Abstract

Conventional views associate microbial biofilm with demineralization in root caries (RC) onset, while research on their collagenases role in the breakdown of collagen matrix has been sporadically developed, primarily in vitro. Recent discoveries, however, reveal proteolytic bacteria enrichment, specially Porphyromonas and other periodontitis-associated bacteria in subgingivally extended lesions, suggesting a potential role in RC by the catabolism of dentin organic matrix. Moreover, genes encoding proteases and bacterial collagenases, including the U32 family collagenases, were found to be overexpressed in both coronal and root dentinal caries. Despite these advancements, to prove microbial collagenolytic proteases’ definitive role in RC remains a significant challenge. A more thorough investigation is warranted to explore the potential of anti-collagenolytic agents in modulating biofilm metabolic processes or inhibiting/reducing the size of RC lesions. Prospective treatments targeting collagenases and promoting biomodification through collagen fibril cross-linking show promise for RC prevention and management. However, these studies are currently in the in vitro phase, necessitating additional research to translate findings into clinical applications. This is a comprehensive state-of-the-art review aimed to explore contributing factors to the formation of RC lesions, particularly focusing on collagen degradation in root tissues by microbial collagenases.

Keywords
Dental caries; Root caries; Collagenases; Microbial collagenase; Matrix metalloproteinases

Introduction

Root caries (RC) is an incident condition linked to life expectancy global increase and edentulism concurrent decline, which is attributed to improved hygiene standards and widespread access to fluoride products.¹1 - Hayes M, Burke F, Allen PF. Incidence, prevalence and global distribution of root caries. Monogr Oral Sci. 2017;26:1-8. doi: 10.1159/000479301
https://doi.org/10.1159/000479301...

2 - Hariyani N, Setyowati D, Spencer AJ, Luzzi L, Do LG. Root caries incidence and increment in the population - a systematic review, meta-analysis and meta-regression of longitudinal studies. J Dent. 2018;77:1-7. doi: 10.1016/j.jdent.2018.06.013
https://doi.org/10.1016/j.jdent.2018.06.... -³3 - Griffin SO, Griffin PM, Swann JL, Zlobin N. Estimating rates of new root caries in older adults. J Dent Res. 2004;83(8):634-8. doi: 10.1177/154405910408300810
https://doi.org/10.1177/1544059104083008... In the clinical scenario, challenges related to operational difficulties in restorative treatments of RC lesions arise due to microanatomical factors. These include issues such as moisture control, contamination with blood or gingival crevicular fluid, high mechanical stress concentration in the area, and cavities broad and shallow shape, which tends to spread horizontally.⁴4 - Damé-Teixeira N, Parolo CC, Maltz M. Specificities of caries on root surface. Monogr Oral Sci. 2017;26:15-25. doi: 10.1159/000479303
https://doi.org/10.1159/000479303... ,⁵5 - Reis A, Soares PV, de Geus J, Loguercio AD. Clinical performance of root surface restorations. Monogr Oral Sci. 2017;26:115-24. doi: 10.1159/000479353
https://doi.org/10.1159/000479353... Additionally, significant amounts of organic material on the surface indicate that these lesions protocol often differs from the one applied to coronal surfaces.⁴4 - Damé-Teixeira N, Parolo CC, Maltz M. Specificities of caries on root surface. Monogr Oral Sci. 2017;26:15-25. doi: 10.1159/000479303
https://doi.org/10.1159/000479303... ,⁶6 - Gostemeyer G, Mata C, McKenna G, Schwendicke F. Atraumatic vs conventional restorative treatment for root caries lesions in older patients: meta- and trial sequential analysis. Gerodontology. 2019;36(3):285-93. doi: 10.1111/ger.12409
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https://doi.org/10.1159/000479352... The available evidence regarding the most effective approaches for disease treatment is characterized by low to moderate certainty.⁸8 - Wierichs RJ, Meyer-Lueckel H. Response to letter to the editor, "Systematic review on noninvasive treatment of root caries lesions". J Dent Res. 94. 2015. doi: 10.1177/0022034515591480
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9 - Meyer-Lueckel H, Machiulskiene V, Giacaman RA. How to intervene in the root caries process? Systematic review and meta-analyses. Caries Res;2019. p. 1-10. doi: 10.1159/000501588
https://doi.org/10.1159/000501588... -¹⁰10 - Paris S, Banerjee A, Bottenberg P, Breschi L, Campus G, Doméjean S, et al. How to intervene in the caries process in older adults: a joint ORCA and EFCD expert Delphi Consensus Statement. Caries Res. 2020;54(5-6):1-7. doi: 10.1159/000510843
https://doi.org/10.1159/000510843... Consequently, there is a need for studying new products and protocols in prevention and treatment of RC lesions.

Understanding RC etiopathogenesis can pave the way for innovative research, identifying potential therapeutics. Novel targets for these approaches can be found within dysbiotic biofilms. For example, recently identified overexpressed genes in RC-associated biofilms have emerged as potential drug targets, including those encoding bacterial collagenases, mobile elements, transcriptional regulators, carbohydrate metabolism enzymes, metabolic activity proteins, sugar transporters, stress tolerance factors, and pH regulators.¹¹11 - Dame-Teixeira N, Parolo CC, Maltz M, Tugnait A, Devine D, Do T. Actinomyces spp. gene expression in root caries lesions. J Oral Microbiol. 2016;8:32383. doi: 10.3402/jom.v8.32383
https://doi.org/10.3402/jom.v8.32383...

12 - Damé-Teixeira N, Parolo C, Maltz M, Rup A, Devine D, Do T. Gene expression of bacterial collagenolytic proteases in root caries. J Oral Microb. 2018;10:1424475. doi: 10.1080/20002297.2018.1424475
https://doi.org/10.1080/20002297.2018.14...

13 - Santos HS, Do T, Parolo CC, Poloni JF, Maltz M, Arthur RA, et al. Streptococcus mutans gene expression and functional profile in root caries: an RNA-Seq study. Caries Res. 2022;56(2):116-28. doi: 10.1159/000524196
https://doi.org/10.1159/000524196... -¹⁴14 - Santos HS, Damé-Teixeira N, Nagano MH, Do T, Parolo CC, Maltz M, et al. Acid tolerance of Lactobacillus spp. on root carious lesions: a complex and multifaceted response. Arch Oral Biol. 2023;156:105820. doi: 10.1016/j.archoralbio.2023.105820
https://doi.org/10.1016/j.archoralbio.20... Given these genes functions, to explore microbial collagenases involvement in the development of RC lesions seems to be particularly promising for developing future biochemical products that aim to preserve the integrity of the root collagen matrix. However, studies in this area face significant challenges. A recent systematic review showed only four studies examining microbial collagenases in clinical samples, either their gene expression or their activity.¹⁵15 - Barbosa CB, Monici Silva I, Cena JA, Stefani CM, Dame-Teixeira N. Presence of host and bacterial-derived collagenolytic proteases in carious dentin: a systematic review of ex vivo studies. Front Cell Infect Microbiol. 2023;13:1278754. doi: 10.3389/fcimb.2023.1278754
https://doi.org/10.3389/fcimb.2023.12787... While bacteria would typically not expend energy to express a gene if it was nonfunctional to the cell, gene expression alone does not guarantee enzyme activity. A more thorough investigation is warranted to explore bacterial collagenolytic activity and enzyme inhibitors potential to modulate biofilm metabolic processes or to inhibit/reduce the size of RC lesions. If confirmed, the biofilm modulation could serve as complementary elements in future RC management in combination with fluoride products.

This state-of-the-art review aims to explore characteristics involved in RC lesions etiopathogenesis, particularly focusing on root collagen degradation by microbial collagenases. We revisited the peculiarities of dental root surfaces, lesion development, and other proteases part in collagen degradation. Finally, we discussed future perspectives for clinically preventing and managing RC.

