Mass loss was significantly higher from low melanin necromass, corresponding with greater 13C and 15N soil inputs. A taxonomically and functionally diverse array of bacteria and fungi was enriched in 13C and/or 15N at all sampling points, with enrichment being consistently higher on low melanin necromass and earlier in decomposition. Similar patterns of preferential C and N enrichment of many bacterial and fungal genera early in decomposition suggest that both microbial groups co-contribute to the rapid assimilation of resource-rich soil organic matter inputs. While overall richness of taxa enriched in C was higher than in N for both bacteria and fungi, there was a significant positive relationship between C and N in co-enriched taxa. Collectively, our results demonstrate that melanization acts as a key ecological trait mediating not only fungal necromass decomposition rate but also necromass C and N release and that both elements are rapidly co-utilized by diverse bacterial and fungal decomposers in natural settings.Microbial necromass contributes significantly to both soil carbon (C) persistence and ecosystem nitrogen (N) availability, but quantitative estimates of C and N movement from necromass into soils and decomposer communities are lacking. Additionally, while melanin is known to slow fungal necromass decomposition, how it influences microbial C and N acquisition as well as elemental release into soils remains unclear. Here, we tracked decomposition of isotopically labeled low and high melanin fungal necromass and measured Recent studies indicate that microbial dead cells, particularly those of fungi, play an important role in long-term carbon persistence in soils. Despite this growing recognition, how the resources within dead fungal cells  move into decomposer communities and soils are poorly quantified, particularly in studies based in natural environments. In this study, we found that the contribution of fungal necromass to soil carbon and nitrogen availability was slowed by the amount of melanin present in fungal cell walls. Further, despite the overall rapid acquisition of carbon and nitrogen from necromass by a diverse range of both bacteria and fungi, melanization also slowed microbial uptake of both elements. Collectively, our results indicate that melanization acts as a key ecological trait mediating not only fungal necromass decomposition rate, but also necromass carbon and nitrogen release into soil as well as microbial resource acquisition. Understanding of how carbon (C) enters and leaves soil, the largest active terrestrial C pool , represek) and the asymptotic (A) mass loss are both strongly influenced by the initial biochemical composition of fungal necromass  has been instrumental in determining which micro-organisms actually assimilate fungal necromass C, with a broad range of bacteria and fungi becoming significantly enriched when grown on boratory , 26 and boratory . Based oboratory  concludeboratory , howeverboratory . This suWorking in a coniferous forest in Minnesota, USA and using low and high melanin fungal necromass types incubated within nylon mesh litter bags, we quantified the accrual of C and N derived from fungal necromass in surrounding soils as well as how much of each element was assimilated by the microbial communities growing on the decaying necromass. We assessed soil C and N inputs and microbial community C and N utilization at both earlier (7 and 14 d) and later (35 and 77 d) stages of decomposition. To estimate taxon-specific C and N enrichment for the bacterial and fungal communities growing on the decaying necromass, we used quantitative stable-isotope probing qSIP, , 30). We. We30]).Meliniomyces bicolor, a mycorrhizal fungus associated with diverse host plants .t plants  and rece bicolor , was groeference ), replicPinus strobus) forest stand . The study site is characterized as a secondary forest, with P. strobus trees approximately 20–40 yr old and an understory composed of sparse shrubs in the genera Prunus and Amelanchier. Climatic conditions are considered continental, with a mean annual temperature of 8°C and a mean annual precipitation of 810 mm. The soils are classified as frigid Udipsamments, with a poorly developed 3–5-cm O-horizon. They are very sandy and contain 4.07% ± 1.72% C and 0.37% ± 0.14% N. At the study site, two 5 × 5 m plots were established ca. 50 m apart in early August 2018. Necromass litter bags were incubated at the litter/soil interface within each plot, each bag was separated from all other bags by 0.5 m. The bags were retrieved after 7, 14, 35 and 77 d. Additionally, bags containing each combination of labeled or unlabeled low or high melanin necromass were taken to the field, placed on the soil, but then immediately harvested to account for any mass loss associated with transport or isotopic transfer into soil during the burial process (none was detected). In total, 54 bags were incubated . In the laboratory, roots were removed, and soils were dried at 60°C for 48 h. Approximately 25-mg aliquots of ground soil were weighed into aluminum foil tins and analyzed for C and N on a Carlo Erba NC2100 elemental analyzer interfaced to a Thermo Electron gas isotope-ratio mass spectrometer at the Colorado Plateau Stable Isotope Laboratory located at Northern Arizona University.Along with the necromass litter bags, soils located directly underneath each litter bag were collected at each timepoint (including day 0). Soils were collected to a depth of 5 cm using sterile 14 × 9.5 × 8 cm plastic containers  for 3 h at room temperature with intermittent agitation. The mass remaining after the strong acid extraction (hereafter referred to as the non-hydrolyzable fraction) is thought to be composed of cell wall melanins and proteins complexed within them  to have sufficient DNA for SIP density separation. While this pooling prevents any tests of block effects, fungal necromass microbial communities have been consistently observed to be a distinct subset of the larger soil microbial community (−1) with a final average density of 1.730 g/mL. The solution was spun in ultracentrifuge tubes  in an Optima L-90K ultracentrifuge  using a VTi65.2 rotor at 44,000 rpm  at 20°C for 109 h, with maximum acceleration and braking of the rotor to maintain the integrity of the density separations. The gradient was then separated initially into 18–33 fractions using a syringe pump delivering light mineral oil at 0.25 mL/min through a 25-G needle inserted through the top of the tube to displace the gradient solution out through a side port needle inserted through the bottom of the tube. These fractions were subsequently composited into nine fractions based on density, with the lowest composite fraction ranging from 1.665 to 1.1685 g/mL and the highest composite fraction ranging 1.765–1.784 g/mL. The density of each fraction was measured using an AR200 digital refractometer  fitted with a prism covering to facilitate measurement from 5 µL volumes, as previously described . Forward and reverse reads were then paired using VSEARCH  for each OTU. The qSIP method relies on tracking the shift in density, calculated by coverage in the different fractions, between unlabeled and labeled samples. An OTU is considered enriched if the density in the heavy isotope samples is significantly higher than in the light isotope samples. This change in density is used to quantify the percent enrichment  from 0% enriched  to 100% enrichment . We quantified AFE values of bacterial and fungal OTUs following a modified version of the procedure described by Hungate et al.  on bacterial and fungal C and N enrichment were tested via multiple linear regressions on each combination of domain (bacteria or fungi) and element (C and N). To further evaluate the AFE values for co-enriched OTUs, we regressed AFE N on AFE C for bacteria and fungi separately, using a type II model regression for accounting for the different variances associated with AFE C and AFE N estimates.To assess the effects of melanin content, time, and their interaction on fungal necromass mass loss, we ran a two-way fully factorial analysis of variance (ANOVA). Ahead of the ANOVA, the percent mass loss values were natural log transformed to reduce heterogeneity. To assess the effects of melanin content, time {here the first two and last two sampling timepoints were combined into “earlier” and “later” groupings given their similarities in mass loss, i.e., day 7 and day 14 samples occurred during the rapid phase of decay, while day 35 and day 77 samples occurred during the slower phase of decay  of the high melanin necromass remained, while only 18 ± 4% of the low melanin necromass remained. There were no significant differences in mass remaining between the unlabeled and 13C- or 15N-labeled necromass treatments .High melanin necromass had greater mass remaining than the low melanin necromass at all timepoints during the 77-day incubation . The amo13C from the fungal necromass was detected in soil cores taken directly below the litter bags at all four sampling times for both necromass types  but did differ in a marginally significant way by necromass type , with greater but also more variable amounts of 13C detected in soils under low melanin necromass. There was also a trend toward greater amounts of 13C being detected from high melanin necromass at the later than earlier sampling times, but the interaction between necromass type and time was not significant . 