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Harvesting bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. However, as stem cells are characterized by self-renewing and commitment in several cellular subtypes (ie, pluripotential differentiation), some concerns may arise as regards their potential uncontrolled proliferation. To screen the behavior of osteoblasts derived from human pulpar stem cells (ODHPSCs), we used microarray techniques to identify genes that are differently regulated in ODHPSC in comparison to normal osteoblasts (NOs). Osteoblasts derived from human pulpar stem cells were obtained from human dental pulp, and cells were selected using a cytometer. The cell profile was c-kit+/CD34+/STRO-1+/CD45−. These cells were capable of differentiation of osteoblasts in vitro. By using DNA microarrays containing 19,200 genes, we identified in ODHPSC some genes whose expression was significantly up- and downregulated compared to NO. The differentially expressed genes have different functional activities: (a) cell differentiation, (b) developmental maturation, (c) cell adhesion, and (d) production of cytoskeleton elements. Thus, some molecular differences exist between NO and ODHPSC, although the previously considered histologic parameters show a normal phenotype.

In this study, we have observed dental pulp stem cells (SBP-DPSCs) performances on different scaffolds, such as PLGA 85:15, hydroxyapatite chips (HA) and titanium. Stem cells were challenged with each engineered surface, either in plane cultures or in a rotating apparatus, for a month. Gingival fibroblasts were used as controls. Results showed that stem cells exerted a different response, depending on the different type of textured surface: in fact, microconcavities significantly affected SBP-DPSC differentiation into osteoblasts, both temporally and quantitatively, with respect to the other textured surfaces. Actually, stem cells challenged with concave surfaces differentiated quicker and showed nuclear polarity, an index of secretion, cellular activity and matrix formation. Moreover, bone-specific proteins were significantly expressed and the obtained bone tissue was of significant thickness. Thus, cells cultured on the concave textured surface had better cell-scaffold interactions and were induced to secrete factors that, due to their autocrine effects, quickly lead to osteodifferentiation, bone tissue formation, and vascularization. The worst cell performance was obtained using convex surfaces, due to the scarce cell proliferation on to the scaffold and the poor matrix secretion. In conclusion, this study stresses that for a suitable and successful bone tissue reconstruction the surface texture is of paramount importance. J. Cell. Physiol. 214:166–172, 2008. © 2007 Wiley-Liss, Inc.

BACKGROUND Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. METHODOLOGY AND FINDINGS In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80-120 microm in diameter and 40-100 microm in depth, which we termed primary; and (ii) smaller microcavities of 10-20 microm in diameter and 3-10 microm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. CONCLUSION In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future.

Stromal stem cells from human dental pulp (SBP-DPSCs) were used to study osteogenic differentiation in vitro and in vivo. We previously reported that SBP-DPSCs are multipotent stem cells able to differentiate into osteoblasts, which synthesize three-dimensional woven bone tissue chips in vitro. In this study, we followed the temporal expression pattern of specific markers in SBP-DPSCs and found that, when differentiating into osteoblasts, they express, besides osteocalcin, also flk-1 (VEGF-R2). In addition, 30% of them expressed specific antigens for endothelial cells, including CD54, von-Willebrand ( domain 1 and 2), CD31 (PECAM-1) and angiotensin-converting enzyme. Interestingly, we found endotheliocytes forming vessel walls, observing that stem cells synergically differentiate into osteoblasts and endotheliocytes, and that flk-1 exerts a pivotal role in coupling osteoblast and endotheliocyte differentiation. When either SBP-DPSCs or bone chips obtained in vitro were transplanted into immunocompromised rats, they generated a tissue structure with an integral blood supply similar to that of human adult bone; in fact, a large number of HLA-1(+) vessels were observed either within the bone or surrounding it in a periosteal layer. This study provides direct evidence to suggest that osteogenesis and angiogenesis mediated by human SBP-DPSCs may be regulated by distinct mechanisms, leading to the organization of adult bone tissue after stem cell transplantion. ispartof: Cell Death and Differentiation vol:14 issue:6 pages:1162-1171 ispartof: location:England status: published

