Stem cells technology as a platform for generating reproductive system organoids and treatment of infertility-related diseases.

In recent years, stem cells have known as a helpful biological tool for the accurate diagnosis, treatment and recognition of diseases. Using stem cells as biomarkers have presented high potential in the early detection of many diseases. Another advancement in stem cell technology includes stem cell derived organoids model that could be a promising platform for diagnosis and modeling different diseases. Furthermore, therapeutic capabilities of stem cell therapy have increased hope in the face of different disability managements. All of these technologies are also widely used in reproductive related diseases especially in today's world that many couples encounter infertility problems. However, with the aid of numerous improvements in the treatment of infertility, over 80% of couples who dreamed of having children could now have children. Due to the fact that infertility has many negative effects on personal and social lives of young couples, many researchers have focused on the treatment of male and female reproductive system abnormalities with different types of stem cells, including embryonic stem cells, bone marrow mesenchymal stem cells (MSCs), and umbilical cord-derived MSCs. Also, design and formation of reproductive system organoids provide a fascinating window into disease modeling, drug screening, personalized therapy, and regeneration medicine. Utilizing these techniques to study, model and treat the infertility-related diseases has drawn attention of many scientists. This review explains different applications of stem cells in generating reproductive system organoids and stem cell-based therapies for male and female infertility related diseases treatment.

Thermo/pH dual-responsive micelles based on the host-guest interaction between benzimidazole-terminated graft copolymer and ß-cyclodextrin-functionalized star block copolymer for smart drug delivery.

Novel temperature and pH dual-sensitive amphiphilic micelles were fabricated exploiting the host-guest interaction between benzimidazole-terminated PHEMA-g-(PCL-BM) and ß-CD-star-PMAA-b-PNIPAM. The fabricated graft copolymer had a brush-like structure with star side chains. The micelles were utilized as dual-responsive nanocarriers and showed the LCST between 40 and 41 °C. The acidic pH promoted the dissociation of the PHEMA-g-(PCL-BM: ß-CD-star-PMAA-b-PNIPAM) micelles. DOX.HCl was loaded into the core of the micelles during self-assembly in an aqueous solution with a high encapsulation efficacy (97.3%). The average size of the amphiphilic micelles was about 80 nm, suitable size for the enhanced permeability and retention effect in tumor vasculature. In an aqueous environment, these micelles exhibited very good self-assembly ability, low CMC value, rapid pH- and thermo-responsiveness, optimal drug loading capacity, and effective release of the drug. The biocompatibility was confirmed by the viability assessment of human breast cancer cell line (MCF-7) through methyl tetrazolium assay. DOX-loaded micelles displayed excellent anti-cancer activity performance in comparison with free DOX.

Evaluation the ability of acellular ovine small intestine submucosa to load and release of mineral pitch and its anti-inflammatory effects.

Injury from the severe burn is exacerbated by a persistent inflammatory response. This response is mediated by cytokines and chemokines, which are released from various immune cells, including mast cells. In this study, the ability of the acellular ovine small intestine submucosa (AOSIS) to load and release of Mineral Pitch (MP) was first investigated, and it was found that the preparation of the scaffold by a modified method enables it to load and release water-soluble drugs. Then, 32 male Wistar rats were divided into four groups, a third-degree burn was created, and except for the control group, the others were treated with: AOSIS, WJ-MSCs seeded AOSIS, or AOSIS loaded with WJ-MSCs and MP. Wound sampling on the 5th day after treatment showed that the number of intact and degranulated mast cells in the treatment groups was associated with a decrease compared to the control group. In the last group, this decrease was the largest (and statically significant (p < 0.05)). Also, by measuring the level of inflammatory factors in blood serum, it was found that in the treatment groups compared to the control group, IL-10 was associated with an increase, and TNF-α was associated with a decrease. The changes in inflammatory factors were more significant (p < 0.05) in the last group. So, our results indicate that AOSIS loaded with WJ-MSCs and MP could be used as an innovative tissue-engineered device to control inflammatory condition during burn wound healing.

