Strategies for modifying the chimeric antigen receptor (CAR) to improve safety and reduce toxicity in CAR T cell therapy for cancer.

Immune cell therapy with chimeric antigen receptor (CAR) T cells, which has shown promising efficacy in patients with some hematologic malignancies, has introduced several successfully approved CAR T cell therapy products. Nevertheless, despite significant advances, treatment with these products has major challenges regarding potential toxicity and sometimes fatal adverse effects for patients. These toxicities can result from cytokine release or on-target off-tumor toxicity that targets healthy host tissue following CAR T cell therapy. The present study focuses on the unexpected side effects of targeting normal host tissues with off-target toxicity. Also, recent safety strategies such as replacing or adding different components to CARs and redesigning CAR structures to eliminate the toxic impact of CAR T cells, including T cell antigen coupler (TAC), switch molecules, suicide genes, and humanized monoclonal antibodies in the design of CARs, are discussed in this review.

Characterization of CAR T Cells Manufactured Using Genetically Engineered Artificial Antigen Presenting Cells.

OBJECTIVE: Chimeric antigen receptor (CAR) T cell therapy has recently emerged as a promising approach for the treatment of different types of cancer. Improving CAR T cell manufacturing in terms of costs and product quality is an important concern for expanding the accessibility of this therapy. One proposed strategy for improving T cell expansion is to use genetically engineered artificial antigen presenting cells (aAPC) expressing a membrane-bound anti-CD3 for T cell activation. The aim of this study was to characterize CAR T cells generated using this aAPC-mediated approach in terms of expansion efficiency, immunophenotype, and cytotoxicity. MATERIALS AND METHODS: In this experimental study, we generated an aAPC line by engineering K562 cells to express a membrane-bound anti-CD3 (mOKT3). T cell activation was performed by co-culturing PBMCs with either mitomycin C-treated aAPCs or surface-immobilized anti-CD3 and anti-CD28 antibodies. Untransduced and CD19-CARtransduced T cells were characterized in terms of expansion, activation markers, interferon gamma (IFN-γ) secretion, CD4/CD8 ratio, memory phenotype, and exhaustion markers. Cytotoxicity of CD19-CAR T cells generated by aAPCs and antibodies were also investigated using a bioluminescence-based co-culture assay. RESULTS: Our findings showed that the engineered aAPC line has the potential to expand CAR T cells similar to that using the antibody-based method. Although activation with aAPCs leads to a higher ratio of CD8+ and effector memory T cells in the final product, we did not observe a significant difference in IFN-γ secretion, cytotoxic activity or exhaustion between CAR T cells generated with aAPC or antibodies. CONCLUSION: Our results show that despite the differences in the immunophenotypes of aAPC and antibody-based CAR T cells, both methods can be used to manufacture potent CAR T cells. These findings are instrumental for the improvement of the CAR T cell manufacturing process and future applications of aAPC-mediated expansion of CAR T cells.

Application of mesenchymal stem cell sheet for regeneration of craniomaxillofacial bone defects.

Bone defects are among the most common damages in human medicine. Due to limitations and challenges in the area of bone healing, the research field has turned into a hot topic discipline with direct clinical outcomes. Among several available modalities, scaffold-free cell sheet technology has opened novel avenues to yield efficient osteogenesis. It is suggested that the intact matrix secreted from cells can provide a unique microenvironment for the acceleration of osteoangiogenesis. To the best of our knowledge, cell sheet technology (CST) has been investigated in terms of several skeletal defects with promising outcomes. Here, we highlighted some recent advances associated with the application of CST for the recovery of craniomaxillofacial (CMF) in various preclinical settings. The regenerative properties of both single-layer and multilayer CST were assessed regarding fabrication methods and applications. It has been indicated that different forms of cell sheets are available for CMF engineering like those used for other hard tissues. By tackling current challenges, CST is touted as an effective and alternative therapeutic option for CMF bone regeneration.

The efficiency of PCL/HAp electrospun nanofibers in bone regeneration: a review.

