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Background: The use of biological scaffolds in regenerative endodontics has gained much attention in recent years. The search for a new biomimetic scaffold that contains tissue-specific cell homing factors could lead to more predictable tissue regeneration. The aim of this study was to prepare and characterize decellularized bovine dental pulp-derived extracellular matrix (P-ECM) hydrogels for regenerative endodontic applications., Methods: Freshly extracted bovine molar teeth were collected. Bovine dental pulp tissues were harvested, and stored at -40º C. For decellularization, a 5-day protocol was implemented incorporating trypsin/EDTA, deionized water and DNase treatment. Decellularization was evaluated by DNA quantification and histological examination to assess collagen and glycosaminoglycans (GAGs) content. This was followed by the preparation of P-ECM hydrogel alone or combined with hyaluronic acid gel (P-ECM + HA). The fabricated scaffolds were then characterized using protein quantification, hydrogel topology and porosity, biodegradability, and growth factor content using Enzyme-linked immunosorbent assay (ELISA): transforming growth factor beta-1(TGF-β1), basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF)., Results: Decellularization was histologically confirmed, and DNA content was below (50 ng/mg tissue). P-ECM hydrogel was prepared with a final ECM concentration of 3.00 mg/ml while P-ECM + HA hydrogel was prepared with a final ECM concentration of 1.5 mg/ml. Total protein content in P-ECM hydrogel was found to be (439.0 ± 123.4 µg/µl). P-ECM + HA showed sustained protein release while the P-ECM group showed gradual decreasing release. Degradation was higher in P-ECM + HA which had a significantly larger fiber diameter, while P-ECM had a larger pore area percentage. ELISA confirmed the retention and release of growth factors where P-ECM hydrogel had higher BMP-2 release, while P-ECM + HA had higher release of TGF-β1, bFGF, and VEGF., Conclusions: Both P-ECM and P-ECM + HA retained their bioactive properties demonstrating a potential role as functionalized scaffolds for regenerative endodontic procedures., (© 2024. The Author(s).)

Background: Regenerative endodontic procedures (REPs) offer the promise of restoring vitality and function to a previously necrotic and infected tooth. However, the nature of regenerated tissues following REPs remains unpredictable and uncontrollable. Decellularized extracellular matrix scaffolds have gained recent attention as scaffolds for regenerative endodontics., Objectives: Preparation and characterization of a bovine dental pulp-derived extracellular matrix (P-ECM) hydrogel for regenerative endodontic applications. Biocompatibility and regenerative capacity of the prepared scaffold were evaluated in vivo in a canine animal model., Methods: Fifteen freshly extracted bovine molar teeth were used to prepare P-ECM hydrogels following approval of the institutional review board of the faculty of dentistry, Alexandria University. Decellularization and lyophilization of the extracted pulp tissues, DNA quantification and histological examination of decellularized P-ECM were done. P-ECM hydrogel was prepared by digestion of decellularized pulps. Prepared scaffolds were evaluated for protein content and release as well as release of VEGF, bFGF, TGF-β1 and BMP2 using ELISA. Rabbit dental pulp stem cells' (rDPSCs) viability in response to P-ECM hydrogels was performed. Finally, proof-of-concept of the regenerative capacity of P-ECM scaffolds was assessed in an infected mature canine tooth model following REPs versus blood clot (BC), injectable platelet-rich fibrin (i-PRF) or hyaluronic acid (HA). Statistical analysis was done using independent t test, the Friedman test and chi-square tests (p value ≤ 0.05)., Results: DNA was found to be below the cut-off point (50 ng/mg tissue). Histological evaluation revealed absence of nuclei, retention of glycosaminoglycans (GAGs) and collagen content, respectively. P-ECM hydrogel had a total protein content of (493.12 µg/µl) and protein release was detected up to 14 days. P-ECM hydrogel also retained VEGF, bFGF, TGF-β1 and BMP2. P-ECM hydrogel maintained the viability of rDPSCs as compared to cells cultured under control conditions. P-ECM hydrogel triggered more organized tissues compared to BC, i-PRF and HA when used in REPs for necrotic mature teeth in dogs. Periapical inflammation was significantly less in HA and P-ECM groups compared to blood-derived scaffolds., Conclusion: Bovine dental pulp-derived extracellular matrix (P-ECM) hydrogel scaffold retained its bioactive properties and demonstrated a promising potential in regenerative endodontic procedures compared to conventional blood-derived scaffolds., (© 2024. The Author(s).)

Seed cells and scaffold materials are essential components of tissue engineering. In this study, we investigated the key pathway of the zirconia/dental pulp stem cell composite scaffold in regulating macrophage polarization by transcriptome sequencing. We established N-rGO/ZrO2 composite scaffold and confirmed its structure using various analytical techniques, including SEM, TEM, FTIR, Raman spectra, XPS, and XRD. DPSCs were seeded onto N-rGO/ZrO2 composite scaffold material, and their proliferation, adhesion, and osteogenic differentiation were evaluated by CCK-8, immunofluorescence staining, ALP staining, and alizarin red staining. We then co-cultured DPSCs combined with N-rGO/ZrO2 as composite material with THP-1 cells in a transwell system to investigate the effect of the composite on macrophage polarization. The levels of pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes were assessed by RT-qPCR and western blot. Through bulk RNA sequencing, we detected the transcriptional characteristics of macrophages under the regulation of the composite materials, and identified the differential genes using the DEseq2 package. We also analyzed the cellular and molecular functions of differentially expressed genes (DEGs) in THP-1 cells with DPSCs combined with N-rGO/ZrO2 treatment using GO enrichment analysis and KEGG pathway enrichment analysis. Our results showed that N-rGO/ZrO2 composite scaffold promoted the proliferation, adhesion, and osteogenic differentiation of DPSCs. Moreover, N-rGO/ZrO2 composite scaffold combined with DPSCs regulated macrophage migration, polarization, and glycolysis. Mechanistically, the combination of N-rGO/ZrO2 composite materials and DPSCs regulated macrophage polarization by activating the TNF signaling pathway. This finding provides a new approach to the clinical preservation of maxillofacial bone defect repair.

Objective . Engineered nerve conduits must simultaneously enhance axon regeneration and orient axon extension to effectively restore function of severely injured peripheral nerves. The dental pulp contains a population of stem/progenitor cells that endogenously express neurotrophic factors (NTFs), growth factors known to induce axon repair. We have previously generated scaffold-free dental pulp stem/progenitor cell (DPSC) sheets comprising an aligned extracellular matrix (ECM). Through the intrinsic NTF expression of DPSCs and the topography of the aligned ECM, these sheets both induce and guide axon regeneration. Here, the capacity of bioactive conduits generated using these aligned DPSC sheets to restore function in critical-sized nerve injuries in rodents was evaluated. Approach . Scaffold-free nerve conduits were formed by culturing DPSCs on a substrate with aligned microgrooves, inducing the cells to align and deposit an aligned ECM. The sheets were then detached from the substrate and assembled into scaffold-free cylindrical tissues. Main results. In vitro analyses confirmed that scaffold-free DPSC conduits maintained an aligned ECM and had uniformly distributed NTF expression. Implanting the aligned DPSC conduits across critical-sized defects in the buccal branch of rat facial nerves resulted in the regeneration of a fascicular nerve-like structure and myelinated axon extension across the injury site. Furthermore, compound muscle action potential and stimulated whisker movement measurements revealed that the DPSC conduit treatment promoted similar functional recovery compared to the clinical standard of care, autografts. Significance. This study demonstrates that scaffold-free aligned DPSC conduits supply trophic and guidance cues, key design elements needed to successfully promote and orient axon regeneration. Consequently, these conduits restore function in nerve injuries to similar levels as autograft treatments. These conduits offer a novel bioactive approach to nerve repair capable of improving clinical outcomes and patient quality of life., (Creative Commons Attribution license.)

Fe-Ca-SAPO-34/CS/PANI, a novel hybrid bio-composite scaffold with potential application in dental tissue engineering, was prepared by freeze drying technique. The scaffold was characterized using FT-IR and SEM methods. The effects of PANI on the physicochemical properties of the Fe-Ca-SAPO-34/CS scaffold were investigated, including changes in swelling ratio, mechanical behavior, density, porosity, biodegradation, and biomineralization. Compared to the Fe-Ca-SAPO-34/CS scaffold, adding PANI decreased the pore size, porosity, swelling ratio, and biodegradation, while increasing the mechanical strength and biomineralization. Cell viability, cytotoxicity, and adhesion of human dental pulp stem cells (hDPSCs) on the scaffolds were investigated by MTT assay and SEM. The Fe-Ca-SAPO-34/CS/PANI scaffold promoted hDPSC proliferation and osteogenic differentiation compared to the Fe-Ca-SAPO-34/CS scaffold. Alizarin red staining, alkaline phosphatase activity, and qRT-PCR results revealed that Fe-Ca-SAPO-34/CS/PANI triggered osteoblast/odontoblast differentiation in hDPSCs through the up-regulation of osteogenic marker genes BGLAP, RUNX2, and SPARC. The significance of this study lies in developing a novel scaffold that synergistically combines the beneficial properties of Fe-Ca-SAPO-34, chitosan, and PANI to create an optimized microenvironment for dental tissue regeneration. These findings highlight the potential of the Fe-Ca-SAPO-34/CS/PANI scaffold as a promising biomaterial for dental tissue engineering applications, paving the way for future research and clinical translation in regenerative dentistry.

