Histochemical localization of putative stem cells in irreversible pulpitis.

OBJECTIVES: Our study aimed to observe the distribution of putative stem cells in irreversible pulpitis and to investigate the expression of specific molecules. SUBJECTS AND METHODS: Extracted third molar teeth were collected and divided into two groups: the normal pulp group and inflamed pulp group. Real-time PCR was applied to detect the expression of several embryonic and dentinogenic genes. The expression of mesenchymal cell markers (STRO-1, CD90, and CD146) and stromal cell-derived factor 1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) proteins was examined by immunohistochemical analysis. RESULTS: The expression levels of most embryonic and dentinogenic genes were not statistically different between the two groups. Immunohistochemical analysis revealed that in inflamed pulp, cells with positive expression for STRO-1, CD90, and CD146 mainly resided in two specific niches, both adjacent to inflammatory sites: one in the pulp core and another in the odontoblast layer. SDF-1α- and CXCR4-positive cells were significantly correlated with STRO-1-positive cells. Double immunofluorescence analysis indicated that STRO-1-positive cells overlapped with SDF-1α- and CXCR4-positive cells near the inflammatory site. CONCLUSIONS: This study gave a direct observation of putative stem cells distributed in irreversible pulpitis and implied a role of SDF-1α/CXCR4 signaling in stem cell-based therapies for reparative dentinogenesis.

A critical appraisal of humanized alternatives to fetal bovine serum for clinical applications of umbilical cord derived mesenchymal stromal cells.

OBJECTIVE: The study is aimed to verify the possibility of using humanized alternatives to fetal bovine serum (FBS) such as umbilical cord blood plasma (CBP) and AB+ plasma to support the long-term growth of mesenchymal stromal cells (MSCs) derived from the umbilical cord. We hypothesized that umbilical CBP would be a potential substitute to FBS, especially for small scale autologous clinical transplantations. METHODS: The MSCs were cultured for six consecutive passages to evaluate xeno-free media's ability to support long-term growth. Cell proliferation rates, colony-forming-unit (CFU) efficiency and population doublings of expanded MSCs, were investigated. Ex vivo expanded MSCs were further characterized using flow cytometry and quantitative PCR. The impact of cryopreservation and composition of cryomedium on phenotype, viability of MSC was also assessed. RESULTS: Our results on cell proliferation, colony-forming unit efficiency suggested that the expansion of the cells was successfully carried out in media supplemented with humanized alternatives. MSCs showed lower CFU counts in FBS (~ 25) than humanized alternatives (~ 35). The gene expression analysis revealed that transcripts showed significant differential expression by two to three folds in the FBS group compared with MSCs grown in medium with humanized alternatives (p < 0.05). In addition, MSCs grown in a medium with FBS had more osteogenic activity, a signature of unwanted differentiation. The majority of ex vivo expanded MSCs at early and late passages expressed CD44+, CD73+, CD105+, CD90+, and CD166+ in all the experimental groups tested (~ 90%). In contrast to the other MSC surface markers, expression levels of STRO-1+ (~ 21-10%) and TNAP+ (~ 29-11%) decreased with the increase in passage number for MSCs cultured in a FBS-supplemented medium (p < 0.05). CONCLUSION: Our results established that CBP supported culture of umbilical cord tissue-derived MSCs and is a safer Xeno free replacement to FBS. The use of CBP also enables the storage of umbilical cord tissue derived MSCs in patient-specific conditions to minimize adverse events if cells are delivered directly to the patient.

Passage-dependent expression of STRO-1 in human gingival mesenchymal stem cells.

The expression of STRO-1, the essential mesenchymal stem cell marker, was found to decrease with advancing passages in few tissues. Because STRO-1 was identified and isolated from human gingiva, we were interested to know its status after a few passages. Human gingival mesenchymal stem cells (HGMSCs) were isolated from human gingiva. Flow cytometry was carried out with STRO-1, mesenchymal stem cell (MSC) positive marker CD73, and negative marker CD34/CD45. Samples were also subjected to CD90 and STRO-1 immunofluorescence staining. Gene expression was carried out for transcription factors OCT-4, NANOG, and NESTIN. The results showed a gradual decrease in STRO-1 and transcription factor expression with an increase in passage numbers. MSC positive marker CD73 was consistently expressed in all the passages. Negative markers were absent in all the passages. We conclude that STRO-1 may be a useful marker to isolate undifferentiated (potent) mesenchymal cells from gingiva.

