Mineralized Matrix Deposition Research Articles

Functionalization of PCL-Based Fiber Scaffolds with Different Sources of Calcium and Phosphate and Odontogenic Potential on Human Dental Pulp Cells.

This study investigated the incorporation of sources of calcium, phosphate, or both into electrospun scaffolds and evaluated their bioactivity on human dental pulp cells (HDPCs). Additionally, scaffolds incorporated with calcium hydroxide (CH) were characterized for degradation, calcium release, and odontogenic differentiation by HDPCs. Polycaprolactone (PCL) was electrospun with or without 0.5% w/v of calcium hydroxide (PCL + CH), nano-hydroxyapatite (PCL + nHA), or β-glycerophosphate (PCL + βGP). SEM/EDS analysis confirmed fibrillar morphology and particle incorporation. HDPCs were cultured on the scaffolds to assess cell viability, adhesion, spreading, and mineralized matrix formation. PCL + CH was also evaluated for gene expression of odontogenic markers (RT-qPCR). Data were submitted to ANOVA and Student's t-test (α = 5%). Added CH increased fiber diameter and interfibrillar spacing, whereas βGP decreased both. PCL + CH and PCL + nHA improved HDPC viability, adhesion, and proliferation. Mineralization was increased eightfold with PCL + CH. Scaffolds containing CH gradually degraded over six months, with calcium release within the first 140 days. CH incorporation upregulated DSPP and DMP1 expression after 7 and 14 days. In conclusion, CH- and nHA-laden PCL fiber scaffolds were cytocompatible and promoted HDPC adhesion, proliferation, and mineralized matrix deposition. PCL + CH scaffolds exhibit a slow degradation profile, providing sustained calcium release and stimulating HDPCs to upregulate odontogenesis marker genes.

Osteosarcoma cell death induced by innovative scaffolds doped with chemotherapeutics.

Osteosarcoma (OS) cancer treatments include systemic chemotherapy and surgical resection. In the last years, novel treatment approaches have been proposed, which employ a drug-delivery system to prevent offside effects and improves treatment efficacy. Locally delivering anticancer compounds improves on high local concentrations with more efficient tumour-killing effect, reduced drugs resistance and confined systemic effects. Here, the synthesis of injectable strontium-doped calcium phosphate (SrCPC) scaffold was proposed as drug delivery system to combine bone tissue regeneration and anticancer treatment by controlled release of methotrexate (MTX) and doxorubicin (DOX), coded as SrCPC-MTX and SrCPC-DOX, respectively. The drug-loaded cements were tested in an in vitro model of human OS cell line SAOS-2, engineered OS cell line (SAOS-2-eGFP) and U2-OS. The ability of doped scaffolds to induce OS cell death and apoptosis was assessed analysing cell proliferation and Caspase-3/7 activities, respectively. To determine if OS cells grown on doped-scaffolds change their migratory ability and invasiveness, a wound-healing assay was performed. In addition, the osteogenic potential of SrCPC material was evaluated using human adipose derived-mesenchymal stem cells. Osteogenic markers such as (i) the mineral matrix deposition was analysed by alizarin red staining; (ii) the osteocalcin (OCN) protein expression was investigated by enzyme-linked immunosorbent assay test, and (iii) the osteogenic process was studied by real-time polymerase chain reaction array. The delivery system induced cell-killing cytotoxic effects and apoptosis in OS cell lines up to Day 7. SrCPC demonstrates a good cytocompatibility and it induced upregulation of osteogenic genes involved in the skeletal development pathway, together with OCN protein expression and mineral matrix deposition. The proposed approach, based on the local, sustained release of anticancer drugs from nanostructured biomimetic drug-loaded cements is promising for future therapies aiming to combine bone regeneration and anticancer local therapy.

Cyclin A1 (CCNA1) inhibits osteoporosis by suppressing transforming growth factor-beta (TGF-beta) pathway in osteoblasts