Decellularized tissues are an attractive scaffolds for 3D tissue engineering. Decellularized animal tissues have certain limitations such as the availability of tissue, high costs and ethical concerns related to the use of animal sources. Plant-based tissue decellularized scaffolds could be a better option to overcome the problem. The leaves of different plants offer a unique opportunity for the development of tissue-specific scaffolds, depending on the reticulate or parallel veination. Herein, we decellularized spinach leaves and employed these for the propagation and osteogenic differentiation of dental pulp stem cells (DPSCs). DPSCs were characterized by using mesenchymal stem cell surface markers CD90, CD105 and CD73 and CD34, CD45 and HLA-DR using flow cytometry. Spinach leaves were decellularized using ethanol, NaOH and HCL. Cytotoxicity of spinach leaf scaffolds were analysed by MTT assay. Decellularized spinach leaves supported dental pulp stem cell adhesion, proliferation and osteogenic differentiation. Our data demonstrate that the decellularized spinach cellulose scaffolds can stimulate the growth, proliferation and osteogenic differentiation of DPSCs. In this study, we showed the versatile nature of decellularized plant leaves as a biological scaffold and their potential for bone regeneration in vitro., (© 2024. The Society for In Vitro Biology.)

This study addresses the morphological and chemical characterization of PGS scaffolds after (6, 12, 18, 24, and 30 min) residence in undoped pyrrole plasma (PGS-PPy) and the evaluation of cell viability with human dental pulp stem cells (hDPSCs). The results were compared with a previous study that used iodine-doped pyrrole (PGS-PPy/I). Analyses through SEM and AFM revealed alterations in the topography and quantity of deposited PPy particles. FTIR spectra of PGS-PPy scaffolds confirmed the presence of characteristic absorption peaks of PPy, with higher intensities observed in the nitrile and -C≡C- groups compared to PGS-PPy/I scaffolds, while raman spectra indicated a lower presence of polaron N + groups. On the other hand, PGS scaffolds modified with PPy exhibited lower cytotoxicity compared to PGS-PPy/I scaffolds, as evidenced by the Live/Dead assay. Furthermore, the PGS-PPy scaffolds at 6 and 12 min, and particularly the PGS-PPy/I scaffold at 6 min, showed the best results in terms of cell viability by the fifth day of culture. The findings of this study suggest that undoped pyrrole plasma modification for short durations could also be a viable option to enhance the interaction with hDPSCs, especially when the treatment times range between 6 min and 12 min., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Aim: To evaluate the role of biomimetic pulp scaffolds derived from the extracellular matrix derived of stem cells from human exfoliated deciduous teeth (SHED-ECM-PS) in promoting pulp-dentine complex regeneration., Methodology: SHED-ECM-PS was prepared through cell aggregation and decellularization techniques. Histological and immunofluorescence analyses, scanning electron microscopy, and DNA quantification assays were used to characterize the SHED-ECM-PS. Additionally, a tooth slice implantation model was established to evaluate the effects of SHED-ECM-PS on regeneration of the pulp-dentine complex in vivo. Extraction medium for SHED-ECM-PS was prepared, and its effect on bone marrow mesenchymal stem cells (BMMSCs) was assessed in vitro. Cell counting kit-8 and Ki-67 staining assays were performed to determine cell proliferation. The rate of apoptosis was evaluated by flow cytometry. Wound healing and transwell assays were conducted to evaluate cell migration. Alizarin red S staining was performed to examine mineralized nodule formation. Western blotting was used to detect the expression of osteogenic and odontogenic markers. The results were analysed using an independent two-tailed Student's t-test. p < .05 was considered statistically significant., Results: SHED-ECM-PS was successfully constructed, exhibiting a striped dental pulp-like shape devoid of nuclear structures or DNA components, and rich in fibronectin, collagen I, DMP1 and DSPP. Notably, SHED-ECM-PS showed no impact on the proliferation or apoptosis of BMMSCs. Histological analysis revealed that dental pulp fibroblasts formed an interwoven mesh in the root canal, and angiogenesis was observed in the SHED-ECM-PS group. Moreover, a continuous, newly formed tubular dentine layer with polarized odontoblast-like cells was observed along the inner wall of the root canal. SHED-ECM-PS promoted the migration, polar alignment and mineralized nodule formation of BMMSCs and specifically elevated the expression levels of odontogenic markers, but not osteogenic markers, compared with the control group in vitro., Conclusion: SHED-ECM-PS exhibited no cytotoxicity and promoted pulp-dentine complex regeneration in vivo as well as cell migration and odontogenic differentiation of BMMSCs in vitro. These findings provide evidence that SHED-ECM-PS, as a novel biological scaffold, has the potential to improve the outcomes of REPs., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Bone tissue regeneration remains a significant challenge in clinical settings due to the complexity of replicating the mechanical and biological properties of bone environment. This study addresses this challenge by proposing a hybrid scaffold designed to enhance both bioactivity and physical stability for bone tissue regeneration. This research is the fisrt to develop a rigid 3D structure composed of polycaprolactone (PCL) and hydroxyapatite nanoparticles (nHA) integrated with a bioink containing human dental pulp stem/stromal cells (hDPSCs), alginate, nHA and collagen (Col). The biofabricated constructs were extensively characterized through cytocompatibility tests, osteogenic differentiation assessment, and biocompatibility evaluation in a rat model. In vitro results demontrated that the hybrid scaffolds presented significantly higher cell viability after 168 h compared to the control group. Furthermore, the hybrid scaffolds showed increased osteogenic differentiation relative to other groups. In vivo evaluation indicated good biocompatibility, characterized by minimal inflammatory response and successful tissue integration. These findings highlight the scaffold's potential to support bone tissue regeneration by combining the mechanical strength of PCL and nHA with the biological activity of the alginate-nHA-Col and hDPSCs bioink. The current study provides a promising foundation for the development of biomaterials aimed at improving clinical outcomes in bone repair and regeneration, particulary for the treatment of critical-size bone defects, targeted drug administration, and three-dimensional models for bone tissue engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: This study aims to compare the application of three types of normal scaffolds-native chitosan, enzymatically modified chitosan, and blood clot (BC)-on pulp regeneration in the teeth of experimental dogs through histological examination, to determine the quantity and type of new tissues formed within the root canal., Materials and Methods: The research sample consisted of 32 root canals from 20 premolars of two male local experimental dogs. The sample was randomly divided into a control group, in which no intervention was performed on the teeth, and three experimental groups based on the type of scaffold used: the BC group, the native chitosan combined with BC (NCS + BC) group, and the enzymatically modified chitosan combined with BC (EMCS + BC) group. Mechanical and chemical cleaning of the canals was performed, followed by the application of the studied scaffolds within the root canals. After 3 months, the teeth were extracted and prepared for histological study, where two variables were studied: the percentage of total vital tissue (soft and hard; VT%) and the percentage of soft vital tissue only (ST%). A one-way ANOVA and Bonferroni tests were used to determine significant differences between the groups at a 95% confidence level., Results: The VT% values were significantly higher in the EMCS + BC group compared to both the NCS + BC and BC groups. The ST% values were also significantly higher in the EMCS + BC group compared to the BC group. However, no significant differences in ST% values were observed between the NCS + BC group and either the BC or EMCS + BC groups., Conclusions: Within the limitations of this study, we conclude that the application of enzymatically modified chitosan scaffolds combined with BC yields superior results in pulp regeneration, which contributes to the formation of pulp-like tissue and cells resembling odontoblasts, as well as apex closure with tissue resembling bone tissue., (© 2024 The Author(s). Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

The demand for macroporous hydrogel scaffolds with interconnected porous and open-pore structures is crucial for advancing research and development in cell culture and tissue regeneration. Existing techniques for creating 3D porous materials and controlling their porosity are currently constrained. This study introduces a novel approach for producing highly interconnected aspartic acid-gelatin macroporous hydrogels (MHs) with precisely defined open pore structures using a one-step emulsification polymerization method with surface-modified silica nanoparticles as Pickering stabilizers. Macroporous hydrogels offer adjustable pore size and pore throat size within the ranges of 50 to 130 μm and 15 to 27 μm, respectively, achieved through variations in oil-in-water ratio and solid content. The pore wall thickness of the macroporous hydrogel can be as thin as 3.37 μm and as thick as 6.7 μm. In addition, the storage modulus of the macroporous hydrogels can be as high as 7250 Pa, and it maintains an intact rate of more than 92% after being soaked in PBS for 60 days, which is also good performance for use as a biomedical scaffold material. These hydrogels supported the proliferation of human dental pulp stem cells (hDPSCs) over a 30 day incubation period, stretching the cell morphology and demonstrating excellent biocompatibility and cell adhesion. The combination of these desirable attributes makes them highly promising for applications in stem cell culture and tissue regeneration, underscoring their potential significance in advancing these fields.

