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Iijima M, Du C, Abbott C, Doi Y, Moradian-Oldak J. Control of apatite crystal growth by the co-operative effect of a recombinant porcine amelogenin and fluoride. Eur J Oral Sci 2006; 114 (Suppl. 1): 304–307 © Eur J Oral Sci, 2006 --> Recently, we used native amelogenins extracted from developing pig enamel to examine the combined effect of fluoride and amelogenins on the growth of octacalcium phosphate (OCP) and apatite crystals. The purpose of the present study was to investigate this combined effect using a highly purified recombinant amelogenin. We applied porcine amelogenin (rP172) and fluoride in a dual‐membrane system as a model for tooth enamel formation. The combination of rP172 and fluoride in this system resulted in the formation of rod‐like apatite crystals. On the other hand, without fluoride, rod‐like OCP crystals of a comparable size were formed, and rather large hexagonal prisms of mixed crystals of OCP and apatite grew without amelogenins. Thus, highly purified and homogeneous recombinant amelogenin, in co‐operation with F, regulated the mineral phase, habit, and size of crystals in the same manner as the extracted heterogeneous porcine amelogenins. We suggest that in both cases the control over the crystal phase and morphology was a direct effect of amelogenin protein serving as a scaffold for apatite mineralization. [ABSTRACT FROM AUTHOR]

Enamel repair is crucial for restoring tooth function and halting dental caries. However, contemporary research often overlooks the retention of organic residues within the repair layer, which hinders the growth of dense crystals and compromises the properties of the repaired enamel. During the maturation of natural enamel, the organic matrix undergoes enzymatic processing to facilitate further crystal growth, resulting in a highly mineralized tissue. Inspired by this process, a biomimetic self-maturation mineralization system is developed, comprising ribonucleic acid-stabilized amorphous calcium phosphate (RNA-ACP) and ribonuclease (RNase). The RNA-ACP induces initial mineralization in the form of epitaxial crystal growth, while the RNase present in saliva automatically triggers a biomimetic self-maturation process. The mechanistic study further indicates that RNA degradation prompts conformational rearrangement of the RNA-ACP, effectively excluding the organic matter introduced earlier. This exclusion process promotes lateral crystal growth, resulting in the generation of denser enamel-like apatite crystals that are devoid of organic residues. This strategy of eliminating organic residues from enamel crystals enhances the mechanical and physiochemical properties of the repaired enamel. The present study introduces a conceptual biomimetic mineralization strategy for effective enamel repair in clinical practice and offers potential insights into the mechanisms of biomineral formation., (© 2024 Wiley‐VCH GmbH.)

Amelogenin protein is involved in organized apatite crystallization during enamel formation. Brushite (CaHPO 4 ·2H 2 O), one of the precursors of hydroxyapatite mineralization in vitro , has been used for fabrication of biomaterials for hard tissue repair. In order to explore its potential application in biomimetic material synthesis, we studied the influence of the enamel protein amelogenin on brushite morphology and phase transformation to monetite. Our results show that amelogenin can adsorb onto the surface of brushite, leading to the formation of layered morphology on the (010) face. Amelogenin promoted the phase transformation of brushite into monetite (CaHPO 4 ) in the dry state, presumably by interacting with crystalline water layers in brushite unit cells. Changes to the crystal morphology mediated by amelogenin continued even after the phase transformation from brushite to monetite, leading to the formation of organized platelets with an interlocked structure. This effect of amelogenin on brushite morphology and the phase transformation to monetite could provide a new approach to developing biomimetic materials., Competing Interests: Notes The authors declare no competing financial interest.

