BACKGROUND: Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects oftherapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to humanhepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and thepresence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this studywas to generate the scalable and functional hepatic micro-tissues (HMTs). METHODS: The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cellsand human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, usingan air-driven droplet generator. RESULTS: The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoringglycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretionlevels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involvedin hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional(2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity tohepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450enzymes confirmed that the HMTs can be used for in vitro drug screening. CONCLUSION Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7,with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.

BACKGROUND: Loss of the dental and paradental tissues resulting from trauma, caries or from systemic diseasesconsidered as one of the most significant and frequent clinical problem to the healthcare professionals. Great attempts havebeen implemented to recreate functionally, healthy dental and paradental tissues in order to substitute dead and diseasedtissues resulting from secondary trauma of car accidents, congenital malformations of cleft lip and palate or due to acquireddiseases such as cancer and periodontal involvements.METHOD: An extensive literature search has been done on PubMed database from 2010 to 2019 about the challenges ofengineering a biomimetic tooth (BioTooth) regarding basic biology of the tooth and its supporting structures, strategies,and different techniques of obtaining biological substitutes for dental tissue engineering. RESULTS: It has been found that great challenges need to be considered before engineering biomimetic individual parts of thetooth such as enamel, dentin-pulp complex and periodontium. In addition, two approaches have been adopted to engineer aBioTooth.The first one was to engineer a BioTooth as an individual unit and the other was to engineer a BioToothwith its supporting structures. CONCLUSION Engineering of BioTooth with its supporting structures thought to be in the future will replace the traditionaland conventional treatment modalities in the field of dentistry. To accomplish this goal, different cell lines and growthfactors with a variety of scaffolds at the nano-scale level are now in use. Recent researches in this area of interest arededicated for this objective, both in vivo and in vitro. Despite progress in this field, there are still many challenges ahead andneed to be overcome, many of which related to the basic tooth biology and its supporting structures and some others related tothe sophisticated techniques isolating cells, fabricating the needed scaffolds and obtaining the signaling molecules.

BACKGROUND: Fetal bovine serum is widely used as a growth supplement for cell culture medium; however, animalbornepathogens increase the risk of transmitting infectious agents. Platelet-rich fibrin is recently considered as a successfulalternative but leukocytes present limits to its allogeneic feasibility. The aim of this study was to explore the effects ofallogeneic fibrin clot (AFC) without leukocytes on inducing odontogenic/cementogenic differentiation of human dentalpulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs) in vitro and in vivo. METHODS: AFC was prepared by high-speed centrifugation and leukocytes were almost removed, and AFC serum wasobtained through three freeze–thaw cycles. hDPSCs and hPDLSCs were treated with AFC serum to investigate theodontogenic or cementogenic associated markers by real-time polymerase chain reaction. hDPSCs were treated with AFCserum and placed inside of dentin canal, hPDLSCs were treated with AFC serum to wrap outside of dentin, the mixture wasthen transplanted into the subcutaneous of nude mice for 12 weeks. RESULTS: AFC serum exhibited enough growth factors and cytokines to induce odontogenic/cementogenic differentiationof hDPSCs and hPDLSCs in vitro. Furthermore, AFC seurum could induce hDPSCs to differentiate into odontoblastslikecells and pulp-like tissues, and hPDLSCs to regenerate cementum-like tissues. CONCLUSION AFC could be an alternative safe source with growth factors for the expansion of human dental mesenchymalstem cells (hDMSCs).