15N from the fungal necromass was also detected in soil cores taken directly below the litter bags at all four sampling times for both necromass types  but did differ marginally significantly by necromass type , with greater amounts of 15N detected in soil under low melanin necromass. The 15N detection patterns were consistent by time and necromass type . The aforementioned trends in 13C and 15N accrual in soil were functionally equivalent when expressed as a percent initial necromass C and N . There was also a significant interaction between time and necromass type on C-enriched bacterial community composition , likely due to greater temporal variation on high than low melanin necromass. Additionally, dispersion in the composition of the C-enriched bacterial communities was significantly higher at later than earlier timepoints regardless of necromass type . The N-enriched bacterial communities followed similar trends, with significant differences in composition  at earlier than later timepoints but no significant differences by necromass type . For the fungi, the C-enriched communities on decomposing M. bicolor necromass differed significantly in composition between the earlier and later timepoints . Furthermore, dispersion in composition of the C-enriched fungal communities was significantly higher on high than low melanin necromass . Finally, for the N-enriched fungal communities, there were only significant differences in composition or dispersion between earlier and later timepoints  of enriched fungal OTUs were enriched with 13C. On the 15N-labeled necromass, 554 (65%) of enriched bacterial taxa were enriched with 15N, while 149 (56%) of enriched fungal OTUs were enriched with 15N. Although more total bacterial and fungal OTUs were enriched in C than N, many bacterial and fungal OTUs were simultaneously enriched in both elements  and 268 (89%) were considered enriched in C and/or N. Specifically, 778 (92%) of these enriched bacterial OTUs were enriched with elements . For tho13C and 15N AFE) on low melanin necromass relative to high melanin necromass , mean bacterial 15N AFE was significantly lower at the later timepoints , but 15N AFE was not significantly different between necromass types  and N   . We reitecromass .Mucilaginibacter, Burkholderia, Sphingomonas, Luteibacter, and Pseudomonas  were enriched in both C and N on both necromass types at both time groupings. There were a number of fungal genera that had significantly greater AFE C and N on low melanin necromass but none with significantly greater AFE C and N on high melanin necromass  had significantly higher enrichment at later timepoints. Like bacteria, however, there was no significant correlation between fungal genera relative abundance (based on sequence read counts) and AFE C or AFE N , multiple resources (C and N), multiple incubation times (earlier and later stages of decomposition), and both soil inputs as well as bacterial and fungal utilization from fungal necromass under field conditions. Our motivations were to: (i) determine the extent to which fungal necromass melanization affected amounts of C and N release into soil at both earlier and later stages of decomposition, (ii) determine the extent to which fungal necromass melanization affected C and N acquisition by decomposer microbial communities at both earlier and later stages of decomposition, and (iii) determine the extent to which fungal necromass serves as a dual source of nutrition and energy for decomposer microbial communities. Overall, we found that melanization did have significant inhibitory effects on C and N inputs into soil as well as microbial acquisition of both resources at earlier and later stages of decomposition. Furthermore, we demonstrated that decaying fungal necromass is frequently used by bacterial and fungal decomposers as a dual source of nutrition and energy. Below we discuss each of these findings in greater detail, particularly in light of our hypotheses.We first hypothesized that the decomposition of low melanin fungal necromass would result in greater overall C and N inputs into soil due to a faster decay rate. This was broadly supported by our data, which showed more C and N accrual in soils under the more rapidly decomposing low melanin necromass. We attribute the differences between necromass types to multiple aspects of their chemical composition. Although not specifically measured here, previous studies have shown that melanin itself is highly resistant to decay , 17, thu13C and 15N enriched bacterial OTUs we identified are classified as copiotrophs and were likely targeting labile C and N sources that were comparatively more available in low melanin necromass during the initial stage of decomposition . In contrast to a previous SIP-based study proposing that fungi play a limited role in fungal necromass decomposition  early in necromass decomposition. These results align with previous research showing these genera can be abundant during fungal necromass decomposition and may be involved in parasitizing fungi participating in the decomposition processes (Similar to previous studies, we found that there were significant shifts in bacterial and fungal community structure over time , 20, 21.ver time . Despitever time , 57, werrocesses , 11. Ourrocesses -60.Our third hypothesis surmised that due to relatively high labilty of the non-structural and structural compounds present in fungal necromass, it would frequently be the target of simultaneous C and N acquisition by both bacteria and fungi. We found general support for this hypothesis, with at least 50% of the enriched bacterial and fungal OTUs being co-enriched in C and N. Moreover, we documented positive overall relationships between the amounts of C and N enrichment for both bacteria and fungi, suggesting their uptake mechanisms are coupled. While we are confident that coupled C and N uptake is likely a common process in microbial utilization of fungal necromass resources, the high variation across OTUs (as shown in Regarding the necromass used in this study, both types were likely richer in initial C and N than naturally occurring fungal necromass due to the resorptive nature of resources within fungal mycelia during senescence . AdditioUsing isotopic labeling, we have demonstrated that C and N release from decaying fungal necromass into soil occurs rapidly, and both elements are frequently assimilated by a diverse range of bacterial and fungal decomposers in natural settings. Furthermore, we show that initial biochemical composition of fungal necromass is a key trait that not only impacts its rates of overall mass loss, but also rates of C and N release as well as resource use by necromass-associated microbial communities. Finally, our results indicate that fungal necromass is consistently targeted by many microbial decomposers as a dual source of C and N, suggesting that future studies of fungal necromass effects on soil biochemical cycling should focus on both elements whenever possible.


Dietary AGEs (dAGEs) have the capacity to bind to the Receptor for Advanced Glycation End Products (RAGE) which is part of the endogenous threat detection network. There are persuasive epidemiological and biochemical arguments that correlate the rise in food allergy in several Western countries with increases in dAGEs. The increased consumption of dAGEs is enmeshed in current theories of the aetiology of food allergy which will be discussed.Sugars can bind non-enzymatically to proteins, nucleic acids or lipids and form compounds called Advanced Glycation End Products (AGEs). Although AGEs can form  Dietary AGEs (dAGEs) have the capacity to bind to the Receptor for Advanced Glycation End Products (RAGE). RAGE is a part of the endogenous threat detection network as it can be activated by amyloid and Danger Associated Molecular Patterns (DAMPs) including High Molecular Group Box 1 (HMGB1) and S100 proteins  to initiate a danger pattern  to a protein, nucleic acid or lipid molecule without enzymatic regulation , 5. The  pattern . Similar pattern , which cUnless otherwise specified, this paper will refer to full-length RAGE, which comprises intracellular, transmembrane and extracellular domains. In addition to this archetypal RAGE there are 19 reportedly splice variants . A splicOther RAGE worth noting are oligosaccharyltransferase 48 (AGER1), 80 K-H phosphoprotein (AGER2), galectin-3 (AGER3), and type I and II scavenger receptors . AGER1 avia four main mechanisms   oxidase) , 30. RAGoxidase) . The infoxidase) , 7, 30 ireceptor , 7, 30, roptosis . TLR2/4 roptosis   including High Molecular Group Box 1 (HMGB1) and S100 proteins . AGE ligIn vitro studies show that AGE-stimulated human dendritic cells lead to greater Th2 responses and increased expression of RAGE  have at least 6 RAGE receptors  compared to younger children or adults . The samtivation . Fructostivation . Fructosrogenase . The latrogenase . Xylose rogenase .Modern common table salt—sodium chloride—is found in abundance in processed foods . This isThe Western diet is commonly associated with UPFs, foods that are dehydrated and having long shelf lives, and food preparation methods like microwaving or frying, all of which increase the formation of AGEs , 87, 88.Heating and the formation of AGEs within food does not always results increased allergenicity. Heating and the formation of AGEs in food does not always result in increased allergenicity. High levels of glycation of bovine lactoglobulin, a common cow's milk protein allergen, results in alteration of the molecular allergen binding sites and less IgE binding .via their alteration of microbiome composition and function  and atopic dermatitis OR 1.7 in US women . This fiDespite many researchers' efforts, the AGEs content of many foods is unknown and calculation of the AGEs scores of composite foods is difficult to standardize. Future studies may benefit from using an AGEs food frequency questionnaire, validated against reliable biomarkers such as serum levels of AGEs. This information will provide us with validated measures of dietary intake .Several hypotheses have been proposed that link allergic disease risk with the microbiome, or more specifically a depletion thereof, such as “missing microbes”, “microbiome depletion”, “microbiome diversity”, “microflora”, “overarching microbiome”, and “old friends” hypotheses . The WesEscherichia coli secrete mAGEs by the energy-dependent efflux pump system and uses this as a local toxin to reduce growth of neighboring competitive bacteria, thus having local inflammatory effects, as well as influencing the microbiome composition  in the jejunum, ileum and colon, and subsequent increased gut permeability . This hafunction , 142. Thfunction .Lactobacillus reuteri, a species associated with reduced Th2 responses , can lead to very high levels of dAGEs. There are suggestive epidemiological and compelling immunological mechanisms which associate AGEs with increased risk of allergy. dAGES and RAGE activation disrupt and alter gut epithelial barriers and the composition of the gut microbiome and their products. These lead to inflammatory responses that can drive food allergy, as there is further injury to the gut epithelium, further inflammation and altered antigen presentation. RAGE may contribute in part to polarisation of CD4 + lymphocytes and the balance of Type 1 and 2 lymphocytes. In sum, these are all conditions unfavourable for dietary tolerance. Multiple dietary modifications and lifestyle interventions have the potential to reduce the formation of AGEs, mitigate their oxidative effects, and reduce expression of the RAGE receptor.


