Synthesis and characterization of injectable thermosensitive hydrogel based on Pluronic-grafted silk fibroin copolymer containing hydroxyapatite nanoparticles as potential for bone tissue engineering.

Injectable hydrogels are promising for bone tissue engineering due to their minimally invasive application and adaptability to irregular defects. This study presents the development of pluronic grafted silk fibroin (PF-127-g-SF), a temperature-sensitive graft copolymer synthesized from SF and modified PF-127 via a carbodiimide coupling reaction. The PF-127-g-SF copolymer exhibited a higher sol-gel transition temperature (34 °C at 16 % w/v) compared to PF-127 (23 °C), making it suitable for injectable applications. It also showed improved flexibility and strength, with a yielding point increase from <10 % to nearly 30 %. Unlike PF-127 gel, which degrades within 72 h in aqueous media, the PF-127-g-SF copolymer maintained a stable gel structure for over two weeks due to its robust crosslinked hydrogel network. Incorporating hydroxyapatite nanoparticles (n-HA) into the hydrogel reduced pore size and decreased swelling and degradation rates, extending structural stability to four weeks. Increasing n-HA concentration from 0 % to 20 % reduced porosity from 80 % to 66 %. Rheological studies indicated that n-HA enhanced the scaffold's strength and mechanical properties without altering gelation temperature. Cellular studies with MG-63 cells showed that n-HA concentration influenced cell viability and mineralization, highlighting the scaffold's potential in bone tissue engineering.

Activation of the BMP2/SMAD4 signaling pathway for enhancing articular cartilage regeneration of mesenchymal stem cells utilizing chitosan/alginate nanoparticles on 3D extracellular matrix scaffold.

This study investigated the efficacy of using chitosan/alginate nanoparticles loaded with recombinant human bone morphogenetic-2 (rhBMP-2) and SMAD4 encoding plasmid to enhance the chondrogenesis of human bone marrow mesenchymal stem cells (hBM-MSCs) seeded on an extracellular matrix (ECM). The research treatments included the stem cells treated with the biological cocktail (BC), negative control (NC), hBM-MSCs with chondrogenic medium (MCM), hBM-MSCs with naked rhBMP-2 and chondrogenic medium (NB/C), and hBM-MSCs with naked rhBMP-2 and chondrogenic medium plus SMAD4 encoding plasmid transfected with polyethyleneimine (PEI) (NB/C/S/P). The cartilage differentiation was performed with real-time quantitative PCR analysis and alizarin blue staining. The data indicated that the biological cocktail (BC) exhibited significantly higher expression of cartilage-related genes compared to significant differences with MCM and negative control (NC) on chondrogenesis. In the (NB/C/S/P), the expression levels of SOX9 and COLX were lower than those in the BC group. The expression pattern of the ACAN gene was similar to COL2A1 changes suggesting that it holds promising potential for cartilage regeneration.

Directed Differentiation of Adipose-Derived Stem Cells Using Imprinted Cell-Like Topographies as a Growth Factor-Free Approach.

The influence of surface topography on stem cell behavior and differentiation has garnered significant attention in regenerative medicine and tissue engineering. The cell-imprinting method has been introduced as a promising approach to mimic the geometry and topography of cells. The cell-imprinted substrates are designed to replicate the topographies and dimensions of target cells, enabling tailored interactions that promote the differentiation of stem cells towards desired specialized cell types. In fact, by replicating the size and shape of cells, biomimetic substrates provide physical cues that profoundly impact stem cell differentiation. These cues play a pivotal role in directing cell morphology, cytoskeletal organization, and gene expression, ultimately influencing lineage commitment. The biomimetic substrates' ability to emulate the native cellular microenvironment supports the creation of platforms capable of steering stem cell fate with high precision. This review discusses the role of mechanical factors that impact stem cell fate. It also provides an overview of the design and fabrication principles of cell-imprinted substrates. Furthermore, the paper delves into the use of cell-imprinted polydimethylsiloxane (PDMS) substrates to direct adipose-derived stem cells (ADSCs) differentiation into a variety of specialized cells for tissue engineering and regenerative medicine applications. Additionally, the review discusses the limitations of cell-imprinted PDMS substrates and highlights the efforts made to overcome these limitations.