BackgroundOsteoporosis is a genetic disease caused by the imbalance between osteoblast-led bone formation and osteoclast-induced bone resorption. However, further gene-related pathogenesis remains to be elucidated.MethodsThe aberrant expressed genes in osteoporosis was identified by analyzing the microarray profile GSE100609. Serum samples of patients with osteoporosis and normal group were collected, and the mRNA expression of candidate genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mouse cranial osteoblast MC3T3-E1 cells were treated with dexamethasone (DEX) to mimic osteoporosis in vitro. Alizarin Red staining and alkaline phosphatase (ALP) staining methods were combined to measure matrix mineralization deposition of MC3T3-E1 cells. Meanwhile, the expression of osteogenesis related genes including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Osterix, and bone morphogenetic protein 2 (BMP2) were evaluated by qRT-PCR and western blotting methods. Then the effects of candidate genes on regulating impede bone loss caused by ovariectomy (OVX) in mice were studied.ResultsCyclin A1 (CCNA1) was found to be significantly upregulated in serum of osteoporosis patients and the osteoporosis model cells, which was in line with the bioinformatic analysis. The osteogenic differentiation ability of MC3T3-E1 cells was inhibited by DEX treatment, which was manifested by decreased Alizarin Red staining intensity, ALP staining intensity, and expression levels of ALP, OCN, OPN, Osterix, and BMP2. The effects of CCNA1 inhibition on regulating osteogenesis were opposite to that of DEX. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that genes negatively associated with CCNA1 were enriched in the TGF-beta signaling pathway. Inhibitor of TGF-beta signaling pathway partly reversed osteogenesis induced by suppressed CCNA1. Furthermore, suppressed CCNA1 relieved bone mass of OVX mice in vivo.ConclusionDownregulation of CCNA1 could activate TGF-beta signaling pathway and promote bone formation, thus playing a role in treatment of osteoporosis.

3D Graphene/silk fibroin scaffolds enhance dental pulp stem cell osteo/odontogenic differentiation

ObjectivesThe current in vitro study aims to evaluate silk fibroin with and without the addition of graphene as a potential scaffold material for regenerative endodontics. Material and MethodsSilk fibroin (SF), Silk fibroin/graphene oxide (SF/GO) and silk fibroin coated with reduced graphene oxide (SF/rGO) scaffolds were prepared (n = 30). The microarchitectures and mechanical properties of scaffolds were evaluated using field emission scanning electron microscopy (FESEM), pore size and water uptake, attenuated total reflectance fourier transformed infrared spectroscopy (ATR-FTIR), Raman spectroscopy and mechanical compression tests. Next, the study analyzed the influence of these scaffolds on human dental pulp stem cell (hDPSC) viability, apoptosis or necrosis, cell adhesion, odontogenic differentiation marker expression and mineralized matrix deposition. The data were analyzed with ANOVA complemented with the Tukey post-hoc test (p < 0.005). ResultsSEM analysis revealed abundant pores with a size greater than 50 nm on the surface of tested scaffolds, primarily between 50 nm and 600 µm. The average value of water uptake obtained in pure fibroin scaffolds was statistically higher than that of those containing GO or rGO (p < 0.05). ATR-FTIR evidenced that the secondary structures did not present differences between pure fibroin and fibroin coated with graphene oxide, with a similar infrared spectrum in all tested scaffolds. Raman spectroscopy showed a greater number of defects in the links in SF/rGO scaffolds due to the reduction of graphene. In addition, adequate mechanical properties were exhibited by the tested scaffolds. Regarding biological properties, hDPSCs attached to scaffolds were capable of proliferating at a rate similar to the control, without affecting their viability over time. A significant upregulation of ALP, ON and DSPP markers was observed with SF/rGO and SF/GO groups. Finally, SF/GO and SF/rGO promoted a significantly higher mineralization than the control at 21 days. SignificanceData obtained suggested that SF/GO and SF/rGO scaffolds promote hDPSC differentiation at a genetic level, increasing the expression of key osteo/odontogenic markers, and supports the mineralization of the extracellular matrix. However, results from this study are to be interpreted with caution, requiring further in vivo studies to confirm the potential of these scaffolds.

Short-term exposure to TNF-α promotes odontogenesis of stem cells of apical papilla on nanofibrous poly(l-lactic acid) scaffolds

The aim of this study was to investigate the effect of short-term (3 days) preconditioning with pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) on proliferation and odontogenic differentiation of stem cells of apical papilla (SCAP) seeded on a 3D scaffold. Our results showed that while TNF-α preconditioning at various concentrations did not affect SCAP proliferation, it enhanced alkaline phosphatase (ALP) activity and mineralized matrix deposition in culture. Preconditioning SCAP using TNF-α at a concentration of about 1 ng/ml optimally enhanced odontogenic gene expression of SCAP in culture. Highly porous nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffolds, with nanofiber features mimicking the environment of the extracellular matrix, were fabricated using a thermally induced phase separation method. TNF-α preconditioning of SCAP at 1 ng/ml promoted ALP activity, calcium deposition and mineralized tissue formation of SCAP cultured on 3D NF-PLLA scaffolds in vitro. Moreover, TNF-α preconditioning at a concentration of 1 ng/ml promoted the formation of more mineralized tissue as well as stronger DSPP expression of SCAP seeded on NF-PLLA scaffolds in vivo. Collectively, this study suggests that short term TNF-α preconditioning, mimicking the transiently low concentration TNF-α present during inflammation, may serve as a feasible approach to enhance the odontogenic differentiation of stem/progenitor cells on a biomimetic scaffold for 3D dentin regeneration. These results present a first step in the development of a dental tissue engineering strategy where odontogenesis is enhanced by modulating inflammation.

