Defining properties of neural crest-derived progenitor cells from the apex of human developing tooth

The connective tissue of the human tooth arises from cells that are derived from the cranial neural crest and, thus, are termed as "ectomesenchymal cells." Here, cells being located in a pad-like tissue adjacent to the apex of the developing tooth, which we designated the third molar pad, were separated by the microexplant technique. When outgrowing from the explant, dental neural crest-derived progenitor cells (dNC-PCs) adhered to plastic, proliferated steadily, and displayed a fibroblast-like morphology. At the mRNA level, dNC-PCs expressed neural crest marker genes like Sox9, Snail1, Snail2, Twist1, Msx2, and Dlx6. Cytofluorometric analysis indicated that cells were positive for CD49d (alpha4 integrin), CD56 (NCAM), and PDGFRalpha, while negative for CD31, CD34, CD45, and STRO-1. dNC-PCs could be differentiated into neurogenic, chondrogenic, and osteogenic lineages and were shown to produce bone matrix in athymic mice. These results demonstrate that human third molar pad possesses neural crest-derived cells that represent multipotent stem/progenitor cells. As a rather large amount of dNC-PCs could be obtained from each single third molar, cells may be used to regenerate a wide range of tissues within the craniofacial region of humans

Three-dimensional perfusion culture of human adipose tissue-derived endothelial and osteoblastic progenitors generates osteogenic constructs with intrinsic vascularization capacity

In this study, we aimed at generating osteogenic and vasculogenic constructs starting from the stromal vascular fraction (SVF) of human adipose tissue as a single cell source. SVF cells from human lipoaspirates were seeded and cultured for 5 days in porous hydroxyapatite scaffolds by alternate perfusion through the scaffold pores, eliminating standard monolayer (two-dimensional [2D]) culture. The resulting cell-scaffold constructs were either enzymatically treated to extract and characterize the cells or subcutaneously implanted in nude mice for 8 weeks to assess the capacity to form bone tissue and blood vessels. SVF cells were also expanded in 2D culture for 5 days and statically loaded in the scaffolds. The SVF yielded 5.9 +/- 3.5 X 10(5) cells per milliliter of lipoaspirate containing both mesenchymal progenitors (5.2 units) and endothelial-lineage cells (54 cells). After 5 days, the total cell number was 1.8-fold higher in 2D than in three-dimensional (31)) cultures, but the percentage of mesenchymaland endothelial-lineage cells was similar (i.e., 65 of CD90(+) cells and 7 implantation, constructs from both conditions contained blood vessels stained for human CD31 and CD34, functionally connected to the host vasculature. Importantly, constructs generated under 3D perfusion, and not those based on 2D-expanded cells, reproducibly formed bone tissue. In conclusion, direct perfusion of human adiposederived cells through ceramic scaffolds establishes a 3D culture system for osteoprogenitor and endothelial cells and generates osteogenic-vasculogenic constructs. It remains to be tested whether the presence of endothelial cells accelerates construct vascularization and could thereby enhance implanted cell survival in larger size implants

In vitro and in vivo validation of human and goat chondrocyte labeling by green fluorescent protein lentivirus transduction

We investigated whether human articular chondrocytes can be labeled efficiently and for long-term with a green fluorescent protein (GFP) lentivirus and whether the viral transduction would influence cell proliferation and tissue-forming capacity. The method was then applied to track goat articular chondrocytes after autologous implantation in cartilage defects. Expression of GFP in transduced chondrocytes was detected cytofluorimetrically and immunohistochemically. Chondrogenic capacity of chondrocytes was assessed by Safranin-O staining, immunostaining for type II collagen, and glycosaminoglycan content. Human articular chondrocytes were efficiently transduced with GFP lentivirus (73.4 +/- 0.5% at passage 1) and maintained the expression of GFP up to 22 weeks of in vitro culture after transduction. Upon implantation in nude mice, 12 weeks after transduction, the percentage of labeled cells (73.6 +/- 3.3%) was similar to the initial one. Importantly, viral transduction of chondrocytes did not affect the cell proliferation rate, chondrogenic differentiation, or tissue-forming capacity, either in vitro or in vivo. Goat articular chondrocytes were also efficiently transduced with GFP lentivirus (78.3 +/- 3.2%) and maintained the expression of GFP in the reparative tissue after orthotopic implantation. This study demonstrates the feasibility of efficient and relatively long-term labeling of human chondrocytes for co-culture on integration studies, and indicates the potential of this stable labeling technique for tracking animal chondrocytes for in cartilage repair studies

Dentistry ― At the Heart of Conflict between Medicine and Technology

As the first Editor-in-Chief of Dentistry Journal, I would like to provide a few comments about this field of research and practice. Evolved from a purely technical profession, dentists are nowadays exposed to a variety of different challenges. [...

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 x 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. Copyright (c) 2012 John Wiley & Sons, Ltd

Fat-Derived Stromal Vascular Fraction Cells Enhance the Bone-Forming Capacity of Devitalized Engineered Hypertrophic Cartilage Matrix

Engineered and devitalized hypertrophic cartilage (HC) has been proposed as bone substitute material, potentially combining the features of osteoinductivity, resistance to hypoxia, capacity to attract blood vessels, and customization potential for specific indications. However, in comparison with vital tissues, devitalized HC grafts have reduced efficiency of bone formation and longer remodeling times. We tested the hypothesis that freshly harvested stromal vascular fraction (SVF) cells from human adipose tissue-which include mesenchymal, endothelial, and osteoclastic progenitors-enhance devitalized HC remodeling into bone tissue. Human SVF cells isolated from abdominal lipoaspirates were characterized cytofluorimetrically. HC pellets, previously generated by human bone marrow-derived stromal cells and devitalized by freeze/thaw, were embedded in fibrin gel with or without different amounts of SVF cells and implanted either ectopically in nude mice or in 4-mm-diameter calvarial defects in nude rats. In the ectopic model, SVF cells added to devitalized HC directly contributed to endothelial, osteoblastic, and osteoclastic populations. After 12 weeks, the extent of graft vascularization and amount of bone formation increased in a cell-number-dependent fashion (up to, respectively, 2.0-fold and 2.9-fold using 12 million cells per milliliter of gel). Mineralized tissue volume correlated with the number of implanted, SVF-derived endothelial cells (CD31+ CD34+ CD146+). In the calvarial model, SVF activation of HC using 12 million cells per milliliter of gel induced efficient merging among implanted pellets and strongly enhanced (7.3-fold) de novo bone tissue formation within the defects. Our findings outline a bone augmentation strategy based on off-the-shelf devitalized allogeneic HC, intraoperatively activated with autologous SVF cells.; This study validates an innovative bone substitute material based on allogeneic hypertrophic cartilage that is engineered, devitalized, stored, and clinically used, together with autologous cells, intraoperatively derived from a lipoaspirate. The strategy was tested using human cells in an ectopic model and an orthotopic implantation model, in immunocompromised animals