Exploring copper (II) porphyrin complexes and their derivatives for electrochemical analysis and biological assessment in the study of breast cancer (MCF-7) cell lines.

In this study, several derivatives of tetraphenylporphyrin were synthesized, each with unique meso-substituent groups including phenyl, methoxyphenyl, butyloxyphenyl, octyloxyphenyl, and dectyloxyphenyl. Additionally, their corresponding copper complexes were prepared and thoroughly characterized. The structural confirmation of all compounds was established through CHN elemental analysis, mass spectrometry, and FT-IR spectroscopy. As the number of carbon atoms in the alkyl long-chain increased, a slight red shift in the electronic absorption band was observed, which was attributed to the electronic influence of the alkyl group. DFT analysis indicated that electron density predominantly localized on the porphyrin ring of both the metal free porphyrins and copper (II) porphyrin complexes, with relatively low electron density in the p orbital of the meso-aryl long-chain substituent group. EPR spectroscopy of the Copper (II) ion complexes revealed signals, indicating their paramagnetic properties. Additionally, the Copper (II) tetraphenylporphyrin (CuTPP) complexes displayed two reversible oxidation peaks at +0.97 V and +1.35 V, whereas other derivatives exhibited lower oxidation potentials. The cytotoxicity of these compounds against MCF-7 cell lines was assessed using MTT assay, revealing cytotoxic effects in all cases. Among them, Copper (II) tetrakis (4-methyloxyphenyl)porphyrin (CuTOMPP) demonstrated the highest potential, with an IC50 value of 32.07 µg/mL.

Effects of Oral Cavity Stem Cell Sources and Serum-Free Cell Culture on Hydrogel Encapsulation of Mesenchymal Stem Cells for Bone Regeneration: An In Vitro Investigation.

INTRODUCTION: To develop a stem cell delivery model and improve the safety of stem cell transplantation for bone regeneration, this study aimed to determine the effects of stem cell sources, serum-free cell culture, and hydrogel cell encapsulation on the growth and osteogenic differentiation of mesenchymal stem cells (MSCs) from the oral cavity. METHODS: The study groups were categorized according to stem cell sources into buccal fat pad adipose (hBFP-ADSCs) (Groups 1, 4, and 7), periodontal ligament (hPDLSCs) (Groups 2, 5, and 8), and dental pulp-derived stem cells (hDPSCs) (Groups 3, 6, and 9). MSCs from each source were isolated and expanded in three types of sera: fetal bovine serum (FBS) (Groups 1-3), human serum (HS) (Groups 4-6), and synthetic serum (SS) (StemPro™ MSC SFM) (Groups 7-9) for monolayer (m) and hydrogel cell encapsulation cultures (e). Following this, the morphology, expression of MSC cell surface antigens, growth, and osteogenic differentiation potential of the MSCs, and the expression of adhesion molecules were analyzed and compared. RESULTS: SS decreased variations in the morphology and expression levels of cell surface antigens of MSCs from three cell sources (Groups 7m-9m). The levels of osteoblastic differentiation of the hPDLSCs and hBFP-ADSCs were increased in SS (Groups 8m and 7m) and the cell encapsulation model (Groups 1e, 4e, 7e-9e), but the promoting effects of SS were decreased in a cell encapsulation model (Groups 7e-9e). The expression levels of the alpha v beta 3 (ITG-αVß3) and beta 1 (ITG-ß1) integrins in the encapsulated cells in FBS (Group 1e) were higher than those in the SS (Group 7e). CONCLUSIONS: Human PDLSCs and BFP-ADSCs were the optimum stem cell source for stem cell encapsulation by using nanohydroxyapatite-calcium carbonate microcapsule-chitosan/collagen hydrogel in serum-free conditions.

Effects of Calcium Carbonate Microcapsules and Nanohydroxyapatite on Properties of Thermosensitive Chitosan/Collagen Hydrogels.

