Osteogenic Potential of Mesenchymal Stem Cells from Adipose Tissue,Bone Marrow and Hair Follicle Outer Root Sheath in a 3D Crosslinked Gelatin-Based Hydrogel

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.     

3D Printed SiOC(N) Ceramic Scaffolds for Bone Tissue Regeneration: Improved Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Bone tissue engineering has developed significantly in recent years as there has been increasing demand for bone substitutes due to trauma, cancer, arthritis, and infections. The scaffolds for bone regeneration need to be mechanically stable and have a 3D architecture with interconnected pores. With the advances in additive manufacturing technology, these requirements can be fulfilled by 3D printing scaffolds with controlled geometry and porosity using a low-cost multistep process. The scaffolds, however, must also be bioactive to promote the environment for the cells to regenerate into bone tissue. To determine if a low-cost 3D printing method for bespoke SiOC(N) porous structures can regenerate bone, these structures were tested for osteointegration potential by using human mesenchymal stem cells (hMSCs). This includes checking the general biocompatibilities under the osteogenic differentiation environment (cell proliferation and metabolism). Moreover, cell morphology was observed by confocal microscopy, and gene expressions on typical osteogenic markers at different stages for bone formation were determined by real-time PCR. The results of the study showed the pore size of the scaffolds had a significant impact on differentiation. A certain range of pore size could stimulate osteogenic differentiation, thus promoting bone regrowth and regeneration.     

Exercise as an Adjuvant to Cartilage Regeneration Therapy

This article provides a brief review of the pathophysiology of osteoarthritis and the ontogeny of chondrocytes and details how physical exercise improves the health of osteoarthritic joints and enhances the potential of autologous chondrocyte implants, matrix-induced autologous chondrocyte implants, and mesenchymal stem cell implants for the successful treatment of damaged articular cartilage and subchondral bone. In response to exercise, articular chondrocytes increase their production of glycosaminoglycans, bone morphogenic proteins, and anti-inflammatory cytokines and decrease their production of proinflammatory cytokines and matrix-degrading metalloproteinases. These changes are associated with improvements in cartilage organization and reductions in cartilage degeneration. Studies in humans indicate that exercise enhances joint recruitment of bone marrow-derived mesenchymal stem cells and upregulates their expression of osteogenic and chondrogenic genes, osteogenic microRNAs, and osteogenic growth factors. Rodent experiments demonstrate that exercise enhances the osteogenic potential of bone marrow-derived mesenchymal stem cells while diminishing their adipogenic potential, and that exercise done after stem cell implantation may benefit stem cell transplant viability. Physical exercise also exerts a beneficial effect on the skeletal system by decreasing immune cell production of osteoclastogenic cytokines interleukin-1β, tumor necrosis factor-α, and interferon-γ, while increasing their production of antiosteoclastogenic cytokines interleukin-10 and transforming growth factor-β. In conclusion, physical exercise done both by bone marrow-derived mesenchymal stem cell donors and recipients and by autologous chondrocyte donor recipients may improve the outcome of osteochondral regeneration therapy and improve skeletal health by downregulating osteoclastogenic cytokine production and upregulating antiosteoclastogenic cytokine production by circulating immune cells.     

The Marine Polysaccharide Ulvan Confers Potent Osteoinductive Capacity to PCL-Based Scaffolds for Bone Tissue Engineering Applications

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications.     

A Guide to Polysaccharide-Based Hydrogel Bioinks for 3D Bioprinting Applications

Three-dimensional (3D) bioprinting is an innovative technology in the biomedical field, allowing the fabrication of living constructs through an approach of layer-by-layer deposition of cell-laden inks, the so-called bioinks. An ideal bioink should possess proper mechanical, rheological, chemical, and biological characteristics to ensure high cell viability and the production of tissue constructs with dimensional stability and shape fidelity. Among the several types of bioinks, hydrogels are extremely appealing as they have many similarities with the extracellular matrix, providing a highly hydrated environment for cell proliferation and tunability in terms of mechanical and rheological properties. Hydrogels derived from natural polymers, and polysaccharides, in particular, are an excellent platform to mimic the extracellular matrix, given their low cytotoxicity, high hydrophilicity, and diversity of structures. In fact, polysaccharide-based hydrogels are trendy materials for 3D bioprinting since they are abundant and combine adequate physicochemical and biomimetic features for the development of novel bioinks. Thus, this review portrays the most relevant advances in polysaccharide-based hydrogel bioinks for 3D bioprinting, focusing on the last five years, with emphasis on their properties, advantages, and limitations, considering polysaccharide families classified according to their source, namely from seaweed, higher plants, microbial, and animal (particularly crustaceans) origin.     