BACKGROUND: The urinary tract can be affected by both congenital abnormalities as well as acquired disorders, such ascancer, trauma, infection, inflammation, and iatrogenic injuries, all of which may lead to organ damage requiring eventualreconstruction. As a gold standard, gastrointestinal segment is used for urinary bladder reconstruction. However, one majorproblem is that while bladder tissue prevents reabsorption of specific solutes, gastrointestinal tissue actually absorbs them.Therefore, tissue engineering approach had been attempted to provide an alternative tissue graft for urinary bladderreconstruction. METHODS: Human adipose-derived stem cells isolated from fat tissues were differentiated into smooth muscle cells andthen seeded onto a triple-layered PLGA sheet to form a bladder construct. Adult athymic rats underwent subtotal urinarybladder resection and were divided into three treatment groups (n = 3): Group 1 (‘‘sham’’) underwent anastomosis of theremaining basal region, Group 2 underwent reconstruction with the cell-free scaffold, and Group 3 underwent reconstructionwith the tissue-engineered bladder construct. Animals were monitored on a daily basis and euthanisation wasperformed whenever a decline in animal health was detected. RESULTS: All animals in Groups 1, 2 and 3 survived for at least 7 days and were followed up to a maximum of 12 weekspost-operation. It was found that by Day 14, substantial ingrowth of smooth muscle and urothelial cells had occurred inGroup 2 and 3. In the long-term follow up of group 3 (tissue-engineered bladder construct group), it was found that theurinary bladder wall was completely regenerated and bladder function was fully restored. Urodynamic and radiologicalevaluations of the reconstructed bladder showed a return to normal bladder volume and function.Histological analysisrevealed the presence of three muscular layers and a urothelium similar to that of a normal bladder. Immunohistochemicalstaining using human-specific myocyte markers (myosin heavy chain and smoothelin) confirmed the incorporation of theseeded cells in the newly regenerated muscular layers. CONCLUSION Implantation of PLGA construct seeded with smooth muscle cells derived from human adipose stemcells can lead to regeneration of the muscular layers and urothelial ingrowth, leading to formation of a completelyfunctional urinary bladder.

BACKGROUND: Mesenchymal stem cell-based treatments are now emerging as a therapy for corneal epithelial damage.Although bone marrow, adipose tissue and umbilical cord blood are the main sources of mesenchymal stem cells (MSCs),other tissues like the peripheral blood also harbor mesenchymal-like stem cells called peripheral blood-derivedmononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasivecollection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneallimbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymalcell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explantconditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiatedPBMNCs were engineered into a cell sheet using an in-house developed thermo-responsive polymer. RESULTS: These blood derived cells were demonstrated to have similar properties to mesenchymal stem cells. Cornealepithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker therebydemonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitrocell sheet that was then demonstrated for cell sheet transfer on an ex vivo excised rabbit eye. CONCLUSION PBMNCs as an alternative autologous cell source for limbal stem cells is envisaged as an effectivetherapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency.

BACKGROUND: Hair loss is a prevalent medical problem in both men and women. Maintaining the potential hair inductivityof dermal papilla cells (DPCs) during cell culture is the main factor in hair follicle morphogenesis and regeneration. Thepresent study was conducted to compare the effects of different concentrations of human hair outer root sheath cell(HHORSC)and platelet lysis (PL) exosomes to maintain hair inductivity of the human dermal papilla cells (hDPCs). METHODS: In this study, hDPCs and HHORSCswere isolated from healthy hair samples. Specific markers of hDPCs (versican,a-SMA) and HHORSCs (K15) were evaluated using flow cytometric and immunocytochemical techniques. The exosomes wereisolated fromHHORSCsand PL with ultracentrifugation technique.Western blot was used to detect specific markers of HHORSCsand PL exosomes. Particle size and distribution of the exosomes were analyzed by NanoSight dynamic light NanoSight DynamicLight Scattering. Different methods such as proliferation test (MTS assay), migration test (Transwell assay) were used to evaluatethe effects of different concentrations of exosomes (2,550,100 lg/ml) derived from HHORSC and PL on hDPCs. Expression ofspecific genes in the hair follicle inductivity, including ALP, versican and a-SMA were also evaluated using real time-PCR. RESULTS: The flow cytometry of the specific cytoplasmic markers of the hDPCs and HHORSCs showed expression ofversican (77%), a-SMA (55.2%) and K15 (73.2%). The result of particle size and distribution of the exosomes wereanalyzed by NanoSight dynamic light NanoSight Dynamic Light Scattering, which revealed the majority of HHORSC andPL exosomes were 30–150 nm. For 100 lg/ml of HHORSC exosomes, the expressions of ALP, versican and a-SMAproteins respectively increased by a factor of 2.1, 1.7and 1.3 compared to those in the control group. CONCLUSION In summary, we applied HHORSC exosomes as a new method to support hair inductivity of dermalpapilla cells and improve the outcome for the treatment of hair loss.

