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X-ray powder diffraction patterns from hMSC aggregates. (a) hMSCs 6 months, (b) hMSCs 5 months, and (c) hMSCs 4 months. (d) The diffraction pattern of a synthetic highly crystalline hydroxyapatite sample is reported as comparison. The diffraction peaks of hydroxyapatite are marked with the (211,112,300) indexes. The vertical axis indicates intensity in arbitrary units.
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Grafts of tissue-engineered bone represent a promising alternative in the treatment of large and small bone defects. Current approaches are often badly tolerated by patients because of invasiveness, ethical problems, culture, and possibility of infection. Autologous grafts have been indicated as a solution to such problems. Because of tissue availa...
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Context 1
... To confirm bone formation and characterize the mineral matrix structure, 4-, 5-, and 6-month osteoinduced hMSC aggregates were subjected to XRD and FTIR spec- troscopy to evaluate the presence and structure of carbonate hydroxyapatite, the bone principal constituent. [6][7][8][9] In Figure 6, powder X-ray diffraction patterns of hMSC ag- gregates obtained after 4, 5, and 6 months of induction are shown. The samples were ground before the analyses. ...
Context 2
... typical of this mineral phase. 7,8 No additional re- flections due to other phases or hydroxyapatite precursors, such as octacalcium phosphate, were observed. In general, in- creasing the osteoinduction time, diffraction patterns showed more evident reflections, although their definition was always very low when compared with that of synthetic HA (Fig. 6). The intensity of the reflection at 25.88, the one corresponding to the diffraction of the (002) plane, clearly increases its intensity going from the 4-to the 6-month sample. In the diffraction pattern of the hMSC 6 sample, a weak reflection (004) is also ...
Citations
... The present study also showed that osteocalcin, an osteogenic differentiation marker, was over-expressed in aggregated cells on PAMPS/PDMAAm DN gels after 14 days of culture. Previous investigators showed that three-dimensional aggregation contributes to osteogenic differentiation of mesenchymal stem cells, as demonstrated by the expression of numerous standard osteoblastic markers, including osteocalcin [11]. Long-term aggregation may induce osteogenic differentiation of C3H10T1/2 cells; therefore, we did not attempt to prolong cultures for more than 14 days. ...
Recently, several animal studies have found that spontaneous hyaline cartilage regeneration can be induced in vivo within a large osteochondral defect by implanting a synthetic double-network (DN) hydrogel, which is composed of poly-(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) and poly-(N,N’-dimethyl acrylamide) (PDMAAm), at the bottom of the defect. However, the effect of hydrogel on hyaline cartilage regeneration remains unexplained. The purpose of this study was to investigate the chondrogenic differentiation of C3H10T1/2 cells on PAMPS/PDMAAm DN gel.
C3H10T1/2 cells of 1.0 × 105 were cultured on PAMPS/PDMAAm DN gel in polystyrene tissue culture dishes or directly on polystyrene tissue culture dishes. We compared cultured cells on PAMPS/PDMAAm DN gel with those on polystyrene dishes by morphology using phase-contrast microscopy, mRNA expression of aggrecan, type I collagen, type II collagen, Sox 9 and osteocalcin using real-time RT-PCR, and local expression of type II collagen using immunocytochemistry.
C3H10T1/2 cells cultured on the PAMPS/PDMAAm DN gels formed focal adhesions, aggregated rapidly and developed into large nodules within 7 days, while the cells cultured on the polystyrene surface did not. The mRNA levels of aggrecan, type I collagen, type II collagen, Sox 9 and osteocalcin were significantly greater in cells cultured on the PAMPS/PDMAAm DN gel than in those cultured on polystyrene dishes. In addition, C3H10T1/2 cells cultured on PAMPS/PDMAAm DN gel expressed more type II collagen at the protein level when compared with cells cultured on polystyrene dishes.
The present study showed that PAMPS/PDMAAm DN gel enhanced chondrogenesis of C3H10T1/2 cells, which are functionally similar to mesenchymal stem cells. This suggests that mesenchymal stem cells from the bone marrow contribute to spontaneous hyaline cartilage regeneration in vivo in large osteochondral defects after implantation of PAMPS/PDMAAm DN gels.
... DPSCs at fifth passage in culture (P5-DPSC) were plated at a density of 4610 4 cells/cm 2 and osteo-induced in a medium already described by Ferro et al. [21,30] . One month old osteodifferentiated DPSC were used for characterization analyses except in X-Ray Diffraction (XRD) and Fourier Transform Infra Red (FTIR) spectroscopy. ...
