Developing Bioactive Dental Resins for Restorative Dentistry.

Despite its reputation as the most widely used restorative dental material currently, resin-based materials have acknowledged shortcomings. As most systematic survival studies of resin composites and dental adhesives indicate, secondary caries is the foremost reason for resin-based restoration failure and life span reduction. In subjects with high caries risk, the microbial community dominated by acidogenic and acid-tolerant bacteria triggers acid-induced deterioration of the bonding interface and/or bulk material and mineral loss around the restorations. In addition, resin-based materials undergo biodegradation in the oral cavity. As a result, the past decades have seen exponential growth in developing restorative dental materials for antimicrobial applications addressing secondary caries prevention and progression. Currently, the main challenge of bioactive resin development is the identification of efficient and safe anticaries agents that are detrimental free to final material properties and show satisfactory long-term performance and favorable clinical translation. This review centers on the continuous efforts to formulate novel bioactive resins employing 1 or multiple agents to enhance the antibiofilm efficacy or achieve multiple functionalities, such as remineralization and antimicrobial activity antidegradation. We present a comprehensive synthesis of the constraints and challenges encountered in the formulation process, the clinical performance-related prerequisites, the materials' intended applicability, and the current advancements in clinical implementation. Moreover, we identify crucial vulnerabilities that arise during the development of dental materials, including particle aggregation, alterations in color, susceptibility to hydrolysis, and loss of physicomechanical core properties of the targeted materials.

Bifunctional Composites for Biofilms Modulation on Cervical Restorations.

Cervical composites treating root carious and noncarious cervical lesions usually extend subgingivally. The subgingival margins of composites present poor plaque control, enhanced biofilm accumulation, and cause gingival irritation. A potential material to restore such lesions should combine agents that interfere with bacterial biofilm development and respond to acidic conditions. Here, we explore the use of new bioresponsive bifunctional dental composites against mature microcosm biofilms derived from subgingival plaque samples. The designed formulations contain 2 bioactive agents: dimethylaminohexadecyl methacrylate (DMAHDM) at 3 to 5 wt.% and 20 wt.% nanosized amorphous calcium phosphate (NACP) in a base resin. Composites with no DMAHDM and NACP were used as controls. The newly formulated 5% DMAHDM-20% NACP composite was analyzed by micro-Raman spectroscopy and transmission electron microscopy. The wettability and surface-free energy were also assessed. The inhibitory effect on the in vitro biofilm growth and the 16S rRNA gene sequencing of survival bacterial colonies derived from the composites were analyzed. Whole-biofilm metabolic activity, polysaccharide production, and live/dead images of the biofilm grown over the composites complement the microbiological assays. Overall, the designed formulations had higher contact angles with water and lower surface-free energy compared to the commercial control. The DMAHDM-NACP composites significantly inhibited the growth of total microorganisms, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum by 3 to 5-log (P < 0.001). For the colony isolates from control composites, the composition was typically dominated by the genera Veillonella, Fusobacterium, Streptococcus, Eikenella, and Leptotrichia, while Fusobacterium and Veillonella dominated the 5% DMAHDM-20% NACP composites. The DMAHDM-NACP composites contributed to over 80% of reduction in metabolic and polysaccharide activity. The suppression effect on plaque biofilms suggested that DMAHDM-NACP composites might be used as a bioactive material for cervical restorations. These results may propose an exciting path to prevent biofilm growth and improve dental composite restorations' life span.

Developing a New Generation of Antimicrobial and Bioactive Dental Resins.