Electrospun polyurethane/poly (ɛ-caprolactone) nanofibers promoted the attachment and growth of human endothelial cells in static and dynamic culture conditions.

In this study, the angiogenic capacity of human endothelial cells was studied after being plated on the surface of polyurethane-poly caprolactone (PU/PCL) scaffolds for 72 h. In this study, cells were designated into five different groups, including PU, PU/PCL (2:1), PU/PCL (1:1); PU/PCL (1:2); and PCL. Data revealed that the PU/PCL (2:1) composition had a higher modulus and breakpoint in comparison with the other groups (p < 0.05). Compared to the other groups, the PU/PCL scaffold with a molar ratio of 2:1 had lower the contact angle θ and higher tensile stress (p < 0.05). The mean size of the PU nanofibers was reduced after the addition of PCL (p < 0.05). Based on our data, the culture of endothelial cells on the surface of PU/PCL (2:1) did not cause nitrosative stress and cytotoxic effects under static conditions compared to cells plated on a conventional plastic surface (p > 0.05). Based on data from the static condition, we fabricated a tubular PU/PCL (2:1) construct for six-day dynamic cell culture inside loop air-lift bioreactors. Scanning electron microscopy showed the attachment of endothelial cells to the luminal surface of the PU/PCL scaffold. Cells were flattened and aligned under the culture medium flow. Immunofluorescence imaging showed the attachment of cells to the luminal surface indicated by blue nuclei on the luminal surface. These data demonstrated that the application of PU/PCL substrate could stimulate endothelial cells activity under static and dynamic conditions.

An improved method in fabrication of smart dual-responsive nanogels for controlled release of doxorubicin and curcumin in HT-29 colon cancer cells.

The combination therapy which has been proposed as the strategy for the cancer treatment could achieve a synergistic effect for cancer therapies and reduce the dosage of the applied drugs. On account of the the unique properties as the high absorbed water content, biocompatibility, and flexibility, the targeting nanogels have been considred as a suitable platform. Herein, a non-toxic pH/thermo-responsive hydrogel P(NIPAAm-co-DMAEMA) was synthesized and characterized through the free-radical polymerization and expanded upon an easy process for the preparation of the smart responsive nanogels; that is, the nanogels were used for the efficient and controlled delivery of the anti-cancer drug doxorubicin (DOX) and chemosensitizer curcumin (CUR) simultaneously like a promising strategy for the cancer treatment. The size of the nanogels, which were made, was about 70 nm which is relatively optimal for the enhanced permeability and retention (EPR) effects. The DOX and CUR co-loaded nanocarriers were prepared by the high encapsulation efficiency (EE). It is important to mention that the controlled drug release behavior of the nanocarriers was also investigated. An enhanced ability of DOX and CUR-loaded nanoformulation to induce the cell apoptosis in the HT-29 colon cancer cells which represented the greater antitumor efficacy than the single-drug formulations or free drugs was resulted through the In vitro cytotoxicity. Overall, according to the data, the simultaneous delivery of the dual drugs through the fabricated nanogels could synergistically potentiate the antitumor effects on the colon cancer (CC).

Multiple functions of microfluidic platforms: Characterization and applications in tissue engineering and diagnosis of cancer.

Microfluidic system, or lab-on-a-chip, has grown explosively. This system has been used in research for the first time and then entered in the clinical section. Due to economic reasons, this technique has been used for screening of laboratory and clinical indices. The microfluidic system solves some difficulties accompanied by clinical and biological applications. In this review, the interpretation and analysis of some recent developments in microfluidic systems in biomedical applications with more emphasis on tissue engineering and cancer will be discussed. Moreover, we try to discuss the features and functions of microfluidic systems.

Design, synthesis, biological evaluation, and docking study of novel dual-acting thiazole-pyridiniums inhibiting acetylcholinesterase and ß-amyloid aggregation for Alzheimer's disease.