Interactions between enamel matrix proteins are important for enamel biomineralization. In recent in situ studies, we showed that the N-terminal proteolytic product of ameloblastin co-localized with amelogenin around the prism boundaries. However, the molecular mechanisms of such interactions are still unclear. Here, in order to determine the interacting domains between amelogenin and ameloblastin, we designed four ameloblastin peptides derived from different regions of the full-length protein (AB1, AB2 and AB3 at N-terminus, and AB6 at C-terminus) and studied their interactions with recombinant amelogenin (rP172), and the tyrosine-rich amelogenin polypeptide (TRAP). A series of amelogenin Trp variants (rP172(W25), rP172(W45) and rP172(W161)) were also used for intrinsic fluorescence spectroscopy. Fluorescence spectra of rP172 titrated with AB3, a peptide encoded by exon 5 of ameloblastin, showed a shift in λ max in a dose-dependent manner, indicating molecular interactions in the region encoded by exon 5 of ameloblastin. Circular dichroism (CD) spectra of amelogenin titrated with AB3 showed that amelogenin was responsible for forming α-helix in the presence of ameloblastin. Fluorescence spectra of amelogenin Trp variants as well as the spectra of TRAP titrated with AB3 showed that the N-terminus of amelogenin is involved in the interaction between ameloblastin and amelogenin. We suggest that macromolecular co-assembly between amelogenin and ameloblastin may play important roles in enamel biomineralization., Competing Interests: The authors declare no competing financial interests.

Bioinspired synthesis of hierarchically structured calcium phosphate (CaP) material is a highly promising strategy for developing improved bone substitute materials. However, synthesis of CaP materials with outstanding mechanical properties still remains an ongoing challenge. Inspired by the formation of lamellar structure in nacre, we designed an organic matrix composed of chitosan and cis-butenediolic acid (maleic acid, MAc) that could assemble into a layered complex and further guide the mineralization of monetite crystals, resulting in the formation of organized and parallel arrays of monetite platelets with a brick-and-mortar structure. Using the layered monetite-chitosan composite as a precursor, we were able to synthesize hydroxyapatite (HAp) with multiscale hierarchically ordered structure via a topotactic phase transformation process. On the nanoscale, needlelike HAp crystallites assembled into organized bundles that aligned to form highly oriented plates on the microscale. On the large-scale level, these plates with different crystal orientations were stacked together to form a layered structure. The organized structures and composite feature yielded CaP materials with improved mechanical properties close to those of bone. Our study introduces a biomimetic approach that may be practical for the design of advanced, mechanically robust materials for biomedical applications., Competing Interests: Notes The authors declare no competing financial interest.

Amelogenin-chitosan (CS-AMEL) hydrogel has shown great potential for the prevention, restoration, and treatment of defective enamel. As a step prior to clinical trials, this study aimed to examine the efficacy of CS-AMEL hydrogel in biomimetic repair of human enamel with erosive or caries-like lesions in pH-cycling systems. Two models for enamel defects, erosion and early caries, were addressed in this study. Two pH-cycling systems were designed to simulate the daily cariogenic challenge as well as the nocturnal pH conditions in the oral cavity. After pH cycling and treatment with CS-AMEL hydrogel, a synthetic layer composed of oriented apatite crystals was formed on the eroded enamel surface. CS-AMEL repaired the artificial incipient caries by re-growing oriented crystals and reducing the depth of the lesions by up to 70% in the pH-cycling systems. The results clearly demonstrate that the CS-AMEL hydrogel is effective at the restoration of erosive and carious lesions under pH-cycling conditions.

Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Petta V, Moradian-Oldak J, Yannopoulos SN, Bouropoulos N. Dynamic light scattering study of an amelogenin gel-like matrix in vitro. Eur J Oral Sci 2006; 114 (Suppl. 1): 308–314 © Eur J Oral Sci, 2006 --> Amelogenin self‐assembly is critical for the structural organization of apatite crystals during enamel mineralization. The aim of the present study was to investigate the influence of temperature and protein concentration on the aggregation of amelogenin nanospheres at high protein concentrations (> 4.4 mg ml−1) in order to obtain an insight into the mechanism of amelogenin self‐assembly to form higher‐order structures. Amelogenins were extracted from enamel scrapings of unerupted mandibular pig molars. The dynamics of protein solutions were measured using dynamic light scattering (DLS) as a function of temperature and at acidic pH. At pH 4–5.5, three kinds of particles were observed, ranging in size from 3 to 80 nm. At pH 6, heating the solution above ≈ 30°C resulted in a drastic change in the solution transparency, from clear to opaque. Low pH showed no aggregation effect, whilst solutions at a slightly acidic pH exhibited diffusion dynamics associated with the onset of aggregation. In addition, at the same temperature range, the hydrodynamic radii of the aggregates increased drastically, by almost one order of magnitude. These observations support the view that hydrophobic interactions are the primary driving force for the pH‐ and temperature‐sensitive self‐assembly of amelogenin particles in a 'gel‐like' matrix. The trend of self‐assembly in a 'gel‐like matrix' is similar to that in solution. [ABSTRACT FROM AUTHOR]

