Enhanced Osteogenesis Potential of MG-63 Cells through Sustained Delivery of VEGF via Liposomal Hydrogel.

The challenges of using VEGF to promote osteoblastic differentiation include a short half-life and a narrow therapeutic window. A carrier system combining hydrogel and liposomes may improve the therapeutic efficacy of VEGF for bone regeneration. This study aimed to investigate the effects of delivery of VEGF via liposomal hydrogel on the osteogenesis of MG-63 cells. Liposomal hydrogel scaffold was fabricated and then characterized in terms of the morphological and chemical properties using FESEM and FTIR. In 2.5D analysis, the MG-63 cells were cultured on liposomal hydrogel + VEGF as the test group. The osteogenic effects of VEGF were compared with the control groups, i.e., hydrogel without liposomes + VEGF, osteogenic medium (OM) supplemented with a bolus of VEGF, and OM without VEGF. Cell morphology, viability, and differentiation and mineralization potential were investigated using FESEM, MTT assay, ALP activity, and Alizarin red staining. The characterization of scaffold showed no significant differences in the morphological and chemical properties between hydrogel with and without liposomes (p > 0.05). The final 2.5D culture demonstrated that cell proliferation, differentiation, and mineralization were significantly enhanced in the liposomal hydrogel + VEGF group compared with the control groups (p < 0.05). In conclusion, liposomal hydrogel can be used to deliver VEGF in a sustained manner in order to enhance the osteogenesis of MG-63 cells.

Osteogenic Potential and Bioactive Profiles of Piper sarmentosum Ethanolic Extract-Treated Stem Cells.

Piper sarmentosum is a well-known traditional herbal plant in various diseases treatments. Multiple scientific studies have also reported various biological activities exhibited by the plant's extract, such as antimicrobial, anticarcinogenic and antihyperglycemic activities, and, in addition, a bone protective effect in ovariectomized rats has been reported. However, no known Piper sarmentosum extract is involved in osteoblast differentiation using stem cells. Our study aims to identify the potential of P. sarmentosum ethanolic extract to induce osteoblast differentiation of human peripheral blood stem cells. Prior to the assay, the proliferation ability of the cells was observed for 14 days and the presence of hematopoietic stem cells in the culture was determined by the expression of SLAMF1 and CD34 genes. During the differentiation assay, the cells were treated with P. sarmentosum ethanolic extract for 14 days. Osteoblast differentiation was examined using an (alkaline phosphatase) ALP assay, by monitoring the expression of osteogenic gene markers and by von Kossa staining. The untreated cells served as the negative control, while cells treated with 50 µg/mL ascorbic acid and 10 mM ß-glycerophosphate acted as the positive control. Finally, the determination of the compound profile was performed using a gas chromatography-mass spectrometry (GC-MS) analysis. The isolated cells were able to proliferate for 14 days during the proliferation assay. The expression of hematopoietic stem cell markers was also upregulated during the 14 days assay. Following the differentiation induction, the ALP activity exhibited a significant increase (p < 0.05) from day 3 of the differentiation assay. A molecular analysis also showed that the osteogenic markers ALP, RUNX2, OPN and OCN were upregulated compared to the positive control. The presence of mineralized cells with a brownish-stained morphology was observed, indicating the mineralization process increased in a time-dependent manner regardless of the concentration used. There were 54 compounds observed in the GC-MS analysis, including ß-asarones, carvacrol and phytol, which have been shown to possess osteoinductive capacities. Our results demonstrate that the ethanolic extract of P. sarmentosum can induce osteoblast differentiation of peripheral blood stem cells. The extract contains potent compounds which can potentially induce the differentiation of bone cells, i.e., osteoblasts.

Label-free Quantitative Proteomic Analysis of Ascorbic Acid-induced Differentially Expressed Osteoblast-related Proteins in Dental Pulp Stem Cells from Deciduous and Permanent Teeth.