New compounds containing thiazole and pyridinium moieties were designed and synthesized. The potency of the synthesized compounds as selective inhibitors of acetylcholinesterase (AChE), and ß-amyloid aggregation (Aß) was evaluated. Compounds 7d and 7j showed the best AChE inhibitory activities at the submicromolar concentration range (IC50 values of 0.40 and 0.69 µM, respectively). Most of the novel compounds showed moderate to low inhibition of butyrylcholinesterase (BChE), which is indicative of their selective inhibitory effects towards AChE. Kinetic studies using the most potent compounds 7d and 7j confirmed a mixed-type of AChE inhibition mechanism in accordance with the docking results, which shows their interactions with both catalytic active (CAS) and peripheral anionic (PAS) sites. The specific binding of 7a, 7j, and 7m to PAS domain of AChE was also confirmed experimentally. In addition, 7d and 7j were able to show ß-amyloid self-aggregation inhibitory effects (20.38 and 42.66% respectively) stronger than donepezil (14.70%) assayed at 10 µM concentration. Moreover, compounds 7j and 7m were shown to be effective neuroprotective agents in H2O2-induced oxidative stress on PC12 cells almost similar to those observed for donepezil. The ability of 7j to pass blood-brain barrier was demonstrated using the PAMPA method. The results presented in this work provide useful information about designing novel anti-Alzheimer agents.

Multifunctional nanoparticles from albumin for stimuli-responsive efficient dual drug delivery.

In this project methotrexate (MTX) conjugated albumin based nanoparticles (MTX-BSA) loaded with curcumin (CUR) drug (CUR-MTX-BSA) for simultaneous delivery of multi-chemotherapeutic drugs and combination cancer therapy were designed. Co-delivery is a new strategy which minimize the amount of each drug, reduce of side effects and also to achieve the synergistic effect for cancer therapies. The MTX was conjugated to albumin via covalent bond. Next, this synthesized prodrug loaded with CUR. Afterward, the formulations were evaluated for physical and chemical properties by DLS, TEM, FTIR, UV/Vis, DSC analysis, in vitro cytotoxicity and in vivo biocompatibility studies. Furthermore, the drug loading and release study were evaluated. Proteinase K enzyme was used to break amid bond between MTX and BSA and also amidic bonds in BSA structure. Administration of up to 2000 mg/kg of BSA to healthy animals was non-toxic and all treated mice were still alive after 24 h. The result of this study proved that CUR-MTX-BSA can be used as a proficient vehicle for effective co-delivery of CUR and MTX in the treatment of cancer.

Retraction Note to: Synthesis, characterization and in vitro studies of doxorubicin-loaded magnetic nanoparticles grafted to smart copolymers on A549 lung cancer cell line.

The Editors have retracted this article [1] because Figs. 6a and 6c have been used in three other publications to represent scanning electron micrographs of different nanoparticles [2-4]. The data reported in this article are therefore unreliable. In addition, Fig. 3 was reproduced from [5] with retrospective permission and the credit line should read as follows: "Reprinted from Acta Biomaterialia, Volume 3, Zhang, J. and Misra, R.D.K., Magnetic drug-targeting carrier encapsulated with thermosensitive smart polymer: core-shell nanoparticle carrier and drug release response, pp. 838-850, copyright (2007) with permission from Acta Materialia Inc. Authors Abolfazl Akbarzadeh, Maryam Anzaby, Soodabeh Davaran, Sang Woo Joo and Mohammad Samiei agree to this retraction. Authors Younes Hanifehpour and Hamid Tayefi Nasrabadi have not responded to any correspondence about this retraction.

Methotrexate-conjugated L-lysine coated iron oxide magnetic nanoparticles for inhibition of MCF-7 breast cancer cells.