Revisiting the particularities of dental root surfaces composition and collagen structure

The cement covering root surfaces is characterized by a highly fibrous matrix comprising well-oriented collagen fiber bundles, which serve as anchorage points for periodontal ligament. It consists of approximately 45%–50% inorganic content and 50% organic content. Similarly, root dentin has high organic content (approximately 18%), with other components including 70% inorganic content and 12% water.⁴4 - Damé-Teixeira N, Parolo CC, Maltz M. Specificities of caries on root surface. Monogr Oral Sci. 2017;26:15-25. doi: 10.1159/000479303
https://doi.org/10.1159/000479303... ,¹⁶16 - Goldberg M, Kulkarni AB, Young M, Boskey A. Dentin: structure, composition and mineralization. Front Biosci (Elite Ed). 2011 Jan 1;3:711-35. doi: 10.2741/e281
https://doi.org/10.2741/e281... The microstructure of the root dentin matrix contains tubules which accommodate odontoblasts cytoplasmic extensions (see Goldberg, et al.¹⁶16 - Goldberg M, Kulkarni AB, Young M, Boskey A. Dentin: structure, composition and mineralization. Front Biosci (Elite Ed). 2011 Jan 1;3:711-35. doi: 10.2741/e281
https://doi.org/10.2741/e281... and Bosshardt, et al.¹⁷17 - Bosshardt DD, Selvig KA. Dental cementum: the dynamic tissue covering of the root. Periodontol 2000. 1997;13:41-75. doi: 10.1111/j.1600-0757.1997.tb00095.x
https://doi.org/10.1111/j.1600-0757.1997... for a comprehensive description of root hard tissues microstructure). In both root hard tissues, an elevated magnesium concentration can enhance hydroxyapatite crystals solubility compared to those found in enamel,¹⁸18 - Hoppenbrouwers PM, Driessens FC, Borggreven JM. The vulnerability of unexposed human dental roots to demineralization. J Dent Res. 1986;65(7):955-8. doi: 10.1177/00220345860650071101
https://doi.org/10.1177/0022034586065007... since magnesium inhibit and regulate crystal growth by replacing calcium ions.¹⁹19 - Klimuszko E, Orywal K, Sierpinska T, Sidun J, Golebiewska M. Evaluation of calcium and magnesium contents in tooth enamel without any pathological changes: in vitro preliminary study. Odontology. 2018;106(4):369-76. doi: 10.1007/s10266-018-0353-6
https://doi.org/10.1007/s10266-018-0353-... ,²⁰20 - Teruel JD, Alcolea A, Hernández A, Ruiz AJ. Comparison of chemical composition of enamel and dentine in human, bovine, porcine and ovine teeth. Arch Oral Biol. 2015;60(5):768-75. doi: 10.1016/j.archoralbio.2015.01.014
https://doi.org/10.1016/j.archoralbio.20... Meanwhile, their organic matrix are primarily composed of type I collagen,²¹21 - Christner P, Robinson P, Clark CC. A preliminary characterization of human cementum collagen. Calcif Tissue Res. 1977;23(2):147-50. doi: 10.1007/BF02012780
https://doi.org/10.1007/BF02012780... although other non-collagen proteins, such as bone sialoproteins and osteopontin are also present in lower abundance.¹⁷17 - Bosshardt DD, Selvig KA. Dental cementum: the dynamic tissue covering of the root. Periodontol 2000. 1997;13:41-75. doi: 10.1111/j.1600-0757.1997.tb00095.x
https://doi.org/10.1111/j.1600-0757.1997... ,²¹21 - Christner P, Robinson P, Clark CC. A preliminary characterization of human cementum collagen. Calcif Tissue Res. 1977;23(2):147-50. doi: 10.1007/BF02012780
https://doi.org/10.1007/BF02012780... ,²²22 - Breschi L, Maravic T, Cunha SR, Comba A, Cadenaro M, Tjäderhane L, et al. Dentin bonding systems: from dentin collagen structure to bond preservation and clinical applications. Dent Mater. 2018;34(1):78-96.

The term “collagen” encompasses 28 proteins, varying in size, function, and tissue distribution (Table 1) (for additional information, consult studies ²³23 - Shoulders MD, Raines RT. Collagen structure and stability. Annu Rev Biochem. 2009;78:929-58. doi: 10.1016/j.dental.2017.11.005
https://doi.org/10.1016/j.dental.2017.11...

24 - Ricard-Blum S, Ruggiero F. The collagen superfamily: from the extracellular matrix to the cell membrane. Pathol Biol (Paris). 2005;53(7):430-42. doi: 10.1016/j.patbio.2004.12.024
https://doi.org/10.1016/j.patbio.2004.12... -²⁵25 - Ricard-Blum S. The collagen family. Cold Spring Harb Perspect Biol. 2011;3(1):a004978. doi: 10.1101/cshperspect.a004978
https://doi.org/10.1101/cshperspect.a004... ). They share a common characteristic: the formation of a supramolecular structure with a triple helix composed of three alpha polypeptide chains within an extracellular matrix.²⁴24 - Ricard-Blum S, Ruggiero F. The collagen superfamily: from the extracellular matrix to the cell membrane. Pathol Biol (Paris). 2005;53(7):430-42. doi: 10.1016/j.patbio.2004.12.024
https://doi.org/10.1016/j.patbio.2004.12... ,²⁶26 - Gelse K, Pöschl E, Aigner T. Collagens--structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003;55(12):1531-46. doi: 10.1016/j.addr.2003.08.002
https://doi.org/10.1016/j.addr.2003.08.0... They are classified according to structure complexity, splice variants, presence of non-helical domains, and their assembly and function. Collagen can be either homotrimer (when formed by three identical chains) or heterotrimer (when formed by two or more different chains).²⁶26 - Gelse K, Pöschl E, Aigner T. Collagens--structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003;55(12):1531-46. doi: 10.1016/j.addr.2003.08.002
https://doi.org/10.1016/j.addr.2003.08.0... In both cases, the three chains supercoil around the central axis, forming an extended helix. Each chain is formed by groups of 18 amino acids.²⁶26 - Gelse K, Pöschl E, Aigner T. Collagens--structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003;55(12):1531-46. doi: 10.1016/j.addr.2003.08.002
https://doi.org/10.1016/j.addr.2003.08.0... ,²⁷27 - Hulmes DJ, Miller A. Quasi-hexagonal molecular packing in collagen fibrils. Nature. 1979;282(5741):878-80. doi: 10.1038/282878a0
https://doi.org/10.1038/282878a0... A structural prerequisite for mounting on a triple helix is a glycine residue (always positioned in the center, the smallest amino acid) in each third position of the polypeptide chains, resulting in a Gly-X-Y repeat structure that characterizes and identifies the “collagen” domains. The X and Y positions are often occupied by proline and hydroxyproline.²⁴24 - Ricard-Blum S, Ruggiero F. The collagen superfamily: from the extracellular matrix to the cell membrane. Pathol Biol (Paris). 2005;53(7):430-42. doi: 10.1016/j.patbio.2004.12.024
https://doi.org/10.1016/j.patbio.2004.12...

The complex molecular structure of type I collagen, predominantly found on tooth root surfaces, is characterized by its ability to gather in oriented supramolecular aggregates with heterotrimeric structure, which contributes to molecular stabilization and dentin mechanical properties. Its fibrils represent a structural pillar and are perpendicularly connected by non-collagen proteins.²⁷27 - Hulmes DJ, Miller A. Quasi-hexagonal molecular packing in collagen fibrils. Nature. 1979;282(5741):878-80. doi: 10.1038/282878a0
https://doi.org/10.1038/282878a0... ,²⁸28 - Varma S, Orgel JP, Schieber JD. Nanomechanics of Type I Collagen. Biophys J. 2016;111(1):50-6. doi: 10.1016/j.bpj.2016.05.038
https://doi.org/10.1016/j.bpj.2016.05.03... This molecular structure is quite complex, formed by a triple helix with approximately 300 nm, comprised of three parallel polypeptide chains coiled together to form fibrils (Figure 1). By this arrangement, the N terminals of two axially adjacent triple helices are separated by the distance of D = 67 nm, and the N terminals of the two triple helices adjacent to the side are axially separated by 0.54 nm.²⁷27 - Hulmes DJ, Miller A. Quasi-hexagonal molecular packing in collagen fibrils. Nature. 1979;282(5741):878-80. doi: 10.1038/282878a0
https://doi.org/10.1038/282878a0... This staggered arrangement creates alternating regions of low and high protein density along the fibril axis with a repetitive unit of length D (67 nm).²⁷27 - Hulmes DJ, Miller A. Quasi-hexagonal molecular packing in collagen fibrils. Nature. 1979;282(5741):878-80. doi: 10.1038/282878a0
https://doi.org/10.1038/282878a0... ,²⁸28 - Varma S, Orgel JP, Schieber JD. Nanomechanics of Type I Collagen. Biophys J. 2016;111(1):50-6. doi: 10.1016/j.bpj.2016.05.038
https://doi.org/10.1016/j.bpj.2016.05.03... For its characteristics, collagen can only be degraded by collagenases. In the aqueous phase, the triple helix is cleaved in its internal structure by digesting the amino group in a ‘Gly-Leu’ bond, enabling intramolecular flexibility, and facilitating specific proteolytic cleavage.²⁶26 - Gelse K, Pöschl E, Aigner T. Collagens--structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003;55(12):1531-46. doi: 10.1016/j.addr.2003.08.002
https://doi.org/10.1016/j.addr.2003.08.0... ,²⁹29 - Takahashi N, Nyvad B. Ecological hypothesis of dentin and root caries. Caries Res. 2016;50(4):422-31. doi: 10.1159/000447309
https://doi.org/10.1159/000447309...

