Alginate microcapsules as nutrient suppliers: an in vitro study

Objective: Alginate, known as a group of anionic polysaccharides extracted from seaweeds, has attracted the attention of researchers because of its biocompatibility and degradability properties. Alginate has shown beneficial effects on wound healing as it has similar function as extracellular matrix. Alginate microcapsules [AM] that are used in tissue engineering as well as Dulbecco's modified Eagle's medium [DMEM] contain nutrients required for cell viability. The purpose of this research was introducing AM in medium and nutrient reagent cells and making a comparison with control group cells that have been normally cultured in long term

Materials and Methods: In this experimental study, AM were shaped in distilled water, it was dropped at 5 mL/hours through a flat 25G5/8 sterile needle into a crosslinking bath containing 0.1 M calcium chloride to produce calcium alginate microspheres. Then, the size of microcapsules [300-350 microm] were confirmed by Scanning Electron Microscopy [SEM] images after the filtration for selection of the best size. Next, DMEM was injected into AM. Afterward, adipose- derived mesenchymal stem cells [ADSCs] and Ringer's serum were added. Then, MTT and DAPI assays were used for cell viability and nucleus staining, respectively. Also, morphology of microcapsules was determined under invert microscopy

Results: Evaluation of the cells performed for spatial media/microcapsules at the volume of 40 microl, showed ADSCs after 1-day cell culture. Also, MTT assay results showed a significant difference in the viability of sustained-release media injected to microcapsules [P<0.05]. DAPI staining revealed living cells on the microcapsules after 1 to 7-day cell culture

Conclusion: According to the results, AM had a positive effect on cell viability in scaffolds and tissue engineering and provide nutrients needed in cell therapy

Expression of odontogenic genes in human bone marrow mesenchymal stem cells

Tooth loss is a common problem and since current tooth replacement methods cannot counter balance with biological tooth structures, regenerating natural tooth structures has become an ideal goal. A challenging problem in tooth regeneration is to find a proper clinically feasible cell to seed. This study was designed to investigate the odontogenic potential of human bone marrow mesenchymal stem cells [HBMSCs] for seeding in tooth regeneration. In this experimental study, three pregnant Sprague Dawley [SD] rats were used at the eleventh embryonic day and rat fetuses were removed surgically using semilunar flap under general anesthesia. The primary mandible was cut using a stereomicroscope. The epithelial and mesenchymal components were separated and the dissected oral epithelium was cultured for 3 days. We used flow cytometry analysis to confirm presence of mesenchymal stem cells and not hematopoietic cells and to demonstrate the presence of oral epithelium. Bone marrow mesenchymal stem cells [BMSCs] and cultured oral epithelium were then co-cultured for 14 days. BMSCs cultured alone were used as controls. Expression of two odontogenic genes Pax9 and DMP1 was assessed using quantitative reverse transcription- polymerase chain reaction [RT-PCR]. Expression of two odontogenic genes, Pax9 and DMP1, were detected in BMSCs co-cultured with oral epithelium but not in the control group. Expression of Pax9 and DMP1 by human BMSCs in the proximity of odontogenic epithelium indicates odontogenic potential of these cells

[Histological evaluation of regeneration in rabbit calvarial bone defects using demineralized bone matrix, mesenchymal stem cells and platelet rich in growth factors]

Regeneration of large bone defects is a major challenge in maxillofacial reconstruction. The present study evaluated the efficacy of simultaneous use of demineralized bone matrix [DBM], mesenchymal stem cells [MSCs] and platelet rich in growth factors [PRGF]. Stem cells were isolated from the femoral bone marrow of rabbits and their stem cell nature was confirmed. PRGF was prepared by drawing 5 cc of blood from the heart of rabbits and its centrifugation. A total of four 8mm defects were surgically created in rabbits calvaria using a trephine bur. Third passage cells were loaded on DBM and stored in an incubator for 24 hours. DBM, DBM+PRGF, DBM+MSCs and DBM+PRGF+MSCs were used in groups 1, 2, 3, and 4, respectively. At 6 and 12 weeks rabbits were sacrificed using vital perfusion. Histomorphometric analysis was performed on 5 micro m sections stained with Hematoxylin and Eosin [H and E]. Data were analyzed using SPSS version 14 software and ANOVA test. Histomorphometric analysis of the sections at 6 and 12 weeks post-operation demonstrated 74.6% and 20.31% osteogenesis in group 1, 14.35% and 28.44% in group 2, 17.75% and 31.33% in group 3 and 18.94% and 37.21% in group 4, respectively. Percentage of new bone formation was higher in group 4. However, this difference was not statistically significant [P>0.05]. It seems that simultaneous use of DBM, PRGF and MSCs results in increased bone regeneration in 8 mm rabbit calvarial bone defects when compared to the application of MSCs or PRGF alone

