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.

High-Throughput Microfluidic Platform for 3D Cultures of Mesenchymal Stem Cells, Towards Engineering Developmental Processes.

The development of in vitro models to screen the effect of different concentrations, combinations and temporal sequences of morpho-regulatory factors on stem/progenitor cells is crucial to investigate and possibly recapitulate developmental processes with adult cells. Here, we designed and validated a microfluidic platform to (i) allow cellular condensation, (ii) culture 3D micromasses of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) under continuous flow perfusion, and (ii) deliver defined concentrations of morphogens to specific culture units. Condensation of hBM-MSCs was obtained within 3 hours, generating micromasses in uniform sizes (56.2 ± 3.9 µm). As compared to traditional macromass pellet cultures, exposure to morphogens involved in the first phases of embryonic limb development (i.e. Wnt and FGF pathways) yielded more uniform cell response throughout the 3D structures of perfused micromasses (PMMs), and a 34-fold higher percentage of proliferating cells at day 7. The use of a logarithmic serial dilution generator allowed to identify an unexpected concentration of TGFß3 (0.1 ng/ml) permissive to hBM-MSCs proliferation and inductive to chondrogenesis. This proof-of-principle study supports the described microfluidic system as a tool to investigate processes involved in mesenchymal progenitor cells differentiation, towards a 'developmental engineering' approach for skeletal tissue regeneration.

An improved cartilage digestion method for research and clinical applications.

Enzymatic isolation of chondrocytes from a cartilage biopsy is the first step to establish in vitro models of chondrogenesis or to generate cell-based grafts for cartilage repair. Such process is based on manually operated procedures and typically results in yields lower than 20% of the total available cells. In this study, we hypothesized that, as compared to conventionally used protocols, the enzymatic digestion of human articular cartilage in the presence of ascorbic acid 2-phosphate (AscA2P) or of sodium chloride (NaCl), in combination with the use of a perfusion bioreactor system, leads to a higher and more reproducible yield of cell populations with high proliferation and chondrogenic capacity. The addition of AscA2P within the enzymatic digestion medium did not significantly increase the cell yield, but resulted in a significant decrease of the intradonor variability in cell yield (-17.8% ± 10.7%, p = 0.0247) and in a significant increase of the proliferation rate of the isolated chondrocytes (+19.0% ± 1.4%, p < 0.05) with respect to the control group. The addition of NaCl during cartilage digestion did not modulate cell yield. When the cartilage digestion was further performed under direct perfusion flow, beneficial synergistic effects were achieved, with an overall increase of 34.7% ± 6.8% (p < 0.001) in the cell yield and an average decrease of 57.8% ± 11.2% (p < 0.01) in the coefficient of variation with respect to the control group. Importantly, by implementing this strategy it was possible to retrieve clonal subpopulations more efficiently capable of undergoing chondrogenesis, both in vitro and in vivo. Our findings bear relevance for the preparation of human chondrocytes for laboratory investigations, and in the perspective of efficient and streamlined manufacturing of cell/tissue grafts for articular cartilage repair.

Re-engineering development to instruct tissue regeneration.

With few exceptions, tissue regeneration strategies based on the conventional combination of cells, scaffolding materials, and soluble factors (tissue engineering) have introduced a rather limited clinical impact. While it is being recognized that the nonconvincing benefits of engineered grafts require more fundamental knowledge on mechanisms of action and potency factors, the attempt to mimic and recapitulate developmental events has inspired an evolution of the paradigm. In the context of skeletal regeneration, a "developmental engineering" approach has been advocated to generate intermediate grafts (i.e., hypertrophic cartilage templates) which, as suggested by limb developmental biology, are capable of autonomous spatial and temporal evolution into fully functional bone organs. However, limited consideration has been given to the fact that the recipient site within adult organisms may not be compatible with well-established developmental processes. This can be due to the possibly restricted function of resident progenitors, to the critical mechanical and physical boundary conditions of mature organs, or to the strong role of inflammatory signals and immune cells at repair sites. We thus propose that predictable, orderly, and durable tissue regeneration should be based on a "developmental RE-engineering" paradigm, with the challenge to instruct the execution of developmental programs in the context of an adult system.

Priming 3D cultures of human mesenchymal stromal cells toward cartilage formation via developmental pathways.