Figure 1
Representation of type I collagen adapted from Gelse, et al. (2003) and Varma, et al. (2016). N=N-terminal telopeptide region with 16 aa residues. C=C-terminal telopeptide region with 26 aa residues D=repeating unit of collagen fibril of length 67mm

Dentinal collagen structure significance becomes apparent in the context of gingival recession. When recession occurs, a new ecological microenvironment emerges on the root surface, transitioning from an anaerobic to an aerobic setting with variable nutrient availability.³⁰30 - Gross EL, Beall CJ, Kutsch SR, Firestone ND, Leys EJ, Griffen AL. Beyond Streptococcus mutans: dental caries onset linked to multiple species by 16S rRNA community analysis. PLoS One. 2012;7(10). doi: 10.1371/journal.pone.0047722
https://doi.org/10.1371/journal.pone.004... The exposed root region, now susceptible to microbial infiltration, is vulnerable to demineralization by the acidic oral microbiota during the carious process.²⁹29 - Takahashi N, Nyvad B. Ecological hypothesis of dentin and root caries. Caries Res. 2016;50(4):422-31. doi: 10.1159/000447309
https://doi.org/10.1159/000447309... In addition, improper brushing of teeth or periodontal treatment itself can often damage or remove cement, rapidly exposing the dentin. This underscores the intricate interplay between root tissues composition and root caries development (Figure 2). Furthermore, due to these unique characteristics, the biomechanical conditions in this area are compromised. A maxillary premolar affected by RC examined with 3D-Finite Element Analysis revealed substantial stress concentration within the carious lesion. This suggests that the cavity resulting from RC may contribute to mechanical stress concentration, thereby influencing lesion development.⁵5 - Reis A, Soares PV, de Geus J, Loguercio AD. Clinical performance of root surface restorations. Monogr Oral Sci. 2017;26:115-24. doi: 10.1159/000479353
https://doi.org/10.1159/000479353...

Figure 2
Representation of collagenases action on the root surface in a dysbiotic environment: upon root surface exposure, rapid demineralization is expected. This demineralization exposes root collagen fibers, which become accessible to microbial biofilm and their acidic metabolites. With the presence of growth factors and cytokines, these metabolites release and activate host matrix metalloproteases (MMPs). The high abundance of bacteria with their own collagenases in this niche, coupled with the expression of genes encoding microbial collagenases in root caries biofilms, suggests collaborative collagen degradation by the biofilm and MMPs

The two phases of the RC lesion development: demineralization and collagen degradation

The characteristics of biofilms on root surfaces has been extensively reviewed elsewhere.³¹31 - Do T, Damé-Teixeira N, Naginyte M, Marsh PD. Root surface biofilms and caries. Monogr Oral Sci. 2017;26:26-34. doi: 10.1159/000479304
https://doi.org/10.1159/000479304... The first ecological concept of caries was proposed by Marsh (1994) and later extended by Takahashi and Nyvad.²⁹29 - Takahashi N, Nyvad B. Ecological hypothesis of dentin and root caries. Caries Res. 2016;50(4):422-31. doi: 10.1159/000447309
https://doi.org/10.1159/000447309... ,³²32 - Marsh PD. Microbiology of dental plaque biofilms and their role in oral health and caries. Dent Clin North Am. 2010;54(3):441-54. doi: 10.1016/j.cden.2010.03.002
https://doi.org/10.1016/j.cden.2010.03.0... These hypotheses underscore that the enrichment of certain species in the oral microbiota, formerly viewed as odontopathogens, occurs in response to environmental changes induced by high consumption of fermentable carbohydrates. In other words, dental caries is not caused by a predetermined set of microorganisms but by composition alterations driven by external factors that shift microbiota balance toward demineralization.²⁹29 - Takahashi N, Nyvad B. Ecological hypothesis of dentin and root caries. Caries Res. 2016;50(4):422-31. doi: 10.1159/000447309
https://doi.org/10.1159/000447309... ,³³33 - Takahashi N, Nyvad B. The role of bacteria in the caries process: ecological perspectives. J Dent Res. 2011;90(3):294-303. doi: 10.1177/0022034510379602
https://doi.org/10.1177/0022034510379602... Regardless of the differences in the teeth crown and root substrates, dental caries initiates on both surfaces due to microbiota imbalance.

There are two successive phases of both coronal dentin and root surfaces lesion development, in which a proteolytic stage occurs after a demineralization stage.²⁹29 - Takahashi N, Nyvad B. Ecological hypothesis of dentin and root caries. Caries Res. 2016;50(4):422-31. doi: 10.1159/000447309
https://doi.org/10.1159/000447309... ,³³33 - Takahashi N, Nyvad B. The role of bacteria in the caries process: ecological perspectives. J Dent Res. 2011;90(3):294-303. doi: 10.1177/0022034510379602
https://doi.org/10.1177/0022034510379602... While demineralization can be reversed, the second stage of collagen degradation is irreversible. These two phases were shown on ultrastructural studies revealing that in early stages, a pH gradient from the outer surface dissolves minerals, maintaining the original cross-links between collagen fibers.³⁴34 - Nyvad B, Fejerskov O. An ultrastructural study of bacterial invasion and tissue breakdown in human experimental root-surface caries. J Dent Res. 1990;69(5):1118-25. doi: 10.1177/00220345900690050101
https://doi.org/10.1177/0022034590069005... ,³⁵35 - Deyhle H, Bunk O, Muller B. Nanostructure of healthy and caries-affected human teeth. Nanomedicine. 2011;7(6):694-701. doi: 10.1016/j.nano.2011.09.005
https://doi.org/10.1016/j.nano.2011.09.0... Demineralized collagen serves as a support for colonizing bacteria (Figure 2). However, in contrast to coronal surfaces, root dentin does not require complete demineralization for bacterial colonization, as the channels of Sharpey’s fibers are possible colonization niches. In more advanced stages of RC lesion development, proteolytic enzymes degrades the exposed collagen, causing its fibers to lose their structural characteristics.²⁹29 - Takahashi N, Nyvad B. Ecological hypothesis of dentin and root caries. Caries Res. 2016;50(4):422-31. doi: 10.1159/000447309
https://doi.org/10.1159/000447309... However, a recent study suggested that cross-links between collagen bands may be degraded during demineralization,³⁶36 - Tjaderhane L, Buzalaf MA, Carrilho M, Chaussain C. Matrix metalloproteinases and other matrix proteinases in relation to cariology: the era of 'dentin degradomics'. Caries Res. 2015;49(3):193-208. doi: 10.1159/000363582
https://doi.org/10.1159/000363582... in which an exposed region of the collagen molecule is degraded by the activity of host-derived collagenolytic proteases (matrix metalloproteinases—MMPs).

