hMSC Proliferation Research Articles

Graded or random – Effect of pore distribution in 3D titanium scaffolds on corrosion performance and response of hMSCs

Researchers agree that the ideal scaffold for tissue engineering should possess a 3D and highly porous structure, biocompatibility to encourage cell/tissue growth, suitable surface chemistry for cell attachment and differentiation, and mechanical properties that match those of the surrounding tissues. However, there is no consensus on the optimal pore distribution. In this study, we investigated the effect of pore distribution on corrosion resistance and performance of human mesenchymal stem cells (hMSC) using titanium scaffolds fabricated by laser beam powder bed fusion (PBF-LB). We designed two scaffold architectures with the same porosities (i.e., 75 %) but different distribution of pores of three sizes (200, 500, and 700 μm). The pores were either grouped in three zones (graded, GRAD) or distributed randomly (random, RAND). Microfocus X-ray computed tomography revealed that the chemically polished scaffolds had the porosity of 69 ± 4 % (GRAD) and 71 ± 4 % (RAND), and that the GRAD architecture had the higher surface area (1580 ± 101 vs 991 ± 62 mm2) and the thinner struts (221 ± 37 vs 286 ± 14 μm). The electrochemical measurements demonstrated that the apparent corrosion rate of chemically polished GRAD scaffold decreased with the immersion time extension, while that for polished RAND was increased. The RAND architecture outperformed the GRAD one with respect to hMSC proliferation (over two times higher although the GRAD scaffolds had 85 % higher initial cell retention) and migration from a monolayer. Our findings demonstrate that the pore distribution affects the biological properties of the titanium scaffolds for bone tissue engineering.

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

Opposite Mechanical Preference of Bone/Nerve Regeneration in 3D-Printed Bioelastomeric Scaffolds/Conduits Consistently Correlated with YAP-Mediated Stem Cell Osteo/Neuro-Genesis.

To systematically unveil how substrate stiffness, a critical factor in directing cell fate through mechanotransduction, correlates with tissue regeneration, novel biodegradable and photo-curable poly(trimethylene carbonate) fumarates (PTMCFs) for fabricating elastomeric 2D substrates and 3D bone scaffolds/nerve conduits, are presented. These substrates and structures with adjustable stiffness serve as a unique platform to evaluate how this mechanical cue affects the fate of human umbilical cord mesenchymal stem cells (hMSCs) and hard/soft tissue regeneration in rat femur bone defect and sciatic nerve transection models; whilst, decoupling from topographical and chemical cues. In addition to a positive relationship between substrate stiffness (tensile modulus: 90-990kPa) and hMSC adhesion, spreading, and proliferation mediated through Yes-associated protein (YAP), opposite mechanical preference is revealed in the osteogenesis and neurogenesis of hMSCs as they are significantly enhanced on the stiff and compliant substrates, respectively. In vivo tissue regeneration demonstrates the same trend: bone regeneration prefers the stiffer scaffolds; while, nerve regeneration prefers the more compliant conduits. Whole-transcriptome analysis further shows that upregulation of Rho GTPase activity and the downstream genes in the compliant group promote nerve repair, providing critical insight into the design strategies of biomaterials for stem cell regulation and hard/soft tissue regeneration through mechanotransduction.

Reversible Host-Guest Crosslinks in Supramolecular Hydrogels for On-Demand Mechanical Stimulation of Human Mesenchymal Stem Cells.

Stem cells are regulated not only by biochemical signals but also by biophysical properties of extracellular matrix (ECM). The ECM is constantly monitored and remodeled because the fate of stem cells can be misdirected when the mechanical interaction between cells and ECM is imbalanced. A well-defined ECM model for bone marrow-derived human mesenchymal stem cells (hMSCs) based on supramolecular hydrogels containing reversible host-guest crosslinks is fabricated. The stiffness (Young's modulus E) of the hydrogels can be switched reversibly by altering the concentration of non-cytotoxic, free guest molecules dissolved in the culture medium. Fine-adjustment of substrate stiffness enables the authors to determine the critical stiffness level E* at which hMSCs turn the mechano-sensory machinery on or off. Next, the substrate stiffness across E* is switched and the dynamic adaptation characteristics such as morphology, traction force, and YAP/TAZ signaling of hMSCs are monitored. These data demonstrate the instantaneous switching of traction force, which is followed by YAP/TAZ signaling and morphological adaptation. Periodical switching of the substrate stiffness across E* proves that frequent applications of mechanical stimuli drastically suppress hMSC proliferation. Mechanical stimulation across E* level using dynamic hydrogels is a promising strategy for the on-demand control of hMSC transcription and proliferation.