Natural polymers are increasingly being used in tissue engineering due to their ability to mimic the extracellular matrix and to act as a scaffold for cell growth, as well as their possible combination with other osteogenic factors, such as mesenchymal stem cells (MSCs) derived from dental pulp, in an attempt to enhance bone regeneration during the healing of a bone defect. Therefore, the aim of this study was to analyze the repair of mandibular defects filled with a new collagen/chitosan scaffold, seeded or not with MSCs derived from dental pulp. Twenty-eight rats were submitted to surgery for creation of a defect in the right mandibular ramus and divided into the following groups: G1 ; G2 ; G3 (defect filled with collagen/chitosan scaffold); and G4 (collagen/chitosan scaffold seeded with DPSCs). The analysis of the scaffold microstructure showed a homogenous material with an adequate percentage of porosity. Macroscopic and radiological examination of the defect area after 6 weeks post-surgery revealed the absence of complete repair, as well as absence of signs of infection, which could indicate rejection of the implants. Histomorphometric analysis of the mandibular defect area showed that bone formation occurred in a centripetal fashion, starting from the borders and progressing towards the center of the defect in all groups. Lower bone formation was observed in G1 when compared to the other groups and G2 exhibited greater osteoregenerative capacity, followed by G4 and G3. In conclusion, the scaffold used showed osteoconductivity, no foreign body reaction, malleability and ease of manipulation, but did not obtain promising results for association with DPSCs. Oral and maxillofacial traumas are common problems in emergency services and mandibular, nasal and zygomatic fractures are the most prevalent maxillofacial traumas . ConsideAlthough several surgical methods are available for the reconstruction of craniomaxillofacial defects caused by trauma, tumor removal and skeletal abnormalities, these methods still have limitations ,4. For ePolymers are macromolecules composed of repeating units of smaller molecules, called monomers, which can be of natural or synthetic origin . NaturalAmong natural polymers, collagen and chitosan matrices obtained from swine or bovine animal tissue and from crustaceans, respectively, are being used. Both materials have shown satisfactory results in experimental studies on bone regeneration, thus justifying their future application in medical and dental clinical practice ,11,18. CThere are about 29 known types of collagen, the most common being types I, II, III and IV. Type I is the most abundant and is the main protein present in bones, skin, tendons and ligaments, corneas and blood vessels. Its basic unit is tropocollagen, which is formed by two identical chains (α1), with about 1055 amino acid residues and a different chain (α2), with about 1029 residues, creating a linear, semi-flexible molecule with 300 nm in length and 1.5 nm in diameter ,25,26. CThe use of collagen in scaffolds also offers a support for cell growth . CollageStudies using bovine osteoblasts cultured on collagen scaffolds have demonstrated excellent in vitro mineralization, alkaline phosphatase production and differentiation into the osteoblastic lineage ,33. FurtChitosan is found in the exoskeleton of crustaceans, mollusks and insects. Its use as a biomaterial is justified by its biocompatibility. In addition, there are no limitations in terms of quantity, it is inexpensive and can be obtained by partial deacetylation of chitin. Thus, chitosan/collagen scaffolds have been indicated for applications in tissue engineering . In addiThe function of DPSCs is to produce odontoblasts. These cells can be obtained from postnatal teeth, extracted wisdom teeth, or exfoliated deciduous teeth ,40. DepeIn addition to their application in tooth structure regeneration, DPSCs are also investigated for the repair of tissues other than teeth. Within this context, the use of mimetic ECM of bone tissue seeded efficiently with DPSCs could provide an ideal microenvironment for specific applications in tissue engineering aimed at bone repair . In the Collagen and chitosan are easy to obtain, but few studies correlate the effectiveness of these natural biopolymers combined with human DPSCs in the treatment of craniomaxillofacial defects, which require rapid recovery in order to morpho-physiologically restore the bone tissue. Considering these uncertainties about the use of DPSCs ,41,42,43This in vivo experimental protocol aimed to analyze the repair of mandibular defects filled with a new collagen/chitosan scaffold, with or without DPSCs. The preparation and characterization of this scaffold was fully described by Massimino (2020) .+, Ca2+ and Na+) for 72 h, followed by treatment with an aqueous salt solution  for 6 h. All salts were removed with deionized water. Anionic collagen was extracted with acetic acid (pH 3.5) and then stored under refrigeration (4 °C). The concentration of anionic collagen was determined by dry mass weighing (n = 3) and adjusted to 1% with acetic acid pH 3.5.Anionic type I collagen was prepared from bovine tendon, as previously described by Horn et al. (2015) . The bovDoryteuthis spp.), as described by Horn et al. (2015) [w/w) at 80 °C for 3 h in nitrogen atmosphere. The chitosan powder was obtained after washing and drying. The chitosan powder was solubilized in acetic acid, pH 3.5, to obtain a 1% chitosan gel (w/w). The degree of acetylation (11.2%) and molecular weight (1.6 × 105 g mol−1) were determined by nuclear magnetic resonance (NMR) spectroscopy and capillary viscosimetry, respectively.Chitosan was extracted from squid pens (. (2015) . The squ®, mod. 715, São Paulo, Brazil). The gel was placed in Teflon® molds , frozen in liquid nitrogen and lyophilized. These scaffolds were neutralized using ammonia vapor for 8 h.The scaffold was prepared by mixing anionic collagen with chitosan (3:1 ratio) under mechanical stirring at 800 rpm . Twenty mg of the sample were placed in a hermetically sealed aluminum support under an N2 flow (80 mL min−1). The temperature range was 25–120 °C, with a heating rate of 10 °C min−1. The temperature of denaturation was given from the inflection point.Differential scanning calorimetry (DSC): Thermal stability of the scaffold was determined by differential scanning calorimetry : Samples of collagen, chitosan and collagen/chitosan (3:1) scaffolds were analyzed in an ATR_FTIR Bruker Alpha Platinum-ATR a range of 4000–400 cm®, Balzers, Liechtenstein), with a chamber pressure of 2.00 × 10−2 mbar, 60 mA current and 0.60 nm s−1 deposition rate. The pore size of the scaffold was determined using Martin’s diameter .Scanning electron microscopy (SEM) analysis: The surface and cross-section were analyzed by SEM using a Zeiss Leo 440 scanning electron microscope  with Oxford detector  operating with a 20 kV electron beam. The scaffold was coated with gold in a BAL-TEC MED 020 Coating System  and the scaffolds were immersed in it; after 24 h, the scaffolds were removed and the ethanol was weighed again (w2). Porosity was determined in triplicate, according to the equation: P(%) = (V2/V1) × 100, where V2 = (w1 − w2)/ρ and ρ is the ethanol density.Porosity was determined following the procedure adapted from Nwe et al. (2009) . The sca® Com. Ind. Ltd., Campinas, Brazil).The samples of the anionic collagen/chitosan scaffolds were sterilized with ethylene oxide at Sterilization Center  derived from human dental pulp were provided and manipulated by R-Crio Células Tronco , where the technicians of that laboratory expanded these cells and, later, associated them with the scaffold. This cell line were chosen solely based on being human cells from an established primary culture.Commercial MSCs , 100 µM ascorbic acid , penicillin–streptomycin solution  and 10% human serum . The flasks with cells were incubated at 37 °C in a 5% CO2 atmosphere and the medium was changed every 48 h.The cells were thawed at room temperature and transferred to a 25-cm² culture flask . The culture medium consisted of Dulbecco’s Modified Eagle’s Medium (DMEM)—low glucose . After complete detachment, the cells were washed and counted by the Trypan Blue exclusion method. Next, 5 × 104 cells suspended in 10 µL of the same expansion medium were seeded on the biomaterial in a 12-well plate , 4 months old and weighing on average 350 g, were used. The animals were maintained under adequate conditions in the Animal House of the Jundiaí Medical School (FMJ), Jundiaí, Brazil. The Ethics Committee on Animal Experimentation of FMJ approved the project . The rats were submitted to a surgical procedure for creation of an experimental defect in the right mandibular ramus and were divided into four groups of seven animals each (G1 (G-C): mandibular defect filled with the blood clot of the defect but did not receive the graft (control group);4 DPSCs suspended in 10 µL culture medium;G2 (G-MSCs): mandibular defect seeded with 5 × 10G3 (G-SCoCh): mandibular defect grafted with the anionic collagen/chitosan gel (3:1) scaffold;4 DPSCs.G4 (G-SCoCh/MSCs): mandibular defect grafted with the anionic collagen/chitosan gel (3:1) scaffold and seeded with 5 × 10Twenty-eight male Wistar rats ®, Fort Dodge Saúde Animal Ltd., Campinas, Brazil) and ketamine  (1:1) at a dose of 0.1 mL/100 g body weight into the gluteus muscle [The animals were anesthetized by intramuscular administration of xylazine  [A circular bone defect measuring 5 mm in diameter was created using a trephine drill coupled to a mini-motor (BELTEC Brazil) . The def®, Campinas, Brazil) for one week [®, Sanofi-Aventis Pharmaceutical Ltd., São Paulo, Brazil) was applied externally to the defect area. Paracetamol diluted in the animal’s water was also provided during the postoperative period and the animals were constantly monitored to assess their behavior. All animals responded adequately to the pre- and postoperative procedures and there was no need for replacement of animals in the groups.During the postoperative period, the rats received daily intramuscular injections of 0.1 mg/100 g body weight of Pentabiotic Veterinário Pequeno Porte antibiotic  intraperitoneally, applied in the abdominal quadrant inferior left of the animal, associated with the local anesthetic lidocaine hydrochloride , at a dosage of 10 mg/kg. The mandible was immediately removed and photodocumented to analyze the clinical conditions of the defect area. The samples were fixed in formaldehyde solution and submitted to image analysis by digital radiography .Six weeks