Culture Of Human Mesenchymal Stem Cells Research Articles

Primary human mesenchymal stem cell culture under xeno-free conditions using surface-modified cellulose nanofiber scaffolds

Stem cell culture often requires various animal-derived components such as serum and collagen. This limits its practical use. Therefore, xeno-free (xenogeneic component-free) culture systems are receiving increased attention. Herein, we propose xeno-free, plant-derived cellulose nanofibers (CNFs) with different surface chemistry: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized CNFs (TOCNFs) with carboxy groups and surface-sulfated CNFs (S-CNFs) for the proliferation of human mesenchymal stem cells (hMSCs) under various serum conditions. We cultured bone marrow-derived hMSCs on CNF scaffolds with various fiber lengths and functional group contents. Original CNFs were bioinert materials that did not contribute to cell adhesion. In contrast, the surface-modified CNFs facilitated the proliferation of immortalized hMSCs under normal and low-serum conditions. The TOCNFs (COONa: 1.47 mmol g−1; length: 0.53 μm), the S-CNFs (OSO3Na: 0.64 mmol g−1; 0.61 μm), and a combination of the two (1:1 by weight) enabled immortalized hMSCs to maintain their multipotency, even under serum-free conditions. Primary cultured hMSCs proliferated well on the TOCNF/S-CNF scaffolds in a completely serum-free medium, comparable to animal-derived type I collagen, although few hMSCs adhered to the standard polystyrene substrate. Our strategy of using surface-modified CNFs will inform the development of xeno-free culture systems to avoid the use of animal-derived materials for both cell culture media and scaffolds.

Electrospun fibers modified with polydopamine for enhancing human mesenchymal stem cell culture

In this study, we coated electrospun polycaprolactone (PCL) fibers with polydopamine (PDA) to modify their hydrophobicity and fabricated a matrix for culturing mesenchymal stem cells (MSCs). Additionally, we incorporated Arg-Gly-Asp (RGD) peptides into PDA to enhance MSCs culture performance on PCL fibers. PDA and RGD were successfully coated in one step by immersing the electrospun fibers in a coating solution, without requiring an additional surface activation process. The characteristics of functionalized PCL fibers were analyzed by scanning electron microscopy with energy-dispersive x-ray analysis, Fourier transform infrared spectroscopy, water contact angle measurement, and fluorescence measurements using a carboxylic-modified fluorescent microsphere. MSCs cultured on the modified PCL fibers demonstrated enhanced cell adhesion, proliferation, and osteogenic- and chondrogenic differentiation. This study provides insight into potential applications for scaffold fabrication in MSCs-based tissue engineering, wound dressing, implantation, and a deeper understanding of MSCs behavior in vitro.

ПОСТРАДИАЦИОННЫЕ ИЗМЕНЕНИЯ КОЛИЧЕСТВА ФОКУСОВ ФОСФОРИЛИРОВАННЫХ БЕЛКОВ H2AX И AТМ В МЕЗЕНХИМАЛЬНЫХ СТВОЛОВЫХ КЛЕТКАХ ЧЕЛОВЕКА, ОБЛУЧЕННЫХ РЕНТГЕНОВСКИМ ИЗЛУЧЕНИЕМ В МАЛЫХ ДОЗАХ

Aim: To study the patterns of changes in the number of foci of phosphorylated DNA double-strand break repair proteins H2AX (γH2AX) and ATM (pATM) in cultured human mesenchymal stem cells (MSCs) 1‒48 hours after exposure to X-ray radiation at doses of 40, 80, 160 and 250 mGy. Material and methods: We used the primary culture of human MSCs, obtained from the collection of LLC “BioloT” (Russia). Cells were irradiated using a RUB RUST-M1 X-ray biological unit (Diagnostika-M LLC, Moscow, Russia) equipped with two X-ray emitters at a dose rate of 40 mGy/min (voltage of 100 kV, an anode current of 8 mA, and a 1.5 mm Al filter) and 4 °C temperature. To quantify the yield of γH2AX and pATM foci immunocytochemical staining was carried out with the use of γH2AX and pATM antibody respectively. Statistical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft). To assess the significance of differences between samples, Student’s t-test was used. Results: It was shown that the kinetics of changes in the number of γH2AX foci after irradiation at doses of 160 and 250 mGy and low (40‒80 mGy) doses are significantly different. In contrast to the significant (50‒60 %) decrease in the number of γH2AX foci observed 6 hours after irradiation at doses of 160 and 250 mGy, after irradiation at low doses, no significant decrease in γH2AX foci was observed at this time point. Analysis of the colocalization of γH2AX foci with pATM foci indicates that the mechanisms for maintaining a high number of γH2AX foci 24‒48 hours after low-dose irradiation are ATM independent. A hypothesis has been put forward to explain the phenomenon of maintaining the number of γH2AX foci 24‒48 hours after irradiation in low doses by replicative stress caused by stimulation of proliferation against the background of hyperproduction of free radicals, resulting in additional formation of DNA double-strand breaks and phosphorylation of H2AX by ATR kinase.