MMPs are an important group of enzymes responsible for extracellular matrix degradation and are strongly related to physiological and pathological oral processes. A recent systematic review showed similar presence of MMP-2 in sound and carious root surfaces, while MMP-13 may be increased in root when compared to coronal carious dentin.¹⁵15 - Barbosa CB, Monici Silva I, Cena JA, Stefani CM, Dame-Teixeira N. Presence of host and bacterial-derived collagenolytic proteases in carious dentin: a systematic review of ex vivo studies. Front Cell Infect Microbiol. 2023;13:1278754. doi: 10.3389/fcimb.2023.1278754
https://doi.org/10.3389/fcimb.2023.12787... The release and production of MMPs occur through cells, such as keratinocytes, polymorphonuclear leukocytes, macrophages, monocytes, fibroblasts, and mesenchymal cells. In the presence of growth factors and cytokines (interleukin-1, TNF-α, and TGF-α), these cells release MMPs into the extracellular environment.³⁷37 - Birkedal-Hansen H. Role of matrix metalloproteinases in human periodontal diseases. J Periodontol. 1993;64(5 Suppl):474-84. doi: 10.1902/jop.1993.64.5s.474
https://doi.org/10.1902/jop.1993.64.5s.4... MMPs are present in dental biofilms, gingival crevicular fluid, and saliva. When dental root is uncovered, bacteria and their acidic metabolites within the exposed collagen fibers can activate MMPs. Bacterial enzymes may be responsible for positive regulation of interleukin-1 present in gingival crevicular fluid and saliva. Interleukin is responsible for stimulating polymorphonuclear leukocytes and macrophages to release MMPs.³⁸38 - Simon-Soro A, Belda-Ferre P, Cabrera-Rubio R, Alcaraz LD, Mira A. A tissue-dependent hypothesis of dental caries. Caries Res. 2013;47(6):591-600. doi: 10.1159/000351663
https://doi.org/10.1159/000351663...

Are microbes involved in root caries collagen degradation?

In addition to MMPs, some well-known oral bacteria involved in oral diseases produce collagenases that could breakdown dentinal collagen (Figure 2). According to Takahashi and Nyvad,²⁹29 - Takahashi N, Nyvad B. Ecological hypothesis of dentin and root caries. Caries Res. 2016;50(4):422-31. doi: 10.1159/000447309
https://doi.org/10.1159/000447309... it is still questionable whether bacteria play a role in the initial stages of teeth organic components degradation. The mechanisms underlying insoluble collagen degradation in overall host tissues by bacterial collagenolytic proteases remain largely unexplored, especially referring to dentinal collagen. However, there is evidence indicating movement towards the collagen N-terminus by C. histolyticum type I collagenase (ColG) along collagen fibrils from rats tails resulting in complete fibril degradation within 20–30 minutes.³⁹39 - Watanabe-Nakayama T, Itami M, Kodera N, Ando T, Konno H. High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils. Sci Rep. 2016;6:28975. doi: 10.1038/srep28975
https://doi.org/10.1038/srep28975... This process depends on a catalytic zinc (Zn) ion, and it is promising in terms of Zn inhibition to stop bacterial collagenolytic activity in these tissues.

Bacterial collagenases are proteinases designed to target collagen, most relying on Zn for enzymatic function. They may possess certain capability to degrade collagen within host organisms,⁴⁰40 - Harrington DJ. Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun. 1996;64(6):1885-91. doi: 10.1128/iai.64.6.1885-1891.1996
https://doi.org/10.1128/iai.64.6.1885-18... with examples including metalloproteases (M9 family) and serine proteases.⁴¹41 - Zhang YZ, Ran LY, Li CY, Chen XL. Diversity, structures, and collagen-degrading mechanisms of bacterial collagenolytic proteases. Appl Environ Microbiol. 2015;81(18):6098-107. doi: 10.1128/AEM.00883-15
https://doi.org/10.1128/AEM.00883-15... All Clostridium histolyticum collagenases are members of the M9B subfamily (EC: 3.4.24.3) ⁴¹41 - Zhang YZ, Ran LY, Li CY, Chen XL. Diversity, structures, and collagen-degrading mechanisms of bacterial collagenolytic proteases. Appl Environ Microbiol. 2015;81(18):6098-107. doi: 10.1128/AEM.00883-15
https://doi.org/10.1128/AEM.00883-15... , while the Porphyromonas and other oral bacteria present U32 family collagenases (EC: 3.4.24.40).¹²12 - Damé-Teixeira N, Parolo C, Maltz M, Rup A, Devine D, Do T. Gene expression of bacterial collagenolytic proteases in root caries. J Oral Microb. 2018;10:1424475. doi: 10.1080/20002297.2018.1424475
https://doi.org/10.1080/20002297.2018.14... As outlined by Zhang, et al.⁴¹41 - Zhang YZ, Ran LY, Li CY, Chen XL. Diversity, structures, and collagen-degrading mechanisms of bacterial collagenolytic proteases. Appl Environ Microbiol. 2015;81(18):6098-107. doi: 10.1128/AEM.00883-15
https://doi.org/10.1128/AEM.00883-15... (٢٠١٥), we adopt the term “bacterial collagenolytic proteases,” which encompasses both bacterial collagenases—enzymes that cleave helical regions of fibrillar collagen molecules under physiological conditions—and bacterial proteases that exclusively hydrolyze denatured collagen or type IV collagen. Therefore, “bacterial collagenolytic proteases” encompasses true bacterial collagenases as well as other proteases exhibiting collagenase activity, although most of them have never been described in oral microorganisms.⁴¹41 - Zhang YZ, Ran LY, Li CY, Chen XL. Diversity, structures, and collagen-degrading mechanisms of bacterial collagenolytic proteases. Appl Environ Microbiol. 2015;81(18):6098-107. doi: 10.1128/AEM.00883-15
https://doi.org/10.1128/AEM.00883-15... Microbial collagenases and MMPs display unique characteristics that could lead to diverse effects on dentinal collagen matrix. Unlike mammalian collagenases, which cleave collagen at a single site, bacterial collagenase from M9 family carries out multiple cleavages ⁴²42 - Kato MT, Leite AL, Hannas AR, Calabria MP, Magalhães AC, Pereira JC, et al. Impact of protease inhibitors on dentin matrix degradation by collagenase. J Dent Res. 2012;91(12):1119-23. doi: 10.1177/0022034512455801
https://doi.org/10.1177/0022034512455801...

43 - Philominathan ST, Koide T, Matsushita O, Sakon J. Bacterial collagen-binding domain targets undertwisted regions of collagen. Protein Sci. 2012;21(10):1554-65. doi: 10.1002/pro.2145
https://doi.org/10.1002/pro.2145... -⁴⁴44 - Vidal CM, Tjäderhane L, Scaffa PM, Tersariol IL, Pashley D, Nader HB, et al. Abundance of MMPs and cysteine cathepsins in caries-affected dentin. J Dent Res. 2014;93(3):269-74. doi: 10.1177/0022034513516979
https://doi.org/10.1177/0022034513516979... and more efficiently than MMPs.⁴⁵45 - Welgus HG, Jeffrey JJ, Eisen AZ. Human skin fibroblast collagenase. Assessment of activation energy and deuterium isotope effect with collagenous substrates. J Biol Chem. 1981;256(18):9516-21.Table 2 describes the classification of bacterial collagenolytic proteases. For more details on their diversity and characteristics, such as molecular mass (kDa) and action mechanisms, see Zhang, et al. ⁴¹41 - Zhang YZ, Ran LY, Li CY, Chen XL. Diversity, structures, and collagen-degrading mechanisms of bacterial collagenolytic proteases. Appl Environ Microbiol. 2015;81(18):6098-107. doi: 10.1128/AEM.00883-15
https://doi.org/10.1128/AEM.00883-15... (2015).

The first sign of oral bacteria involvement in RC can be a grand isolation of bacteria with collagenolytic proteases from root lesions, such as Prevotella and Propionibacterium, although not directly linked to their role in dentinal collagen degradation.⁴⁰40 - Harrington DJ. Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun. 1996;64(6):1885-91. doi: 10.1128/iai.64.6.1885-1891.1996
https://doi.org/10.1128/iai.64.6.1885-18... ,⁴⁶46 - Aas JA, Griffen AL, Dardis SR, Lee AM, Olsen I, Dewhirst FE, et al. Bacteria of dental caries in primary and permanent teeth in children and young adults. J Clin Microbiol. 2008;46(4):1407-17. doi: 10.1128/JCM.01410-07
https://doi.org/10.1128/JCM.01410-07...

