A Hyaluronan-Based Scaffold for the in Vitro Construction of Dental Pulp-Like Tissue

Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyaluronan-based non-woven meshes in the presence of differentiation factors to induce the commitment of stem cells to neuronal, glial, endothelial and osteogenic phenotypes. In vitro experiments, among which were gene expression profiling and immunofluorescence (IF) staining, proved the commitment of DPSCs to the main components of dental pulp tissue. In particular, the hyaluronan-DPSCs construct showed a dental pulp-like morphology consisting of several specialized cells growing inside the hyaluronan fibers. Furthermore, these constructs were implanted into rat calvarial critical-size defects. Histological analyses and gene expression profiling performed on hyaluronan-DPSCs grafts showed the regeneration of osteodentin-like tissue. Altogether, these data suggest the regenerative potential of the hyaluronan-DPSC engineered tissue.     

Distinct Expression Patterns of Cxcl12 in Mesenchymal Stem Cell Niches of Intact and Injured Rodent Teeth

Specific stem cell populations within dental mesenchymal tissues guarantee tooth homeostasis and regeneration throughout life. The decision between renewal and differentiation of stem cells is greatly influenced by interactions with stromal cells and extracellular matrix molecules that form the tissue specific stem cell niches. The Cxcl12 chemokine is a general marker of stromal cells and plays fundamental roles in the maintenance, mobilization and migration of stem cells. The aim of this study was to exploit Cxcl12-GFP transgenic mice to study the expression patterns of Cxcl12 in putative dental niches of intact and injured teeth. We showed that endothelial and stromal cells expressed Cxcl12 in the dental pulp tissue of both intact molars and incisors. Isolated non-endothelial Cxcl12⁺ dental pulp cells cultured in different conditions in vitro exhibited expression of both adipogenic and osteogenic markers, thus suggesting that these cells possess multipotent fates. Taken together, our results show that Cxcl12 is widely expressed in intact and injured teeth and highlight its importance as a key component of the various dental mesenchymal stem cell niches.     

MiR-132-3p Regulates the Osteogenic Differentiation of Thoracic Ligamentum Flavum Cells by Inhibiting Multiple Osteogenesis-Related Genes

Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.     

Cartilage Oligomeric Matrix Protein Gene Multilayers Inhibit Osteogenic Differentiation and Promote Chondrogenic Differentiation of Mesenchymal Stem Cells

There are still many challenges to acquire the optimal integration of biomedical materials with the surrounding tissues. Gene coatings on the surface of biomaterials may offer an effective approach to solve the problem. In order to investigate the gene multilayers mediated differentiation of mesenchymal stem cells (MSCs), gene functionalized films of hyaluronic acid (HA) and lipid-DNA complex (LDc) encoding cartilage oligomeric matrix protein (COMP) were constructed in this study via the layer-by-layer self-assembly technique. Characterizations of the HA/DNA multilayered films indicated the successful build-up process. Cells could be directly transfected by gene films and a higher expression could be obtained with the increasing bilayer number. The multilayered films were stable for a long period and DNA could be easily released in an enzymatic condition. Real-time polymerase chain reaction (RT-PCR) assay presented significantly higher (p < 0.01) COMP expression of MSCs cultured with HA/COMP multilayered films. Compared with control groups, the osteogenic gene expression levels of MSCs with HA/COMP multilayered films were down-regulated while the chondrogenic gene expression levels were up-regulated. Similarly, the alkaline phosphatase (ALP) staining and Alizarin red S staining of MSCs with HA/COMP films were weakened while the alcian blue staining was enhanced. These results demonstrated that HA/COMP multilayered films could inhibit osteogenic differentiation and promote chondrogenic differentiation of MSCs, which might provide new insight for physiological ligament-bone healing.     

Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.     

Effects of Helioxanthin Derivative-Treated Human Dental Pulp Stem Cells on Fracture Healing

Bone defects affect patients functionally and psychologically and can decrease quality of life. To resolve these problems, a simple and efficient method of bone regeneration is required. Human dental pulp stem cells (DPSCs) have high proliferative ability and multilineage differentiation potential. In our previous study, we reported a highly efficient method to induce osteogenic differentiation using DPSC sheets treated with a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH)) in a mouse calvarial defect model. However, the localization of the DPSCs after transplantation remains unknown. Therefore, in this study, we investigated the localization of transplanted DPSCs in a mouse fracture model. DPSCs were collected from six healthy patients aged 18–29 years, cultured in normal medium (NM), osteogenic medium (OM), or OM with TH, and fabricated them into cell sheets. To evaluate the efficacy of fracture healing using DPSCs treated with OM+TH, and to clarify the localization of the transplanted DPSC sheets in vivo, we transplanted OM+TH-treated DPSC sheets labeled with PKH26 into mouse tibiae fractures. We demonstrated that transplanted OM+TH-treated DPSCs sheets were localized to the fracture site and facilitated bone formation. These results indicated that transplanted OM+TH-treated DPSCs were localized at fracture sites and directly promoted fracture healing.     

Isolation and Multiple Differentiation Potential Assessment of Human Gingival Mesenchymal Stem Cells

The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.     

