Response Of Mesenchymal Stem Cells Research Articles

Carboxymethyl Chitosan Promotes Migration and Inhibits Lipopolysaccharide-Induced Inflammatory Response in Canine Bone Marrow-Derived Mesenchymal Stem Cells

Carboxymethyl Chitosan Promotes Migration and Inhibits Lipopolysaccharide-Induced Inflammatory Response in Canine Bone Marrow-Derived Mesenchymal Stem Cells

Effect of Low-Dose Ionizing Radiation on the Expression of Mitochondria-Related Genes in Human Mesenchymal Stem Cells.

The concept of hormesis describes a phenomenon of adaptive response to low-dose ionizing radiation (LDIR). Similarly, the concept of mitohormesis states that the adaptive program in mitochondria is activated in response to minor stress effects. The mechanisms of hormesis effects are not clear, but it is assumed that they can be mediated by reactive oxygen species. Here, we studied effects of LDIR on mitochondria in mesenchymal stem cells. We have found that X-ray radiation at a dose of 10 cGy as well as oxidized fragments of cell-free DNA (cfDNA) at a concentration of 50 ng/mL resulted in an increased expression of a large number of genes regulating the function of the mitochondrial respiratory chain complexes in human mesenchymal stem cells (MSC). Several genes remained upregulated within hours after the exposure. Both X-ray radiation and oxidized cfDNA resulted in upregulation of FIS1 and MFN1 genes, which regulated fusion and fission of mitochondria, within 3–24 h after the exposure. Three hours after the exposure, the number of copies of mitochondrial DNA in cells had increased. These findings support the hypothesis that assumes oxidized cell-free DNA as a mediator of MSC response to low doses of radiation.

PPIA and YWHAZ Constitute a Stable Pair of Reference Genes during Electrical Stimulation in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2. We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2. In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

Autologous versus allogeneic mesenchymal stem cell therapy: The pros and cons

Mesenchymal stem cells (MSCs) have been the subject of countless clinical studies for a wide variety of pathologies, from Crohn’s disease to graft-versus-host disease (GVHD). The results from these MSC-based clinical studies have established that MSCs are safe, but MSC efficacy has not been as well established, with very few studies progressing to phase 2 or 3. The varied outcomes of MSCs in clinical studies is an area of intense and active investigation, and several factors have been identified as critical determinants of MSC response patterns.

Bone morphogenetic protein 9 enhances osteogenic and angiogenic responses of human amniotic mesenchymal stem cells cocultured with umbilical vein endothelial cells through the PI3K/AKT/m-TOR signaling pathway.

Background: Neovascularization plays an essential part in bone fracture and defect healing, constructing tissue engineered bone that targets bone regeneration. Bone morphogenetic protein 9 (BMP9) is a regular indicator that potentiates osteogenic and angiogenic differentiation of MSCs.Objectives: To investigate the effects of BMP9 on osteogenesis and angiogenesis of human amniotic mesenchymal stem cells (hAMSCs) cocultured with human umbilical vein endothelial cells (HUVECs) and determine the possible underlying molecular mechanism.Results: The isolated hAMSCs expressed surface markers of MSCs. hAMSCs cocultured with HUVECs enhance osteogenic differentiation and upregulate the expression of angiogenic factors. BMP9 not only potentiates angiogenic signaling of hAMSCs cocultured with HUVECs also increases ectopic bone formation and subcutaneous vessel invasion. Mechanically, the coupling effect between osteogenesis and angiogenesis induced by BMP9 was activated by the BMP/Smad and PI3K/AKT/m-TOR signaling pathways.Conclusions: BMP9-enhanced osteoblastic and angiogenic differentiation in cocultivation with hAMSCs and HUVECs in vitro and in vivo also provide a chance to harness the BMP9-regulated coordinated effect between osteogenic and angiogenic pathways through BMP/Smad and PI3K/AKT/m-TOR signalings.Materials and Methods: The ALP and Alizarin Red S staining assay to determine the effects of osteoblastic differentiation. RT-qPCR and western blot was measured the expression of angiogenesis-related factors. Ectopic bone formation was established and retrieved bony masses were subjected to histochemical staining. The angiogenesis ability and vessel invasion were subsequently determined by immunofluorescence staining. Molecular mechanisms such as the BMP/Smad and PI3K/AKT/m-TOR signaling pathways were detected by ELISA and western blot analysis.