after surgery, euthanasia was performed in a silent environment and away from other animals, using barbiturate thiopental at the dosage for rats (150 mg/kg) as follows: Sodium Thiopental 2.5%  and 0.44% sodium hydroxide . The decalcifying solution was changed weekly and, to assess the complete decalcification, radiographs were taken with periapical film Insight Adult IP-21 F-Speed Carestream  for an approximate period of 6 weeks. After completing this phase, the pieces were dehydrated in an increasing series of ethyl alcohol, cleared in xylene and embedded in Histosec paraffin .After digital radiography, the collected bone pieces were washed in water for 24 h and submitted to the demineralization process using a solution of ethylenediaminetetraacetic acid (EDTA), a solution containing 4.13% of tritiplex III (Merck KGaA®) for analysis of the fibrillar components of ECM by polarized light microscopy. The histological sections used for staining and subsequent qualitative and quantitative analysis were made in the center of the 5 mm diameter defect and also at the medium distance between the center and the peripheral region  were cut along the bone defect. The histological sections were stained with Masson’s trichrome for the characterization of new bone formation and with Picrosirius red  for the collagen present in the scaffold was 48.7 °C. The collagen extraction procedure used promotes selective hydrolysis of the collagen carboxyamide groups, increasing the negative charges of the biopolymer . This in−1 shows the integrity of the collagen triple helix, with values close to 1.0 indicating that the collagen did not undergo denaturation; and in this case, the bovine collagen had a ratio of 0.96. The typical amide bands can be observed, like amide A band in the 3720–3120 cm−1 region, amide B in the 3115–2815 cm−1 region, amide I at 1633 cm−1, amide II at 1549 cm−1 and amide III at 1243 cm−1. These bands agree with the collagen extraction processes shown in −1 and 1033 cm−1, referring to the −CO group in CH2−OH and overlap with the −CO group in the ether−COC−bond (pyranose ring) [The ATR-FTIR spectra of the bovine collagen, chitosan and collagen/chitosan scaffold are shown in se ring)   , pores bPores smaller than 100 μm contribute to cell adhesion and bone regeneration . The scaIn addition to pore size and interconnectivity, porosity is also an important factor for bone regeneration. The percentage of porosity influences the absorption of exudates, the colonization rate, the cell structure and the process of angiogenesis . High poThis study evaluated the capacity of polymeric collagen/chitosan scaffolds seeded with DPSCs to provide an osteogenic stimulus for bone neoformation during treatment of mandibular bone defects experimentally induced in rats. The results obtained were compared with data reported by other researchers and some important differences and similarities were observed. Within this context, we hope that the results obtained can contribute to tissue engineering in regenerative medicine and dentistry and help surgeons choose the best scaffold for the treatment of craniomaxillofacial injuries. For this purpose, the availability and favorable use properties of natural polymers in tissue engineering must be considered , as wellThe results regarding the clinical conditions of the animals 6 weeks after surgery showed good recovery of all rats as demonstrated by adequate skin healing and normal hair growth in the defect area, absence of clinical signs of inflammatory processes, and normal behavior and feeding of the animals. In the implanted groups , there were no signs of subcutaneous infection or infection in deeper anatomical planes that would indicate immune rejection of DPSCs or of the scaffolds used as implants. The soft tissues in the defect area had healed well, with no formation of adhesions .The positive clinical results of the animals can be attributed to the fact that all animal use experimental protocols were followed and to the pre- and postoperative care and sterilization of the scaffolds and surgical field, as carried out in other studies on the regeneration of craniofacial injuries ,17,65,66Successfully used in different regenerative medicine applications, ECM scaffolds can be obtained by decellularization of manipulated tissue . Thus, cAccording to Horn et al. (2009) , the durBiomaterials used in tissue engineering must meet certain requisites when implanted into the host tissue, which include being non-toxic, non-immunogenic, biodegradable and able to simulate the microenvironment necessary for cell growth, proliferation and migration in order to promote the formation of new tissues . ConfirmBased on the above considerations and the macroscopic, radiological and histological results that indicate biocompatibility of the scaffolds and DPSCs used in this study, histomorphometric analysis of new bone formation in the defect area was performed to confirm the osteogenic efficacy of the standardized protocol.Polymeric membranes with or without cellular or biological mediators have received special attention in biomedical engineering and dental, oral and craniofacial regenerative medicine for their role as scaffolds in cell therapies and as slow-release devices of drugs, biomolecules and growth factors that are important for the in vitro development of tissue and organs ,15,70. Cw/w). Chitosan was obtained by deacetylation of squid chitin and porcine collagen was obtained from the serosa using alkaline hydrolysis of different durations. The authors demonstrated that this protocol interferes with pore size. In the present study, we prepared collagen from bovine tendon and subjected it to alkaline hydrolysis [The combined use of anionic collagen biopolymer and chitosan has been shown to stimulate vascular formation and to improve the three-dimensional structure of the scaffold, enabling cell migration . In addidrolysis  for 72 hConsidering the advantages of alkaline hydrolysis for natural polymers described by Bet et al. (2001) , studiesIn this experimental protocol using rat mandible, bone formation occurred from the borders of the defect towards the polymer in the group grafted with anionic collagen/chitosan scaffolds G3); however, there was no direct bone-implant interaction due to the presence of interposed connective tissue. In G4 that received the same scaffold but seeded with DPSCs in vitro, greater bone formation closer to the biomaterial was observed, with little connective tissue at the bone-implant interface . However; howeverIn G2 animals seeded only with DPSCs, bone formation occurred towards the center of the defect, probably because these cells were introduced into the entire space of the defect. In the control group (G1), there was little bone formation at the borders of the defect, with the largest part of the defect being filled predominantly with connective tissue . These rSoares et al. (2016)  tested aIn all groups studied, the newly formed bone showed histological characteristics of immature tissue and the presence of osteoid matrix , with anTo confirm the stimulus potential of the implants used in this study, the percentage of newly formed bone in the defect area was analyzed morphometrically. Histological sections were made in the center of the 5 mm diameter defect and also in the middle distance between the center and the peripheral region . The perp < 0.05). These results suggest that a stimulus is necessary for bone repair in critical mandibular defects since the lowest value was observed in the control group (G1). Among the groups studied, G2 exhibited the greatest bone formation at the periphery and in the center of the defect, suggesting that MSCs exert an osteogenic effect which, however, is not sufficient to promote complete regeneration within the period of 6 weeks after surgery standardized in this study. Jégoux et al. (2010) [Statistically, all possible comparisons showed significant differences between groups (. (2010)  used bon. (2010) , who eva. (2010) ,80.In the present study, despite the good results observed in the grafted groups G2, G3, G4) compared to the control group (G1), complete repair did not occur probably because a postoperative period of 6 weeks was standardized in order to determine the speed of bone formation in response to the protocol used ,82. Thus, G4 compMechanical properties, degradation capacity and the porosity necessary for vascular and bone growth must also be analyzed when selecting a biomaterial ,85,86. IAccording to the concept of guided bone regeneration in maxillofacial surgeries, biomaterials are applied to bone defects in order to maintain a space between the bone defect and the periosteum and to assist with bone formation. Ideally, osteoprogenitor cells should colonize the space beneath the bone defect; however, these cells have a slower growth rate than epithelial and connective tissue cells. To prevent the bone defect from being occupied by epithelial cells and connective tissue, a membrane (resorbable or non-resorbable) is used to cover the graft ,89.Polymeric materials are considered osteoconductive and maintain space for cell migration to the bone defect in order to induce the regeneration process . The chiIn In addition, the combination of guided bone regeneration technique with DPSCs would help develop a treatment protocol capable of enhancing cell function and mimicking the physiological reactions of bone tissue in order to accelerate the process of bone regeneration in maxillofacial traumas. All of these factors will contribute to more personalized treatment and a faster and more satisfactory recovery of the patient.A possible limitation of the study can be considered, immunohistochemical reactions with bone markers for evaluation of protein expression could correlate with the histological findings of the repair process. Also, the lack of analyses prior to this study on the biocompatibility of the scaffold for seeded DPSCs can be considered a limitation.This pre-clinical experimental protocol was carried out in order to evaluate the in vivo effects of the combination of anionic collagen/chitosan scaffold with or without DPSCs in the repair of mandibular bone defects in rats. The scaffold used showed osteoconductivity, no foreign body reaction, malleability and ease of manipulation, but did not obtain promising results for association with DPSCs. The scaffold-filled groups (G3 and G4) had a higher percentage of new bone formed in the defect area than the control , but the presence of this type of scaffold is less effective than DPSC alone in formation of new bone (G2).