... Then DPSC at P5 were exposed for 1 month to differentiation conditions to induce osteoblastic phenotype373839, during this period DPSC changed their morphology developing an asymmetric shape with an enlarged end (Fig. 2A). Subsequently, cells were analyzed by rt-PCR to evidence the expression of the osteospecific markers: Cbfa-1; alkaline phosphatase (Alp); Bone Morphonetic Proteins (BMPs), (Coll-I); osteocalcin (Osc), osteonectin (Osn) and osteopontin (Osp), distal less homeobox (Dlx-5), and moreover Dentin SialoPhosphoProtein (DSPP) [30] . Semiquantitative rt-PCR on osteo-differentiated over undifferentiated DPSC showed up-regulation of Cbfa-I type II (1.5360.069) ...
... Instead DSPP m-RNAs were not expressed by undifferentiated or differentiated cells (Data not shown) [39]. Osteocalcin (Fig. 2C), a late osteoblastic marker, as well as the early osteoblastic differentiation marker, osteopontin (Fig. 2D) were expressed after induction as shown by IF [30]. We used XRD and FTIR to evaluate the presence of hydroxyapatite (HA) in three month osteo-differentiated DPSC aggregates. ...
Adult stem cells have been proposed as an alternative to embryonic stem cells to study multilineage differentiation in vitro and to use in therapy. Current culture media for isolation and expansion of adult stem cells require the use of large amounts of animal sera, but animal-derived culture reagents give rise to some questions due to the real possibility of infections and severe immune reactions. For these reasons a clinical grade substitute to animal sera is needed. We tested the isolation, proliferation, morphology, stemness related marker expression, and osteoblastic differentiation potential of Dental Pulp Stem Cells (DPSC) in a chemically defined medium containing a low percentage of human serum, 1.25%, in comparison to a medium containing 10% Fetal Bovine Serum (FBS). DPSCs cultured in presence of our isolation/proliferation medium added with low HS percentage were obtained without immune-selection methods and showed high uniformity in the expression of stem cell markers, proliferated at higher rate, and demonstrated comparable osteoblastic potential with respect to DPSCs cultured in 10% FBS. In this study we demonstrated that a chemically defined medium added with low HS percentage, derived from autologous and heterologous sources, could be a valid substitute to FBS-containing media and should be helpful for adult stem cells clinical application.
... XRD diffraction pattern shows the typical diffraction peaks of HA around 25.8° and 32° of 2θ and sodium chloride at 27.3°, 31.6° and 45.4° of 2θ (Fig. 2E) [21], [30]. The FTIR spectrum (Fig. 2F) shows absorption peaks characteristic of phosphate group (ν3 = 1037 cm-1; ν4 = 603 cm−1 and 564 cm−1) and carbonate group (ν3 = 1421 cm-1) [21], [30]. ...
... Instead DSPP m-RNAs were not expressed by undifferentiated or differentiated cells (Data not shown) [39]. Osteocalcin (Fig. 2C), a late osteoblastic marker, as well as the early osteoblastic differentiation marker, osteopontin (Fig. 2D) were expressed after induction as shown by IF [30]. We used XRD and FTIR to evaluate the presence of hydroxyapatite (HA) in three month osteo-differentiated DPSC aggregates. ...
... Then DPSC at P5 were exposed for 1 month to differentiation conditions to induce osteoblastic phenotype [37]–[39], during this period DPSC changed their morphology developing an asymmetric shape with an enlarged end (Fig. 2A). Subsequently, cells were analyzed by rt-PCR to evidence the expression of the osteo-specific markers: Cbfa-1; alkaline phosphatase (Alp); Bone Morphonetic Proteins (BMPs), (Coll-I); osteocalcin (Osc), osteonectin (Osn) and osteopontin (Osp), distal less homeobox (Dlx-5), and moreover Dentin SialoPhosphoProtein (DSPP) [30]. Semiquantitative rt-PCR on osteo-differentiated over undifferentiated DPSC showed up-regulation of Cbfa-I type II (1.53±0.069) ...
Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES) pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A). Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.
... (14,19) In addition, MSCs are capable of producing adenosine because they all express CD73. (28) Recently, adenosine also has been shown to help the localization of human MSCs and promote their differentiation into hepatocyte-like cells, (29) suggesting that adenosine also has a role in the differentiation of MSCs into several possible lineages. Thus we have explored how adenosine receptors are modulated as MSCs are induced to differentiate into osteoblasts and adipocytes and how these processes may be influenced by adenosine. ...