Microstructure Manipulation of Polyurethane‐Based Macromolecular Scaffold for Tendon/Ligament Tissue Engineering

AbstractIn spite of improvements in tissue engineering approaches, tendon and ligament (T/L) regeneration is still a clinical challenge. The fabrication of functional scaffolds that possess the structural and biological properties of native T/L tissues is critical to a successful healing. Herein, the effect of scaffold microstructure on mesenchymal stem cells (MSCs) response is evaluated. Electrospun polyurethane (EPU) scaffolds with different alignment degrees of nanofibers are fabricated using five rotating speeds of the collector. Although the constructs display similarity in terms of porosity (85%), the increasing degree of alignment results in a notable reduction of fiber diameter. Accordingly, it also improves the tensile strength from 26.44 ± 0.50 to 35.76 ± 1.50 MPa and tensile modulus from 18.94 ± 1.02 to 31.10 ± 1.11 MPa. Furthermore, despite similarities of cell supporting behavior among all types of scaffolds in terms of cell attachment, spreading, proliferation, and infiltration, the aligned nanofibers noticeably direct cell orientation parallel to their alignment direction, resulting in significant upregulation of tendon‐related markers expression. Taken together, the results highlight that the aligned EPU scaffolds are a promising candidate for T/L regeneration, which deserves to be further investigated with long‐term studies.

Induction of Premature Cell Senescence Stimulated by High Doses of Antioxidants Is Mediated by Endoplasmic Reticulum Stress.

In our previous study, we found that high doses of several substances with antioxidant capacities (Tempol, resveratrol, diphenyleneiodonium) can cause genotoxic stress and induce premature senescence in the human mesenchymal stem cells (MSCs). Here, using whole-transcriptome analysis, we revealed the signs of endoplasmic reticulum stress and unfolded protein response (UPR) in MSCs stressed with Tempol and resveratrol. In addition, we found the upregulation of genes, coding the UPR downstream target APC/C, and E3 ubiquitin ligase that regulate the stability of cell cycle proteins. We performed the molecular analysis, which further confirmed the untimely degradation of APC/C targets (cyclin A, geminin, and Emi1) in MSCs treated with antioxidants. Human fibroblasts responded to antioxidant applications similarly. We conclude that endoplasmic reticulum stress and impaired DNA synthesis regulation can be considered as potential triggers of cell damage and premature senescence stimulated by high-dose antioxidant treatments.

BMP-2 Induced Signaling Pathways and Phenotypes: Comparisons Between Senescent and Non-senescent Bone Marrow Mesenchymal Stem Cells.

The use of BMP-2 in orthopedic surgery is limited by uncertainty surrounding its effects on the differentiation of mesenchymal stem cells (MSCs) and how this is affected by cellular aging. This study compared the effects of recombinant human BMP-2 (rhBMP-2) on osteogenic and adipogenic differentiation between senescent and non-senescent MSCs. Senescent and non-senescent MSCs were cultured in osteogenic and adipogenic differentiation medium containing various concentrations of rhBMP-2. The phenotypes of these cells were compared by performing a calcium assay, adipogenesis assay, staining, real-time PCR, western blotting, and microarray analysis. rhBMP-2 induced osteogenic differentiation to a lesser extent (P < 0.001 and P = 0.005 for alkaline phosphatase activity and Ca2+ release) in senescent MSCs regardless of dose-dependent increase in both cells. However, the induction of adipogenic differentiation by rhBMP-2 was comparable between them. There was no difference between these two groups of cells in the adipogenesis assay (P = 0.279) and their expression levels of PPARγ were similar. Several genes such as CHRDL1, NOG, SMAD1, SMAD7, and FST encoding transcription factors were proposed to underlie the different responses of senescent and non-senescent MSCs to rhBMP-2 in microarray analyses. Furthermore, inflammatory, adipogenic, or cell death-related signaling pathways such as NF-kB or p38-MAPK pathways were upregulated by BMP-2 in senescent MSCs, whereas bone forming signaling pathways involving BMP, SMAD, and TGF- ß were upregulated in non-senescent MSCs as expected. This phenomenon explains bone forming dominance by non-senescent MSCs and possible frequent complications such as seroma, osteolysis, or neuritis in senescent MSCs during BMP-2 use in orthopedic surgery.