Lapine periodontal ligament stem cells for musculoskeletal research in preclinical animal trials.

BACKGROUND: Human periodontal ligament stem cells (hPDLSCs) have been shown to be a reliable source of mesenchymal stem cells (MSCs). On the other hand, rabbits have been commonly used in preclinical trials for musculoskeletal research. However, there is a lack of sufficient data on using rabbit periodontal ligament stem cells (rPDLSCs) for regenerative dentistry. This study, for the first time, comprehensively compared rPDLSCs against hPDLSCs in terms of clonogenicity, growth potential, multi-differential capacity and surface antigens. METHODS: Periodontal ligament (PDL) was obtained from the rabbit and human teeth. rPDL and hPDL cells were isolated from PDL using enzymatic digestion method. After culturing for 2 weeks, the cells were first analyzed microscopically. STRO-1+CD146+ PDLSCs were then sorted from PDL cells by fluorescence-activated cell sorting (FACS) followed by examination of CD34, CD45, CD90, vimentin and desmin markers. The cells were also evaluated by immunohistocytochemical and multi-differentiation potential tests. The clonogenicity and growth of PDL cells were analyzed by Independent T test and 2-way repeated measures ANOVA respectively. RESULTS: rPDL cells were broader and less elongated as compared to hPDL cells. STRO-1+CD146+ hPDLSCs were isolated from hPDL cells but not from the rPDL cells. Therefore, heterogeneous population of rabbit and human PDL cells were subsequently used for latter comparative studies. FACS analysis and immunohistocytochemistry revealed that rPDL cells were partially positive for STRO-1 as compared to hPDL cells. Furthermore, both rPDL cells and hPDL cells were positive for CD146, CD90, vimentin, and desmin, while negative for CD34 and CD45. No difference in clonogenicity between rPDL and hPDL cells was found (p > 0.05). The proliferative potential of rPDL cells displayed significantly slower growth as compared to hPDL cells (p < 0.05). Osteogenic, adipogenic, and chondrogenic differentiation potential was comparatively less in rPDL cells than that of hPDL cells, but the neurogenic differential potential was similar. CONCLUSION: Although rPDL cells manifested variable differences in expression of stem cell markers and multi-differential potential as compared to hPDL cells, they demonstrated the attributes of stemness. Further studies are also required to validate if the regenerative potential of rPDL cells is similar to rPDLSCs.

The Mesenchymal Precursor Cell Marker Antibody STRO-1 Binds to Cell Surface Heat Shock Cognate 70.

Since its discovery more than 25 years ago, the STRO-1 antibody has played a fundamental role in defining the hierarchical nature of mesenchymal precursor cells (MPC) and their progeny. STRO-1 antibody binding remains a hallmark of immature pluripotent MPC. Despite the significance of STRO-1 in the MPC field, the identity of the antigen has remained elusive. Using a combination of two-dimensional gel electrophoresis, coupled with Western blotting and Tandem mass spectroscopy, we have identified the STRO-1 antigen as heat shock cognate 70 (HSC70;HSPA8). STRO-1 binds to immune-precipitated HSC70 and siRNA-mediated knock down of HSPA8 reduced STRO-1 binding. STRO-1 surface binding does not correlate with HSC70 expression and sequestration of cholesterol reduces STRO-1 surface binding, suggesting that the plasma membrane lipid composition may be an important determinant in the presentation of HSC70 on the cell surface. HSC70 is present on the surface of STRO-1+ but not STRO-1- cell lines as assessed by cell surface biotinylation and recombinant HSC70 blocks STRO-1 binding to the cell surface. The STRO-1 epitope on HSC70 was mapped to the ATPase domain using a series of deletion mutants in combination with peptide arrays. Deletion of the first four amino acids of the consensus epitope negated STRO-1 binding. Notably, in addition to HSC70, STRO-1 cross-reacts with heat shock protein 70 (HSP70), however all the clonogenic cell activity is restricted to the STRO-1BRIGHT /HSP70- fraction. These results provide important insight into the properties that define multipotent MPC and provide the impetus to explore the role of cell surface HSC70 in MPC biology. Stem Cells 2017;35:940-951.

