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Detecting Calcified stones and cracks in tooth

Detecting Calcified stones and cracks in tooth



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I had a dental problem when my first molar broke some months ago and after some decay and pain I am undergoing a dental treatment. The doctor was performing a root canal and kept on talking about calcified stones being formed and looking for a 'tak' sound inspite of having x-rays. Doctor did not point to the calcified stones earlier and suddenly came out with this new issue. In the mean time when looking for stones doctor actually managed to break my tooth and has blamed grinding and clenching for a crack that caused the tooth to break.

I am shocked that neither the calcified stone nor the crack showed up in x-ray and the preliminary examination. With current technology, can the cracks in tooth and calcified stones not be detected.


Cracks on your teeth are hard to detect if they are cracks on the mesial or distal surfaces (surface of the tooth closest to and farthest from the midline respectively) through x-rays. Please check this link for illustrations and very good explanations from the University of Maryland dentistry department. The different methods on the detection of cracks as well as the uses of radiographic evidence can be found in this link.

Now pulp stones can be identified through x-rays unless they are too small or not too dense. There has been researches done specifically using radiographs for detection like "A radiographic assessment of the prevalence of pulp stones in Australians". It could also be human error that your doctor was unable to detect it. A small explanation on pulp stones can be found here.


Cracked Teeth

Whether your tooth cracks from an injury or general wear and tear, you can experience a variety of symptoms ranging from erratic pain when you chew your food to sudden pain when your tooth is exposed to very hot or cold temperatures. In many cases, the pain may come and go and your dentist may have difficulty locating the tooth causing the discomfort. If you experience these symptoms or suspect a cracked tooth, it’s best to see an endodontist as soon as possible.

Endodontists specialize in saving cracked teeth and will cater treatment to the type, location, and extent of the crack. The sooner your tooth is treated, the better the outcome. Once treated, most cracked teeth continue to function as they should, for many years of pain-free biting and chewing.

Endodontists are specialists in saving teeth. Learn more about why you should see an endodontist.


Calcium in the Body

You’ve probably heard that calcium grows strong bones to keep you healthy as you age. But what else does it do for your body? According to the National Osteoporosis Foundation, calcium builds your bones and muscles and enables blood to clot 12.

About 99 percent of the calcium found in your body is in the bones and teeth.

The body does not produce calcium on its own. Instead, it’s found in foods like yogurt, milk, salmon and even kale — and it can also be consumed as a dietary supplement.

The amount you need per day depends on your age, as well as your biological sex, and can range anywhere from 200 milligrams for babies to 1,000 milligrams for breastfeeding adults. The National Institutes of Health warns that vegans, people with lactose sensitivities, postmenopausal women, and people with amenorrhea often need higher levels of calcium than others 2.

  • You’ve probably heard that calcium grows strong bones to keep you healthy as you age.
  • About 99 percent of the calcium found in your body is in the bones and teeth.

Introduction

The term 'cuspal fracture odontalgia' was first used by Gibbs in 1954, 1 to describe a condition which is better now known as 'cracked tooth syndrome' or 'cracked cusp syndrome'. The latter concept was coined by Cameron in 1964, 2 who proceeded to define the condition as 'an incomplete fracture of a vital posterior tooth that involves the dentine and occasionally extends to the pulp'. In more recent times the definition has been amended to include, 'a fracture plane of unknown depth and direction passing through tooth structure that, if not already involving, may progress to communicate with the pulp and or periodontal ligament'. 3

The term 'incomplete fracture of posterior teeth' is often used interchangeably with that of cracked tooth syndrome, 4 while the terms 'green-stick fracture' or 'split tooth syndrome' have also been used synonymously. 5

Patients suffering from cracked tooth syndrome (CTS) classically present with a history of sharp pain when biting, or when consuming cold food/beverages. 6 It has been suggested that the symptom of pain on biting increases as the applied occlusal force is raised. 7 A detailed assessment of the symptoms may reveal a history of discomfort that may have been present for several months previously. Other symptoms may include pain on release of pressure when fibrous foods are eaten, 'rebound pain'. 8 Pain may also be elicited by the consumption of sugar containing substances 5 and also by the act of tooth grinding or during the undertaking of excursive mandibular movements. 9 While some patients are able to specify the precise tooth from which the symptoms may be arising, the latter is not a consistent feature. The absence of heat induced sensitivity may also be a feature.

Where the fracture line may eventually propagate into the pulp chamber ('complete fracture'), symptoms of irreversible pulpitis or apical periodontitis may ensue, while fractures which progress further towards the root may be associated with areas of localised periodontal breakdown or at worst culminate in vertical tooth fracture 5 as shown by Figure 1. Table 1 provides a summary of the commonly associated signs and symptoms associated with CTS.

Any delays in instituting therapy may result in such an outcome, which may happen where there is doubt over the diagnosis of the condition

The physiological basis of pain on chewing has been hypothesised by Brannstrom et al. 10 to be accounted for by the sudden movement of fluid present in dentinal tubules which occurs when the fractured portions of the tooth move independently of one another. It is thought that the latter results in the activation of myelinated A-type fibres within the dental pulp, thereby accounting for the acute nature of the pain. It has also been suggested that the perception of hypersensitivity to cold may occur as a result of the seepage of noxious irritants through the crack, which results in the subsequent release of neuropeptides which cause a concomitant lowering in the pain threshold of unmyleinated C-type fibres within the dental pulp. 11

An alternative hypothesis has been proposed, whereby it has been postulated that the symptoms are caused by the alternating stretching and compressing of the odontoblast processes located within the crack. 12

The aim of this article is to provide an overview of the condition of 'cracked tooth syndrome', with regards to its epidemiology, aetiology and diagnosis.

Figure 2 illustrates a tooth which has an incomplete fracture, which was revealed upon the removal of an existing silver amalgam restoration. An incomplete tooth fracture is often difficult to visualise before a restoration is removed, but transillumination can be used from different aspects to show the presence of an interface within the tooth (Fig. 3). Tooth fractures can be highlighted by the use of stains although this may be difficult to remove and colour the final aesthetic restoration.

Shows an example of a tooth which has an incomplete fracture (a) which was revealed upon removal of an existing silver amalgam restoration (b) the arrows illustrates the path of the fracture line running around the mesiopalatal cusp

The transmission of the light beam has been 'stopped' mesio-lingually by the presence of the fracture


The Fundamental Thermodynamics of Calcium-Phosphate Precipitation

Key Concepts

There are key facts in calcification that derive straightforward from the laws of thermodynamics, and therefore must always be obeyed. The most basic concept is the second law of thermodynamics, stating that the entropy of an isolated system can never decrease (see Box 1). This law sets forth the condition for spontaneous chemical processes governed by the Gibbs expression (Gibbs, 1874�): DG < 0, where DG is the variation of the Gibbs free energy, which it is related to the entropy change. In this review we use often the term precipitation concerning the process of calcium phosphate solid formation from its free ions in a solution state. With this term we refer to its classic definition: 𠇊 relatively rapid formation of a sparingly soluble crystalline—or sometimes amorphous—solid phase from a liquid solution phase” (Karpiński and Jerzy Baᐭyga, 2019). Thus, translating the Gibbs condition to the precipitation of ions from solutions or biological matrices, it turns out that the activity product of free ions in solution must by higher than the corresponding activity in the solid. This relationship, expressed in terms of supersaturation (S), characterizes the thermodynamic conditions for all spontaneous calcifications as S > 1 (Myerson et al., 2019), with no exceptions. Thus, this expression means that the precipitate will be formed only if the product of the activities of free ions in the fluid state is higher than the product of the activities of ions in the solid state given by the solubility product, Ksp when S < 1, the solid will dissolve. Thus, the key point for determining whether MVC is likely to occur is to determine the activity of the free ions in the tissues that compose the medial layer. However, this is not a trivial task, given that there are many factors that affect the activities of free ions in a fluid, such as ionic strength, phosphate proton dissociation equilibria, the sequestration of free ions by ligands, the precipitation of different calcium phosphate solids, and the viscosity of the medium. Due to such high level of biological complexity, the available values of Ksp and other related thermodynamic constants needed for these calculations that have been derived from in-vitro conditions may be only approximates. In addition, it is important to realize that the thermodynamic condition for solid growth is related to ionic activities and not the ionic concentration. While the latter can be determined by chemical analysis, the former are far more difficult to determine due to the biological and the structural complexity of the medial layer. Concentrations and activities are related by the activity coefficient, γ(a = γ⋅ conc), which, in turn, is related to the ionic strength, I (see Box 2 and Table 1).