Enhancing wettability and adhesive properties of PVDF-based substrates through non-thermal helium plasma surface modification

Polyvinylidene fluoride (PVDF) has gained attention as a promising material for tissue engineering due to its biocompatibility and piezoelectricity. However, achieving proper cell adhesion to PVDF surfaces remains a challenge. This study focuses on the preparation and characterization of PVDF-based substrates, including PVDF thin films and nanocomposites incorporating CoFe2O4 magnetic nanoparticles. Helium plasma treatment is employed to modify the surface properties of these substrates. The plasma treatment induces significant changes in the topography of the PVDF-based nanocomposites. The roughness values of the samples increase from 2–3 nm to 14–17 nm after 90 s of plasma treatment, leading to enhanced hydrophilicity with an average contact angle below 60°. Importantly, the helium plasma treatment preserves the magnetic and structural properties of the substrates, suggesting the retention of their magnetoelectric properties and, thus treated composites hold potential for remote stem cell activation. We provide evidence of cytocompatibility and improved adhesive properties of plasma-treated substrates using human mesenchymal stem cell cultures.

Synergistic Therapeutic Potential of Dual 3D Mesenchymal Stem Cell Therapy in an Ischemic Hind Limb Mouse Model.

Three-dimensional (3D) culture systems have been widely used to promote the viability and metabolic activity of mesenchymal stem cells (MSCs). The aim of this study was to explore the synergistic benefits of using dual 3D MSC culture systems to promote vascular regeneration and enhance therapeutic potential. We used various experimental assays, including dual 3D cultures of human adipose MSCs (hASCs), quantitative reverse transcription polymerase chain reaction (qRT-PCR), in vitro cell migration, Matrigel tube network formation, Matrigel plug assay, therapeutic assays using an ischemic hind limb mouse model, and immunohistochemical analysis. Our qRT-PCR results revealed that fibroblast growth factor 2 (FGF-2), granulocyte chemotactic protein-2 (GCP-2), and vascular endothelial growth factor-A (VEGF-A) were highly upregulated in conventional 3D-cultured hASCs (ASC-3D) than in two-dimensional (2D)-cultured hASCs. Hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and stromal-cell-derived factor-1 (SDF-1) showed higher expression levels in cytokine-cocktail-based, 3D-cultured hASCs (ASC-3Dc). A conditioned medium (CM) mixture of dual 3D ASCs (D-3D; ASC-3D + ASC-3Dc) resulted in higher migration and Matrigel tube formation than the CM of single 3D ASCs (S-3D; ASC-3D). Matrigel plugs containing D-3D contained more red blood cells than those containing S-3D. D-3D transplantation into ischemic mouse hind limbs prevented limb loss and augmented blood perfusion when compared to S-3D transplantation. Transplanted D-3D also revealed a high capillary density and angiogenic cytokine levels and transdifferentiated into endothelial-like cells in the hind limb muscle. These findings highlight the benefits of using the dual 3D culture system to optimize stem-cell-based therapeutic strategies, thereby advancing the therapeutic strategy for ischemic vascular disease and tissue regeneration.

Conditioned Media from Human Pulp Stem Cell Cultures Improve Bone Regeneration in Rat Calvarial Critical-Size Defects.

The aim of this study was to test whether lyophilized conditioned media from human dental pulp mesenchymal stem cell cultures promote the healing of critical-size defects created in the calvaria of rats. Prior to the surgical procedure, the medium in which dental pulp stem cells were cultured was frozen and lyophilized. After general anesthesia, an 8 mm diameter bone defect was created in the calvaria of twenty-four rats. The defects were filled with the following materials: xenograft alone (G1) or xenograft associated with lyophilized conditioned medium (G2). After 14 or 42 days, the animals were euthanized, and the specimens processed for histologic and immunohistochemical analysis. Bone formation at the center of the defect was observed only in the G2 at 42 days. At both timepoints, increased staining for VEGF, a marker for angiogenesis, was observed in G2. Consistent with this, at 14 days, G2 also had a higher number of blood vessels detected by immunostaining with an anti-CD34 antibody. In conclusion, conditioned media from human dental pulp mesenchymal stem cell cultures had a positive effect on the regenerative process in rat critical-size bone defects. Both the formation of bone and enhancement of vascularization were stimulated by the conditioned media.