B. abortus Infection Promotes an Imbalance in the Adipocyte-Osteoblast Crosstalk Favoring Bone Resorption.

Osteoarticular injury is the most common presentation of active brucellosis in humans. Osteoblasts and adipocytes originate from mesenchymal stem cells (MSC). Since those osteoblasts are bone-forming cells, the predilection of MSC to differentiate into adipocytes or osteoblasts is a potential factor involved in bone loss. In addition, osteoblasts and adipocytes can be converted into each other according to the surrounding microenvironment. Here, we study the incumbency of B. abortus infection in the crosstalk between adipocytes and osteoblasts during differentiation from its precursors. Our results indicate that soluble mediators present in culture supernatants from B. abotus-infected adipocytes inhibit osteoblast mineral matrix deposition in a mechanism dependent on the presence of IL-6 with the concomitant reduction of Runt-related transcription factor 2 (RUNX-2) transcription, but without altering organic matrix deposition and inducing nuclear receptor activator ligand kβ (RANKL) expression. Secondly, B. abortus-infected osteoblasts stimulate adipocyte differentiation with the induction of peroxisome proliferator-activated receptor γ (PPAR-γ) and CCAAT enhancer binding protein β (C/EBP-β). We conclude that adipocyte-osteoblast crosstalk during B. abortus infection could modulate mutual differentiation from its precursor cells, contributing to bone resorption.

Calcium silicate-coated porous chitosan scaffold as a cell-free tissue engineering system for direct pulp capping.

This study aimed to develop and characterize different formulations of porous chitosan scaffolds (SCH) associated with calcium silicate (CaSi) and evaluate their chemotactic and bioactive potential on human dental pulp cells (hDPCs). Different concentrations of CaSi suspensions (0.5%, 1.0%, and 2.0%, w/v) were incorporated (1:5; v/v) /or not, into 2% chitosan solution, giving rise to the following groups: SCH (control); SCH+0.5CaSi; SCH+1.0CaSi; SCH+2.0 CaSi. The resulting solutions were submitted to thermally induced phase separation followed by freeze-drying procedures to obtain porous scaffolds. The topography, pH, and calcium release kinetics of the scaffolds were assessed. Next, the study evaluated the influence of these scaffolds on cell migration (MG), viability (VB), proliferation (PL), adhesion and spreading (A&S), and on total protein synthesis (TP), alkaline phosphatase (ALP) activity, mineralized matrix deposition (MMD), and gene expression (GE) of odontogenic differentiation markers (ALP, DSPP, and DMP-1). The data were analyzed with ANOVA complemented with the Tukey post-hoc test (α=5%). Incorporation of the CaSi suspension into the chitosan scaffold formulation increased pore diameter when compared with control. Increased amounts of CaSi in the CH scaffold resulted in higher pH values and Ca release. In Groups SCH+1.0CaSi and SCH+2.0CaSi, increased VB, PF, A&S, GE of DSPP/DMP-1 and MMD values were shown. However, Group SCH+2.0CaSi was the only formulation capable of enhancing MG and showed the highest increase in TP, MMD, and GE of DMP-1 and DSPP values. SCH+2.0CaSi formulation had the highest chemotactic and bioactive potential on hDPCs and may be considered a potential biomaterial for pulp-dentin complex regeneration.

TiO2 nanoparticles added to dense bovine hydroxyapatite bioceramics increase human osteoblast mineralization activity