Modified-Hydroxyapatite-Chitosan Hybrid Composite Interfacial Coating on 3D Polymeric Scaffolds for Bone Tissue Engineering.

Three dimensional (3D) scaffolds have huge limitations due to their low porosity, mechanical strength, and lack of direct cell-bioactive drug contact. Whereas bisphosphonate drug has the ability to stimulate osteogenesis in osteoblasts and bone marrow mesenchymal stem cells (hMSC) which attracted its therapeutic use. However it is hard administration low bioavailability, and lack of site-specificity, limiting its usage. The proposed scaffold architecture allows cells to access the bioactive surface at their apex by interacting at the scaffold's interfacial layer. The interface of 3D polycaprolactone (PCL) scaffolds has been coated with alendronate-modified hydroxyapatite (MALD) enclosed in a chitosan matrix, to mimic the native environment and stupulate the through interaction of cells to bioactive layer. Where the mechanical strength will be provided by the skeleton of PCL. In the MALD composite's hydroxyapatite (HAP) component will govern alendronate (ALD) release behavior, and HAP presence will drive the increase in local calcium ion concentration increases hMSC proliferation and differentiation. In results, MALD show release of 86.28±0.22. XPS and SEM investigation of the scaffold structure, shows inspiring particle deposition with chitosan over the interface. All scaffolds enhanced cell adhesion, proliferation, and osteocyte differentiation for over a week without in vitro cell toxicity with 3.03±0.2kPa mechanical strength.

Long non-coding RNA CASC2 regulates osteoblasts matrix mineralization.

Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases in the bone tissue, including osteoporosis and tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (hMSCs). CASC2 knockdown, using small interfering RNA against CASC2 (siCASC2), increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining level nor the expression of early osteogenic transcripts, including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at both transcriptional and protein level. On the other hand, inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP. The results indicate that lncRNA CASC2 regulates late osteogenic differentiation and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests that targeting lncRNA CASC2 could be a potential approach for modulating bone mineralization.

Nanofiber matrix formulations for the delivery of Exendin-4 for tendon regeneration: In vitro and in vivo assessment.

Tendon and ligament injuries are the most common musculoskeletal injuries, which not only impact the quality of life but result in a massive economic burden. Surgical interventions for tendon/ligament injuries utilize biological and/or engineered grafts to reconstruct damaged tissue, but these have limitations. Engineered matrices confer superior physicochemical properties over biological grafts but lack desirable bioactivity to promote tissue healing. While incorporating drugs can enhance bioactivity, large matrix surface areas and hydrophobicity can lead to uncontrolled burst release and/or incomplete release due to binding. To overcome these limitations, we evaluated the delivery of a peptide growth factor (exendin-4; Ex-4) using an enhanced nanofiber matrix in a tendon injury model. To overcome drug surface binding due to matrix hydrophobicity of poly(caprolactone) (PCL)-which would be expected to enhance cell-material interactions-we blended PCL and cellulose acetate (CA) and electrospun nanofiber matrices with fiber diameters ranging from 600 to 1000nm. To avoid burst release and protect the drug, we encapsulated Ex-4 in the open lumen of halloysite nanotubes (HNTs), sealed the HNT tube endings with a polymer blend, and mixed Ex-4-loaded HNTs into the polymer mixture before electrospinning. This reduced burst release from ∼75% to ∼40%, but did not alter matrix morphology, fiber diameter, or tensile properties. We evaluated the bioactivity of the Ex-4 nanofiber formulation by culturing human mesenchymal stem cells (hMSCs) on matrix surfaces for 21 days and measuring tenogenic differentiation, compared with nanofiber matrices in basal media alone. Strikingly, we observed that Ex-4 nanofiber matrices accelerated the hMSC proliferation rate and elevated levels of sulfated glycosaminoglycan, tendon-related genes (Scx, Mkx, and Tnmd), and ECM-related genes (Col-I, Col-III, and Dcn), compared to control. We then assessed the safety and efficacy of Ex-4 nanofiber matrices in a full-thickness rat Achilles tendon defect with histology, marker expression, functional walking track analysis, and mechanical testing. Our analysis confirmed that Ex-4 nanofiber matrices enhanced tendon healing and reduced fibrocartilage formation versus nanofiber matrices alone. These findings implicate Ex-4 as a potentially valuable tool for tendon tissue engineering.