A Bilayer Osteochondral Scaffold with Self‐Assembled Monomeric Collagen Type‐I, Type‐II, and Polymerized Chondroitin Sulfate Promotes Chondrogenic and Osteogenic Differentiation of Mesenchymal Stem Cells

Osteochondral (OC) injuries are suffered by over 40 million patients in Europe alone. Tissue‐engineering approaches may provide a more promising alternative over current treatments by potentially eliminating the need for revision surgery and creating a long‐term substitute. Herein, the goal is to capture the natural biological and mechanical properties of the joint by developing a bilayer scaffold that is novel in two ways: first, a biomimetic bottom‐up approach is used to improve production precision and reduce immunogenicity; monomeric collagen type I and II are self‐assembled to fibrils and then processed to 3D scaffolds. Second, to induce a tissue‐specific response in mesenchymal stem cells (MSCs), polymerized chondroitin sulfate (PCS) is synthesized and grafted to collagen II and hydroxyapatite (HA) is added to collagen I. Incorporation of PCS into collagen II induces a chondrogenic response by upregulation of COL2A1 and ACAN expression, and incorporation of HA into collagen I stimulates osteogenesis and upregulates the expression of COL1A2 and RUNX2. It is remarkable that MSCs give rise to distinct behavior of chondrogenesis and osteogenesis in the two different regions of the bilayer scaffold. This hybrid scaffold of collagen II‐PCS and collagen I‐HA offers a great potential treatment for OC injuries.

Impact of High-Dose Anti-Infective Agents on the Osteogenic Response of Mesenchymal Stem Cells.

Treatment of infected nonunions and severe bone infections is a huge challenge in modern orthopedics. Their treatment routinely includes the use of anti-infective agents. Although frequently used, little is known about their impact on the osteogenesis of mesenchymal stem cells. In a high- and low-dose set-up, this study evaluates the effects of the antibiotics Gentamicin and Vancomycin as well as the antifungal agent Voriconazole on the ability of mesenchymal stem cells to differentiate into osteoblast-like cells and synthesize hydroxyapatite in a monolayer cell culture. The osteogenic activity was assessed by measuring calcium and phosphate concentrations as well as alkaline phosphatase activity and osteocalcin concentration in the cell culture medium supernatant. The amount of hydroxyapatite was measured directly by radioactive 99mTechnetium-HDP labeling. Regarding the osteogenic markers, it could be concluded that the osteogenesis was successful within the groups treated with osteogenic cell culture media. The results revealed that all anti-infective agents have a cytotoxic effect on mesenchymal stem cells, especially in higher concentrations, whereas the measured absolute amount of hydroxyapatite was independent of the anti-infective agent used. Normed to the number of cells it can therefore be concluded that the above-mentioned anti-infective agents actually have a positive effect on osteogenesis while high-dose Gentamycin, in particular, is apparently capable of boosting the deposition of minerals.