STRO-1 confers myofibroblast transdifferentiation in fibroblasts derived from oral submucous fibrosis.

BACKGROUND: STRO-1 is a mesenchymal cell marker present on all clonogenic stromal precursors. Current evidence has indicated that the pathogenesis of fibrotic changes may be mediated by stemness properties. The aim of this study was to investigate the role of STRO-1 in areca quid chewing-associated oral submucous fibrosis (OSF). METHODS: Thirty OSF specimens and ten normal buccal mucosae were examined by immunohistochemistry. The activity of STRO-1 from fibroblasts cultured from normal buccal mucosa (BMFs) and OSF (OSFFs) were measureed and the effect of arecoline, a major areca nut alkaloid, on STRO-1 in BMFs was evaluated. Compared the activities between sorted STRO-1+ cells and STRO-1- cells from OSFF were measured by collagen gel contraction, migration, invasion abilities, and the expression of α-smooth muscle actin (α-SMA) and pro-α1 (I) chain of type I collagen. RESULTS: Our results first showed that the expression of STRO-1 was more evident in areca quid chewing-associated OSF than normal buccal mucosa tissues (P < .05). Arecoline dose-dependently activated the level of STRO-1 in BMFs (P < .05). The relative expression of STRO-1 was significantly higher in OSFFs compared with BMFs (P < .05). In addition, the sorted STRO-1+ cells from OSFFs exhibited higher collagen gel contraction, migration, and invasion abilities as well as elevated expression of α-SMA and pro-α1 (I) chain of type I collagen than the negative subset (P < .05). CONCLUSION: These findings suggested that the stemness marker STRO-1 may be a crucial factor in the pathogenesis of areca quid chewing-associated OSF.

Role of Stro1+/CD44+ stem cells in myometrial physiology and uterine remodeling during pregnancy.

Regulation of myometrial functions during pregnancy has been considered the result of the integration of endocrine and mechanical signals. Nevertheless, uterine regeneration is poorly understood, and the cellular source within the gravid uterus is largely unexplored.In this study, we isolated and quantified the myometrial stem cells (MSC) population from pregnant female Eker rat uteri, by using Stro1/CD44 surface markers. We demonstrated that prior parity significantly increased the percentage of Stro1+/CD44+ MSC because of injured tissue response. Interestingly, we established that Stro1+/CD44+ MSC respond efficiently to physiological cues when they were treated in vitro under different dose-dependent pregnant rat serum.Previous studies reveal strong regulatory links between O2 availability and stem cell function. Based on these premises, cell proliferation assays showed that isolated Stro1+/CD44+ MSC possess a higher proliferative rate under hypoxic versus normoxic conditions. We also detected a total of 37 upregulated and 44 downregulated hypoxia-related genes, which were differentially expressed in Stro1+/CD44+ MSC, providing an alternative approach to infer into complex molecular mechanisms such as energy metabolism, inflammatory response, uterine expansion, and/or remodeling.Since these cells preferentially grow under low oxygen conditions, we propose that the increase of the rat uterus during pregnancy involves myometrial oxygen consumption, thereby enhancing MSC proliferation. Moreover, pregnancy-induced mechanical stretching results in hypoxic conditions, ultimately creating an environment that promotes stem cell proliferation and further uterine enlargement, which is essential for a successful pregnancy. In summary, all of these data support that rat Stro1+/CD44+ MSC contribute to uterine enlargement during pregnancy.

Establishing criteria for human mesenchymal stem cell potency.