Dental caries is prevalent, and secondary caries causes restoration failures. This article reviews recent studies on developing a new generation of bioactive resins with anticaries properties. Extensive effects were made to develop new antimicrobial composites, bonding agents, and other resins containing quaternary ammonium methacrylates to suppress plaque buildup and bacterial acid production. The effects of alkyl chain length and charge density and the antimicrobial mechanisms for chlorhexidine, nano-silver, quaternary ammonium methacrylates, and protein-repellent agents were discussed. Synergistic effects of contact-killing and protein-repellent properties were shown to yield the greatest biofilm-inhibition effects. The combination of antimicrobial, protein-repellent, and calcium phosphate nanoparticle remineralization was suggested to provide maximal anticaries effects. In addition, for use orally, cytotoxicity and biocompatibility were important considerations for the new bioactive materials. Furthermore, rather than kill all bacteria, it would be more desirable to modulate the oral biofilm compositions via bioactive resins to suppress cariogenic/pathogenic species and promote benign species. For widespread clinical use of the new antimicrobial and therapeutic materials, whether they would induce bacterial drug resistance needs to be determined, which requires further study. Nonetheless, the new generation of bioactive anticaries resins with therapeutic and biofilm acid-inhibiting properties has the potential to substantially benefit oral health.

Effect of antibacterial dental adhesive on multispecies biofilms formation.

Antibacterial adhesives have favorable prospects to inhibit biofilms and secondary caries. The objectives of this study were to investigate the antibacterial effect of dental adhesives containing dimethylaminododecyl methacrylate (DMADDM) on different bacteria in controlled multispecies biofilms and its regulating effect on development of biofilm for the first time. Antibacterial material was synthesized, and Streptococcus mutans, Streptococcus gordonii, and Streptococcus sanguinis were chosen to form multispecies biofilms. Lactic acid assay and pH measurement were conducted to study the acid production of controlled multispecies biofilms. Anthrone method and exopolysaccharide (EPS):bacteria volume ratio measured by confocal laser scanning microscopy were performed to determine the EPS production of biofilms. The colony-forming unit counts, scanning electron microscope imaging, and dead:live volume ratio decided by confocal laser scanning microscopy were used to study the biomass change of controlled multispecies biofilms. The TaqMan real-time polymerase chain reaction and fluorescent in situ hybridization imaging were used to study the proportion change in multispecies biofilms of different groups. The results showed that DMADDM-containing adhesive groups slowed the pH drop and decreased the lactic acid production noticeably, especially lactic acid production in the 5% DMADDM group, which decreased 10- to 30-fold compared with control group (P < 0.05). EPS was reduced significantly in 5% DMADDM group (P < 0.05). The DMADDM groups reduced the colony-forming unit counts significantly (P < 0.05) and had higher dead:live volume ratio in biofilms compared with control group (P < 0.05). The proportion of S. mutans decreased steadily in DMADDM-containing groups and continually increased in control group, and the biofilm had a more healthy development tendency after the regulation of DMADDM. In conclusion, the adhesives containing DMADDM had remarkable antimicrobial properties to serve as "bioactive" adhesive materials and revealed its potential value for antibiofilm and anticaries clinical applications.

Stem Cells and Calcium Phosphate Cement Scaffolds for Bone Regeneration.

Calcium phosphate cements (CPCs) have excellent biocompatibility and osteoconductivity for dental, craniofacial, and orthopedic applications. This article reviews recent developments in stem cell delivery via CPC for bone regeneration. This includes: (1) biofunctionalization of the CPC scaffold, (2) co-culturing of osteoblasts/endothelial cells and prevascularization of CPC, (3) seeding of CPC with different stem cell species, (4) human umbilical cord mesenchymal stem cell (hUCMSC) and bone marrow MSC (hBMSC) seeding on CPC for bone regeneration, and (5) human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) seeding with CPC for bone regeneration. Cells exhibited good attachment/proliferation in CPC scaffolds. Stem-cell-CPC constructs generated more new bone and blood vessels in vivo than did the CPC control without cells. hUCMSCs, hESC-MSCs, and hiPSC-MSCs in CPC generated new bone and blood vessels similar to those of hBMSCs; hence, they were viable cell sources for bone engineering. CPC with hESC-MSCs and hiPSC-MSCs generated new bone two- to three-fold that of the CPC control. Therefore, this article demonstrates that: (1) CPC scaffolds are suitable for delivering cells; (2) hUCMSCs, hESCs, and hiPSCs are promising alternatives to hBMSCs, which require invasive procedures to harvest with limited cell quantity; and (3) stem-cell-CPC constructs are highly promising for bone regeneration in dental, craniofacial, and orthopedic applications.