Osteoblasts and adipocytes differentiate from a common precursor cell, the mesenchymal stem cell (MSC). Adenosine is known to signal via four adenosine receptor subtypes, and significantly, recent findings indicate that these may play a role in MSC differentiation. We therefore investigated adenosine receptor expression and activation during the differentiation of MSCs to osteoblasts and adipocytes. The A(2B) R was dominant in MSCs, and its expression and activity were transiently upregulated at early stages of osteoblastic differentiation. Both activation and overexpression of A(2B) R induced the expression of osteoblast-related genes [Runx2 and alkaline phosphatase (ALP)], as well as ALP activity, and stimulation increased osteoblast mineralization. The expression of A(2A) R was upregulated during later stages of osteoblastic differentiation, when its activation stimulated ALP activity. Differentiation of MSCs to adipocytes was accompanied by significant increases in A(1) R and A(2A) R expression, and their activation was associated with increased adipogenesis. Enhanced A(2A) R expression was sufficient to promote expression of adipocyte-related genes (PPARγ and C/EBPα), and its activation resulted in increased adipocytic differentiation and lipid accumulation. In contrast, the A(1) R was involved mainly in lipogenic activity of adipocytes rather than in their differentiation. These results show that adenosine receptors are differentially expressed and involved in lineage-specific differentiation of MSCs. We conclude, therefore, that fruitful strategies for treating diseases associated with an imbalance in the differentiation and function of these lineages should include targeting adenosine receptor signal pathways. Specifically, these research avenues will be useful in preventing or treating conditions with insufficient bone or excessive adipocyte formation.
The application of mesenchymal stromal cells (MSCs) from different sources, including bone marrow (BM, bmMSCs), adipose tissue (atMSCs), and human term placenta (hPSCs) has been proposed for various clinical purposes. Accumulated evidence suggests that the activity of the different MSCs is indirect and associated with paracrine release of pro-regenerative and anti-inflammatory factors. A major limitation of bmMSCs-based treatment for autologous application is the limited yield of cells harvested from BM and the invasiveness of the procedure. Similar effects of autologous and allogeneic MSCs isolated from various other tissues were reported. The easily available fresh human placenta seems to represent a preferred source for harvesting abundant numbers of human hPSCs for allogenic use. Cells derived from the neonate tissues of the placenta (f-hPSC) can undergo extended expansion with a low risk of senescence. The low expression of HLA class I and II on f-hPSCs reduces the risk of rejection in allogeneic or xenogeneic applications in normal immunocompetent hosts. The main advantage of hPSCs-based therapies seems to lie in the secretion of a wide range of pro-regenerative and anti-inflammatory factors. This renders hPSCs as a very competent cell for therapy in humans or animal models. This review summarizes the therapeutic potential of allogeneic applications of f-hPSCs, with reference to their indirect pro-regenerative and anti-inflammatory effects and discusses clinical feasibility studies.
Bone defects are common and are associated with a significant burden of disease threatening the health of many people around the globe. Since the last decade, data obtained from case studies have demonstrated that 20% of patients who experience an osteoporotic hip break are unable to endure the primary year after medical treatment. Many similar cases suggest that there is a huge requirement for better treatment of unhealthy and broken bones. Human bone comprises of about 70% of calcium phosphate (CaP) mineral, therefore CaPs are possible alternative materials to fix a broken bone. CaP is broadly utilized for bone fixation because of its bioactive properties like osteoinductivity, osteoconductivity, and biodegradability. Therefore, examination of these properties and the impact of their different affecting factors are crucial for balancing CaP during the fabrication procedure to maximally fulfill required clinical prerequisites. The aim of this chapter is to highlight the systems behind the CaP-assisted bone development in the initial phase, specifically as a biocompatible bone graft substitute. In this study, the latest developments in the biological properties of CaP biomaterials, including hydroxyapatite (HA), tricalcium phosphate (TCP), and biphasic CaP (BCP), have been summarized. Moreover, recent advances on how their properties are altered by different factors are reviewed. Finally, perspectives regarding future developments of CaP materials are provided.