47 - Preza D, Olsen I, Aas JA, Willumsen T, Grinde B, Paster BJ. Bacterial profiles of root caries in elderly patients. J Clin Microbiol. 2008;46(6):2015-21. doi: 10.1128/JCM.02411-07
https://doi.org/10.1128/JCM.02411-07... -⁴⁸48 - Hashimoto K, Sato T, Shimauchi H, Takahashi N. Profiling of dental plaque microflora on root caries lesions and the protein-denaturing activity of these bacteria. Am J Dent. 2011;24(5):295-9. Interestingly, a recent study showed that the bacterial composition of RC lesions located under the gingival margin is likely to have periodontal pathobionts: Porphyromonas, Selenomonas, Filifactor, Peptococcus and Tannerela inhabit RC lesions that extend beyond gingival margin.⁴⁹49 - Takenaka S, Edanami N, Komatsu Y, Nagata R, Naksagoon T, Sotozono M, et al. Periodontal pathogens inhabit root caries lesions extending beyond the gingival margin: a next-generation sequencing analysis. Microorganisms. 2021;9(11). doi: 10.3390/microorganisms9112349
https://doi.org/10.3390/microorganisms91... This suggests that microbiome in RC lesions expanding across the gingival margin would show an increase in bacterial proteolytic diversity. The expression of P. gingivalis collagenases-related genes in RC lesions has also been demonstrated.¹²12 - Damé-Teixeira N, Parolo C, Maltz M, Rup A, Devine D, Do T. Gene expression of bacterial collagenolytic proteases in root caries. J Oral Microb. 2018;10:1424475. doi: 10.1080/20002297.2018.1424475
https://doi.org/10.1080/20002297.2018.14... The integrated ecological hypothesis for caries and periodontitis ⁵⁰50 - Nyvad B, Takahashi N. Integrated hypothesis of dental caries and periodontal diseases. J Oral Microbiol. 2020;12(1):1710953. doi: 10.1080/20002297.2019.1710953
https://doi.org/10.1080/20002297.2019.17... points to a common risk factor for both diseases, which are originated in the dynamic stability stage and emerges in response to nutritional imbalances in microbiota. According to authors of the integrated hypothesis, when inflammatory nutrients are intense and prolonged, the microbiota pH can move from dynamic stability to stages of proteolytic degradation, whereas the more intense and prolonged the episodes of exposure to dietary carbohydrates, the greater the pH shift to acidogenic and aciduric stages associated with caries.⁵⁰50 - Nyvad B, Takahashi N. Integrated hypothesis of dental caries and periodontal diseases. J Oral Microbiol. 2020;12(1):1710953. doi: 10.1080/20002297.2019.1710953
https://doi.org/10.1080/20002297.2019.17... Given its physical connection with periodontal tissues, RC likely occupies an intermediate stage between acidogenic and proteolytic stages, harboring a diverse array of saccharolytic, aciduric, acidogenic, and proteolytic organisms.³¹31 - Do T, Damé-Teixeira N, Naginyte M, Marsh PD. Root surface biofilms and caries. Monogr Oral Sci. 2017;26:26-34. doi: 10.1159/000479304
https://doi.org/10.1159/000479304... Additionally, microbes within RC can generate acid or ammonia through the catabolism of nitrogenous substrates, whether obtained exogenously or derived from the organic matrix of dentin.¹¹11 - Dame-Teixeira N, Parolo CC, Maltz M, Tugnait A, Devine D, Do T. Actinomyces spp. gene expression in root caries lesions. J Oral Microbiol. 2016;8:32383. doi: 10.3402/jom.v8.32383
https://doi.org/10.3402/jom.v8.32383... ,³¹31 - Do T, Damé-Teixeira N, Naginyte M, Marsh PD. Root surface biofilms and caries. Monogr Oral Sci. 2017;26:26-34. doi: 10.1159/000479304
https://doi.org/10.1159/000479304... ,⁵¹51 - Syed SA, Loesche WJ, Pape HL Jr, Grenier E. Predominant cultivable flora isolated from human root surface caries plaque. Infect Immun. 1975;11(4):727-31. doi: 10.1128/iai.11.4.727-731.1975
https://doi.org/10.1128/iai.11.4.727-731...

Microorganism significance and oral environment impact on dentin matrix collagen were investigated by van Strijp and colleagues through a series of in situ studies. These studies assessed the extent of denatured collagen following an experimental period under various cariogenic conditions.⁵²52 - Van Strijp AJ, Klont B, Ten Cate JM. Solubilization of dentin matrix collagen in situ. J Dent Res. 1992;71(8):1498-502. doi: 10.1177/00220345920710080701
https://doi.org/10.1177/0022034592071008...

53 - van Strijp AJ, Jansen DC, DeGroot J, ten Cate JM, Everts V. Host-derived proteinases and degradation of dentine collagen in situ. Caries Res. 2003;37(1):58-65. doi: 10.1159/000068223
https://doi.org/10.1159/000068223...

54 - van Strijp AJ, Takatsuka T, Sono R, Iijima Y. Inhibition of dentine collagen degradation by hesperidin: an in situ study. Eur J Oral Sci. 2015;123(6):447-52. doi: 10.1111/eos.12225
https://doi.org/10.1111/eos.12225... -⁵⁵55 - van Strijp AJ, van Steenbergen TJ, de Graaff J, ten Cate JM. Bacterial colonization and degradation of demineralized dentin matrix in situ. Caries Res. 1994;28(1):21-7. doi: 10.1159/000261615
https://doi.org/10.1159/000261615... For this purpose, completely demineralized dentin specimens were positioned on the buccal surfaces of partial dentures worn by volunteers. Following an intraoral period of seven weeks, dentin samples were analyzed for denatured collagen. Intra-individual and inter-individual differences in collagen loss were found, likely attributed to microbiota composition differences, which colonized the demineralized specimens. In addition, differences in microorganisms ability to degrade the collagen matrix were credited for deviations in collagen loss.⁵²52 - Van Strijp AJ, Klont B, Ten Cate JM. Solubilization of dentin matrix collagen in situ. J Dent Res. 1992;71(8):1498-502. doi: 10.1177/00220345920710080701
https://doi.org/10.1177/0022034592071008... Gelatinolytic activity of isolated strains related to dentin matrix degradation was evaluated after identifying the colonizing microbiota of decalcified dentinal matrix. The predominant species found were Streptococcus mitis, Peptostreptococcus spp., Lactobacillus casei, Propionibacterium species, and Veillonella parvula. Although no correlation was found with the severity of dentin matrix degradation, some gelatinolytic activity was observed in both saliva and dentin collagen.⁵⁵55 - van Strijp AJ, van Steenbergen TJ, de Graaff J, ten Cate JM. Bacterial colonization and degradation of demineralized dentin matrix in situ. Caries Res. 1994;28(1):21-7. doi: 10.1159/000261615
https://doi.org/10.1159/000261615... However, no correlation was observed between enzyme activity levels and collagen loss in dentin specimens.⁵³53 - van Strijp AJ, Jansen DC, DeGroot J, ten Cate JM, Everts V. Host-derived proteinases and degradation of dentine collagen in situ. Caries Res. 2003;37(1):58-65. doi: 10.1159/000068223
https://doi.org/10.1159/000068223...

After these studies developed by van Strijp, et al., little has been published regarding oral bacteria involvement in root collagen degradation. More recently, few molecular studies have contributed to researching the role of microorganisms and their proteases in the progression of caries. Simon-Sóro, et al.³⁸38 - Simon-Soro A, Belda-Ferre P, Cabrera-Rubio R, Alcaraz LD, Mira A. A tissue-dependent hypothesis of dental caries. Caries Res. 2013;47(6):591-600. doi: 10.1159/000351663
https://doi.org/10.1159/000351663... (٢٠١٣) proposed a “tissue-dependent hypothesis” of caries by comparing the metagenomics of carious biofilms from enamel and dentin caries. The study showed different metabolic events occurring in each carious tissue, in which genes involved in acid stress tolerance and dietary sugar fermentation were overexpressed at the enamel caries biofilms, whereas collagenases and proteases were overexpressed in dentin cavities.³⁸38 - Simon-Soro A, Belda-Ferre P, Cabrera-Rubio R, Alcaraz LD, Mira A. A tissue-dependent hypothesis of dental caries. Caries Res. 2013;47(6):591-600. doi: 10.1159/000351663
https://doi.org/10.1159/000351663... Interestingly, genes for fermenting sugar in the diet and pH stress appeared at very low levels in dentin lesions, presenting an overrepresentation of genes involved in monosaccharides and disaccharides metabolism. Regarding deep dentin samples, genes related to host immune response were also overrepresented.³⁸38 - Simon-Soro A, Belda-Ferre P, Cabrera-Rubio R, Alcaraz LD, Mira A. A tissue-dependent hypothesis of dental caries. Caries Res. 2013;47(6):591-600. doi: 10.1159/000351663
https://doi.org/10.1159/000351663...