BOX 1. The driving force of precipitation.

Similar to all chemical processes, the precipitation of calcium phosphates is governed by the Gibbs free energy of the component ions in the solid and the solution. 1 A chemical system is at equilibrium when the Gibbs free energy is zero.

which at constant pressure and temperature is reduced to:

where μi is the chemical potential of component i, and ni is its number of moles. The chemical potential is defined as:

where ai is the activity of component i.

For a binary solid in equilibrium with its component ions in aqueous solution.

the equilibrium condition would be:

considering that the activity of a solid is zero, the equation is reduced to:

From that expression, the solubility product is defined as

Ksp is a constant that varies with the temperature as:

For a crystal to grow from a solution:

From that expression, the ionic activity product is defined as

Thus, a solution would be supersaturated with respect to a given solid when S > 1.

S is the driving force for crystallization, and it is the key factor in precipitation processes.

In order to compare salts with different stoichiometry, it is better to use an expression of supersaturation weighted by the number of ions in the chemical formula of the compound, n,

G, Gibbs function V, volume P, pressure S (Niskanen et al., 1994), entropy T, temperature μ, chemical potential n, number of moles R, gas constant a, activity coefficient aq, aqueous Ksp, solubility Product IAP, ionic activity product S (McCrindle et al., 2017 Seethapathy and Noureddine, 2019), supersaturation.

BOX 2. Activity coefficient and ionic strength, I.

The ionic strength, I, is the measure of the total amount of all the ions present in solution:

where zi is the charge of ion i. The main ionic components of human serum are listed in Table 1. Applying this equation, the resulting ionic strength would be: I = 0.141 M. However, in the following we will consider a value of 0.15 M for the ionic strength in blood, because this value is commonly accepted and generally used in the manufacturing of the blood replacement media (Nel et al., 2009).

Among the several expressions for calculating the activity coefficient based on the ionic strength the most commonly accepted one is the Debye–H࿌kel expression, as proposed by Davies: 6

where A is 0.52, at 37ଌ. 7 At an ionic strength of I = 0.15, for Ca 2+ and HPO4 2– ions, γ = 0.33, and for PO4 3– , γ = 0.08.

Table 1. Molar concentrations of ionic components in human serum with a concentration above 0.1 mM.

For a given ion concentration, ion activity decreases with the ionic strength. For example, for I = 0.15, the activity of the Ca 2+ ion is reduced threefold with respect to the Ca 2+ concentration, and the activity of the PO4 3– ion decreases 10-fold. Such reductions ultimately prevent spontaneous crystallization in blood and, thus also, for example, invalidate the hypothesis that CKD- or ESRD-related hyperphosphatemia per se causes precipitations seen in MVC.

Calcium Phosphate Precipitation System

Calcium ions in aqueous solution are not totally free rather they are solvated with water (Zavitsas, 2005). In other words, they form a coordination sphere of water molecules. This coordination sphere is composed of an inner shell of six water molecules with strong binding energy and an outer shell of 0𠄶 water molecules with weaker binding energy. The strong hydration of calcium is an important factor in the crystallization of calcium salts from water. Actually, the detachment of water molecules from calcium ions occurring on a crystal surface before they are incorporated into the crystal lattice is often the rate-limiting factor in crystal growth kinetics (van der Voort and Hartman, 1991). This is the case, for example, in calcium oxalate crystallization, for which trihydrate and dihydrate salts are kinetically favored and precipitate earlier than the monohydrate polymorph that has lower solubility and higher thermodynamic stability (Grases et al., 1990).

There are three different phosphate ions derived from the dissociation of phosphoric acid (H3PO4): H2PO4 – , HPO4 2– , and PO4 3– . In aqueous solution produces several species of phosphate ions according to the following equilibria (Vega et al., 1994):

Their relative presence in a medium depends on the pH (Figure 1A). At the physiological pH = 7.4, the concentrations of H2PO4 – , HPO4 2– , and PO4 3– are 0.049, 0.95, and 1.08 × 10 𠄵 , respectively. In solutions with a pH of 6.8, the concentrations change to 0.17, 0.83, and 0.27 × 10 𠄵 , respectively. In solutions with pH of 7.6, the concentrations become 0.031, 0.97, and 1.71 × 10 𠄵 . Thus, in this pH – range of 6.8𠄷.6, the variations of HPO4 2– are minimal, but the concentration of H2PO4 – is reduced more than fivefold and that of PO4 3– is increased in a similar proportion (see Box 3).

Figure 1. Parameters affecting precipitation. (A) La Mer plots representing the different stages of precipitation from solution in relation to supersaturation under different conditions are shown. Initial conditions (left panel): the supersaturation limit value (Virchow, 1858) and a metastable range of ion activities in which the solution does not precipitate, despite being supersaturated, are depicted. This metastable region represents an energy barrier for nucleation. Once a critical concentration is reached (Sc), homogeneous precipitation will take place in the solution. The limits can be reduced in the presence of nucleation promoters (central panel). Promoters in solution have a high affinity for any of the crystal component ions and can induce nucleation even below the critical supersaturation level through a process called heterogeneous nucleation. On the other hand, other molecules, termed inhibitors of nucleation, have the capacity to increase the energy barrier for nucleation, thereby increasing both the critical supersaturation value and the induction time for nucleation (right panel). (B) Effect of pH on the formation of the different species of phosphate and carbonate. Left, molar ratio of the phosphate species H2PO4 – (orange), HPO4 2– (green), and PO4 3– (violet) in solution as a function of pH, within the pH range of interest: 5.5𠄸.5. Right, variation of the concentrations of H2CO3 (orange), HCO3 – (green), and CO3 2– (violet) within the same pH range in DMEM medium. The shaded area corresponds to the expected physiological pH. (C) Effect of pH on the supersaturation of ACP and HAP in blood at the lowest and highest concentrations of Ca and phosphate (Pi), at normal concentration limits and under hyperphosphatemic conditions. (D) Effect of the total concentration of phosphate (Pi) on the supersaturation of ACP and HAP in blood, at pH = 7.4. The lower limit corresponds to the Ca and Pi concentrations of 1.02 and 1.00 mM, respectively the upper limit corresponds to 1.23 and 1.5 mM and hyperphosphatemia corresponds to 1.23 and 2.00 mM.

BOX 3. Dissociation constants of calcium and phosphate ions.

The amount of free calcium and phosphate ions in solution available for precipitation is reduced by the formation of soluble calcium phosphate. The equilibrium equations and corresponding association constants of these complexes are as follows (Chughtai et al., 1968):

Ca 2 + + H 2 ⁢ PO 4 - ⟺ CaH 2 ⁢ PO 4 +     K = 31.9 l/mol

Ca 2 + + HPO 4 2 - ⟺ CaHPO 4 ( aq . )      K = 6.81 × 10 2 l/mol

The different phosphate ions can combine with calcium ions into 12 different compounds, although only six can be produced directly from solution. The empirical formulas and solubility products of these six compounds are given in Table 2.