Since hydroxyapatite and bone morphogenetic protein-2 (BMP-2) can regulate chondrogenesis and osteogenesis, their individual and combined effects on endochondral ossification within human bone marrow-derived stem cell (hMSC) aggregates were investigated. Hydroxyapatite was presented in the form of mineral-coated hydroxyapatite microparticles (MCM) capable of controlled BMP-2 delivery. Aggregates were treated with varied BMP-2 concentrations supplemented in the media and loaded onto MCM to examine the influence of BMP-2 amount and spatial presentation on regulating chondrogenesis and osteogenesis. MCM alone induced GAG and type II collagen production by week 5 for two of three donors, and BMP-2 may have accelerated MCM-induced chondrogenesis. ALP activity and calcium content of cells-only aggregates suggest that the BMP-2-induced osteogenic response may be concentration-dependent. Treatment with MCM and BMP-2 resulted in chondrogenesis as early as week 2, which may have promoted additional mineralization by week 5, suggesting the induction of endochondral ossification. Released BMP-2 had similar if not higher levels of bioactivity compared to that of exogenous BMP-2 with regard to chondrogenesis and osteogenesis. In addition to providing localized and sustained BMP-2 delivery, MCM incorporation within aggregates yields a self-sustaining system that may be injected or implanted more rapidly to heal bone defects through endochondral ossification without extended in vitro culture.
Context: Early development of many organs shows many morphological and molecular similarities (teeth, lung, hair, kidney and etc.). Fundamental questions in organogenesis are related to the identification of a simplified model which is able to mimic the molecular mechanisms involved in pattern organization and cell fate determination as well. Evidence Acquisition: It is widely accepted that cells behave more natively if cultured in three-dimensional conditions. Advances in 3D non-destructive, non-invasive analysis methods and improvements in the multi-scale techniques and bioreactors to obtain test and culture 3D cell aggregates have been made. On the other hand, even if 3D aggregate culture methods are able to recapitulate in vitro the cell-extracellular matrix interactions properly observed in vivo, and the synthetic/natural matrix and scaffolds have biochemical and mechanical properties, in order to mimic the native extracellular matrix, both of these systems possesses some limitations and some methodological improvements are needed. Results: The processes by which re-aggregated adult single cell types or adult and embryonic explanted tissues are able to recapitulate embryogenesis in vitro, when cultured in adhesion or embedded in 3D gels, is not surprising and is clearly under the control of a reminiscent cellular memory which recapitulates early developmental stages. Conclusions: Our underlying hypothesis is that recapitulating the three-dimensional early embryonic structure, in order to reproduce
Dental pulp stem cells (DPSC) have been proposed as an alternative to pluripotent stem cells to study multilineage differentiation in vitro and for therapeutic application. Standard culture media for isolation and expansion of stem cells includes animal sera or animal-derived matrix components (e.g., Matrigel(®)). However, animal-derived reagents raise significant concerns with respect to the translational ability of these cells due to the possibility of infection and/or severe immune reaction. For these reasons clinical grade substitutes to animal components are needed in order for stem cells to reach their full therapeutic potential. In this chapter we detail a method for isolation and proliferation of DPSC in a chemically defined medium containing a low percentage of human serum. We demonstrate that in this defined culture medium a 1.25 % human serum component sufficiently replaces fetal bovine serum. This method allows for isolation of a morphologically and phenotypically uniform population of DPSCs from dental pulp tissue. DPSCs represent a rapidly proliferating cell population that readily differentiates into the osteoblastic, neuronal, myocytic, and hepatocytic lineages. This multilineage capacity of these DPSCs suggests that they may have a more broad therapeutic application than lineage-restricted adult stem cell populations such as mesenchymal stem cells. Further the culture protocol presented here makes these cells more amenable to human application than current expansion techniques for other pluripotent stem cells (embryonic stem cell lines or induced pluripotent stem cells).
A modeling approach to identify sets of culture conditions to promote homogeneous growth of cells in perfusion bioreactors equipped with regular shape scaffolds is proposed. We identify cases in which dynamic culturing is necessary using a zero-dimensional mass transport and reaction model. Then, based on the three-dimensional (3-D) rendering of the flow field inside the bioreactor, we identify regions where cellular growth may become critical; finally, using a 1-D mass transport and reaction model, we calculate the minimal perfusion flow necessary to maintain the cellular growth rate above a target threshold. The developed approach is used to analyze culturing conditions inside an indirect perfusion bioreactor equipped with a lattice scaffold. Regions where the perfusion flow is inadequate to foster cellular growth at the desired rate are identified. The perfusion flow required to maintain the target growth rate inside the bioreactor is calculated. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3131–3144, 2013
