Cell and Tissue Journal

Abstract Introduction: Cartilage, a tissue without blood vessels and nerves, possesses inherently limited regenerative capacity following injury, often leading to progressive joint degeneration and conditions like osteoarthritis (OA) if left untreated. Current clinical interventions, such as surgical microfracture or autologous chondrocyte implantation (ACI), face significant challenges, including donor site morbidity, immune rejection, and the formation of fibrocartilage with inferior biomechanical properties. These limitations underscore the urgent need for novel therapeutic strategies that can effectively stimulate hyaline cartilage regeneration. In this context, mesenchymal stem cell-derived exosomes (MSC-Exos) have garnered attention as a cell-free regenerative tool, leveraging their cargo of bioactive molecules (e.g., miRNAs, cytokines, and growth factors) to modulate chondrogenesis, suppress inflammation, and enhance extracellular matrix (ECM) synthesis. Concurrently, glucosamine, a natural amino sugar and precursor for glycosaminoglycan (GAG) biosynthesis, has demonstrated dual functionality in joint health: not only does it serve as a building block for proteoglycans critical to cartilage integrity, but it also exhibits chondroprotective effects by mitigating ECM degradation and promoting stem cell chondrogenic differentiation. The potential synergy between MSC-Exos and glucosamine could thus address multiple facets of cartilage repair, combining anabolic stimulation (via exosomal signaling) with metabolic support (via glucosamine supplementation), offering a promising combinatorial approach to halt OA progression and restore functional cartilage.
Aims: This study aimed to investigate the combined effect of mouse bone marrow stem cell-derived exosomes and glucosamine on the expression of cartilage-specific genes, including Sox9, Acan, Col2a1, and Col10a1.
Materials and Methods: Bone marrow mesenchymal stem cells were prepared from NMRI mice. The mice were euthanized by cervical dislocation, the femoral heads were removed, and the bone marrow contents were transferred into a cell culture flask using a syringe containing culture medium. The bone marrow cells were cultured and were ready for use after 3 to 5 passages. The cell supernatant was separated, and exosomes were extracted from it by successive rounds of centrifugation followed by ultracentrifugation. Mesenchymal stem cell viability and determining the appropriate concentration of exosomes and glucosamine were performed using the MTT assay. The experiments were performed on mesenchymal stem cells in 4 groups: control, exosome, glucosamine, and exosome + glucosamine. The effects of exosomes and glucosamine on the expression of Sox9, Acan, Col2a1, and Col10a1 genes in mesenchymal stem cells were investigated in the presence of chondrogenic medium.
Results: According to the MTT assay results demonstrating the synergistic effect of exosomes and glucosamine, the combined concentrations of 15 μg/mL exosomes and 25 μg/mL glucosamine were chosen for subsequent applications. Real-time PCR results showed that the expression of Sox9, Acan, and Col2a1 genes in stem cells treated with exosomes and glucosamine significantly increased compared to the other groups after 14 days, while the expression of the Col10a1 gene significantly decreased compared to the other groups.
Discussion: The combined treatment of bone marrow–derived mesenchymal stem cell (BMSC) exosomes and glucosamine significantly upregulated the expression of key chondrogenic markers, including Sox9, Acan, and Col2a1, while downregulating the hypertrophic marker Col10a1. This gene expression profile suggests a dual beneficial effect: (1) promotion of chondrogenic differentiation and extracellular matrix (ECM) synthesis, and (2) suppression of hypertrophic differentiation, a critical factor in preventing cartilage calcification and osteoarthritis progression. These findings highlight the synergistic potential of BMSC exosomes and glucosamine as a combinatorial therapy for cartilage regeneration. By enhancing anabolic processes (Sox9-mediated chondrogenesis and aggrecan/collagen II deposition) and concurrently inhibiting catabolic pathways (Col10a1-associated hypertrophy), this strategy may offer a promising approach to delay or reverse early-stage cartilage degeneration in degenerative joint diseases
Conclusion: Our study reveals that combining bone marrow stem cell-derived exosomes with glucosamine synergistically enhances chondrogenesis by upregulating key cartilage markers (Sox9, Acan, Col2a1) while suppressing hypertrophy-related Col10a1. This dual action suggests that exosomes promote cartilage matrix synthesis through their bioactive cargo (e.g., miRNAs/growth factors), while glucosamine likely inhibits hypertrophic differentiation, potentially via modulation of the Wnt/β-catenin pathway. These findings support this combination as a promising strategy for improving cartilage repair and preventing OA progression, though further in vivo validation is needed.