However, no difference was observed between 1 w and 5 m post-MSA. Bone graft displacement was observed in eight patients; the reduction rate from 1 w to 5 m post-MSA was 8.38% ± 4.88%. Sex, septa, maxillary sinus floor–palatal bone distance, and maxillary sinus floor–maxillary ostium distance were associated with bone graft displacement (p < 0.05). The height from the maxillary sinus floor to the palatal bone and the sinus angle influenced the augmentation degree (p < 0.05). The PLA/PCL membrane is compared favorably with other membranes and may be useful as a barrier membrane for the MSA open window.Maxillary sinus augmentation with a lateral approach (MSA) is a well-established treatment. In this prospective study, we evaluated risk factors for postoperative bone graft displacement and reported the clinical application of long-term resorbable L-lactic acid/-caprolactone (PLA/PCL) as a barrier membrane to cover the open window in the lateral wall in MSA. Twenty-four patients underwent MSA according to the relevant criteria; CT data obtained before and 1 week (1 w) and 5–6 months (5 m) post-MSA, bone height changes, bone height reduction rates at 1 w and 5 m post-MSA, bone graft displacement measurements, and risk factors were examined. All patients showed bone height increments ( In patients undergoing implant therapy for maxillary molar defects, the lack of existing bone height in the maxillary molars often prevents ideal implant placement. Maxillary sinus augmentation with a lateral approach (MSA) is performed to solve this problem. MSA has become an established treatment method for maxillary molars. Long-term prognosis data show good outcomes, with systematic reviews showing a high implant survival rate of 92–100%. No differences in implant survival rates have been observed based on graft material type  or the initial bone height (less than 5 mm and 5 mm or greater) ,4,5. On Complications related to MSA include sinus membrane perforation, maxillary sinusitis, and graft material displacement. Sinus membrane perforation occurs in a relatively high proportion of patients (20–25%), and its development is influenced by anatomical characteristics such as thin sinus mucosa, residual bone height, narrow sinus angle, the presence of septa, and thick lateral walls. Additional factors contributing to perforation include the presence of smears, smoking habits, surgical techniques involving sinus floor instruments, and the surgeons’ expertise. Maxillary sinusitis following MSA occurs in approximately 4.2–8.4% of cases. This is associated with preoperative issues in the osteomeatal complex, intraoperative large membrane perforations, overfilling graft material, and mucosal swelling post-surgery. The incidence of such cases is high in the UK. Moreover, graft material displacement is caused by overfilling of the graft material and inadequate barrier membrane coverage at the lateral open window, which leads to reduced bone height due to MSA. Bacterial infection can cause facial pain or swelling and the previously mentioned maxillary sinusitis, often necessitating the removal of the graft material and the implant body. Placing a barrier membrane in the lateral open window has been shown to be important in solving problems caused by graft material displacement. This approach improves new bone formation rates and reduces the amount of residual graft material. However, the differences in effectiveness between different types of barrier membranes (resorbable or non-resorbable) and materials  have not been investigated to date ,8,9,10.The placement of a barrier membrane has been reported to promote bone formation and increase the implant survival rate because the barrier membrane covering the open window in MSA can prevent fibroblast invasion from connective tissue during wound healing and promote bone regeneration around the bone graft material ,14,15,16In the present study, we prospectively evaluated the effectiveness of L-lactic acid/ε-caprolactone (PLA/PCL), a long-term resorbable guided bone regeneration membrane, as a barrier membrane for lateral approach maxillary sinus augmentation and examined the risk factors associated with postoperative bone graft displacement and reduced bone height.This prospective study was conducted at Showa University Dental Hospital Implant Center. The clinical trial, including patient recruitment, was conducted from October 2018 to May 2022, according to the protocol in This study was approved by the Ethics Committee of SHOWA University Clinical Research Review Board  and by the Institutional Review Board of Showa University Dental Hospital . It was conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent for this study. Informed consent was obtained from all subjects involved in the study.®, GC Corporation, Tokyo, Japan: hereafter CO3Ap). Carbonated apatite is synthesized by a dissolution–precipitation reaction and has the chemical formula Ca10-a(CO3)b(PO4)6-c. CO3Ap contains 6–9% carbonate by weight in an apatite crystal structure, and the particle size (short diameter) of CO3Ap ranges from 600 to 1000 μm. Carbonate apatite is thermodynamically stable in a neutral environment in vivo. In contrast, in a weakly acidic environment (pH 5), where osteoclasts form, it becomes unstable and dissolves, exhibiting excellent bone replacement properties [The artificial bone substitute was carbonate apatite  was used as the barrier membrane for the open window in lateral approach MSA. The PLA/PCL membrane has a white membrane structure molded from a bioabsorbable polymer. It is a rectangular film measuring approximately 30 mm × 40 mm and consists of two layers, a dense layer and a porous layer, with a thickness of approximately 200 μm. The dense layer is placed on the soft tissue side, and the porous layer is on the hard tissue side. The dense layer blocks fibroblast invasion, while the porous layer promotes osteoblast differentiation. The GC Corporation provided PLA/PCL  of amoxicillin hydrate  1 h before surgery. MSA was conducted under local infiltration anaesthesia . The bony window of the lateral maxillary wall was designed according to the planned location(s) of the implant(s) and the anatomy of the maxillary sinus. A horizontal incision was made on the buccal side of the alveolar bone at the maxillary molar defect site, and the mucoperiosteal flap was turned over to expose the lateral wall of the maxillary sinus. A small diamond bar was used to cut 360° around the entire circumference of the bone to form a bony window, which was separated from the sinus membrane. The mucosa of the maxillary sinus floor was carefully dissected and elevated using a mucosa elevator. The sinus floor mucosa was carefully checked for perforation. After elevation of the basilar membrane of the sinus, the elevated space was filled with CO3Ap granules. Finally, the bony window was repositioned; a PLA/PCL membrane was placed over it; and the mucoperiosteal flap was sutured . All maxTo prevent postoperative infection, patients received amoxicillin 250 mg four times a day for five days and 0.2% benzethonium chloride mouthwash four times a day for two weeks, as well as loxoprofen sodium 60 mg three times a day for three days as an analgesic. Sutures were removed two weeks after surgery.Cone beam computed tomography (CBCT) examinations were performed preoperatively (pre–MSA), within one week postoperatively (post–MSA1W), and at 5–6 months postoperatively (post–MSA5M), in accordance with the protocol shown in The following items were evaluated to measure the effectiveness of the PLA/PCL membrane in MSA: presence or absence of infection and sinusitis, exposure of barrier membrane, bone height post–MSA1W and post–MSA5M, rate of reduction of bone height post–MSA1W and post–MSA5M, presence and amount of bone graft displacement mm; .Inflammation, inflection, and sinusitis were assessed by the presence or absence of persistent pain for at least one week postoperatively; the presence or absence of redness, swelling, and drainage of pus in the oral cavity; the condition of the maxillary sinus by CBCT two weeks postoperatively; and the patient’s nasal symptoms.The following factors were evaluated concerning bone graft displacement and the percentage decrease in bone height : sex; agThe same oral and maxillofacial radiologist, with over 20 years of experience, performed all measurements.p–values of <0.05 were considered statistically significant .The effectiveness of the PLA/PCL membrane was evaluated using the Wilcoxon signed-rank test. The factors related to bone graft displacement and the percentage decrease in bone height were evaluated using the Mann–Whitney U test, Spearman’s correlation, and the chi-square test. Moreover, multiple comparisons among the three groups were performed using the Friedman test. In addition, Approximately 6–7 months after MSA, implants with a diameter of 4.1 mm and a length of 10 mm were placed in all cases, with or without bone displacement , and thep < 0.005). However, no difference was observed in bone height between post–MSA1W and post–MSA5M , in those without a septum (p = 0.040), and in those with smaller distances from the maxillary sinus floor to the palatal bone and from the maxillary sinus floor to the maxillary ostium .Bone displacement was more likely in women than in men  as a control group that was similar to the present study but did not use barrier membranes, Nagata et al. [In the present study, the mean bone height pre–MSA was 2.02 ± 0.81 mm, whereas the mean height post–MSA1W was 16.26 ± 3.09 mm and post-MSA5M was 15.05 ± 2.99 mm, indicating a mean reduction of 8.38% ± 4.88% in augmentation. In a similar study that did not use barrier membranes, Nakagawa et al.  investiga et al.  evaluatea et al.  investiga et al. ; (see TaThe results of our study also showed that the sinus angle and the height from the palatal bone to the maxillary sinus floor were factors related to reduced maxillary sinus elevation, with smaller distances from the palatal bone to the maxillary sinus floor and larger sinus angles being associated with larger decrease rates. Factors related to bone graft displacement included sex, presence of a septum, height from the maxillary sinus floor to the palatal bone, and height from the maxillary sinus floor to the maxillary ostium. Bone displacement was more likely to occur in women, those without a septum, and those with smaller distances from the palatal bone to the maxillary sinus floor and smaller distances from the maxillary sinus floor to the maxillary ostium. In the study by Ohayon et al.  on bone Limitations of this study include the lack of pathological evaluation and investigation, the lack of control for the position and size of the open window, and the lack of comparison with other membranes or other graft materials because this was a single-arm study. In particular, the position and size of the open window need to be thoroughly examined, including the results of this study, as there are reports of differences in augmented bone volume and the percentages of newly formed bone ,40,41. FCompared to other previously reported membranes, the PLA/PCL membrane could serve as a helpful barrier membrane for MSA. Factors related to bone graft displacement included sex, presence of a septum, height from the maxillary sinus floor to the palatal bone, and height from the maxillary sinus floor to the maxillary ostium. Therefore, focusing on these factors and the usual measurement points as a preoperative diagnosis with CT imaging would result in a higher quality MSA.