Methotrexate (MTX), a stoichiometric inhibitor of dihydrofolate reductase enzyme, is a chemotherapeutic agent for treating a diversity of neoplasms. In this study, we design and developed a new formulation of MTX that serves as drug carrier and examined its cytotoxic effect in vitro. This target drug delivery system is dependent on the release of the MTX within the lysosomal compartment. The iron oxide magnetic nanoparticles (IONPs) were first surface-coated with L-lysine and subsequently conjugated with MTX through amidation between the carboxylic acid end groups on MTX and the amine groups on the IONPs surface. MTX-conjugated L-lysine coated IONPs (F-Lys-MTX NPs) was characterized by X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy, vibrating sample magnetometer, and transmission electron microscopy techniques. The cytotoxicity of the void of MTX and F-Lys-MTX NPs were compared to each other by MTT assay of the treated MCF-7 cell lines. The results showed that the ζ-potential of F-Lys-MTX NPs was about -5.49 mV and the average size was 43.72 ± 4.73 nm. Model studies exhibited the release of MTX via peptide bond cleavage in the presence of proteinase K and at low pH. These studies specify that F-Lys-MTX NPs have a very remarkable anticancer effect, for breast cancer cell lines.

Theranostic nanoparticles based on magnetic nanoparticles: design, preparation, characterization, and evaluation as novel anticancer drug carrier and MRI contrast agent.

In this work, we reported the synthesis of curcumin (CUR)-loaded hydrophilic and hydrophobic natural amino acids (AAs)-modified iron oxide magnetic nanoparticles (IONPs). Two types of AAs, l-lysine (Lys) and l-phenylalanine (PhA), were selected to study their effects on loading capacity, release profile of CUR, biocompatibility, and anticancer activity. CUR-loaded AAs-modified IONPs (F@AAs@CUR NPs) were characterized by X-ray diffraction (XRD), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM), and transmission electron microscopy (TEM) techniques. Next, the various kinetic equations were fitted to the release data of CUR from F@Lys@CUR NPs and F@PhA@CUR NPs. Additionally, hemolysis test and MTT assays on HFF-2 and HEK-293 cell lines were performed for determination of biocompatibility of AAs-coated IONPs. Finally, the anticancer activity of F@AAs@CUR NPs examined on MCF-7 breast cancer cell line. The results indicate that these nanocarriers are nontoxic and biocompatible and also F@AAs@CUR NPs are suitable carriers for delivery of curcumin and even other hydrophobic drugs. Also, the MRI training established the effectiveness of IONPs as contrast agent for the revealing of tumor as evidenced from the phantom images as well as higher T2 relaxivity.

pH- and thermo-sensitive MTX-loaded magnetic nanocomposites: synthesis, characterization, and in vitro studies on A549 lung cancer cell and MR imaging.

In the current study, we proposed a facile method for fabrication of multifunctional pH- and thermo-sensitive magnetic nanocomposites (MNCs) as a theranostic agent for using in targeted drug delivery and magnetic resonance imaging (MRI). To this end, we decorated Fe3O4 magnetic nanoparticles (MNPs) with N,N-dimethylaminoethyl methacrylate (DMAEMA) and N-isopropylacrylamide (NIPAAm), best known for their pH- and thermo-sensitive properties, respectively. We also conjugated mesoporous silica nanoparticles (MSNs) to polymer matrix acting as drug container to enhance the drug encapsulation efficacy. Methotroxate (MTX) as a model drug was successfully loaded in MNCs (M-MNCs) via surface adsorption onto MSNs and electrostatic interaction between drug and carrier. The pH- and temperature-triggered release of MTX was concluded through the evaluation of in vitro release at both physiological and simulated tumor tissue conditions. Based on in vitro cytotoxicity assay results, M-MNCs significantly revealed higher antitumor activity compared to free MTX. In vitro MR susceptibility experiment showed that M-MNCs relatively possessed high transverse relaxivity (r2) of about 0.15 mM-1·ms-1 and a linear relationship between the transverse relaxation rate (R2) and the Fe concentration in the M-MNCs was also demonstrated. Therefore, the designed MNCs can potentially become smart drug carrier, while they also can be promising MRI negative contrast agent.

Preparation, characterization, and evaluation of amino acid modified magnetic nanoparticles: drug delivery and MRI contrast agent applications.