Table 2. Main calcium phosphate compounds appearing in precipitates from aqueous solutions.

The important question is which of those species may be relevant in MVC? An analysis of in vitro and in vivo calcifications shows that from the six different calcium phosphate solid compounds (Bohner, 2010), only amorphous calcium phosphate (ACP) and hydroxyapatite (HAP) are generally present in MVC (Chernov, 1984 Mullin, 2001 Klaus-Werner, 2019).

ACP is usually the first phase to precipitate under physiological conditions (Manas et al., 2012 Hortells et al., 2017 Lotsari et al., 2018), and therefore it is the relevant early phase in pathophysiology of ectopic calcifications. Importantly, the ACP and HAP are mainly composed of PO4 3– ions as confirmed by IR analysis (Vecstaudza et al., 2019). Therefore, although the concentrations of H2PO4 – and HPO4 2– ions are substantially higher than that of PO4 3– ions at physiological systemic pH values, it appears that in MVC the concentration of PO4 3ions will be the most critical one.

Given that ACP is the first compound to be observed in the MVC process (Hortells et al., 2017), the supersaturation of this compound (SACP) within the media will likely predict and trigger the appearance of MVC. To calculate SACP, reliable values of Ksp(ACP) in biological tissues would be needed. However, the only value of Ksp available at present Ksp(37ଌ) = a C ⁢ a 2 + × a H + 0.22 × a P ⁢ O 4 3 - 0.74 = 3.35 × 10 � was determined in vitro, while allowing for a wide variation of pH values (Christoffersen et al., 1990). Such pH variation is unlikely occur in the living tissues closely controlling the pH homeostasis. Therefore, the Ksp(ACP) would need to be determined to reflect the conditions within the media. Meanwhile, we have used the in vitro Ksp (ACP) value in our calculations of ACP supersaturation keeping in mind that the results can be only approximates. The results are provided and compared with those of dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP), and HAP in normal subjects in Table 3. These calculations show that within the biological range of minimum and maximum calcium and phosphate concentrations in humans, the supersaturation should vary from S = 0.65 to S = 0.83. During the hyperphosphatemic conditions in patients with CKD, S equals 0.92, which is still below the precipitation level (S ≥ 1). However, the local changes in pH within the media or imbalance between the promoters and inhibitors could favor precipitation, as shown below. The changes in local pH within the media could explain the fact that MVC appears in CKD before hyperphosphatemia is observed (Hortells et al., 2017), may be as a consequence of VSMC transdifferentiation, and possibly also the occurrence of MVC observed in non-CKD conditions, such as diabetes or aging.

Table 3. Supersaturation, S, of the various CaP species (ACP, DCPD, OCP, HAP) at several experimental conditions, at 37 ଌ.

Molecular Processes in CaP Precipitation

The knowledge of the molecular mechanisms of the physiological bone and teeth mineralization may be helpful to understand ectopic calcifications, as they may share common mechanisms (Allen and Moe, 2020). In summary, physiological calcification is a complex phenomenon carried out by specialized cells, and it involves a variety of actors, mainly proteins that in the bone constitute the osteoid: (a) collagenous proteins and elastin that, in the vascular wall, create close compartments to facilitate confined crystallization and crystal growth (Veis, 2005) (b) amphiphilic proteins self-assembled to form scaffolds to provide potential nucleation sites before mineralization begins (Beniash et al., 2000) and (c) non-collagenous proteins that promote nucleation and control crystal growth morphology through interactions with certain crystal faces (Veis, 2005). In the first stage of biomineralization, an amorphous inorganic compound is produced in an environment occupied by a majority of organic compounds (60% of the total volume 82). In the second stage, the inorganic compound crystallizes, and the crystals are organized into highly hierarchical superstructures. This complex biological machinery exercises complete control over the crystallization process, including crystal size, crystalline purity, growth orientation, shape molding, and hierarchical superstructuring.

Recent analyses of the early stages of CaP precipitation using high-resolution molecular techniques and ab initio molecular dynamics simulations (Lin and Chiu, 2018 Zhao et al., 2018) provide better insights into the molecular processes involved in CaP precipitation (Habraken et al., 2016). During the initial step, calcium and phosphate ions associate to form dinuclear and trinuclear complexes and subsequently polynuclear Posner clusters (Beniash et al., 2000) composed of nine Ca 2+ ions and six phosphate PO4 3– anions, surrounded by 30 water molecules, as depicted in Figures 2A, 3. After that, Posner clusters agglomerate to form hydrated precipitates with a low density and a Ca/P ratio of 1:1 (Zhang et al., 2015 Jiao et al., 2016 Niu et al., 2017). In the third step, the precipitate becomes denser by losing water and increasing the Ca-O-P connectivity to form ACP, with a Ca/P ratio of 1:1.35. Finally, through slow transformation in the solid state, crystalline HAP nanoparticles are formed within the precipitate (Figure 3). This mechanism does not follow classical nucleation theory that is based on the one by one incorporation of add-atoms to the growing nuclei. This also applies to the process of CaCO3 nucleation (Smeetsa et al., 2017). One of the consequences features of this non-classical behavior is the lowering of the nucleation energy barrier (see below) (Yang et al., 2019).

Figure 2. Promoters of CaP mineralization. (A) Structure of major biomolecules that as promoters of calcification: phosphorylated proteins, sulfated glycosaminoglycans, carboxyglutamic proteins and phospholipid membranes. (B) CaP mineralization in the presence of nucleation promoters: accumulation of Ca by absorption on promoters, precipitation of ACP, and crystallization of HAP.

Figure 3. Hypothetical molecular processes in calcium phosphate medial vascular mineralization. At pH up to 7.40 Ca 2+ ions are hydrated, and the majority of the phosphate molecules are in the form of HPO4- 2 - ions. The precipitation is triggered by a local increase of pH to above 7.90, associated with a marked increase in PO4 3– ions the main component in ACP and HAP. Ca 2+ and PO4 3– ions form complexes of increasing coordination number, eventually forming multinuclear clusters, which contain nine Ca 2+ ions and six PO4 3– ions. Subsequently, promoter macromolecules with charged Ca 2+ -ligand groups (phosphate, carboxylate, and/or sulfate) produce a local accumulation of Posner clusters and the appearance of ACP precipitates. Finally, as the process progresses, the precipitate clusters rearrange into dense crystalline HAP nanoparticles.

Amorphous calcium phosphate precipitates gradually transform into HAP (Hortells et al., 2015). The amorphous ACP solid evolves by slow reorganization of the loosely packed clusters into crystalline, rod-like domains of nanometer size, with a HAP structure (Figure 2). The appearance of HAP implies a dramatic change of the calcification regime. Because HAP is virtually insoluble, the crystallization process turns largely irreversible. It should be noted that the blood and fluids contained in biological tissues are highly supersaturated in HAP. The fact that HAP does not precipitate spontaneously in tissues appears to be due to a high nucleation barrier of this calcium phosphate phase keeping the medium in a metastable state. Once HAP has been formed, there is no energy barrier to stop growth, provided S > 1, unless inhibitors are present on site. Thus, in this sense at pH of 7.40 and at the lowest level of blood Ca concentration (1.02 mM), calcifications composed of HAP would be allowed to grow even when the concentration of Pi would be 1,000 times less than the lowest value in blood (see Figures 1C,D).