Electrospinning is a method which produces various nanofiber scaffolds for different tissues was attractive for researchers. Nanofiber scaffolds could be made from several biomaterials and polymers. Quality and virtues of final scaffolds depend on used biomaterials (even about single substance, the origin is effective), additives (such as some molecules, ions, drugs, and inorganic materials), electrospinning parameter (voltage, injection speed, temperature, …), etc. In addition to its benefits, which makes it more attractive is the possibility of modifications. Common biomaterials in bone tissue engineering such as poly-caprolactone (PCL), hydroxyapatite (HAp), and their important features, electrospinning nanofibers were widely studied. Related investigations indicate the critical role of even small parameters (like the concentration of PCL or HAp) in final product properties. These changes also, cause deference in cell proliferation, adhesion, differentiation, and in vivo repair process. In this review was focussed on PCL/HAp based nanofibers and additives that researchers used for scaffold improvement. Then, reviewing properties of gained nanofibers, their effect on cell behaviour, and finally, their valency in bone tissue engineering studies (in vitro and in vivo).

Evaluation of bone regeneration in mandible large defect using undifferentiated adipose stem cells loaded on gelatin carrier: An animal model case study.

Background. Large mandibular defects are considered difficult reconstructive challenges for oral and maxillofacial surgeons. Cell therapy, as an alternative technique, might increase the speed of bone regeneration. This study aimed to investigate bone regeneration in large defects of dog mandibles using allogenic adipose-derived stem cells on gelatin foam as a cell carrier. Methods. The tissue engineering phase consisted of the sampling of adult dogs' adipose tissue that can easily be isolated from adipose stem cells (ASCs) of the dogs, ASCs were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) with low glucose, containing 10% fetal bovine serum (FBS) (Sigma, USA) and 1% penicillin-streptomycin (Gibco, USA), with the characterization of dog ASCs and gelatin-transplanted ASCs. Six dogs were included in this experimental study in the next step and randomly assigned to the treatment and control groups. The samples in both groups underwent surgery under general anesthesia to create uniform 3-cm bony defects. The samples in both groups were reconstructed with titanium reconstruction plates and screws. A large bone gap filled with ASCs (5×106 ) was seeded on gelatin (ASCs) in the treatment group. In the control group, bony defects were filled with a cell delivery carrier without ASCs. Six months after transplantation, the animals' mandibles were evaluated by CT scan imaging, and the results were quantified through the Hounsfield unit (HU). The data were analyzed with t-test. Results. Before transplantation, the nature of the stem cells was confirmed by the expression of CD44 and CD105 cell markers at 71.9% and 89.3%, respectively, and a lack of the CD45 cell marker expression at 2.2%. Evaluation of CT scan images showed significantly higher bone repair in the ASCs group (920.25±572.92 HU) than in the control group (-94.746± 08.42). Conclusion. The bone regeneration of the ASCs group was significantly higher than that in the control group.

Application of neurotrophic factor-secreting cells (astrocyte - Like cells) in the in-vitro Alzheimer's disease-like pathology on the human neuroblastoma cells.