Sun Z, Ahsan MM, Wang HJ, Du C, Abbott C, Moradian-Oldak J. Assembly and processing of an engineered amelogenin proteolytic product (rP148). Eur J Oral Sci 2006; 114 (Suppl. 1): 59–63 © Eur J Oral Sci, 2006 --> The purpose of this study was to express, characterize, and investigate the self‐assembly of a recombinant porcine amelogenin lacking the hydrophilic 24 C‐terminal amino acids (rP148). To gain further insight into the function of amelogenin processing during enamel mineralization, this protein was also used as a substrate to examine the action of matrix metalloproteinase‐20 (MMP‐20). The assembly properties of rP148 were monitored by dynamic light scattering (DLS). In general, rP148 molecules assemble into monomers, dimers, oligomers, and some nanosphere‐like particles. Depending on the solution conditions, large aggregates were also observed. Matrix metalloproteinase‐20 cleaved the rP148 molecule at a few sites, creating a number of different products, including the tyrosine‐rich amelogenin polypeptide (TRAP). Our data suggest that although rP148 self‐assembles into small particles, its assembly properties are different from those of the full‐length rP172, indicating that the C‐terminal 24 amino acids play a critical role in nanosphere assembly. We further demonstrate that MMP‐20 digests rP148 in a manner that generates a similar proteolytic pattern, as would be expected to occur in vivo. [ABSTRACT FROM AUTHOR]

Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.

We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed that they are structurally similar to the wild-type amelogenin. Utilizing the intrinsic fluorescence of single tryptophan residue and fluorescence resonance energy transfer [FRET], we analyzed the accessibility and strength of their binding with an ameloblast cell membrane-mimicking model membrane (ACML) and a negatively charged liposome used as a membrane model. We found that amelogenin has membrane-binding ability mainly via its N-terminal, close to residues W25 and W45. Significant blue shift was also observed in the fluorescence of a N-terminal peptide following addition of liposomes. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta (AI) with mutations at the N-terminal may be the result of defective amelogenin-cell interactions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The host immune system is known to influence mesenchymal stem cell (MSC)-mediated bone tissue regeneration. However, the therapeutic capacity of hydrogel biomaterial to modulate the interplay between MSCs and T-lymphocytes is unknown. Here it is shown that encapsulating hydrogel affects this interplay when used to encapsulate MSCs for implantation by hindering the penetration of pro-inflammatory cells and/or cytokines, leading to improved viability of the encapsulated MSCs. This combats the effects of the host pro-inflammatory T-lymphocyte-induced nuclear factor kappaB pathway, which can reduce MSC viability through the CASPASE-3 and CAS-PASE-8 associated proapoptotic cascade, resulting in the apoptosis of MSCs. To corroborate rescue of engrafted MSCs from the insult of the host immune system, the incorporation of the anti-inflammatory drug indomethacin into the encapsulating alginate hydrogel further regulates the local microenvironment and prevents pro-inflammatory cytokine-induced apoptosis. These findings suggest that the encapsulating hydrogel can regulate the MSC-host immune cell interplay and direct the fate of the implanted MSCs, leading to enhanced tissue regeneration.

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities., (© 2014 Wiley Periodicals, Inc.)