LIM Mineralization Protein-1 Inhibits the Malignant Phenotypes of Human Osteosarcoma Cells

Osteosarcoma (OS), also known as osteogenic sarcoma, is the most common primary malignancy of bone tumor in children and adolescents. However, its underlying molecular pathogenesis is still only vaguely understood. Recently, LIM mineralization protein-1 (LMP-1) was reported to be an essential positive regulator of osteoblast differentiation. In the present study, we found that the expression of LMP-1 is downregulated in OS tissues compared with adjacent normal tissues. Moreover, we restored the expression of LMP-1 through a recombinant adenovirus. Overexpression of LMP-1 inhibited cell proliferation and invasion, arrested cell cycle progression, and induced apoptosis in vitro. Finally, ectopic LMP-1 expression suppressed the expression of Runx2 and BMP-2 in OS cells. These data demonstrate that LMP-1 is an essential tumor suppressor in the OS pathological process, which will provide a new opportunity for discovering and identifying novel effective treatment strategies.     

Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation

The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271⁺, CD271⁻ mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271⁺ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271⁺ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.     

The odontogenic differentiation of human dental pulp stem cells on hydroxyapatite-coated biodegradable nanofibrous scaffolds

The authors aimed to design nanofibrous (NF) scaffolds that facilitate odontogenic and osteogenic differentiation of human dental pulp-derived mesenchymal stem cells (DPSCs) in vitro. For this purpose, hydroxyapatite (HA)–loaded poly (L-lactic acid)/poly (?-caprolactone) (PLLA:PCL 2;1) blend NFs were prepared using the electrospinning method. Alizarin red activity and cell viability were evaluated by MTT assay, and SEM revealed the proliferation properties of NF scaffolds. QRT-PCR results demonstrated that HA-loaded PLLA/PCL can lead to osteoblast/odontoblast differentiation in DPSCs through the up-regulation of related genes, thus indicating that electrospun biodegradable PCL/PLA/HA has remarkable prospects as scaffolds for bone and tooth tissue engineering.     

The Selective Histone Deacetylase Inhibitor MI192 Enhances the Osteogenic Differentiation Efficacy of Human Dental Pulp Stromal Cells

The use of human dental pulp stromal cells (hDPSCs) has gained increasing attention as an alternative stem cell source for bone tissue engineering. The modification of the cells’ epigenetics has been found to play an important role in regulating differentiation, with the inhibition of histone deacetylases 3 (HDAC3) being linked to increased osteogenic differentiation. This study aimed to induce epigenetic reprogramming using the HDAC2 and 3 selective inhibitor, MI192 to promote hDPSCs osteogenic capacity for bone regeneration. MI192 treatment caused a time–dose-dependent change in hDPSC morphology and reduction in viability. Additionally, MI192 successfully augmented hDPSC epigenetic functionality, which resulted in increased histone acetylation and cell cycle arrest at the G₂/M phase. MI192 pre-treatment exhibited a dose-dependent effect on hDPSCs alkaline phosphatase activity. Quantitative PCR and In-Cell Western further demonstrated that MI192 pre-treatment significantly upregulated hDPSCs osteoblast-related gene and protein expression (alkaline phosphatase, bone morphogenic protein 2, type I collagen and osteocalcin) during osteogenic differentiation. Importantly, MI192 pre-treatment significantly increased hDPSCs extracellular matrix collagen production and mineralisation. As such, for the first time, our findings show that epigenetic reprogramming with the HDAC2 and 3 selective inhibitor MI192 accelerates the osteogenic differentiation of hDPSCs, demonstrating the considerable utility of this MSCs engineering approach for bone augmentation strategies.     

Comparison of Osteogenic Potentials of Dental Pulp and Bone Marrow Mesenchymal Stem Cells Using the New Cell Transplantation Platform,CellSaic, in a Rat Congenital Cleft-Jaw Model

Scaffolds stimulate cell proliferation and differentiation and play major roles in providing growth and nutrition factors in the repair of bone defects. We used the recombinant peptide Cellnest™ to prepare the three-dimensional stem cell complex, CellSaic, and evaluated whether CellSaic containing rat dental pulp stem cells (rDPSCs) was better than that containing rat bone marrow stem cells (rBMSCs). rDPSC-CellSaic or rBMSC-CellSaic, cultured with or without osteogenic induction medium, formed the experimental and control groups, respectively. Osteoblast differentiation was evaluated in vitro and transplanted into a rat model with a congenital jaw fracture. Specimens were collected and evaluated by microradiology and histological analysis. In the experimental group, the amount of calcium deposits, expression levels of bone-related genes (RUNX2, ALP, BSP, and COL1), and volume of mineralized tissue, were significantly higher than those in the control group (p < 0.05). Both differentiated and undifferentiated rDPSC-CellSaic and only the differentiated rBMSC-CellSaic could induce the formation of new bone tissue. Overall, rBMSC-CellSaic and rDPSC-CellSaic made with Cellnest™ as a scaffold, provide excellent support for promoting bone regeneration in rat mandibular congenital defects. Additionally, rDPSC-CellSaic seems a better source for craniofacial bone defect repair than rBMSC-CellSaic, suggesting the possibility of using DPSCs in bone tissue regenerative therapy.