Effect assessment of a type of xeno-free and serum-free human adipose-derived mesenchymal stem cells culture medium by proliferation and differentiation capacities.

Human mesenchymal stem cells (hMSCs) possess broad prospects in pre-clinical research. In vitro amplification of hMSCs requires appropriate medium to reach the number of seed cells with clinical significance. However, the uncertainty of the heterologous components of the traditional fetal bovine serum (FBS) culture medium has great safety risks. Moreover, existing commercial hMSCs medium is very expensive, therefore a safer and more optimal hMSCs medium is urgently needed. Accordingly, we developed five components adipose-derived hMSCs (hADMSCs) medium without xenogenic components, named E5 SFM. which is mainly composed of knockout serum replacement (KSR), and additionally four components such as fibroblast growth factor and transferrin. Here, we mainly compared the E5 SFM with traditional FBS-containing medium and a commercial medium by surface markers testing, proliferation assay as well as osteogenic, adipogenicand chondrogenic differentiation assessment. We demonstrated that hADMSCs cultured in the E5 SFM showed similar morphological characteristics and immunophenotypes to those in other media. Notably, cell proliferative capability was similar to that in the commercial medium, but higher than that in the FBS-containing medium and other media. Additionally, their capabilities of adipogenic and osteogenic differentiation were significantly higher than those of other media. Consequently, we concluded that the E5 SFM medium can not only effectively promote cell proliferation of hMSCs, but also has optimal differentiative capacity and clear and simple ingredients.

Osteoinductive and Osteogenic Capacity of Freeze-Dried Bovine Bone Compared to Deproteinized Bovine Bone Mineral Scaffold in Human Umbilical Cord Mesenchymal Stem Cell Culture: An In Vitro Study.

Freeze-dried bovine bone scaffold (FDBB) or decellularized FDBB (dc-FDBB) was developed as an ideal scaffold with osteoinductive properties. This research aims to compare the osteoinductive properties marked by the expression of runt-related transcription factor-2 (RUNX2) and Osterix (OSX) and the osteogenic capacity of these scaffolds imbued with human umbilical cord mesenchymal stem cells (hUCMSCs). This study was performed in five experimental groups: a negative control group (C-) of hUCMSCs with a normal growth medium, a positive control group (C + ) of hUCMSCs with an osteogenic medium, experimental group 1 (E1) with an FDBB conditioned medium (CM), and experimental group 2 (E2) with a dc-FDBB-CM, and a third experimental group (E3) consisting of a DBBM-CM. Alizarin red staining was performed to qualitatively assess osteoinductive capacity. RUNX2 and OSX expression was quantified using real-time quantification polymerase chain reaction with two replications on day six (D6) and day 12 (D12) as fold changes. This experiment revealed that hUCMSCs were positively expressed by CD73, CD90, and CD105 but were not expressed by CD34. Alizarin red staining showed that E1 had the most calcium deposition on D6 and D12, followed by E3 and then E2 The RUNX2 and OSX expression was higher in E1 but this difference was not significant. The OSX expression in E1,E2,E3 was lower on D12 and C+ of OSX had the highest expression. There was a significant difference of fold change measured between all groups (p < 0.05), and there was no significant difference between any of the groups treated with OSX and RUNX2 on D6 and D12. FDBB osteoinduction and osteogenic capacity were higher when compared with DBBM and dc-FDBB.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance Angiogenesis Through Upregulation of the VWF and Flk1 Genes in Endothelial Cells.