Mature tooth enamel is acellular and does not regenerate itself. Developing technologies that rebuild tooth enamel and preserve tooth structure is therefore of great interest. Considering the importance of amelogenin protein in dental enamel formation, its ability to control apatite mineralization in vitro , and its potential to be applied in fabrication of future bio-inspired dental material this review focuses on two major subjects: amelogenin and enamel biomimetics. We review the most recent findings on amelogenin secondary and tertiary structural properties with a focus on its interactions with different targets including other enamel proteins, apatite mineral, and phospholipids. Following a brief overview of enamel hierarchical structure and its mechanical properties we will present the state-of-the-art strategies in the biomimetic reconstruction of human enamel.

Purpose: Erosive tooth wear (ETW) is characterized by subsurface demineralization and tooth substance loss with crater formation. Remineralization of subsurface demineralization has previously been demonstrated; however, repair of the eroded surface is still under investigation. This study investigated the effectiveness of mouthwashes containing hydrolyzed wheat protein (HWP) in repairing ETW through promotion of organized crystal growth. Methods: Enamel Erosion was created on 210 enamel blocks by 10-minute demineralization in 1% Citric Acid (pH 3.5). Then, blocks were randomly assigned to seven groups (30/group); (A) 0.2% HWP, B) 1% HWP, (C) 2% HWP, (D) 1% HWP + 0.05% NaF, (E) Listerine™ mouthwash, (F) 0.02% NaF Crest™ Pro-health mouthwash and (G) artificial saliva (AS) only. Groups were subjected to daily pH-cycling consisting of one 5-minute erosive challenge with citric acid, three 1-minute mouthwash treatment periods, and then storage in AS for the rest of the time for 28 days. Treatment effects were assessed using SEM-EDX. Statistical analysis was by ANOVA and Tukey's multiple comparison. Results: In groups exposed to HWP-containing mouthwashes, there was growth of fiber-like crystals that increased in packing density in a dose-dependent manner (0.2%, 1%, 2%) on the eroded enamel surfaces, with increased calcium and phosphate contents on the treated surfaces. The non-HWP-containing groups had the eroded surfaces covered by structureless deposit layer firmly attached to the surface. Conclusion: Treating eroded enamel surface with HWP-containing mouthwash resulted in repair of the damaged tissue by formation of a protective layer of crystal deposits within and on the eroded enamel tissue. [ABSTRACT FROM AUTHOR]

Dentin phosphophoryn (DPP) an extracellular matrix protein activates Wnt signaling in DPSCs (dental pulp stem cells). Wnt/β catenin signaling is essential for tooth development but the role of DPP-mediated Wnt5a signaling in odontogenesis is not well understood. Wnt5a is typically considered as a non-canonical Wnt ligand that elicits intracellular signals through association with a specific cohort of receptors and co-receptors in a cell and context–dependent manner. In this study, DPP facilitated the interaction of Wnt5a with Frizzled 5 and LRP6 to induce nuclear translocation of β-catenin. β-catenin has several nuclear binding partners that promote the activation of Wnt target genes responsible for odontogenic differentiation. Interestingly, steady increase in the expression of Vangl2 receptor suggest planar cell polarity signaling during odontogenic differentiation. In vitro observations were further strengthened by the low expression levels of Wnt5a and β-catenin in the teeth of DSPP KO mice which exhibit impaired odontoblast differentiation and defective dentin mineralization. Together, this study suggests that the DPP-mediated Wnt5a signaling could be exploited as a therapeutic approach for the differentiation of dental pulp stem cells into functional odontoblasts and dentin regeneration. [ABSTRACT FROM AUTHOR]