ObjectivesThis study evaluated the effect of TiO2 nanoparticles + dense hydroxyapatite (HA) on human osteoblast cells (SAOS-2). MethodsParticulate bovine HA powder with or without the addition of either 5 or 8 % TiO2 (HA, HA/TiO2Np5 % or HA/TiO2Np8 %) were pressed into disks (Ø = 12.5 mm; thickness = 1.3 mm) uniaxially (100 MPa) and isostatically (200 MPa/1 min) and sintered at 1300 °C. Y-TZP disks were used as control. The following tests were performed: Scanning Electron Microscopy and Dispersive Energy Spectroscopy (SEM/EDS), Atomic Force Microscopy (AFM), cell viability assay (Alamar Blue-AB) and mineralized matrix deposition (Alizarin Red-AR). AB and AR data were submitted to 2-way ANOVA/Tukey tests and ANOVA/Tukey tests, respectively. ResultsSEM revealed that the surface of HA/TiO2Np5% resembles DPBHA surface, but also contains smaller granules. HA/TiO2Np8% characteristics resembles HA/TiO2Np5% surface, but with irregular topography. Y-TZP showed a typical oxide ceramic surface pattern. EDS revealed Ca, O, and P in all samples. C, O, and Zr appeared in Y-TZP samples. AFM data corroborates SEM analysis. AB test revealed excellent cellular viability for HA/TiO2Np5% group. AR test showed that all groups containing TiO2np had more mineralized matrix deposition than all other groups, with statistically differences between HA/TiO2Np8% and HA cultivated in non-osteogenic medium. Culture in osteogenic medium exhibited much more mineralized matrix deposition by TiO2np groups. SignificanceIn conclusion, the addition of TiO2np showed chemical, superficial, and biological changes in the reinforced materials. HA/TiO2Np5% showed the best results for cell viability and HA/TiO2Np8% for mineralized matrix deposition.

The Impact of Graphene Oxide on Polycaprolactone PCL Surfaces: Antimicrobial Activity and Osteogenic Differentiation of Mesenchymal Stem Cell

In dentistry, bone regeneration requires osteoinductive biomaterial with antibacterial properties. Polycaprolactone (PCL) may be combined with different nanofillers including reduced graphene oxide (rGO). Here, the amount of rGO filler was defined to obtain a biocompatible and antibacterial PCL-based surface supporting the adhesion and differentiation of human mesenchymal stem cells (MSCs). Compounds carrying three different percentages of rGO were tested. Among all, the 5% rGO-PCL compound is the most bacteriostatic against Gram-positive bacteria. All scaffolds are biocompatible. MSCs adhere and proliferate on all scaffolds; however, 5% rGO-PCL surface supports the growth of cells and implements the expression of extracellular matrix components necessary to anchor the cells to the surface itself. Moreover, the 5% rGO-PCL surface has superior osteoinductive properties confirmed by the improved alkaline phosphatase activity, mineral matrix deposition, and osteogenic markers expression. These results suggest that 5% rGO-PCL has useful properties for bone tissue engineering purposes.

3D bioprinted gelatin/gellan gum-based scaffold with double-crosslinking network for vascularized bone regeneration

Three-dimensional (3D) bioprinting holds promise for precise repair of bone defects, but rapid formation of effective vascularized tissue by 3D-printed construct is still a challenge. In this study, deferoxamine (DFO)-loaded ethosomes (Eth) were combined with gelatin methacrylate (GelMA)/gellan gum methacrylate (GGMA) hybrid bioink to fabricate 3D-printed scaffold by photo- and ion-crosslinking. The GelMA/GGMA bioinks showed excellent printability and improved mechanical property through the double-crosslinking method. In vitro experiments showed that Eth-DFO@GelMA/GGMA scaffold had good cytocompatibility while achieved sustained release of DFO, which significantly promoted endothelial cells migration and tube formation, mineralized matrix deposition and alkaline phosphatase expression of osteoblast. In vivo experiments of rat cranial defect model demonstrated that composite scaffold could promote angiogenesis and bone regeneration by activating the hypoxia-inducible factor 1-α (HIF1-α) signaling pathway. In conclusion, this 3D bioprinted Eth-DFO@GelMA/GGMA scaffold can couple angiogenesis and osteogenesis, and will be a promising candidate for the bone defects treatment.

An amelogenin-based peptide hydrogel promoted the odontogenic differentiation of human dental pulp cells

Amelogenin can induce odontogenic differentiation of human dental pulp cells (HDPCs), which has great potential and advantages in dentine-pulp complex regeneration. However, the unstability of amelogenin limits its further application. This study constructed amelogenin self-assembling peptide hydrogels (L-gel or D-gel) by heating-cooling technique, investigated the effects of these hydrogels on the odontogenic differentiation of HDPCs and explored the underneath mechanism. The critical aggregation concentration, conformation, morphology, mechanical property and biological stability of the hydrogels were characterized, respectively. The effects of the hydrogels on the odontogenic differentiation of HDPCs were evaluated via alkaline phosphatase activity measurement, quantitative reverse transcription polymerase chain reaction, western blot, Alizarin red staining and scanning electron microscope. The mechanism was explored via signaling pathway experiments. Results showed that both the L-gel and D-gel stimulated the odontogenic differentiation of HDPCs on both Day 7 and Day 14, while the D-gel showed the highest enhancement effects. Meanwhile, the D-gel promoted calcium accumulation and mineralized matrix deposition on Day 21. The D-gel activated MAPK-ERK1/2 pathways in HDPCs and induced the odontogenic differentiation via ERK1/2 and transforming growth factor/smad pathways. Overall, our study demonstrated that the amelogenin peptide hydrogel stimulated the odontogenic differentiation and enhanced mineralization, which held big potential in the dentine-pulp complex regeneration.