Regulation of cell fate by cell imprinting approach in vitro.

Cell culture-based technologies are widely utilized in various domains such as drug evaluation, toxicity assessment, vaccine and biopharmaceutical development, reproductive technology, and regenerative medicine. It has been demonstrated that pre-adsorption of extracellular matrix (ECM) proteins including collagen, laminin and fibronectin provide more degrees of support for cell adhesion. The purpose of cell imprinting is to imitate the natural topography of cell membranes by gels or polymers to create a reliable environment for the regulation of cell function. The results of recent studies show that cell imprinting is a tool to guide the behavior of cultured cells by controlling their adhesive interactions with surfaces. Therefore, in this review we aim to compare different cell cultures with the imprinting method and discuss different cell imprinting applications in regenerative medicine, personalized medicine, disease modeling, and cell therapy.

Cellulose nanocrystals-reinforced dual crosslinked double network GelMA/hyaluronic acid injectable nanocomposite cryogels with improved mechanical properties for cartilage tissue regeneration.

Improvement of mechanical properties of injectable tissue engineering scaffolds is a current challenge. The objective of the current study is to produce a highly porous injectable scaffold with improved mechanical properties. For this aim, cellulose nanocrystals-reinforced dual crosslinked porous nanocomposite cryogels were prepared using chemically crosslinked methacrylated gelatin (GelMA) and ionically crosslinked hyaluronic acid (HA) through the cryogelation process. The resulting nanocomposites showed highly porous structures with interconnected porosity (>90%) and mean pore size in the range of 130-296 µm. The prepared nanocomposite containing 3%w/v of GelMA, 20 w/w% of HA, and 1%w/v of CNC showed the highest Young's modulus (10 kPa) and excellent reversibility after 90% compression and could regain its initial shape after injection by a 16-gauge needle in the aqueous media. The in vitro results demonstrated acceptable viability (>90%) and migration of the human chondrocyte cell line (C28/I2), and chondrogenic differentiation of human adipose stem cells. A two-month in vivo assay on a rabbit's ear model confirmed that the regeneration potential of the prepared cryogel is comparable to the natural autologous cartilage graft, suggesting it is a promising alternative for autografts in the treatment of cartilage defects.

Macrophage cell morphology-imprinted substrates can modulate mesenchymal stem cell behaviors and macrophage M1/M2 polarization for wound healing applications.

Mesenchymal stem cells and macrophages (MQ) are two very important cells involved in the normal wound healing process. It is well understood that topological cues and mechanical factors can lead to different responses in stem cells and MQ by influencing their shape, cytoskeleton proliferation, migration, and differentiation, which play an essential role in the success or failure of biomaterial implantation and more importantly wound healing. On the other hand, the polarization of MQ from proinflammatory (M1) to prohealing (M2) phenotypes has a critical role in the acceleration of wound healing. In this study, the morphology of different MQ subtypes (M0, M1, and M2) was imprinted on a silicon surface (polydimethylsiloxane [PDMS]) to prepare a nano-topography cell-imprinted substrate with the ability to induce anti-inflammatory effects on the mouse adipose-derived stem cells (ADSCs) and RAW264.7 monocyte cell line (MO). The gene expression profiles and flow cytometry of MQ revealed that the cell shape microstructure promoted the MQ phenotypes according to the specific shape of each pattern. The ELISA results were in agreement with the gene expression profiles. The ADSCs on the patterned PDMS exhibited remarkably different shapes from no-patterned PDMS. The MOs grown on M2 morphological patterns showed a significant increase in expression and section of anti-inflammatory cytokine compared with M0 and M1 patterns. The ADSCs homing in niches heavily deformed the cytoskeletal, which is probably why the gene expression and phenotype unexpectedly changed. In conclusion, wound dressings with M2 cell morphology-induced surfaces are suggested as excellent anti-inflammatory and antiscarring dressings.