Abstract Introduction: Mesenchymal stem cells (MSCs) are  important cellular components in the microenvironment of various types of cancers, including multiple myeloma (MM); their dynamic interactions with malignant cells can control the tumor microenvironment (TME) to favor the progression, drug resistance, and survival of cancer cells. It is shown that not only are MM cells influenced by the environmental cells, but also malignant cells affect the behavior of other cells in the TME. Secreted signaling factors are important players in this scenario. Accordingly, MSCs derived from TME, affected by cancer cells, are different from normal MSCs; therefore, in cases of disease modeling and drug screening studies, optimization of MSCs culture condition and their priming with cancer cell-derived secretome are crucial considerations to become more similar to the patient-derived MSCs. In this regard, conditioned medium, which is derived from the supernatant culture medium of cultured cells, contains cell-secreted factors and can be used as a source of cell secretome.
Aims: This study attempted to evaluate the effect of conditioned medium derived from the myeloma cell line U266 (U266-CM) on the proliferation, cell cycle, and differentiation of bone marrow-derived MSCs (BMMSCs).
Materials and methods: U266 cells were cultured in RPMI-1640 complete culture medium, and their conditioned medium was collected after two days of culture and stored. Since the basal culture medium of the U266 cell line (RPMI-1640) was different from the basal culture medium of BMMSCs (DMEM), in the first step, the impact of changing the culture medium from DMEM to RPMI-1640 on the proliferation and viability of BMMSCs was evaluated. In the next step, the effects of U266-CM treatment on the proliferation, cell cycle, and differentiation of BMMSCs into osteoblasts and adipocytes were assessed. Alkaline phosphatase activity assessment and Alizarin red staining were performed to evaluate the osteogenic differentiation, and Oil red O staining was carried out to assess the adipogenic differentiation. BMMSCs cultured in DMEM complete media were considered as control group.
Results: Changing the culture media from DMEM complete media to RPMI-1640 complete media affected the viability and proliferation rate of BMMSCs; however, up to 48 hours, the viability and proliferation rate of cells cultured in both culture media were maintained. Therefore, 48 hours was selected as the optimum incubation time of BMMSCs with U266-CM. Treatment with U266-CM decreased the proliferation rate of BMMSCs, assessed by cell counting and cell cycle analysis. Moreover, BMMSCs cultured in U266-CM showed slightly increased osteogenic differentiation and maintained their adipogenic propensity.
Discussion: Our results showed that CM derived from the MM cell line affected the proliferation rate and the differentiation potential of BMMSCs. These observations highlighted the importance of a more similar recapitulation of BMMSCs culture condition to the TME, for disease modeling and drug screening studies. Further experiments, including providing dynamic interactions of MSCs with MM cells in direct or indirect co-culture systems, are required to better recapitulate TME conditions for MSC priming. Moreover, for mechanistic analysis, evaluation of the secreted factors and assessment of expressed transcripts and proteins are highly recommended for future studies.
Conclusion: In summary, U266-CM influenced the proliferation rate and the differentiation potential of BMMSCs.