Additional microorganisms also exhibited overexpression of genes associated with collagenases in RC, indicating their potential contribution to protein degradation, such as Veillonella parvula DSM 2008 (VPAR_RS05935 and VPAR_RS05390), Veillonella dispar ATCC 17748 (VEIDISOL_RS04770), and Leptotrichia buccalis (LEBU_RS05040). Those genes encode the collagenase-type protease family PrtC of the peptidase family U32.⁵⁶56 - Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, Carson MB, et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A. 2002;99(22):14434-9. doi: 10.1073/pnas.172501299
https://doi.org/10.1073/pnas.172501299... The U32 family of peptidases is a broad family of enzymes with little known structure and catalytic mechanism. This family presence has also been described in other pathogenic bacteria such as P. gingivalis, Proteus mirabilis, Helicobacter pylori, and Aeromonas veronii. In all cases, these bacteria hold putative collagenases, which are generally related to bacterial infections.¹²12 - Damé-Teixeira N, Parolo C, Maltz M, Rup A, Devine D, Do T. Gene expression of bacterial collagenolytic proteases in root caries. J Oral Microb. 2018;10:1424475. doi: 10.1080/20002297.2018.1424475
https://doi.org/10.1080/20002297.2018.14... ,⁵⁷57 - Navais R, Méndez J, Pérez-Pascual D, Cascales D, Guijarro JA. The yrpAB operon of Yersinia ruckeri encoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions. Virulence. 2014;5(5):619-24. doi: 10.4161/viru.29363
https://doi.org/10.4161/viru.29363... For example, studies report the potential role of U32 collagenase in P. gingivalis virulence, which can degrade soluble fibrillar collagen type I and reconstitute at or below body temperature.⁴¹41 - Zhang YZ, Ran LY, Li CY, Chen XL. Diversity, structures, and collagen-degrading mechanisms of bacterial collagenolytic proteases. Appl Environ Microbiol. 2015;81(18):6098-107. doi: 10.1128/AEM.00883-15
https://doi.org/10.1128/AEM.00883-15... A purified protease characterization, expressed from the bacterium’s prtC gene, enabled these analyses .⁵⁸58 - Kato T, Takahashi N, Kuramitsu HK. Sequence analysis and characterization of the Porphyromonas gingivalis prt C gene, which expresses a novel collagenase activity. J Bacteriol. 1992;174(12):3889-95. doi: 10.1128/jb.174.12.3889-3895.1992
https://doi.org/10.1128/jb.174.12.3889-3... PrtC peptidase has been documented to breakdown soluble and reconstituted fibrillar type I collagen, as well as heat-denatured type I collagen and azocoll.⁵⁸58 - Kato T, Takahashi N, Kuramitsu HK. Sequence analysis and characterization of the Porphyromonas gingivalis prt C gene, which expresses a novel collagenase activity. J Bacteriol. 1992;174(12):3889-95. doi: 10.1128/jb.174.12.3889-3895.1992
https://doi.org/10.1128/jb.174.12.3889-3... PrtC inhibitors include ethylenediaminetetraacetic acid (EDTA), thiol-blocking agents, and salivary histatin.⁵⁸58 - Kato T, Takahashi N, Kuramitsu HK. Sequence analysis and characterization of the Porphyromonas gingivalis prt C gene, which expresses a novel collagenase activity. J Bacteriol. 1992;174(12):3889-95. doi: 10.1128/jb.174.12.3889-3895.1992
https://doi.org/10.1128/jb.174.12.3889-3...

Overexpression of putative prtC genes in S. mutans (SMU_761 and SMU_759 - S. mutans UA159) were also related to proteolytic activity and collagenases in RC lesions.¹²12 - Damé-Teixeira N, Parolo C, Maltz M, Rup A, Devine D, Do T. Gene expression of bacterial collagenolytic proteases in root caries. J Oral Microb. 2018;10:1424475. doi: 10.1080/20002297.2018.1424475
https://doi.org/10.1080/20002297.2018.14... SMU_761 encodes a 428 aa protein, whereas SMU_759 encodes a 308 aa protein. These findings are relevant due to this organism’s higher abundance and activity in RC lesions.¹³13 - Santos HS, Do T, Parolo CC, Poloni JF, Maltz M, Arthur RA, et al. Streptococcus mutans gene expression and functional profile in root caries: an RNA-Seq study. Caries Res. 2022;56(2):116-28. doi: 10.1159/000524196
https://doi.org/10.1159/000524196... However, as previously stated, gene expression alone does not ensure enzyme activity. When investigating enzymatic activity, some studies contradict the theory of bacterial collagenases involvement in RC. One study suggested that bacterial collagenases are highly sensitive to pH and unable to withstand acidic conditions (pH 4.3) during the dental demineralization stage, such as demonstrated for MMPs, not playing a substantial role to the development of carious lesions.⁵⁹59 - Kawasaki K, Featherstone JD. Effects of collagenase on root demineralization. J Dent Res. 1997;76(1):588-95. doi: 10.1177/00220345970760011001
https://doi.org/10.1177/0022034597076001... Consistent with this, Tjaderhane, et al.⁶⁰60 - Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T. The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions. J Dent Res. 1998;77(8):1622-9. doi: 10.1177/00220345980770081001
https://doi.org/10.1177/0022034598077008... (2015) observed no gelatinolytic or collagenolytic activity in bacterial samples and concluded its inactivity in dentinal caries. Although these results demonstrated the pH-dependent activation mechanism of human MMPs, they did not provide evidence for a similar mechanism in bacterial enzymes. Nevertheless, our current results contradict their findings. We showed no differences in S. mutans activity when using the synthetic collagen FALGPA (N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala) at acidic and neutral pH levels. This indicates that bacteria could exert some collagenolytic function even in lower pH environments, suggesting an intrinsic collagenolytic capability of S. mutans (unpublished data).