Fabrication and multiscale modeling of polycaprolactone/amniotic membrane electrospun nanofiber scaffolds for wound healing.

BACKGROUND: Enhancing the efficiency of cell-based skin tissue engineering (TE) approaches is possible via designing electrospun scaffolds possessing natural materials like amniotic membrane (AM) with wound healing characteristics. Concentrating on this aim, we fabricated innovative polycaprolactone (PCL)/AM scaffolds through the electrospinning process. METHODS: The manufactured structures were characterized by employing scanning electron microscope (SEM), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, tensile testing, Bradford protein assay, etc. In addition, the mechanical properties of scaffolds were simulated by the multiscale modeling method. RESULTS: As a result of conducting various tests, it was concluded that the uniformity and distribution of fibers decreased with an increase in the amniotic content. Furthermore, PCL-AM scaffolds contained amniotic and PCL characteristic bands. In the case of protein release, greater content of AM led to the release of higher amounts of collagen. Tensile testing revealed that scaffolds' ultimate strength increased when the AM content augmented. The multiscale modeling demonstrated that the scaffold had elastoplastic behavior. In order to assess cellular attachment, viability, and differentiation, human adipose-derived stem cells (ASCs) were seeded on the scaffolds. In this regard, SEM and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays showed significant cellular proliferation and viability on the proposed scaffolds, and these analyses illustrated that higher cell survival and adhesion could be achieved when scaffolds possessed a larger amount of AM. After 21 days of cultivation, particular keratinocyte markers, such as keratin I and involucrin, were identified through utilizing immunofluorescence and real-time polymerase chain reaction (PCR) tests. The markers' expressions were higher in the PCL-AM scaffold with a ratio of 90:10 v v-1 compared with the PCL-epidermal growth factor (EGF) structure. Moreover, the presence of AM in the scaffolds resulted in the keratinogenic differentiation of ASCs even without employing EGF. Consequently, this state-of-the-art experiment suggests that the PCL-AM scaffold can be a promising candidate in skin bioengineering. CONCLUSION: This study showed that mixing AM with PCL, a widely used polymer, in different concentrations can overcome PCL disadvantages such as high hydrophobicity and low cellular compatibility.

Biomimetic vascular tissue engineering by decellularized scaffold and concurrent cyclic tensile and shear stresses.

Decellularization by chemical approaches has harmful effects on extracellular matrix (ECM) proteins, and damages lots of functional peptides and biomolecules present in the ultrastructure. In this study, we employed a combination of chemical and physical decellularization methods to overcome these disadvantages. The induced osmotic pressure by hypertonic/hypotonic solutions dissociated and removed most of cellular membranes significantly without any detergent or chemical agent. In total, 0.025% trypsin solution was found adequate to remove the remaining debrides, and ultimately 1% Triton X-100 was utilized for final cleansing. In addition, conducting all the decellularization processes at 4 °C yielded an ECM with least damages in the ultrastructure which could be inferred by close mechanical strength and swelling ratio to the native vessel, and high quality and quantity of cell attachment, migration and proliferation which were examined by optical microscopy and scanning electron microscopy (SEM) of the histology samples. Moreover, the obtained biological scaffold (BS) had no cytotoxicity according to the MTT assay, and this scaffold is storable at -20 °C. Employing bioreactor for concurrent cyclic tensile and shear stresses improved the cell migration into pores of the BS and made the cells and the scaffold compact in analogous to native tissue. As opening angle test showed by decellularizing of the blood vessel, the residual stress dropped significantly which revealed the role of cells in the amount of induced stress in the structure. However, intact and healthy ECM explicitly recovered upon recellularization and beat the initial residual stress of the native tissue. The tensile test of the blood vessels in longitudinal and radial directions revealed orthotropic behavior which can be explained by collagen fibers direction in the ECM. Furthermore, by the three regions of the stress-strain curve can be elucidated the roles of cells, elastin and collagen fibers in mechanical behavior of the vascular tissues.