Development of a Self‐Assembled Peptide/Methylcellulose‐Based Bioink for 3D Bioprinting

The introduction of 3D bioprinting to fabricate living constructs with tailored architecture has provided a new paradigm for biofabrication, with the potential to overcome several drawbacks of conventional scaffold‐based tissue regeneration strategies. Hydrogel‐based materials are suitable candidates regarding cell biocompatibility but often display poor mechanical properties. Self‐assembling peptides are a promising source of biomaterials to be used as 3D scaffolds based on their similarity to extracellular matrices (structurally and mechanically). In this study, an advanced bioink for biofabrication is presented based on the optimization of a RAD16‐I‐based biomaterial. The strategy followed to build 3D predefined structures by 3D printing is based on an enhancement of bioink viscosity by adding methylcellulose (MC) to a RAD16‐I solution. The resultant constructs display high shape fidelity and stability and embedded human mesenchymal stem cells present high viability after 7 days of culture. Moreover, cells are also able to differentiate to the adipogenic lineage, suggesting the suitability of this novel biomaterial for soft tissue engineering applications.     

Hyaluronic Acid and a Short Peptide Improve the Performance of a PCL Electrospun Fibrous Scaffold Designed for Bone Tissue Engineering Applications

Bone tissue engineering is a rapidly developing, minimally invasive technique for regenerating lost bone with the aid of biomaterial scaffolds that mimic the structure and function of the extracellular matrix (ECM). Recently, scaffolds made of electrospun fibers have aroused interest due to their similarity to the ECM, and high porosity. Hyaluronic acid (HA) is an abundant component of the ECM and an attractive material for use in regenerative medicine; however, its processability by electrospinning is poor, and it must be used in combination with another polymer. Here, we used electrospinning to fabricate a composite scaffold with a core/shell morphology composed of polycaprolactone (PCL) polymer and HA and incorporating a short self-assembling peptide. The peptide includes the arginine-glycine-aspartic acid (RGD) motif and supports cellular attachment based on molecular recognition. Electron microscopy imaging demonstrated that the fibrous network of the scaffold resembles the ECM structure. In vitro biocompatibility assays revealed that MC3T3-E1 preosteoblasts adhered well to the scaffold and proliferated, with significant osteogenic differentiation and calcium mineralization. Our work emphasizes the potential of this multi-component approach by which electrospinning, molecular self-assembly, and molecular recognition motifs are combined, to generate a leading candidate to serve as a scaffold for bone tissue engineering.     

In Situ Forming Hydrogel Based on Chondroitin Sulfate–Hydroxyapatite for Bone Tissue Engineering

In this research, tissue engineered hydrogels consist of chondroitin sulfate and hydroxyapatite capable of light curing and cell encapsulation were prepared. Chemical and physical characteristics were analyzed by FTIR spectroscopy, compressive strength and water uptake measurements. Observations with fluorescence microscopy as well as MTT viability assay revealed no toxicity for encapsulated cells. Moreover, alkaline phosphatase activity, calcium deposition, and gene expression analysis were determined to prove the osteogenic potential of the prepared hydrogel. In conclusion, this in situ forming hydrogel can be utilized as filler for bone deficiencies as well as carrier for cell encapsulation and osteogenic differentiation.     

Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.     