Biological properties of human periodontal ligament cell spheroids cultivated on chitosan and polyvinyl alcohol membranes

Multicellular spheroid cultures have attracted increasing attention in the field of periodontal regeneration. However, very few studies have reported the periodontal ligament (PDL) cell spheroid formation via biomaterials-induced processes. This study investigated the biological characteristics of human PDL cell spheroids formed on two hydrophilic polymer-based biomaterials, namely chitosan and polyvinyl alcohol. The expressions of periostin, paxillin, hypoxia-inducible factor 1-α (HIF-1α), and vascular endothelial growth factor (VEGF) were analyzed. Cell migration ability was assessed using a scratch assay. Furthermore, PDL cell spheroids were cultured in 3D-printed polylactic acid scaffolds to evaluate mineralizing capability. Western blot analysis revealed increased expressions of periostin, HIF-1α, and VEGF in the 3D spheroids. After the spheroids were reseeded, the cells gradually migrated outward from the spheroids and time-dependent distribution of paxillin was observed. The cells migrating outward from the 3D spheroids demonstrated greater migration ability than that of 2D monolayer cells. Compared to the dissociated cells from a monolayer culture, the cell spheroids formed on the chitosan membrane exhibited elevated alkaline phosphatase activity and an increase in mineralized matrix deposition. The biomaterial-induced formation of PDL cell spheroids suggests a novel strategy for cell delivery in research and clinical applications of periodontal regeneration.

Chitosan in association with osteogenic factors as a cell-homing platform for dentin regeneration: Analysis in a pulp-in-a-chip model

ObjectiveIn this paper we propose the association of β-glycerophosphate (βGP) and calcium-hydroxide with chitosan (CH) to formulate a porous bioactive scaffold suitable as a cell-homing platform for dentin regeneration. MethodsCalcium hydroxide and βGP solutions were incorporated into chitosan to modulate scaffold architecture and composition by a phase separation technique. Architecture, chemical composition, and degradability were evaluated, and biological characterizations were performed by the seeding of dental pulp cells (DPCs) onto scaffolds, or by cultivating them in contact with leachable components (extracts), to determine cytocompatibility and odontoblastic differentiation. Cell-free scaffolds were then positioned in intimate contact with a 3D culture of DPCs in a pulp-in-a-chip platform under simulated pulp pressure. Cell mobilization and odontoblastic marker expression were evaluated. Deposition of mineralized matrix was assessed in direct contact with dentin, in the absence of osteogenic factors. ResultsIncorporation of calcium hydroxide and βGP generated a stable porous chitosan scaffold containing Ca-P nanoglobule topography (CH-Ca-βGP), which favored cell viability, alkaline phosphatase activity, and mineralized matrix deposition by cells seeded onto the scaffold structure and at a distance. The pulp-in-a-chip assay denoted its chemotactic and bioactive potential, since dentin sialoprotein-positive DPCs from 3D culture adhered to CH-Ca-βGP more than to plain chitosan. The higher deposition of mineralized matrix onto the scaffold and surrounding dentin was also observed. SignificanceA CH-Ca-βGP scaffold creates a microenvironment capable of mobilizing DPC migration toward its structure, harnessing the odontogenic potential and culminating in the expression of a highly mineralizing phenotype, key factors for a cell-homing strategy.

Mineral-induced bubbling effect and biomineralization as strategies to create highly porous and bioactive scaffolds for dentin tissue engineering.