This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age- and sex-matched donors. Adherence to plastic was not indicative of potency, yet capacity for long-term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high-growth capacity or low-growth capacity. Using this grouping strategy, high-growth capacity MSCs were smaller in size, had greater colony-forming efficiency, and had longer telomeres. Cell-surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high-growth capacity and low-growth capacity MSCs, whereas STRO-1 and platelet-derived growth factor receptor alpha were preferentially expressed on high-growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST-1 and DERMO-1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high-growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low-growth capacity MSCs when assessed for ectopic bone-forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application.

Cell and matrix modulation in prenatal and postnatal equine growth cartilage, zones of Ranvier and articular cartilage.

Formation of synovial joints includes phenotypic changes of the chondrocytes and the organisation of their extracellular matrix is regulated by different factors and signalling pathways. Increased knowledge of the normal processes involved in joint development may be used to identify similar regulatory mechanisms during pathological conditions in the joint. Samples of the distal radius were collected from prenatal and postnatal equine growth plates, zones of Ranvier and articular cartilage with the aim of identifying Notch signalling components and cells with stem cell-like characteristics and to follow changes in matrix protein localisation during joint development. The localisation of the Notch signalling components Notch1, Delta4, Hes1, Notch dysregulating protein epidermal growth factor-like domain 7 (EGFL7), the stem cell-indicating factor Stro-1 and the matrix molecules cartilage oligomeric matrix protein (COMP), fibromodulin, matrilin-1 and chondroadherin were studied using immunohistochemistry. Spatial changes in protein localisations during cartilage maturation were observed for Notch signalling components and matrix molecules, with increased pericellular localisation indicating new synthesis and involvement of these proteins in the formation of the joint. However, it was not possible to characterise the phenotype of the chondrocytes based on their surrounding matrix during normal chondrogenesis. The zone of Ranvier was identified in all horses and characterised as an area expressing Stro-1, EGFL7 and chondroadherin with an absence of COMP and Notch signalling. Stro-1 was also present in cells close to the perichondrium, in the articular cartilage and in the fetal resting zone, indicating stem cell-like characteristics of these cells. The presence of stem cells in the articular cartilage will be of importance for the repair of damaged cartilage. Perivascular chondrocytes and hypertrophic cells of the cartilage bone interface displayed positive staining for EGFL7, which is a novel finding and suggests a role of EGFL7 in the vascular infiltration of growth cartilage.

Co-culture of STRO1 + human gingival mesenchymal stem cells and human umbilical vein endothelial cells in 3D spheroids: enhanced in vitro osteogenic and angiogenic capacities.

Stem cell spheroid is a promising graft substitute for bone tissue engineering. Spheroids obtained by 3D culture of STRO1+ Gingival Mesenchymal Stem Cells (sGMSCs) (sGMSC spheroids, GS) seldom express angiogenic factors, limiting their angiogenic differentiation in vivo. This study introduced a novel stem cell spheroid with osteogenic and angiogenic potential through 3D co-culture of sGMSCs and Human Umbilical Vein Endothelial Cells (HUVECs) (sGMSC/HUVEC spheroids, GHS). GHS with varying seeding ratios of sGMSCs to HUVECs (GHR) were developed. Cell fusion within the GHS system was observed via immunofluorescence. Calcein-AM/PI staining and chemiluminescence assay indicated cellular viability within the GHS. Furthermore, osteogenic and angiogenic markers, including ALP, OCN, RUNX2, CD31, and VEGFA, were quantified and compared with the control group comprising solely of sGMSCs (GS). Incorporating HUVECs into GHS extended cell viability and stability, initiated the expression of angiogenic factors CD31 and VEGFA, and upregulated the expression of osteogenic factors ALP, OCN, and RUNX2, especially when GHS with a GHR of 1:1. Taken together, GHS, derived from the 3D co-culture of sGMSCs and HUVECs, enhanced osteogenic and angiogenic capacities in vitro, extending the application of cell therapy in bone tissue engineering.

Immunohistochemical characterization of stem cell, vascular, neural, and differentiation markers in the apical papilla and dental pulp of human teeth at various stages of root development.