BACKGROUND: Proteomic is capable of elucidating complex biological systems through protein expression, function, and interaction under a particular condition. OBJECTIVE: This study aimed to determine the potential of ascorbic acid alone in inducing differentially expressed osteoblast-related proteins in dental stem cells via the liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) approach. METHODS: The cells were isolated from deciduous (SHED) and permanent teeth (DPSC) and induced with 10 µg/mL of ascorbic acid. Bone mineralisation and osteoblast gene expression were determined using von Kossa staining and reverse transcriptase-polymerase chain reaction. The label-free protein samples were harvested on days 7 and 21, followed by protein identification and quantification using LC-MS/MS. Based on the similar protein expressed throughout treatment and controls for SHED and DPSC, overall biological processes followed by osteoblast-related protein abundance were determined using the PANTHER database. STRING database was performed to determine differentially expressed proteins as candidates for SHED and DPSC during osteoblast development. RESULTS: Both cells indicated brownish mineral stain and expression of osteoblast-related genes on day 21. Overall, a total of 700 proteins were similar among all treatments on days 7 and 21, with 482 proteins appearing in the PANTHER database. Osteoblast-related protein abundance indicated 31 and 14 proteins related to SHED and DPSC, respectively. Further analysis by the STRING database identified only 22 and 11 proteins from the respective group. Differential expressed analysis of similar proteins from these two groups revealed ACTN4 and ACTN1 as proteins involved in both SHED and DPSC. In addition, three (PSMD11/RPN11, PLS3, and CLIC1) and one (SYNCRIP) protein were differentially expressed specifically for SHED and DPSC, respectively. CONCLUSION: Proteome differential expression showed that ascorbic acid alone could induce osteoblastrelated proteins in SHED and DPSC and generate specific differentially expressed protein markers.

Gene expression profiles for in vitro human stem cell differentiation into osteoblasts and osteoclasts: a systematic review.

Background: There have been promising results published regarding the potential of stem cells in regenerative medicine. However, the vast variety of choices of techniques and the lack of a standard approach to analyse human osteoblast and osteoclast differentiation may reduce the utility of stem cells as a tool in medical applications. Therefore, this review aims to systematically evaluate the findings based on stem cell differentiation to define a standard gene expression profile approach. Methods: This review was performed following the PRISMA guidelines. A systematic search of the study was conducted by retrieving articles from the electronic databases PubMed and Web of Science to identify articles focussed on gene expression and approaches for osteoblast and osteoclast differentiation. Results: Six articles were included in this review; there were original articles of in vitro human stem cell differentiation into osteoblasts and osteoclasts that involved gene expression profiling. Quantitative polymerase chain reaction (qPCR) was the most used technique for gene expression to detect differentiated human osteoblasts and osteoclasts. A total of 16 genes were found to be related to differentiating osteoblast and osteoclast differentiation. Conclusion: Qualitative information of gene expression provided by qPCR could become a standard technique to analyse the differentiation of human stem cells into osteoblasts and osteoclasts rather than evaluating relative gene expression. RUNX2 and CTSK could be applied to detect osteoblasts and osteoclasts, respectively, while RANKL could be applied to detect both osteoblasts and osteoclasts. This review provides future researchers with a central source of relevant information on the vast variety of gene expression approaches in analysing the differentiation of human osteoblast and osteoclast cells. In addition, these findings should enable researchers to conduct accurately and efficiently studies involving isolated human stem cell differentiation into osteoblasts and osteoclasts.

Comparison between hydroxyapatite and polycaprolactone in inducing osteogenic differentiation and augmenting maxillary bone regeneration in rats.