It is not known whether cells derived from stem cells retain their differentiation and morpho-functional properties after long-term cryopreservation. This information is of importance to evaluate their potential for long-term storage with a view to subsequent use in therapy. Here, we describe the morpho-functional properties of dental pulp stem cells (SBP-DPSCs), and of their differentiated osteoblasts, recovered after long-term cryopreservation. After storage for 2 years, we found that stem cells are still capable of differentiation, and that their differentiated cytotypes proliferate and produce woven bone tissue. In addition, cells still express all their respective surface antigens, confirming cellular integrity. In particular, SBP-DPSCs differentiated into pre-osteoblasts, showing diffuse positivity for ALP, BAP, RUNX-2, and calcein. Recovered osteoblasts expressed bone-specific markers and were easily recognizable ultrastructurally, with no alterations observed at this level. In addition, after in vivo transplantation, woven bone converted into a 3D lamellar bone type. Therefore, dental pulp stem cells and their osteoblast-derived cells can be long-term cryopreserved and may prove to be attractive for clinical applications.

Objective: Emdogain (EMD) is a protein extract purified from porcine enamel and has been introduced in clinical practice to obtain periodontal regeneration. EMD is composed mainly of amelogenins (90%), while the remaining 10% is composed of non-amelogenin enamel matrix proteins such as enamelins, tuftelin, amelin and ameloblastin. Enamel matrix proteins seem to be involved in root formation. EMD has been reported to promote proliferation, migration, adhesion and differentiation of cells associated with healing periodontal tissues in vivo. Design: How this protein acts on osteoblasts is poorly understood. We therefore attempted to address this question by using a microarray technique to identify genes that are differently regulated in osteoblasts exposed to enamel matrix proteins. Results: By using DNA microarrays containing 20 000 genes, we identified several upregulated and downregulated genes in the osteoblast-like cell line (MG-63) cultured with enamel matrix proteins (Emd). The differentially expressed genes cover a broad range of functional activities: (i) signaling transduction, (ii) transcription, (iii) translation, (iv) cell cycle regulation, proliferation and apoptosis, (v) immune system, (vi) vesicular transport and lysosome activity, and (vii) cytoskeleton, cell adhesion and extracellular matrix production. Conclusions: The data reported are the first genome-wide scan of the effect of enamel matrix proteins on osteoblast-like cells. These results can contribute to our understanding of the molecular mechanisms of bone regeneration and as a model for comparing other materials with similar clinical effects.

Stem cells were obtained from deciduous dental pulp of healthy subjects, aged 6-10 years. This stem cell population was cultured, expanded, and specifically selected, detecting using a FACsorter, c-kit, CD34, and STRO-1 antigen expression. Then, c-kit+/ CD34+/STRO-1+cells were replaced in the culture medium added of 20% FBS, leading to osteoblast differentiation. In fact, these cells, after a week, showed a large positivity for CD44, osteocalcin, and RUNX-2 markers. To achieve an adipocytic differentiation, cells, after sorting, were challenged with dexamethason 10-8mM in the same culture medium. To obtain myotube fusion, sorted cells were co-cultured in ATCC medium with mouse myogenic C2C12 cells and, after a week, human stem cell nuclei were found to be able to fuse, forming myotubes. Differentiated osteoblasts, as assessed by a large positivity to several specific antibodies, after 30 days of culture and already in vitro, started to secrete an extracellular mineralized matrix, which, 2 weeks later, built a considerable number of 3D woven bone samples, which showed a strong positivity to alkaline phosphatase (ALP), alizarin red, calcein, other than to specific antibodies. These bone samples, after in vivo transplantation into immunosuppressed rats, were remodeled in a lamellar bone containing entrapped osteocytes. Therefore, this study provides strong evidence that human deciduous dental pulp is an approachable "niche" of stromal stem cells, and that it is an ideal source of osteoblasts, as well as of mineralized tissue, ready for bone regeneration, transplantation, and tissue-based clinical therapies. © 2005 Wiley-Liss, Inc.