Effects of quaternary ammonium chain length on antibacterial bonding agents.

The objectives of this study were to synthesize new quaternary ammonium methacrylates (QAMs) with systematically varied alkyl chain lengths (CL) and to investigate, for the first time, the CL effects on antibacterial efficacy, cytotoxicity, and dentin bond strength of bonding agents. QAMs were synthesized with CL of 3 to 18 and incorporated into Scotchbond Multi-Purpose (SBMP) bonding agent. The cured resins were inoculated with Streptococcus mutans. Bacterial early attachment was investigated at 4 hrs. Biofilm colony-forming units (CFU) were measured after 2 days. With CL increasing from 3 to 16, the minimum inhibitory concentration and minimum bactericidal concentration were decreased by 5 orders of magnitude. Incorporating QAMs into SBMP reduced bacterial early attachment, with the least colonization at CL = 16. Biofilm CFU for CL = 16 was 4 log lower than SBMP control (p < .05). All groups had similar dentin bond strengths (p > .1). The new antibacterial materials had fibroblast/odontoblast viability similar to that of commercial controls. In conclusion, increasing the chain length of new QAMs in bonding agents greatly increased the antibacterial efficacy. A reduction in Streptococcus mutans biofilm CFU by 4 log could be achieved, without compromising bond strength and cytotoxicity. New QAM-containing bonding agents are promising for a wide range of restorations to inhibit biofilms.

Remineralization of demineralized enamel via calcium phosphate nanocomposite.

Secondary caries remains the main problem limiting the longevity of composite restorations. The objective of this study was to investigate the remineralization of demineralized human enamel in vitro via a nanocomposite containing nanoparticles of amorphous calcium phosphate (NACP). NACP were synthesized by a spray-drying technique and incorporated into a dental resin. First, caries-like subsurface enamel lesions were created via an acidic solution. Then, NACP nanocomposite or a commercial fluoride-releasing control composite was placed on the demineralized enamel, along with control enamel without a composite. These specimens were then treated with a cyclic demineralization/remineralization regimen for 30 days. Quantitative microradiography showed typical enamel subsurface demineralization before cyclic demineralization/remineralization treatment, and significant remineralization in enamel under the NACP nanocomposite after the demineralization/remineralization treatment. The NACP nanocomposite had the highest enamel remineralization (mean ± SD; n = 6) of 21.8 ± 3.7%, significantly higher than the 5.7 ± 6.9% for fluoride-releasing composite (p < 0.05). The enamel group without composite had further demineralization of -26.1 ± 16.2%. In conclusion, a novel NACP nanocomposite was effective in remineralizing enamel lesions in vitro. Its enamel remineralization was 4-fold that of a fluoride-releasing composite control. Combined with the good mechanical and acid-neutralization properties reported earlier, the new NACP nanocomposite is promising for remineralization of demineralized tooth structures.

Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering.

Human embryonic stem cells (hESC) are promising for use in regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there have been no reports on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESC were cultured to form embryoid bodies (EB), and the MSC were then migrated out of the EB. hESCd-MSC had surface markers characteristic of MSC, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSC were encapsulated in alginate at a density of 1millioncellsml(-1), with an average microbead size of 207µm. CPC contained mannitol porogen to create a porosity of 64% and 218-µm macropores, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSC encapsulated in microbeads in CPC had good viability from 1 to 21days. ALP gene expression at 21days was 25-fold that at 1day. Osteocalcin (OC) at 21days was two orders of magnitude of that at 1day. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21days was fivefold that at 1day. Mineral synthesis by the encapsulated hESCd-MSC at 21days was sevenfold that at 1day. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSC were encapsulated in alginate microbeads in macroporous CPC, showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications.

Anti-biofilm dentin primer with quaternary ammonium and silver nanoparticles.