Background: The selection of appropriate scaffold plays an important role in ensuring the success of bone regeneration. The use of scaffolds with different materials and their effect on the osteogenic performance of cells is not well studied and this can affect the selection of suitable scaffolds for transplantation. Hence, this study aimed to investigate the comparative ability of two different synthetic scaffolds, mainly hydroxyapatite (HA) and polycaprolactone (PCL) scaffolds in promoting in vitro and in vivo bone regeneration. Method: In vitro cell viability, morphology, and alkaline phosphatase (ALP) activity of MC3T3-E1 cells on HA and PCL scaffolds were determined in comparison to the accepted model outlined for two-dimensional systems. An in vivo study involving the transplantation of MC3T3-E1 cells with scaffolds into an artificial bone defect of 4 mm length and 1.5 mm depth in the rat's left maxilla was conducted. Three-dimensional analysis using micro-computed tomography (micro-CT), hematoxylin and eosin (H&E), and immunohistochemistry analyses evaluation were performed after six weeks of transplantation. Results: MC3T3-E1 cells on the HA scaffold showed the highest cell viability. The cell viability on both scaffolds decreased after 14 days of culture, which reflects the dominant occurrence of osteoblast differentiation. An early sign of osteoblast differentiation can be detected on the PCL scaffold. However, cells on the HA scaffold showed more prominent results with intense mineralized nodules and significantly (p < 0.05) high levels of ALP activity with prolonged osteoblast induction. Micro-CT and H&E analyses confirmed the in vitro results with bone formation were significantly (p < 0.05) greater in HA scaffold and was supported by IHC analysis which confirmed stronger expression of osteogenic markers ALP and osteocalcin. Conclusion: Different scaffold materials of HA and PCL might have influenced the bone regeneration ability of MC3T3-E1. Regardless, in vitro and in vivo bone regeneration was better in the HA scaffold which indicates its great potential for application in bone regeneration.

Multivariate versus univariate spectrum analysis of dentine sialophosphoprotein (DSPP) for root resorption prediction: a clinical trial.

BACKGROUND: A force applied during orthodontic treatment induces inflammation to root area and lead to root resorption known as orthodontically induced inflammatory root resorption (OIIRR). Dentine sialophosphoprotein (DSPP) is one of the most abundant non-collagenous proteins in dentine that was released into gingival crevicular fluid (GCF) during OIIRR. The aim of this research is to compare DSPP detection using the univariate and multivariate analysis in predicting classification level of root resorption. METHODS: The subjects for this study consisted of 30 patients in 3 group classified as normal, mild, and severe groups of OIIRR. The GCF samples were taken from upper permanent central incisors in the normal and mild group while the upper primary second molars in the severe group. The DSPP qualitative detection limit was determined by analyzing the whole absorption spectrum utilizing multivariate analysis embedded with different preprocessing method. The multivariate analysis represents the multi-wavelength spectrum while univariate analyzes the absorption of a single wavelength. RESULTS: The results showed that the multivariate analysis technique using partial least square-discriminate analysis (PLS-DA) with the preprocess method has successfully improved in classification prediction for the normal and mild group at 0.88 percent accuracy. The multivariate using PLS-DA algorithm with Mean Center preprocess method was able to predict normal and mild tooth resorption classes better than the univariate analysis. The classification parameters have improved in term of the specificity, precision and accuracy. CONCLUSION: Therefore, the multivariate analysis helps to predict an early detection of tooth resorption complimenting the sensitivity of the univariate analysis. Trial registration NCT05077878 (14/10/2021).

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection.

AIMS: We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on Prostate- Specific Antigen (PSA). BACKGROUND: High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based Enzyme-Linked Lectin Sorbent Assays (ELISAs), as they can mask specific binding signals and cause false-positive results. METHODS: In this study, we constructed an ELISA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELISA. RESULTS: The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELISA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. CONCLUSION: The ELISA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.

Label-free quantitative proteomic analysis of gingival crevicular fluid to identify potential early markers for root resorption.