Positive effect of acellular amniotic membrane dressing with immobilized growth factors in skin wound healing.

The human amniotic membrane dressing has been shown to accelerate the wound healing process in the clinic. In this study, heparin was conjugated to a human Acellular Amniotic Membrane (hAAM) to provide affinity binding sites for immobilizing growth factors. To study the acceleration of the wound healing process, we bound epidermal growth factor and fibroblast growth factor 1 to heparinized hAAMs (GF-Hep-hAAMs). The heparinized hAAMs (Hep-hAAMs) were characterized by toluidine blue staining and infrared spectroscopy. The quality control of hAAM was performed by hematoxylin staining, swelling capacity test and biomechanical evaluation. The cytotoxicity, adhesion, and migration in vitro assays of GF-Hep-hAAMs on L-929 fibroblast cells were also studied by MTT assay, scanning electron microscopy, and scratch assay, respectively. Finally, in vivo skin wound healing study was performed to investigate the wound closure rate, re-epithelization, collagen deposition, and formation of new blood vessels. The results showed that GF-Hep-hAAMs enhance the rate of wound closure and epidermal regeneration in BALB/c mice. In conclusion, GF-Hep-hAAMs could accelerate the wound healing process, significantly in the first week.

Engineered substrates incapable of induction of chondrogenic differentiation compared to the chondrocyte imprinted substrates.

It is well established that surface topography can affect cell functions. However, finding a reproducible and reliable method for regulating stem cell behavior is still under investigation. It has been shown that cell imprinted substrates contain micro- and nanoscale structures of the cell membrane that serve as hierarchical substrates, can successfully alter stem cell fate. This study investigated the effect of the overall cell shape by fabricating silicon wafers containing pit structure in the average size of spherical-like chondrocytes using photolithography technique. We also used chondrocyte cell line (C28/I2) with spindle-like shape to produce cell imprinted substrates. The effect of all substrates on the differentiation of adipose-derived mesenchymal stem cells (ADSCs) has been studied. The AFM and scanning electron microscopy images of the prepared substrates demonstrated that the desired shapes were successfully transferred to the substrates. Differentiation of ADSCs was investigated by immunostaining for mature chondrocyte marker, collagen II, and gene expression of collagen II, Sox9, and aggrecan markers. C28/I2 imprinted substrate could effectively enhanced chondrogenic differentiation compared to regular pit patterns on the wafer. It can be concluded that cell imprinted substrates can induce differentiation signals better than engineered lithographic substrates. The nanostructures on the cell-imprinted patterns play a crucial role in harnessing cell fate. Therefore, the patterns must include the nano-topographies to have reliable and reproducible engineered substrates.

Biomimetic engineered approaches for neural tissue engineering: Spinal cord injury.

The healing process for spinal cord injuries is complex and presents many challenges. Current advances in nerve regeneration are based on promising tissue engineering techniques, However, the chances of success depend on better mimicking the extracellular matrix (ECM) of neural tissue and better supporting neurons in a three-dimensional environment. The ECM provides excellent biological conditions, including desirable morphological features, electrical conductivity, and chemical compositions for neuron attachment, proliferation and function. This review outlines the rationale for developing a construct for neuron regrowth in spinal cord injury using appropriate biomaterials and scaffolding techniques.

Defined Physicochemical Cues Steering Direct Neuronal Reprogramming on Colloidal Self-Assembled Patterns (cSAPs).