Thermosensitive chitosan/collagen hydrogels are osteoconductive and injectable materials. In this study, we aimed to improve these properties by adjusting the ratio of nanohydroxyapatite particles to calcium carbonate microcapsules in a ß-glycerophosphate-crosslinked chitosan/collagen hydrogel. Two hydrogel systems with 2% and 5% nanohydroxyapatite particles were studied, each of which had varying microcapsule content (i.e., 0%, 1%, 2%, and 5%). Quercetin-incorporated calcium carbonate microcapsules were prepared. Calcium carbonate microcapsules and nanohydroxyapatite particles were then added to the hydrogel according to the composition of the studied system. The properties of the hydrogels, including cytotoxicity and biocompatibility, were investigated in mice. The calcium carbonate microcapsules were 2-6 µm in size, spherical, with rough and nanoporous surfaces, and thus exhibited a burst release of impregnated quercetin. The 5% nanohydroxyapatite system is a solid particulate gel that supports homogeneous distribution of microcapsules in the three-dimensional matrix of the hydrogels. Calcium carbonate microcapsules increased the mechanical and physical strength, viscoelasticity, and physical stability of the nanohydroxyapatite hydrogels while decreasing their porosity, swelling, and degradation rates. The calcium carbonate microcapsules-nanohydroxyapatite hydrogels were noncytotoxic and biocompatible. The properties of the hydrogel can be tailored by adjusting the ratio of calcium carbonate microcapsules to the nanohydroxyapatite particles. The 1% calcium carbonate microcapsules containing 5% nanohydroxyapatite particle-chitosan/collagen hydrogel exhibited mechanical and physical strength, permeability, and prolonged release profiles of quercetin, which were superior to those of the other studied systems and were optimal for promoting bone regeneration and delivering natural flavonoids.

Property and biological effects of the cuttlebone derived calcium phosphate particles, a potential bioactive bone substitute material.

Cuttlebone (CB) is a marine waste-derived biomaterial and a rich source of calcium carbonate for the biosynthesis of the calcium phosphate (CaP) particles. The current study aimed to synthesize CB derived biphasic calcium phosphate (CB-BCP) and investigate biological activity of the CB-CaP: hydroxyapatite (CB-HA), beta-tricalcium phosphate (CB-b-TCP) and biphasic 60:40 (w/w) HA/b-TCP (CB-BCP) with the human dental pulp stem cells (hDPSCs). The particles were synthesized using solid state reactions under mild condition and properties of the particles were compared with a commercial BCP as a reference material. Morphology, particle size, physicochemical properties, mineral contents, and the ion released patterns of the particles were examined. Then the particle/cell interaction, cell cytotoxicity and osteogenic property of the particles were investigated in the direct and indirect cell culture models. It was found that an average particles size of the CB-HA was 304.73 ± 4.19 nm, CB-b-TCP, 503.17 ± 23.06 nm and CB-BCP, 1394.67 ± 168.19 nm. The physicochemical characteristics of the CB-CaP were consistent with the HA, b-TCP and BCP. The highest level of calcium (Ca) was found in the mineral contents and the preincubated medium of the CB-BCP and traces of fluoride, magnesium, strontium, and zinc were identified in the CB-CaP. The cell cytotoxicity and osteogenic property of the particles were dose dependent. The particles adhered on cell surface and were internalized into the cell cytoplasm. The CB-BCP and CB-HA indirectly and directly promote osteoblastic differentiations of the hDPSCs in stronger levels than other groups. The CB-BCP and CB-HA were potential bioactive bone substitute materials.

Characteristics and biologic effects of thermosensitive quercetin-chitosan/collagen hydrogel on human periodontal ligament stem cells.

Thermosensitive hydrogels could function as scaffolds and delivery vehicle of natural flavonoids. The current study aimed to investigate effects of chitosan/collagen ratios on properties of thermosensitive beta-glycerophosphate (bGP) chitosan/collagen hydrogels as delivery vehicle of quercetin and then examined effects of quercetin-hydrogels on growth and cell viability of human periodontal ligament stem cells (hPDLSCs). Microstructure and physical, mechanical and antioxidant properties and quercetin release profiles of the hydrogels were investigated. Fourier transform infrared spectroscopy and X-ray powder diffraction analyses were performed to examine gelation process of the hydrogels. Antioxidant assays were conducted to measure antioxidant capacity of quercetin-hydrogels. It was found that bGP-chitosan/collagen hydrogels exhibited porous structures with interconnected pore architecture and could sustain quercetin release. Chitosan content improved well defined porous structure, increased porosity of the hydrogels and decreased releasing rate of quercetin from the hydrogels. The quercetin-bGP-2:1 (wt/wt) chitosan/collagen hydrogels exhibited antioxidant capacity and were able to promote growth of hPDLSCs in a dose dependent manner. In conclusion, the thermosensitive quercetin-bGP-2:1 (wt/wt) chitosan/collagen hydrogel demonstrated optimal properties of scaffolds for bone tissue engineering and sustained release of natural flavonoids. Incorporating quercetin in the chitosan/collagen hydrogel enhanced bioactive microenvironment that supported stem cell encapsulation.

Effects of beta-tricalcium phosphate nanoparticles on the properties of a thermosensitive chitosan/collagen hydrogel and controlled release of quercetin.