Abstract Aim: Cartilage tissue has limited capacity for spontaneous repair and self-renew due to the absence of vascularization and progenitor cells, thus requiring a therapeutic approach to repair tissue damage. Recently, extracellular vesicles (EVs) have attracted attentions because of their major roles in cell communication and tissue repair by regulating cellular processes such as cell growth, differentiation, and proliferation. Therefore, it is necessary to isolate extracellular vesicles with chondrogenic potential from an appropriate cellular source for cartilage regeneration. This study aims to compare the chondrogenic ability of extracellular vesicles derived from cocultured -chondrocytes / mesenchymal stem cells (CHO / MSC) at ratios of 1/2 and 1/4.
Material and Methods: Towards this goal, chondrocytes and mesenchymal stem cells were isolated from rabbit articular cartilage and bone marrow, respectively. Mesenchymal phenotype of isolated MSCs were characterized based on their surface markers and by differentiation into mesenchymal lineages. Chondrocytes and mesenchymal stem cells were co-cultured with defined ratios, their conditioned media were collected and extracellular vesicles were extracted with an ultracentrifuge. Concentrations of extracellular vesicles were determined using BCA Protein Assay Kit, then EVs were characterized in terms of size, morphology, and expression of surface markers by dynamic light scattering (DLS), scanning electron microscope (SEM) and western blotting, respectively. Mesenchymal stem cells were treated with different concentrations of extracellular vesicles (50, 100, and 150 ug/ml) for 21 days. Quantitative real time-PCR (qRT-PCR) and histological analysis were subsequently performed to assess the quality of chondrogenic differentiation among experimental groups. The differentiation of MSCs to osteogenic and adipogenic lineages  was demonstrated by Oil Red and Alizarin Red staining after 21 days.
Results: The results of flow cytometry showed that CD90 was expressed by 88.6% of the cell population as a positive marker and CD34 as a negative marker was only expressed in 5.48% of the cell population. SEM micrographs confirmed the spherical shape of CHO/ MSC-EV 1/2 and CHO/MSC-EV 1/4 and the mean particle size of isolated EVs were 51.66 ± 8.15 and 19.11 ± 6.03 nm, respectively. Special surface markers for extracellular vesicles containing CD9 and CD81 were expressed in both groups. At concentrations of 100 and 150 ug/ml of CHO/MSC-EV 1/2, higher cartilage - specific markers, including Col II were observed compared to CHO/MSC-EV 1/4. Similarly, safranin O and toluidine blue staining revealed the more deposition of aminoglycan and proteoglycan at concentrations of 100 and 150 ug/ml of CHO/ MSC-EV 1/2, compared to CHO/ MSC-EV 1/4. This study demonstrated the chondrogenesis potential of extracellular vesicles, especially in CHO /MSC-EV 1/2 and extracellular vesicles derived from co-culturing chondrocytes/MSCs improve matrix production and chondrogenesis.
Conclusion: Our research shows that chondrocyte-MSCs proximity is essential for the response as improved viability, chondrogenesis, and matrix formation in recipient MSCs. It is also proposed that co-cultured derived EVs with the ability to promote chondrogenesis have the potential to be utilized in cartilage regeneration.  Due to their immunogenicity and their low tumorigenic potential, extracellular vesicles can be used to help repair cartilage tissue.