This study is a report about the synthesis iron oxide magnetic nanoparticles (IONPs) which modified with positive and negative charged amino acids (AAs). l-Arginine (Arg) and l-aspartic acid (Asp) which have of guanidinium and carboxylic acid groups, respectively, were selected for this study. After loading chrysin in amino acids modified iron oxide magnetic nanoparticles (F@AAs@Chrysin NPs), it was characterized by XRD, TGA, FTIR, VSM, and TEM techniques. Finally, MTT assays on HFF-2 and HEK-293 cell lines were performed for determination of biocompatibility of AA coated IONPs. The results show that, the ζ-potential and average size of F@Arg@chrysin NPs and F@Asp@chrysin NPs were to -3.87, -2.12 mV, 18.75 ± 2.40 (mean ± SD (n = 50)) nm, and 19.86 ± 2.22 (mean ± SD (n = 48)) nm, respectively. Also, the results indicated that these F@AAs@Chrysin NPs were appropriate for delivery of chrysin. Furthermore, the phantom MRI studies showed the IONPs can be used as contrast agent for the revealing of tumor.

Co-delivery of hydrophilic and hydrophobic drugs by micelles: a new approach using drug conjugated PEG-PCLNanoparticles.

Co-delivery strategy has been proposed to minimize the amount of each drug and to achieve the synergistic effect for cancer therapies. A conjugate of the antitumor drug, doxorubicin, with diblock methoxy poly (ethylene glycol)-poly caprolactone (mPEG-PCL) copolymer was synthesized by the reaction of mPEG-PCL copolymer with doxorubicin in the presence of p-nitrophenylchloroformate. The conjugated copolymer was characterized in vitro by 1H-NMR, FTIR, DSC and GPC techniques. Then, the doxorubicin conjugated mPEG-PCL(DOX-mPEG-PCL) was self-assembled into micelles in the presence of curcumin in aqueous solution. The resulting micelles were characterized further by various techniques such as dynamic light scattering (DLS) and atomic force microscopy (AFM).The encapsulation efficiency of doxorubicin and curcumin were 82.31 ± 3.32 and 78.15 ± 3.14%, respectively. The results revealed that the micelles formed by the DOX-mPEG-PCL with and without curcumin have spherical structure with average size of 116 and 134 nm respectively. The release behavior of curcumin and doxorubicin loaded to micelles were investigated in a different media. The release rate of micelles consisted of the conjugated copolymer was pH dependent as it was higher at lower pH than in neutral condition. Another feature of the conjugated micelles was a sustained release profile. The cytotoxicity of micelles were evaluated by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, atetrazole) assay on lung cancer A549 cell lines. In vitro cytotoxicity assay showed that the mPEG-PCL copolymer did not affect the growth of A549 cells. The cytotoxic activity of the micelles against A549 cells was greater than free doxorubicin and free curcumin.

Fluorescent multi-responsive cross-linked P(N-isopropylacrylamide)-based nanocomposites for cisplatin delivery.

Magnetic, pH and temperature-sensitive, poly(N-isopropylacrylamide) (PNIPAM)-based nanocomposites with fluorescent properties were synthesized by free radical copolymerization-cross linking of NIPAM, N,N-dimethylaminoethyl methacrylate (DMAEMA) and 4-acrylamidofluorescein (AFA). The model anti-cancer drug, cisplatin (CDDP), was loaded into the resulted nanogel. For the production of CDDP-loaded nanocomposite, Fe3O4 magnetic nanoparticles (MNPs) and CDDP were loaded into the nanogel. Field-emission scanning electron microscopy (FE-SEM) indicated that the size of nanogel and CDDP-loaded nanocomposite were about 90 and 160 nm, respectively. The encapsulation efficiency of CCDP was found up to 65%. The loaded CCDP showed sustained thermal and pH-responsive drug release. A high level of drug release was observed under the conditions of low pH and high temperature. The lower critical solution temperature (LCST) of synthesized nanogel was about 40 °C. CDDP-loaded nanocomposite showed a volume phase transition from 282 to 128 nm at its LCST. Accordingly, in this study, the synthesized nanocomposite can be employed as a stimuli-responsive anti-cancer drug delivery system and the pH and temperature of solution have the potential to monitor the drug release.