Objectives: To synthesize casein enzymatic hydrolysate (CEH)-laden gelatin methacryloyl (GelMA) fibrous scaffolds and evaluate the cytocompatibility and anti-inflammatory effects on dental pulp stem cells (DPSCs)., Materials and Methods: GelMA fibrous scaffolds with 10%, 20%, and 30% CEH (w/w) and without CEH (control) were obtained via electrospinning. Chemo-morphological, degradation, and mechanical analyses were conducted to evaluate the morphology and composition of the fibers, mass loss, and mechanical properties, respectively. Adhesion/spreading and viability of DPSCs seeded on the scaffolds were also assessed. The anti-inflammatory potential on DPSCs was tested after the chronic challenge of cells with lipopolysaccharides (LPS), followed by treatment with extracts obtained after immersing the scaffolds in α-MEM. The synthesis of the pro-inflammatory cytokines IL-6, IL-1α, and TNF-α was measured by ELISA. Data were analyzed by ANOVA/post-hoc tests (α = 5%)., Results: CEH-laden electrospun fibers had a larger diameter than pure GelMA (p ≤ 0.036). GelMA scaffolds laden with 20% and 30% CEH had a greater mass loss. Tensile strength was reduced for the 10% CEH fibers (p = 0.0052), whereas no difference was observed for the 20% and 30% fibers (p ≥ 0.6736) compared to the control. Young's modulus decreased with CEH (p < 0.0001). Elongation at break increased for the 20% and 30% CEH scaffolds (p ≤ 0.0038). Over time, DPSCs viability increased across all groups, indicating cytocompatibility, with CEH-laden scaffolds exhibiting greater cell viability after seven days (p ≤ 0.0166). Also, 10% CEH-GelMA scaffolds decreased the IL-6, IL-1α, and TNF-α synthesis (p ≤ 0.035)., Conclusion: CEH-laden GelMA scaffolds facilitated both adhesion and proliferation of DPSCs, and 10% CEH provided anti-inflammatory potential after chronic LPS challenge., Clinical Relevance: CEH incorporated in GelMA fibrous scaffolds demonstrated the potential to be used as a cytocompatible and anti-inflammatory biomaterial for vital pulp therapy., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Most of the conditions involving cartilaginous tissues are irreversible and involve degenerative processes. The aim of the present study was to fabricate a biocompatible fibrous and film scaffolds using electrospinning and casting techniques to induce chondrogenic differentiation for possible application in cartilaginous tissue regeneration. Polycaprolactone (PCL) electrospun nanofibrous scaffolds and PCL film were fabricated and incorporated with multi-walled carbon nanotubes (MWCNTs). Thereafter, coating of chondroitin sulfate (CS) on the fibrous and film structures was applied to promote chondrogenic differentiation of human dental pulp stem cells (hDPSCs). First, the morphology, hydrophilicity and mechanical properties of the scaffolds were characterized by scanning electron microscopy (SEM), spectroscopic characterization, water contact angle measurements and tensile strength testing. Subsequently, the effects of the fabricated scaffolds on stimulating the proliferation of human dental pulp stem cells (hDPSCs) and inducing their chondrogenic differentiation were evaluated via electron microscopy, flow cytometry and RT‒PCR. The results of the study demonstrated that the different forms of the fabricated PCL-MWCNTs scaffolds analyzed demonstrated biocompatibility. The nanofilm structures demonstrated a higher rate of cellular proliferation, while the nanofibrous architecture of the scaffolds supported the cellular attachment and differentiation capacity of hDPSCs and was further enhanced with CS addition. In conclusion, the results of the present investigation highlighted the significance of this combination of parameters on the viability, proliferation and chondrogenic differentiation capacity of hDPSCs seeded on PCL-MWCNT scaffolds. This approach may be applied when designing PCL-based scaffolds for future cell-based therapeutic approaches developed for chondrogenic diseases., (© 2024. The Author(s).)

Bone tissue regeneration strategies have incorporated the use of natural polymers, such as hydroxyapatite (nHA), chitosan (CH), gelatin (GEL), or alginate (ALG). Additionally, platelet concentrates, such as platelet-rich fibrin (PRF) have been suggested to improve scaffold biocompatibility. This study aimed to develop scaffolds composed of nHA, GEL, and CH, with or without ALG and lyophilized PRF, to evaluate the scaffold's properties, growth factor release, and dental pulp stem cells (DPSC), and osteoblast (OB) derived from DPSC viability. Four scaffold variations were synthesized and lyophilized. Then, degradation, swelling profiles, and morphological analysis were performed. Furthermore, PDGF-BB and FGF-B growth factors release were quantified by ELISA, and cytotoxicity and cell viability were evaluated. The swelling and degradation profiles were similar in all scaffolds, with pore sizes ranging between 100 and 250 μm. FGF-B and PDGF-BB release was evidenced after 24 h of scaffold immersion in cell culture medium. DPSC and OB-DPSC viability was notably increased in PRF-supplemented scaffolds. The nHA-CH-GEL-PRF scaffold demonstrated optimal physical-biological characteristics for stimulating DPSC and OB-DPSC cell viability. These results suggest lyophilized PRF improves scaffold biocompatibility for bone tissue regeneration purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Bone tissue injuries within oral and dental contexts often present considerable challenges because traditional treatments may not be able to fully restore lost or damaged bone tissue. Novel approaches involving stem cells and targeted 3D scaffolds have been investigated in the search for workable solutions. The use of scaffolds in stem cell-assisted bone regeneration is a crucial component of tissue engineering techniques designed to overcome the drawbacks of traditional bone grafts. This study provides a detailed review of scaffold applications for bone regeneration with stem cells in dentistry. This review focuses on scaffolds and stem cells while covering a broad range of studies explaining bone regeneration in dentistry through the presentation of studies conducted in this field. The role of different stem cells in regenerative medicine is covered in great detail in the reviewed literature. These studies have addressed a wide range of subjects, including the effects of platelet concentrates during dental surgery or specific combinations, such as human dental pulp stem cells with scaffolds for animal model bone regeneration, to promote bone regeneration in animal models. Noting developments, research works consider methods to improve vascularization and explore the use of 3D-printed scaffolds, secretome applications, mesenchymal stem cells, and biomaterials for oral bone tissue regeneration. This thorough assessment outlines possible developments within these crucial regenerative dentistry cycles and provides insights and suggestions for additional study. Furthermore, alternative creative methods for regenerating bone tissue include biophysical stimuli, mechanical stimulation, magnetic field therapy, laser therapy, nutritional supplements and diet, gene therapy, and biomimetic materials. These innovative approaches offer promising avenues for future research and development in the field of bone tissue regeneration in dentistry.

Cell-based tissue engineering often requires the use of scaffolds to provide a three-dimensional (3D) framework for cell proliferation and tissue formation. Polycaprolactone (PCL), a type of polymer, has good printability, favorable surface modifiability, adaptability, and biodegradability. However, its large-scale applicability is hindered by its hydrophobic nature, which affects biological properties. Composite materials can be created by adding bioactive materials to the polymer to improve the properties of PCL scaffolds. Osteolectin is an odontogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR+ cells into osteoblasts. Therefore, the aim of this study was to evaluate whether 3D-printed PCL/osteolectin scaffolds supply a suitable microenvironment for the odontogenic differentiation of human dental pulp cells (hDPCs). The hDPCs were cultured on 3D-printed PCL scaffolds with or without pores. Cell attachment and cell proliferation were evaluated using EZ-Cytox. The odontogenic differentiation of hDPCs was evaluated by alizarin red S staining and alkaline phosphatase assays. Western blot was used to evaluate the expression of the proteins DSPP and DMP-Results: The attachment of hDPCs to PCL scaffolds with pores was significantly higher than to PCL scaffolds without pores. The odontogenic differentiation of hDPCs was induced more in PCL/osteolectin scaffolds than in PCL scaffolds, but there was no statistically significant difference. 3D-printed PCL scaffolds with pores are suitable for the growth of hDPCs, and the PCL/osteolectin scaffolds can provide a more favorable microenvironment for the odontogenic differentiation of hDPCs., (© 2024 IOP Publishing Ltd.)