Direct neuronal reprogramming of somatic cells into induced neurons (iNs) has been recently established as a promising approach to generating neuron cells. Previous studies have reported that the biophysical cues of the in vitro microenvironment are potent modulators in the cell fate decision; thus, the present study explores the effects of a customized pattern (named colloidal self-assembled patterns, cSAPs) on iN generation from human fibroblasts using small molecules. The result revealed that the cSAP, composed of binary particles in a hexagonal-close-packed (hcp) geometry, is capable of improving neuronal reprogramming efficiency and steering the ratio of the iN subtypes. Cells exhibited distinct cell morphology, upregulated cell adhesion markers (i.e., SDC1 and ITGAV), enriched signaling pathways (i.e., Hippo and Wnt), and chromatin remodeling on the cSAP compared to those on the control substrates. The result also showed that the iN subtype specification on cSAP was surface-dependent; therefore, the defined physicochemical cue from each cSAP is exclusive. Our findings show that direct cell reprogramming can be manipulated through specific biophysical cues on the artificial matrix, which is significant in cell transdifferentiation and lineage conversion.

Enhancement of keratinocyte growth factor potential in inducing adipose-derived stem cells differentiation into keratinocytes by collagen-targeting.

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.

Neuroprotective Effect of Root Extracts of Berberis Vulgaris (Barberry) on Oxidative Stress on SH-SY5Y Cells.

Objectives: Oxidative stress plays a key role in chronic and acute brain disorders and neuronal damage associated with Alzheimer disease (AD) and other neurodegeneration symptoms. The neuroprotective effects of berberine and Berberis vulgaris (barberry) root extract against apoptosis induced by hydrogen peroxide (H2O2) in the human SH-SY5Y cell line were studied. Methods: The methanolic extraction of barberry root was performed using a maceration procedure. Oxidative stress was induced in SH-SY5Y cells by H2O2, and an MTT assay was applied to evaluate the neuroprotective effects of berberine and barberry root extract. The cells were pretreated with the half maximal inhibitory concentration (IC50) of each compound (including berberine, barberry root extract, and H2O2), and the anti-apoptotic effects of all components were investigated using RT-PCR. Results: The SH-SY5Y cell viability increased in both groups exposed to 75 and 150 ppm barberry extract compared with that in the H2O2-treated group. The data showed that exposing SH-SY5Y cells to 30 ppm berberine significantly increased the cell viability compared with the H2O2-treated group; treatment with 150 and 300 ppm berberine and H2O2 significantly decreased the SH-SY5Y cell viability and was associated with berberine cytotoxicity. The mRNA levels of Bax decreased significantly under treatment with berberine at 30 ppm compared with the control group. A significant increase in Bcl-2 expression was observed only after treatment with the IC50 of berberine. The expression level of Bcl-2 in cells exposed to both berberine and barberry extracts was also significantly higher than that in cells exposed to H2O2. Conclusion: The outcomes of this study suggest that treatment of SH-SY5Y cells with barberry extract and berberine could suppress apoptosis by regulating the actions of Bcl-2 family members.

Bioactivation of 3D Cell-Imprinted Polydimethylsiloxane Surfaces by Bone Protein Nanocoating for Bone Tissue Engineering.

Physical and chemical parameters that mimic the physiological niche of the human body have an influence on stem cell fate by creating directional signals to cells. Micro/nano cell-patterned polydimethylsiloxane (PDMS) substrates, due to their ability to mimic the physiological niche, have been widely used in surface modification. Integration of other factors such as the biochemical coating on the surface can achieve more similar microenvironmental conditions and promote stem cell differentiation to the target cell line. Herein, we investigated the effect of physical topography, chemical functionalization by acid bone lysate (ABL) nanocoating, and the combined functionalization of the bone proteins' nanocoated surface and the topographically modified surface. We prepared four distinguishing surfaces: plain PDMS, physically modified PDMS by 3D cell topography patterning, chemically modified PDMS with bone protein nanocoating, and chemically modified nano 3D cell-imprinted PDMS by bone proteins (ABL). Characterization of extracted ABL was carried out by Bradford staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, followed by the MTT assay for evaluation of cell viability on ABL-coated PDMS. Moreover, field emission scanning electron microscopy and profilometry were used for the determination of optimal coating thickness, and the appropriate coating concentration was identified and used in the study. The binding and retention of ABL to PDMS were confirmed by Fourier transform infrared spectroscopy and bicinchoninic acid assay. Sessile drop static water contact angle measurements on substrates showed that the combined chemical functionalization and nano 3D cell-imprinting on the PDMS surface improved surface wettability by 66% compared to plain PDMS. The results of ALP measurement, alizarin red S staining, immunofluorescence staining, and real-time PCR showed that the nano 3D cell-imprinted PDMS surface functionalized by extracted bone proteins, ABL, is able to guide the fate of adipose derived stem cellss toward osteogenic differentiation. Eventually, chemical modification of the cell-imprinted PDMS substrate by bone protein extraction not only improved the cell adhesion and proliferation but also contributed to the topographical effect itself and caused a significant synergistic influence on the process of osteogenic differentiation.