Bone Healing Materials in the Treatment of Recalcitrant Nonunions and Bone Defects

The usual treatment for bone defects and recalcitrant nonunions is an autogenous bone graft. However, due to the limitations in obtaining autogenous bone grafts and the morbidity associated with their procurement, various bone healing materials have been developed in recent years. The three main treatment strategies for bone defects and recalcitrant nonunions are synthetic bone graft substitutes (BGS), BGS combined with bioactive molecules, and BGS and stem cells (cell-based constructs). Regarding BGS, numerous biomaterials have been developed to prepare bone tissue engineering scaffolds, including biometals (titanium, iron, magnesium, zinc), bioceramics (hydroxyapatite (HA)), tricalcium phosphate (TCP), biopolymers (collagen, polylactic acid (PLA), polycaprolactone (PCL)), and biocomposites (HA/MONs@miR-34a composite coating, Bioglass (BG)-based ABVF-BG (antibiotic-releasing bone void filling) putty). Bone tissue engineering scaffolds are temporary implants that promote tissue ingrowth and new bone regeneration. They have been developed to improve bone healing through appropriate designs in terms of geometric, mechanical, and biological performance. Concerning BGS combined with bioactive molecules, one of the most potent osteoinductive growth factors is bone morphogenetic proteins (BMPs). In recent years, several natural (collagen, fibrin, chitosan, hyaluronic acid, gelatin, and alginate) and synthetic polymers (polylactic acid, polyglycolic acid, polylactic-coglycolide, poly(e-caprolactone) (PCL), poly-p-dioxanone, and copolymers consisting of glycolide/trimethylene carbonate) have been investigated as potential support materials for bone tissue engineering. Regarding BGS and stem cells (cell-based constructs), the main strategies are bone marrow stromal cells, adipose-derived mesenchymal cells, periosteum-derived stem cells, and 3D bioprinting of hydrogels and cells or bioactive molecules. Currently, significant research is being performed on the biological treatment of recalcitrant nonunions and bone defects, although its use is still far from being generalized. Further research is needed to investigate the efficacy of biological treatments to solve recalcitrant nonunions and bone defects.     

3D Printable Composite Biomaterials Based on GelMA and Hydroxyapatite Powders Doped with Cerium Ions for Bone Tissue Regeneration

The main objective was to produce 3D printable hydrogels based on GelMA and hydroxyapatite doped with cerium ions with potential application in bone regeneration. The first part of the study regards the substitution of Ca²⁺ ions from hydroxyapatite structure with cerium ions (Ca_10-xCe_x(PO₄)₆(OH)₂, xCe = 0.1, 0.3, 0.5). The second part followed the selection of the optimal concentration of HAp doped, which will ensure GelMA-based scaffolds with good biocompatibility, viability and cell proliferation. The third part aimed to select the optimal concentrations of GelMA for the 3D printing process (20%, 30% and 35%). In vitro biological assessment presented the highest level of cell viability and proliferation potency of GelMA-HC5 composites, along with a low cytotoxic potential, highlighting the beneficial effects of cerium on cell growth, also supported by Live/Dead results. According to the 3D printing experiments, the 30% GelMA enriched with HC5 was able to generate 3D scaffolds with high structural integrity and homogeneity, showing the highest suitability for the 3D printing process. The osteogenic differentiation experiments confirmed the ability of 30% GelMA-3% HC5 scaffold to support and efficiently maintain the osteogenesis process. Based on the results, 30% GelMA-3% HC5 3D printed scaffolds could be considered as biomaterials with suitable characteristics for application in bone tissue engineering.     

Antiosteoporotic Nanohydroxyapatite Zoledronate Scaffold Seeded with Bone Marrow Mesenchymal Stromal Cells for Bone Regeneration: A 3D In Vitro Model