Enhancement of hMSC In Vitro Proliferation by Surface Immobilization of a Heparin-Binding Peptide.

The use of human Mesenchymal Stem Cells (hMSC) as therapeutic agents for advanced clinical therapies relies on their in vitro expansion. Over the last years, several efforts have been made to optimize hMSC culture protocols, namely by mimicking the cell physiological microenvironment, which strongly relies on signals provided by the extracellular matrix (ECM). ECM glycosaminoglycans, such as heparan-sulfate, sequester adhesive proteins and soluble growth factors at the cell membrane, orchestrating signaling pathways that control cell proliferation. Surfaces exposing the synthetic polypeptide poly(L-lysine, L-leucine) (pKL) have previously been shown to bind heparin from human plasma in a selective and concentration-dependent manner. To evaluate its effect on hMSC expansion, pKL was immobilized onto self-assembled monolayers (SAMs). The pKL-SAMs were able to bind heparin, fibronectin and other serum proteins, as demonstrated by quartz crystal microbalance with dissipation (QCM-D) studies. hMSC adhesion and proliferation were significantly increased in pKL-SAMs compared to controls, most probably related to increased heparin and fibronectin binding to pKL surfaces. This proof-of-concept study highlights the potential of pKL surfaces to improve hMSC in vitro expansion possible through selective heparin/serum protein binding at the cell-material interface.

Synergistic effects of growth factor-based serum-free medium and tendon-like substrate topography on tenogenesis of mesenchymal stem cells

Addressing clinical challenges for tendon injuries requires a deeper understanding of the effects that biological and biophysical cues have on tenogenesis. Although prior studies have identified tenogenic growth factors (GFs) or elucidated the effects of substrate topography on tenocyte behavior, few have characterized their combined effect in the presence of a tendon-like substrate. In this study, we assessed the effect of biological (GFs) and biophysical (substrate topography) cues on tenogenic proliferation and differentiation under defined, serum-free conditions. Specifically, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured in a serum-free culture medium containing a GF cocktail comprised of fibroblast growth factor-2 (FGF-2), transforming growth factor-beta 3 (TGF-β3), and insulin-like growth factor-1 (IGF-1), either alone or in combination with tendon-like substrate topography produced by replica casting of tendon tissue sections. Our data demonstrated that the use of serum-free GF cocktail medium alone promoted hMSC proliferation, as shown via DNA staining as well as Ki67 protein levels and gene expression. In particular, gene expression of Ki67 was increased by 8.46-fold in all three donors relative to serum-free medium control. Also, serum-free GF cocktail promoted tenogenic differentiation, on the basis of expression of tendon-associated gene and protein markers, scleraxis (SCX), tenascin C (TNC), and collagen type I (COL1A1) including increased normalized collagen production by 1.4-fold in two donors relative to serum-free medium control. Interestingly, hMSCs cultured on a tendon-like substrate exhibited highly oriented cell morphology and extracellular matrix (ECM) alignment reminiscent of tendon. In particular, when this GF cocktail was combined with tendon-like topography, they showed a synergistically increased expression of tendon-related markers and anisotropic organization of ECM proteins with moderate-to-large effect sizes. Together, in addition to showing the utility of a GF cocktail for expansion and differentiation of tenocyte-like cells, our findings clearly demonstrate the synergistic relationship between GF-mediated and substrate topography-related effects on hMSC tenogenic differentiation. This information provides insights into the design of strategies that combine biological and biophysical cues for ex vivo tenocyte production and tendon tissue engineering.

Crosslinker structure modulates bulk mechanical properties and dictates hMSC behavior on hyaluronic acid hydrogels.