Magnesium-incorporated sol-gel Ta2O5 coating on Ti6Al4V and in vitro biocompatibility

Tantalum (Ta) has been used to modify the surface of titanium (Ti) due to its superior biological response under some clinically compromised situations such as osteoporosis and diabetes; however, the techniques used presented technical disadvantages. The currently study prepared Ta2O5 and Mg-incorporated Ta2O5 coatings on Ti6Al4V by a sol-gel method and compared in vitro response of rabbit bone marrow mesenchymal stem cells. Both coatings were submicrometer-thick and comprised nano-sized particles, the former being crystalline whereas the latter amorphous. Compared with uncoated Ti6Al4V, both coatings increased the corrosion resistance and water wettability, and improved the viability, proliferation, osteoblastic differentiation of the cells. Moreover, they also upregulated the expression of an angiogenesis-related gene (HIF-1α). With the exception of corrosion rate, greatest improvements were observed from the Mg-incorporated Ta2O5 coating. These improvements were probably due to the nanostructures and elements release of Mg and Ta from the coatings. All above results suggest that, Ta2O5-based coatings are promising tools for improving the biological response of Ti-based implants under compromised conditions.

The surface modification of long carbon fiber reinforced polyether ether ketone with bioactive composite hydrogel for effective osteogenicity

Long carbon fiber reinforced polyether ether ketone (LCFRPEEK) is fabricated using a three-dimensional (3D) needle-punched method in our previous work, which is considered as a potential orthopedic implant due to its high mechanical strength and isotropic properties, as well as having an elastic modulus similar to human cortical bone. However, the LCFRPEEK has inferior integration with bone tissue, limiting its clinical application. Thus, a facile surface modification method, using gelatin methacrylate/polyacrylamide composite hydrogel coating (GelMA/PAAM) loading with dexamethasone (Dex) on our newly-developed LCFRPEEK composite via concentrated sulfuric acid sulfonating and ultraviolet (UV) irradiation grafting methods, has been developed to tackle the problem. The results demonstrate that the GelMA/PAAM/Dex coating modified sulfonated LCFRPEEK (SCP/GP/Dex) has a hydrophilicity surface, a long-term Dex release capability and forms more bone-like apatite nodules in SBF. The SCP/GP/Dex also displays enhanced cytocompatibility and osteogenic differentiation in terms of rat bone marrow mesenchymal stem cells (rBMSCs) responses in vitro assay. The in vivo rat cranial defect assay confirms that SCP/GP/Dex boosts bone regeneration/osseointegration, which significantly improves osteogenic fixation between the implant and bone tissue. Therefore, the newly-developed LCFRPEEK modified via GelMA/PAAM/Dex bioactive coating exhibits improved biocompatibility and osteogenic integration capability, which has the basis for an orthopedic implant for clinical application.

Hitchhiking Nanoparticles: Mesenchymal Stem Cell-Mediated Delivery of Theranostic Nanoparticles.

Nanotechnology has emerged as a promising solution to permanent elimination of cancer. However, nanoparticles themselves lack specificity to tumors. Due to enhanced migration to tumors, mesenchymal stem cells (MSCs) were suggested as cell-mediated delivery vehicles of nanoparticles. In this study, we have constructed a complex composed of photoluminescent quantum dots (QDs) and a photosensitizer chlorin e6 (Ce6) to obtain multifunctional nanoparticles, combining cancer diagnostic and therapeutic properties. QDs serve as energy donors-excited QDs transfer energy to the attached Ce6 via Förster resonance energy transfer, which in turn generates reactive oxygen species. Here, the physicochemical properties of the QD-Ce6 complex and singlet oxygen generation were measured, and the stability in protein-rich media was evaluated, showing that the complex remains the most stable in protein-free medium. In vitro studies on MSC and cancer cell response to the QD-Ce6 complex revealed the complex-loaded MSCs' potential to transport theranostic nanoparticles and induce cancer cell death. In vivo studies proved the therapeutic efficacy, as the survival of tumor-bearing mice was statistically significantly increased, while tumor progression and metastases were slowed down.

Transduction of rat and human adipose-tissue derived mesenchymal stromal cells by adeno-associated viral vector serotype DJ.

