Harnessing the Regenerative Potential of Purified Bovine Dental Pulp and Dentin Extracellular Matrices in a Chitosan/Alginate Hydrogel.

When a tooth is diseased or damaged through caries, bioactive molecules are liberated from the pulp and dentin as part of the natural response to injury and these are key molecules for stimulating stem cell responses for tissue repair. Incorporation of these extracellular-matrix (ECM)-derived molecules into a hydrogel model can mimic in vivo conditions to enable dentin-pulp complex regeneration. Here, a chitosan/alginate (C/A) hydrogel is developed to sequester bovine ECM extracts. Human dental pulp cells (hDPCs) are cultured with these constructs and proliferation and cytotoxicity assays confirm that these C/A hydrogels are bioactive. Sequential z-axis fluorescent imaging visualizes hDPCs protruding into the hydrogel as it degraded. Alizarin red S staining shows that hDPCs cultured with the hydrogels display increased calcium-ion deposition, with dentin ECM stimulating the highest levels. Alkaline phosphatase activity is increased, as is expression of transforming growth factor-beta as demonstrated using immunocytochemistry. Directional analysis following phase contrast kinetic image capture demonstrates that both dentin and pulp ECM molecules act as chemoattractants for hDPCs. Data from this study demonstrate that purified ECM from dental pulp and dentin when delivered in a C/A hydrogel stimulates dental tissue repair processes in vitro.

In Vitro Models Used in the Formation of Root Caries Lesions-A Review of the Literature.

The management of root caries remains a challenge for clinicians due to its unique anatomical location and structure. There is increasing interest in utilising artificial root caries lesions to develop new strategies for remineralisation. An ideal protocol has not yet been agreed upon. The aim of this review is to provide a structured overview of previously reported in vitro root caries models. The literature was screened and mined for information mainly on substrate selection, model systems utilised, and variables used in the models. Human roots (60%) were the most frequently used substrates, followed by bovine roots (40%). Chemical models (69%) were the most frequently utilised model systems, followed by microbiological models (27%), to form root caries lesions. Acetate buffer solution (80%), pH 5.0 or above (40%), and a demineralisation time of five days (25%) were the common variables used in the chemical systems, while mono-species biofilm was most frequently used (73%) in microbiological models and Streptococcus mutans was the most common bacterial strain utilised in these models (80%). This review highlights the variability amongst the experimental approaches, discusses the advantages and limitations of these approaches, and emphasises that standardisation of experimental conditions along with sustained research will benefit root caries research.

In vitro effects of wool-derived keratin on human dental pulp-derived stem cells for endodontic applications.

In this study we examine the influence of wool-derived keratin intermediate filament proteins (kIFPs) on human dental pulp-derived stem cells (hDPSCs). kIFPs were diluted (10 mg/mL to 0.001 mg/mL) in cell culture media. Effects on hDPSCs proliferation were measured using Alamar blue assay. Keratin concentrations of 1 mg/mL and 0.1 mg/mL were tested for odontogenic differentiation and mineralisation. Alkaline phosphatase (ALP) quantification (7th, 14th, and 21st days), alizarin red S (AR-S) staining and calcium quantification (21st day), reverse transcription polymerase chain reaction (RT-PCR, collagen expression), and immunocytochemical staining for dentin matrix protein (DMP) were performed. hDPSCs showed higher proliferation with kIFPs of 0.1 mg/mL or less (p < 0.0001). The 0.1 mg/mL keratin concentration promoted odontogenic differentiation, confirmed by increased ALP activity, significant calcium deposits (AR-S staining, p < 0.05), up-regulated collagen expression (RT-PCR, p < 0.05), and positive DMP staining. These results suggest that kIFPs could be a potential biomaterial for pulp-dentin regeneration.

A critical review of in vitro research methodologies used to study mineralization in human dental pulp cell cultures.