This study investigated physical proximity and paracrine activity of neurotrophic factor-secreting cells (NTF-SCs) on beta-amyloid treated cells. Mesenchymal stem cells (MSCs) - to-NTF-SCs (Astrocyte -like cells) trans-differentiation was confirmed using immunofluorescence staining of GFAP. BDNF and NGF levels were measured by ELISA. To mimic AD-like condition, SH-SY5Y cells were exposed to 10 µM Aß1-42. SH-SY5Y cells were allocated into Control; and Aß1-42-treated cells. Treated cells were further classified into three subgroups including Aß1-42 cells, Aß1-42 cells + NTF-SCs (CM) and Aß1-42 cells + NTF-SCs co-culture. Cell viability was measured by MTT assay. Anti-inflammatory and anti-tau hyperphosphorylation effects of NTF-SCs were assessed via monitoring TNF-α and hyperphosphorylated Tau protein expression level respectively. To explore the impact of NTF-SCs on synaptogenesis and synaptic functionality, real-time PCR assay was performed to measure the expression of synapsine 1, homer 1 and ZIF268. The level of synaptophysin was monitored via immunofluorescence staining. Data showed MSCs potential in trans-differentiating toward NTF-SCs indicated with enhanced GFAP expression (p < 0.05). ELISA assay confirmed the superiority of NTF-SCs in releasing NGF and BDNF compared to the MSCs (p < 0.05). Aß significantly induced SH-SY5Y cells death while juxtacrine and paracrine activity of NTF-SCs significantly blunted these conditions (p < 0.05). Trans-differentiated cells had potential to reduce Tau hyperphosphorylation and TNF-α level after treatment with Aß through juxtacrine and paracrine mechanisms (p < 0.05). Moreover, NTF-SCs significantly increased the expression rate of synapsin 1, homer 1 and zif 268 genes in Aß-treated cells compared to matched-control group coincided with induction of synaptophysin at the protein level(p < 0.05). NTF-SCs reversed AD-like neuropathological alterations in SH-SY5Y cells via paracrine and juxtacrine mechanisms.

Understanding breast cancer heterogeneity through non-genetic heterogeneity.

Intricacy in treatment and diagnosis of breast cancer has been an obstacle due to genotype and phenotype heterogeneity. Understanding of non-genetic heterogeneity mechanisms along with considering role of genetic heterogeneity may fill the gaps in landscape painting of heterogeneity. The main factors contribute to non-genetic heterogeneity including: transcriptional pulsing/bursting or discontinuous transcriptions, stochastic partitioning of components at cell division and various signal transduction from tumor ecosystem. Throughout this review, we desired to provide a conceptual framework focused on non-genetic heterogeneity, which has been intended to offer insight into prediction, diagnosis and treatment of breast cancer.

Efficacy of mesenchymal stromal cells and cellular products in improvement of symptoms for COVID-19 and similar lung diseases.

At the end of 2019, respiratory coronavirus diseases 2019 (COVID-19) appeared and spread rapidly in the world. Besides several mutations, the outcome of this pandemic was the death up to 15% of hospitalized patients. Mesenchymal stromal cell therapy as a therapeutic strategy seemed successful in treatment of several diseases. Not only mesenchymal stromal cells of several tissues, but also their secreted extracellular vesicles and even secretome indicated beneficial therapeutic function. All of these three options were studied for treatment of COVID-19 as well as those respiratory diseases that have similar symptom. Fortunately, most of the outcomes were promising and optimistic. In this paper, we review in-vivo and clinical studies which have been used different sources of mesenchymal stromal cell, secreted extracellular vesicles, and secretome to improve and treat symptoms of COVID-19 and similar lung diseases.

Treatment of cartilage defects by Low-intensity pulsed ultrasound in a sheep model.

Aim of this study was to evaluate effects of Low-intensity pulsed ultrasound on repair of articular cartilage defects. Low-intensity pulsed ultrasound (Lipus) can induce the differentiation and activation of chondrocytes. This study was designed to evaluate the effect of Lipus on articular cartilage defects in a sheep. Eight sheep were divided in to two groups. The animals received bilateraly, articular cartilage defects 4 mm in diameter and 2 mm in deep on the patellar groove and experimental groups were treated with intensity 200 mW/cm2, 20 min/day with low-intensity pulsed ultrasound for 2 month. Then both knee joints underwent surgery for remove of formed tissue sample from defects.The samples were evaluated by Quantitative real-time polymerase chain reaction (qRT-PCR), Safranin-o staining, Immunofluorescence Staining and Morphological characterization. The best and worst sample per group according to Macroscopic and micriscopic scoring were icentified. The results showed that the operated groups with-Lipus-treatment and without-Lipus treatment had considered statistically significant. Gross photography revealed that the defects in experimental groups were filled with proliferative tissue, while in control groups, a thin layer of proliferative tissue was formed in defects. qRT-PCR results showed the expression of coll2, sox9, aggrecan and Osteocalcin in experimental groups. Intense safranin-O staining show the formation cartilage tissue in ultrasound treated group, while loose safranin-o-staining were observed at the control groups. Immunofluorescence staining showed the type 2 Collagen protein expression. We suggest that low-intensity pulsed ultrasound provide the mechanistic basis force for articular cartilage repair and effective treatment modality for improving of articular cartilage defects.