Preparation and in vivo evaluation of in situ gel system as dual thermo-/pH-responsive nanocarriers for sustained ocular drug delivery.

OBJECTIVE: Ciprofloxacin (CIP) was effective in treating bacterial keratitis. The purpose of this study was to prepare an effective prolonged-release of CIP by both temperature and pH-triggered in situ nanogels for the treatment of keratitis. MATERIALS AND METHODS: Poly(N-isopropylacrylamide-methacrylicacide-vinylpyrrolidone) [P (NIPAAm-MAA-VP)] nanoparticles was synthesised and used for preparation of CIP-loaded nanogels. Antimicrobial and in vivo animal studies of the CIP-loaded nanoformulation were performed. RESULTS: Nanoformulation with a mean particle size between 10 and 50 nm and higher than 95% encapsulation efficiency was obtained. Ciprofloxacin released from the nanoparticles showed an enhanced antibacterial effect as determined by minimal inhibitory concentrations. In vivo studies demonstrated reasonable efficacy in severe keratitis using the developed nanoformulation. CONCLUSIONS: Nanoformulation had acceptable efficacy in treating bacterial keratitis in an animal model. Therefore, the developed system has the potential to be used in localised application for the treatment of keratitis.

Staphylococcal entorotoxin B anchored exosome induces apoptosis in negative esterogen receptor breast cancer cells.

Exosomes (EXO) are acellular vehicles used for cancer immunotherapy due to their immune inducing properties. To identify whether designed structure based on tumoral EXO have a cytotoxic effect together with a potent immunological property, we synthesized a novel structure based on EXO and staphylococcal entrotoxin B (SEB), two immune inducer substances, and surveyed its cytostatic effect on the breast cancer cell line. EXO were purified from tumor cells and SEB was anchored on it by protein transfer method. To determine the cytotoxic and apoptosis inducing effect of this structure, treated cells with different concentrations of EXO/SEB were examined by MTT assay and Hoechst staining method. In addition, the expression rate of bcl-2, bax, bak, fas, bcl-xl and the activity of caspase-3 and caspase-9 were assessed. We observed that EXO/SEB significantly decreased the cell proliferation and stimulated apoptosis (P < 0.001) at all concentration after 24 h (P < 0.001). Furthermore, EXO/SEB raised the expression rate of bax and bak (P < 0.001) but no impact on fas and bcl-xl after 48 h. We observed reducing effect of EXO/SEB on the mRNA expression of bcl-2. After 24 h of exposing the cell with the EXO/SEB, a significant increase was found in the activity of caspase at the concentration of 2.5, 5 and 10 µg/100 µl for caspase-9 and at all concentrations for caspase-3 (P < 0.001). Our designed structure, the EXO/SEB, is a novel model for apopto-immunotherapy being able to induce apoptosis in ER(-) breast cancer cells.

Preparation of pH sensitive insulin-loaded nano hydrogels and evaluation of insulin releasing in different pH conditions.

In the recent years, temperature and pH-sensitive hydrogels were developed as suitable carriers for drug delivery. In this study, four different pH-sensitive nanohydrogels were designed for an oral insulin delivery modeling. NIPAAm-MAA-HEM copolymers were synthesized by radical chain reaction with 80:8:12 ratios respectively. Reactions were carried out in four conditions including 1,4-dioxan and water as two distinct solution under nitrogen gas-flow. The copolymers were characterized with FT-IR, SEM and TEM. Copolymers were loaded with regular insulin by modified double emulsion method with ratio of 1:10. Release study carried out in pH 1.2 and pH 6.8 at 37 °C. For pH 6.8 and pH 1.2, 2 mg of the insulin loaded nanohydrogels was float in a beaker containing 100 mL of PBS with pH 6.8 and 100 mL of HCl solution with pH 1.2, respectively. Sample collection was done in different times and HPLC was used for analysis of samples using water/acetonitrile (65/35) as the mobile phase. Nanohydrogels synthesis reaction yield was 95 %, HPLC results showed that loading in 1,4-dioxan without cross-linker nanohydrogels was more than others, also indicated that the insulin release of 1,4-dioxan without cross-linker nanohydrogels at acidic pH is less, but in pH 6.8 is the most. Results showed that by opting suitable polymerization method and selecting the best nanohydrogels, we could obtain a suitable insulin loaded nanohydrogels for oral administration.