Selective biofunctionalization of 3D cell-imprinted PDMS with collagen immobilization for targeted cell attachment.

Cell-imprinted polydimethylsiloxane substrates, in terms of their ability to mimic the physiological niche, low microfabrication cost, and excellent biocompatibility were widely used in tissue engineering. Cells inside the mature cells' cell-imprinted PDMS pattern have been shown in previous research to be capable of being differentiated into a specific mature cell line. On the other hand, the hydrophobicity of PDMS substrate leads to weak cell adhesion. Moreover, there was no guarantee that the cells would be exactly located in the cavities of the cells' pattern. In many studies, PDMS surface was modified by plasma treatment, chemical modification, and ECM coating. Hence, to increase the efficiency of cell-imprinting method, the concavity region created by the cell-imprinted pattern is conjugated with collagen. A simple and economical method of epoxy silane resin was applied for the selective protein immobilization on the desired regions of the PDMS substrate. This method could be paved to enhance the cell trapping into the cell-imprinted pattern, and it could be helpful for stem cell differentiation studies. The applied method for selective protein attachment, and as a consequence, selective cell integration was assessed on the aligned cell-imprinted PDMS. A microfluidic chip created the aligned cell pattern. After Ar+ plasma and APTES treatment of the PDMS substrate, collagen immobilization was performed. The immobilized collagen was removed by epoxy silane resin stamp from the ridge area where the substrate lacked cell pattern and leaving the collagen only within the patterned areas. Coomassie brilliant blue staining was evaluated for selective collagen immobilization, and the collagen-binding stability was assessed by BCA analysis. MTT assay for the evaluation of cell viability on the modified surface was further analyzed. Subsequently, the crystal violet staining has confirmed the selective cell integration to the collagen-immobilized site on the PDMS substrate. The results proved the successfully selective collagen immobilization on the cell-imprinted PDMS and showed that this method increased the affinity of cells to attach inside the cell pattern cavity.

Molecular imprinting as a simple way for the long-term maintenance of the stemness and proliferation potential of adipose-derived stem cells: an in vitro study.

Cells are smart creatures that respond to every signal after isolation and in vitro culture. Adipose-derived stem cells (ADSCs) gradually lose their characteristic spindle shape, multi-lineage differentiation potential, and self-renewal ability, and enter replicative senescence after in vitro expansion. This loss of cellular function is a serious impediment to clinical applications that require huge numbers of cells. It has been proven that substrates with cell imprints can be applied for stem cells' differentiation into desired cells or to re-culture any cell type while maintaining its ordinary activity. This study demonstrated the application of cell-imprinted substrates as a novel method in the long-term expansion of ADSCs while maintaining their stemness. Here we used molecular imprinting of stem cells as a physical signal to maintain stem cells' stemness. First, ADSCs were isolated and cultured on the tissue culture plate. Then, cells were fixed, and stem cell-imprinted substrates were fabricated using PDMS. Afterward, ADSCs were cultured on these substrates and subjected to osteogenic and adipogenic differentiation signals. The results were compared with ADSCs cultured on a polystyrene tissue culture plate and non-patterned PDMS. Morphology analysis with optical and fluorescence microscopy and SEM images illustrated that ADSCs seeded on imprinted substrates kept ADSC morphology. Alizarin Red S and Oil Red O staining, flow cytometry, and qPCR results showed that ADSC-imprinted substrates could reduce the differentiation of stem cells in vitro even if the differentiating stimulations were applied. Also, cell cycle analysis revealed that ADSCs could maintain their proliferation potential. So this method can maintain stem cells' stemness for a long time and reduce the unwanted stem cell differentiation that occurs in conventional cell culture on tissue culture plates.