Epigenetic change has been found to play an important role in cell differentiation and regulation and the dental pulp stem cell in tissue engineering is gaining attention due to the ability of cells to differentiate into odontoblast and other cells. This study evaluated the influence of poly L- lactic acid with hydroxyapatite-coated with polyaniline scaffold (PLLA/HA/PANI) on dental pulp stem cell (DPSC) proliferation and differentiation. After scaffold preparation and DPSCs seeding, the cells proliferation and differentiation were evaluated by immunocytochemistry assay and cell viability was measured by cytotoxicity / MTT assay. The results showed (PLLA/HA/PANI) scaffold facilitates DPSC proliferation and differentiation with gene expression. This finding underscores the promise of this biomaterial combination as a scaffold for dental tissue regeneration and application.

The integration of precision medicine principles into bone tissue engineering has ignited a wave of research focused on customizing intricate scaffolds through advanced 3D printing techniques. Bioceramics, known for their exceptional biocompatibility and osteoconductivity, have emerged as a promising material in this field. This article aims to evaluate the regenerative capabilities of a composite scaffold composed of 3D-printed gelatin combined with hydroxyapatite/tricalcium phosphate bioceramics (G/HA/TCP), incorporating human dental pulp-derived stem cells (hDPSCs). Using 3D powder printing, we created cross-shaped biphasic calcium phosphate scaffolds with a gelatin layer. The bone-regenerating potential of these scaffolds, along with hDPSCs, was assessed through in vitro analyses and in vivo studies with 60 rats and critical-sized calvarial defects. The assessment included analyzing cellular proliferation, differentiation, and alkaline phosphatase activity (ALP), and concluded with a detailed histological evaluation of bone regeneration. Our study revealed a highly favorable scenario, displaying not only desirable cellular attachment and proliferation on the scaffolds but also a notable enhancement in the ALP activity of hDPSCs, underscoring their pivotal role in bone regeneration. However, the histological examination of calvarial defects at the 12-wk mark yielded a rather modest level of bone regeneration across all experimental groups. The test and cell group exhibited significant bone formation compared to all other groups except the control and cell group. This underscores the complexity of the regenerative process and paves the way for further in-depth investigations aimed at improving the potential of the composite scaffolds., (© 2024. The Society for In Vitro Biology.)

Hybrid nanohydroxyapatite/carboxymethyl chitosan (nHAp-CMC) scaffolds have garnered significant attention in the field of regenerative engineering. The current study comparatively analyzed the physicochemical and biological properties of synthetic nanohydroxyapatite (SnHA)- and eggshell-sourced nanohydroxyapatite (EnHA)- based CMC biocomposites for pulp-dentin regeneration. EnHA and CMC were synthesized through a chemical process, whereas SnHA was commercially obtained. Composite scaffolds of SnHA-CMC and EnHA-CMC (1:5 w/w) were prepared using freeze-drying method. All biomaterials were characterized by FTIR, micro-Raman, XRD, HRSEM-EDX, and TEM analyses, and their in vitro bioactivity was assessed by immersing them in simulated body fluid for 21 days. The biological properties of the composite scaffolds were evaluated by assessing cytocompatibility using MTT assay and biomineralization potential by analyzing the odontogenic gene expressions (ALP, DSPP, DMP-1 and VEGF) in human dental pulp stem cells (DPSCs) using RT-qPCR method. Characterization studies revealed that EnHA displayed higher crystallinity and superior surface morphology compared to SnHA. The composite scaffolds showed a highly interconnected porous microstructure with pore sizes ranging between 60 and 220 μm, ideal for cell seeding. All tested materials, SnHA, EnHA, and their respective composites, displayed high cytocompatibility, increased ALP activity and degree of mineralization with significant upregulation of odontogenic-related genes on DPSCs (p < 0.05). Nevertheless, the odontogenic differentiation potential of EnHA-CMC on DPSCs was significantly higher when compared to SnHA-CMC. The findings from this study highlight the potential of EnHA-CMC as a promising candidate for pulp-dentin engineering., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bioprinting technology promotes innovation of fabricating tissue engineered constructs. Dental pulp stem cells (DPSCs) have significant advantages over classical bone mesenchymal stem cells (BMSCs) and are a promising seed cell candidate for bone engineering bioprinting. However, current reports about bioprinted DPSCs for bone regeneration are incomprehensive. The objective of this study was to investigate the osteogenic potential of DPSCs in methacrylate gelatin (GelMA) hydrogels bioprinted scaffolds in vitro and in vivo. Firstly, we successfully bioprinted GelMA with different concentrations embedded with or without DPSCs. Printability, physical features and biological properties of the bioprinted constructs were evaluated. Then, osteogenic differentiation levels of DPSCs in bioprinted constructs with various concentrated GelMA were compared. Finally, effects of bioprinted constructs on cranial bone regeneration were evaluated in vivo. The results of our study demonstrated that 10% GelMA had higher compression modulus, smaller pores, lower swelling and degradation rate than 3% GelMA. Twenty-eight days after printing, DPSCs in three groups of bioprinted structures still maintained high cell activities (>90%). Moreover, DPSCs in 10% GelMA showed an upregulated expression of osteogenic markers and a highly activated ephrinB2/EphB4 signaling, a signaling involved in bone homeostasis. In vivo experiments showed that DPSCs survived at a higher rate in 10% GelMA, and more new bones were observed in DPSC-laden 10% GelMA group, compared with GelMA of other concentrations. In conclusion, bioprinted DPSC-laden 10% GelMA might be more appropriate for bone regeneration application, in contrast to GelMA with other concentrations., Competing Interests: Declaration of Competing Interest No competing financial interests exist., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this study, we propose a spatially patterned 3D-printed nanohydroxyapatite (nHA)/beta-tricalcium phosphate (β-TCP)/collagen composite scaffold incorporating human dental pulp-derived mesenchymal stem cells (hDP-MSCs) for bone regeneration in critical-sized defects. We investigated angiogenesis and osteogenesis in a rabbit critical-sized mandibular defect model treated with this engineered construct. The critical and synergistic role of collagen coating and incorporation of stem cells in the regeneration process was confirmed by including a cell-free uncoated 3D-printed nHA/β-TCP scaffold, a stem cell-loaded 3D-printed nHA/β-TCP scaffold, and a cell-free collagen-coated 3D-printed nHA/β-TCP scaffold in the experimental design, in addition to an empty defect. Posteuthanasia evaluations through X-ray analysis, histological assessments, immunohistochemistry staining, histomorphometry, and reverse transcription-polymerase chain reaction (RT-PCR) suggest the formation of substantial woven and lamellar bone in the cell-loaded collagen-coated 3D-printed nHA/β-TCP scaffolds. Histomorphometric analysis demonstrated a significant increase in osteoblasts, osteocytes, osteoclasts, bone area, and vascularization compared to that observed in the control group. Conversely, a significant decrease in fibroblasts/fibrocytes and connective tissue was observed in this group compared to that in the control group. RT-PCR indicated a significant upregulation in the expression of osteogenesis-related genes, including BMP2, ALPL, SOX9, Runx2, and SPP1. The findings suggest that the hDP-MSC-loaded 3D-printed nHA/β-TCP/collagen composite scaffold is promising for bone regeneration in critical-sized defects.

The decellularized matrix has a great potential for tissue remodeling and regeneration; however, decellularization could induce host immune rejection due to incomplete cell removal or detergent residues, thereby posing significant challenges for its clinical application. Therefore, the selection of an appropriate detergent concentration, further optimization of tissue decellularization technique, increased of biosafety in decellularized tissues, and reduction of tissue damage during the decellularization procedures are pivotal issues that need to be investigated. In this study, we tested several conditions and determined that 0.1% Sodium dodecyl sulfate and three decellularization cycles were the optimal conditions for decellularization of pulp tissue. Decellularization efficiency was calculated and the preparation protocol for dental pulp decellularization matrix (DPDM) was further optimized. To characterize the optimized DPDM, the microstructure, odontogenesis-related protein and fiber content were evaluated. Our results showed that the properties of optimized DPDM were superior to those of the non-optimized matrix. We also performed the 4D-Label-free quantitative proteomic analysis of DPDM and demonstrated the preservation of proteins from the natural pulp. This study provides a optimized protocol for the potential application of DPDM in pulp regeneration., (Creative Commons Attribution license.)