Phosphate dosing is the principle strategy used in theUnited Kingdomto reduce the concentration of lead in tap waters supplied by leadwater pipes. The mechanisms of phosphate-mediated lead control arenot fully understood, but solid solutions of lead calcium apatiteare thought to play an important role. This study investigated themicrostructure of a lead pipe, supplied with high-alkalinity tap water,in which the lead calcium apatite crystals were spherulitic havingrounded and dumb-bell-shaped morphologies. XRD, Fourier transforminfrared spectroscopy, optical microscopy, Raman spectroscopy, scanningelectron microscopy, and energy-dispersive spectroscopy showed thatthe lead pipe had a well-established inner layer of litharge; a middlelayer containing lead calcium apatite spherulites, plumbonacrite,and some hydrocerussite; and an outer layer containing iron, lead,phosphorus, calcium, silicon, and aluminum. It was found that spheruliticlead calcium apatite could be grown in the laboratory by adding hydrocerussiteto synthetic soft and hard water-containing phosphate, chloride, andcitrate ions at pH 5.5 but not when the citrate was absent. This suggeststhat dissolved organic molecules might play a role in spherulite formationon lead water pipes. These molecules might inhibit the formation oflead calcium apatite, reducing the effectiveness of phosphate dosingin lead water pipes. Thisstudy investigates the spherical shaped lead phosphatecrystals that are sometimes found in lead water pipes when phosphateis added to tap water to control lead release and goes on to discusstheir wider application within the context of the mineral scale. Precise figures are not available, buthousing data3 The principle strategy adoptedby all U.K. water utilities to control lead at customers’ tapsis to dose the tap water with phosphate (orthophosphate).6 Most U.K. utilities use 3–6 mg/L phosphate (1–2 mg/Las P) at a pH between 7 and 8. The practice of phosphate dosing beganin the 1970s and 80s with a gradual increase in the proportion ofsupplies treated until 2000 when dosing became widely adopted. Since2000, compliance with the 10 μg/L limit increased from 95.0to 99.5% .7 Therefore, from a regulatory and public health perspective,the practice of phosphate dosing has been very successful.8The harmful effects of lead are well known, and in the United Kingdom,the current regulatory limit for a random daytime sample of 1 L oftap water, taken from a customer’s kitchen tap, is 10 μg/Ltotal lead.5(PO4)3Cl] and hydroxylpyromorphite[Pb5(PO4)3OH],9 which would lower the concentration of lead. It was thoughtthat a layer of these minerals would grow on top of the existing leadcarbonate layer, possibly acting as a physical barrier against furtherrelease of lead and would take up to a year to provide full protection.10The original theory was that phosphatedosing would promote theformation of highly insoluble lead phosphate minerals pyromorphite.14 Lead phosphate minerals occurredin only some of the cases in which orthophosphate and blended phosphatehave been added to tap waters.14 In thiscase, hydroxylpyromorphite [Pb5(PO4)3OH], lead phosphate [Pb9(PO4)6],and a mixed lead–calcium phosphate mineral [PbxCax5–(PO4)3] were observed.12 A similar set of minerals have been observed in pipes from the UnitedKingdom except that plattnerite has not been observed.15Most of the studies on the mineral scalesin lead water pipes havebeen undertaken in the United States and Canada, where a greater varietyof methods are used for mitigating corrosion. Ongoing studies of leadwater pipes from Midwestern and Northeast states of the United Stateshave found that approximately 25% of operators use orthophosphate,another 25% use blended phosphate (a mixture of orthophosphate andpolyphosphates), and the remainder use pH and alkalinity control.17 have shown that the mineral phases within the scale are spatiallydistributed within discrete layers. For the purpose of this study,a simplified schematic diagram has been created by the authors .18The chemistry of lead pipe mineral scaleis complex, and the mechanismsof corrosion and mineralization are not well understood. Some leadpipes are over 100 years old, and their scale might reflect conditionsthat are very different from those of today. In the United Kingdom,the formation of lead phosphate minerals must have occurred withinthe last 20–40 years, the time period of phosphate dosing.Recent studies authors 1a. The i19 although theywere not identified as spherulites. Spherulites are not uncommon incrystal growth but are not widely reported in the literature. Theyare polycrystals with a radial structure of needles or fibers thatbranch at small angles20 and can be looselygrouped into category 1 spherulites, where branching occurs from acentral core, and category 2 spherulites, where branching occurs offa central needle .Preliminary analyses of these micron-sized crystals indicated thatthey were spherulitic in morphology and comprised lead calcium apatite.Crystals that look spherulitic have been reported in a small numberof lead pipe rig studies,l needle 1b.21 AltThe occurrenceof lead calcium apatite crystals with unusual shapesimplies that the mineral might not be growing in an expected manner.The thermodynamic and kinetic properties of these spherulites mightdiffer from those of single crystals with well-defined planar crystalfaces. This difference is of interest to U.K. water utilities whowould like to have a better understanding of how phosphate mitigateslead in tap water.This study focused on a pipe obtained fromthe city of Kingstonupon Hull, East Yorkshire, that had been supplied with high-alkalinitywater and contained spherulitic crystals. A comprehensive analysisof both the mineral scale and the spherulites within was undertakenusing powder X-ray diffraction (XRD), Fourier transform infrared (FTIR)spectroscopy, optical microscopy (OM), SEM, energy-dispersive spectroscopy(EDS), and Raman spectroscopy. A small number of lead water pipesfrom other locations that contained spherulites were also investigated.Spherulitic crystals of lead calcium apatite were grown in the laboratoryfrom the dissolution and reprecipitation of the lead carbonate mineralhydrocerussite in synthetic soft and hard tap waters that containeda mixture of calcium, phosphate, chloride, and citrate ions.The aims of this study were to understand the phenomena of spheruliteformation on lead water pipes to discuss this in the wider contextof the lead pipe mineral scale and to understand more about how phosphateinfluences the chemistry of the lead pipe mineral scale. The objectiveswereto (1) characterize the structure of the mineral scale; (2) confirmwhether the mineral scale comprised an inner, a middle, and an outerlayer; (3) characterize the structure of the spherulites; (4) confirmwhether the unusual rounded crystals in lead water pipes were spheruliticand comprised lead calcium apatite; (5) determine whether spheruliticlead calcium apatite could be grown in the laboratory; and (6) determinefactors that give rise to lead calcium apatite spherulites.Thelead pipe used in this study was a communication pipe that connectedthe mains pipe under the street to the stop tap in the pavement andwas owned by the utility. The supply pipe, which connected the stoptap in the pavement to the home was not analyzed.The lead communicationpipe was extracted in 2010 from a terraced property (built in 1901–1911)in Kingston upon Hull, East Yorkshire (postcode HU3), and analyzedbetween 2015 and 2020. It was drained on site and sealed at both endswith plastic bags before being sent to the laboratory, whereupon itwas air-dried. It was then stored dry at room temperature and 40–60%humidity. A rachet pipe cutter with a clean blade was used to cutthe pipe into shorter transverse sections (length 1.5 cm). Smallerlongitudinal sections were then cut with a clean, stainless steel,utility knife (Stanley).3,pH 7.2).The transverse sections were embeddedin under vacuum using theStruers Epofix cold mounting resin. The purpose of the vacuum wasto remove air bubbles in order to improve contact between the pipeand the resin. The embedded samples were covered and left to hardenin air for a day at room temperature. Cross-sections of the pipe werethen ground and polished. The sequence was (1) manual polishing with240 SiC paper on a granite grinding plate with a gentle flow of tapwater and rotating by 90° every 30 s, (2) manual polishing withp600 SiC paper for 5 min on a polishing wheel, (3) manual polishingwith p1200 SiC paper for 5 min, (4) gently rubbing the surface witha piece of selvyt cloth coated in 1 μm diamond suspension spray,and (5) rinsing the surface with tap water and then methanol beforebeing air-dried. The tap water used during this sequence was not fromthe sampling site but from the mains supply at the University of Huddersfield.The water contained phosphate, which would have limited the amountof mineral dissolution  and hasshown that the size of the litharge peak increases with depth. However,other changes in mineral composition have not been seen. Therefore,complete scrapings rather than successive scrapings were used on thepipes in this study. It is worth noting that the thickness of themineral scale on U.K. lead water pipes extends over a short distance,making it hard to remove and separate discrete layers.3 and pH 7.1, respectively. A moredetailed analysis is given in Table S1.The water in the city of Kingston uponHull is a high-alkalinity tap water extracted from a chalk bedrock.The pipe from Hull had been exposed to this type of water for over100 years. During this time, chlorine was always used as a disinfectant,and flocculants were never used. Phosphate dosing was initiated in2001. Since then, the average alkalinity and average pH of the waterhave been 240 mg/L CaCO–1.Powder XRD was carried out usinga Bruker D2 PHASER fitted with a LYNXEYE detector and Cu Kαradiation (=1.5418 Å). Samples of scraped mineral scale wereground in isopropanol using a pestle and mortar and loaded into thecenter of a silicon crystal sample holder with no cavity. Scans wereperformed between the diffraction angles 10 and 60° 2θ.FTIR of the scraped mineral scale was undertaken using a Thermo ScientificNicolet iS5 spectrometer. Scans were performed between 500 and 2000cmText S1). The samples of pipe were attached to aluminum stubs witha conducting carbon sticky tape, and the sides were coated with theconducting paint. Where necessary, the samples were then coated witha thin layer of gold/palladium (15–20 nm). Raman microscopywas performed on the polished sections of the pipe using a ThermoScientific DXR Raman microscope. A green laser of