Synthetic hydrogels are attractive platforms due in part to their highly tunable mechanics, which impact cell behavior and secretory profile. These mechanics are often controlled by altering the number of crosslinks or the total polymer concentration in the gel, leading to structure-property relationships that inherently couple network connectivity to the overall modulus. In contrast, the native extracellular matrix (ECM) contains structured biopolymers that enable stiff gels even at low polymer content, facilitating 3D cell culture and permeability of soluble factors. To mimic the hierarchical order of natural ECM, this work describes a synthetic hydrogel system in which mechanics are tuned using the structure of sequence-defined peptoid crosslinkers, while fixing network connectivity. Peptoid crosslinkers with different secondary structures are investigated: 1) a helical, molecularly stiff peptoid, 2) a non-helical, less stiff peptoid, and 3) an unstructured, relatively flexible peptoid. Bulk hydrogel storage modulus increases when crosslinkers of higher chain stiffness are used. In-vitro studies assess the viability, proliferation, cell morphology, and immunomodulatory activity of human mesenchymal stem cells (hMSCs) on each hydrogel substrate. Matrix mechanics regulate the morphology of hMSCs on the developed substrates, and all of the hydrogels studied upregulate IDO production over culture on TCP. Softer substrates further this upregulation to a plateau. Overall, this system offers a biomimetic strategy for decoupling hydrogel storage modulus from network connectivity, enabling systematic study of biomaterial properties on hMSC behavior and enhancement of cellular functionality for therapeutic applications. STATEMENT OF SIGNIFICANCE: Various strategies to tune hydrogel mechanics have been developed to control human mesenchymal stem cell (hMSC) behavior and regulate their immunomodulatory potential. However, these strategies typically couple mechanics to network connectivity, which in turn changes other hydrogel properties such as permeability that may have unintended effects on hMSC behavior. This work presents a strategy to tune hydrogel mechanics using crosslinkers with different secondary structure and molecular rigidity. This strategy successfully decouples hydrogel moduli from crosslinker stoichiometry and mimics the hierarchical nature of the native extracellular matrix. The moduli of the developed hydrogels led to significant impacts on hMSC morphology and proliferation, and increased immunomodulatory potential, indicating that molecular rigidity is a promising avenue to control engineered ECM mechanics for therapeutic applications.

Micromechanical property mismatch between pericellular and extracellular matrices regulates stem cell articular and hypertrophic chondrogenesis

<h2>Summary</h2> Within the complex microarchitecture of native cartilage tissue, the micromechanical properties of pericellular and extracellular matrices (PCM and ECM) potentially play important roles in developmental, physiological, and pathological processes. Here, we report a unique biomaterial-based engineering strategy to create cartilage-tissue equivalents possessing PCM-ECM microarchitecture of native cartilage, where human mesenchymal stem cell (hMSC)-laden soft microgels representing PCM are encapsulated in stiff hydrogels representing ECM. Mechanical property mismatches between soft PCM and stiff ECM under cyclic compression regulates hMSC proliferation and chondrogenesis. High PCM-ECM mechanical mismatch (softer PCM) and the presence of PCM degradation under cyclic compression individually or synergistically direct hMSC articular chondrogenesis through the proliferation-associated protein kinase C signaling pathway, whereas low PCM-ECM mechanical mismatch (stiffer PCM) is solely responsible for hMSC hypertrophic chondrogenesis through the yes-associated protein signaling pathway. Our findings highlight PCM-ECM mechanical property mismatch as a critical design cue under dynamic compression for hMSC-based cartilage repair.

Substrate Stiffness Controls the Cell Cycle of Human Mesenchymal Stem Cells Via Cellular Traction

The microenvironment of human mesenchymal stem cells (hMSCs) regulates their self-renewal and differentiation properties. Previously, it was shown that hMSCs remain quiescent on soft (0.25 kPa) polyacrylamide (PA) gels, but re-enter the cell cycle on a stiff (7.5 kPa) gel. However, how cells behave on intermediate stiffness and what intracellular factors transmit mechanical changes to cell interior, thereby regulating cell cycle, remained unknown. In this work, we have demonstrated that PA gels of stiffness between 1 kPa and 5 kPa act as a mechanical switch from quiescence to proliferation in the cell cycle of hMSCs. Further studies with ROCK inhibitor Y-27632 have revealed that contractile proteins, but not cell spread area, account for the sensitivity of hMSCs towards substrate stiffness, and hence correlates with their changes in cell cycle. These observations therefore suggest that substrate stiffness regulates hMSC proliferation through contractile forces, as generated by cellular contractile proteins in a unique pattern that is distinct from the two other cell types studied here, C2C12 and 3T3.

Enhanced proliferation and differentiation of human mesenchymal stem cells in the gravity-controlled environment.