[ effect of amniotic membrane transplantation in healing of primary stages of pseudomonas keratitis]

Human amniotic membrane [HAM] forms the inner wall of the membranous sac that surrounds and protects the embryo during gestation. The main advantages of amniotic membrane transplantation [AMT] in the treatment of bacterial keratitis are its epithelial bandage properties. Previous studies have documented the presence of some antimicrobial proteins and peptides in amniotic fluid such as lactoferrin, lysozyme, bactericidal or permeability increasing protein, calprotectin [MRP8/14 protein complex], LL37, and neutrophil defensins [Human Neutrophil Peptides, HNP 1-3]. Furthermore, the amniotic membrane does not express HLA-A, B, C or DR surface antigens, which may help avoid rejection after its transplantation. Thus, it can be used as a biological immune barrier. The purpose of this study was to evaluate the effectiveness of the amniotic membrane's healing properties in rabbits with pseudomonas keratitis. By using an animal model, 14 rabbits were divided into two groups of controls and cases. A syringe was used to inoculate the corneal stroma of the animals by Pseudomonas aeruginosa ATCC27853. After 20 hours pseudomonas keratitis was created and amniotic membrane was transplanted to the cornea of the case group. The infiltration size were observed on the first, third and seventh days after the experiment. Corneal perforation was seen in the controls [P<0.001] but amniotic membrane prevented perforation in the case group [P=0.02]. Transplantation of amniotic membrane in the primary stages of pseudomonas keratitis treatment remarkably prevents corneal perforation and it can be used to control the disease process

Effect of amniotic membrane combined with ciprofloxacin in curing the primary stages of pseudomonal keratitis

Keratitis caused by Pseudomonas aeruginosa is often resulted in severe corneal ulcers and perforation, which leads to losses of vision. Human amniotic membrane [HAM] forms the inner wall of the membranous sac which surrounds and protects the embryo during gestation. The purpose of this study was to evaluate the effectiveness of the amniotic membrane's healing in rabbits with pseudomonas keratitis. In total 14 rabbits divided in 2 groups of: 1 as Control and 2 as experimental amniotic membrane combined with ciprofloxacin. A 0.05 ml suspension of Pseudomonas aeruginosa ATCC 27853 was injected into rabbit's corneal stroma, with no interference in control group. In the second group, the amniotic membrane in pieces of 1.5 x 1.5 cm transplanted to the entire corneal surface by eight interrupted 10.0 nylon sutures. In the first day ciprofloxacin drop was injected to the second group every 30 minutes and through second to seventh days every 2 hours. The results of perforation in cornea and the amount of infiltration were registered. The results showed that amniotic membrane transplantation [AMT] + ciprofloxacin group had 0% perforation and the control group 85.6%. Average infiltrations were 5 mm in AMT + ciprofloxacin groups and 23.75 mm in control. The use of amniotic membrane with ciprofloxacin was effective in prevention of cornea perforation and controlling the process of pseudomonal keratitis remission. The improvement of inflammation rapidly happened in ciprofloxacin + AMT group.