This study explores the recent advances of and functional insights into hydrogel composites, materials that have gained significant attention for their versatile applications across various fields, including contemporary dentistry. Hydrogels, known for their high water content and biocompatibility, are inherently soft but often limited by mechanical fragility. Key areas of focus include the customization of hydrogel composites for biomedical applications, such as drug delivery systems, wound dressings, and tissue engineering scaffolds, where improved mechanical properties and bioactivity are critical. In dentistry, hydrogels are utilized for drug delivery systems targeting oral diseases, dental adhesives, and periodontal therapies due to their ability to adhere to the mucosa, provide localized treatment, and support tissue regeneration. Their unique properties, such as mucoadhesion, controlled drug release, and stimuli responsiveness, make them ideal candidates for treating oral conditions. This review highlights both experimental breakthroughs and theoretical insights into the structure–property relationships within hydrogel composites, aiming to guide future developments in the design and application of these multifunctional materials in dentistry. Ultimately, hydrogel composites represent a promising frontier for advancing materials science with far-reaching implications in healthcare, environmental technology, and beyond. [ABSTRACT FROM AUTHOR]

Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter‐ and intra‐molecular interactions. Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes. Furthermore, the principle of protein assembly gives guidelines for new biomimetic materials with potential applications in medicine, energy, and nanotechnology. Atomic force microscopy (AFM) is a powerful tool for investigating protein assembly and interactions across spatial scales (single molecules to cells) and temporal scales (milliseconds to days). It has significantly contributed to understanding nanoscale architectures, inter‐ and intra‐molecular interactions, and regulatory elements that determine protein structures, assemblies, and functions. This review describes recent advancements in elucidating protein assemblies with in situ AFM. We discuss the structures, diffusions, interactions, and assembly dynamics of proteins captured by conventional and high‐speed AFM in near‐native environments and recent AFM developments in the multimodal high‐resolution imaging, bimodal imaging, live cell imaging, and machine‐learning‐enhanced data analysis. These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research. [ABSTRACT FROM AUTHOR]

Hydroxyapatite (HA) forms an essential constituent of human teeth and bone. Its distinctive characteristic features, such as bioactivity and osteoconductivity, make it an ideal candidate to be used as an implant coating in restorative dentistry and maxillofacial surgery for bone regeneration. However, low fracture toughness and brittleness are a few of the inherent features of HA, which limit its application in load-bearing areas. The potential of HA to engage its lattice structure with either partial or complete substitution with external ions has become an increasing area of research as this phenomenon has the potential to enhance the biological and functional properties of the material. Consequently, this review aimed to highlight the role of various substituted ions in dental applications. Data indicate that the newly formed HA-substituted biomaterials demonstrate enhanced remineralization and antimicrobial activity along with improved hardness. Ion-substituted HA offers a promising strategy for future clinical research as these materials may be incorporated into various dental products for therapeutic treatments. [ABSTRACT FROM AUTHOR]

Stem cell-mediated bio-root regeneration is an alternative tooth replacement strategy; however, physiologically functional bio-root regeneration with distinctive dentin structure remains challenging. In this study, the distinct arrangements of collagen fibril bundles were identified that account for hierarchical structural differences between dentin, cementum, and alveolar bone. Thus, an "engineered pre-dentin" was fabricated, which was a dentin hierarchical structure mimicking collagen (MC) scaffold, with well-aligned hierarchical mineralized collagen fibril bundles. The results revealed that it has a stronger effect on promoting biological root regeneration in nude mice and miniature pigs with dental pulp stem cell (DPSC) and periodontal ligament stem cell (PDLSC) sheets compared to hydroxyapatite tricalcium phosphate (HA/TCP). The success rate in the MC group was also higher than that in the HA/TCP group (67% and 33%, respectively). In conclusion, the hierarchical dentin-mimicking scaffold can enhance the regeneration of bio-roots, which provides a promising strategy for tooth regeneration. [ABSTRACT FROM AUTHOR]