BACKGROUND: Orthodontically-induced root resorption is an iatrogenic effect and it cannot be examined regularly due to the harmful effects of sequential doses of radiation with more frequent radiography. This study aims to compare protein abundance (PA) of pre-treatment and during orthodontic treatment for root resorption and to determine potential early markers for root resorption. METHODS: Ten subjects (n = 10) who had upper and lower fixed appliances (MBT, 3 M Unitek, 0.022″ × 0.028″) were recruited for this study. Human gingival crevicular fluid (GCF) was obtained using periopaper strips at pre-treatment (T0), 1 month (T1), 3 months (T3), and 6 months (T6) of orthodontic treatment. Periapical radiographs of the upper permanent central incisors were taken at T0 and T6 to measure the amount of root resorption. Identification of changes in PA was performed using liquid chromatography-tandem mass spectrometry. Student's t-test was then performed to determine the significance of the differences in protein abundance before and after orthodontic treatment. RESULTS: Our findings showed that all ten subjects had mild root resorption, with an average resorption length of 0.56 ± 0.30 mm. A total of 186 proteins were found to be commonly present at T0, T1, T3, and T6. There were significant changes in the abundance of 16 proteins (student's t-test, p ≤ 0.05). The increased PA of S100A9, immunoglobulin J chain, heat shock protein 1A, immunoglobulin heavy variable 4-34 and vitronectin at T1 suggested a response to stress that involved inflammation during the early phase of orthodontic treatment. On the other hand, the increased PA of thymidine phosphorylase at T3 suggested growth promotion and, angiogenic and chemotactic activities. CONCLUSIONS: The identified proteins can be potential early markers for root resorption based on the increase in their respective PA and predicted roles during the early phase of orthodontic treatment. Non-invasive detection of root resorption using protein markers as early as possible is extremely important as it can aid orthodontists in successful orthodontic treatment.

The effects of Piper sarmentosum aqueous extracts on zebrafish (Danio rerio) embryos and caudal fin tissue regeneration.

In Malaysia, Piper sarmentosum or 'kaduk' is commonly used in traditional medicines. However, its biological effects including in vivo embryonic toxicity and tissue regenerative properties are relatively unknown. The purpose of this study was to determine zebrafish (Danio rerio) embryo toxicities and caudal fin tissue regeneration in the presence of P. sarmentosum aqueous extracts. The phytochemical components and antioxidant activity of the extract were studied using GC-MS analysis and DPPH assay, respectively. Embryo toxicity tests involving survival, heartbeat, and morphological analyses were conducted to determine P. sarmentosum extract toxicity (0-60 µg/mL); concentrations of 0-400 µg/mL of the extract were used to study tissue regeneration in the zebrafish caudal fin. The extract contained several phytochemicals with antioxidant activity and exhibited DPPH scavenging activity (IC50 = 50.56 mg/mL). Embryo toxicity assays showed that a concentration of 60 µg/mL showed the highest rates of lethality regardless of exposure time. Slower embryogenesis was observed at 40 µg/mL, with non-viable embryos first detected at 50 µg/mL. Extracts showed significant differences (p < 0.01) for tissue regeneration at all concentrations when compared to non-treated samples. In conclusion, Piper sarmentosum extracts accelerated tissue regeneration, and extract concentrations at 60 µg/mL showed the highest toxicity levels for embryo viability.

Effects of the Sintering Process on Nacre-Derived Hydroxyapatite Scaffolds for Bone Engineering.