Human bone marrow mesenchymal stem cells (hMSCs) present a promising cell source with the potential to be used for curing various intractable diseases, and it is expected that the development of regenerative medicine employing cell-based therapy would be significantly accelerated when such methods are established. For that, powerful methods for selective growth and differentiation of hMSCs should be developed. We developed an efficient method for hMSC proliferation and differentiation into osteoblasts and adipocytes using gravity-controlled environments. The results indicate that the average doubling time of hMSCs cultured in a regular maintenance medium under microgravity conditions (0.001 G) was 1.5 times shorter than that of cells cultured under natural gravity conditions (1.0G). Furthermore, 99.2% of cells grown in the microgravity environment showed the expression of hMSC markers, as indicated by flow cytometry analysis. Osteogenic and adipogenic differentiation of hMSCs expanded in the microgravity environment was enhanced under microgravity and hypergravity conditions, respectively, as evidenced by the downregulation of hMSC markers and upregulation of osteoblast and adipocyte markers, respectively. Most cells differentiated into osteoblasts in the microgravity environment after 14 days (~80%) and adipocytes in the hypergravity environment after 12 days (~90%). Our results indicate that hMSC proliferation and selective differentiation into specific cell lineages could be promoted under microgravity or hypergravity conditions, suggesting that cell culture in the gravity-controlled environment is a useful method to obtain cell preparations for potential clinical applications.

Three-dimensional Printing of Biomimetic Titanium Mimicking Trabecular Bone Induces Human Mesenchymal Stem Cell Proliferation: An In-vitro Analysis.

In vitro analysis. The aim of this study was to assess the effect of three-dimensional (3D) printing of porous titanium on human mesenchymal stem cell (hMSC) adhesion, proliferation, and osteogenic differentiation. A proprietary implant using three-dimensional porous titanium (3D-pTi) that mimics trabecu-lar bone structure, roughness, porosity, and modulus of elasticity was created (Ti-LIFE technology™, Spineart SA Switzerland). Such implants may possess osteoinductive properties augmenting fusion in addition to their structural advantages. However, the ability of 3D-pTi to affect in vitro cellular proliferation and osteogenic differentiation remains undefined. Disks of 3D-pTi with a porosity of 70% to 75% and pore size of 0.9 mm were produced using additive manufacturing technology. 2D Ti6Al4V (2D-Ti) and 2D polyetheretherketone (2D-PEEK) disks were prepared using standard manufacturing process. Tissue culture plastic (TCP) served as the control surface. All discs were characterized using 2D-micros-copy, scanning electron microscopy (SEM), and x-ray micro-computed tomography. Forty thousand hMSCs were seeded on the disks and TCP and cultured for 42 days. hMSC morphology was assessed using environmental SEM and confocal imaging following phalloidin staining. hMSC proliferation was evaluated using DNA fluorescent assay. hMSC differentiation was assessed using RT-qPCR for genes involved in hMSC osteogenic differentiation and biochemical assays were performed for alkaline phosphatase activity (ALP) and calcium content. 3D-pTi lead to a higher cell number as compared to 2D-Ti and 2D-PEEK at D21, D28 and D42. ALP activity of hMSCs seeded into 3D-pTi scaffolds was as high as or higher than that of hMSCs seeded onto TCP controls over all time points and consistently higher than that of hMSCs seeded onto 2D-Ti scaffolds. However, when ALP activity was normalized to protein content, no statistical differences were found between all scaffolds tested and TCP controls. 3D-pTi provides a scaffold for bone formation that structurally mimics cancellous bone and improves hMSC adhesion and proliferation compared to 2D-Ti and PEEK.

Dentin Matrix Protein 1 on Titanium Surface Facilitates Osteogenic Differentiation of Stem Cells.

Dentin matrix protein 1 (DMP1) contains a large number of acidic domains, multiple phosphorylation sites, a functional arginine-glycine-aspartate (RGD) motif, and a DNA binding domain, and has been shown to play essential regulatory function in dentin and bone mineralization. DMP1 could also orchestrate bone matrix formation, but the ability of DMP1 on Ti to human mesenchymal stem cell (hMSC) conversion to osteoblasts has not been studied. There is importance to test if the DMP1 coated Ti surface would promote cell migration and attachment to the metal surface and promote the differentiation of the attached stem cells to an osteogenic lineage. This study aimed to study the human mesenchymal stem cells (hMSCs) attachment and proliferation on DMP1 coated titanium (Ti) disks compared to non-coated disks, and to assess possible osteoblastic differentiation of attached hMSCs. Sixty-eight Ti disks were divided into two groups. Group 1 disks were coated with dentin matrix protein 1 and group 2 disks served as control. Assessment with light microscopy was used to verify hMSC attachment and proliferation. Cell viability was confirmed through fluorescence microscopy and mitochondrial dehydrogenase activity. Real-time polymerase chain reaction analysis was done to study the gene expression. The proliferation assay showed significantly greater cell proliferation with DMP1 coated disks compared to the control group (p-value < 0.001). Cell vitality analysis showed a greater density of live cells on DMP1 coated disks compared to the control group. Alkaline phosphatase staining revealed higher enzyme activity on DMP1 coated disks and showed itself to be significantly higher than the control group (p-value < 0.001). von Kossa staining revealed higher positive areas for mineralized deposits on DMP1 coated disks than the control group (p-value < 0.05). Gene expression analysis confirmed upregulation of runt-related transcription factor 2, osteoprotegerin, osteocalcin, osteopontin, and alkaline phosphatase on DMP1 coated disks (p-value < 0.001). The dentin matrix protein promoted the adhesion, proliferation, facilitation differentiation of hMSC, and mineralized matrix formation.