Exosomes derived from mesenchymal stem cells (MSCs) are crucial mediators of the paracrine effects as well as tissue repair and have promising clinical applications. They enhance tissue regeneration by reducing inflammatory responses, enhancing proliferation, inhibiting apoptosis, and stimulating angiogenesis. This study aimed to evaluate the mechanism of angiogenesis supported by exosomes derived from MSCs. Exosomes were isolated via ultracentrifugation of a conditioned medium collected from human umbilical cord MSC (hUCMSC) cultures. These exosomes were characterized using transmission electron microscopy, and the expression of specific markers (CD9, CD81, and CD63) was evaluated. To understand the mechanism of angiogenesis, we evaluated the effects of exosomes in endothelial cells (HUVECs). The obtained exosomes were supplemented at a dose of 20μg/mL into two kinds of culture media for HUVECs (M200 medium and endothelial cell growth medium), while phosphate-buffered saline was added to these media as a control. The effects of the exosomes were evaluated based on the formation of a tubular structure in the culture and the expression of angiogenic genes (MMP-2, Ephrin B2, Ephrin B4, Flk1, Flt1, VWF, VE-cadherin, CD31, ANG1, ANG2, and HGF) via RT-PCR. The exosomes were obtained from the hUCMSCs at a concentration of 0.7±0.029μg/mL. They accelerated the formation of new blood vessels by upregulating HGF, VWF, CD31, Flt1, and Flk1 (especially VWF and Flt1). Exosomes derived from hUCMSCs can promote angiogenesis through upregulation of VWF and Flt1 in endothelial cells.

Processing by Laser Stereolithography and &lt;i&gt;In Vitro&lt;/i&gt; Biological Evaluation of Hydroxyapatite Scaffolds Mimicking Human Trabecular Bone Architecture

Hydroxyapatite (HA) ceramic scaffolds are commonly used as bone graft substitutes. Design of such scaffolds is a challenge to improve biological properties and extend the applications of HA ceramics in the field of bone tissue engineering. In this work, we investigated the processing and the in vitro properties of HA ceramic scaffolds mimicking human trabecular bone architecture. Samples of human tibial trabecular bone were collected (University Hospital Center of Limoges) and scanned by X-Ray μ-computed tomography (μ-CT) to generate 3D model database. From this computer-aided design, HA ceramic scaffolds were shaped layer-by-layer by additive manufacturing using laser stereolithography (SLA). Then, green parts were sintered to obtain dense ceramic scaffolds. The shaped parts were compared to the model (wall thickness, size, and geometry of the porous network) using image analysis. A good agreement was found. Only small differences were detected due to a light overpolymerization or to some unprinted very small details that were not linked to a polymerized area of the previous layer. Due to part shrinkage during sintering a magnifying factor has to be applied to the scanned CAO model to match the real dimensions of the trabecular bone sample. Human mesenchymal stem cell (hMSC) cultures were performed to investigate the biological properties of these scaffolds (cell attachment and proliferation of hMSC). These preliminary biological evaluations show the good biocompatibility and cell adhesion of the HA substitute. This work evidences the efficiency of SLA to produce ceramic scaffold architectures mimicking that of the natural trabecular bone with promising biological behavior.

Therapeutic Effects of Human Pluripotent Stem Cell-Derived Mesenchymal Stem Cells on a Murine Model of Acute Type-2-Dominated Airway Inflammation

Allergic rhinitis and allergic asthma are the most common type-2 inflammatory diseases, which are hardly curable and cause heavy burden to general well-being. Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells with potential immunomodulatory effects that have been showning to have a therapeutic effect on allergic diseases. Here, we investigated the effects of human induced pluripotent stem cell (iPSC)-derived MSCs on airway hyperresponsiveness and acute type-2-dominated inflammation throughout the upper and lower airways. In this study, human MSCs, MSC cell culture supernatant, and culture medium (control) was injected into the acute airway inflammatory model via the tail vein. Mouse behavioristics were recorded immediately and mouse lung function was measured 24hours after the last ovalbumin (OVA) challenge. Histological staining, Luminex, Elisa and flow cytometry were employed to evaluate the effects on the production of total/OVA-specific IgG1 and IgE, cytokines expression in lung tissues, and inflammatory cells infiltration in the lung and spleen of the experimental mice. Expressions of eotaxin, IL-4, IL-5, IL-13, IL-33 in nasal and lung lavage were evaluated by Luminex and Elisa. We found that for this acute inflammatory mouse model, human MSC transplantation significantly mitigated the decreased motoring time and the increased lung function Rrs caused by OVA challenge. Serum OVA-IgG1, OVA-IgE, and eosinophil percentages in the splenocytes were significantly decreased. Injection of the MSC supernatant also showed the same trend, but not significantly changed. After treatment, IL-4 and IL-13 were significantly decreased in the lung tissue, and IL-5 and IL-13 were significantly decreased in lung lavage. In conclusion, both human MSC culture supernatant and cell transplantation could alleviate AHR and inflammation in acute inflammatory experimental animals, which demonstrated their potential for clinical therapeutics. Human iPSC-MSCs, MSC cell culture supernatant, or culture medium (control) was injected into the OVA-induced acute airway inflammatory model via the tail vein. Behavioral changes, AHR, serum OVA-specific IgG1 and IgE concentrations, and type-2 inflammations were alleviated.