BACKGROUND: The extracellular vesicles (EVs) secreted by bone marrow-derived mesenchymal stem cells (MSCs)hold significant potential as a novel alternative to whole-cell therapy. We herein compare the therapeutic potential of BMMSCsversus their EVs (MSC-EVs) in an experimental Carbon tetrachloride (CCl4)-induced liver damage rat model. METHODS: Rats with liver damage received a single IV injection of MSC-EVs, 1 million MSCs, or 3 million MSCs. Thetherapeutic efficacy of each treatment was assessed using liver histopathology, liver function tests and immunohistochemistryfor liver fibrosis and hepatocellular injury. RESULTS: Animals that received an injection of either MSCs-EVs or 3 million MSCs depicted significant regression ofcollagen deposition in the liver tissue and marked attenuation of hepatocellular damage, both structurally and functionally. CONCLUSION Similar to high doses of MSC-based therapy (3 million MSCs), MSC-EVs mitigated the fibrogenesis andhepatocellular injury in a rat model of CCl4-induced liver fibrosis. The anti-fibrinogenic effect was induced by attenuatinghepatic stellate cell activation. Therefore, the administration of MSC-EVs could be considered as a candidate cell-freetherapeutic strategy for liver fibrosis and hepatocellular damage.

BACKGROUND: Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bonedefects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelialgrowth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1a). This studyassessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derivedmesenchymal stem/stromal cells (AT-MSCs). METHODS: Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response.Immunostaining and western-blots served to verify the HIF-1a stabilization response. The optimized concentrations forlong-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiationof AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogeneticprotein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secretedphosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4(KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. RESULTS: PHIs stabilized HIF-1a in a dose-dependent manner and showed evident dose- and time dependent antiproliferativeeffects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic inductionon the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressedosteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. CONCLUSION PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemnessrelatedgenes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on theosteogenic differentiation of AT-MSCs.

BACKGROUND: Introduction of pro-angiogenic cells into tissue-engineered (TE) constructs (prevascularisation) is apromising approach to overcome delayed neovascularisation of such constructs post-implantation. Accordingly, in thisstudy, we examined the contribution of human dermal microvascular endothelial cells (HDMECs) and human dermalfibroblasts (HDFs) alone and in combination on the formation of new blood vessels in ex-ovo chick chorioallantoicmembrane (CAM) assay. METHODS: Poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) and polycaprolactone (PCL) were first examined interms of their physical, mechanical, and biological performances. The effect of gelatin coating and co-culture conditions onenhancing endothelial cell viability and growth was then investigated. Finally, the angiogenic potential of HDMECs andHDFs were assessed macroscopically and histologically after seeding on simple electrospun PHBV scaffolds either inisolation or in indirect co-culture using an ex-ovo CAM assay. RESULTS: The results demonstrated that PHBV was slightly more favourable than PCL for HDMECs in terms of cellmetabolic activity. The gelatin coating of PHBV scaffolds and co-culture of HDMECs with HDFs both showed a positiveimpact on HDMECs viability and growth. Both cell types induced angiogenesis over 7 days in the CAM assay either inisolation or in co-culture. The introduction of HDMECs to the scaffolds resulted in the production of more blood vessels inthe area of implantation than the introduction of HDFs, but the co-culture of HDMECs and HDFs gave the most significantangiogenic activity. CONCLUSION Our findings showed that the in vitro prevascularisation of TE constructs with HDMECs and HDFs aloneor in co-culture promotes angiogenesis in implantable TE constructs.