Differential Biological Behavior of Fibroblasts and Endothelial Cells under Aloe vera Gel Culturing.

Aloe vera is used for its large variety of biological activities such as wound healing, anti-fungal, anti-inflammatory, hypoglycemic, immunomodulatory, gastroprotective, and anti-cancer. Although the beneficial effects of Aloe vera on wound healing have been proven, little is known about its effects at the cellular level. In this study, we evaluated the angiogenic and migrative effects of Aloe vera gel on fibroblasts and endothelial cells. Fibroblasts and endothelial cells were cultured in monolayer conditions with low glucose DMEM with 10% serum and 1% penicillin-streptomycin. Fresh and mature leaves of Aloe vera were used for gel preparation. Cell proliferation and morphology were studied by an inverted microscope. The migration of fibroblasts was assessed by scratch assay. MTT assay was performed for cell viability assessment, and real-time RT-PCR was used for evaluation of PECAM-1, integrin α1 and ß1 transcription. After two days, the protein level of PECAM-1 was detected by flow cytometry. Our results showed that Aloe vera has a higher proliferative effect on fibroblasts in comparison with endothelial cells. Aloe vera also induced the migration of fibroblasts. The viability of both types of cells was similar to control ones. Integrin α1, ß1 and PECAM-1 gene expression increased significantly (P <0.005) in Aloe vera treated fibroblasts and endothelial cells in comparison with the control groups. However, the expression of these genes was significantly higher in fibroblasts in comparison with endothelial cells. Protein levels of PECAM-1 showed no change in both cell types upon Aloe vera treatment. Aloe vera gel induced angiogenic and cell adhesion properties in fibroblasts more than endothelial cells. Further investigations are needed to show the main role of fibroblasts rather than endothelial cells in wound healing by Aloe vera administration.

Effect of preheating on the cytotoxicity of bulk-fill composite resins.

Background. Due to the effect of pre-heating on the degree of conversion of composite resins and the possible effect on cytotoxicity, the effect of pre-heating of bulk-fill composite resins was investigated on cytotoxicity in this study. Methods. In this study, three different types of composite resin were used, including Tetric N-Ceram Bulk-Fil, Xtrafil, and Xtrabase. From each composite resin, 10 cylindrical samples (5 mm in diameter and 4 mm in height) were prepared, with five samples preheated to 68°C, and the other five samples polymerized at room temperature (25°C). Twenty-four hours after polymerization, cytotoxicity was assessed by MTT assay on human fibroblasts. Statistical analysis of data was carried out with two-way ANOVA and Sidak Post-Hoc. The significance level of the test was determined at 0.05. Results. There was no statistically significant difference between the mean percentage of cytotoxicity in terms of pre-heating (P>0.05), but the cytotoxicity of the studied composite resins was significantly different (P<0.001). The cytotoxicity of Tetric N-Ceram Bulk-fil composite resin was higher than that of the two other composite resins. Conclusion. Pre-heating of bulk-fill composite resin did not affect their cytotoxicity. In addition, the cytotoxicity of different bulk-fill composite resins was not the same.

Adipose-derived stem cells: An appropriate selection for osteogenic differentiation.

Mesenchymal stem cells (MSCs) are a major component of various forms of tissue engineering. MSCs have self-renewal and multidifferential potential. Osteogenic differentiation of MSCs is an area of attention in bone regeneration. One form of MSCs are adipose-derived stem cells (ASCs), which can be simply harvested and differentiated into several cell lineages, such as chondrocytes, adipocytes, or osteoblasts. Due to special properties, ASCs are frequently used in vitro and in vivo bone regeneration. Identifying factors involved in osteogenic differentiation of ASCs is important for better understanding the mechanism of osteogenic differentiation. Different methods are used to stimulate osteogenesis of ASCs in literature, including common osteogenic media, growth factors, hormones, hypoxia, mechanical and chemical stimuli, genetic modification, and nanotechnology. This review article provides an overview describing the isolation procedure, characterization, properties, current methods for osteogenic differentiation of ASCs, and their basic biological mechanism.