Abstract Aim: In recent years using of Bone marrow mesenchymal stem cells (BMSC) in regenerative medicine, tissue engineering and gene therapy is highly regarded. Convenient access, ability to expand and MSC differentiation capacity along with the ability of adhesion to plastic surfaces and in-vitro growth and development are considered as the characteristic feature of these cells. Bone marrow mesenchymal stem cells possess the ability to differentiate into mesodermal lineage, among other adult cells, can be used in tissue engineering and are good candidates for transplantation. Lovastatin as a lowering cholesterol agent and reducing inflammation, as well as antioxidant and, in particular, neuroprotective effects can be effective in the treatment of neurogenic diseases. The aim of this study was to evaluate the effect of lovastatin on survival, proliferation and expression of GDNF and oct4 genes of Bone marrow mesenchymal stem cells. Lovastatin is presumed to exert their neuroprotective effects by inducing neurotrophic factor gene expression and cell proliferation. Material and methods: In this experimental study, we used 4-6 week adult Wistar rats. The BMSCs were isolated from rat femurs and tibias and cultured in α-MEM. The cell pellet was resuspended in α-MEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and cultured in 25-cm2 culture flasks at a density of 2 × 104 cells and incubated at 37°C and 5% CO2.  For lovastatin treatment, we exposed MSCs to 1 μM, 5 μm, 10 μm and 15 μm of lovastatin for 24 h. The survival rate of cells was measured by MTT assay. The growth rate and proliferation of cells at 24 hours after culture were assessed by staining with DAPI. The expression of Oct4 and GDNF factors was also evaluated by RT-PCR.
 Results: MSCs were attached to culture plate and were quickly proliferated. In the culture plate, these cells were usually appeared in three forms: small spherical, fusiform and fibroblast-like and flattened. The results of this study indicate that the proliferation rate at 5, 10 and 15 µM lovastatin showed a significant increase compared to control groups (P<0.5). Gene expression density of gelial derived neurotrophic factors (GDNF) and oct4 genes showed that, there were significant differences between MSCs treatment groups and control group (P<0.5). Cell viability and proliferation rate indicate that experimental groups has a higher proliferation rate than control group. Moreover, results showed an increase in mRNA expression for GDNF and Oct4 compared to the control group (P <0.05).
 Conclusion:Therefore, lovastatin can be used to improve the culture of mesenchymal stem cells, which is used for transplantation and cell therapy. BMSCs may be a useful therapeutic agent for the treatment of neurodegenerative disorders.

Abstract Aim: Cell therapy using mesenchymal stem cells (MSCs) can be a promising tool in regenerative medicine. One of the richest sources of mesenchymal stem cells is fetal bone marrow. Silymarin has strong antioxidant and anti-inflammatory activities with a positive effect on the proliferation of some cells as well as anti-osteoporosis properties. This study aimed to show the effect of silymarin on the differentiation of mesenchymal stem cells derived from the bone marrow of sheep embryos into the osteogenic line.
Materials and Methods: Mesenchymal stem cells were isolated from the bone marrow of sheep embryos. MTT test was performed to investigate the cytotoxicity of silymarin on cells at different concentrations for 24 and 72 hours. Then, cells in one of 8 groups 1: negative control; 2: treated with 10 μmol/liter silymarin in the usual environment, 3: treated with 20 μmol/liter silymarin in the usual environment, 4: treated with 100 μmol/liter estradiol in the usual environment, 5: positive control, 6: treatment treated with 10 μmol/liter silymarin in the differentiation medium, 7: treated with 20 μmol/liter silymarin in the differentiation medium, 8: treated with 100 μmol/liter in the differentiation medium, were cultured for 21 days. To determine the osteogenic differentiation of cells, the deposition of hydroxyapatite ions was examined using alizarin staining, and also, the amount of ALP enzyme secretion was also measured in the studied groups. Results: Comparing the average optical absorption of cells at different concentrations between 24 and 72 hours after treatment showed that the average optical absorption of cells at zero concentration of silymarin after 72 hours of treatment decreased in comparison with those treated for 24 hours (P<0.05), but no significant difference was observed in other concentrations (P>0.05). Examining the level of ALP enzyme secretion, 21 days after treatment with silymarin in the studied groups showed that the highest level of enzyme secretion was in group 8 (P≤0.05). The lowest amount of enzyme secretion was observed in group 1 (negative control) and then in group 2 and group 3 respectively (P<0.05). No significant difference was observed between groups 4, 5, and 6 (P>0.05). Based on the alizarin red staining results, calcium ions deposition was observed in all the groups related to the differentiation medium, which increased in groups 8, 7, 6, and 5, respectively. In the groups cultured in the usual environment, there was no calcification in group 1 and the amount of calcification increased in groups 2, 3, and 4, respectively. In total, the amount of calcification in the differentiation environment groups was higher in comparison with the usual environment.
Conclusion: During this study, Silymarin had no toxic effect on the mesenchymal stem cells derived from the bone marrow of sheep embryos in the studied concentrations after 24 and 72 hours of treatment. It increased the differentiation of the cells into the osteogenic lineage in a concentration-dependent manner. Therefore, it seems that with further studies and identification of the molecular pathways of silymarin's effect, it can be used in cell therapy in order to repair bone lesions.