3D Confinement Regulates Cell Life and Death

AbstractBiophysical properties of the cellular microenvironment, including stiffness and geometry, have been shown to influence cell function. Recent findings have implicated 3D confinement as an important regulator of cell behavior. The understanding of how mechanical signals direct cell function is based primarily on 2D studies. To investigate how the extent of 3D confinement affects cell function, a single cell culture platform is fabricated with geometrically defined and fully enclosed microwells and it is applied to investigate how niche volume and stiffness affect human mesenchymal stem cells (hMSC) life and death. The viability and proliferation of hMSCs in confined 3D microniches are compared with unconfined cells in 2D. Confinement biases hMSC viability and proliferation, and this influence depends on the niche volume and stiffness. The rate of cell death increases and proliferation markedly decreases upon 3D confinement. The observed differences in hMSC behavior are correlated to changes in nuclear morphology and YES‐associated protein (YAP) localization. In smaller 3D microniches, hMSCs display smaller and more rounded nuclei and primarily cytoplasmic YAP localization, indicating reduced mechanical activation upon confinement. Interestingly, these effects scale with the extent of 3D confinement. These results demonstrate that the extent of confinement in 3D can be an important regulator of cell function.

Synergistic effect of sericin and keratin in gelatin based nanofibers for in vitro applications

Protein-based nanomaterials are gaining growing interest in biomedical field. The present paper evaluates the physico-chemical properties of electrospun nanofibers resulting from the combination of gelatin with keratin (from wool) and sericin (from silk) to validate their use for in vitro interaction studies. We demonstrated that that presence of sericin influences the fiber morphology at macroscopic level – i.e., wide diameter distributions by SEM and image analysis – with effects on chemical – i.e., a decrease of hydrogen bonds of NH groups verified by infrared spectroscopy – and thermal behavior of electrospun nanofibers, in comparison with gelatin-based ones. Moreover, we verified that sericin, in combination with keratin macromolecules, can amplify the biochemical signal of gelatin, improving the in-vitro stability of gelatin-based nanofibers. In vitro results confirm a synergistic effect of sericin and keratin on human Mesenchymal Stem Cells (hMSC) proliferation – increase over 50% respect to other types – associated to the enhancement of in vitro stability directly ascribable to the peculiar physical interaction among the proteins. These findings suggest the use of sericin/keratin/gelatin enriched electrospun fibers as nanostructured platforms for interface tissue engineering.

3D Bioprinting of Polythiophene Materials for Promoting Stem Cell Proliferation in a Nutritionally Deficient Environment.

3D printing of stem cells provides a tremendous opportunity to tissue engineering in regenerative medicine. However, developing new bioactive materials to rationally augment stem cell viability is still an enormous challenge owing to the nutritionally deficient environment caused by the limited-penetration distance of nutrition when cells are encapsulated within biomaterials. In this work, a cationic conjugated polythiophene derivative, poly[3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT), is designed and integrated into an anionic gelatin/alginate matrix to develop a new 3D bioprintable conjugated polymer ink Gel/Alg/PMNT, while the electrostatic interaction can assist PMNT to anchor inside ink without severe diffusional loss. In principle, PMNT is confirmed to promote human umbilical cord-derived mesenchymal stem cell (hMSC) proliferation in a serum-free medium by driving cell cycles and up-regulating gene expression in the pathways of biosynthesis and the metabolism. By employing the 3D bioprinting strategy together with hMSCs, the accelerated healing of full-thickness excisional wounds is further realized through the augmented-stem cell therapeutics utilizing Gel/Alg/PMNT ink, in which hMSC proliferation can be effectively promoted upon inductive stimulation of PMNT. The inherent highly bioactive and robust proliferation-promoted nature of the developed conjugated polymer ink Gel/Alg/PMNT significantly overcomes the nutritionally deficient environment, especially in 3D-printed large-scale architectures. The bioactive polythiophene material exhibits a unique capacity to promote stem cell proliferation without the need of serum, providing a new bioink for 3D bioprinting in tissue reconstructions.