ABSTRACTEx vivo, gene therapy is a powerful approach holding great promises for the treatment of both genetic and acquired diseases. Adeno-associated virus (AAV) vectors are a safe and efficient delivery system for modification of mesenchymal stem cells (MSC) that could maximize their therapeutic benefits. Assessment of MSC viability and functional activity after infection with new AAV serotypes is necessary, due to AAV tropism to specific cell types. We infected human and rat adipose-tissue MSC with hybrid AAV-DJ serotype vectors carrying GFP and SCF genes. GFP expression from AAV-DJ was about 1.5-fold superior to that observed with AAV-2 and lasted for at least 21 days as was evaluated by flow cytometry and fluorescence microscopy. AAV-DJ proves to be suitable for the infection of rat and human MSC with a similar efficiency. Infected MSC were still viable but showed a 25-30% growth-rate slowdown. Moreover, we found an increase of SERPINB2 mRNA expression in human MSC while expression of other oxidative stress markers and extracellular matrix proteins was not affected. These results suggest that there is a differential cellular response in MSC infected with AAV viral vectors, which should be taken into account as it can affect the expected outcome for the therapeutic application.

“Viscotaxis”- directed migration of mesenchymal stem cells in response to loss modulus gradient

Directed cell migration plays a crucial role in physiological and pathological conditions. One important mechanical cue, known to influence cell migration, is the gradient of substrate elastic modulus (E). However, the cellular microenvironment is viscoelastic and hence the elastic property alone is not sufficient to define its material characteristics. To bridge this gap, in this study, we investigated the influence of the gradient of viscous property of the substrate, as defined by loss modulus (G″) on cell migration. We cultured human mesenchymal stem cells (hMSCs) on a collagen-coated polyacrylamide gel with constant storage modulus (G′) but with a gradient in the loss modulus (G″). We found hMSCs to migrate from high to low loss modulus. We have termed this form of directional cellular migration as “Viscotaxis”. We hypothesize that the high loss modulus regime deforms more due to creep in the long timescale when subjected to cellular traction. Such differential deformation drives the observed Viscotaxis. To verify our hypothesis, we disrupted the actomyosin contractility with myosin inhibitor blebbistatin and ROCK inhibitor Y27632, and found the directional migration to disappear. Further, such time-dependent creep of the high loss material should lead to lower traction, shorter lifetime of the focal adhesions, and dynamic cell morphology, which was indeed found to be the case. Together, findings in this paper highlight the importance of considering the viscous modulus while preparing stiffness-based substrates for the field of tissue engineering. Statement of significanceWhile the effect of substrate elastic modulus has been investigated extensively in the context of cell biology, the role of substrate viscoelasticity is poorly understood. This omission is surprising as our body is not elastic, but viscoelastic. Hence, the role of viscoelasticity needs to be investigated at depth in various cellular contexts. One such important context is cell migration. Cell migration is important in morphogenesis, immune response, wound healing, and cancer, to name a few. While it is known that cells migrate when presented with a substrate with a rigidity gradient, cellular behavior in response to viscoelastic gradient has never been investigated. The findings of this paper not only reveal a completely novel cellular taxis or directed migration, it also improves our understanding of cell mechanics significantly.

Autophagy stimulation delayed biological aging and decreased cardiac differentiation in rabbit mesenchymal stem cells.