Abstract Aim: In the present study, the effects of hydroalcoholic extract of Berberis integrrima root on the induction of differentiation in mesenchymal stem cells derived from adipose tissue of male Wistar rats towards osteoblasts were investigated.
Material and Methods: In this study, stem cells derived from male Wistar rat tissue were isolated and flow cytometry was performed to confirm the surface markers. Stem cells were treated with 10, 20, 40 and 80 μg/ml hydroalcoholic extract of B. integrrima root. The cell toxicity of the extract was evaluated by MTT assay and its differentiated effects was evaluated by alizarin red staining and alkaline phosphatase activity, calcium deposition assay. Results were analyzed using one-way ANOVA at a significance level of P <0.05.
Results: Stem cells markers including CD105, CD44, and CD73 had positive, and CD45 and CD34 had negative expression in these cells. The results of the MTT assay showed Concentrations less than 20 µg/ml do not have significant toxic effects on cells. Higher alkaline phosphatase activity was observed in the treatment group compared to the control group on day 10. The results of Alizarin red staining and measurement of calcium content for 21days showed that this extract in a concentration dependent manner leads to the differentiation of stem cells into osteoblasts.
Conclusion: The findings of this study showed that mesenchymal stem cells derived from adipose tissue of male Wistar rats treated with hydroalcoholic extract of Berberis integrrima root enter osteoblasts in the differentiation pathway.

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Abstract Aim: This study was aimed to preparation of sheep urinary bladder derived from biological scaffold by a combined (physical and chemical) method and evaluation of scaffold biocompatibility.
 Materials and Methods: Urinary bladder decellularization was performed by physical and chemical methods. In the physical method, the bladder fragments were incubated at -4 °C for 24 h and intervaled the fragments each 6 h by placing for 10 min in 0.1% sodium azide solution. After 24 h, the samples were placed at -20 and -40 °C for 2 and 1 h, respectively. The bladder fragments were held in a cryotube and emerged in liquid nitrogen by five two-minute steps, and finally washed by the PBS buffer containing 0.1% sodium azide. In the chemical decellularization, all of the physically treated bladder fragments were placed in a sodium dodecyl sulphate solution under slow stirring for 24 h. The samples were rinsed with sterile distilled water, sterilized with 75% ethanol and 0.2% peracetic acid and finally were placed in PBS for 24 h.
Results: The light and electron microscopey studies revealed the biocompatibility of seeded stem cells on the sheep bladder bioscaffold, in 3rd, 5th and 7th days. The most biocompatibility was observed in the end of 7th day.
Conclusion: Decellularized urinary bladder scaffold revealed biocompatibility which could be considered as a potential nontoxic and biocompatible bioscaffold for application in tissue engineering regenerative medicine.
 

Abstract Aim: In this study, we use combination of enzymatic and explant methods, for isolation and culture of ADMSCs.
Materials and Method: Adipose tissues dissected out from the abdominal region of male Wistar rats. Tissues were minced into small pieces (1-2 mm), and then treated with trypsin-EDTA 0.25% for 30 minutes at 37 ̊C in shaker-incubator. Enzymatic treated tissues were centrifuged and floating parts were cultured. Statistical analysis of the cells number was investigated using GraphPad Prismv6 software.
Results: Results showed that ADMSCs isolated with our methods have growth characteristics similar to conventional methods. ADMSCs were maintained up to 10th passage and exhibited a homogeneous population in terms of appearance and morphology. We demonstrated that ADMSCs by these combination methods have mesenchymal characteristic markers (positive for CD90, CD44, CD73, CD105 and negative for CD35, CD45, CD11b).
Conclusion: In the current study, we showed that the combination of enzymatic and explant methods creates a simple, inexpensive and reproducible method for isolation and culture of ADMSCs.
 