A hydroxyapatite scaffold is a suitable biomaterial for bone tissue engineering due to its chemical component which mimics native bone. Electronic states which present on the surface of hydroxyapatite have the potential to be used to promote the adsorption or transduction of biomolecules such as protein or DNA. This study aimed to compare the morphology and bioactivity of sinter and nonsinter marine-based hydroxyapatite scaffolds. Field emission scanning electron microscopy (FESEM) and micro-computed tomography (microCT) were used to characterize the morphology of both scaffolds. Scaffolds were co-cultured with 5 × 104/cm2 of MC3T3-E1 preosteoblast cells for 7, 14, and 21 days. FESEM was used to observe the cell morphology, and MTT and alkaline phosphatase (ALP) assays were conducted to determine the cell viability and differentiation capacity of cells on both scaffolds. Real-time polymerase chain reaction (rtPCR) was used to identify the expression of osteoblast markers. The sinter scaffold had a porous microstructure with the presence of interconnected pores as compared with the nonsinter scaffold. This sinter scaffold also significantly supported viability and differentiation of the MC3T3-E1 preosteoblast cells (p < 0.05). The marked expression of Col1α1 and osteocalcin (OCN) osteoblast markers were also observed after 14 days of incubation (p < 0.05). The sinter scaffold supported attachment, viability, and differentiation of preosteoblast cells. Hence, sinter hydroxyapatite scaffold from nacreous layer is a promising biomaterial for bone tissue engineering.

Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells.

BACKGROUND: Stem cells are normally isolated from dental pulps using the enzymatic digestion or the outgrowth method. However, the effects of the isolation method on the quality of the isolated stem cells are not studied in detail in murine models. The aim of this study was to compare the matrices secreted by osteoblast and chondrocytes differentiated from dental pulp stem cells isolated through different means. METHOD: DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG) or the enzymatic digestion (DPSC-ED) method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells' doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey's multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted. RESULTS: Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC-ED with the shortest doubling time was 5 × 102 cells/cm2 (11.49 ± 2.16 h) and 1 × 102 cells/cm2 (10.55 h ± 0.50), respectively. Chondrocytes differentiated from DPSC-ED produced  2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria. DISCUSSION: Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells with better characteristics than outgrowth.

Analyses of basal media and serum for in vitro expansion of suspension peripheral blood mononucleated stem cell.

Transplantation of stem cells requires a huge amount of cells, deeming the expansion of the cells in vitro necessary. The aim of this study is to define the optimal combination of basal medium and serum for the expansion of suspension peripheral blood mononucleated stem cells (PBMNSCs) without resulting in loss in the differentiation potential. Mononucleated cells were isolated from both mice and human peripheral blood samples through gradient centrifugation and expanded in α-MEM, RPMI, MEM or DMEM supplemented with either NBCS or FBS. The suspension cells were then differentiated to osteoblast. Our data suggested that α-MEM supplemented with 10 % (v/v) NBCS gives the highest fold increase after 14 days of culture for both mice and human PBMNSCs, which were ~1.51 and ~2.01 times, respectively. The suspension PBMNSCs in the respective medium were also able to maintain osteoblast differentiation potential as supported by the significant increase in ALP specific activity. The cells are also viable during the differentiated states when using this media. All these data strongly suggested that α-MEM supplemented with 10 % NBCS is the best media for the expansion of both mouse and human suspension PBMNSCs.

Enzyme activity profiles and ELISA analysis of biomarkers from human saliva and gingival crevicular fluid during orthodontic tooth movement using self-ligating brackets.

AIM: Profiles of orthodontic tooth movement biomarkers, i.e., Lactate Dehydrogenase (LDH), Aspartate Aminotransferase (AST), Tartrate-resistant Acid Phosphatase (TRAP) and Alkaline Phosphatase (ALP), using Self-ligating Brackets (SLBs) and possible relationships among their activities and total enzymes produced were determined. METHODS: Saliva and Gingival Crevicular Fluid (GCF) were collected from 19 subjects (n=19) before and during orthodontic treatment (5 weeks). The subjects were bonded with SLBs with 100 g or 150 g of orthodontic force. Enzyme assays, ELISA and tooth movement measurements were performed. RESULTS: A statistical analysis (paired t-test) showed that compared to baseline values, significant differences (p<0.05) were observed in the saliva levels of AST at week 5, the levels of TRAP at week 2, and the levels of ALP at weeks 1 to 5. In the GCF, LDH showed significant differences (p<0.05) at weeks 2, 3 and 4 (100 g) and at weeks 1, 2 and 3 (150 g). AST showed significant differences (p<0.05) at weeks 4 and 5 (100 g) and at weeks 3 and 4 (150 g), while TRAP exhibited a significant difference at week 5 (100 g). Pearson's correlation test revealed a weak correlation between enzyme activities and total enzymes. The use of 100 g compared to 150 g of force for tooth movement was not significant (p>0.05). CONCLUSION: Therefore, 100 g is recommended as a better force for patient comfort. AST, TRAP and ALP in the saliva and LDH, AST and TRAP in the GCF are potential biomarkers in orthodontic tooth movement using SLB systems.