Transmission Electron Microscopy analyses of CaP calcifications in cell cultures are consistent with the precipitation mechanism described above (Hortells et al., 2015). Fresh precipitates appear as low-density sheets having a Ca/P ratio of one and showing some areas of higher electron density. After some aging time, precipitates show spherulite formations, which do not exhibit any crystalline order in electron diffraction and have a Ca/P ratio of 1.35, like ACP. After longer aging periods, precipitates show rod-like nanoparticles that yield electron diffraction rings, and they show crystal planes at high resolution. Based on observations of in a rat model using scanning electron microscopy (SEM), the arterial calcifications are localized and not uniformly distributed, suggesting a heterogeneous nucleation induced by a promoter, or a local increase in supersaturation, or both mechanisms as the origin of the calcification (Hortells et al., 2017). A chemical analysis of the deposits yielded mostly a Ca/P ratio of 1.35, close to that of ACP, although occasionally higher values were found, approaching that of HAP (1.67). Rat arteries that did not show any sign of calcification in optical microscope, at closer observation by SEM revealed small areas with a strong presence of Ca, but no detectable phosphate (Hortells et al., 2017). Although this preliminary observation should be studied in greater detail, it appears possible that these Ca aggregates may actually trigger CaP deposition in similar fashion as observed in physiological calcification in bones and/or teeth with calcium-rich proteins serving as promoters of mineralization.

Nucleation and the Activation Barrier

It is important to note that solubility products are calculated based on the activities of ions in solution that are in direct contact with the solid phase. However, in the absence of the solid phase, precipitation does not occur immediately after the ionic product exceeds the solubility product. In other words, S > 1 is a necessary but not sufficient condition for precipitation because there is an energy barrier for nucleation, similar to the majority of chemical reactions (Vallence, 2017) that has to be overcome before precipitation takes place. The onset of nucleation occurs when the size of the nuclei reaches a certain critical value, due to the large surface energy of small nuclei (Chernov, 1984 Mullin, 2001 Klaus-Werner, 2019). Below that critical size, the process of ion association is transient and reversible. Below that critical size, the process of ion association is transient and reversible. This is due to the fact that ions on the particle surface have greater energy than those in the bulk, given that the number of nearest neighbors of ions on the surface is obviously lower than that of ions in the bulk. The total Gibbs free energy of a particle growing from its component free ions is the sum of that of bulk ions and surface ions, and for small sizes, the number of surface ions is relatively large, so they tend to re-dissolve until the supersaturation in solution reaches the critical value. The process of formation of stable nuclei requires a definite induction time defined by the period between the time the critical supersaturation has been established and the first nuclei were detected (Chernov, 1984 Mullin, 2001 Klaus-Werner, 2019). As the size of the nuclei increases, they can grow with decreasing levels of supersaturation. Finally, large crystals will continue to grow even at very low levels of supersaturation, as shown in Figure 2. Thus, while induction of nucleation requires high levels of supersaturation, once larger particles have been formed, the growth will proceed with ever decreasing levels of supersaturation.

Consequently, a supersaturated solution can remain in a metastable state until the activation barrier has been overcome, i.e., the critical supersaturation value, Sc, has been reached, therefore enabling the formation of stable nuclei (Christoffersen et al., 1990 Figure 1B). Thus, S (ACP) > 1 is the necessary condition for growth, and S (ACP) > Sc is the necessary condition to trigger precipitation. Using data from spontaneous nucleation experiments in water (Strates et al., 1957 Fleish and Neuman, 1961), we have estimated the Sc values of 1.03 (Fleish and Neuman, 1961) and 1.05 (Strates et al., 1957) for ACP. These values represent early estimates and require validation using well-controlled experimental models emulating in vivo conditions. Nevertheless, these early Sc (ACP) estimates indicate that the critical supersaturation for MVC should be rather low, underscoring the non-classical character of ACP nucleation (Smeetsa et al., 2017 Yang et al., 2019). Thus indeed, the molecular mechanism of ACP nucleation appears to proceed through the gradual formation of soluble CaP complexes of increasing size, eventually forming multinuclear clusters with the formula, Ca9(PO4)6⋅nH2O (Mancardi et al., 2016). These clusters having the same structure as HAP are the likely precursors of ACP. initially forming the low-density amorphous precipitates with a Ca/P ratio of approximately 1 (ACP1) and eventually evolving into denser particles (ACP2) with a Ca/P ratio of 1.35, typically found in MVC, in bones, and other physiological and ectopic calcifications.

Nucleation Promoters

The process of CaP precipitation described above can be altered by the presence of promoters and/or inhibitors in the system. Promoters are agents that favor the precipitation of compounds in solutions that otherwise would remain metastable. Conversely, nucleation inhibitors restrain the formation of precipitates from solutions that otherwise would experience spontaneous homogeneous nucleation. In thermodynamic terms, promoters decrease Sc and induce heterogeneous nucleation located at the promoter site, whereas inhibitors increase Sc and retard the calcification process (Figure 1B). Thus, promoters and inhibitors may control the spontaneous crystallization processes and appear to dominate MVC.

Calcification promoters typically harbor abundant Ca 2+ -ligand groups in their chemical structure, responsible for the local accumulation of these ions. In this calcium-rich environment created by the promoter, nucleation might occur with minor increases in phosphate ion activities calcification mechanism responsible for the formation of bones and teeth (Addadi and Weiner, 1992 Beniash et al., 2000 Veis, 2005). However, the disordered texture of the deposits observed in MVC suggests that the mechanism of mineralization and the agents involved in the process are likely different. Yet the two processes, bone formation and MVC, may have in common that the nucleation promoters can induce the onset of precipitation, because in both cases, the occurrence of deposits is discrete and localized, both being typical features of heterogeneous nucleations. Disseminated foci of calcifications within the medial layers are the outstanding hallmark of the MVC. However, the nature of promoters in MVC has not yet been studied in detail and requires further clarification.

CaP nucleation promoters are molecules or macromolecules with a high affinity for calcium and/or phosphate ions. To interact with phosphate ions, promoters must have positive charges, which in biomolecules are mostly provided by ammonium ions or quaternary amines. In any case, however, the interactions are weak. Figure 4A shows the typical Ca 2+ -ligand groups in biomolecules. In descending order according to their binding strength, these groups include phosphonate, phosphate > carboxylate > sulfonate = sulfate > alkoxide ≈ water (Zhao et al., 2018). Several kinds of biomolecules may contain large numbers of these groups, as it is the case of phosphate in multiphosphorylated proteins (Figure 4Aa), such as phosvitin (Greengard et al., 1964), involved in physiological calcifications (Sarem et al., 2017). Phosvitin consists of approximately 50% serine residues, which carry a phosphate group (Greengard et al., 1964) that gives this protein an extraordinary capacity to accumulate Ca 2+ ions. Sarem et al. (2017) were able to show that the precipitation of CaP proceeded by the incorporation of Ca 2+ ions into phosvitin, followed by the precipitation of ACP, and subsequent recrystallization into HAP by aging. The experimental conditions included HEPES buffer (pH = 8), 1.25 mM Ca(NO3)2, and 1.5 mM (NH4)3PO4. Under these conditions, the supersaturations of ACP and HAP correspond to 1.21 and 8, respectively. Furthermore, recently, it has been shown that the disordered secondary structure of phosvitin orchestrates the nucleation and growth of biomimetic bone such as apatite (Sarem et al., 2017).