Aligned Gelatin Microribbon Scaffolds with Hydroxyapatite Gradient for Engineering the Bone-Tendon Interface.

Injuries of the bone-to-tendon interface, such as rotator cuff and anterior cruciate ligament tears, are prevalent musculoskeletal injuries, yet effective methods for repair remain elusive. Tissue engineering approaches that use cells and biomaterials offer a promising potential solution for engineering the bone-tendon interface, but previous strategies require seeding multiple cell types and use of multiphasic scaffolds to achieve zonal-specific tissue phenotype. Furthermore, mimicking the aligned tissue morphology present in native bone-tendon interface in three-dimensional (3D) remains challenging. To facilitate clinical translation, engineering bone-tendon interface using a single cell source and one continuous scaffold with alignment cues would be more attractive but has not been achieved before. To address these unmet needs, in this study, we develop an aligned gelatin microribbon (μRB) hydrogel scaffold with hydroxyapatite nanoparticle (HA-np) gradient for guiding zonal-specific differentiation of human mesenchymal stem cell (hMSC) to mimic the bone-tendon interface. We demonstrate that aligned μRBs led to cell alignment in 3D, and HA gradient induced zonal-specific differentiation of mesenchymal stem cells that resemble the transition at the bone-tendon interface. Short chondrogenic priming before exposure to osteogenic factors further enhanced the mimicry of bone-cartilage-tendon transition with significantly improved tensile moduli of the resulting tissues. In summary, aligned gelatin μRBs with HA gradient coupled with optimized soluble factors may offer a promising strategy for engineering bone-tendon interface using a single cell source. Impact statement Our 3D macroporous microribbon hydrogel platform with alignment cues zonally integrated with hydroxyapatite nanoparticles enables differentiation across the bone-tendon interface within a continuous scaffold. While most interfacial scaffolds heretofore rely on composites and multilayer approaches, we present a continuous scaffold utilizing a single cell source. The synergy of niche cues with human mesenchymal stem cell (hMSC) culture leads to an over 45-fold enhancement in tensile modulus in culture. We further demonstrate that priming hMSCs towards the chondrogenic lineage can enhance the differential osteogenesis. Relying on a single cell source could enhance zone integration and scaffold integrity, along with practical benefits.

Mesenchymal Stem Cell Behavior on Soft Hydrogels with Aligned Surface Topographies.

Human mesenchymal stem cells (HMSCs) are important for cell-based therapies. However, the success of HMSC therapy requires large-scale in vitro expansion of these multipotent cells. The traditional expansion of HMSCs on tissue-culture-treated stiff polystyrene induces significant changes in their shape, multipotency, and secretome, leading to early senescence and subdued paracrine activity. To enhance their therapeutic potential, here, we have developed two-dimensional soft hydrogels with imprinted microscale aligned grooves for use as HMSC culture substrates. We showed that, depending on the dimensions of the topographical features, these substrates led to lower cellular spreading and cytoskeletal tension, maintaining multipotency and osteogenic and adipogenic differentiate potential, while lowering cellular senescence. We also observed a greater capacity of HMSCs to produce anti-inflammatory cytokines after short-term priming on these hydrogel substrates. Overall, these soft hydrogels with unique surface topography have shown great promise as in vitro culture substrates to maximize the therapeutic potential of HMSCs.

Optimizing Barium Titanate Nanocomposite Bone Scaffolds for Biomineralization in Dynamic Compression Bioreactors Using Time-Lapsed Microstructural Imaging and Smart Thresholding

