells with three-dimensional (3D) porous biomaterials of pre-defined size and shape. Various strategies have been reported for the engineering of osteochondral composites, which result from the use of one or more cell types cultured into single-component or composite scaffolds in a broad spectrum of compositions and biomechanical properties. The variety of concepts and models proposed by different groups for the generation of osteochondral grafts reflects that understanding of the requirements to restore a normal joint function is still poor. In order to introduce the use of engineered osteochondral composites in the routine clinical practice, it will be necessary to comprehensively address a number of critical issues, including those related to the size and shape of the graft to be generated, the cell type(s) and properties of the scaffold(s) to be used, the potential physical conditioning to be applied, the degree of functionality required, and the strategy for a cost-effective manufacturing. The progress made in material science, cell biology, mechanobiology and bioreactor technology will be key to support advances in this challenging field.

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.

Cartilage tissue engineering for degenerative joint disease.

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.

Use of hydrodynamic forces to engineer cartilaginous tissues resembling the non-uniform structure and function of meniscus.

The aim of this study was to demonstrate that differences in the local composition of bi-zonal fibrocartilaginous tissues result in different local biomechanical properties in compression and tension. Bovine articular chondrocytes were loaded into hyaluronan-based meshes (HYAFF-11) and cultured for 4 weeks in mixed flask, a rotary Cell Culture System (RCCS), or statically. Resulting tissues were assessed histologically, immunohistochemically, by scanning electron microscopy and mechanically in different regions. Local mechanical analyses in compression and tension were performed by indentation-type scanning force microscopy and by tensile tests on punched out concentric rings, respectively. Tissues cultured in mixed flask or RCCS displayed an outer region positively stained for versican and type I collagen, and an inner region positively stained for glycosaminoglycans and types I and II collagen. The outer fibrocartilaginous capsule included bundles (up to 2 microm diameter) of collagen fibers and was stiffer in tension (up to 3.6-fold higher elastic modulus), whereas the inner region was stiffer in compression (up to 3.8-fold higher elastic modulus). Instead, molecule distribution and mechanical properties were similar in the outer and inner regions of statically grown tissues. In conclusion, exposure of articular chondrocyte-based constructs to hydrodynamic flow generated tissues with locally different composition and mechanical properties, resembling some aspects of the complex structure and function of the outer and inner zones of native meniscus.

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.

Effects of in vitro preculture on in vivo development of human engineered cartilage in an ectopic model.

We investigated whether, and under which conditions (i.e., cell-seeding density, medium supplements), in vitro preculture enhances in vivo development of human engineered cartilage in an ectopic nude mouse model. Monolayer-expanded adult human articular chondrocytes (AHACs) were seeded into Hyalograft C disks at 1.3 x 10(7) cells/cm3 (low density) or 7.6 x 10(7) cells/cm3 (high density). Constructs were directly implanted subcutaneously in nude mice for up to 8 weeks or precultured for 2 weeks before implantation. Preculture medium contained either transforming growth factor-beta1 (TGF-beta1, 1 ng/mL), fibroblast growth factor-2, and platelet-derived growth factor (proliferating medium) or TGF-beta1 (10 ng/mL) and insulin (differentiating medium). Both in vitro and after in vivo implantation, constructs derived by cell seeding at high versus low density and precultured in differentiating versus proliferating medium generated more cartilaginous tissues containing higher amounts of glycosaminoglycan and collagen type II and lower amounts of collagen type I, and with higher equilibrium moduli. As compared with direct implantation of freshly seeded scaffolds, preculture of AHAC-Hyalograft C constructs in differentiating medium, but not in proliferating medium, supported enhanced in vivo development of engineered cartilage. The effect of preculture was more pronounced when constructs were seeded at low density as compared with high density. This study indicates that preculture of human engineered cartilage in differentiating medium has the potential to provide grafts with higher equilibrium moduli and enhanced in vivo developmental capacity than freshly seeded scaffolds. These findings need to be validated in an orthotopic model system.

Bioreactor-based engineering of osteochondral grafts: from model systems to tissue manufacturing.

Osteochondral defects (i.e., those that affect both the articular cartilage and underlying subchondral bone) are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. The in vitro fabrication of osteochondral grafts of predefined size and shape, starting from autologous cells combined with three-dimensional porous biomaterials, is a promising approach for the treatment of osteochondral defects. However, the quality of ex vivo generated cartilage and bone-like tissues is currently restricted by a limited understanding of the regulatory role of physicochemical culture parameters on tissue development. By allowing reproducible and controlled changes in specific biochemical and biomechanical factors, bioreactor systems provide the technological means to reveal fundamental mechanisms of cell function in a three-dimensional environment and the potential to improve the quality of engineered tissues. In addition, by automating and standardizing the manufacturing process in controlled closed systems, bioreactors could reduce production costs and thus facilitate broader clinical impact of engineered osteochondral grafts.

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.