Abstract Aim: Electromagnetic field as a physical stimulus, willingly or unwillingly are affected cellular processes. In this study morphology changing and proliferation rate of mesenchymal stem cells were investigated in presence of electromagnetic field (EMF) and nitric oxide (NO). Material and methods: The stromal stem cells were isolated from the Rat bone marrow and incubated. After several passages of the harvested cells, Deta-NO as a donor of nitric oxide was then added to cell culture. These cells were also treated by EMF (50 Hz and 20 mT). The MTT test was used for estimating proliferation rate of the cells. To estimate the proportion of the cell line in different phases of cell cycle due to treatment with NO and EMF, cellular DNA contents were measured by flow cytometry. Result: The results demonstrated decreasing proliferation rate of the stem cells exposed with EMF and NO compared with the control group. We found that the rate of decrease is highly related to the concentration of NO. The double treatment of EMF and nitric oxide was yielded to an obvious arrest in the cell cycle at G2/M phase. Nitric oxide associated with EMF also changed the cell morphology and increased the cell motilities. Conclusion: The changing of cell morphology and reduction of proliferation rate of the stem cell can be considered as a symptom showing the beginning of cell differentiation.

Abstract Aim: The aim of the present study was to evaluate the effects of different concentrations of Chir99021 on ovine fetal marrow-derived mesenchymal stem cells (BM-MSCs) expansion in culture.  Material and Methods: BM-MSCs were isolated from ovine fetal and cultured. Passaged-3 cells were examined for their differentiation potential into osteocytes and adipocytes. In the present study, BM-MSCs from ovine fetal were plated in the presence of 0, 0.5, 1, 1.5, 3 and 5 μM of Chir9902. During the cultivation period, the cultures were statistically compared in terms of induces of cell growth including the number of colonies, population doubling number (PDN), doubling time (DT) and the number of viable cells. Furthermore, expression of the beta-catenin was evaluated. The culture without Chir99021 was taken as the control group.  Results: Our findings indicated that, addition of 0.5 and 1 µM of Chir99021 to medium significantly improved overall proliferation compared to that of control group as well as 5µM of Chir99021(p < 0.05). Furthermore, Five days after treatment with small molecule showed that the treatments with 0.5 and 1 µM Chir99021 formed higher Colony Forming Unit-Fibroblast (CFU-F) compared to control and 5 µM of Chir99021. The expression of beta-catenin was significantly higher in culture supplemented with 1 μM of Chir99021 compared to that in groups containing 0.5 and 1.5 μM (p < 0.05). Conclusion: In conclusion, using Chir99021 at concentration of 1 μM could enhance in vitro proliferation of ovine fetal BM-MSC; however administration of high concentration of this small molecule had toxic effects on these cells proliferation.

Abstract Aim: In this study, it was done to evaluate the efficiency of both PRP and Fibrin Glue scaffolds in producing suitable environment for the growth of mesenchymal stem cells.
Material and Methods: In this study, the preparation of PRP and Fibrin Glue Scaffold were carry out and Mesenchymal Stem Cells (MSCs) isolated from adipose tissue. The mesenchymal phenotype of these cells was determined by mesenchymal surface marker using flow cytometry. Then, MSCs were cultured, and on the third passage (P3)stage, they were seeded separately on the two scaffolds and after 48 hours of cell culture, the ability of the scaffolds seeded cells was evaluated by MTT assay for cells viability.
Results:Flow cytometry results showed that human adipose-derived mesenchymal stem cell expressed CD44, CD90 and CD105 surface markers. Also, the results of this study showed that the active PRP could be creating a more suitable environment for the survival and proliferation of mesenchymal stem cells in compare with Fibrin glue
Conclusion:It is suggested that the PRP as a protective scaffold could be used for mesenchymal stem cells growth to provide effective strategies in order to tissue engineering development and regenerative medicine.