Aloe Vera/Collagen Mixture Induces Integrin α1ß1 and PECAM-1 Genes Expression in Human Adipose-Derived Stem Cells.

Purpose: Natural biomaterials are a key base in tissue engineering, and collagen, as the main content of the extracellular matrix (ECM), is frequently used in tissue engineering. Aloe vera has some therapeutic effects on ulcers, therefore, the use of this natural resource has always been considered for improving collagen function. We aimed to evaluate the effect of Aloe vera/ Collagen blended on cell viability, cell attachment, and angiogenic potential by determining of integrin α1ß1 and platelet endothelial cell adhesion molecule (PECAM-1) genes expression in human adipose-derived stem cells (hASCs). Methods: In this study, hASCs after harvesting of adipose tissues from abdominal subcutaneous adipose tissue and isolation, were cultured in four groups of control, collagen gel, Aloe vera gel, and Aloe vera/collagen blended in vitro environment at 24h and then cell viability was assessed by MTT (3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium) assay. Integrin α1ß1 and PECAM-1 genes expression were evaluated by real-time RT-PCR. Results: The results of MTT showed that the combination of Aloe vera/collagen was retained the cell viability at the normal range and improved it. In real-time RT-PCR results, integrin α1ß1 and PECAM-1 gene expression were increased in the Aloe vera/collagen blended group compared to the control group. Conclusion: For tissue engineering purposes, Aloe vera improves collagen properties in the culture of hASCs by increasing the expression of the integrin α1ß1 and PECAM-1 genes.

In vivo articular cartilage regeneration through infrapatellar adipose tissue derived stem cell in nanofiber polycaprolactone scaffold.

In this study, we report the development of a nanofiber polycaprolactone scaffold that can act as a stem cell carrier to induce chondrogenesis and promote cartilage repair in vivo. Infrapatellar fat pads were obtained from sheep knee and the stem cells were isolated and characterized by flow cytometry. Defects were created in sheep knee, two defects received adipose tissue derived stem cells (ASCs)-polycaprolactone construct, second group received polycaprolactone (PCL), the third group was chosen as the ASCs group and the fourth group was control group. Morphological evaluation showed that defects treated with ASCs-scaffold constructs were completely filled with cartilage-like tissue, while other groups revealed the formation of a thin layer of cartilage-like tissue in the defects. Real-Time RT-PCR showed the increase in collagen type 2 mRNA levels, aggrecan and Sox9 in ASCs/PCL groups in comparison with the other groups. Immunofluorescence and toluidine blue staining results showed the protein expression of collagen type 2 and formation of round and polygonal clusters of chondrocytes in ASCS/PCL group. According to our results nanofiber polycaprolactone promoted the chondrogenesis of infrapatellar adipose tissue derived stem cells in vivo and could offer significant promise in the biological functionality of stem cell tissue engineering in clinical practice.

An Investigation on the Regenerative Effects of Intra Articular Injection of Co-Cultured Adipose Derived Stem Cells with Chondron for Treatment of Induced Osteoarthritis.