Besides, there is some evidence on S. mutans collagenolytic activity, such as its capacity to degrade collagen from rodent tendons.⁶¹61 - Rosengren L, Winblad B. Proteolytic activity of Streptococcus mutans (GS-5). Oral Surg Oral Med Oral Pathol. 1976;42(6):801-9. doi: 10.1016/0030-4220(76)90103-1
https://doi.org/10.1016/0030-4220(76)901... Still, another study showed that the same strain of S. mutans GS-5 collected from human carious lesions was able not only to induce caries in rodents, but also to promote bone tissue degradation, reinforcing the microbial role hypothesis in RC collagen degradation.⁶¹61 - Rosengren L, Winblad B. Proteolytic activity of Streptococcus mutans (GS-5). Oral Surg Oral Med Oral Pathol. 1976;42(6):801-9. doi: 10.1016/0030-4220(76)90103-1
https://doi.org/10.1016/0030-4220(76)901... Although studies have claimed that S. mutans constitutes only a small proportion of the microbiota,⁶²62 - Bowden GH. Microbiology of root surface caries in humans. J Dent Res. 1990;69(5):1205-10. doi: 10.1177/00220345900690051701
https://doi.org/10.1177/0022034590069005... this microorganism is strongly related to the disease. In addition it is present at higher frequencies on decayed root surfaces than on biofilms on root surfaces ⁶³63 - Ellen RP, Banting DW, Fillery ED. Streptococcus mutans and Lactobacillus detection in the assessment of dental root surface caries risk. J Dent Res. 1985;64(10):1245-9. doi: 10.1177/00220345850640101301
https://doi.org/10.1177/0022034585064010... ,⁶⁴64 - Nyvad B, Kilian M. Microflora associated with experimental root surface caries in humans. Infect Immun. 1990;58(6):1628-33. doi: 10.1128/iai.58.6.1628-1633
https://doi.org/10.1128/iai.58.6.1628-16... and can play an important role in RC progression.¹³13 - Santos HS, Do T, Parolo CC, Poloni JF, Maltz M, Arthur RA, et al. Streptococcus mutans gene expression and functional profile in root caries: an RNA-Seq study. Caries Res. 2022;56(2):116-28. doi: 10.1159/000524196
https://doi.org/10.1159/000524196... ,³¹31 - Do T, Damé-Teixeira N, Naginyte M, Marsh PD. Root surface biofilms and caries. Monogr Oral Sci. 2017;26:26-34. doi: 10.1159/000479304
https://doi.org/10.1159/000479304... ,⁶³63 - Ellen RP, Banting DW, Fillery ED. Streptococcus mutans and Lactobacillus detection in the assessment of dental root surface caries risk. J Dent Res. 1985;64(10):1245-9. doi: 10.1177/00220345850640101301
https://doi.org/10.1177/0022034585064010... Yet, these results remain inconclusive, largely because of methodological challenges. For example, we have been striving to provide a more comprehensive description of S. mutans collagenase activity. Despite attempts to knockout SMU_761 and SMU_759 using various methods and strategies, the transformation proved to be inefficient.⁶⁵65 - Barbosa CB, Salles LP, Dame-Teixeira N. Isolamento e construção de cassetes de inativação de genes codificadores de colagenases de Streptococcus mutans com possível envolvimento na degradação colagenolítica em Cárie Radicular [Dissertação]: University of Brasilia; 2020. Although FALGPA and gelatin substrates exhibited degradation, we observed only minimal activity against type I collagen (unpublished data). It is crucial to note that results do not consider the natural composition of dental biofilms, which inherently comprise a diverse and multifunctional microbiota. Biofilm components assume different functions compared to when they are isolated, due to these communities multidimensional and highly complex nature.⁴⁶46 - Aas JA, Griffen AL, Dardis SR, Lee AM, Olsen I, Dewhirst FE, et al. Bacteria of dental caries in primary and permanent teeth in children and young adults. J Clin Microbiol. 2008;46(4):1407-17. doi: 10.1128/JCM.01410-07
https://doi.org/10.1128/JCM.01410-07... This indicates that studying isolates may yield different results in terms of collagenolytic activity compared to natural biofilms.

To summarize, so far we can state that host-derived MMPs initiates partial breakdown of the telopeptide region of dentin collagen molecules. Regarding bacterial collagenolytic proteases, there is controversy regarding the scientific evidence for their activity in carious lesions, alongside a notably small number of clinical studies.¹⁵15 - Barbosa CB, Monici Silva I, Cena JA, Stefani CM, Dame-Teixeira N. Presence of host and bacterial-derived collagenolytic proteases in carious dentin: a systematic review of ex vivo studies. Front Cell Infect Microbiol. 2023;13:1278754. doi: 10.3389/fcimb.2023.1278754
https://doi.org/10.3389/fcimb.2023.12787... Nevertheless, U32 bacterial collagenases can hypothetically cleave collagen at multiple sites at the same time. Then, collagen molecules, now solubilized, could be irreversibly denatured under acidic conditions and body temperature. The presence of proteolytic bacteria and the activation of genes encoding collagenolytic proteases, along with the ones encoding collagen-binding proteins for highly active bacteria in RC,¹¹11 - Dame-Teixeira N, Parolo CC, Maltz M, Tugnait A, Devine D, Do T. Actinomyces spp. gene expression in root caries lesions. J Oral Microbiol. 2016;8:32383. doi: 10.3402/jom.v8.32383
https://doi.org/10.3402/jom.v8.32383... indicates potential contribution to matrix degradation.

Future perspectives for translational research in prevention and management of root caries

A significant challenge in addressing RC lies in its clinical treatment. Invasive treatments pose various operational difficulties, including challenges in moisture control particularly in subgingivally extended lesions.⁶⁶66 - Schwendicke F, Gostemeyer G. Cost-effectiveness of root caries preventive treatments. J Dent. 2017;56:58-64. doi: 10.1016/j.jdent.2016.10.016
https://doi.org/10.1016/j.jdent.2016.10.... Promoting remineralization is an important strategy for RC control.⁶⁷67 - Mellberg JR, Sanchez M. Remineralization by a monofluorophosphate dentifrice in vitro of root dentin softened by artificial caries. J Dent Res. 1986;65(7):959-62. doi: 10.1177/00220345860650071201
https://doi.org/10.1177/0022034586065007...

68 - Clarkson BH, Rafter ME. Emerging methods used in the prevention and repair of carious tissues. J Dent Educ. 2001;65(10):1114-20.-⁶⁹69 - Baysan A, Lynch E, Ellwood R, Davies R, Petersson L, Borsboom P. Reversal of primary root caries using dentifrices containing 5,000 and 1,100 ppm fluoride. Caries Res. 2001;35(1):41-6. doi: 10.1159/000047429
https://doi.org/10.1159/000047429... Non-invasive therapies with high concentrations of fluoride, such as 5000 ppm/F toothpastes and silver diamine fluoride, have a good body of evidence on controlling lesion progression and preventing new lesions.⁷⁰70 - Wierichs RJ, Meyer-Lueckel H. Systematic review on noninvasive treatment of root caries lesions. J Dent Res. 2015;94(2):261-71. doi: 10.1177/0022034514557330
https://doi.org/10.1177/0022034514557330... However, current studies emphasize collagen integrity preservation as a potential strategy for new adjunctive treatments in RC.

There is very limited clinical research on the use of collagenase inhibitors in root surfaces. For instance, the application of 2% chlorhexidine after acid-etching in restorative procedures has not demonstrated any significant effect in the longevity of non-carious root lesions.⁷¹71 - Araújo MS, Souza LC, Apolonio FM, Barros LO, Reis A, Loguercio AD, et al. Two-year clinical evaluation of chlorhexidine incorporation in two-step self-etch adhesive. J Dent. 2015;43(1):140-8. doi: 10.1016/j.jdent.2014.07.010
https://doi.org/10.1016/j.jdent.2014.07.... ,⁷²72 - Montagner AF, Perroni AP, Corrêa MB, Masotti AS, Pereira-Cenci T, Cenci MS. Effect of pre-treatment with chlorhexidine on the retention of restorations: a randomized controlled trial. Braz Dent J. 2015;26(3):234-41. doi: 10.1590/0103-6440201300009
https://doi.org/10.1590/0103-64402013000... However, these studies utilized short follow-up periods and did not provide information on collagen stabilization in exposed roots. Furthermore, in both root and coronal caries, most investigated adjunctive therapeutic agents function as antimicrobials so far. Due to the inherent dysbiotic nature in RC etiopathogenesis, antimicrobials are ineffective in caries control, therefore its clinical application is meaningless. However, substances with phytochemical effects on dental tissue or the ability to modulate biofilm metabolic processes present some potential.⁷³73 - Cai L, Wu CD. Compounds from Syzygium aromaticum possessing growth inhibitory activity against oral pathogens. J Nat Prod. 1996;59(10):987-90. doi: 10.1021/np960451q
https://doi.org/10.1021/np960451q...