In the present study, an inorganic matrix of beta-tricalcium phosphate (bTCP) nanoparticles and quercetin was incorporated into an organic matrix of 2:1 (w/w) chitosan/collagen composite to fabricate thermosensitive bTCP-chitosan/collagen-quercetin hydrogels. A sol-gel transition of the hydrogels was stimulated by beta-glycerophosphate (bGP) and temperature changes at physiological temperature and pH levels. Thereafter, the effects of 1%-3% (w/v) bTCP on properties of the bTCP-bGP-2:1 (w/w) chitosan/collagen hydrogels were investigated. Notably, the incorporation of 1%-3% (w/v) bTCP in the hydrogels did not interfere with the gelation process and time of the hydrogels at physiological temperature and pH levels. The bTCP-hydrogels exhibited a porous structure, interconnecting pore architecture, and median pore size of 100-200 µm. The incorporation of 3% bTCP increased the mechanical strength but decreased the swelling and degradation rates, pore size, permeability, and quercetin release rate of the hydrogels. The hydrogels were noncytotoxic and able to support cell encapsulation. A sustained quercetin release profile of the 3% bTCP-hydrogel further suggested the applicability of the hydrogel as a delivery vehicle of natural flavonoids for bone regeneration.

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds.

Allogenic human serum, a clinical grade serum supplement for promoting human periodontal ligament stem cell expansion.

Exposing human periodontal ligament stem cells (hPDLSCs) to animal proteins during cell expansion would compromise quality and safety of the hPDLSCs for clinical applications. The current study aimed to evaluate the replacement of animal-based serum by human serum for the expansion of hPDLSCs. hPDLSCs were cultured in culture media supplemented with four types of serums: Group A: fetal bovine serum (FBS); Group B: allogeneic human male AB serum (HS); Group C: in-house autologous (Auto-HS); and Group D: in-house allogeneic human serums (Allo-HS). Exhibitions of mesenchymal stem cell characteristics of hPDLSCs were examined. Then, growth and osteogenic (OS) differentiation potential of hPDLSCs in FBS and HS at passages 5 and 15 were compared to investigate the effects of serum supplements on growth and expansion stability of the expanded hPDLSCs. After that, growth and OS differentiation of hPDLSCs in Auto- and Allo-HS were investigated. Flow cytometrical analyses, functional differentiations, cell growth kinetic, cytogenetic analysis, alkaline phosphatase and calcium content assays, and oil red O and von Kossa staining were performed. Results showed that at passage 5, HS promoted growth and OS differentiation of hPDLSCs and extensive cell expansion, decreased growth and differentiation potential of the expanded hPDLSCs, particularly in HS. Growth and OS differentiation of hPDLSCs in Auto-HS and Allo-HS were not different. In summary, allogeneic human serum could be a replacement to FBS for hPDLSC expansion. In vitro cell expansion of hPDLSCs should be minimal to ensure optimal cell quality. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of Titanium Surface Microtopography and Simvastatin on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells in Estrogen-Deprived Cell Culture.

PURPOSE: This study aimed to investigate the effects of titanium surface topography and simvastatin on growth and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in estrogen-deprived (ED) cell culture. MATERIALS AND METHODS: Human BMSCs were seeded on cell culture plates, smooth-surface titanium (Ti) disks, and sandblasted with large grits and acid etched (SLA)-surface Ti disks; and subsequently cultured in regular (fetal bovine serum [FBS]), ED, and ED-with 100 nM simvastatin (ED-SIM) culture media for 14 to 21 days. Live/dead cell staining, scanning electron microscope examination, and cell viability assay were performed to determine cell attachment, morphology, and growth. Expression levels of osteoblast-associated genes, Runx2 and bone sialoprotein and levels of alkaline phosphatase (ALP) activity, calcium content, and osteocalcin in culture media were measured to determine osteoblastic differentiation. Expression levels of bone morphogenetic protein-2 (BMP-2) were investigated to examine stimulating effects of simvastatin (n = 4 to 5, mean ± SD). In vitro mineralization was verified by calcein staining. RESULTS: Human BMSCs exhibited different attachment and shapes on smooth and SLA titanium surfaces. Estrogen-deprived cell culture decreased cell attachment and growth, particularly on the SLA titanium surface, but cells were able to grow to reach confluence on day 21 in the ED-osteogenic (OS) culture medium. Promoting effects of the SLA titanium surface in ED-OS were significantly decreased. Simvastatin significantly increased osteogenic differentiation of human BMSCs on the SLA titanium surface in the ED-OS medium, and the promoting effects of simvastatin corresponded with the increasing of BMP-2 gene expression on the SLA titanium surface in ED-OS-SIM culture medium. CONCLUSION: The ED cell culture model provided a well-defined platform for investigating the effects of hormones and growth factors on cells and titanium surface interaction. Titanium, the SLA surface, and simvastatin synergistically promoted osteoblastic differentiation of hBMSCs in ED condition and might be useful to promote osteointegration in osteoporotic bone.