Introduction: Cardiovascular disease (CVD) is a type of disease that affects the function of cardiac-vascular tissues. This study aimed to consider the possible effects of autophagy, as an intrinsic catabolic pathway of cells, on the differentiation and aging process of mesenchymal stem cells (MSCs). Methods: In this study, bone marrow-derived MSCs were obtained from rabbit bone marrow aspirates. The stemness feature was confirmed by using flow cytometry analysis Cells at passage three were treated with 50 μM Metformin and 15μM hydroxychloroquine (HCQ) for 72 hours. The intracellular accumulation of autophagolysosomes was imaged using LysoTracker staining. Protein levels of autophagy (LC3II/I ratio), aging (Klotho, PARP-1, and Sirt-1) effectors, and cardiomyocyte-like phenotype (α-actinin) were studied by western blotting. Results: Based on our findings, flow cytometry analysis showed that the obtained cells expressed CD44 and CD133 strongly, and CD31 and CD34 dimly, showing a typical characteristic of MSCs. Our data confirmed an increased LC3II/I ratio in the metformin-received group compared to the untreated and HCQ-treated cells (P < 0.05). Besides, we showed that the incubation of rabbit MSCs with HCQ increased cellular aging by induction of PARP-1 while Metformin increased rejuvenating factor Sirt-1 comparing with the normal group (P < 0.05). Western blotting data showed that the autophagy stimulation response in rabbit MSCs postponed the biological aging and decreased the differentiation potential to the cardiac cells by diminishing α-actinin comparing with control cells (P < 0.05). Conclusion: In summary, for the informants in this study, it could be noted that autophagy inhibition/stimulation could alter rabbit MSCs aging and differentiation capacity.

Basic responses of mesenchymal stem cells exposed to bovine biomaterial and platelet rich fibrin

The scaffolds and their interaction with mesenchymal stem cells are objects of study in bioengineering and tissue repair. Mechanisms such as surface adhesion, proliferation, viability, and cytotoxicity are essential for the development of therapies. The present study analyzed the influence of platelet-rich fibrin (PRF) in viability, cytotoxicity, and proliferation of stem cells from human exfoliated deciduous teeth (SHED) exposed to bovine biomaterial surfaces. The studied groups were divided and analyzed as follows: (S) only SHED as control Group; (SB) SHED + biomaterial; (SBP) SHED + biomaterial + PRF. Analyses of cells seeded in 24-well plates were performed after 24, 48 and 72 hours. Individual groups were subjected to viability, cytotoxicity and cell proliferation tests using neutral red, MTT and crystal violet, respectively; and in the 72-hour group, scanning electron microscopy (SEM) was performed to record cell ultra-morphology. Data were submitted to statistical analysis by two-factor ANOVA with a significance level of 5%. The results demonstrated a better performance in the viability/cytotoxicity and proliferation of stem cells for the group (SBP) in comparison to the group (SB) and the group (S). The applied statistical tests showed that the biomaterial factor, time, and interaction between them gave rise to results with statistical significance. SHED submitted to bovine biomaterial were more viable, proliferative and with lower toxicity when associated with PRF. PRF seemed to activate the metabolism of stem cells in culture, indicating that such an association can bring an effective benefit in clinical outcome.

Immobilizing type I collagen via natural cross-linker genipin to enhance the osteogenic responses to titanium implant surface

Titanium (Ti) surface, with an essentially bioinert character, is not easy to actively initiate interactions with surrounding biological tissue. In this study, we developed a novel surface modification to enhance the osteogenic response to Ti surface in orthopedic and dental implant applications. The proposed surface modification combined alkaline treatment and type I collagen immobilization through using a natural cross-linker genipin. A variety of experiments were used to characterize the resulting surfaces in terms of morphology, chemistry, roughness, hydrophilicity, and bioactivity. We also assessed the responses of human bone marrow mesenchymal stem cells (hMSCs) to Ti surfaces in terms of adhesion, migration, proliferation, mineralization, and differentiation. Results showed that the modified Ti surfaces revealed a hydrophilic (water and diiodomethane contact angles <22°) nano-/submicron-scaled porous network porosity with surface roughness Ra ~0.1 μm and good bioactivity (Ca/P formation ability). The collagen-immobilized Ti surfaces with good coating adhesion was shown to facilitate the mineralization of the extracellular matrix and the expressions of specific osteogenic markers (osteopontin, bone sialoprotein, and osteocalcin) in hMSCs, thanks to improved cell adhesion and migration. Overall, the proposed Ti implant surface modification proved highly effective in terms of promoting osteogenic responses in vitro.