Figure 4. Promoters and inhibitors of CaP mineralization. (A) Structure of major biomolecules that as promoters of calcification: phosphorylated proteins, sulfated glycosaminoglycans, carboxyglutamic proteins and phospholipid membranes. (B) CaP mineralization in the presence of nucleation promoters: accumulation of Ca by absorption on promoters, precipitation of ACP, and crystallization of HAP. (C) Mechanism of crystal growth inhibition. (a) Representation of a Kossel crystal, with the different positions of adatoms on the crystal surface: flat site (surface nucleation) (Virchow, 1858), step site (Niskanen et al., 1994), kink site (Lehto et al., 1996), inhibitor molecule blocking a kink site (I) (b) a screw dislocation, and blocking of face growth by an inhibitor molecule.

Phospholipids (Figure 4Ad), the main component of cell membranes, matrix vesicles and exosomes, have also been proposed as promoters of pathogenic calcifications (Felix and Fleisch, 1976 Boskey, 1978 Schoen et al., 1988 Anderson, 2007 Wuthier and Lipscomb, 2011). Phospholipids are arranged in layers with a high density of phosphate-charged groups endowed with the capacity to concentrate Ca 2+ ions. Indirect evidence of their role as promoters has been found in analyses of medial sclerosis of large arteries, aortic valves, and atherosclerotic plaques (Felix and Fleisch, 1976 Boskey, 1978 Schoen et al., 1988 Anderson, 2007 Wuthier and Lipscomb, 2011). Phospholipids may be released following degradation of cells or matrix vesicles (Felix and Fleisch, 1976). In addition, their phosphate groups may be also released as PO4 3– ions by alkaline phosphatase, potentially also facilitating calcium precipitation (Towler, 2005).

Promoter biomolecules containing sulfate include glycosaminoglycans (GAGs) (Figure 4Ab) or mucopolysaccharides (Kepa et al., 2015), such as chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, and heparin. In combination with proteins, form mucoproteins are formed. Mucopolysaccharides and mucoproteins have been the object of intense research based on their role as a matrix for the nucleation and structuring of calcium oxalate renal calculi. Among the mucopolysaccharides, dermatan appears the most interesting one, because its presence in the skin, blood vessels, and heart valves.

Biomolecules with a large number of carboxylic residues include glutamic- and carboxy-glutamic-rich proteins (Figure 4Ac). Collagen and elastin neutral proteins have also been proposed as promoters of VC (Urry, 1971). This hypothesis is based on the observation that Ca 2+ ions may interact strongly with protein carbonyl groups from glycine amino acids arranged in a helix conformation. However, it should be noted that the carbonyl groups can barely compete in Ca 2+ binding with charged groups, such as phosphate, carboxylate, and sulfate ions, because these groups interact through strong electrostatic forces, in a bidentate (also named chelate and it means bonded by to atoms to the central atom) manner. Instead, most likely, carbonyl groups appear to bind to Ca 2+ ions by means of hydrogen bonds with hydration water (see section “Key Concepts”). Therefore, glycoproteins such as bone acidic glycoprotein-75, or bone phosphoproteins such as osteopontin or bone sialoprotein (Chen et al., 1992), appear to be more suitable promoters than collagen and elastin. These proteins not only have a higher capacity to bind Ca 2+ , but they interact with collagen itself in the presence of Ca 2+ in a concentration-dependent manner. This Ca 2+ -mediated interaction with collagen has also been observed in matrix vesicles (Kirsch and von der Mark, 1991). Furthermore, studies on Ca 2+ -collagen interactions have shown that they take place through electrostatic interactions with the carboxylate groups (Glu and Asp) present in collagen (Pang et al., 2017).

All of these biomolecules have demonstrated the capacity to promote CaP nucleation in metastable solutions in vitro and appear to be suitable potential candidates for triggering and sustaining the calcification process in MVC, whether they are present in exosomes, apoptotic bodies, long-life proteins or transdifferentiated cells. A scheme of CaP deposition induced by promoters is shown in Figure 4C. In the first step the promoter expedites an accumulation of Ca 2+ ions that are trapped by the high density of calcium ligand groups in the promoter structure. In the presence of phosphate, Ca 2+ ions will form soluble CaP clusters, eventually ending up as precipitates, as described above.

Inhibition of Calcium Phosphate Nucleation and Crystal Growth

In addition to the promoters the onset and propagation of VC is also determined by the presence of agents that retard or restrain CaP precipitation. These agents comprise the class of inhibitors. Based on the mechanisms of action, four main groups of agents may be distinguished. The first group consists of agents precluding or retarding the formation of stable nuclei by increasing the activation barrier (nucleation inhibitors) (Giocondi et al., 2010). The second consists of agents that are in small amounts capable of restraining the crystals’ growth by attaching to the crystals’ surfaces (crystal growth inhibitors) (Dobberschuütz et al., 2018). The third constitute agents slowing the maturation from ACP to HAP and the fourth compounds that, without being truly inhibitors, can reduce supersaturation by sequestering calcium ions in the form of soluble complexes (Reznikov et al., 2016) or, in the case of renal disease, reduce hyperphosphatemia with the use of phosphate binders (Floege, 2016) or renal Pi reabsorption (Tsuboi et al., 2020), thus reducing the risk of calcification.

Nucleation Inhibition

Albeit the precise value of critical supersaturation required for the nucleation of ACP in vivo is not known and data on nucleation inhibition are sparse (Strates et al., 1957 Fleish and Neuman, 1961) approximate value can be calculated based on in vitro data available in the literature (Strates et al., 1957). Accordingly, the critical supersaturation for the nucleation of ACP would be Sc = 1.05 at pH = 7.4. Interestingly, analogous calculations based on experimental data of other authors (Fleish and Neuman, 1961) yields Sc = 1.03 at pH = 7.4. According to this value, and considering the average concentration of Ca 2+ = 1.18 mM found in healthy subjects, the expected concentration of Pi needed for spontaneous calcification in blood would be 2.75 mM a value far above the regular blood levels (0.97𠄱.45 mM) blood levels, or even of patients with severe hyperphosphatemia.

It has been suggested that active collagen may act as a promoter of calcification (Strates et al., 1957 Fleish and Neuman, 1961). However, this proposition appears rather unlikely because the calculated Sc = 0.73 based on the solubility product of ACP reported by Christoffersen et al. (1990), would not fulfill the conditions required for precipitation S ≥ 1. These discrepancies emphasize the need for precise calculations of thermodynamic parameters based on well-controlled experimental studies in vivo. Nevertheless, Fleish’s experiment has also demonstrated the nucleation inhibition capacity of pyrophosphate (PPi) and polyphosphate by increasing the Sc(ACP) for spontaneous (homogeneous) precipitation up to 1.07 and 1.10, respectively. Apparently, the critical supersaturation values for nucleation are rather low, but this conclusion should be considered with caution, given that both experiments𠅊s well as the determination of Ksp(ACP)—were performed at different ionic strengths and under changing pH conditions (pH has a huge effect on CaP precipitation). Actually, the Ksp was calculated at I = 0.035, and the pH changed from 7.4 to 5.7 during precipitation while in Strates et al. (1957) the ionic strength was 0.165 and the final pH was around 5.9. Nevertheless, despite of the obvious shortcomings of calculations of thermodynamic parameters based on in vitro data, these calculations offer an important impetus to explore further the role of collagen in CaP nucleation promotion by studying the relevant thermodynamic parameters in vivo. Furthermore, they also appear to explain the ability of pyrophosphate and organic polyphosphates to inhibit or to retard the onset of CaP nucleation. Strates et al. (1957) also described that HAP seed crystals can grow in normal blood serum, a proposal that supports our hypothesis that the nucleation of CaP deposits is controlled by ACP precipitation kinetics, but once the transformation to HAP occurs, the kinetics are driven by HAP crystal growth, which will progress even when the alterations that provoked the mineralization event return to normal. Actually, the threshold of Pi concentration for HAP supersaturation at pH = 7.40 and at the lower limit of the total calcium concentration in blood (1.01 mM) could be as low as 0.85 mM.