Purpose: Adipose tissue derived stem cells (ASCs) and chondrocytes are best cells for articular cartilage regeneration. Chondrocyte with peri-cellular matrix (PCM) is called chondron provides ideal microenviroment than chondrocytes. We aimed to evaluate the regenerative effects of intra-articular injection of ASCs co-cultures with chondron in induced osteoarthritis (OA). Methods: ASC, from the peri-renal fat of male rat and chondron from primary newborn rat hyaline cartilage were isolated. ASCs were cultured for at least three passages in vitro. Six weeks after OA induction, rats were randomly distributed in five groups of control, osteoarthritic, ASC, chondron and co-cultured. ASCs (107), chondrons (107) and combination of chondrons and ASCs (107) were injected into intra-articular space of the rat knee. The effect of treatments was evaluated by macroscopic and microscopic examinations. The expression levels of collagen type ΙΙ was studied by immunohistochemistry. Results: Macroscopic appearance of the co-cultured group, showed much enhanced articular cartilage regeneration compared to ASC and chondron groups. H&E showed evidence of repair site of articular surface without erosion and fibrillation versus OA group which showed thin layer of hyaline cartilage over tidemark and spontaneous fibrocartilage formation. Metachromatic regions stained with toluidine blue were larger in treatment groups versus OA group. Strong intensity of type ΙΙ collagen staining was observed in co-culture group compared to other groups. Conclusion: Co-culture of chondrons and ASCs increased articular hyaline cartilage formation and provides a useful tool to improve limitations of each of applied cells in this model.

High Quality of Infant Chondrocytes in Comparison with Adult Chondrocytes for Cartilage Tissue Engineering.

BACKGROUND: Tissue engineering is used for the treatment of many diseases, and the ideal cell source for cartilage tissue engineering is chondrocytes. The main limitation of chondrocyte is the low number of cells in cartilage tissue engineering. This study investigated a suitable cell source with high proliferation rate to obtain a large number of chondrocytes. METHODS: Adult cartilage tissue samples were obtained from adult patients undergoing surgical procedure, and infant cartilage tissue samples were obtained from polydactyly surgical waste. After isolation and expansion of chondrocytes, the proliferation rate was evaluated by calculating population doubling time (PDT) and MTT assay for both types of cells. Cartilage film was prepared with sheets of over confluent chondrocytes. The cartilage tissue film from infant and adult chondrocytes were evaluated histologically and by immunefluorescent staining collagen type 2. RESULTS: PDT and MTT assays revealed that the growth rate of the infant chondrocytes was significantly higher than adult chondrocytes. Histological findings showed that sheets were thicker in the cartilage film of infant chondrocytes and they had more extracellular matrix between the sheets of cells than the cartilage film of adult chondrocytes. The findings of the immunofluorescent staining of cartilage film indicated that collagen type II film of polyductily was more positive than adult chondrocytes. CONCLUSION: The recent study presented a new cell source to overcome the limitation of low number of chondrocytes for cell therapy of cartilage defects in adults and also sheets of cells able to overcome the problems of scaffolds.

Comparison of Characteristics of Human Amniotic Membrane and Human Adipose Tissue Derived Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are ideal candidates for treatment of diseases. Amniotic membranes are an inexpensive source of MSCs (AM-MSC) without any donor site morbidity in cell therapy. Adipose tissue derived stem cells (ASCs) are also suitable cells for cell therapy. There is discrepancy in CD271 expression among MSCs from different sources. In this study, the characteristics of AM-MSC and ASCs and CD271 expression were compared. METHODS: Adult adipose tissue samples were obtained from patients undergoing elective surgical procedure, and samples of amniotic membrane were collected immediately after caesarean operation. After isolation and expansion of MSCs, the proliferation rate and viability of cells were evaluated through calculating DT and MTT assay. Expression of routine mesenchymal specific surface antigens of MSCs and CD271 was evaluated by flow cytometry for both types of cells. RESULTS: The growth rate and viability of the MSCs from the amniotic membrane was significantly higher compared with the ASCs. The low expression of CD14 and CD45 indicated that AM-MSC and ASCs are non hematopoietic cells, and both cell types expressed high percentages of CD44, CD105. The results revealed that AM-MSC and ASCs expressed no CD271 on their surfaces. CONCLUSION: This study showed that amniotic membrane is a suitable cell source for cell therapy, and CD271 is a negative marker for MSCs identification from amniotic membrane and adipose tissue.

Importance of Floating Chondrons in Cartilage Tissue Engineering.