Distraction osteogenesis (DO) is a mechanotransduction process capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Several variables are implicated in DO: magnitude of mechanical strain, distraction rate, and type of distracted bone. The combination of these factors acts on different types of cells inducing apoptosis, cell proliferation, and differentiation. The elucidation of the molecular mechanisms has important clinical implications because it may facilitate the use of recombinant proteins or gene therapy to accelerate bone regeneration. Previous reports have analyzed several molecules such as extracellular matrix proteins, cytokines, bone morphogenetic proteins, hormones, and angiogenic factors. Moreover, a single protein can have multifunctional roles. With such a huge number of mechanical, histologic, cellular, and molecular variables, there is the need to have a cell culture model that enables the selection of the effect of a specific strength to a single cell type at different time points and with or without cytokines. The analysis of the genetic profiling of a cell line cultured on an equibiaxial stretch device has such characteristic. Because there is a recruitment and commitment of preosteoblastic cells during bone lengthening and no previous report has focus on them, the authors used a preosteoblast MC3T3-E1 cell line to detect the early molecular effects of distraction on mesenchymal cells. By using DNA microarrays containing 15,000 clones, the authors identified several genes the expression of which was significantly up- or down-regulated. The differentially expressed genes cover a broad range of biological processes: cell growth, metabolism, morphogenesis, cell communication, response to stress, and cell death. The data reported are the first genetic portrait of stretched preosteoblasts. They can be relevant in the better understanding of the molecular mechanism of DO and as a model for comparing the effect of distraction on different cell lines and primary cultures, rate and strength of distraction, and with or without cytokines.

Ascorbic acid (AA), also known as Vitamin C, is a cofactor required for the function of several hydroxylases. It is not synthesised in humans and has to be provided by diet. Its absence is responsible for scurvy, a condition related to the defective synthesis of collagen by the reduced function of prolylhydroxylase. AA is also a risk factor for periodontal disease. Recently, it has been shown that AA induces embryonic stem cells to differentiate into osteoblasts. The mechanism by which AA sustains pre-osteoblast proliferation and commitment is mediated through the synthesis of collagen type I, interaction with alpha2- and beta1-integrin, activation of the mitogen-activated protein kinase pathway, and phosphorylation of osteoblast-specific transcription factors. However, the multifunctional role of AA is not fully elucidated. MC3T3-E1 mouse calvaria-derived cell line is a well-defined in vitro model of pre-osteoblast differentiation, and AA is essential for the proliferation and differentiation of MC3T3-E1. By using DNA micro-arrays containing 15,000 genes, we identified several genes in MC3T3-E1 cultured with AA for 24h whose expression was significantly up or downregulated. The differentially expressed genes covered a broad range of functional activities: (1) cell growth; (2) metabolism; (3) morphogenesis; (4) cell death; (5) cell communication. The data reported are, to our knowledge, the first genetic portrait of early stage stimulation of pre-osteoblasts by AA, and may be relevant to better understand the molecular mechanism of pre-osteoblast proliferation and commitment. Elucidation of the molecular mechanism has important clinical implications because it may facilitate the correct use of AA to accelerate bone regeneration.

Calcium sulfate (CaS) is an highly biocompatible material that has the characteristic of being one of the simplest as well as one of the synthetic bone-like graft with the longest clinical history, spanning more than 100 years. Solidified or crystallized CaS is very osteogenic in vivo. As the surface CaS dissolves in body fluid, the calcium ions form calcium phosphate that reprecipitates on the surface forming an osteoblast "friendly" environment. How this "friendly" environment alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identified genes that are differently regulated in osteoblasts exposed to CaS. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) cultured with CaS (Surgiplaster, Classimplant, Roma, Italy) several genes that expression was significantly upregulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) lysosomal enzymes production, (c) cell cycle regulation, (d) and signaling transduction. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of CaS effects. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

To harvest bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. The authors describe their preliminary results in this new field with its potential application in craniomaxillofacial surgery. Dental pulp was gently extracted from 34 human permanent teeth (all third molars) of patients 19 to 37 years of age. After they were digested, the cells were selected using a cytometer for c-kit, STRO-1, CD34, CD45, and then for CD44 and RUNX-2. This study, made on a considerable number of cases, provided evidence that dental pulp is extremely rich in stem cells, which were c-kit+/CD34+/STRO-1+/ CD45j, capable of differentiation toward several stromal-derived differentiated cells and mainly osteoblasts. These findings, supported by the large number of cases, are of great interest for tissue regeneration, tissue-based clinical therapies, and transplantation.