Bone scaffolds made of calcium phosphate polymer nanocomposites have limited osteoinductive properties. Piezoelectric materials have attracted considerable interest in bone tissue engineering due to their potential to promote osteogenesis through additional electrical stimulation. Time-lapsed micro-CT imaging is a time-effective tool for in vitro optimization of such scaffolds but is challenged by nanocomposites with a high attenuation coefficient, such as one containing high amounts of piezoelectric barium titanate. We used high-resolution end-point micro-CT scans combined with histology and Raman spectroscopy to screen polydopamine functionalized nanocomposites containing 3–27 vol% barium titanate for collagenous extracellular matrix formation and mineralization. All compositions showed well-connected extracellular matrix and birefringent matured collagen after seven weeks of static human mesenchymal stem cell cultures. Nevertheless, high-resolution micro-CT analysis combined with smart thresholding during image processing enabled us to observe modest differences in ECM mineralization between groups suggesting that a volume fraction of 9–21% barium titanate facilitated the formation of dense mineral clusters in the pores even in the absence of mechanical stimuli, further corroborated by Raman spectroscopy. The same image processing approach facilitated the analysis of time-lapsed micro-CT images of scaffold cultures in dynamic compression bioreactors where 9 vol% barium titanate was the best nanocomposite composition, resulting in a significant twofold increased maturation rate under dynamic conditions. On the other hand, barium titanate content of ≥15 vol% did not improve mineralization. At 27 vol%, the biomineralization of the collagenous extracellular matrix was even impeded in the nanocomposite scaffolds, as evidenced by histology stainings. Overall, our approach enables time-lapsed quantitative assessment of high X-ray absorbing nanocomposite scaffolds for biomineralization under dynamic compression, facilitating the optimization of such mechanically responsive scaffolds.

Optimization of human umbilical cord blood-derived mesenchymal stem cell isolation and culture methods in serum- and xeno-free conditions

BackgroundAlthough umbilical cord blood (UCB) is identified as a source of mesenchymal stem cells (MSCs) with various advantages, the success in cell isolation is volatile. Therefore, it is necessary to optimize methods of cord blood-derived MSC (UCB-MSC) isolation and culture. In this study, we evaluated the efficiency of UCB-MSC isolation and expansion using different commercially available serum- and xeno-free media and investigated the capacity of autologous serum and plasma as a supplement to support cell proliferation. Additionally, we defined the presence of multilineage-differentiating stress-enduring (Muse) cells in the UCB-MSC population. Functions of UCB-MSC in in vitro angiogenesis processes and anti-cancer were also verified.MethodsMononuclear cells were isolated using density gradient separation and cultured in four commercial media kits, as well as four surface coating solutions. UCB-MSCs were characterized and tested on tube formation assay, and co-cultured with SK-MEL cells in a transwell system.ResultsThe results showed that only StemMACS™ MSC Expansion Media is more appropriate to isolate and culture UCB-MSCs. The cells exhibited a high cell proliferation rate, CFU forming capability, MSC surface marker expression, trilineage differentiate potential, and chromosome stability. In addition, the culture conditions with autologous serum coating and autologous plasma supplement enhanced cell growth and colony forming. This cell population contained Muse cells at rate of 0.3%. Moreover, UCB-MSCs could induce the tube formation of human umbilical vein endothelial cells and inhibit more than 50% of SK-MEL cell growth.ConclusionsUCB-MSCs could be high-yield isolated and expanded under serum- and xeno-free conditions by using the StemMACS™ MSC Expansion Media kit. Autologous serum coating and plasma supplement enhanced cell proliferation. These UCB-MSCs had effected the tube formation process and an anti-cancer impact.

Effect of Hydroxyapatite Microspheres, Amoxicillin-Hydroxyapatite and Collagen-Hydroxyapatite Composites on Human Dental Pulp-Derived Mesenchymal Stem Cells.

In this study, the preparation and characterization of three hydroxyapatite-based bioactive scaffolds, including hydroxyapatite microspheres (HAps), amoxicillin–hydroxyapatite composite (Amx–HAp), and collagen–hydroxyapatite composite (Col–HAp) were performed. In addition, their behavior in human dental pulp mesenchymal stem cell (hDPSC) culture was investigated. HAps were synthesized through the following methods: microwave hydrothermal, hydrothermal reactor, and precipitation, respectively. hDPSCs were obtained from samples of third molars and characterized by immunophenotypic analysis. Cells were cultured on scaffolds with osteogenic differentiation medium and maintained for 21 days. Cytotoxicity analysis and migration assay of hDPSCs were evaluated. After 21 days of induction, no differences in genes expression were observed. hDPSCs highly expressed the collagen IA and the osteonectin at the mRNA. The cytotoxicity assay using hDPSCs demonstrated that the Col–HAp group presented non-viable cells statistically lower than the control group (p = 0.03). In the migration assay, after 24 h HAps revealed the same migration behavior for hDPSCs observed compared to the positive control. Col–HAp also provided a statistically significant higher migration of hDPSCs than HAps (p = 0.02). Migration results after 48 h for HAps was intermediate from those achieved by the control groups. There was no statistical difference between the positive control and Col–HAp. Specifically, this study demonstrated that hydroxyapatite-based bioactive scaffolds, especially Col-Hap, enhanced the dynamic parameters of cell viability and cell migration capacities for hDPSCs, resulting in suitable adhesion, proliferation, and differentiation of this osteogenic lineage. These data presented are of high clinical importance and hold promise for application in therapeutic areas, because Col–HAp can be used in ridge preservation, minor bone augmentation, and periodontal regeneration. The development of novel hydroxyapatite-based bioactive scaffolds with clinical safety for bone formation from hDPSCs is an important yet challenging task both in biomaterials and cell biology.