Inhibitors of ACP Conversion Into HAP

Some molecules are capable of stabilizing ACP and preventing or retarding the maturation into HAP, thereby keeping the door open to reversion of mineralization, which would become extremely improbable once HAP has been formed. Two known inhibitors of calcification acting by the ACP stabilization are pyrophosphate (PPi) (Ibsen and Birkedal, 2018) and magnesium (Boskey and Posner, 1974 Ter Braake et al., 2018). For details, see Box 4.

BOX 4. Crystal growth inhibition.

Inhibitors of crystal growth are molecules or ions that attach firmly to the crystal surface, thereby making it difficult for the growing crystal units to displace them thus preventing or retarding the attachment of new adatoms. The effect of inhibitors on crystal growth is briefly explained. If the ions in a solution remain above the nucleation rate, then the supersaturation will remain above the critical value for nucleation, Sc, allowing the process to go on. Initially, crystals have a rounded shape and a rough surface, but gradually they develop flat faces to minimize their surface energy. Using the model of a Kossel crystal (Kossel, 1927 Stranski, 1928 Figure 4C) with cubic adatoms that bind to the nearest neighbors on each crystal face, the energy gain of an adatom incorporating on a completely flat face will be obviously low (Figure 4Ca, adatom 1). Yet once attached, it will create a stairs-like step facilitating the adsorption of the next adatom to the two nearest neighbors (Figure 4Ca, adatom 2). Once an adatom incorporates into a step of the stair it forms a kink site, such that the next adatom will bind to the three nearest neighbors (Figure 4Ca, adatom 3). Some inhibitor molecules may block the kink sites (Figure 4Ca, I), therefore restraining the growth of the face. This mechanism of surface nucleation and the spreading of steps to form new layers require high levels of supersaturation. However, if the face has a defect, called a screw dislocation (Figure 4Cb), permanent step site is created, and the supersaturation required for crystal growth decreases considerably. In this manner, only a small number of inhibitor molecules attached to the screw dislocations may very effectively restrain the growth of a crystal face. Inhibitors will be less effective on a face growing through a secondary nucleation mechanism. Furthermore, in the case of rough faces that consist mostly of kink sites, large amount of inhibitor molecules will be required to block the growth. It should be kept in mind that, ultimately, the type of the prevailing mechanism will depend largely on the level of supersaturation in the following order of importance: rough growth > surface nucleation > screw dislocation.

Available reports on CaP crystal growth inhibition refer mostly to HAP. The best well-known inhibitor of HAP crystallization is PPi. This ion is firmly adsorbed on a HAP crystal surface (Fleisch, 1978 Ibsen and Birkedal, 2018) affecting the progression of the mineralization process in a number ways including an increase in the critical supersaturation for both homogeneous and heterogeneous nucleation, lengthening of the nucleation induction period, change of the crystal morphology, and crystal stabilization by reduction of the growth and the dissolution rate. Although less effective, other molecules such as citrate (Mekmene et al., 2012 Shao et al., 2018) and magnesium (Boskey and Posner, 1974) can also be considered HAP crystal growth inhibitors. Thus, while PPi and Mg 2+ appear to limit the growth of HAP deposits, they are unable to reverse them. An ambitious in vivo experiment of CKD in rat seemed to have suggested the ability of Mg 2+ to revert VC (D໚z-Tocados et al., 2017), however, the experimental design appears to favor the accumulation of the reversible amorphous CaP rather than HAP crystal formation.

The list of potential inhibitors of HAP crystal growth can be further extended to phosphocitrate, polyphosphates, bisphosphonates, carboxyphosphonates, phytate (Thomas and Tilden, 1972), and other not yet identified plasma components (Rufenacht and Fleisch, 1984). However, because these in vitro studies on nucleation progression inhibition have been performed at pH, temperature, and concentration values far from physiological or even pathophysiological conditions, their validity for in vivo processes is limited (Hunter and Goldberg, 1993) yet potentially useful in specific clinical settings such as renal calciphylaxis (Millan, 1990 Perelló et al., 2018 Calciphyx, 2019).

The majority of the available reports focus on the role of PPi in the prevention of mineralization (Lomashvili et al., 2004) and the role of alkaline phosphatase (AP) responsible for the hydrolysis of the PPi into phosphate (Towler, 2005 Haarhaus et al., 2017). However, due to the significant variability of the experimental in vitro and in vivo study designs, only tentative conclusions regarding the promotion and inhibition of CaP crystallization are feasible at present. To obtain full insight into the mechanisms of calcifications, standard protocols compatible with the in vivo conditions within the medial layer, preferably those in encountered in humans, will be required.

Nucleation Promoters That Act as Growth Inhibitors

In some cases, nucleation promoters may possibly also act as crystal growth inhibitors based on their capacity to bind and accumulate Ca 2+ and to adsorb on CaP crystal surfaces. This is the case of GAGs with respect to calcium phosphate brushite (Zhai et al., 2019). Occasionally, the inhibition of crystal growth by typical nucleation promoters [i.e., chondroitin sulfate (CS) and mucoproteins] has been claimed in batch precipitation reports (Hunter and Goldberg, 1993). But this apparent inhibition effect may just be the result of Ca 2+ absorption and the consequent decrease of the supersaturation. In constant composition studies, which more accurately reflect the conditions in blood vessels, CS has promoted precipitation. Phosvitin is a special case, which behaves like a nucleation inhibitor in dissolution but promotes nucleation when it is immobilized on a collagen surface (Onuma, 2005). Protein immobilization is also decisive in the promoter-inhibitor behavior of osteocalcin, mucoproteins, phosvitin, and phosphophoryn.

Effect of pH on Phosphate and Carbonate Nucleation

Of the three phosphate ion species, only PO4 3– participates in all stages of precipitation, as evidenced by: (i) PO4 3– is the main component of soluble precursor clusters (ii) PO4 3– is the main phosphate ion in the first precipitating phase, ACP and (iii) PO4 3– is also the phosphate component of HAP. As outlined above, the concentration of PO4 3– in solution is very low at pH = 6.9, (Figure 1A), but it increases rapidly with increasing pH. Consequently, the supersaturation of ACP, and the risk of CaP precipitation, is highly dependent on the pH (Figure 1C): the pH has a much greater impact than a variation in the phosphate concentration. Actually, an increase in local pH to 7.90 would be enough to reach the critical supersaturation value (S = 1.03) estimated for the average blood Ca and Pi concentration in healthy subjects. In physiological Ca 2+ and pH conditions, for example, it would be necessary to increase the local concentration of Pi to 3.0 mM to reach that S value, which is far above the usual level in hyperphosphatemic patients. In vitro experiments assessing the effect of cell activity onto the calcification process have also shown a notable importance of pH, mainly as a consequence of the used of highly bicarbonate media and low CO2 concentration in the atmosphere (Hortells et al., 2015). Significant changes in local pH in vivo in contrast to the serum’s strictly regulated pH homeostasis, could arise, as explained below, through a modification of the activities of Na + /H + - and bicarbonate exchangers and of proton pumps, carbonic anhydrases, and other factors related to VSMC’ metabolism (Leibrock et al., 2016 Yuan et al., 2019). Although local alkalinity has not been demonstrated to play a role in the pathogenesis of MVC, it could be hypothesized by combination of mechanisms, such as increased presence of promoters, depletion of inhibitors possibly orchestrated by a perfect metabolic storm signifying the transdifferentation of VSMC.