Antibacterial bonding agents could combat recurrent caries at the tooth-composite margins. The objectives of this study were to develop novel antibacterial dentin primers containing quaternary ammonium dimethacrylate (QADM) and nanoparticles of silver (NAg), and to investigate the effects on dentin bond strength and dental plaque microcosm biofilms for the first time. Scotchbond Multi-Purpose ("SBMP") bonding agent was used. QADM and NAg were incorporated into SBMP primer, yielding 4 primers: SBMP primer (control), control + 10% QADM (mass), control + 0.05% NAg, and control + 10% QADM + 0.05% NAg. Human saliva was collected to grow microcosm biofilms. The NAg particle size (mean ± SD; n = 100) was 2.7 ± 0.6 nm. Dentin shear bond strengths (n = 10) with human third molars were approximately 30 MPa for all groups (p > 0.1). QADM-NAg-containing primer increased the bacteria inhibition zone by 9-fold, compared with control primer (p < 0.05). QADM-NAg-containing primer reduced lactic acid production and colony-forming units of total micro-organisms, total streptococci, and mutans streptococci by an order of magnitude. In conclusion, novel QADM-NAg-containing primers were strongly antibacterial without compromising dentin bond strength, and hence are promising to inhibit biofilms and secondary caries. The processing method of incorporating QADM and NAg together into the same primer produced the strongest antibacterial effect, which could have a wide applicability to other bonding systems.

Antibacterial nanocomposite with calcium phosphate and quaternary ammonium.

Secondary caries is a frequent reason for restoration failure, resulting from acidogenic bacteria and their biofilms. The objectives of this study were to: (1) develop a novel nanocomposite containing nanoparticles of amorphous calcium phosphate (NACP) and quaternary ammonium dimethacrylate (QADM); and (2) investigate its mechanical and antibacterial durability. A spray-drying technique yielded NACP with particle size of 116 nm. The nanocomposite contained NACP and reinforcement glass fillers, with QADM in the resin. Two commercial composites were tested as controls. Composites were inoculated with Streptococcus mutans. After 180-day water-aging, NACP+QADM nanocomposite had flexural strength and elastic modulus matching those of commercial controls (p > 0.1). NACP+QADM nanocomposite reduced the biofilm colony-forming units (CFU) by 3-fold, compared with commercial composites (p < 0.05). Metabolic activity and lactic acid production of biofilms on NACP+QADM were much less than those on commercial composites (p < 0.05). The antibacterial properties of NACP+QADM were maintained after water-aging for 30, 90, and 180 d (p > 0.05). In conclusion, the novel NACP-QADM nanocomposite greatly decreased biofilm metabolic activity, CFU, and lactic acid, while matching the load-bearing capability of commercial composites without antibacterial properties. The NACP-QADM nanocomposite with strong and durable antibacterial properties, together with its previously reported Ca-PO(4) release capability, may render it useful for caries-inhibiting restorations.

Stem cell-calcium phosphate constructs for bone engineering.

While human bone-marrow-derived mesenchymal stem cells (hBMSCs) have been investigated, human umbilical cord mesenchymal stem cells (hUCMSCs) are a relatively new cell source. Little has been reported on hUCMSC encapsulation in scaffolds for bone tissue engineering. The objective of this study was to encapsulate hBMSCs and hUCMSCs in calcium phosphate cement (CPC) scaffolds for dental, craniofacial, and orthopedic applications. Stem-cell-encapsulating CPC construct with chitosan and fiber reinforcement reached the strength of cancellous bone, which was much stronger than previous injectable carriers for cell delivery. hUCMSCs and hBMSCs inside the constructs showed excellent viability and osteo-differentiation. The encapsulated hUCMSCs synthesized nearly three-fold more bone minerals than the hBMSCs in vitro. Hence, stem-cell-encapsulating CPC-chitosan-fiber construct may be promising for dental and orthopedic applications. This study indicated that the hUCMSCs were a potent alternative to the gold-standard hBMSCs, which may have a broad impact on regenerative medicine and dental tissue engineering.

Strong nanocomposites with Ca, PO(4), and F release for caries inhibition.