The field of regenerative medicine has increasingly recognized the importance to be inspired by developmental processes to identify signaling pathways crucial for 3D organogenesis and tissue regeneration. Here, we aimed at recapitulating the first events occurring during limb development (ie, cell condensation and expansion of an undifferentiated mesenchymal cell population) to prime 3D cultures of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) toward the chondrogenic route. Based on embryonic development studies, we hypothesized that Wnt3a and fibroblast growth factor 2 (FGF2) induce hBM-MSC to proliferate in 3D culture as an undifferentiated pool of progenitors (defined by clonogenic capacity and expression of typical markers), retaining chondrogenic potential upon induction by suitable morphogens. hBM-MSC were responsive to Wnt signaling in 3D pellet culture, as assessed by significant upregulation of main target genes and increase of unphosphorylated ß-catenin levels. Wnt3a was able to induce a five-fold increase in the number of proliferating hBM-MSC (6.4% vs. 1.3% in the vehicle condition), although total DNA content of the 3D construct was decreasing over time. Preconditioning with Wnt3a improved transforming growth factor-ß1 mediated chondrogenesis (30% more glycosaminoglycans/cell in average). In contrast to developmental and 2D MSC culture models, FGF2 antagonized the Wnt-mediated effects. Interestingly, the CD146⁺ subpopulation was found to be more responsive to Wnt3a. The presented data indicate a possible strategy to prime 3D cultures of hBM-MSC by invoking a "developmental engineering" approach. The study also identifies some opportunities and challenges to cross-fertilize skeletal development models and 3D hBM-MSC culture systems.

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.

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.

Surface-dependent modulation of proliferation, bone matrix molecules, and inflammatory factors in human osteoblasts.

Surface properties affect the biological properties of cells modulating the expression of different factors. Osteoblasts contribute both to new bone formation and controlling haematopoiesis through cytokines and growth factors. We analyzed the effect of bone (calcium-phosphate bone slides), cartilaginous (hyaluronan-based scaffold), and plastic substrate culture on human osteoblast proliferation, bone matrix molecule, and inflammatory factor expression. Osteoblast proliferation increased to a greater extent when the cells were grown for 14 days on plastic and bone slides, whereas hyaluronan-based scaffold (HA-scaffold) induced only a minimal increase. Collagen type I, osteonectin, alkaline phosphatase and osteocalcin were expressed on osteoblasts grown on plastic and bone slides and down-modulated at mRNA and protein level by HA-scaffold. Bone slides showed the ability to increase osteopontin mRNA expression. The expression of CXCR4 and CXCL13 was upregulated by bone slides and HA-scaffold, while CXCL12 and CXCR5 expression was down-modulated. These data suggest a substrate-dependent modulation of human osteoblast proliferation, bone matrix and inflammatory factor expression, which might help to understand the active role played by osteoblasts in bone microenvironment by coupling bone extracellular matrix, chemokines and the haematopoietic system.

CCL20 chemokine induces both osteoblast proliferation and osteoclast differentiation: Increased levels of CCL20 are expressed in subchondral bone tissue of rheumatoid arthritis patients.

We evaluated the role of CCL20 (MIP-3alpha) chemokine in cells directly involved in the remodeling of bone tissue (osteoblasts and osteoclasts) and we confirmed its expression in the subchondral bone tissue of rheumatoid arthritis (RA) patients. The expression of CCL20 and of its receptor CCR6 was evaluated in osteoblasts isolated from bone tissue of post-traumatic (PT) patients. Functional tests were performed to evaluate osteoblast proliferation and matrix protein modulation. Immunohistochemical analysis for CCR6, CCL20, and RANKL was performed on bone samples from RA patients. The role of CCL20 was then analyzed in osteoclast differentiation. We found that in basal conditions CCR6, but not its ligand CCL20, was highly expressed by osteoblasts. Functional analysis on osteoblasts showed that CCL20 significantly increased cellular proliferation but did not affect matrix protein expression. Pro-inflammatory cytokines significantly induced the release of CCL20 and RANKL by human osteoblasts but did not modulate CCR6 expression. Increased expression of CCR6, CCL20, and RANKL was confirmed in RA subchondral bone tissue biopsies. We demonstrated that CCL20 was also an earlier inducer of osteoclast differentiation by increasing the number of pre-osteoclasts, thus favoring cell fusion and MMP-9 release. Our results add new insight to the important role of the CCL20/CCR6, RANKL system in the bone tissue of RA. The contemporary action of CCL20 on osteoblasts and osteoclasts involved in the maintenance of bone tissue homeostasis demonstrates the important role of this compartment in the evolution of RA, by showing a clear uncoupling between new bone formation and bone resorption.