Precision 3D printed meniscus scaffolds to facilitate hMSCs proliferation and chondrogenic differentiation for tissue regeneration

BackgroundThe poor regenerative capability and structural complexity make the reconstruction of meniscus particularly challenging in clinic. 3D printing of polymer scaffolds holds the promise of precisely constructing complex tissue architecture, however the resultant scaffolds usually lack of sufficient bioactivity to effectively generate new tissue.ResultsHerein, 3D printing-based strategy via the cryo-printing technology was employed to fabricate customized polyurethane (PU) porous scaffolds that mimic native meniscus. In order to enhance scaffold bioactivity for human mesenchymal stem cells (hMSCs) culture, scaffold surface modification through the physical absorption of collagen I and fibronectin (FN) were investigated by cell live/dead staining and cell viability assays. The results indicated that coating with fibronectin outperformed coating with collagen I in promoting multiple-aspect stem cell functions, and fibronectin favors long-term culture required for chondrogenesis on scaffolds. In situ chondrogenic differentiation of hMSCs resulted in a time-dependent upregulation of SOX9 and extracellular matrix (ECM) assessed by qRT-PCR analysis, and enhanced deposition of collagen II and aggrecan confirmed by immunostaining and western blot analysis. Gene expression data also revealed 3D porous scaffolds coupled with surface functionalization greatly facilitated chondrogenesis of hMSCs. In addition, the subcutaneous implantation of 3D porous PU scaffolds on SD rats did not induce local inflammation and integrated well with surrounding tissues, suggesting good in vivo biocompatibility.ConclusionsOverall, this study presents an approach to fabricate biocompatible meniscus constructs that not only recapitulate the architecture and mechanical property of native meniscus, but also have desired bioactivity for hMSCs culture and cartilage regeneration. The generated 3D meniscus-mimicking scaffolds incorporated with hMSCs offer great promise in tissue engineering strategies for meniscus regeneration.Graphical

Human platelet-rich plasma as a biological stimulant for proliferation and differentiation of mesenchymal stem cells

Platelet is one of the cells within the blood that have potential in regenerative therapy. Recently platelet-related products got special attention due to the abundance of growth factors and easy availability and processing. With the robust in cell-based therapy, platelet-rich plasma (PRP) has been intensively studied for its potential to substitute the fetal bovine serum (FBS) as the supplement in cell culture. Our study aimed to investigate the use of human PRP in mesenchymal stem cells (MSC) culture and its related effect on stem cell biology. We searched in vitro studies that used human PRP as a supplementing factor on human MSCs culture. From the initial 172 studies, 14 studies fulfilled the selection criteria and were analysed. The results showed that the sources of MSCs were varied, including adipose tissue, bone marrow, and dental tissue. The PRP concentration showing the best effects was ranged from 10 to 20%. In addition, most of the studies demonstrated the superiority of PRP to FBS in promoting the proliferation and differentiation of MSCs in vitro. Therefore, PRP could be an alternative to FBS in supporting a xeno-free culture system. Studies are needed to reveal the mechanism of PRP in maintaining the physiology of MSCs.

Influence of a macroporous β-TCP structure on human mesenchymal stem cell proliferation and differentiation in vitro

This work aims at studying the links between a porous scaffold in β-TCP and its impact on cell differentiation and proliferation in vitro. β-TCP was synthesized by aqueous precipitation. The shaping methods were chosen for their ability to generate original structures with the potential to positively influence cell behaviour. The impregnation of polymeric structure (PS) yielded an interconnected network of spherical pores, stereolithography (3D) yielded a network of interconnected diamond shaped pores and freeze casting (FC) yielded a network of parallel ellipsoidal channel-like pores. Two different trends emerged from the human mesenchymal stem cell culture: 3D and PS seems to promote surface proliferation whereas the freeze casted samples promoted cell penetration, bulk colonization, expression of key osteoblastic genes (RunX-2, ALP, BMP-2, BGLAP and Collagen) and alkaline phosphatase activity. The architectural features created by freeze casting positively influenced human mesenchymal stem cells behaviour in vitro.