Effects of autogenous growth factors on heterotopic bone formation of osteogenic cells in small animal model.

AIMS: This study used a new approach to investigate the effective concentrations of growth factors released from platelet concentrate (PC) on the bone formation capacity of osteogenically differentiated rat bone marrow stromal cells (rBMSCs). MATERIALS AND METHODS: Rat BMSCs and whole blood were harvested from 40 adult male Spraque-Dawly rats. Rat BMSCs were expanded in an osteogenic medium and seeded on inert collagenous bovine bone matrix (ICBM). Growth factors released from degranulated PC (GFs) containing TGF-ß1 1 (25ng/ml)-10ng (250ng/ml) and rhBMP-2 400ng (10µg/ml) were suspended in 40µl platelet poor plasma (PPP) and applied on the ICBM-rBMSC constructs or ICBM only, respectively. The constructs were then transplanted in autologous hosts for 4 weeks. Concurrently, osteoblastic differentiation of rBMSCs on ICBM-rBMSC-PPP constructs was characterized in vitro. RESULTS: Rat BMSCs in osteogenic medium exhibited phenotypes of mature osteoblasts. The amount of newly formed bone among groups of ICBM-rBMSC-PPP with and without GFs was not significantly different (p>0.05) and was significantly lower than a group of ICBM-PPP-BMP-2 (p<0.05). CONCLUSIONS: Autogenous GFs had no effect on the capacity of rBMSCs to form new bone. The ability to measure the bone formation capacity of transplanted autologous cells and growth factors in a small animal model was demonstrated.

Phenotypic and differentiation stability of human embryonic stem cell-derived osteoblasts.

PURPOSE: To ensure the efficiency and safety of transplanted human embryonic stem cell (hESC)-derived osteoblast-like cells (hESC-OS) for bone regeneration, this study was designed to determine the effects of continuous cell expansion on the osteoblastic differentiation stability, pluripotency, and tumorigenic potential of long-term expanded hESC-OS. METHODS: hESCs manually harvested as cell aggregates or enzymatically dissociated as single cells were directly incubated in osteogenic medium and serially passaged to passage 25. Expression of osteoblast-related genes, pluripotent regulator genes, and genes related to tumorigenesis were examined at the primary passage and every 5 passages thereafter. hESC-OS were subcutaneously transplanted into nude mice for 4-24 weeks to test for teratoma formation. hESC-OS were recultivated in hESC culture conditions to evaluate the extent to which reverse differentiation back to the undifferentiated stage may occur. RESULTS: hESC-OS derived from hESC aggregates and dissociated cells exhibited comparable osteoblast differentiation patterns. Expression levels of osteoblast-related genes reached plateau levels at passages 5-10 before declining in higher passages. Expression of tumor-associated genes was not significantly increased. Only hESC-OS at primary and first passages formed teratomas after 4 weeks in vivo. The hESC-OS were not able to revert to hESCs. CONCLUSIONS: Expanded hESC-OS demonstrated lineage-specific differentiation stability, did not maintain the pluripotency of hES cells, and were genetically stable. Thus, hESC-OS may be considered for large animal preclinical studies.

Expansion and characterization of human embryonic stem cell-derived osteoblast-like cells.

Human embryonic stem cells (hESCs) have the potential to serve as a repository of cells for the replacement of damaged or diseased tissues and organs. However, to use hESCs in clinically relevant scenarios, a large number of cells are likely to be required. The aim of this study was to demonstrate an alternative cell culture method to increase the quantity of osteoblast-like cells directly derived from hESCs (hESCs-OS). Undifferentiated hESCs were directly cultivated and serially passaged in osteogenic medium (hESC-OS), and exhibited similar expression patterns of osteoblast-related genes to osteoblast-like cells derived from mesenchymal stem cells derived from hESCs (hESCs-MSCs-OS) and human bone marrow stromal cells (hBMSCs-OS). In comparison to hESCs-MSCs-OS, the hESCs-OS required a shorter expansion time to generate a homogenous population of osteoblast-like cells that did not contain contaminating undifferentiated hESCs. Identification of human specific nuclear antigen (HuNu) in the newly formed bone in calvarial defects verified the role of the transplanted hESCs-OS as active bone forming cells in vivo. Taken together, this study suggests that osteoblast-like cells directly derived from hESCs have the potential to serve as an alternative source of osteoprogenitors for bone tissue engineering strategies.