Endodontic therapy, while generally successful, is primarily limited to mature teeth, hence the pressing need to explore regenerative approaches. Gelatin methacryloyl (GelMA) hydrogels have emerged as pivotal biomaterials, promising a bright future for dental pulp regeneration. Despite advancements in tissue engineering and biomaterials, achieving true pulp tissue regeneration remains a formidable task. GelMA stands out for its injectability, rapid gelation, and excellent biocompatibility, serving as the cornerstone of scaffold materials. In the pursuit of dental pulp regeneration, GelMA holds significant potential, facilitating the delivery of stem cells, growth factors, and other vital substances crucial for tissue repair. Presently, in the field of dental pulp regeneration, researchers have been diligently utilizing GelMA hydrogels as engineering scaffolds to transport various effective substances to promote pulp regeneration. However, existing research is relatively scattered and lacks comprehensive reviews and summaries. Therefore, the primary objective of this article is to elucidate the application of GelMA hydrogels as regenerative scaffolds in this field, thereby providing clear direction for future researchers. Additionally, this article provides a comprehensive discussion on the synthesis, characterization, and application of GelMA hydrogels in root canal therapy regeneration. Furthermore, it offers new application strategies and profound insights into future challenges, such as optimizing GelMA formulations to mimic the complex microenvironment of pulp tissue and enhancing its integration with host tissues., (© 2024 Wiley Periodicals LLC.)

Background: Decellularized extracellular matrix (dECM) from several tissue sources has been proposed as a promising alternative to conventional scaffolds used in regenerative endodontic procedures (REPs). This systematic review aimed to evaluate the histological outcomes of studies utilizing dECM-derived scaffolds for REPs and to analyse the contributing factors that might influence the nature of regenerated tissues., Methods: The PRISMA 2020 guidelines were used. A search of articles published until April 2024 was conducted in Google Scholar, Scopus, PubMed and Web of Science databases. Additional records were manually searched in major endodontic journals. Original articles including histological results of dECM in REPs and in-vivo studies were included while reviews, in-vitro studies and clinical trials were excluded. The quality assessment of the included studies was analysed using the ARRIVE guidelines. Risk of Bias assessment was done using the (SYRCLE) risk of bias tool., Results: Out of the 387 studies obtained, 17 studies were included for analysis. In most studies, when used as scaffolds with or without exogenous cells, dECM showed the potential to enhance angiogenesis, dentinogenesis and to regenerate pulp-like and dentin-like tissues. However, the included studies showed heterogeneity of decellularization methods, animal models, scaffold source, form and delivery, as well as high risk of bias and average quality of evidence., Discussion: Decellularized ECM-derived scaffolds could offer a potential off-the-shelf scaffold for dentin-pulp regeneration in REPs. However, due to the methodological heterogeneity and the average quality of the studies included in this review, the overall effectiveness of decellularized ECM-derived scaffolds is still unclear. More standardized preclinical research is needed as well as well-constructed clinical trials to prove the efficacy of these scaffolds for clinical translation., Other: The protocol was registered in PROSPERO database #CRD42023433026. This review was funded by the Science, Technology and Innovation Funding Authority (STDF) under grant number (44426)., (© 2024. The Author(s).)

Background: In recent years, the electrospinning method has received attention because of its usage in producing a mimetic nanocomposite scaffold for tissue regeneration. Hydroxyapatite and gelatin are suitable materials for producing scaffolds, and curcumin has the osteogenesis induction effect., Aims: This study aimed to evaluate the toxicity and early osteogenic differentiation stimulation of nanofibrous gelatin-hydroxyapatite scaffold containing curcumin on dental pulp stem cells (DPSCs)., Objective: The objective of the present investigation was the evaluation of the proliferative effect and primary osteogenic stimulation of DPSCs with a nanofibrous gelatin-hydroxyapatite scaffold containing curcumin. Hydroxyapatite and gelatin were used as suitable and biocompatible materials to make a scaffold suitable for stimulating osteogenesis. Curcumin was added to the scaffold as an osteogenic differentiation- enhancing agent., Methods: The effect of nano-scaffold on the proliferation of DPSCs was evaluated. The activity of alkaline phosphatase (ALP) as the early osteogenic marker was considered to assess primary osteogenesis stimulation in DPSCs., Results: The nanofibrous gelatin-hydroxyapatite scaffold containing curcumin significantly increased the proliferation and the ALP activity of DPSCs (P<0.05). The proliferative effect was insignificant in the first 2 days, but the scaffold increased cell proliferation by more than 40% in the fourth and sixth days. The prepared scaffold increased the activity of the ALP of DPSCs by 60% compared with the control after 14 days (p<0.05)., Conclusion: The produced nanofibrous gelatin-hydroxyapatite scaffold containing curcumin can be utilized as a potential candidate in tissue engineering and regeneration of bone and tooth., Future Prospects: The prepared scaffold in the present study could be a beneficial biomaterial for tissue engineering and the regeneration of bone and tooth soon., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The prevalence of central nervous system (CNS) dysfunction as a result of disease or trauma remains a clinically unsolved problem which is raising increased awareness in our aging society. Human Dental Pulp Stem Cells (hDPSCs) are excellent candidates to be used in tissue engineering and regenerative therapies of the CNS due to their neural differentiation ability and lack of tumorigenicity. Accordingly, they have been successfully used in animal models of spinal cord injury, stroke and peripheral neuropathies. The ideal therapy in brain injury should combine strategies aiming to protect the damaged lesion and, at the same time, accelerate brain tissue regeneration, thus promoting fast recovery while minimizing side or long-term effects. The use of bioresorbable nanopatterned poly(lactide-co-ɛ-caprolactone) (PLCL) polymeric scaffolds as hDPCSs carriers can represent an advantage for tissue regeneration. In this chapter, we describe the surgical procedures to implant functionalized bioresorbable scaffolds loaded with hDPSCs to improve the brain lesion microenvironment in an intracranial stab wound injury model severing the rostral migratory stream (RMS) that connects the brain subventricular zone (SVZ) and the olfactory bulb in nude mice. Additionally, we also describe the technical steps after animal sacrifice for histological tissue observation and characterization., Competing Interests: Disclosures I.M.R., Y.P., B.P.R., J.L., R.B.T., D.M.A., I.R., S.M.C., J.M., L.G.S., G.I., F.L., Y.H., C.E., A.L., J.R.P. have no conflict of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Introduction: Chitosan is a natural biopolymer that attracted enormous attention in biomedical fields. The main components of regenerative endodontic procedures (REPs), as well as tissue engineering, are scaffolds, stem cells, and growth factors. As one of the basic factors in the REPs is maintaining vascularization, this study was aimed at developing basic fibroblast growth factor- (bFGF-) loaded scaffolds and investigating their effects on the angiogenic induction in human dental pulp stem cells (hDPSCs)., Methods: Poly ( ε -caprolactone) (PCL)/chitosan- (CS-) based highly porous scaffold (PCL/CS) was prepared and evaluated by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) analyses. The adhesion and survival potency of seeded cells were assessed by SEM and MTT assays, respectively. The amount of angiogenic markers was investigated in gene and protein levels by real-time PCR and western blotting assays, respectively., Results: Based on our findings, the SEM and FTIR tests confirmed the appropriate structure of synthesized scaffolds. Besides, the adhesion and survival rate of cells and the levels of VEGFR-2, Tie2, and Angiopoietin-1 genes were increased significantly in the PCL/CS/bFGF group. Also, the western blotting results showed the upregulation of these markers at protein levels, which were considerably higher at the PCL/CS/bFGF group ( P < 0.05)., Conclusions: On a more general note, this study demonstrates that the bFGF-loaded PCL/CS scaffolds have the potential to promote angiogenesis of hDPSCs, which could provide vitality of dentin-pulp complex as the initial required factor for regenerative endodontic procedures., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Baharak Divband et al.)

The main goal of regenerative endodontics procedures (REPs) is to revitalize teeth by the regeneration of healthy dental pulp. In this study, we evaluated the potential of combining a natural and accessible biomaterial based on Platelet Poor Plasma (PPP) as a support for dental pulp stem cells (DPSC) and umbilical cord mesenchymal stem cells (UC-MSC). A comparison study between the two cell sources revealed compatibility with the PPP based scaffold with differences noted in the proliferation and angiogenic properties in vitro. Additionally, the release of growth factors including VEGF, HGF and DMP-1, was detected in the media of cultured PPP and was enhanced by the presence of the encapsulated MSCs. Dentin-Discs from human molars were filled with PPP alone or with MSCs and implanted subcutaneously for 4 weeks in mice. Histological analysis of the MSC-PPP implants revealed a newly formed dentin-like structure evidenced by the expression of Dentin sialophosphoprotein (DSPP). Finally, DPSC induced more vessel formation around the dental discs. This study provides evidence of a cost-effective, xenofree scaffold that is compatible with either autologous or allogenic strategy for dental pulp regeneration. This attempt if successfully implemented, could make REPs treatment widely accessible, contributing in improving global health conditions., (© 2022. The Author(s).)