Background: Bisphosphonates are widely employed drugs for the treatment of pathologies with high bone resorption, such as osteoporosis, and display a great affinity for calcium ions and apatitic substrates. Here, we aimed to investigate the potentiality of zoledronate functionalized hydroxyapatite nanocrystals (HAZOL) to promote bone regeneration by stimulating adhesion, viability, metabolic activity and osteogenic commitment of human bone marrow derived mesenchymal stromal cells (hMSCs). Methods: we adopted an advanced three-dimensional (3D) in vitro fracture healing model to study porous scaffolds: hMSCs were seeded onto the scaffolds that, after three days, were cut in halves and unseeded scaffolds were placed between the two halves. Scaffold characterization by X-ray diffraction, transmission and scanning electron microscopy analyses and cell morphology, viability, osteogenic differentiation and extracellular matrix deposition were evaluated after 3, 7 and 10 days of culture. Results: Electron microscopy showed a porous and interconnected structure and a uniform cell layer spread onto scaffolds. Scaffolds were able to support cell growth and cells progressively colonized the whole inserts in absence of cytotoxic effects. Osteogenic commitment and gene expression of hMSCs were enhanced with higher expressions of ALPL, COL1A1, BGLAP, RUNX2 and Osterix genes. Conclusion: Although some limitations affect the present study (e.g., the lack of longer experimental times, of mechanical stimulus or pathological microenvironment), the obtained results with the adopted experimental setup suggested that zoledronate functionalized scaffolds (GHAZOL) might sustain not only cell proliferation, but positively influence osteogenic differentiation and activity if employed in bone fracture healing.     

Isolation and Multiple Differentiation Potential Assessment of Human Gingival Mesenchymal Stem Cells

The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.     

3D Bioprinting of Human Adipose-Derived Stem Cells and Their Tenogenic Differentiation in Clinical-Grade Medium

Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 10⁶ ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-β3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE.     

Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion

Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated and then characterized by flow cytometry, morphological observation, and immunofluorescence staining. The LF cells were also cultivated in polyethylene (glycol) diacrylate (PEGDA) hydrogels that incorporated bone morphogenetic protein-2 (BMP-2) growth factor, to evaluate their proliferation and secretion of ECM and differentiation in vitro. The experimental results thus obtained that the proliferation, ECM secretion, and differentiation of the PEGDA-BMP-2 group exceeded those of the PEGDA group during the period of cultivation. The mineralization and histological staining results differed similarly. A nude mice model was utilized to prove that LF cells on hydrogels could undergo osteogenic differentiation in vivo. These experimental results also revealed that the PEGDA-BMP-2 group had better osteogenic effects than the PEGDA group following a 12 weeks after transplantation. According to all of these experimental results, LF cells are a source of cells for spinal fusion and PEGDA-BMP-2 hydrogel is a candidate biomaterial for spinal fusion by tissue engineering.     

Facile fabrication of near-infrared light-responsive shape memory nanocomposite scaffolds with hierarchical porous structures

In this work, the near-infrared (NIR) light-responsive shape memory scaffolds with hierarchical porous structures are designed and facilely formed by freeze drying of 3D printed viscous gel-like pickering emulsions, which are stabilized by hydrophobically modified graphene oxide (g-GO) and silica nanoparticles, and contain thermo-responsive poly(d , l -lactic acid-co-trimethylene carbonate) (PLMC) in the oil phase. The prepared scaffolds display an interconnected filament structure with hierarchical pores and high porosity. The incorporation of g-GO nanoparticles into PLMC matrix prompts that the scaffold shape memory can be triggered by NIR light with fast shape recovery. Moreover, the in vitro mineralization experiment shows that the scaffolds have biological activity, and the drug release study demonstrates that the scaffolds can be used as drug carriers with efficient drug release capacity. Furthermore, cell culture assays based on mouse bone mesenchymal stem cells exhibit that the scaffolds own good cytocompatibility. Therefore, the facile preparation and remote activation of the shape memory nanocomposite scaffolds with hierarchical porous structure and multifunctionality represents a highly attractive candidate as minimally invasive implantation scaffolds for bone tissue engineering applications.     

Evaluation of a Novel Thiol–Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells

Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications.     

Polymeric Microspheres/Cells/Extracellular Matrix Constructs Produced by Auto-Assembly for Bone Modular Tissue Engineering