This article reviews recent studies on: (1) the synthesis of novel calcium phosphate and calcium fluoride nanoparticles and their incorporation into dental resins to develop nanocomposites; (2) the effects of key microstructural parameters on Ca, PO(4), and F ion release from nanocomposites, including the effects of nanofiller volume fraction, particle size, and silanization; and (3) mechanical properties of nanocomposites, including water-aging effects, flexural strength, fracture toughness, and three-body wear. This article demonstrates that a major advantage of using the new nanoparticles is that high levels of Ca, PO(4), and F release can be achieved at low filler levels in the resin, because of the high surface areas of the nanoparticles. This leaves room in the resin for substantial reinforcement fillers. The combination of releasing nanofillers with stable and strong reinforcing fillers is promising to yield a nanocomposite with both stress-bearing and caries-inhibiting capabilities, a combination not yet available in current materials.

Effects of calcium phosphate nanoparticles on Ca-PO4 composite.

Nano-particles of dicalcium phosphate anhydrous (DCPA) were synthesized for the first time. The objectives of this study were to incorporate DCPA nano-particles into resin for Ca-PO(4) release to combat dental caries, and to investigate the filler level effects. Nano-DCPA and nano-silica-fused silicon nitride whiskers at a 1:1 ratio were used at filler mass fractions of 0-75%. The flexural strengths in MPa (mean +/- SD; n = 6) of DCPA-whisker composites ranged from (106 +/- 39) at 0% fillers to (114 +/- 23) at 75% fillers, similar to (112 +/- 22) of a non-releasing composite (TPH) (p > 0.1). The composite with 75% fillers in a NaCl solution (133 mmol/L, pH = 7.4, 37 degrees C) yielded a Ca concentration of (0.65 +/- 0.02) mmol/L and PO(4) of (2.29 +/- 0.07) mmol/L. Relationships were established between ion-release and DCPA volume fraction V(DCPA): Ca = 4.46 V(DCPA)(1.6,) and = 66.9 V(DCPA)(2.6). Nano-DCPA-whisker PO(4) composites had high strength and released high levels of Ca-PO(4) requisite for remineralization. These new nano-composites could provide the needed combination of stress-bearing and caries-inhibiting capabilities.

Nano DCPA-whisker composites with high strength and Ca and PO(4) release.

The main challenges facing composite restorations are secondary caries and bulk fracture. The objective of this study was to develop nano DCPA (dicalcium phosphate anhydrous)-whisker composites with high strength and Ca and PO(4) ion release to combat caries. Flexural strength for the nano DCPA-whisker composites at a nano DCPA:whisker mass ratio of 1:2 ranged from (148 +/- 9) MPa to (167 +/- 23) MPa, significantly higher than the (103 +/- 32) MPa of an inlay/onlay commercial control composite without Ca-PO(4) release. The nano DCPA-whisker composite released PO(4) to a concentration of (1.95 +/- 0.13) mmol/L and Ca of (0.68 +/- 0.05) mmol/L. Compared with previous conventional Ca- and PO(4)-releasing composites, the nano DCPA-whisker composites had strengths two-fold higher, and released comparable or higher levels of Ca and PO(4). In conclusion, combining nano-DCPA with whiskers yielded novel composites that released high levels of Ca and PO(4) requisite for remineralization. These high-strength composites may provide a unique combination of stress-bearing and caries-inhibiting capabilities.

Distribution of glutamine synthetase and glial fibrillary acidic protein and correlation of glutamine synthetase with glutamate decarboxylase in different regions of the rat central nervous system.