With respect to calcium carbonate, it is present in considerable amounts in pathological calcifications (Bazin et al., 2012) and in in vitro calcifications (our own experiments). The concentration of CO3 2– ions also increases rapidly above a pH of 7 (Figure 1A), and the supersaturation of CaCO3 in blood can be higher than that of ACP under physiological conditions (Table 4) supporting the hypothesis that VC may be, at least in part, related to pH rather than phosphate concentration’s changes. In culture media, MEM or DMEM (media commonly used in the in vitro calcification procedures) maintained at 5% CO2 atmosphere at given concentrations of bicarbonate and the pH (Hortells et al., 2015), the supersaturation of calcium carbonate (CaCO3) exceeds that of CaP in both media. This finding may not only explain the co-precipitations of CaCO3 with CaP but may also suggest its role in seeding calcium phosphate nucleation sites.

Table 4. Supersaturation of ACP and CaCO3 in blood and in MEM [(NaHCO3) = 2.2 g/L], and DMEM media with different concentrations of calcium and phosphate ions.

Precipitation in Intracellular or Extracellular Matrix Environments

To date, studies in the real in vivo or ex vivo medial layer environment are not available. Thus, the hypothesis charting the pathogenesis of CaP precipitation thermodynamics in the medial layer must be based only on the currently available incomplete evidence derived largely from in vitro observations.

Medial layer consists of ECM comprising matrisome with a number of different glycoproteins and proteoglycans with embedded networks of collagen and elastic fibers (Hynes and Naba, 2012) and VSMC. Due to the extensive extra- and intracellular compartmentalization and due to the higher viscosity of both ECM and VSMC cytoplasm compared with the water solutions and other solvents’ the mobility of the ions will be less predictable and more restricted. However, based on the available data, the viscosity of cytoplasm is approximately 1.2𠄱.5 times higher compared to water (Fushimi and Verkman, 1991 Bicknese et al., 1993 Kao et al., 1993 Luby-Phelps et al., 1993 Chang H. C. et al., 2008). To our knowledge, no data on viscosity of the ECM are available. Thus, as a matter of approximation, we assume that the environment of the media corresponds to viscous solution rather than solid gel. Based on these scanty reports we tentatively conclude that an increase in viscosity as suggested in the medial layer will mainly affect the ion transport, leading to a diffusion-controlled CaP crystal growth, possibly the single most important difference to the crystal growth in vitro solutions.

However, it is important to understand that in any biological system regardless of the composition and physical-chemical properties such as viscosity, the laws of thermodynamics and the links between supersaturation and CaP crystals’ growth retain their validity.

It is well known that electrochemical gradient between the micromolar concentration of Ca 2+ in the VSMC cytoplasm and the millimolar extracellular Ca 2+ concentration can be only maintained at high energy cost. This extremely low [Ca 2+ i] in cytosol along with the presence of PPi prevent spontaneous CaP precipitation inside the cell under physiological conditions [the cytosol also contains 3𠄵 mM free Pi (Peacock, 2021)] and normal metabolic states. Disruption of energy supply by the mitochondria, oxidative stress, electrophilic insults, etc., will cause uncontrolled influx of [Ca 2+ i] from the extracellular space or endoplasmic reticulum followed by breakdown of intracellular homeostasis and cell death (Orrenius et al., 2013). Apoptotic bodies or cell debris could become nucleation sites and thus contribute to calcification.

The hypothesis of the medial CaP crystallization’s stepwise process outlined in the section “Molecular Processes in CaP Precipitation” has received support by experimental data from Transmission Electron Microscopy analysis of CaP deposits from cell cultures and rat arteries demonstrating that early CaP deposits consist of ACP undergoing progressive densification process and resulting in the crystallization of HAP nanoparticles (Villa-Bellosta et al., 2011 Hortells et al., 2015, 2017). The fact that the two methods of VC research, in vitro and in vivo, show similar early deposits but both calcification processes are unrelated (in vitro being alkali-mediated homogeneous precipitation, whereas in vivo is heterogeneous precipitation caused by promoter nucleation) clearly shows that deposit formation and crystal maturation thermodynamics are constant and similar, independently of the environmental setup (Hortells et al., 2015).

Given the impact of pH on the supersaturation of ACP and therefore potentially initiation of calcifications, changes in pH should be considered as a potential important factor in MVC, in both intra- and extracellular milieu. The pH in both milieus is determined by a number of factors including the metabolic production of the acid moieties, the transmembrane protons’ transport, the activity of bicarbonate transporters and exchangers, and the enzymatic synthesis of bicarbonate. While the intravasal homeostasis of pH is strictly controlled within narrow boundaries, the local tissue pH appears less stable depending on the type of the tissue and specific metabolic activities (Martin and Jain, 1994). Osteoblasts, for example, thrive in vitro at alkaline pH (Galow et al., 2017), in agreement with the effect of metabolic alkalosis increasing osteoblastic collagen synthesis and reducing bone resorption related to a decrease in osteoclastic beta-glucuronidase release (Bushinsky, 1996). Albeit it has never been demonstrated as yet, it is tempting to conclude that local changes in tissue pH possibly caused by contractile or partially transdifferentiated VSMC could be involved in triggering MVC, particularly if combined with nucleation promotion and VC inhibitor depletion.

Local interstitial-intracellular pH changes are interdependent and determined by both local and systemic factors. In VSMC, several transporters participate in the movement of protons and bicarbonate to quickly control intracellular pH (pHi) and local extracellular pH (pHo). Sodium-proton exchanger 1 (NHE1, Slc9a1) eliminates protons from the cell, whereas the bicarbonate transporter, NBCn1 (Slc4a7), inwardly co-transports sodium and bicarbonate anions. With an intracellular excess of bicarbonate, this is eliminated by anion exchanger 2 (AE2, Slc4a2) (Boedtkjer et al., 2012). More recently, additional bicarbonate transporter transcripts—Slc4a3, Slc26a2, Slc26a6, Slc26a8, and Slc26a11—have been identified in rat aortic SMC in vitro, further increasing the complexity of intracellular pH control in VSMC (Hortells et al., 2020). In addition, carbonic anhydrases also present in VSMC can increase the concentration of bicarbonate and also alter the intracellular acid-base equilibrium. Interestingly, the inhibition of these enzymes with acetazolamide prevents the soft tissue calcification of klotho-hypomorphic mice (Leibrock et al., 2016) and in apolipoprotein E (ApoE –/– ) mice (Yuan et al., 2019).

Furthermore, changes in pH in VSMC have been implicated in several physiological and pathological states. For example, alkaline pHi is necessary for VSMC proliferation (Beniash et al., 2000 Boedtkjer et al., 2012), as well as for ECM remodeling and the activation of matrix metalloproteinases (Harrison et al., 1992 Stock et al., 2005). NHE1 inhibits apoptosis by increasing pHi, cell volume, and sodium content and by reducing the activity of enzymes required for apoptosis (Pedersen, 2006). Conversely, the inhibition of NHE1 or of NBCn1 should cause intracellular acidification and the reciprocal local extracellular alkalinity, consequently promoting MVC by promoting apoptosis associated with calcium nucleation (Shroff et al., 2008). This local alkalinity could also create an optimal TNAP environment for hydrolyzing PPi and organic phosphates such as phospholipids. In turn, the increased expression of TNAP seems to be one of the first steps of MVC in CKD (Hortells et al., 2017). In addition, the local alkalinity will also supersaturate the medium with respect to ACP and calcium carbonate. These examples illustrate the potential importance of pH regulation of activities in VSMC, potentially applicable to MVC pathogenesis.