BACKGROUND: Dedifferentiation of chondrocytes remains a major problem for cartilage tissue engineering. Chondrocytes loss differentiated phenotype in in vitro culture that is undesired for repair strategies. The chondrocyte is surrounded by a pericellular matrix (PCM), together forming the chondron. PCM has a positive effect on the maintenance of chondrocyte phenotype during culture in comparison to uncovered chondrocyte. Studies suggest that the PCM influence on functional properties of the chondrocytes. However there is no study to show gene expression phenotype differences between round chondron and fibroblastic chondrocytes. We aimed to investigate the effect of pericellular matrix in maintaining of chondrogenic gene expression to solve dedifferentiation problem of chondrocyte. METHODS: In this study enzymatically isolated chondrons were cultured for 7 days. Morphology of chondrons were assessed by microscopic examination. Chondrogenic gene expression of Sox9, aggrecan (AGG), cartilage oligomeric matrix protein (COMP), Link protein and chondro-osteogenic gene expression (Runx2, Col1, Col 10 and MMP13) of attached and float chondrons were assessed by real time RT PCR. RESULTS: Microscopic observation showed that round shape of chondron observed at day 7 in floating chondrocytes. Gene expression results showed that attached chondrons significantly dedifferentiated by low gene expression of Sox9 and COMP and high MMP13 versus floating cells. CONCLUSION: Our results showed that PCM of chondrocyte could restore differentiated state of chondrocytes at day 7. Using unattached form of chondron in cartilage tissue PCM in maintenance of chondrogenic gene expression engineering could be a novel method to solve dedifferentiation problem of chondrocyte.

Indirect coculture of stem cells with fetal chondrons using PCL electrospun nanofiber scaffolds.

In vitro coculture system provides a powerful tool for tissue engineering. In this study, we evaluated the gene expressions of human adipose-derived stem cells (ASCs) on polycaprolactone (PCL) scaffold in coculture model with fetal chondrons. Electrospun PCL scaffolds (900 nm fiber diameter) were created and human infrapatellar fat pad-adipose-derived stem cells (IPFP-ASCs) were seeded on these scaffolds. Scanning electron microscopy (SEM) showed attachment of human IPFP-ASCs to scaffold. IPFP-ASCs on scaffolds were cocultured with fetal chondrons in transwell. Gene expressions were investigated using real-time polymerase chain reaction (real-time PCR). In comparison with control group, the expression level of collagen type 2 and aggrecan were significantly decreased but Indian Hedgehog(IHH) significantly increased (P < 0.05).These findings may interpreted that IPFP-ASCs seeded on PCL scaffold, in cocultures with fetal chondrons are tending toward osteogenesis rather than chondrogenesis.

Study of chondrogenic potential of stem cells in co-culture with chondrons.

OBJECTIVES: Three-dimensional biomimetic scaffolds have widespread applications in biomedical tissue engineering due to similarity of their nanofibrous architecture to native extracellular matrix. Co-culture system has stimulatory effect on chondrogenesis of adult mesenchymal stem cells. This work presents a co-culture strategy using human articular chondrons and adipose-derived stem cells (ASCs) from infrapatellar fat pad (IPFP) for cartilage tissue production. MATERIALS AND METHODS: Isolated stem cells were characterized by flowcytometry. Electrospun and polycaprolactone (PCL) scaffolds (900 nm fiber diameter) was obtained from Bon Yakhteh (Tehran-Iran) and human infrapatellar fat pad-derived stem cells (IPFP-ASCs) were seeded on them. IPFP-ASCs on scaffolds were co-cultured with articular chondrons using transwell. After 21 day, chondrogenic differentiation of stem cell was evaluated by determining the genes expression of collagen2, aggrecan and Indian hedgehog using real-time RT-PCR. RESULTS: Genes expression of collagen2, aggrecan by IPFP-ASCs did not alter significantly in comparison with control group. Howevers, expression of Indian hedgehog decreased significantly compared to control group (P< 0.05). CONCLUSION: These findings indicate that chondrons obtained from osteoarthritic articular cartilage did not stimulate chondrogenic differentiation of IPFP-ASCs in co-culture.