Ascorbic acid induces osteoblast differentiation of human suspension mononuclear cells.

BACKGROUND AIMS: Suspension mononuclear cells (MNCs) can be differentiated into osteoblasts with the induction of ascorbic acid and ß-glycerophosphate. The aim of this study was to determine the ability of suspension MNCs to differentiate into osteoblasts using ascorbic acid only. METHODS: Suspension MNCs were obtained by a combination of gradient centrifugation and culture selection. Suspension MNCs were subjected to differentiation assay by culturing them inside proliferation medium supplemented with 10 µg/mL, 30 µg/mL, 50 µg/mL, 60 µg/mL, 90 µg/mL and 500 µg/mL of ascorbic acid. Proliferation medium supplemented with 50 µg/mL ascorbic acid and 10 mmol/L ß-glycerophosphate was used as a positive control for osteoblast induction, and proliferation medium without ascorbic acid was used as a negative control. Differentiation analysis was performed using alkaline phosphatase (ALP) assay, von Kossa staining and expression of osteoblast-related genes. RESULTS: With all concentrations of ascorbic acid used, there was a significant increase (P < 0.05) in ALP-specific activity and mineralized nodule formation throughout the differentiation course compared with negative control. Ascorbic acid was also able to activate the expression of osteopontin (SPP1), osteonectin (SPARC) and runt-related transcription factor 2 (RUNX2) messenger RNA in positive control and ascorbic acid-induced MNCs (30 µg/mL and 90 µg/mL) but not in negative control. CONCLUSIONS: Ascorbic acid alone was sufficient to induce osteoblast differentiation from suspension MNCs; 30-90 µg/mL of ascorbic acid was found to be the optimal concentration. Ascorbic acid can be used as a nutritional supplement for cellular therapy of bone-related disease.

Differentiation of dental pulp stem cells into neuron-like cells in serum-free medium.

Dental pulp tissue contains dental pulp stem cells (DPSCs). Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells) are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146(+) , Cd166(+) , and Cd31(-) in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

Crevicular Alkaline Phosphatase Activity and Rate of Tooth Movement of Female Orthodontic Subjects under Different Continuous Force Applications.

Purpose. This study is aimed to compare the effects of two different orthodontic forces on crevicular alkaline phosphatase activity, rate of tooth movement, and root resorption. Materials and Methods. Twelve female subjects of class II division 1 malocclusion participated. Maxillary canines with bonded fixed appliances acted as the tested teeth, while their antagonists with no appliances acted as the controls. Canine retraction was performed using nickel titanium coil spring that delivered forces of 100 gm or 150 gm to either side. Crevicular fluid was analyzed for ALP activity, and study models were casted to measure tooth movements. Root resorption was assessed using periapical radiographs before and after the force application. Results. ALP activity at the mesial sites peaked at week 1 for 150 gm group with significant differences when compared with the 100 gm group. Cumulative canine movements were significantly greater in the 150 gm force (2.10 ± 0.50 mm) than in the 100 gm force (1.57 ± 0.44 mm). No root resorption was in the maxillary canines after retraction. Conclusions. A force of 150 gm produced faster tooth movements and higher ALP activity compared with the 100 gm group and had no detrimental effects such as root resorption.