Calcium phosphates (CaPs) and their substituted derivatives encompass a large number of compounds with a vast presence in nature that have aroused a great interest for decades. In particular, hydroxyapatite (HAp, Ca10(OH)2(PO4)6) is the most abundant CaP mineral and is significant in the biological world, at least in part due to being a major compound in bones and teeth. HAp exhibits excellent properties, such as safety, stability, hardness, biocompatibility, and osteoconductivity, among others. Even some of its drawbacks, such as its fragility, can be redirected thanks to another essential feature: its great versatility. This is based on the compound's tendency to undergo substitutions of its constituent ions and to incorporate or anchor new molecules on its surface and pores. Thus, its affinity for biomolecules makes it an optimal compound for multiple applications, mainly, but not only, in biological and biomedical fields. The present review provides a chemical and structural context to explain the affinity of HAp for biomolecules such as proteins and nucleic acids to generate hybrid materials. A size-dependent criterium of increasing complexity is applied, ranging from amino acids/nucleobases to the corresponding macromolecules. The incorporation of metal ions or metal complexes into these functionalized compounds is also discussed. [ABSTRACT FROM AUTHOR]

Finding biomolecules in fossils is a challenging task due to their degradation over time from physical, chemical, and biological factors. The primary hypothesis for explaining the presence of biomolecules in fossilized bones tissues suggests their survival in the fossilization process. In contrast, some of these biomolecules could either derive from bacteria biofilm, thus without a direct relationship with the fossil record or could be an artifact from measurement procedures. Raman spectroscopy studies across various fossil ages and environments have detected multiple bands ranging from 1200 to 1800 cm−1 associative of organic compounds. However, the significance of these bands remains elusive. Our research aims to address this issue through a deep Raman spectroscopy investigation on Pleistocene teeth from Tayassu and Smilodon populator. These fossils were obtained from a well-preserved stratigraphic succession in Toca de Cima do Pilão cave, near the National Park of Serra da Capivara in semiarid Brazil. We propose two hypotheses to explain the presence of organic compounds related to 1200 to 1800 cm−1 Raman spectral range in fossil tissues: (i) these bands are biological signatures of preserved fossil biomolecules, or (ii) they are exogenous biological signatures associated with the bacterial biofilm formation during post-depositional processes. Our results align with the latter hypothesis, followed by biofilm degradation. However, the specific mechanisms involved in the natural biofilm degradation in fossil records remain unexplored in this study. In our case, the formation of biofilm on fossil bones is attributed to the oligotrophic conditions of the cave sediment matrix. We present a comprehensive model to elucidate the existence of biofilm on fossilized tissues, emphasizing the pivotal role of post-depositional processes, especially water action, in the cave environment. As the fossils were discovered in a cave setting, post-depositional processes significantly contribute to the formation of the biofilm matrix. Although our study provides insights into biofilm formation, further research is needed to delve into the specific mechanisms driving natural biofilm degradation in fossils. [ABSTRACT FROM AUTHOR]

Background: Tyrosine-rich amelogenin peptide (TRAP) is the main amelogenin digestion product in the developmental enamel matrix. It has been shown to promote remineralization of demineralized enamel in our previous study. However, direct evidence of the effect of TRAP on the morphology and nanostructure of crystal growth on an enamel surface has not been reported. This study aimed to examine the effect of TRAP on the morphology of calcium phosphate crystals grown on early enamel erosion using a pH-cycling model. Methods: Eroded lesions were produced in human premolars by 30-second immersion in 37% phosphoric acid. Forty-five samples of eroded human premolar enamel blocks were selected and randomly divided into 3 groups: deionized water (DDW, negative control); 100 µg/mL TRAP, and 2 ppm sodium fluoride (NaF, positive control group). For 14 days, the specimens were exposed to a pH-cycling model. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods, the surface morphology, calcium-phosphorus ratio, and enamel surface roughness were examined. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were used to assess crystal characteristics. Results: After pH-cycling, compared to the two control groups, the surface of the eroded enamel of the peptide TRAP group shows a large number of new, densely arranged rod-like crystals, parallel to each other, regularly arranged, forming an ordered structure, with crystal morphology similar to that of natural enamel. The crystals are mostly hydroxyapatite (HA). Conclusion: This study demonstrates that the peptide TRAP modulates the formation of hydroxyapatite in eroded enamel and that the newly formed crystals resemble natural enamel crystals and promote the remineralization of enamel, providing a promising biomaterial for remineralization treatment of enamel lesions. [ABSTRACT FROM AUTHOR]