Stem cells of the lamina propria of human oral mucosa and gingiva develop into mineralized tissues in vivo.

AIMS: To characterize the mineralized tissue formed constitutively in the supracalvarial region of scid mice by a primitive stem cell population (hOMSC) derived from the lamina propria of the human oral mucosa and gingiva. MATERIAL AND METHODS: Fibrin-hOMSC constructs were cultured for 14 days at which time point they were analysed for the expression of osteoblastic/cementoblastic markers and implanted between the skin and calvaria bones into scid mice. After 8 weeks, the animals were sacrificed and the implantation sites analysed. RESULTS: Two-week-old cultures of fibrin-hOMSC constructs expressed osteogenic/cementogenic markers at the gene level. Macroscopic and radiographic examinations revealed mineralized masses at the implantation sites of fibrin-hOMSC constructs. Histology, histochemistry and immunofluorescence showed mineralized masses consisting of avascular cellular and acellular matrices that stained positively for collagen, Ca, cementum attachment protein, cementum protein 1, bone sialoprotein, alkaline phosphatase, osteocalcin, amelogenin and ameloblastin. Positive anti-human nuclear antigen indicated the human origin of the cells. Atomic force microscopy depicted long prismatic structures organized in lamellar aggregates. CONCLUSIONS: Within the limitation of this study, the results indicate for the first time that fibrin-hOMSC constructs are endowed with the constitutive capacity to develop into mineralized tissues that exhibit certain similarities to cementum and bone.

Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells.

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth-anchoring structure. Periodontal ligament is a unique non-mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)-positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over-expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast-related cells, but it also regulates cementoblast commitment in periodontal ligament cells.

Soluble ECM promotes organotypic formation in lung alveolar model.

Micropatterned suspension culture creates consistently sized and shaped cell aggregates but has not produced organotypic structures from stable cells, thus restricting its use in accurate disease modeling. Here, we show that organotypic structure is achieved in hybrid suspension culture via supplementation of soluble extracellular matrix (ECM). We created a viable lung organoid from epithelial, endothelial, and fibroblast human stable cell lines in suspension culture. We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of lumen-like structures with airspace showing feasible gas exchange units, formation of branching, perfusable vasculature, and long-term 70-day maintenance of lumen structure. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We also demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease.

Characterization of recombinant human cementum protein 1 (hrCEMP1): primary role in biomineralization.

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombinant CEMP1 protein in a human gingival fibroblast cell line. The purified protein (hrCEMP1) has a M(r) 50,000. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of beta-sheet (55%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration.

Human recombinant cementum attachment protein (hrPTPLa/CAP) promotes hydroxyapatite crystal formation in vitro and bone healing in vivo.

Cementum extracellular matrix is similar to other mineralized tissues; however, this unique tissue contains molecules only present in cementum. A cDNA of these molecules, cementum attachment protein (hrPTPLa/CAP) was cloned and expressed in a prokaryotic system. This molecule is an alternative splicing of protein tyrosine phosphatase-like A (PTPLa). In this study, we wanted to determine the structural and functional characteristics of this protein. Our results indicate that hrPTPLa/CAP contains a 43.2% α-helix, 8.9% ß-sheet, 2% ß-turn and 45.9% random coil secondary structure. Dynamic light scattering shows that this molecule has a size distribution of 4.8 nm and aggregates as an estimated mass of 137 kDa species. AFM characterization and FE-SEM studies indicate that this protein self-assembles into nanospheres with sizes ranging from 7.0 to 27 nm in diameter. Functional studies demonstrate that hrPTPLa/CAP promotes hydroxyapatite crystal nucleation: EDS analysis revealed that hrPTPLa/CAP-induced crystals had a 1.59 ± 0.06 Ca/P ratio. Further confirmation with MicroRaman spectrometry and TEM confirm the presence of hydroxyapatite. In vivo studies using critical-size defects in rat cranium showed that hrPTPLa/CAP promoted 73% ± 2.19% and 87% ± 1.97% new bone formation at 4 and 8 weeks respectively. Although originally identified in cementum, PTPLa/CAP is very effective at inducing bone repair and healing and therefore this novel molecule has a great potential to be used for mineralized tissue bioengineering and tissue regeneration.

Bone regeneration in rat cranium critical-size defects induced by Cementum Protein 1 (CEMP1).

Gene therapy approaches to bone and periodontal tissue engineering are being widely explored. While localized delivery of osteogenic factors like BMPs is attractive for promotion of bone regeneration; method of delivery, dosage and side effects could limit this approach. A novel protein, Cementum Protein 1 (CEMP1), has recently been shown to promote regeneration of periodontal tissues. In order to address the possibility that CEMP1 can be used to regenerate other types of bone, experiments were designed to test the effect of hrCEMP1 in the repair/regeneration of a rat calvaria critical-size defect. Histological and microcomputed tomography (µCT) analyses of the calvaria defect sites treated with CEMP1 showed that after 16 weeks, hrCEMP1 is able to induce 97% regeneration of the defect. Furthermore, the density and characteristics of the new mineralized tissues were normal for bone. This study demonstrates that hrCEMP1 stimulates bone formation and regeneration and has therapeutic potential for the treatment of bone defects and regeneration of mineralized tissues.

Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro.

The dental follicle (DF) surrounding the developing tooth germ is an ectomesenchymal tissue composed of various cell populations derived from the cranial neural crest. Human dental follicle cells (HDFC) are believed to contain precursor cells for cementoblasts, periodontal ligament cells, and osteoblasts. Bone morphogenetic proteins (BMPs) produced by Hertwig's epithelial root sheath or present in enamel matrix derivatives (EMD) seem to be involved in the control of DF cell differentiation, but their precise function remains largely unknown. We report the immunolocalization of STRO-1 (a marker of multipotential mesenchymal progenitor cells) and BMP receptors (BMPR) in DF in vivo. In culture, HDFC co-express STRO-1/BMPR and exhibit multilineage properties. Incubation with rhBMP-2 and rhBMP-7 or EMD for 24 h increases the expression of BMP-2 and BMP-7 by HDFC. Long-term stimulation of these cells by rhBMP-2 and/or rhBMP-7 or EMD significantly increases alkaline phosphatase activity (AP) and mineralization. Expression of cementum attachment protein (CAP) and cementum protein-23 (CP-23), two putative cementoblast markers, has been detected in EMD-stimulated whole DF and in cultured HDFC stimulated with EMD or BMP-2 and BMP-7. RhNoggin, a BMP antagonist, abolishes AP activity, mineralization, and CAP/CP-23 expression in HDFC cultures and the expression of BMP-2 and BMP-7 induced by EMD. Phosphorylation of Smad-1 and MAPK is stimulated by EMD or rhBMP-2. However, rhNoggin blocks only Smad-1 phosphorylation under these conditions. Thus, EMD may activate HDFC toward the cementoblastic phenotype, an effect mainly (but not exclusively) involving both exogenous and endogenous BMP-dependent pathways.

Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

Cyclosporin A promotes mineralization by human cementoblastoma-derived cells in culture.

OBJECTIVE: The immunosuppressive drug cyclosporin A has been shown to induce cementum deposition in vivo in experimental animals. Using cementoblastoma-derived cells, we have studied whether this drug will be useful to study cementum mineralization and differentiation in vitro. METHODS: Human cementoblastoma cells and gingival fibroblasts (controls) were cultured and treated with 0.5, 1.0 and 5.0 microg/ml of cyclosporin A. Cell proliferation was evaluated by MTT (tetrazolium) assay and cell number, and cell viability was assessed by trypan blue dye exclusion. Induction of mineralization was evaluated by alizarin red S staining to detect mineralized nodules and by reverse transcription-polymerase chain reaction (RT-PCR) to assess the expression of bone differentiation markers alkaline phosphatase, osteocalcin, bone sialoprotein and core-binding factor a1 (Cbfa1). RESULTS: Cyclosporin A at 5.0 microg/ml concentration reduced significantly the increase in the number of cementoblastoma cells. A dose-dependent increase in the number of mineralized nodules occurred in cultures of cementoblastoma-derived cells treated with cyclosporin A, and RT-PCR analyses showed significantly higher levels of expression of alkaline phosphatase, bone sialoprotein, type I collagen, matrix metalloproteinase-1, osteocalcin, osteopontin, and Cbfa1. Human gingival fibroblast proliferation and cell number were not affected. Mineralized nodules were not detected in gingival fibroblasts and bone specific proteins were not expressed. CONCLUSIONS: Presence of cyclosporin A during 14-day culture period appears to suppress the proliferation of cementoblastoma cells and induce the formation mineralized-like tissue by these cells.

Progenitor cells from dental follicle are able to form cementum matrix in vivo.

To address the molecular mechanisms of cementogenesis, we have isolated dental follicle cells and examined them to see if they contain cementoblast progenitors. Dental follicle tissue was dissected from the root surface of bovine tooth germ and cells were released by digestion with bacterial collagenase. The released cells were maintained as a bovine dental follicle cells (BDFC). To elucidate the differentiation capacity of BDFC, they were transplanted into severe combined immunodeficiency (SCID) mice for 4 weeks. Transplanted BDFC formed cementumlike matrix; in contrast, bovine alveolar osteoblast (BAOB) transplants formed bonelike matrix, and bovine periodontal ligament cells (BPDL) formed a small amount of the cementumlike matrix. Immunohistochemical analysis showed that cementumlike matrix was positive for anti-cementum attachment protein monoclonal antibody, whereas bone-like matrix was negative. These results indicated that the BDFC contained cementoblast progenitors that were able to differentiate to cementoblasts in vivo. They also indicated that the BDFC are phenotypically distinct from BAOB and BPDL, and provide a useful model for investigating molecular mechanisms of cementogenesis.