The objective of the study was to assess the biological and mechanical characteristics of chitosan-based scaffolds enriched by mineral phases and biomineralized in simulated body fluid (SBF) as a possible biomaterial for dentin regeneration. Thus, porous chitosan scaffolds were prepared by the mineral-induced bubbling-effect technique and subjected to biomineralization to create biomimetic scaffolds for dentin tissue engineering. Suspensions containing calcium hydroxide, nanohydroxyapatite, or β-tricalcium phosphate were added to the chitosan (CH) solution and subjected to gradual freezing and freeze-drying to obtain CHCa, CHnHA, and CHβTCP porous scaffolds, respectively, by the bubbling effect. Then, scaffolds were incubated in SBF for 5 days at 37°C, under constant stirring, to promote calcium-phosphate (CaP) biomineralization. Scanning electron microscopy revealed increased pore size and porosity degree on mineral-containing scaffolds, with CHCa and CHnHA presenting as round, well-distributed, and with an interconnected pore network. Nevertheless, incubation in SBF disrupted the porous architecture, except for CHCaSBF , leading to the deposition of CaP coverage, confirmed by Fourier Transform Infrared Spectroscopy analyses. All mineral-containing and SBF-treated formulations presented controlled degradation profiles and released calcium throughout 28 days. When human dental pulp cells (HDPCs) were seeded onto scaffold structures, the porous and interconnected architecture of CHCa, CHnHA, and CHCaSBF allowed cells to infiltrate and spread throughout the scaffold structure, whereas in other formulations cells were dispersed or agglomerated. It was possible to determine a positive effect on cell proliferation and odontogenic differentiation for mineral-containing formulations, intensely improved by biomineralization. A significant increase in mineralized matrix deposition (by 8.4 to 18.9 times) was observed for CHCaSBF , CHnHASBF , and CHβTCPSBF in comparison with plain CH. The bioactive effect on odontoblastic marker expression (ALP activity and mineralized matrix) was also observed for HDPCs continuously cultivated with conditioned medium obtained from scaffolds. Therefore, biomineralization of chitosan scaffolds containing different mineral phases was responsible for increasing the capacity for mineralized matrix deposition by pulpal cells, with potential for use in dentin tissue engineering.

P1 - Calcium Silicate Loaded-Porous Chitosan Scaffold for Pulp-Dentin Complex Regeneration

Innovative pulp therapy strategies based on the use of biomaterials capable of stimulating dental pulp cells to participate effectively of the complete pulp-dentin complex regeneration is a challenge in Dentistry. Therefore, the aim of this study was to develop and characterize several formulations of calcium silicate (SiCa)-loaded porous chitosan (CH) scaffolds, and evaluate their bioactive and chemotactic potential on human dental pulp cells (hDPCs). Different concentrations of SiCa suspensions (0.5, 1.0 and 2.0%, w/v) were either incorporated (1:5; v/v) or not to 2% CH solution, establishing the following groups: CH (control); CH+0.5SiCa; CH+1.0SiCa; CH+2.0SiCa. The resulting solutions were submitted to thermally induced phase separation followed by freeze drying to obtain porous scaffolds. The topography and chemical characterization (SEM/EDS) of the scaffolds, as well as their pH and calcium release kinetics were assessed. Next, hDPCs were seeded onto the scaffolds to assess their bioactivity potential according with the parameters of cell viability (Live/Dead assay; 1, 7 and 14-days), proliferation (AlamarBlue assay; 1, 7 and 14-days), adhesion and spreading (F-actin assay; 1, 7 and 14-days). The pulp cells were also evaluated for total protein synthesis (Lowry Protein assay; 14-days), alkaline phosphatase activity (ALP Assay Kit; 14-days), mineralized matrix deposition (Alizarin Red assay; 21-days), and gene expression of odontogenic differentiation markers (ALP, DSPP, and DMP-1; RT-qPCR assay, 21-days). The chemotactic stimulus of the scaffolds on pulp cells (Transwell assay; 24 h) was also assessed. Data were analyzed by ANOVA and Tukey post-hoc (?=5%). The incorporation of SiCa in the chitosan scaffold formulations increased the pore diameter in comparison to control (p<0.05). High amounts of SiCa changed the surface nano-topography of the chitosan scaffolds (Image 1) and increased the pH and Ca release (p<0.05). Greater cell viability, proliferation, adhesion and spreading, as well as expression of odontogenic markers and mineralized matrix deposition was observed in the CH+1.0SiCa and CH+2.0SiCa groups (p<0.05). However, increased synthesis of total protein and mineralized matrix, as well as expression of DMP-1 and DSPP occurred only in group CH+2.0SiCa (p<0.05). Based on the fact that porous chitosan scaffolds enriched with 2% of calcium silicate showed improved bioactive and chemotactic potential on hDPCs, it should be considered as a potential biomaterial for cell-free tissue engineering strategy to drive pulp-dentin complex regeneration.

Electrospun Fiber Alignment Guides Osteogenesis and Matrix Organization Differentially in Two Different Osteogenic Cell Types.