Bioactive and biodegradable porous scaffolds can hasten the healing of bone defects; moreover, patient stem cells seeded onto scaffolds can enhance the osteoinductive and osteoconductive properties of these biomaterials. In this work, porous alginate/hydroxyapatite scaffolds were functionalized with a bioactive coating of a lactose-modified chitosan (CTL). The highly interconnected porous structure of the scaffold was homogeneously coated with CTL. The scaffolds showed remarkable stability up to 60 days of aging. Human Dental Pulp Stem Cells (hDPSCs) cultured in the presence of CTL diluted in culture medium, showed a slight and negligible increase in terms of proliferation rate; on the contrary, an effect on osteogenic differentiation of the cells was observed as a significant increase in alkaline phosphatase activity. hDPSCs showed higher cell adhesion on CTL-coated scaffolds than on uncoated ones. CTL coating did not affect cell proliferation, but stimulated cell differentiation as shown by alkaline phosphatase activity analysis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Neural tissue engineering aims to restore the function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial composition, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of three-dimensional (3D) graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that the addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p < 0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of the scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly- l-lysine was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favorable effects on the biological responses of DPSCs., (© 2021 Wiley Periodicals LLC.)

Mineral trioxide aggregate (MTA) is a common biomaterial used in endodontics regeneration due to its antibacterial properties, good biocompatibility and high bioactivity. Surface modification technology allows us to endow biomaterials with the necessary biological targets for activation of specific downstream functions such as promoting angiogenesis and osteogenesis. In this study, we used caffeic acid (CA)-coated MTA/polycaprolactone (PCL) composites and fabricated 3D scaffolds to evaluate the influence on the physicochemical and biological aspects of CA-coated MTA scaffolds. As seen from the results, modification of CA does not change the original structural characteristics of MTA, thus allowing us to retain the properties of MTA. CA-coated MTA scaffolds were shown to have 25% to 55% higher results than bare scaffold. In addition, CA-coated MTA scaffolds were able to significantly adsorb more vascular endothelial growth factors ( p < 0.05) secreted from human dental pulp stem cells (hDPSCs). More importantly, CA-coated MTA scaffolds not only promoted the adhesion and proliferation behaviors of hDPSCs, but also enhanced angiogenesis and osteogenesis. Finally, CA-coated MTA scaffolds led to enhanced subsequent in vivo bone regeneration of the femur of rabbits, which was confirmed using micro-computed tomography and histological staining. Taken together, CA can be used as a potently functional bioactive coating for various scaffolds in bone tissue engineering and other biomedical applications in the future.

This research aimed to design innovative therapeutic bio-composites that enhance odontogenic and osteogenic differentiation of human dental pulp-derived mesenchymal stem cells (h-DPSCs) in-vitro regeneration. Herein, we report the fabrication of scaffolds containing chitosan, Ca-SAPO-34 monometallic and/or Fe-Ca-SAPO-34 bimetallic nanoparticles by freeze-drying technique. The scaffolds and nanoparticles were characterized using ICP-AES, FT-IR, XRD, TGA, TEM, BET, SEM, and EDS methods. The effects of SAPO-34 and nanoparticles were investigated by changes on the physicochemical properties of scaffolds including swelling ratio, density, porosity, bio-degradation, mechanical behavior, and biomineralization. Cell viability, cell adhesion and cytotoxicity of Ca-SAPO-34/CS and Fe-Ca-SAPO-34 scaffolds were investigated by MTT assay and SEM on h-DPSCs which revealed cell proliferation no toxicity on scaffolds. Cell tests demonstrated that Ca-SAPO-34/CS scaffold clearly displayed a positive effect on differentiation of hDPSCs into osteogenic/odontogenic cells and moderate effect on cell proliferation. Moreover, the incorporation of Fe 2 O 3 to Ca-SAPO-34/CS scaffold promoted the proliferation of hDPSCs and osteogenic differentiation. Alizarin red, Alkaline phosphatase and QRT-PCR results showed that Fe-Ca-loaded SAPO-34/CS can lead to osteoblast/odontoblast differentiation in DPSCs through the up-regulation of related genes, thus indicating that Fe-Ca-SAPO-34/CS has remarkable prospects as a biomaterial for hard tissue engineering., (Copyright © 2021. Published by Elsevier B.V.)

The demand for bone grafting procedures in various fields of medicine is increasing. Existing substitutes in clinical practice do not meet all the criteria required for an ideal bone scaffold, so new materials are being sought. This study evaluated bone regeneration using a critical-size Wistar rat's calvarial defect model. 12 male and 12 female rats were evenly divided into 3 groups: 1. Negative and positive (Geistlich Bio-Oss®) controls; 2. polylactic acid (PLA) and PLA/hydroxyapatite (HA); 3. PLA/HA cellularised with dental pulp stem cells (DPSC) and PLA/HA extracellular matrix (ECM) scaffolds. PLA/HA filament was created using hot-melt extrusion equipment. All scaffolds were fabricated using a 3D printer. DPSC were isolated from the incisors of adult Wistar rats. The defects were evaluated by micro-computed tomography (µCT) and histology, 8 weeks after surgery. µCT revealed that the Bio-Oss group generated 1.49 mm3 and PLA/HA ECM 1.495 mm3 more bone volume than the negative control. Histology showed a statistically significant difference between negative control and both (Bio-Oss and PLA/HA ECM) groups in rats of both genders. Moreover, histology showed gender-specific differences in all experimental groups and a statistically significant difference between cellularised PLA/HA and PLA/HA ECM groups in female rats. Qualitative histology showed the pronounced inflammation reaction during biodegradation in the PLA group. In conclusion, the bone-forming ability was comparable between the Bio-Oss and PLA/HA ECM scaffolds. Further research is needed to analyse the effects of ECM and PLA/HA ratio on osteoregeneration.

Background: The regeneration of dental pulp, especially in cases of pulp death of immature teeth, is the goal of the regenerative endodontic procedures (REPs) that are based on tissue engineering principles, consisting of stem cells, growth factors, and scaffolds. Photobiomodulation therapy (PBMT) showed to improve dental pulp regeneration through cell homing approaches in preclinical studies and has been proposed as the fourth element of tissue engineering. However, when a blood clot was used as a scaffold in one of these previous studies, only 30% of success was achieved. The authors pointed out the instability of the blood clot as the regeneration shortcoming. Then, to circumvent this problem, a new scaffold was developed to be applied with the blood clot. The hypothesis of the present study was that an experimental injectable chitosan hydrogel would facilitate the three-dimensional spatial organization of endogenous stem cells in dental pulp regeneration with no interference on the positive influence of PBMT., Methods: For the in vitro analysis, stem cells from the apical papilla (SCAPs) were characterized by flow cytometry and applied in the chitosan scaffold for evaluating adhesion, migration, and proliferation. For the in vivo analysis, the chitosan scaffold was applied in a rodent orthotopic dental pulp regeneration model under the influence of PBMT (660 nm; power output of 20 mW, beam area of 0.028 cm 2 , and energy density of 5 J/cm 2 )., Results: The scaffold tested in this study allowed significantly higher viability, proliferation, and migration of SCAPs in vitro when PBMT was applied, especially with the energy density of 5 J/cm 2 . These results were in consonance to those of the in vivo data, where pulp-like tissue formation was observed inside the root canal., Conclusion: Chitosan hydrogel when applied with a blood clot and PBMT could in the future improve previous results of dental pulp regeneration through cell homing approaches., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Maria Stella Moreira et al.)

The repair of skeletal defects in maxillofacial region remains an intractable problem, the rising technology of bone tissue engineering provides a new strategy to solve it. Scaffolds, a crucial element of tissue engineering, must have favorable biocompatibility as well as osteoinductivity. In this study, we prepared berberine/polycaprolactone/collagen (BBR/PCL/COL) scaffolds with different concentrations of berberine (BBR) (25, 50, 75 and 100 μg/mL) through electrospinning. The influence of dosage on scaffold morphology, cell behavior and in vivo bone defect repair were systematically studied. The results indicated that scaffolds could release BBR stably for up to 27 days. Experiments in vitro showed that BBR/PCL/COL scaffolds had appropriate biocompatibility in the concentration of 25-75 μg/mL, and 50 and 75 μg/mL scaffolds could significantly promote osteogenic differentiation of dental pulp stem cells. Scaffold with 50 μg/mL BBR was implanted into the critical bone defect of rats to evaluate the ability of bone repair in vivo. It was found that BBR/PCL/COL scaffold performed more favorable than polycaprolactone/collagen (PCL/COL) scaffold. Overall, our study is the first to evaluate the capability of in vivo bone repair of BBR/PCL/COL electrospun scaffold. The results indicate that BBR/PCL/COL scaffold has prospective potential for tissue engineering applications in bone regeneration therapy.