Derivation of adipocytes from human endometrial stem cells [EnSCs]

Due to increasing clinical demand for adipose tissue, a suitable cell for reconstructive adipose tissue constructs is needed. In this study, we investigated the ability of Human Endometrial-derived stem cells [EnSCs] as a new source of mesenchymal stem cells to differentiate into adipocytes. EnSCs are the abundant and easy available source with no immunological response, for cell replacement therapy. Single-cell suspensions of EnSCs were obtained from endometrial tissues from 10 women experiencing normal menstrual cycles, and were cultured at clonal density [10 cells/cm[2]] or limiting dilution. Endometrial mesenchymal stem cell markers were examined flow cytometry. These cells were treated with adipogenicinducing medium for 28 days. The adipogenic differentiation of the EnSC was assessed by cellular morphology and further confirmed by Oil Red O staining and RTPCR. The BM-MSC differentiated into adipocytes in the presence of adipogenic stimuli for 3 weeks. The flow cytometric analysis showed that the cells were positive for CD90, CD105, CD146 and were negative for CD31, CD34.We showed that the key adipocytes marker PPARa was expressed in mRNA level after 28 days post treatment [PT]. According to our finding, it can be concluded that EnSCs represent a useful in vitro model for human adipogenesis, and provide opportunities to study the stages prior to commitment to the adipocyte lineage

Radiographic and histopathological analysis in calvarias bone regeneration process by platelet-rich plasma, platelet-rich plasma-gel and auto bone chips in rat

A functional treatment for skeletal damages in orthopedic and oral maxillofacial surgeries is required. Platelet growth factors such as Platelet Derived Growth Factor [PDGF], Bone Morphogenic Factor [BMP], Transferring Growth Factor-beta [TGF-beta] and Insulin-like Growth Factor-1 [IGF-1] precede wound healing and bone regeneration. In the present study we focused on the effect of platelet rich plasma [PRP], platelet rich plasma gel [PRP-Gel] and auto bone chips on this process. 30 male, 22 weeks old, Sprague-Dawley rats weighing 525 g were used. They were divided in three groups consisting of PRP [treated by Platelet-Rich Plasma], PRP-Gel [treated by it], Bone chips and Control [two cavities created in each animal in this group]. After 16 weeks they were histologically investigated while in the periods of 40, 60, 90and 120 days, the radiography had been done. The radiographic analysis showed complete treatment in all groups; however, by the histo-pathological investigations by auto bone chips complete and PRP-Gel partial healing has been observed. By histo-morphometric surveys [100+/-25] % in bone chips and [50+/-25] % in PRP-Gel groups bone bridging were observed, whereas in PRP it was not noticeable. The Present study suggests that neither PRP, nor PRP-Gel could be as beneficial as bone chips. Statistically, in PRP-Gel group, due to the existence of fibrin and thrombin, solid bone bridging at the treated site is indicated. According to the previous studies, in which the key role of both inhibitory and stimulatory signals in controlling the bone regeneration were proven, we suggest that auto bone chips could completely enhance healing due to signals among blood factors, environmental tissues and skeletal particles

Evaluation of pluripotency gene expression in mouse embryonic stem cell cultured on the human feeder layer

Embryonic stem [ES] cells are derived from the pluripotent inner cell mass [ICN] cells of blastocysts with the potential to maintain an undifferentiated state indefinitely. The derivation process involves plating of the blastocysts on mouse embryonic fibroblast [MEF] and expansion of the outgrowth in to established ES cell line. ES cell are capable of unlimited self-renewal by symmetric division and differentiated cells to all primitive embryonic germ layers. The capacity of ES cells to differentiate in to almost all the cell types of human body highlights their potential to play a promising role in cell replacement therapies for treatment of human diseases. In this study, MEFs have been replaced with human mesenchymal stem cells [hMSCs]. C4 mES cell [mouse embryonic stem cell line] colonies are cultured on inactivated hMSCs amplified >/= 600-folds during the 30 days of continuous culture. The longest continuous expansion of C4 mES cells on hMSC was 30 passages. In this study the gene expression for Oct-4, Nanog, Rex1, Brachyury, LIF, LIFR, TERT, B2M, Stat3, Sox2, Fgf4 in mES cells using reverse transcriptase polymerase chain reaction [RT-PCR] and in which genes expression for Stat3, Sox2, Fgf4 genes was negative whilst the gene expansion for Oct-4, Nanog, Rex1, Brachyury, LIF, LIFR, TERT, B2M genes was positive. There was also a karyotype analysis for ES which showed normal result. The immunocytochemical analysis of Oct4 transcriptional factor for ES cells was made which showed positive result for this factor. These genes may be novel candidates to play critical roles in the regulation of ES Cell pluripotency and self-renewal