SDF-1α/OPF/BP Composites Enhance the Migrating and Osteogenic Abilities of Mesenchymal Stem Cells.

In situ cell recruitment is a promising regenerative medicine strategy with the purpose of tissue regeneration without stem cell transplantation. This chemotaxis-based strategy is aimed at ensuring a restorative environment through the release of chemokines that promote site-specific migration of healing cell populations. Stromal cell-derived factor-1α (SDF-1α) is a critical chemokine that can regulate the migration of mesenchymal stem cells (MSCs). Accordingly, here, SDF-1α-loaded microporous oligo[poly(ethylene glycol) fumarate]/bis[2-(methacryloyloxy)ethyl] phosphate composites (SDF-1α/OPF/BP) were engineered and probed. SDF-1α/OPF/BP composites were loaded with escalating SDF-1α concentrations, namely, 0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml, and were cocultured with MSC. Scratching assay, Transwell assay, and three-dimensional migration model were utilized to assess the migration response of MSCs. Immunofluorescence staining of Runx2 and osteopontin (OPN), ELISA assay of osteocalcin (OCN) and alkaline phosphatase (ALP), and Alizarin Red S staining were conducted to assess the osteogenesis of MSCs. All SDF-1α/OPF/BP composites engendered a release of SDF-1α (>80%) during the first four days. SDF-1α released from the composites significantly promoted migration and osteogenic differentiation of MSCs documented by upregulated expression of osteogenic-related proteins, ALP, Runx2, OCN, and OPN. SDF-1α at 100 ng/ml was optimal for enhanced migration and osteogenic proficiency. Thus, designed SDF-1α/OPF/BP composites were competent in promoting the homing and osteogenesis of MSCs and thus offer a promising bioactive scaffold candidate for on-demand bone tissue regeneration.

Electrical stimulation of titanium to promote stem cell orientation, elongation and osteogenesis

Electrical stimulation of cells allows exogenous electric signals as stimuli to manipulate cell growth, preferential orientation and bone remodelling. In this study, commercially pure titanium discs were utilised in combination with a custom-built bioreactor to investigate the cellular responses of human mesenchymal stem cells via in-vitro functional assays. Finite element analysis revealed the homogeneous delivery of electric field in the bioreactor chamber with no detection of current density fluctuation in the proposed model. The custom-built bioreactor with capacitive stimulation delivery system features long-term stimulation with homogeneous electric field, biocompatible, sterilisable, scalable design and cost-effective in the manufacturing process. Using a continuous stimulation regime of 100 and 200 mV/mm on cp Ti discs, viability tests revealed up to an approximately 5-fold increase of cell proliferation rate as compared to non-stimulated controls. The human mesenchymal stem cells showed more elongated and differentiated morphology under this regime, with evidence of nuclear elongation and cytoskeletal orientation perpendicular to the direction of electric field. The continuous stimulation did not cause pH fluctuations and hydrogen peroxide production caused by Faradic reactions, signifying the suitability for long-term toxic free stimulation as opposed to the commonly used direct stimulation regime. An approximate of 4-fold increase in alkaline phosphatase production and approximately 9-fold increase of calcium deposition were observed on 200 mV/mm exposed samples relative to non-stimulated controls. It is worth noting that early stem cell differentiation and matrix production were observed under the said electric field even without the presence of chemical inductive growth factors. Statement of significanceThis manuscript presents a study on combining pure titanium (primarily preferred as medical implant materials) and electrical stimulation in a purpose-built bioreactor with capacitive stimulation delivery system. A continuous capacitive stimulation regime on titanium disc has resulted in enhanced stem cell orientation, nuclei elongation, proliferation and differentiation as compared to non-stimulated controls. We believe that this manuscript creates a paradigm for future studies on the evolution of healthcare treatments in the area of targeted therapy on implantable and wearable medical devices through tailored innovative electrical stimulation approach, thereby influencing therapeutic conductive and electroactive biomaterials research prospects and development.