Osteogenic differentiation driven by osteoclasts and macrophages

IntroductionOsteoclasts are bone-resorbing cells closely related to bone turnover, whereas different macrophage subtypes contribute to bone fracture healing. As osteoclasts and macrophages share the same hematopoietic origin, the difference between both cell types on osteoblast coupling, crosstalk extent and consequent bone formation remains poorly understood. This study compares the potential of primary cells that are routinely considered as osteoclast and macrophage cultures on their ability to support osteogenic differentiation of human mesenchymal stromal cells (hMSCs). MethodsHuman Peripheral Blood Mononuclear Cells (hPBMCs) were used to obtain macrophage or osteoclast cultures using appropriate stimulatory factors. With different seeding densities of hPBMCs, conditioned media from macrophage or osteoclast cultures were harvested for comparative evaluation of effects thereof on the osteogenic differentiation of hMSCs. Specific cytological staining was used to qualitatively evaluate macrophage and osteoclast cultures. Additionally, quantitative data on hMSC proliferation, osteogenic differentiation and mineralization were obtained via biochemical assays. ResultsConditioned medium from osteoclast cultures obtained via low hPBMCs seeding densities, but not from high hPBMCs seeding densities or macrophages, stimulated hMSC osteogenic differentiation and mineralization. Upon cellular crosstalk, both pre-differentiated osteoclasts and non-polarized macrophages equally supported early hMSC osteogenic differentiation and mineralization, as confirmed by increased alkaline phosphatase levels within 7 days and increased calcium content within 14 days in comparison with undifferentiated controls. Initial hPBMCs seeding density strongly influences osteoclastogenesis and the paracrine effect of the resultant osteoclast population on the osteogenic differentiation of hMSCs. In addition, only in indirect coculture, macrophages provide similar stimulatory effects as pre-differentiated osteoclasts on the osteogenic differentiation of MSCs and mineralization. ConclusionOur results demonstrate stimulatory effects of osteoclast conditioned medium on hMSC osteogenic differentiation, depending on initial hPBMC seeding density. In addition, we show that osteoclast and macrophage cultures contain pools of polarized macrophages, which may be involved in the osteogenic effects. Our data provide insight into bone tissue engineering approaches by using multicellular interactions related to bone remodeling and healing for the in vitro modulation of osteogenic differentiation.

A chondrogenesis induction system based on a functionalized hyaluronic acid hydrogel sequentially promoting hMSC proliferation, condensation, differentiation, and matrix deposition

Hydrogel scaffolds are widely used in cartilage tissue engineering as a natural stem cell niche. In particular, hydrogels based on multiple biological signals can guide behaviors of mesenchymal stem cells (MSCs) during neo-chondrogenesis. In the first phase of this study, we showed that functionalized hydrogels with grafted arginine-glycine-aspartate (RGD) peptides and lower degree of crosslinking can promote the proliferation of human mesenchymal stem cells (hMSCs) and upregulate the expression of cell receptor proteins. Moreover, grafted RGD and histidine-alanine-valine (HAV) peptides in hydrogel scaffolds can regulate the adhesion of the intercellular at an early stage. In the second phase, we confirmed that simultaneous use of HAV and RGD peptides led to greater chondrogenic differentiation compared to the blank control and single-peptide groups. Furthermore, the controlled release of kartogenin (KGN) can better facilitate cell chondrogenesis compared to other groups. Interestingly, with longer culture time, cell condensation was clearly observed in the groups with RGD and HAV peptide. In all groups with RGD peptide, significant matrix deposition was observed, accompanied by glycosaminoglycan (GAG) and collagen (Coll) production. Through in vitro and in vivo experiments, this study confirmed that our hydrogel system can sequentially promote the proliferation, adhesion, condensation, chondrogenic differentiation of hMSCs, by mimicking the cell microenvironment during neo-chondrogenesis.