Proteomic analysis of saliva identifies potential biomarkers for orthodontic tooth movement.

Orthodontic treatment has been shown to induce inflammation, followed by bone remodelling in the periodontium. These processes trigger the secretion of various proteins and enzymes into the saliva. This study aims to identify salivary proteins that change in expression during orthodontic tooth movement. These differentially expressed proteins can potentially serve as protein biomarkers for the monitoring of orthodontic treatment and tooth movement. Whole saliva from three healthy female subjects were collected before force application using fixed appliance and at 14 days after 0.014'' Niti wire was applied. Salivary proteins were resolved using two-dimensional gel electrophoresis (2DE) over a pH range of 3-10, and the resulting proteome profiles were compared. Differentially expressed protein spots were then identified by MALDI-TOF/TOF tandem mass spectrometry. Nine proteins were found to be differentially expressed; however, only eight were identified by MALDI-TOF/TOF. Four of these proteins-Protein S100-A9, immunoglobulin J chain, Ig alpha-1 chain C region, and CRISP-3-have known roles in inflammation and bone resorption.

In vitro chondrogenesis transformation study of mouse dental pulp stem cells.

A major challenge in the application of mesenchymal stem cells in cartilage reconstruction is that whether the cells are able to differentiate into fully mature chondrocytes before grafting. The aim of this study was to isolate mouse dental pulp stem cells (DPSC) and differentiate them into chondrocytes. For this investigation, morphological, molecular, and biochemical analyses for differentiated cells were used. To induce the chondrocyte differentiation, DPSC were cultured in chondrogenic medium (Zen-Bio, Inc.). Based on morphological analyses using toluidine blue staining, proteoglycan products appear in DPSC after 21 days of chondrocyte induction. Biochemical analyses in differentiated group showed that alkaline phosphatase activity was significantly increased at day 14 as compared to control (P < 0.05). Cell viability analyses during the differentiation to chondrocytes also showed that these cells were viable during differentiation. However, after the 14th day of differentiation, there was a significant decrease (P < 0.05) in the viability proportion among differentiated cells as compared to the control cells. In RT-PCR molecular analyses, mouse DPSC expressed Cd146 and Cd166 which indicated that these cells belong to mesenchymal stem cells. Coll I and Coll II markers showed high expression after 14 and 21 days, respectively. In conclusion, this study showed that DPSC successfully differentiated into chondrocytes.

Characterization of mononucleated human peripheral blood cells.

Unspecialized cells that can renew themselves and give rise to multiple differentiated cell types are termed stem cells. The objective of this study was to characterize and investigate, through molecular and biochemical analyses, the stemness of cells derived from isolated mononucleated cells that originated from peripheral blood. The isolated mononucleated cells were separated according to their physical characteristics (adherent and suspension), after 4 to 7 days into a 14-day culturing period in complete medium. Our results revealed that adherent and suspension cells were positive for mesenchymal stem cell (MSC) and hematopoietic stem cell (HSC) markers, respectively. Differentiation of adherent cells into osteoblasts was associated with expression of the OPN gene and increasing ALP enzyme activity, while differentiation of suspension cells into osteoclasts was associated with expression of the TRAP gene and increasing TRAP enzyme activity. In conclusion, molecular and biochemical analyses showed that mononucleated cells consist of MSC (adherent) and HSC (suspension), and both cell types are able to differentiate into specialized cells from their respective lineage: osteoblast (MSC) and osteoclast (HSC).

Molecular markers of dental pulp tissue during orthodontic tooth movement: a pilot study.

Three specific orthodontic tooth movement genes, that is, FCRL1, HSPG2, and LAMB2 were detected at upper first premolar (with appliance) dental pulp tissue by using GeneFishing technique as compared to lower first premolar (without appliance). These three differentially expressed genes have the potential as molecular markers during orthodontic tooth movement by looking at molecular changes of pulp tissue.