74 - Li XC, Cai L, Wu CD. Antimicrobial compounds from Ceanothus americanus against oral pathogens. Phytochemistry. 1997;46(1):97-102. doi: 10.1016/s0031-9422(97)00222-7.
https://doi.org/10.1016/s0031-9422(97)00... -⁷⁵75 - Chu JP, Li JY, Hao YQ, Zhou XD. Effect of compounds of Galla chinensis on remineralisation of initial enamel carious lesions in vitro. J Dent. 2007;35(5):383-7. doi: 10.1016/j.jdent.2006.11.007
https://doi.org/10.1016/j.jdent.2006.11.... Studies have investigated extracts abundant in phenols and polyphenols to assess their impact on the interplay of cross-linking, resistance to digestion, the activities of MMPs and other collagenases.⁷⁶76 - Wang Y, Green A, Yao X, Liu H, Nisar S, Gorski JP, et al. Cranberry juice extract rapidly protects demineralized dentin against digestion and Inhibits Its Gelatinolytic Activity. Materials (Basel). 2021;14(13). doi: 10.3390/ma14133637
https://doi.org/10.3390/ma14133637... Also, once the participation of U32 proteases in RC is confirmed, inhibition using metal ion-chelating substances like Zn and Fe²2 - Hariyani N, Setyowati D, Spencer AJ, Luzzi L, Do LG. Root caries incidence and increment in the population - a systematic review, meta-analysis and meta-regression of longitudinal studies. J Dent. 2018;77:1-7. doi: 10.1016/j.jdent.2018.06.013
https://doi.org/10.1016/j.jdent.2018.06.... could be considered. For instance, a hemi-synthetic anacardic acid compound derived from cashew nut shell liquid was capable of inhibiting about 90% of the P. gingivalis collagenase activity in vitro, probably due to its selective chelation of Fe²2 - Hariyani N, Setyowati D, Spencer AJ, Luzzi L, Do LG. Root caries incidence and increment in the population - a systematic review, meta-analysis and meta-regression of longitudinal studies. J Dent. 2018;77:1-7. doi: 10.1016/j.jdent.2018.06.013
https://doi.org/10.1016/j.jdent.2018.06.... .⁷⁷77 - Dame-Teixeira N, El-Gendy R, Monici Silva I, Holanda CA, Oliveira AS, Romeiro LAS, et al. Sustainable multifunctional phenolic lipids as potential therapeutics in Dentistry. Sci Rep. 2022;12(1):9299. doi: 10.1038/s41598-022-13292-0
https://doi.org/10.1038/s41598-022-13292...

Another adjunctive treatment option to prevent collagen breakdown involves the biomodification of dental root surface. Dentin biomodification strategy is based on the formation of inter- and intramolecular cross-links, which result in stabilization of the collagen constituting dentin organic matrix. Extensive in vitro studies have explored the capacity of various substances to promote cross-links in root collagen, which could reduce dentin matrix degradation and increase the biomechanical properties of healthy tissue.⁷⁸78 - Walter R, Miguez PA, Arnold RR, Pereira PN, Duarte WR, Yamauchi M. Effects of natural cross-linkers on the stability of dentin collagen and the inhibition of root caries in vitro. Caries Res. 2008;42(4):263-8. doi: 10.1159/000135671
https://doi.org/10.1159/000135671... ,⁷⁹79 - Knott L, Bailey AJ. Collagen cross-links in mineralizing tissues: a review of their chemistry, function, and clinical relevance. Bone. 1998;22(3):181-7. doi: 10.1016/s8756-3282(97)00279-2
https://doi.org/10.1016/s8756-3282(97)00... Several agents have been recognized by studies as cross-linkers, including glutaraldehyde (GA), formaldehyde, carbodiimide and epoxy compounds.⁷⁶76 - Wang Y, Green A, Yao X, Liu H, Nisar S, Gorski JP, et al. Cranberry juice extract rapidly protects demineralized dentin against digestion and Inhibits Its Gelatinolytic Activity. Materials (Basel). 2021;14(13). doi: 10.3390/ma14133637
https://doi.org/10.3390/ma14133637... ,⁷⁸78 - Walter R, Miguez PA, Arnold RR, Pereira PN, Duarte WR, Yamauchi M. Effects of natural cross-linkers on the stability of dentin collagen and the inhibition of root caries in vitro. Caries Res. 2008;42(4):263-8. doi: 10.1159/000135671
https://doi.org/10.1159/000135671... ,⁸⁰80 - Bedran-Russo AK, Castellan CS, Shinohara MS, Hassan L, Antunes A. Characterization of biomodified dentin matrices for potential preventive and reparative therapies. Acta Biomater. 2011;7(4):1735-41. doi: 10.1016/j.actbio.2010.12.013
https://doi.org/10.1016/j.actbio.2010.12...

81 - Tezvergil-Mutluay A, Mutluay MM, Agee KA, Seseogullari-Dirihan R, Hoshika T, Cadenaro M, et al. Carbodiimide cross-linking inactivates soluble and matrix-bound MMPs, in vitro. J Dent Res. 2012;91(2):192-6. doi: 10.1177/0022034511427705
https://doi.org/10.1177/0022034511427705... -⁸²82 - Scheffel DL, Hebling J, Scheffel RH, Agee KA, Cadenaro M, Turco G, et al. Stabilization of dentin matrix after cross-linking treatments, in vitro. Dent Mater. 2014;30(2):227-33. doi: 10.1016/j.dental.2013.11.007
https://doi.org/10.1016/j.dental.2013.11... However, adverse effects such as toxicity and/or instability limited these substances use in vivo. To the best of our knowledge, no relevant clinical data is currently available to either prevent or impede RC lesions.

Recently, natural agents available in fruits, bark, leaves, and seeds, have been considered biocompatible and stable for a long period of time in animals. Natural agents are characterized by lower toxicity compared to synthetics and can be considered renewable. Most of these agents derive from plants and are their secondary metabolites. Polyphenols, for example, can be subdivided into phenolic acids, flavonoids, stilbenes, and lignans. These agents are capable of forming cross-links through covalent bonds and hydrogen bonds.⁸³83 - Han B, Jaurequi J, Tang BW, Nimni ME. Proanthocyanidin: a natural crosslinking reagent for stabilizing collagen matrices. J Biomed Mater Res A. 2003;65(1):118-24. doi: 10.1002/jbm.a.10460
https://doi.org/10.1002/jbm.a.10460... Examples include proanthocyanidins (PAC), genipin, and cranberry .⁷⁶76 - Wang Y, Green A, Yao X, Liu H, Nisar S, Gorski JP, et al. Cranberry juice extract rapidly protects demineralized dentin against digestion and Inhibits Its Gelatinolytic Activity. Materials (Basel). 2021;14(13). doi: 10.3390/ma14133637
https://doi.org/10.3390/ma14133637... Promising results have shown that PAC could efficiently stabilize collagen by increasing its resistance against caries in vitro under artificial lesion formation.⁷⁸78 - Walter R, Miguez PA, Arnold RR, Pereira PN, Duarte WR, Yamauchi M. Effects of natural cross-linkers on the stability of dentin collagen and the inhibition of root caries in vitro. Caries Res. 2008;42(4):263-8. doi: 10.1159/000135671
https://doi.org/10.1159/000135671... Similarly, other study used an in vitro pH cycling model to evaluate the effect of GSE (grape seed extract that contains PAC) on artificial root caries remineralization, showing a positive effect in demineralization and/or remineralization processes.⁸⁴84 - Xie Q, Bedran-Russo AK, Wu CD. In vitro remineralization effects of grape seed extract on artificial root caries. J Dent. 2008;36(11):900-6. doi: 10.1016/j.jdent.2008.07.011.
https://doi.org/10.1016/j.jdent.2008.07.... Nevertheless, it is important to bear in mind that structure modifications using cross-linking agents could potentially involve not only collagen stabilization but also remineralization effects.

It is crucial to note that the positive outcomes of anti-collagenases and cross-linkers are currently derived from in vitro experiments that simulate demineralizing conditions using cyclic pH models. While these conditions pose challenges for immediate clinical applicability, if these treatments effectiveness are confirmed, they might represent low-cost and easy-to-apply adjunctive non-invasive therapies for controlling RC lesions.

Conclusion

Root caries lesion formation is likely a two-stage process, where collagen breakdown follows mineral loss. MMPs presence, particularly MMP-13, must be associated with collagen denaturation in RC. Given the prevalence of proteolytic bacteria in RC-associated biofilms, their contribution to the second stage of lesion formation is plausible. Nevertheless, proving the role of microbial collagenases in RC remains a significant challenge. A more in-depth investigation into microbial collagenases role in these lesions is warranted. Modulating biofilm by inhibiting collagenases or protecting the collagen matrix from degradation by using cross-linking agents could be a targeted approach for RC treatments and prevention.

Acknowledgments

The authors acknowledge the Coordination of Higher Education and Graduate Training (CAPES) and the Federal District Research Foundation (FAP/DF) for the scholarships provided to CBB and IMS, respectively. NDT thanks the Academy of Medical Sciences for Newton International Fellowship.

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