This study aimed to evaluate the expression of several differentiation markers in the apical papilla (AP) and dental pulp (DP) of human permanent teeth. Twenty young human teeth were extracted and classified according to three Moorrees tooth development stages: initial root formation (Ri), root length ½ (R1/2), and root length complete (Rc). Immunohistochemical assays were performed using STRO-1, VEGF Receptor-2, Neurofilament heavy (NFH), and Nestin antibodies and analyzed under light microscopy. Decalcified, formalin fixed paraffin embedded tooth sections stained with hematoxylin and eosin showed an apical cell rich zone between the DP and AP. The AP revealed fewer vascular and cellular components than the DP. STRO-1 was expressed on vascular and neuronal elements beneath the odontoblast (OB) and in the sub-odontoblastic (SOB) zone, and VEGFR-2 positive cells were observed in the endothelium, arterioles, and blood vessels. Neuroepithelial stem cell protein (Nestin) was highly expressed in differentiated odontoblasts in the predentin odontotoblast and odontoblast cell processes. Neurofilament heavy (NFH) was expressed in mature axons throughout the DP. STRO-1 and VEGFR-2 microvascular expression was higher at the stages Ri and R1/2 while STRO-1 and NFH expression showed strong spatial distribution of Rc neuronal elements as compared to Ri and R1/2. Differentiated OB and SOB cells showed Nestin expression, indicating a reservoir of newly differentiated odontoblast-like cells.

Fibronectin Conformations after Electrodeposition onto 316L Stainless Steel Substrates Enhanced Early-Stage Osteoblasts' Adhesion but Affected Their Behavior.

The implantation of metallic orthopedic prostheses is increasingly common due to an aging population and accidents. There is a real societal need to implement new metal implants that combine durability, good mechanical properties, excellent biocompatibility, as well as affordable costs. Since the functionalization of low-cost 316L stainless steel substrates through the successive electrodeposition of a polypyrrole film (PPy) and a calcium phosphate deposit doped with silicon was previously carried out by our labs, we have also developed a bio-functional coating by electrodepositing or oxidating of fibronectin (Fn) coating. Fn is an extracellular matrix glycoprotein involved in cell adhesion and differentiation. Impacts of either electrodeposition or oxidation on the structure and functionality of Fn were first studied. Thus, electrodeposition is the technique that permits the highest deposition of fibronectin, compared to adsorption or oxidation. Furthermore, electrodeposition seems to strongly modify Fn conformation by the formation of intermingled long fibers, resulting in changes to the accessibility of the molecular probes tested (antibodies directed against Fn whole molecule and Fn cell-binding domain). Then, the effects of either electrodeposited Fn or oxidized Fn were validated by the resulting pre-osteoblast behavior. Electrodeposition reduced pre-osteoblasts' ability to remodel Fn coating on supports because of a partial modification of Fn conformation, which reduced accessibility to the cell-binding domain. Electrodeposited Fn also diminished α5 integrin secretion and clustering along the plasma membrane. However, the N-terminal extremity of Fn was not modified by electrodeposition as demonstrated by Staphylococcus aureus attachment after 3 h of culture on a specific domain localized in this region. Moreover, the number of pre-osteoblasts remains stable after 3 h culture on either adsorbed, oxidized, or electrodeposited Fn deposits. In contrast, mitochondrial activity and cell proliferation were significantly higher on adsorbed Fn compared with electrodeposited Fn after 48 h culture. Hence, electro-deposited Fn seems more favorable to pre-osteoblast early-stage behavior than during a longer culture of 24 h and 48 h. The electrodeposition of matrix proteins could be improved to maintain their bio-activity and to develop this promising, fast technique to bio-functionalize metallic implants.

STRO-1 positive cell expansion during osteogenic differentiation: A comparative study of three mesenchymal stem cell types of dental origin.