Background: Recently a greater interest in tissue engineering for the treatment of large bone defect has been reported. The aim of the present systematic review and meta-analysis was to investigate the effectiveness of dental pulp stem cells and synthetic block complexes for bone defect treatment in preclinical in vivo articles., Methods: The electronic database and manual search was conducted on Pubmed, Scopus, and EMBASE. The papers identified were submitted for risk-of-bias assessment and classified according to new bone formation, bone graft characteristics, dental pulp stem cells (DPSCs) culture passages and amount of experimental data. The meta-analysis assessment was conducted to assess new bone formation in test sites with DPSCs/synthetic blocks vs. synthetic block alone., Results: The database search identified a total of 348 papers. After the initial screening, 30 studies were included, according to the different animal models: 19 papers on rats, 3 articles on rabbits, 2 manuscripts on sheep and 4 papers on swine. The meta-analysis evaluation showed a significantly increase in new bone formation in favor of DPSCs/synthetic scaffold complexes, if compared to the control at 4 weeks (Mean Diff: 17.09%, 95% CI: 15.16-18.91%, p < 0.01) and at 8 weeks (Mean Diff: 14.86%, 95% CI: 1.82-27.91%, p < 0.01) in rats calvaria bone defects., Conclusion: The synthetic scaffolds in association of DPSCs used for the treatment of bone defects showed encouraging results of early new bone formation in preclinical animal studies and could represent a useful resource for regenerative bone augmentation procedures.

Here, we fabricated thermosensitive injectable hydrogel containing poly (N-isopropylacrylamide) (PNIPAAm)-based copolymer/graphene oxide (GO) composite with different feed ratio to chitosan (CS) as a natural polymer through physical and chemical crosslinking for the proliferation and differentiation of the human dental pulp stem cells (hDPSCs) to the osteoblasts. The PNIPAAm copolymer/GO composite was synthesized by free-radical copolymerization of (N-isopropylacrylamide) (NIPAAm), itaconic acid (IA) and maleic anhydride-modified poly(ethylene glycol) (PEG) in the presence of GO and used for the preparation of the hydrogels. The formulated hydrogels were evaluated for the porous architecture, rheological behavior, compressive strength, swelling property, in vitro degradation, hemocompatibility, biocompatibility, and differentiation. The hydrogel could enhance the deposition of minerals and the activity of alkaline phosphatase (ALP), in large part attributable to the oxygen and amine-containing functional groups of GO and CS. The engineered hydrogel could also upregulate the expression of the Runt-related transcription factor 2 and osteocalcin in the hDPSCs cultivated in both the normal and osteogenic media. It seems to promote the absorption of osteogenic inducer too. Based on our findings, the engineered hydrogel demonstrated the osteogenic potential, upon which it is proposed as a constructing scaffold in bone tissue engineering for the transplantation of hDPSCs., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2020. Published by Elsevier B.V.)

Polyglycerol sebacate (PGS) scaffolds obtained using a leaching technique were modified with iodine-doped polypyrrole (PPy-I) in a plasma reactor in order to study the effect of exposure time on the cell viability of hDPSCs. SEM analysis showed the formation and growth of PPy-I particles as the exposure time was increased, while FTIR and XPS analysis revealed the presence of -NH- and N+ groups in the chemical composition of the surfaces, relating to the increase in the amount of PPY-I particles. The water contact angle measurements showed an increase in the scaffold's hydrophilicity with greater exposure times which was also attributed to the rising of PPy-I particles. It was also observed that PPy-I promotes the rigidity of the treated PGS scaffolds. when in direct contact with treated PGS scaffolds, cell viability improved with respect to non-treated scaffolds, however only at shorter time exposures. Extracts of plasma-treated PGS scaffolds showed high cytotoxicity as the time exposure to plasma treatment was increased.

The aim of this study was to develop a highly porous calcium-containing chitosan scaffold suitable for dentin regeneration. A calcium hydroxide (Ca[OH] 2 ) suspension was used to modulate the degree of porosity and chemical composition of chitosan scaffolds. The chitosan solution concentration and freezing protocol were adjusted to optimize the porous architecture using the phase-separation technique. Scanning electron microscopy/energy-dispersive spectroscopy demonstrated the fabrication of a highly porous calcium-linked chitosan scaffold (CH-Ca), with a well-organized and interconnected porous network. Scaffolds were cross-linked on glutaraldehyde (GA) vapor. Following a 28-day incubation in water, cross-linked CH scaffold had no changes on humid mass, and CH-Ca featured a controlled degradability profile since the significant humid mass loss was observed only after 21 (26.0%) and 28 days (42.2%). Fourier-transform infrared spectroscopy indicated the establishment of Schiff base on cross-linked scaffolds, along with calcium complexation for CH-Ca. Cross-linked CH-Ca scaffold featured a sustained Ca 2+ release up to 21 days in a humid environment. This porous and stable architecture allowed for human dental pulp cells (HDPCs) to spread throughout the scaffold, with cells exhibiting a widely stretched cytoplasm; whereas, the cells seeded onto CH scaffold were organized in clusters. HDPCs seeded onto CH-Ca featured significantly higher ALP activity, and gene expressions for ALP, Col1, DMP-1, and DSPP in comparison to CH, leading to a significant 3.5 times increase in calcium-rich matrix deposition. In sum, our findings suggest that CH-Ca scaffolds are attractive candidates for creating a highly porous and bioactive substrate for dentin tissue engineering., (© 2020 Wiley Periodicals, Inc.)

The aim of this study was to synthesize an innovative composite scaffold, which structured of clinoptilolite-nanohydroxyapatite/chitosan-gelatin (CLN-nHA/CS-G) with enhanced attributes for utilization in the bone tissue engineering. This composite scaffold was prepared by blending the CLN, nHA, chitosan, and gelatin solution followed by a freeze-drying step. The fabricated composite scaffolds were studied using BET, FTIR, XRD, and SEM techniques. The highly porous composite scaffolds with a pore size of 200 ± 100 μm were synthesized. Moreover, the effects of CLN and nHA on the physicochemical features of the scaffold such as density, swelling ratio, biomineralization, biodegradation, and mechanical behavior were studied. Compared with CS-G scaffold, the presence of CLN and nHA leads to an increased surface area, increased biomineralization, and low rate of degradation in simulated body fluid solution (SBF) and mechanical strength. Cytotoxicity of the CLN-nHA/CS-G scaffold was studied by MTT assay on human dental pulp stem cells (h-DPSCs). The biological response of h-DPSCs showed no toxicity and studied cells proliferated and attached on the pore surfaces of the scaffold. Results indicated that introducing CLN and nHA to composite improves the scaffold characteristics in a way that makes it suitable for bone tissue engineering., (© 2019 Wiley Periodicals, Inc.)

Background/purpose: In recent years, 3D printing technology has flourished and applied to tissue engineering regeneration. The purpose of this study is to investigate the effects of gap width between struts (GWbS) of three-dimensional-printed polylactic acid scaffolds (3DP-PLASs) on neural differentiation of human dental pulp stem cells (hDPSCs)., Methods: Both the 3DP-PLASs with the GWbS of 150 μm and 200 μm were experimental groups and the 3DP-PLAS without microfilament struts was the control group. Properties of 3DP-PLASs were observed by water contact angles (WCA), atomic force microscope (AFM), and differential scanning calorimeter (DSC). The cell culture of hDPSCs on 3DP-PLASs was performed, and cytotoxicities were measured with Alamar Blue assay. The neural differentiation of hDPSCs on different 3DP-PLASs was compared after neural induction. Expressions of neural markers Nestin, MAP2, beta III tubulin, and GFAP were evaluated with immunocytochemical staining., Results: Our results demonstrated no cytotoxicities among scaffolds, whereas they may differ in crystal sizes and directions resulting from different orders of cooling time, contact surface, and temperature distribution during the building process. In addition, hDPSCs could successfully adhere to 3DP-PLAS modified by alcohol or poly-l-Lysine and demonstrate morphological change and related protein performance., Conclusion: We conclude that 3DP-PLASs with 150 μm gaps can induce cellular orientations more easily than those with 200 μm gaps. In addition, 3DP-PLASs seem to improve cell adhesion after being coated with poly-l-lysine or soaked with alcohol., (Copyright © 2019. Published by Elsevier B.V.)