BACKGROUND: The pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains. OBJECTIVES: The purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes. METHOD: The literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review. RESULTS: Most published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent. DISCUSSION: While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach. CONCLUSION: Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

Effect of chitosan infiltration on hydroxyapatite scaffolds derived from New Zealand bovine cancellous bones for bone regeneration.

Hydroxyapatite (HA) derived from bovine bones garnered wider interest as a bone substitute due to their abundant availability as meat wastes and similarities in morphology and mineral composition to human bone. In our previous work, we developed an easy and reproducible method to prepare xenograft HA scaffolds from NZ bovine cancellous bones (BHA). However, the processing methodology rendered the material mechanically weak. The present study investigated the infiltration of chitosan (CS) into the bovine HA scaffolds (CSHA) to improve the mechanical properties of BHA. The presence of characteristic functional groups of HA and CS as detected by infrared spectroscopy confirmed the infiltration of CS into the BHA scaffolds. X-ray Diffraction study confirmed the presence of the hydroxyapatite phase in both BHA and CSHA scaffolds. SEM and µCT analyses showed the CSHA scaffolds presented adequate porosity and an interconnected porous architecture required for cell migration and attachment. CSHA scaffolds presented good thermal, chemical and structural stability while demonstrating sustained biodegradability in simulated body fluid. CSHA scaffolds presented mechanical properties significantly higher than the BHA scaffolds. CSHA scaffolds were biocompatible with Saos-2 osteoblast cells and supported cell proliferation significantly better than the BHA scaffolds indicating their potential in bone tissue engineering.

Development and characterization of a xenograft material from New Zealand sourced bovine cancellous bone.

A xenograft (bovine hydroxyapatite [BHA]) was developed from New Zealand sourced bovine cancellous bone by a successful defatting and deproteinizing procedure. The BHA was chemically, compositionally and structurally characterized. Fourier transform infrared spectroscopy confirmed the removal of organic matter from the bone matrix and the presence of carbonate ( CO32-), hydroxyl (OH- ), and phosphate ( PO43-) functional groups. X-ray diffraction analysis suggested that the processed bone corresponds characteristically to hydroxyapatite (HA). SEM analysis showed that the BHA has an interconnected porous architecture with a pore diameter ranging from 100 to 700 µm while µCT analysis calculated the total porosity as 73.46% ± 1.08. Furthermore, the BHA was stable up to 1000°C and lost only 1.8% of its weight. The Ca/P molar ratio of the BHA was 1.58, which is comparable with commercially available natural HA-Endobon® . After 28 days of incubation in simulated body fluid (SBF), the pH value only fluctuated between 7.1 and 7.5 and the BHA scaffold did not degrade significantly by weight indicating the scaffold had excellent chemical and structural stability. In vitro studies showed the BHA was cytocompatible and supported the proliferative growth of Saos-2 osteoblast cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1054-1062, 2017.

Substituted hydroxyapatites for bone regeneration: A review of current trends.

At present hydroxyapatite (HA) is been extensively investigated for biomedical applications, largely as a result of its similarity in composition to the mineral portion of bone. Although HA undergoes osseointegration and is bioactive and osteoconductive, the inherent brittleness and low fracture toughness limits its use under load bearing conditions, also once implanted in the body, HA takes a long time to resorb. The crystal structure of HA is conducive to a variety of ionic substitutions. To accurately mimic the calcium deficient and carbonate-containing nature of HA in bone, both cationic and anionic substituents have been incorporated to synthetic HA. This article focuses on the incorporation of both the well established (Zn, Si, Sr, F, and carbonate) and latest ions (Ag, citrate, iron, niobate, and tantalates) into the HA structure and aims to highlight the key effects of these substitutions in terms of their chemical, physical, and biological properties. It can be shown that a minor substituent cannot only alter the microstructure, stability and crystallinity of the HA structure in an implant, but also have a significant effect on bone cells colonizing the implant, which in turn can influence the new bone formation and bone remodeling processes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1285-1299, 2017.