wavelength 532 nmwas used. Cross-sections of the mineral scale were very sensitiveto the Raman laser, and there was a playoff between power, resolution,and damage. Each point was photobleached for 2 min after which five30 s frames were taken. A relatively low power of 0.4 mW was foundto give optimum results, although the signal-to-noise ratio for manyof the peaks was high.Microscopy was undertaken looking downon the mineral scale in a plan view and across the mineral scale ina cross-sectional view. Optical microscopy was carried out using adigital Keyence VHX-2000 microscope equipped with a VHX-J250 lens.SEM and elemental analysis (EDS) of the lead pipe minerals scale wereperformed using a FEI Quanta 250 FEG microscope equipped with an OxfordInstruments X-Max detector, a Zeiss Supra 40VP FEG microscope equippedwith a EDAX Inc, an Apollo 40 SDD, and a Zeiss Evo MA10 tungsten filamentmicroscope equipped with an Oxford Instruments X-Max detector. Theimages from the different microscopes were taken at an acceleratingvoltage of 10–20 kV at a working distance of 4.5–12mm. Plan view images were taken in the secondary electron mode, andcross-sectional images were taken in the backscattered mode. EDS pointanalyses and area analyses were recorded to see what elements werepresent. EDS maps were recorded to see where elements were concentrated.The lighter shades were probably background radiation  and sodium hydrogencarbonate solution  to ultrapure water  with rapid stirring. The two solutions were addeddropwise over 30 min, and the pH was continually adjusted to pH 8.0by the addition of 0.1 M carbon dioxide-free NaOH. The resulting whitesuspension was filtered off using a 0.45 μm Millipore filter,and the damp solid was transferred to a sample vial containing 50mL of ultrapure water. The mass of hydrocerussite in the vial was200 mg (based on >95% yield). Powder XRD of a sample of a driedsolidgave a single large peak at 2θ = 34.0° corresponding tothe {110} planes of hydrocerussite . The single large peak was due to the orientation of the hydrocerussitecrystals. These were shown by SEM to be thin hexagonal plates of diameter1–3 μm, stacked with their large faces on top of eachother .The reagents usedwere of analytical grade and are listed in 3 powder  in ultrapure water while bubbling with CO2 gas. To these were added stock solutions of NaH2PO4, NaCl, and trisodium citrate (Na3C6H5O7), and the pH was increased to 5.5by bubbling with air to remove CO2(g). The soft water contained20 mg/L dissolved CaCO3, and the hard water contained 200mg/L CaCO3. Both finished solutions contained 30 mg/L phosphate(10 mg/L as P), 10 mg/L chloride, and 0.5 mM citrate . The suspensions of hydrocerussite in softand hard water were sealed in 1 L of high-density polyethylene samplebottles with minimum air space. They were then stirred with a magneticstirrer and sampled after 1 day. The samples were dried and analyzedby XRD, FTIR, SEM, and EDS. The relatively low pH of 5.5, the highconcentration of phosphate of 30 mg/L (10 mg/L as P), and the highconcentration of citrate (0.5 mM) were chosen to maximize the likelihoodof precipitating spherulites in a small period of time, not to mimicthe conditions within a lead water pipe. Control experiments werealso undertaken in which the citrate was omitted.The spheruliteswere prepared by adding 25 mL of the hydrocerussite suspension (100mg of solid) into a 1 L solution of synthetic soft water or hard waterand at pH 5.5. The soft and hard waters were made by dissolving CaCOThis section considers the analyses of thelead pipe mineral scale,the lead pipe spherulites, and the laboratory-grown spherulites.Figure S3). Some of the fragmentation must have been artifactual dueto the drying down process and to the stresses on the pipe duringcutting, and some may have occurred when the pipe was in operation.A light brown, partly fragmented mineral scale with cracks andvalleys was observed within the lead pipe (xCax5–(PO4)3], hydroxyapatite [Ca5(PO4)3OH], hydrocerussite [Pb3(CO3)2(OH)2], and/or plumbonacrite[Pb5(CO3)3O(OH)2] werepresent. The minerals were identified by comparing the pattern  with the standard patterns . The most prominent peakswere from the {101} and {110} crystal planes of litharge. Less prominentwere the peaks at 2θ = 22.1 and 30.8°, which were similarbut not the same as those associated with phosphohedyphane [Ca2Pb3(PO4)3Cl], which has peaksat 2θ = 21.9 and 30.9° . Both hydrocerussite and plumbonacrite appeared to be present,but it was difficult to separate the two sets of peaks. Peaks forplattnerite (β-PbO2), which have been observed inpipes in North America supplied with chlorinated tap water,13 were not observed.XRD of complete scrapingsof the mineral scale indicated that litharge (α-PbO), lead–calciumapatite [PbFigure S6). The doublet at 541 and 586 cm–1 was characteristic of lead apatite minerals.15The presence of carbonate and phosphatecomponents in the bulk mineral scale was confirmed by FTIR , the red innerlayer can be seen at the base of the valley, the white middle layercan be seen at the edges of the valley, and the brown outer layercan be seen on the surface. The border of the valley is marked in Figure S7b by the white line.Figure S7a,b are shown in Figure S7c–e. Theouter layer  contained irregular-shapedaggregates of diameter <5 μm, whose fine structure was notresolved by SEM. The middle layer  contained spherulitic-shaped particles that were 5 μm indiameter. These had a textured surface. The inner layer containedinterlocking particles of size 0.2–5 μm . They were plate like, and some hada hexagonal outline suggestive of crystals. It was not clear whetherthese particles were on the surface of the inner layer or whetherthey were part of the inner layer.Magnifiedimages of the boxed areas in Figure S8.The inner, middle, and outer layers I, M,and O, respectively, were clearly visible when the scale was viewedin cross-section by OM and SEM 2, but unText S1. Raman spectroscopy provided furtherevidence of the distribution of the minerals within the three layers.The spectra coupled with the OM images showed that phosphate and carbonatewere concentrated in the middle layers and that litharge was concentratedin the inner layer .The elemental maps 2d–g showFigure S9) indicated that Al and Si were alsopresent in theouter layer; that Sn was a minor component of the metal, inner layer,and middle layer; and that there was no P in the inner layer.Individual EDS spectra across the mineral scale (S13). Althoughthe surfaces of the particulates looked smooth in plan view, theywere found to comprise many small needle-like protrusions in cross-section indicated that it containedPb, P, Ca, and O, which is consistent with the spherulite being madeof lead calcium apatite. Al was also detected. The elements were notspatially segregated, indicating that the composition of the spherulitewas uniform throughout. Plate-like crystals were often in close proximityto the spherulites, which were probably the crystals of hydrocerussiteand/or plumbonacrite .Wheatsheaf-shaped crystalswere observed in experiments conducted using soft and hard waterscontaining citric acid 5. Those Figure S15) and closely resembled those of pyromorphite and hydroxypyromorphitestandards . It wasnot possible to tell which was the best fit without more detailedanalysis. Unlike the standards, the peaks were broad, indicating thatsmall nanosized particles or misaligned domains were present, whichis consistent with the needle-like structure found in spherulites.Hydrocerussite crystals, which were added at the beginning of bothexperiments , were absent andhad completely dissolved.The XRD patterns for spherulites grown in syntheticsoft and hardwaters were similar to each other . Thesecrystals were not spherulitic. The XRD patterns indicated that thesmall crystals comprised lead apatite .The control experiments in which citratewas omitted from the softand hard waters did not produce spherulites. Instead, very small crystalswere observed that appeared to grow off the remaining hydrocerussitecrystals . However, in others, there is no core, and instead, themisalignment of the adjacent elongated crystallites and branches causesthe fibers to spread out like a fan, giving rise to wheatsheaf-shapedcrystals, dumbbells, and spheres (category 2).21 The two types are shown in It is generally accepted that the spheruliticshape 1c isa r21 Computational models ofthe solid–liquid interface using phase field theory have beenused to investigate this mechanism and applied to spherulitic growthin metal solidification and polymer crystallization.31 However, non-classical crystal growth mechanisms have also beenproposed in which the needles and fibers are regarded as mesocrystalsformed by the aggregation and alignment of nano-sized particles.36 The two competing theories have led to heated debate.37A growth front nucleation mechanismin which slightly misalignednuclei grow into branches has been used to explain the shapes of thespherulites.Spherulites are often found in systemsin which there is a highthermodynamic driving force for crystallization coupled with a kineticbarrier. The latter gives rise to slower than expected crystal growth.Examples of kinetic barriers include the presence of impurities, surfaceinhomogeneities, and lattice defects.35 strontium sulfate (celestite) spherulites thatgrow on calcium sulfate (gypsum) crystals as they dissolve,38 fluorapatite spherulites that grow in the presenceof additives such as citric acid or gelatin,40 hydroxyapatite spherulites that grow in a collagen gel,41 and calcium carbonate  spherulitesthat grow in the feces of ruminant herbivores42 and fish.43 Spherulitic minerals havealso been grown in solutions containing bacteria,44 where control of crystal growth has been attributed tothe release of an extracellular material such as polysaccharides (EPSs).Spherulites can form in any crystal system, but the likelihood isincreased if the crystal is insoluble and if it grows with a needle-likemorphology.20Examples of mineral spherulitesinclude calcium carbonate (vaterite)spherulites that grow at moderate supersaturation without impurities,Figures S19–S21, andothers are reported elsewhere.17The spherulites observed in this study were not an isolated case,and various spherulite morphologies of lead calcium apatite spherulitehave been observed by the authors in other lead water pipes from theUnited Kingdom. The SEM and EDS images of some of these spherulitesare shown in 45 or to surface inhomogeneities. The latter might arise from the latticestrain caused by the incorporation of Ca2+ and Pb2+ ions of different size or by the incorporation of other ions suchas CO32– and