Abstract Aim: In this study the effect of vitreous humor on the mesenchymal stem cells (MSCs) derived from bone marrow, adipose tissue and amniotic fluid to lens like cells was investigated.
Material and methods: Inthis experimental study collected stem cells from femurs bone marrow and inguinal fat pads of mature NMRI mice and amniotic fluid of 13 days embryos of NMRI mice were cultured. The mesenchymal character of these cells was proven by flow cytometry markers like CD90, CD31. This experiment took place for 14 days with different dosages 10, 15, 20, 30, 40, 50 % of bovine vitreous humor. Express of crystalline markers were detected by immunocytochemistry in experimental and control groups.
Results: The flow cytometeric analyses of surface markers were shown expression of CD90 by bone marrow and amniotic fluid stem cells and adipose tissue stem cells (29/29%, 0/57%, 82%, respectively). Moreover, mesenchymal stem cells from these 3 sources had expression of CD31 (3/44%, 1/53%, 0/1%, respectively). Immunocytochemistry  results revealed that 40 % vitreous humor in culture media fluid more inducing effect on adipose and amniotic fluid mesenchymal stem cells and 15 % vitreous humor on bone marrow stem cells in order to differentiate.
Conclusion: According to the findings of this study, it can be concluded that MSCs derived from all sources can differentiate into lens fiber like cells by inducing effect of vitreous humor.

Abstract Aim: The main goal of this research was to prepare three-dimensional matrix of gingival palate tissues and compare the behavior of bone marrow mesenchymal stem cells and blastema on the scaffolds.
Material and methods: The tissues obtained from gingival surgeries
in periodontal clinic were decellularized using detergents including sodium dodecyl sulfate and Triton X-100 then after washing and sterilization, were used as scaffold for culturing the rat’s mesenchymal stem cells. The scaffolds were studied by light and electron microscopy before and after 1, 2, and 4 weeks of culture. In addition the prepared scaffolds were assembled within the rings of blastema tissues from rabbit^,s pinna. Then the scaffolds were similarly studied by histological techniques after 1, 2, and 4 weeks of culture.
Results: Scanning electron microscopy showed that epithelial matrix and collagen fibers in connective tissue remained intact. In both samples epithelium-like structures were observed. Also in the blastema cells migrating to the scaffolds cell secretion was also observed.
Conclusion: Human gingival matrix can be a suitable scaffold for studying the cell behaviors. More research should be carried out to determine the identity of differentiated cells, which finally can improve our knowledge regarding the cell-matrix interactions.
 

Abstract Aim: The aim of this study was to compare the morphological and biochemical character of mesenchymal stem cells and osteoblasts in vitro. 
Material and Methods: After extraction of rat bone marrow mesenchymal stem cells and culturing them till the 3^rd passage, these cells were cultured in media containing beta-glycerol phosphate, dexamethasone and ascorbic acid for 21 days. Then morphology of the mesenchymal cells and differentiated one were investigated using Hoechst and acridine orange. In addition the level of matrix deposition, level of calcium, activity of alkaline phosphatase in mesenchymal cells and differentiated on as well as metabolic activity of osteoblasts with the help of MTT was determined.
Results: Morphological study showed delocalization of nuclei and roundness of cytoplasm in differentiated cells as compared to mesenchymal stem cells. In addition mineralization of the matrix started from the 10^th day and reached the maximum level at the 21^st day. Also from the day 10 the level of calcium increased significantly (p < 0.05) but activity of alkaline phosphatase decreased significantly (p < 0.05) in differentiated cells. Where as during the differentiation process the metabolic activity of the cells increased significantly (p < 0.05).
Conclusion: In addition to morphological differences, calcium content, alkaline phosphatase activity and metabolic activity of the mesenchymal cells and osteoblasts also differs. Therefore in addition to alizarin red staining these factors also can be used to investigate the differentiation processes in vitro.