Biomimetic replication of the structural anisotropy of musculoskeletal tissues is important to restore proper tissue mechanics and function. Physical cues from the local micro-environment, such as matrix fiber orientation, may influence the differentiation and extracellular matrix (ECM) organization of osteogenic progenitor cells. This study investigates how scaffold fiber orientation affects the behavior of mature and progenitor osteogenic cells, the influence on secreted mineralized-collagenous matrix organization, and the resulting construct mechanical properties. Gelatin-coated electrospun poly(caprolactone) fibrous scaffolds were fabricated with either a low or a high degree of anisotropy and cultured with mature osteoblasts (MLO-A5s) or osteogenic mesenchymal progenitor cells (hES-MPs). For MLO-A5 cells, alkaline phosphatase (ALP) activity was highest, and more calcium-containing matrix was deposited onto aligned scaffolds. In contrast, hES-MPs, osteogenic mesenchymal progenitor cells, exhibited higher ALP activity, collagen, and calcium deposition on randomly orientated fibers compared with aligned counterparts. Deposited matrix was isotropic on random fibrous scaffolds, whereas a greater degree of anisotropy was observed in aligned fibrous constructs, as confirmed by second harmonic generation (SHG) and scanning electron microscope (SEM) imaging. This resulted in anisotropic mechanical properties on aligned constructs. This study indicates that mineralized-matrix deposition by osteoblasts can be controlled by scaffold alignment but that the early stages of osteogenesis may not benefit from culture on orientated scaffolds.

Chitosan-Calcium-Simvastatin Scaffold as an Inductive Cell-Free Platform

The development of biomaterials based on the combination of biopolymers with bioactive compounds to develop delivery systems capable of modulating dentin regeneration mediated by resident cells is the goal of current biology-based strategies for regenerative dentistry. In this article, the bioactive potential of a simvastatin (SV)–releasing chitosan-calcium-hydroxide (CH-Ca) scaffold was assessed. After the incorporation of SV into CH-Ca, characterization of the scaffold was performed. Dental pulp cells (DPCs) were seeded onto scaffolds for the assessment of cytocompatibility, and odontoblastic differentiation was evaluated in a microenvironment surrounded by dentin. Thereafter, the cell-free scaffold was adapted to dentin discs positioned in artificial pulp chambers in direct contact with a 3-dimensional (3D) culture of DPCs, and the system was sealed to simulate internal pressure at 20 cm/H2O. In vivo experiments with cell-free scaffolds were performed in rats’ calvaria defects. Fourier-transform infrared spectroscopy spectra proved incorporation of Ca and SV into the scaffold structure. Ca and SV were released upon immersion in a neutral environment. Viable DPCs were able to spread and proliferate on the scaffold over 14 d. Odontoblastic differentiation occurred in the DPC/scaffold constructs in contact with dentin, in which SV supplementation promoted odontoblastic marker overexpression and enhanced mineralized matrix deposition. The chemoattractant potential of the CH-Ca scaffold was improved by SV, with numerous viable and dentin sialoprotein–positive cells from the 3D culture being observed on its surface. Cells at 3D culture featured increased gene expression of odontoblastic markers in contact with the SV-enriched CH-Ca scaffold. CH-Ca-SV led to intense mineralization in vivo, presenting mineralization foci inside its structure. In conclusion, the CH-Ca-SV scaffold induces differentiation of DPCs into a highly mineralizing phenotype in the presence of dentin, creating a microenvironment capable of attracting pulp cells to its surface and inducing the overexpression of odontoblastic markers in a cell-homing strategy.

A Composite Lactide-Mineral 3D-Printed Scaffold for Bone Repair and Regeneration

Orthopedic tumor resection, trauma, or degenerative disease surgeries can result in large bone defects and often require bone grafting. However, standard autologous bone grafting has been associated with donor site morbidity and/or limited quantity. As an alternate, allografts with or without metallic or polyether-etherketone have been used as grafting substitutes. However, these may have drawbacks as well, including stress shielding, pseudarthrosis, disease-transmission, and infection. There is therefore a need for alternative bone substitutes, such as the use of mechanically compliant three-dimensional (3D)-printed scaffolds. Several off-the-shelf materials are available for low-cost fused deposition 3D printing such as polylactic acid (PLA) and polycaprolactone (PCL). We have previously described the feasibility of 3D-printed PLA scaffolds to support cell activity and extracellular matrix deposition. In this study, we investigate two medical-grade filaments consistent with specifications found in American Society for Testing and Materials (ASTM) standard for semi-crystalline polylactide polymers for surgical implants, a pure polymer (100M) and a copolymeric material (7415) for their cytocompatibility and suitability in bone tissue engineering. Moreover, we assessed the impact on osteo-inductive properties with the addition of beta-tricalcium phosphate (β-TCP) minerals and assessed their mechanical properties. 100M and 7415 scaffolds with the additive β-TCP demonstrated superior mesenchymal stem cells (MSCs) differentiation detected via increased alkaline phosphatase activity (6-fold and 1.5-fold, respectively) and mineralized matrix deposition (14-fold and 5-fold, respectively) in vitro. Furthermore, we evaluated in vivo compatibility, biosafety and bone repair potential in a rat femur window defect model. 100M+β-TCP implants displayed a positive biosafety profile and showed significantly enhanced new bone formation compared to 100M implants evidenced by μCT (39 versus 25% bone volume/tissue volume ratio) and histological analysis 6 weeks post-implantation. These scaffolds are encouraging composite biomaterials for repairing bone applications with a great potential for clinical translation. Further analyses are required with appropriate evaluation in a larger critical-sized defect animal model with long-term follow-up.