Chemical and Biological Sensor Capsules for Real-Time Measurement of Cell Properties in Bioreactors

Introduction Real-time bioprocess monitoring and control is needed for the scalable production and deployment of cancer cell therapies at reasonable cost. For example, CAR-T cell therapy shows promise as an effective treatment for cancer with high (83%) remission rates.1 However, the cost of these treatments is too expensive for their mass adoption.2 One reason for the steep price is the difficulty scaling production while ensuring delivery of an efficacious therapy to the patient. One factor preventing scalability is the lack of effective process analytical technology. As a potential solution to this issue, we have developed a fully integrated, wireless, 3D-printed sensor capsule to be used for multiplexed sensing of critical quality attributes (CQAs), namely pH, glucose, and lactate concentrations within cell bioreactors. CQAs are used not only as metrics for cell viability, but also as determinants of a cell’s ability to deliver efficacious treatment. Unlike current process monitoring technology, capsule sensors are buoyant and can be propelled using impellers within the bioreactor. This allows for measurements uniformly inside the bioreactor. To our knowledge, this is the first time that 3D-printed, wireless sensor capsules have been developed for use in cell bioreactors for cancer cell therapy. Methods The capsule consists of electrochemical sensors, and read-out and wireless transmission electronics integrated into a capsule made of a biocompatible polymer (Figure 1). The different sensors for glucose, lactate, and pH along with an Ag on-chip reference electrodes were fabricated using standard lithographic techniques on silicon substrates. The reference electrode consisted of a thin film of Ag deposited by e-beam evaporation that was later coated with polyvinyl butyral (PVB) to improve stability.3 Glucose and lactate sensing was achieved amperometrically using enzymes, glucose oxidase or lactase oxidase respectively, immobilized on a Pt working electrode biased to 0.7 V. pH sensing was achieved by depositing a pH-sensitive oxide on a gold electrode. A pH-sensitive Al2O3 or HfO2 layer was deposited through atomic layer deposition.Figure 1 shows the capsule and sensor design. The capsule packaging was 3D printed using a Stratasys Connex 350 and was designed to be fully insulated from solution except for an opening on the top revealing the various sensors. The sensor chip is installed within the cap of the capsule which can be screwed off, but with wired connection to the electronics housed in the body of the capsule. This allows for simple replacement of the multiplexed sensor chip while retaining the capsule electronics, rechargeable battery, and packaging for reuse. Results and Conclusions Before testing within the capsule, the sensors were tested using microfluidics. Figure 2 shows the performance of the Ag/PVB on-chip pseudo reference electrode (pseudo-RE). The pseudo-RE shows a stable potential in different pH buffer solutions and shows similar sensitivity results for pH sensing when compared to a commercial reference electrode. By having a stable microfabricated pseudoRE, the sensing components will not be a limiting factor for miniaturization of the capsule. Figure 3 shows the response of the pH, lactate, and glucose sensors at different concentrations. The pH sensor shows high sensitivity (~53 mV/pH) over the range of pH 3 to 7. Glucose and lactate sensing was tested in 1X PBS. The glucose sensor shows a linear response to glucose concentrations from 0 to 25 mM validating the use of the sensor within cell culture media.4 The glucose sensor shows the same linear range when tested in human mesenchymal stem cell culture. In this case, the glucose concentration was first tested using a commercial sensor and then diluted to targeted glucose concentrations by dilution with PBS. The lactate sensor shows sensitivity in the concentration range of 1 to 5 mM with saturation occurring around ~6 mM. The capsule’s wireless sensing capability was validated using pH sensing in a beaker. The pH sensing results in Figure 4 were obtained wirelessly from the capsule and were nearly identical to the results measured using our microfluidic system and conventional parameter analyzer.This work has successfully demonstrated wireless sensing using a 3D-printed sensor capsule and functionality of electrochemical sensors for multiple CQAs for cell growth in bioreactors. These sensor capsules can enable scalability of the cell manufacturing process while ensuring delivery of an efficacious treatment. Future work will focus on capsule sensor validation in cell growth media and eventually monitoring of cell growth in bioreactors.