Comparison of engineered cartilage based on BMSCs and chondrocytes seeded on PVA-PPU scaffold in a sheep model.

In this study, polyvinyl alcohol hydrogel chains were crosslinked by polyurethane in order to synthesize a suitable substrate for cartilage lesions. The substrate was fully characterized, and in vitro and in vivo investigations were conducted based on a sheep model. In vitro tests were performed based on the chondrocyte cells with the Alcian Blue and safranin O staining in order to prove the presence of proteoglycan on the surface of the synthesized substrate, which has been secreted by cultures of chondrocytes. Furthermore, the expression of collagen type I, collagen type II, aggrecan, and Sox9 was presented in the chondrocyte cultures on the synthesized substrate through RT-PCR. In addition, the H&E analysis and other related tests demonstrated the formation of neocartilage tissue in a sheep model. The results were found to be promising for cartilage tissue engineering and verified that the isolated chondrocyte cultures on the synthesized substrate retain their original composition.

Optimization of a PDMS-Based Cell Culture Substrate for High-Density Human-Induced Pluripotent Stem Cell Adhesion and Long-Term Differentiation into Cardiomyocytes under a Xeno-Free Condition.

Despite the numerous advantages of PDMS-based substrates in various biomedical applications, they are limited by their highly hydrophobic surface that does not optimally interact with cells for attachment and growth. Hence, the lack of lengthy and straightforward procedures for high-density cell production on the PDMS-based substrate is one of the significant challenges in cell production in the cell therapy field. In this study, we found that the PDMS substrate coated with a combination of polydopamine (PDA) and laminin-511 E8 fragments (PDA + LME8-coated PDMS) can support human-induced pluripotent stem cell (hiPSC) attachment and growth for the long term and satisfy their demands of differentiation into cardiomyocytes (iCMs). Compared with prior studies, the density of hiPSCs and their adhesion time on the PDMS surface were increased during iCM production. Although the differentiated iCMs beat and produce mechanical forces, which disturb cellular attachments, the iCMs on the PDA + LME8-coated PDMS substrate showed dramatically better attachment than the control condition. Further, the substrate required less manipulation by enabling one-step seeding throughout the process in iCM formation from hiPSCs under animal-free conditions. In light of the results achieved, the PDA + LME8-coated PDMS substrate will be an up-and-coming tool for cardiomyocyte production for cell therapy and tissue engineering, microfluidics, and organ-on-chip platforms.

Synergistic effect of cell and molecule - imprinted substrates for bone tissue engineering.

BACKGROUND: The purpose of this research was to investigate the in vitro osteogenic induction of MG-63 cells using topography and collagen protein printed on polydimethyl siloxane (PDMS). METHODS: ALKALINE PHOSPHATASE (ALP) assay, calcium content, alizarin red staining, immunocytochemistry (ICC), and real-time polymerase chain reaction (PCR) were used to evaluate the osteo-differentiation of human adipose stem cells on the MG-63 cell pattern, MG-63 cells/collagen pattern, and collagen pattern. Also, the differentiated cell shape was studied by crystal violet staining and scanning electron microscopy (SEM). RESULTS: Our results showed that calcium content and ALP activity increased significantly on the MG-63 cells /collagen pattern (P < 0.05). The gene expression analysis (ALKALINPHOSPHATASE, COLLAGEN1 and OSTEOCALCIN) and bone marker protein expression (OSTEOCALCIN) confirmed the osteo differentiation of adipose stem cells (ADSCs) seeded on the imprinting substrate. DISCUSSION: Cell and molecular printing enhanced osteogenic development of adipose stem cells, according to our findings.