The concentration of soluble glial fibrillary acidic (GFA) protein and the specific activity of glutamine synthetase (GS) were estimated in 11 central nervous system (CNS) regions of the 90-day-old rat. Marked differences were observed in the regional distribution of these astrocyte marker proteins. The striatum and spinal cord contained the lowest concentration (per g wet weight) of GFA protein and GS activity, respectively, while the olfactory bulbs had the highest level of both astrocytic proteins. Differences between the lowest and the highest values were 3-fold for GS and 4-fold for GFA protein. More significant was the marked variation in the ratio of GS to GFA protein in different CNS regions; the highest and lowest values were in the striatum and the spinal cord respectively, and the difference between the highest and the lowest value was about 5-fold. The spinal cord contained low GS and high GFA protein; on the other hand, the colliculi had high GS and relatively low GFA protein. Immunochemical detection of GS and GFA proteins in whole homogenates of different regions showed that the variation of the specific activities of GS and the concentration of soluble GFA protein were due to the differences in their absolute protein concentrations. In different regions of the brain the activity of GS was significantly correlated with that of glutamate decarboxylase, but not with that of choline acetyltransferase. These observations provide further evidence for differing biochemical properties of astrocytes from various CNS regions and for the involvement of GS in processes associated with amino acid neurotransmission.

Developmental changes in the amount of glial fibrillary acidic protein in three regions of the rat brain.

Glial fibrillary acidic (GFA) protein, extractable in 50 mM phosphate buffer, pH 8, was measured in the olfactory bulbs, forebrain and cerebellum of the rat during development using a double antibody radioimmunoassay. Each brain region showed a different pattern of development for GFA protein. At birth GFA protein per mg protein was highest in olfactory bulbs followed by forebrain and cerebellum, and these amounted to 15, 10 and 8% of the adult values, respectively. The relative increase in GFA protein was more marked during the first 2 postnatal weeks than in the following 7 weeks after birth. When values were expressed per brain region, the developmental increase in the amount of GFA protein from birth to adulthood was about 100-fold in olfactory bulbs, 85-fold in forebrain and 485-fold in cerebellum. The patterns of developmental increases in GFA protein and in glutamine synthetase activity, another protein enriched in astrocytes, were similar in the forebrain and olfactory bulbs, but differed markedly in the cerebellum. The major increase in content of the GFA protein during development was found to correspond with the maturation of astrocytes rather than with their proliferation; however, a small but significant amount of GFA protein acquired at an early age may be related to increase in astroglial cell numbers in the cerebellum.

Effect of dexamethasone on glutamine synthetase and glial fibrillary acidic protein in normal and transformed astrocytes.

The effect of dexamethasone on astrocyte differentiation was investigated in vitro, using cultures of normal and transformed astrocytes. The astrocyte-enriched proteins glutamine synthetase (GS) and glial fibrillary acidic (GFA) protein were used as markers of astrocyte differentiation. Ethanol, the vehicle for dexamethasone, decreases GS activity and increases GFA protein concentration in cultures of the established cell line U251MG and in the majority of cultures of transformed astrocytes derived from varying grades of astrocytoma. Ethanol has no effect on primary cultures of astrocytes derived from immature rat brain. Dexamethasone increases GS activity and decreases GFA protein concentration in cultures of U251MG and a grade IV astrocytoma-derived culture, in comparison with ethanol control. Our results show differential effects of two factors on cell-specific proteins in normal and transformed astrocytes.

Changes in the metabolism of glial fibrillary acid protein (GFAP) during chronic relapsing experimental allergic encephalomyelitis in the strain 13 guinea-pig.

Spinal cords were removed from strain 13 guinea pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CREAE). Levels of glial fibrillary acidic protein (GFAP) in cord extracts were determined by radioimmunoassay and protein synthesis was monitored by incubating tissue prisms with [(35)S]methionine. Analysis of cytoskeletal enriched preparations from these incubations by SDS-polyacrylamide electrophoresis and fluorography permitted evaluation of the metabolism of discrete polypeptides; the identity of a labelled band at 51,000 daltons as GFAP was confirmed by immunoprecipitation with a specific antibody. Total protein synthesis increased 4-fold during the acute phase of CREAE but fell to control values with clinical recovery, while over the same period GFAP synthesis increased by 6-7-fold and remained elevated in the post-acute phase at about 200% of control values. During this time there was no increase in the GFAP content of the cord indicating an increased turnover of this protein rather than net synthesis. In later stages of CREAE however, GFAP levels were raised, correlating with a further increase in the incorporation of precursor into GFAP, this being most pronounced in animals in clinical relapse.