Modular tissue engineering (MTE) is a novel “bottom-up” approach to create engineered biological tissues from microscale repeating units. Our aim was to obtain microtissue constructs, based on polymer microspheres (MSs) populated with cells, which can be further assembled into larger tissue blocks and used in bone MTE. Poly(L-lactide-co-glycolide) MS of 165 ± 47 µm in diameter were produced by oil-in-water emulsification and treated with 0.1 M NaOH. To improve cell adhesion, MSs were coated with poly-L-lysine (PLL) or human recombinant collagen type I (COL). The presence of oxygenated functionalities and PLL/COL coating on MS was confirmed by X-ray photoelectron spectroscopy (XPS). To assess the influence of medium composition on adhesion, proliferation, and osteogenic differentiation, preosteoblast MC3T3-E1 cells were cultured on MS in minimal essential medium (MEM) and osteogenic differentiation medium (OSG). Moreover, to assess the potential osteoblast–osteoclast cross-talk phenomenon and the influence of signaling molecules released by osteoclasts on osteoblast cell culture, a medium obtained from osteoclast culture (OSC) was also used. To impel the cells to adhere and grow on the MS, anti-adhesive cell culture plates were utilized. The results show that MS coated with PLL and COL significantly favor the adhesion and growth of MC3T3-E1 cells on days 1 and 7, respectively, in all experimental conditions tested. On day 7, three-dimensional MS/cell/extracellular matrix constructs were created owing to auto-assembly. The cells grown in such constructs exhibited high activity of early osteogenic differentiation marker, namely, alkaline phosphatase. Superior cell growth on PLL- and COL-coated MS on day 14 was observed in the OSG medium. Interestingly, deposition of extracellular matrix and its mineralization was particularly enhanced on COL-coated MS in OSG medium on day 14. In our study, we developed a method of spontaneous formation of organoid-like MS-based cell/ECM constructs with a few millimeters in size. Such constructs may be regarded as building blocks in bone MTE.     

The Selective Histone Deacetylase Inhibitor MI192 Enhances the Osteogenic Differentiation Efficacy of Human Dental Pulp Stromal Cells

The use of human dental pulp stromal cells (hDPSCs) has gained increasing attention as an alternative stem cell source for bone tissue engineering. The modification of the cells’ epigenetics has been found to play an important role in regulating differentiation, with the inhibition of histone deacetylases 3 (HDAC3) being linked to increased osteogenic differentiation. This study aimed to induce epigenetic reprogramming using the HDAC2 and 3 selective inhibitor, MI192 to promote hDPSCs osteogenic capacity for bone regeneration. MI192 treatment caused a time–dose-dependent change in hDPSC morphology and reduction in viability. Additionally, MI192 successfully augmented hDPSC epigenetic functionality, which resulted in increased histone acetylation and cell cycle arrest at the G₂/M phase. MI192 pre-treatment exhibited a dose-dependent effect on hDPSCs alkaline phosphatase activity. Quantitative PCR and In-Cell Western further demonstrated that MI192 pre-treatment significantly upregulated hDPSCs osteoblast-related gene and protein expression (alkaline phosphatase, bone morphogenic protein 2, type I collagen and osteocalcin) during osteogenic differentiation. Importantly, MI192 pre-treatment significantly increased hDPSCs extracellular matrix collagen production and mineralisation. As such, for the first time, our findings show that epigenetic reprogramming with the HDAC2 and 3 selective inhibitor MI192 accelerates the osteogenic differentiation of hDPSCs, demonstrating the considerable utility of this MSCs engineering approach for bone augmentation strategies.     

Patients’ Stem Cells Differentiation in a 3D Environment as a Promising Experimental Tool for the Study of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease (NDD) that affects motor neurons, causing weakness, muscle atrophy and spasticity. Unfortunately, there are only symptomatic treatments available. Two important innovations in recent years are three-dimensional (3D) bioprinting and induced pluripotent stem cells (iPSCs). The aim of this work was to demonstrate the robustness of 3D cultures for the differentiation of stem cells for the study of ALS. We reprogrammed healthy and sALS peripheral blood mononuclear cells (PBMCs) in iPSCs and differentiated them in neural stem cells (NSCs) in 2D. NSCs were printed in 3D hydrogel-based constructs and subsequently differentiated first in motor neuron progenitors and finally in motor neurons. Every step of differentiation was tested for cell viability and characterized by confocal microscopy and RT-qPCR. Finally, we tested the electrophysiological characteristics of included NSC34. We found that NSCs maintained good viability during the 3D differentiation. Our results suggest that the hydrogel does not interfere with the correct differentiation process or with the electrophysiological features of the included cells. Such evidence confirmed that 3D bioprinting can be considered a good model for the study of ALS pathogenesis.