OBJECTIVE: Although the osteogenic differentiation potential of mesenchymal stem cells of dental origin is well established, the roles of different marker proteins in this process remain to be clarified. Our aim was to compare the cellular and molecular changes, focusing in particular on mesenchymal stem cell markers, during in vitro osteogenesis in three dental stem cell types: dental follicle stem cells (DFSCs), periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs). DESIGN: Human DFSCs, PDLSCs and DPSCs were isolated, cultured and their osteogenic differentiation was induced for 3 weeks. Mineralization was assessed by von Kossa staining and calcium concentration measurements. The expression of mesenchymal and osteogenic markers was studied by immunocytochemistry and qPCR techniques. Alkaline phosphatase (ALP) activity and the frequency of STRO-1 positive cells were also quantified. RESULTS: The three cultures all showed abundant mineralization, with high calcium content by day 21. The expression of vimentin and nestin was sustained after osteogenic induction. The osteogenic medium induced a considerable elevation of STRO-1 positive cells. By day 7, the ALP mRNA level had increased more than 100-fold in DFSCs, PDLSCs, and DPSCs. Quantitative PCR results indicated dissimilarities of osteoblastic marker levels in the three dental stem cell cultures. CONCLUSIONS: DFSCs, PDLSCs and DPSCs have similar functional osteogenic differentiation capacities although their expressional profiles of key osteogenic markers show considerable variations. The STRO-1 positive cell fraction expands during osteogenic differentiation while vimentin and nestin expression remain high. For identification of stemness, functional studies rather than marker expressions are needed.

The local concentration of Ca2+ correlates with BMP7 expression and osseointegration in patients with total hip arthroplasty.

BACKGROUND: A successful osseointegration of total hip arthroplasty (THA) relies on the interplay of implant surface and bone marrow microenvironment. This study was undertaken to investigate the impact of perioperative biochemical molecules (Ca2+, Mg2+, Zn2+, VD, PTH) on the bone marrow osteogenetic factors (BMP2, BMP7, Stro-1+ cells) in the metaphyseal region of the femoral head, and further on the bone mineral density (BMD) of Gruen R3. METHODS: Bone marrow aspirates were obtained from the discarded metaphysis region of the femoral head in 51 patients with THA. Flow cytometry was used to measure the Stro-1+ expressing cells. ELISA was used to measure the concentrations of bone morphologic proteins (BMP2 and BMP7) and the content of TRACP5b in serum. TRAP staining was used to detect the osteoclast activity in the hip joint. The perioperative concentrations of the biochemical molecules above were measured by radioimmunoassay. The BMD of Gruen zone R3 was examined at 6 months after THA, using dual-energy X-ray absorptiometry (DEXA). RESULTS: Our data demonstrated that the concentration of Ca2+ was positively correlated with BMP7 expression, and with the postoperative BMD of Gruen zone R3. However, the concentration of Mg2+ had little impact on the R3 BMD, although it was negatively correlated with the expression of BMP7. Osteoclast activity in hip joint tissue of patients with femoral neck fractures was increased. Compared with the patients before THA, the levels of TRACP5b in serum of patients after THA were decreased. The data also suggested that the other biochemical molecules, such as Zn2+, VD, and PTH, were not significantly correlated with any bone marrow osteogenetic factors (BMP2, BMP7, Stro-1+ cells). The postoperative R3 BMD of patients of different gender and age had no significant difference. CONCLUSIONS: These results indicate the local concentration of Ca2+ may be an indicator for the prognosis of THA patients.

Effects of concentrated growth factors (CGF) on the quality of the induced membrane in Masquelet's technique - An experimental study in rabbits.