Al3+.Lead calcium apatites have the potential to form spherulites becausethey are very insoluble in the presence of phosphate and because theytend to form needles. However, for spherulites to form, the needlesmust undergo small-angle branching. In other systems, this has beenattributed to the adsorption of inorganic or organic impurities4 = 30 mg/L) was 5–10 times that found in thelead pipe (PO4 = 3–6 mg/L) because the laboratorytime available for the experiments was short. For the same reason,the pH was reduced to pH 5.5 to enhance the rate of dissolution ofthe hydrocerussite crystals. Calcium was added as dissolved calciumcarbonate, which meant that both Pb2+ and Ca2+ ions were present. Citric acid was used because it had been shownto promote spherulitic growth in calcium carbonate27 and fluorapatite precipitation.39 The laboratory experiments indicated that spherulites of lead calciumapatite could be grown when hydrocerussite was added to soft and hardwaters containing citric acid at pH 5.5. The control experiments withoutcitric acid did not produce spherulites, and so citric acid was afactor in spherulite crystal growth. It is not clear at this stagewhether a mixture of soluble Ca and Pb is needed for spherulite formationat pH 5.5, but it might be important at the higher pH values foundin tap waters.Thelaboratory experiments were undertaken to acquire a betterunderstanding of spherulite crystal growth. The aim was not to mimicconditions on the lead pipe but to explore whether lead calcium apatitespherulites could be grown. Hydrocerussite was used as the sourceof Pb because lead carbonate minerals occur in the mineral scale andbecause they are more soluble than lead calcium apatite minerals,when phosphate is present. In addition, hydrocerussite provided asurface for reprecipitation reactions. The concentration of phosphate thatforms stable complexes with divalent metal ions. At pH 5.5, and inthe presence of lead, most of the citrate forms PbCit– ions. Solubility and speciation calculations using PHREEQC  indicate that the amount of leadsolubilized in a suspension of hydrocerussite in soft water at pH5.5 will be ≈100 mg/L and that in hard water will be ≈60mg/L. Such high concentrations of soluble lead are unlikely in themineral scale of a lead water pipe. This is one reason why reducingthe citric acid concentration and increasing the pH will be neededin future experiments to reduce the concentration of soluble lead.Spherulites might still grow under these conditions, especially ifstrong adsorption of the citrate molecules onto the crystal surfaceis more important than the concentration of PbCit– ions.One consequence of using citric acid at 0.5 mMwas that a largeamount of lead would have been solubilized. Citric acid is a smalltriprotic organic acid, H46 Other candidates include organic molecules suchas extracellular polymeric substances, EPS, that are released by bacteria,as biofilms have been observed on lead water pipes.47 Spherulitic lead phosphate crystals have been grown inthe laboratory in the presence of aerobic Acetobacter sp.,48 although the media was rich inorganics, which is not the case for tap waters. Regardless of thesource, dissolved organic molecules can act as crystal growth inhibitors.In lead pipes, they might reduce the rate of lead calcium apatitegrowth, thereby reducing the effectiveness of phosphate dosing. Thisis why the spherulites are so interesting as they might help to identifydissolved organic molecules that effect the chemistry of lead calciumapatite and hence effect the release of lead. Without further study,it is not yet possible to say when the spherulites grew on the leadpipe. They may have grown soon after the lead pipe was first exposedto phosphate in 2001 when the dissolution equilibria for the existinglead carbonate phases would have changed.The results from the citric acid experiments suggestthat dissolvedorganic molecules are important for spherulite formation in lead waterpipes. Possible candidates include humic and fulvic acids that arepresent in tap waters. These molecules, which contain carboxylic groups,are already known to affect the concentration of lead in tap water.49 With this in mind, there is a small possibility that the spherulitesgrew when the pipe was transported after it was drained on site. However,a simple calculation of the pipe geometry and the number of molesof lead and phosphate indicates that there was insufficient concentration of soluble phosphatein a 1 mm film of tap water (a worst-case scenario) to account forthe amount of lead calcium apatite seen. Furthermore, spheruliteshave since been seen in pipes that are emptied and immediately air-dried.Recently, the importanceof lead pipe sample methodology for ensuringaccurate microscopy results has been raised.If the mineral scale was homogeneous, then only one mineral phasewould be present, and according to thermodynamics, this would be themost insoluble mineral phase. In lead pipes supplied with phosphate-dosedtap waters, lead calcium apatite minerals would grow, eventually replacingthe more soluble minerals until only lead calcium apatite was present.However, lead water pipes such as those in the United Kingdom areheterogeneous with a three-layer structure, and the amount of leadcalcium apatite in the scales of these pipes varies considerably frompipe to pipe. Full conversion to a lead phosphate mineral has nottaken place despite 20 years of phosphate dosing.43– and Ca2+ ions in the inner layer is consistent with thelatter. The fact that one of the two prominent XRD peaks was broadand the other narrow, which suggested that the crystals of PbO wereanisotropic in shape, either thin plates or thin needles.The innerlayer on this lead pipe was very prominent comprising the highly solublemineral litharge, PbO. The mechanism for PbO growth is not known.It might be solution-mediated or involve a solid-state transitionfrom lead metal to lead oxide. The absence of POThemiddle layer was a mixture of lead carbonate minerals (hydrocerussiteand plumbonacrite) and lead calcium apatite spherulites. The leadcarbonate minerals are much more soluble than the lead calcium apatiteswhen phosphate is present, which means that the carbonates shoulddissolve and lead calcium apatite should precipitate. This is a precipitationdissolution process. The addition of phosphate causes the water incontact with the lead carbonate minerals to become supersaturatedwith respect to lead calcium apatites and these minerals grow. Thisremoves soluble lead, causing the lead carbonate minerals to becomeundersaturated and dissolve.52 Studies on ICPD tend to use relatively large crystals, typically0.1–5.0 mm in diameter. Because of this, the more insolublemineral may have time to form a protective outer layer on the surfaceof the dissolving mineral before complete dissolution takes place,thereby reducing the rate of dissolution. This outer layer can slowthe diffusion of ions to and from the reaction interface, especiallyif its porosity is low. In the case of lead water pipes, the combinedinner and middle layers are typically 50–200 μm thick,and so protective surface layers can also develop. This would explainwhy the litharge, PbO, inner layer is stable despite being highlysoluble. It follows that the porosity of the mineral scales may beimportant. The difference in porosity may explain why some U.K. leadpipes contain more lead calcium apatite minerals than other pipeseven when they are exposed to similar pH, alkalinity, and phosphateconcentrations. More porous layers would result in more conversionof the soluble lead oxide and lead carbonate minerals to lead calciumapatite.The development of the layeredstructure can be attributed to interfacecoupled dissolution precipitation processes (ICPD) in which nucleationand growth of the more insoluble mineral occurs on or near a dissolvingcrystal. Precipitation and dissolution are spatially coupled, andone mineral replaces by another. This process, also called a mineralreplacement reaction, has been extensively studied in geochemistry.2. The cavities are interesting asthey might provide local environments in which the water chemistryis different from that of the bulk water flowing through the pipe.Cavities have been observed in mineral formations that are in contactwith biofilms,53 and therefore, it is conceivablethat bacteria might play a role in lead water pipes. The presenceof phosphate in tap water would help in this regard.Other heterogeneous features of the mineral scale werethe presenceof mineralized channels (plumes) through the inner layer, which connectedthe middle layer to the lead metal, and the presence of cracks andcavities. All are features of minerals undergoing ICPD. In ICPD, thechannels and cracks provide an easier route by which ions can diffusethrough the mineral layers. The simplest explanation for the cavitiesis that the rate of dissolution was faster than the rate of precipitation.However, it is not clear when this event took place. The cavitiesmay predate phosphate dosing. Cavities have also been observed inlead pipe scales from North America, where they are associated withthe dissolution of PbOSpherulitesrepresent a pathway by which phosphate is mineralized in the mineralscale of the lead pipe, and this mineralization may account for thereduction in lead concentration seen after phosphate dosing.This study confirmed that the unusual rounded, wheatsheaf- or, dumb-bell-shapedcrystals seen in some lead water pipes, after phosphate dosing, werespherulitic, comprising a radial arrangement of needles. The spheruliteswere present in the middle layer of the mineral scale and must haveformed by mineral replacement reactions in which lead carbonate orlead oxide minerals, formed prior to phosphate dosing, were dissolvedand more insoluble lead calcium apatite minerals were precipitated.Dissolution precipitation reactions were replicated in the laboratoryand showed that spherulites of lead calcium apatite could be grownby adding the lead carbonate mineral hydrocerussite to soft and hardwaters containing phosphate, chloride, and citric acid at pH 5.5.However, no spherulites formed when citric acid was absent, whichindicated that dissolved organic molecules might play a role in spheruliticlead calcium apatite formation on lead water pipes.Future workwill investigate whether laboratory spherulites canbe grown under conditions closer to those found in tap waters . It will also look at the impactof supersaturation and reaction rate on spherulite formation and willidentify the origin of the dissolved organic molecules, whether theyare from humic and fulvic acids in tap water or from biofilms attachedto the walls of the lead pipe.