Ivan Martin

Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro

Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF‐2, TGFβ1, or FGF‐2/TGFβ1). Cells were then induced to redifferentiate in 3D pellets using serum‐supplemented medium (SSM), serum‐free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGFβ1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real‐time PCR. FGF‐2/TGFβ1‐expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR‐expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF‐, PDGFbb‐ or FGF‐2‐expanded chondrocytes. Upon supplementation of SFM with TGFβ and dexamethasone (SFM TD), CII/CI ratios decreased 4.4‐fold for EGF‐ and PDGFbb‐expanded chondrocytes, but increased 96‐fold for FGF‐2/TGFβ1‐expanded cells. Chondrocytes expanded with FGF‐2/TGFβ1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment. J. Cell. Biochem. 81:368–377, 2001.

Real‐time quantitative RT‐PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real‐time RT‐PCR assays to investigate how the expression of typical osteoblast‐related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor‐2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, β‐glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein‐2 (BMP‐2), bone sialo protein‐II (BSP), osteopontin (OP) and to a lower extent cbfa‐1 increased with culture time in osteogenic medium (OM), both in CTR‐ and FGF/Dex‐expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP‐2, BSP, and OP were more expressed in FGF/Dex than in CTR‐expanded BMSC (mRNA levels were, respectively, 9.5‐, 14.9‐, and 5.8‐fold higher), unlike all the other investigated genes. Analysis of single‐colony‐derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex‐expanded clones expressed higher mRNA levels of BMP‐2, BSP, and OP than CTR‐expanded clones. In conclusion, we provide evidence that mRNA levels of BMP‐2, BSP, and OP, quantified using real‐time RT‐PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion. J. Cell. Biochem. 85: 737–746, 2002.

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.

Three-Dimensional Culture of Melanoma Cells Profoundly Affects Gene Expression Profile: A High Density Oligonucleotide Array Study

Growth in three‐dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10‐30,000 cells were generated upon overnight culture in poly‐Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long‐term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with ≥threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, ≥threefold, upregulation of the expression of >100 transcripts while 73 were ≥threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO‐α, ‐β, and γ), IL‐8, CCL20 (MIP‐3α), and Angiopoietin‐like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real‐time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process.

Design of graded biomimetic osteochondral composite scaffolds

With the ultimate goal to generate suitable materials for the repair of osteochondral defects, in this work we aimed at developing composite osteochondral scaffolds organized in different integrated layers, with features which are biomimetic for articular cartilage and subchondral bone and can differentially support formation of such tissues. A biologically inspired mineralization process was first developed to nucleate Mg-doped hydroxyapatite crystals on type I collagen fibers during their self-assembling. The resulting mineral phase was non-stoichiometric and amorphous, resembling chemico-physical features of newly deposited, natural bone matrix. A graded material was then generated, consisting of (i) a lower layer of the developed biomineralized collagen, corresponding to the subchondral bone, (ii) an upper layer of hyaluronic acid-charged collagen, mimicking the cartilaginous region, and (iii) an intermediate layer of the same nature as the biomineralized collagen, but with a lower extent of mineral, resembling the tidemark. The layers were stacked and freeze-dried to obtain an integrated monolithic composite. Culture of the material for 2 weeks after loading with articular chondrocytes yielded cartilaginous tissue formation selectively in the upper layer. Conversely, ectopic implantation in nude mice of the material after loading with bone marrow stromal cells resulted in bone formation which remained confined within the lower layer. In conclusion, we developed a composite material with cues which are biomimetic of an osteochondral tissue and with the capacity to differentially support cartilage and bone tissue generation. The results warrant testing of the material as a substitute for the repair of osteochondral lesions in orthotopic animal models.

Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005

Multipotent mesenchymal stromal cells isolated from bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/or management of autoimmune diseases. In vitro studies have shown that they exhibit a dose-dependent antiproliferative effect on T and B lymphocytes, dendritic cells, natural killer cells and various B cell tumour lines – an effect that is both cell contact and soluble factor dependent. Animal models of autoimmune disease treated with multipotent mesenchymal stromal cells have mostly exhibited a positive clinical response, as have a limited number of patients suffering from acute graft versus host disease. This review summarizes the findings of a 1-day meeting devoted to the subject with the aim of coordinating efforts.

A Nude Mouse Model for Human Bone Formation in Unloaded Conditions

We describe an experimental model for human bone formation in unloaded conditions. Bone formation has been assessed by implanting in vivo human bone marrow stromal cells (BMSC) on porous hydroxyapatite (HA) bioceramics subcutaneously in nude mice. In this system, human bone formation and remodeling occurs and can be studied in unloaded conditions, i.e., with no influence of muscle tension. Using this model system, we have been also studying the effects of dexamethasone (Dex) in combination with fibroblast growth factor-2 (FGF-2) on the osteogenic potential of human BMSC. A colony-forming unit-fibroblastoid (CFU-F) formed in clonal conditions were significantly larger when Dex/FGF-2 was present in the culture medium. The cell proliferation rate was also increased by the combination Dex/FGF-2 at a higher extent than Dex or FGF-2 alone. BMSC expanded with Dex/FGF-2 displayed alkaline phosphatase levels lower (56%) than Dex expanded cells, but significantly higher than FGF-2 expanded cells. Our results suggest that Dex/FGF-2 expanded BMSC are able to form more bone than BMSC expanded in the presence of FGF-2 alone.

Tissue engineering for total meniscal substitution : Animal study in sheep model

OBJECTIVE The aim of the study was to investigate the use of a novel hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Two different surgical implantation techniques in a sheep model were evaluated. METHODS Twenty-four skeletally mature sheep were treated with total medial meniscus replacements, while two meniscectomies served as empty controls. The animals were divided into two groups: cell-free scaffold and scaffold seeded with autologous chondrocytes. Two different surgical techniques were compared: in 12 animals, the implant was sutured to the capsule and to the meniscal ligament; in the other 12 animals, also a transtibial fixation of the horns was used. The animals were euthanized after 4 months. The specimens were assessed by gross inspection and histology. RESULTS All implants showed excellent capsular ingrowth at the periphery. Macroscopically, no difference was observed between cell-seeded and cell-free groups. Better implant appearance and integrity was observed in the group without transosseous horns fixation. Using the latter implantation technique, lower joint degeneration was observed in the cell-seeded group with respect to cell-free implants. The histological analysis indicated cellular infiltration and vascularization throughout the implanted constructs. Cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. CONCLUSION The current study supports the potential of a novel HYAFF/polycaprolactone scaffold for total meniscal substitution. Seeding of the scaffolds with autologous chondrocytes provides some benefit in the extent of fibrocartilaginous tissue repair.

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.

New dimensions in tumor immunology: what does 3D culture reveal?

Experimental models indicate that tumor cells in suspension, unlike solid tumor fragments, might be unable to produce life-threatening cancer outgrowth when transferred to animal models, irrespective of the number of cells transferred, although they induce specific immune responses. Human tumor cells cultured in three dimensions display increased pro-angiogenic capacities and resistance to interferons, chemotherapeutic agents or irradiation, as compared with cells cultured in two-dimensional (2D) monolayers. Tumor cells cultured in three dimensions were also shown to be characterized by defective immune recognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens (TAAs) and by a capacity to inhibit CTL proliferation and dendritic cell (DC) functions. Downregulation of human leukocyte antigen (HLA) or TAA expression and high production of lactic acid might play a role in the elicitation of these effects. Here, we propose that growth in 3D architectures might provide new insights into tumor immunology and could represent an integral missing component in pathophysiological tumor immune escape mechanisms.