AIMS: The aim of the study was to test if the addition of CGF to the Masquelet technique contributes to the quality of the membrane formed surrounding the polymethylmethacrylate (PMMA), in terms of inflammation, proliferation and vasculazition in the Masquelet technique in the early and late phases in a rabbit model. MATERIALS AND METHODS: A critical bone defect of 15 mm was created in radius diaphysis, leaving 3 cm of intact bone to the joint. To mimic the Masquelet technique and to increase stability, a 6-hole 1.5 mm plate with two screws was applied, although it was initialy stable because of the inherently fixed ulna and radius both proximally and distally in the rabbits. Group 1 and Group 3, were soleley treated with the Masquelet technique as control groups, and were sacrificed at 3 and 6 weeks, respectively. Group 2 and Group 4, were treated with the Masquelet technique + CGF prepared from the rabbit blood groups, and were sacrificed at 3 and 6 weeks, respectively. The groups were compared histopathologically and immunohistochemically, in respect of the means of thickness of the membrane and ratio of elastic fibers, membrane vascularization (CD31), inflammation (MAC387), proliferation (Ki67), and presence of stem cells (STRO-1). RESULTS: Thickness of the membrane and CD31 values were significantly higher in Group 4 than Group 3 (p = 0.004 for both). MAC387 was statistically significantly higher in Group 2 compared to Group 1 and Group 4 compared to Group 3 (p = 0.04 for both). Ki67 was significantly higher in Group 2 compared to Group 1 and Group 4 compared to Group 3 (p = 0.05 and p = 0.006, respectively). Proliferation in the membrane was statistically significantly higher in Group 2 compared to Group 1 (p = 0.05). Likewise, the proliferation index of Group 4 was statistically significantly higher than Group 3 (p = 0.06). STRO-1 was significantly higher in Group 2 compared to Group 1 (p = 0036). CONCLUSION: The addition of CGF to the Masquelet technique contributes to the quality of the membrane formed, in respect of inducing inflammation and proliferation, maintaining vascularization on large diaphyseal bone defects, and increasing the number of stem cells.

Stem Cells From the Apical Papilla (SCAP) as a Tool for Endogenous Tissue Regeneration.

Stem cells extracted from developing tissues possibly exhibit not only unique but also superior traits against their developed counterparts. Indeed, stem cells from the apical papilla (SCAP); a unique group of dental stem cells related to developing roots have been shown to be a promising tool for regenerative endodontic procedures and regeneration in general. Studies have characterized the phenotypic traits as well as other regenerative potentials of these cells. Specific sub-populations have been highlighted as well as their neurogenic and angiogenic properties. Nevertheless, in light of the previously discussed features and potential applications of SCAP, there is still much to understand and a lot of information to unravel. The current review will discuss the role of specific markers for detection of different functional populations of SCAP; including CD146 and STRO-1, as well as their true multilineage differentiation potential. In particular, the role of the secretome in association with paracrine signaling in inflammatory microenvironments is also tackled. Additionally, the role of SCAP both in vitro and in vivo during regenerative approaches and in response to different growth factors and biologic scaffolds is highlighted. Finally, this review will shed light on current knowledge regarding the clinical translational potential of SCAP and elucidate possible areas for future research applications.

Role of STRO-1 sorting of porcine dental germ stem cells in dental stem cell-mediated bone tissue engineering.

Stem cells of dental origin emerged as a new source for the regeneration of tissues with advantages mainly including non-invasive collection procedures and lack of ethical contraversies with their harvest or use. In this study, porcine TGSCs (pTGSCs) were isolated from mandibular third molar tooth germs of 6-month-old domestic pigs. This is the first study that reports the isolation and characterization of TGSCs from porcine third molars and their differentiation depending on STRO-1 expression. PTGSCs were sorted according to their STRO-1 expression as STRO-1(+) and STRO-1(-). Sorted and unsorted heterogenous cells (US) were characterized by their osteogenic, chondrogenic and adipogenic differentiation capabilities. STRO-1(+) cells exhibited a higher proliferation rate owing to their clonogenic properties. All three groups of cells were found differentiated into osteogenic lineage as shown by ALP activity, calcium deposition assay, detection of osteogenic mRNAs and, proteins and mineralization staining. According to differentiation analysis, STRO-1(+) cells did not show a better performance for osteogenesis compared to STRO-1(-) and US cells. This might indicate that STRO-1(+) cells might require a heterogeneous population of cells including STRO-1(-) in their niche to perform their proposed role in osteogenesis.

Human dental pulp stem cells expressing STRO-1, c-kit and CD34 markers in peripheral nerve regeneration.

Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The application of stem cells able to differentiate in Schwann cell-like cells in vitro and in vivo, could represent an attractive therapeutic approach for the treatment of nerve injuries. Further, stem cells sources sharing the same embryological origin as Schwann cells might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal subpopulation of human STRO-1+ /c-Kit+ /CD34+ DPSCs, expressing P75NTR , nestin and SOX-10, to differentiate into Schwann cell-like cells in vitro and to promote axonal regeneration in vivo, which led to functional recovery as measured by sustained gait improvement, in animal rat model of peripheral nerve injury. Transplanted human dental pulp stem cells (hDPSCs) engrafted into sciatic nerve defect, as revealed by the positive staining against human nuclei, showed the expression of typical Schwann cells markers, S100b and, noteworthy, a significant number of myelinated axons was detected. Moreover, hDPSCs promoted axonal regeneration from proximal to distal stumps 1 month after transplantation. This study demonstrates that STRO-1+ /c-Kit+ /CD34+ hDPSCs, associated with neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues. Copyright © 2016 John Wiley & Sons, Ltd.

Influence of STRO-1 selection on osteogenic potential of human tooth germ derived mesenchymal stem cells.

Mesenchymal stem cells derived from the human tooth germ (hTGSCs) are a heterogeneous cell population that can differentiate into osteogenic, neurogenic, and adipogenic lineages. The aim of this study was to compare the osteogenic differentiation capacity of STRO-1 positive (STRO-1+) hTGSCs and unsorted heterogeneous hTGSCs and to establish if STRO-1+ cells are more committed to osteogenic differentiation. HTGSCs were isolated from impacted third molar tooth germ tissues of adolescents, and a subpopulation of STRO-1+ hTGSCs was obtained by fluorescence-activated cell sorting. STRO-1+, STRO-1 negative (STRO-1-), and unsorted cells were cultured in osteogenic and standard culture media to compare their capacity to differentiate towards osteoblastic lineage. Cells were tested for proliferation rates, alkaline phosphatase activity, and amounts of accumulated calcium. Gene expression levels of the RUNX2, osteocalcin, and osteonectin genes were analyzed with real time PCR. Mineralization and osteogenic protein expression were examined by using von Kossa staining and confocal microscopy. Our results indicated that osteogenically induced cell populations showed greater mineralization capacity than non-induced cells. However, expression levels of early and late osteogenic markers were not significantly different between STRO-1+ and unsorted cells. In conclusion, the selection by STRO-1 expression does not yield cells with osteogenic capacity higher than that of the heterogeneous hTGSC population. Cell sorting using osteogenic markers other than STRO-1 might be beneficial in obtaining a more sensitive osteogenic sub-population from unsorted heterogenous hTGSCs.

Effects of fibroblast growth factor-2 on cell proliferation of cementoblasts.

BACKGROUND/PURPOSE: Fibroblast growth factor (FGF)-2 is known as a signaling molecule that induces tissue regeneration. Little is known about the effect of FGF-2 on cementoblasts for periodontal and periapical regeneration. The aim of this study was to investigate the effects of FGF-2 on murine immortalized cementoblast cell line (OCCM.30). MATERIALS AND METHODS: Cell growth and proliferation was judged by using alamar blue reduction assay. Flow cytometry analysis was used to evaluate Stro-1 positive cells expression with or without FGF-2. Western blot was used to evaluate the expression of phosphorylated serine-threonine kinase Akt (p-Akt) and extracellular signal-regulated protein kinase (p-ERK) in cementoblasts. RESULTS: FGF-2 was found to increase cell growth in a dose-dependent manner (P < 0.05). The concentration of 10 ng/mL FGF-2 enhanced cell proliferation in a time-dependent manner (P < 0.05). In addition, 10 ng/mL FGF-2 significantly increased the number of Stro-1 positive cells in the first 24 hours (P < 0.05). Moreover, 10 ng/mL FGF-2 was found to upregulate p-Akt and p-ERK in a time-dependent manner (P < 0.05). CONCLUSION: Taken together, FGF-2 could increase cementoblast growth, proliferation, and Stro-1 positive cells. These enhancements are associated with the upregulation of p-Akt and p-ERK expression. The application of FGF-2 may provide benefit for periodontal and periapical regeneration during the early phase of wound healing.