In Vitro Osteoinductivity Assay of Hydroxylapatite Scaffolds, Obtained with Biomorphic Transformation Processes, Assessed Using Human Adipose Stem Cell Cultures.

In this study, the in vitro biocompatibility and osteoinductive ability of a recently developed biomorphic hydroxylapatite ceramic scaffold (B-HA) derived from transformation of wood structures were analyzed using human adipose stem cells (hASCs). Cell viability and metabolic activity were evaluated in hASCs, parental cells and in recombinant genetically engineered hASC-eGFP cells expressing the green fluorescence protein. B-HA osteoinductivity properties, such as differentially expressed genes (DEG) involved in the skeletal development pathway, osteocalcin (OCN) protein expression and mineral matrix deposition in hASCs, were evaluated. In vitro induction of osteoblastic genes, such as Alkaline phosphatase (ALPL), Bone gamma-carboxyglutamate (gla) protein (BGLAP), SMAD family member 3 (SMAD3), Sp7 transcription factor (SP7) and Transforming growth factor, beta 3 (TGFB3) and Tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11)/Receptor activator of NF-κB (RANK) ligand (RANKL), involved in osteoclast differentiation, was undertaken in cells grown on B-HA. Chondrogenic transcription factor SRY (sex determining region Y)-box 9 (SOX9), tested up-regulated in hASCs grown on the B-HA scaffold. Gene expression enhancement in the skeletal development pathway was detected in hASCs using B-HA compared to sintered hydroxylapatite (S-HA). OCN protein expression and calcium deposition were increased in hASCs grown on B-HA in comparison with the control. This study demonstrates the biocompatibility of the novel biomorphic B-HA scaffold and its potential use in osteogenic differentiation for hASCs. Our data highlight the relevance of B-HA for bone regeneration purposes.

The Myokine Irisin Promotes Osteogenic Differentiation of Dental Bud-Derived MSCs.

Simple SummaryIrisin is a recently discovered protein, mainly produced in the muscle tissue, whose action is proving effective in many other tissues. The crosstalk between muscle and bone has been long since demonstrated, and physical activity has shown to have an impressive positive effect in both tissues. Irisin production increases with exercising and drops with sedentariness and aging, indicating that the molecule is involved in sarcopenia and in bone mass reduction. Although skeleton is target of irisin, its mechanism of action on bone cells has not yet been completely elucidated. The aim of this work is to analyze the effect of irisin on osteoblast differentiation; to this purpose, we used a stem cell model reproducing the osteoblastogenesis and the bone-forming processes. We performed an in vitro study exploring the main osteoblast markers in the presence of irisin. We found that irisin has an impressive effect on the most peculiar osteoblast feature: the bone mineral matrix secretion process. Moreover, irisin demonstrated an inductive effect on osteoblast osteocalcin production. Both results suggest a stimulating effect of irisin in bone formation. The association we observed between irisin addition and osteoblast osteocalcin production should be further investigated.The myokine irisin, well known for its anabolic effect on bone tissue, has been demonstrated to positively act on osteoblastic differentiation processes in vitro. Mesenchymal stem cells (MSCs) have captured great attention in precision medicine and translational research for several decades due to their differentiation capacity, potent immunomodulatory properties, and their ability to be easily cultured and manipulated. Dental bud stem cells (DBSCs) are MSCs, isolated from dental tissues, that can effectively undergo osteoblastic differentiation. In this study, we analyzed, for the first time, the effects of irisin on DBSC osteogenic differentiation in vitro. Our results indicated that DBSCs were responsive to irisin, showed an enhanced expression of osteocalcin (OCN), a late marker of osteoblast differentiation, and displayed a greater mineral matrix deposition. These findings lead to deepening the mechanism of action of this promising molecule, as part of osteoblastogenesis process. Considering the in vivo studies of the effects of irisin on skeleton, irisin could improve bone tissue metabolism in MSC regenerative procedures.