Bone defects due to trauma or diseases still pose a clinical challenge to be resolved in the current tissue engineering approaches. As an alternative to traditional methods to restore bone defects, such as autografts, bone tissue engineering aims to achieve new bone formation via novel biomaterials used in combination with multipotent stem cells and bioactive molecules. Mesenchymal stem cells (MSCs) can be successfully isolated from various dental tissues at different stages of development including dental pulp, apical papilla, dental follicle, tooth germ, deciduous teeth, periodontal ligament and gingiva. A wide range of biomaterials including polymers, ceramics and composites have been investigated for their potential as an ideal bone scaffold material. This article reviews the properties and the manufacturing methods of biomaterials used in bone tissue engineering, and provides an overview of bone tissue regeneration approaches of scaffold and dental stem cell combinations as well as their limitations.

Dental pulp stem cells (DPSCs) are adult multipotent stem cells of neuroectodermal origin; they provide an encouraging perspective in the domain of nerve tissue engineering. DPSCs could be transplanted in biodegradable electrospun neuro-supportive scaffold (optimized in various 3D geometries like coating on the surface of titanium implant, hollow/solid tubes, etc.) for enhanced in vivo recovery of peripheral nerves. Herein, we describe the fabrication of uniform bead-free nanofibrous scaffold which supports DPSCs, proliferation, and their subsequent neural differentiation and thus could be utilized for enhanced regeneration of peripheral nervous system.

Repairing the lost or damaged mandible is very difficult and time-consuming, so there is a great hope for tissue engineering to accelerate it. At the present study, electrospinning was applied to fabricate polyvinylidene fluoride (PVDF) and PVDF-polyaniline (PANI) composite scaffolds. In addition, extremely low frequency pulsed electromagnetic field (PEMF) was applied for treating the stem cells derived from dental pulp (DPSCs) when cultured on the nanofibrous scaffolds. Osteoinductive property of the fabricated PVDF, PVDF-PANI scaffold at the presence and absence of the PEMF was investigated by evaluating the common osteogenic differentiation markers in seeded-DPSCs on the scaffold. Results demonstrated that cell attachment, protein adsorption and cells viability were increased when PEMF was applied. In addition, ALP activity, calcium content, osteogenic genes and protein evaluations confirmed that PEMF could significantly increase osteoinductivity of the PVDF while composite with PANI. According to the results, the use of polymers with piezoelectricity and conductivity features plus PEMF exposure has a promising potential to improve the current treatment methods in bone and mandibular defects.

The aim of this study was to prepare a promising biomaterial for dental pulp tissue repair and regeneration. The ultrasmall superparamagnetic iron oxide (USPIO)-labeled hydroxyapatite (HA)/silk fibroin (SF) scaffold loaded dental pulp stem cells (DPSCs) was designed. The odontogenic differentiation of DPSCs was investigated, and USPIO was used as magnetic resonance imaging (MRI) contrast agent. The results indicated that scaffolds freeze-dried from SF solution with 0.025 mg/mL USPIO and 5 mg/mL HA showed stable physical properties, accurate MRI images, and low cytotoxicity in vitro. The composite scaffolds were characterized and implanted to the subcutaneous space under the nude mice with tooth fragment. After 2 W of implantation, the relaxation rate (R2) values increased because of incorporation of USPIO, then decreased with the degradation of scaffolds. The tissue regeneration and scaffolds degradation behaviors were monitored by MRI. The expression of dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein 1 (DMP1) indicated that DPSCs differentiated into odontoblast-like cells. Revascularization and mineralization evaluated by HE-stained images and alizarin red staining images showed good formation. The USPIO/HA/SF composite scaffold loaded DPSCs promoted the repair and regeneration of dental pulp tissue and provided the ability of imaging noninvasively., (Copyright © 2019. Published by Elsevier B.V.)

The objectives of this study were to incorporate iron oxide nanoparticles (IONPs) into calcium phosphate cement (CPC) to enhance bone engineering, and to investigate the effects of IONPs as a liquid or powder on stem cells using IONP-CPC scaffold for the first time. IONP-CPCs were prepared by adding 1% IONPs as liquid or powder. Human dental pulp stem cells (hDPSCs) were seeded. Subcutaneous implantation in mice was investigated. IONP-CPCs had better cell spreading, and greater ALP activity and bone mineral synthesis, than CPC control. Subcutaneous implantation for 6 weeks showed good biocompatibility for all groups. In conclusion, incorporating IONPs in liquid or powder form both substantially enhanced hDPSCs on IONP-CPC scaffold and exhibited excellent biocompatibility. IONP incorporation as a liquid was better than IONP powder in promoting osteogenic differentiation of hDPSCs. Incorporating IONPs and chitosan lactate together in CPC enhanced osteogenesis of hDPSCs more than using either alone., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Clinoptilolite (Cpt)-nanohydroxyapatite (HA) (Cpt-HA) scaffolds were fabricated as a potential material for loadbearing orthopaedic applications. Cpt-HA materials were successfully synthesized by using microwave assisted reflux method followed by the fabrication of three-dimensional (3D) porous scaffold via thermal decomposition process using polyethylene glycol (PEG)/ polyvinyl alcohol (PVA) as porogens. The scaffold materials were characterized using x-ray diffraction, Fourier transform Infra-red, Scanning electron microscopy and Energy dispersive spectroscopy techniques. Incorporation of Cpt in HA scaffold significantly increased the compressive strength and surface hardness while scaffolds retained an interconnected porous structure with 64% porosity. Human dental pulp stem cells (DPSCs) were isolated from the third molar and used as pluripotent-like cell model to evaluate the biological properties of Cpt-HA scaffolds. Highest cellular attachment and proliferation were observed for DPSCs seeded on 2.0 g Cpt-HA scaffolds compare to pure HA. Similarly, significantly higher ALP activity of cells was observed on Cpt-HA scaffolds compared to pure HA. The enhanced proliferation and osteogenic response of the DPSCs cultured on Cpt-HA scaffolds suggest that the fabricated scaffolds can be used in bone tissue engineering. In this work, we have successfully shown that the interconnected porous Cpt-HA scaffolds have superior mechanical biological properties compared to pure HA scaffold.

Dental caries is a dental disease affecting public health, which results in many socio-economic consequences. This disease causes loss of tooth hard tissue and subsequent inflammation and loss of the dental pulp. In this study, it was aimed to develop and characterize boron (B) modified bioactive glass nanoparticles (BG-NPs) containing cellulose acetate/oxidized pullulan/gelatin (CA/ox-PULL/GEL) three dimensional scaffolds with tubular morphology for dentin regeneration. 3D nanobiocomposite structures were prepared by thermally induced phase separation and porogen leaching methods and characterized by in vitro degradation analysis, water absorption (WA) capacity measurement, SEM, in vitro biomineralization analysis, porosity measurement and mechanical tests. Scaffolds lost about (30-40)% of their weight during one month and WA capacity decreased with increase in immersion time in phosphate buffer solution (PBS) during one month. According to SEM, aligned and tubular structures were formed with mean diameter of about 11 μm, and BG-NPS were distributed evenly in all parts of the scaffolds. Scaffolds (without BG-NPs) possessed the highest porosity percentage. Addition of 10% BG-NPs improved the mechanical properties of scaffolds. Scaffolds surfaces were fully covered by calcium phosphate (Ca-P) deposits after conditioning in simulated body fluid for 14 days with higher quantity of deposition in groups with inclusion of B-BG-NPs. Human dental pulp stem cells (hDPSCs) were isolated from third molar teeth and used in cell culture studies. In all groups, cells adhered well 1 day after culture. Group B14-10 showed a slight increase of proliferation than group (without BG-NPs) after 7 days of incubation. Alkaline phosphatase activity (ALP) and intracellular calcium amounts increased significantly 14 days after incubation with highest values in B14-10 and B14-20 groups. Confocal laser scanning microscopy (CSLM) analysis, showed that cells on B14-10 and B21-10 scaffold groups, spread more 14 days after culture, and they also possessed extended processes specific to odontoblasts. Alizarin Red quantification showed that the highest calcium deposition was observed on B14-10 scaffolds. Immunohistochemical and Von Kossa stainings showed that scaffolds positively affected the odontoblastic differentiation of the hDPSCs. In this work, results showed that boron modified BG-NPs (B-BG-NPs) incorporated dentin-like constructs bring a new approach for dental tissue engineering applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nowadays, regenerative medicine has paid special attention to research (in vitro and in vivo) related to bone regeneration, specifically in the treatment of bone fractures or skeletal defects, which is rising worldwide and is continually demanding new developments in the use of stem cells, growth factors, membranes and scaffolds based on novel nanomaterials, and their applications in patients by using advanced tools from molecular biology and tissue engineering. Strontium (Sr) is an element that has been investigated in recent years for its participation in the process of remodeling and bone formation. Based on these antecedents, this is a review about the Strontium Folate (SrFO), a recently developed non-protein based bone-promoting agent with interest in medical and pharmaceutical fields due to its improved features in comparison to current therapies for bone diseases.