A Systematic Review of Stem Cell Differentiation into Keratinocytes for Regenerative Applications.

To improve wound healing or treatment of other skin diseases, and provide model cells for skin biology studies, in vitro differentiation of stem cells into keratinocyte-like cells (KLCs) is very desirable in regenerative medicine. This study examined the most recent advancements in in vitro differentiation of stem cells into KLCs, the effect of biofactors, procedures, and preparation for upcoming clinical cases. A range of stem cells with different origins could be differentiated into KLCs under appropriate conditions. The most effective ways of stem cell differentiation into keratinocytes were found to include the co-culture with primary epithelial cells and keratinocytes, and a cocktail of growth factors, cytokines, and small molecules. KLCs should also be supported by biomaterials for the extracellular matrix (ECM), which replicate the composition and functionality of the in vivo extracellular matrix (ECM) and, thus, support their phenotypic and functional characteristics. The detailed efficient characterization of different factors, and their combinations, could make it possible to find the significant inducers for stem cell differentiation into epidermal lineage. Moreover, it allows the development of chemically known media for directing multi-step differentiation procedures.In conclusion, the differentiation of stem cells to KLCs is feasible and KLCs were used in experimental, preclinical, and clinical trials. However, the translation of KLCs from in vitro investigational system to clinically valuable cells is challenging and extremely slow.

Development of a Composite Hydrogel Containing Statistically Optimized PDGF-Loaded Polymeric Nanospheres for Skin Regeneration: In Vitro Evaluation and Stem Cell Differentiation Studies.

Platelet-derived growth factor-BB (PDGF-BB) is a polypeptide growth factor generated by platelet granules faced to cytokines. It plays a role in forming and remodeling various tissue types, including epithelial tissue, through interaction with cell-surface receptors on most mesenchymal origin cells. However, it breaks down quickly in biological fluids, emphasizing the importance of preserving them from biodegradation. To address this challenge, we formulated and evaluated PDGF-encapsulated nanospheres (PD@PCEC) using polycaprolactone-polyethylene glycol-polycaprolactone. PD@PCECs were fabricated through the triple emulsion methodology and optimized by using the Box-Behnken design. The encapsulation efficiency (EE) of nanoencapsulated PDGF-BB was investigated concerning four variables: stirring rate (X1), stirring duration (X2), poly(vinyl alcohol) concentration (X3), and PDGF-BB concentration (X4). The selected optimized nanospheres were integrated into a gelatin-collagen scaffold (PD@PCEC@GC) and assessed for morphology, biocompatibility, in vitro release, and differentiation-inducing activity in human adipose-derived stem cells (hADSCs). The optimized PD@PCEC nanospheres exhibited a particle size of 177.9 ± 91 nm, a zeta potential of 5.2 mV, and an EE of 87.7 ± 0.44%. The release profile demonstrated approximately 85% of loaded PDGF-BB released during the first 360 h, with a sustained release over the entire 504 h period, maintaining bioactivity of 87.3%. The study also included an evaluation of the physicochemical properties of the scaffolds and an assessment of hADSC adhesion to the scaffold's surface. Additionally, hADSCs cultivated within the scaffold effectively differentiated into keratinocyte-like cells (KLCs) over 21 days, evidenced by morphological changes and upregulation of keratinocyte-specific genes, including cytokeratin 18, cytokeratin 19, and involucrin, at both transcriptional and protein levels.