Phenotypic characterization, osteoblastic differentiation, and bone regeneration capacity of human embryonic stem cell-derived mesenchymal stem cells.

To enhance the understanding of differentiation patterns and bone formation capacity of hESCs, we determined (1) the temporal pattern of osteoblastic differentiation of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs), (2) the influence of a three-dimensional matrix on the osteogenic differentiation of hESC-MSCs in long-term culture, and (3) the bone-forming capacity of osteoblast-like cells derived from hESC-MSCs in calvarial defects. Incubation of hESC-MSCs in osteogenic medium induced osteoblastic differentiation of hESC-MSCs into mature osteoblasts in a similar chronological pattern to human bone marrow stromal cells and primary osteoblasts. Osteogenic differentiation was enhanced by culturing the cells on three-dimensional collagen scaffolds. Fluorescent-activated cell sorting of alkaline phosphatase expressing cells was used to obtain an enriched osteogenic cell population for in vivo transplantation. The identification of green fluorescence protein and expression of human-specific nuclear antigen in osteocytes in newly formed bone verified the role of transplanted human cells in the bone regeneration process. The current cell culture model and osteogenic cell enrichment method could provide large numbers of osteoprogenitor cells for analysis of differentiation patterns and cell transplantation to regenerate skeletal defects.

A comparison of the stability of single-piece and segmental Le Fort I maxillary advancements.

This study retrospectively evaluated the stability of Le Fort I maxillary advancements and compared segmental and one-piece maxillary osteotomy procedures. A cephalometric analysis was performed on 26 cases of maxillary advancement. The sample comprised 11 cases of one-piece and 15 cases of segmental maxillary procedures. The tracings were superimposed and digitized by computer software, and the skeletal changes were analyzed before surgery, immediately after surgery, and at a minimum of 1 year of follow-up. Different values were compared by the paired and nonpaired t tests and were correlated by the Pearson correlation test. The significant value was set at a 95% confidence interval. The maxilla was advanced by a mean of 5.0 +/- 1.6 mm (P < 0.001), and the anterior maxilla was repositioned inferiorly by a mean of 1.5 +/- 3.3 mm (P < 0.05). The maxilla relapsed posteriorly by a mean of 0.6 +/- 1.2 mm (P < 0.05) and superiorly at the anterior maxilla by a mean of 0.8 +/- 1.1 mm (P < 0.001). Overjet and overbite did not significantly change (P > 0.05). It was concluded that maxillary advancement using rigid fixation and interpositional bone grafting in both groups was a stable procedure, particularly in the horizontal plane. In the one-piece group, there was a significantly higher relapse in the vertical plane than in the segmental group (P < 0.05), however. Minor skeletal relapse was compensated for by postoperative tooth movement, and segmental procedures are recommended when required to enhance occlusal results.

Stability of combined Le Fort I maxillary advancement and mandibular reduction.

BACKGROUND: There have been reports that correction of severe Class III abnormality by single jaw surgery may invite relapse in the long-term. The purpose of this study was to retrospectively evaluate the stability of combined Le Fort I maxillary advancement and bilateral sagittal split osteotomies for mandibular reduction. METHODS: Thirty patients with a skeletal Class III malocclusion underwent bimaxillary surgery using rigid fixation and interpositional bone grafting of the maxilla. The average age was 24.4 years, and the mean follow-up period was 20 months (Range: 12-63 months). Post-operative changes were measured on lateral cephalometric radiographs using an anatomical best-fit technique. RESULTS: The maxilla was advanced, on average, 6.1 mm (SD: 1.8 mm) and repositioned superiorly at PNS 1.9 mm (SD: 2.1 mm). The mandible was repositioned posteriorly 5.6 mm ISD: 4.2 mm) at menton, which also auto-rotated superiorly. At follow-up, the maxilla relapsed horizontally 0.6 mm (SD: 1.1 mm, p < 0.01) with no significant vertical change. The maxillary central incisors were proclined and the interincisal angle was reduced. Menton relapsed anteriorly 1.4 mm (SD: 2.7 mm, p < 0.01), and gonion rotated superiorly 1.5 mm (SD: 2.3 mm, p < 0.001). In 67 per cent of cases menton moved anteriorly less than 2.5 mm. The overjet and overbite did not change significantly. CONCLUSIONS: The data show that 12-months post-operatively, maxillary advancement combined with mandibular setback was relatively stable in the horizontal and vertical planes.