Role of Cell Transdifferentiation

Changes in local pH, TNAP overexpression and other factors, could accompany transdifferentiation of VSMC into osteo/chondroblastic-like cells. Following the detection of the bone forming gene expression in atherosclerotic lesions (Boström et al., 1993), the active role of VSMC in VC process has been extensively studied (e.g., 153�). Osteo/chondrogenic transdifferentiation of VSMC has been mainly studied in CKD-related MVC. In these patients, the observed uremic and hyperphosphatemic conditions have tempted to the use of a simple research model accounting for a design of a fairly complete pathogenetic proposal based on direct effects of Pi as follows. The highly abundant phosphate would either permeate directly into the VSMC or it would be sensed through the sodium-phosphate cotransporters (PiT1/2), activating a signal transduction pathway resulting in a phenotypic transformation into osteochondroblast-like cells. This transformation could be mediated by the expression of Pi-induced transcription factors such as Msx2 (msh homeobox 2), Runx2 (Runt-related transcription factor 2), osterix, etc., that, in turn, would increase the expression of bone-forming proteins, such as Bmp2 (bone morphogenetic protein-2), TNAP, osteocalcin, collagen type I, etc., causing PPi depletion, increased number of nucleating sites, formation of calciprotein particles, etc., therefore facilitating, initiating, and stimulating calcification (Voelkl et al., 2019 Lee et al., 2020 Tyson et al., 2020).

Whereas VSMC transdifferentiation and the thermodynamics of CaP precipitation belong to different scientific fields, they complement each other in exploring MVC pathogenesis. While the former provides hypothesis, the later accounts for hypothesis testing. Consequently, the above outlined pathogenetical scenario has still to be considered with caution, for several reasons. Firstly, in the model or 5/6-nephrectomized rats, hyperphosphatemia is observed only after the first deposits have been formed and not before (Hortells et al., 2017), therefore, hyperphosphatemia should not be considered a necessary cause of calcification, but as an accelerator and complicating agent. Secondly, MVC may require interplay of different systemic and local factors absent in a cellular culture such as VSMC. Thirdly, thermodynamic principles suggest that calcium phosphates are not supersaturated in the normal or even CKD patients and therefore homogenous precipitation is an excluded possibility. Fourthly, in VSMC cultures, nanoparticles of calcium phosphate (or possibly calcium carbonate) rather than soluble Pi are responsible for osteo-/chondrogenic transdifferentiation. This can be easily observed by incubating VSMC cultures with high Pi concentrations in the presence of nucleating inhibitors, such as PPi, phosphonoformic acid or bisphosphonates: such nanoprecipitates are not formed and, consequently, no bone-related genes are expressed in VSMC despite the high Pi concentration (Villa-Bellosta et al., 2011 Lee et al., 2020). Fifthly, the nanoparticles do not appear to be caused by the native or transdifferentiated VSMC that would nucleate calcium heterogeneously, but rather by an alkaline pH-mediated supersaturation that causes homogeneous precipitation in media when using highly bicarbonated media in the presence of low CO2, along with the extremely high concentrations of Pi (Hortells et al., 2015). It is important to note that homogeneous precipitation does not occur in vivo. If pH is set at pH 7.4 in culture medium, no precipitates are formed, and no transdifferentiation of VSMC occurs. In fact, calcification also occurs using non-transdifferentiated, dead cells (Villa-Bellosta and Sorribas, 2009). Thus, the above in vitro experimental model represents an extreme case of biomedical research reductionism, misrepresenting the multifactorial complexity of the MVC. For example, because fluoride prevents the growth of calcium deposits in vitro it could be considered calcification inhibitor, yet, in contrast, fluoride promotes MVC in in vivo experimental rat model (5/6-nephrectomy) likely due to multitude of effects, mainly nephrotoxicity (Martín-Pardillos et al., 2014).

It has been established that osteo/chondrogenic transformation of VSMC does occur in the medial layer in MVC in vivo, as shown by the expression of several bone forming genes. Nevertheless, if CaP homogeneous precipitation in blood and direct Pi effect on bone forming gene expression rather effected by the calcium nanoprecipitate depositions can be excluded, s are then osteo/chondrogenic transformation could be considered a consequence of preceding calcium depositions caused for example by factors such as the local ACP supersaturation, AP overexpression and/or abundance of nucleation promoter (Hortells et al., 2017 Hruska et al., 2017), depending of the processes associated with CKD, DM, aging, or other. Therefore, we suggest, that any hypothesis of pathogenesis of MVC needs to comply with the thermodynamic principles and pass successfully the filter of energetic plausibility.


Materials and Methods

The Neolithic remains studied were excavated in 1994 at the Camí de Can Grau site (Granollers, Barcelona, Spain). The site comprised 23 tombs that have been C14 dated to between 3,500 and 3,000 years BC. As with most archeological excavations of human samples that have previously been used in aDNA analyses, no particular precautions were taken by the excavators to prevent direct contact between the handlers' skin or other sources of DNA (e.g., sweat) with the material. Immediately after excavation, the remains were washed under running water and allowed to dry naturally. Subsequently, the remains underwent an anthropological investigation, before being stored for 10 years in sealed plastic bags, within closed boxes, in a storage room of the local museum of Granollers. Roser Pou (R.P.) and Miquel Martí (M.M.) were the archeologists who excavated, cleaned, and washed the remains. Elisenda Vives (E.V.) undertook the anthropological study ( table 1), during which the cranial and dental fragments were glued together and the bones and skulls measured with standard anthropological instruments.

Mitochondrial Haplotypes of the Only 6 Researchers Who Have Been in Contact with the Samples


Detecting Calcified stones and cracks in tooth - Biology

Dental pulp is an unmineralized oral tissue composed of soft connective tissue, vascular, lymphatic and nervous elements that occupies the central pulp cavity of each tooth. Pulp has a soft, gelatinous consistency. Figure 1 (adjacent), indicates that by either weight or volume, the majority of pulp (75-80%) is water. Aside from the presence of pulp stones, found pathologically within the pulp cavity of aging teeth, there is no inorganic component in normal dental pulp. There are a total of 32 pulp organs in adult dentition. The pulp cavities of molar teeth are approximately four times larger than those of incisors.

The pulp cavity extends down through the root of the tooth as the root canal which opens into the periodontium via the apical foramen. The blood vessels, nerves etc. of dental pulp enter and leave the tooth through this foramen. This sets up a form of communication between the pulp and surrounding tissue - clinically important in the spread of inflammation from the pulp out into the surrounding periodontium.

Developmentally and functionally, pulp and dentin are closely related. Both are products of the neural crest-derived connective tissue that formed the dental papilla.

Dental pulp is a loose connective tissue with an appearance similar to mucoid CT. It contains the components common to all connective tissues:

  • Cells: fibroblasts and undifferentiated mesenchymal cells ( Lab Image 1 ) as well as other cell types (macrophages, lymphocytes, etc.) required for the maintenance and defense of the tissue .
  • F ibrous matrix: collagen fibers, type I and II, are present in an unbundled and randomly dispersed fashion, higher in density around blood vessels and nerves. Type I collagen is thought to be produced by the odontoblasts as dentin, secreted by these cells, is composed of type I collagen. Type II is probably produced by the pulp fibroblasts as this type increases in frequency with the age of the tooth. Older pulp contains more collagen of both the bundled and diffuse types.
  • Ground substance: the environment that surrounds both cells and fibers of the pulp ( Lab Image 2 ) is rich in proteoglycans, glycoproteins and large amounts of water.

The large number of undifferentiated mesenchymal cells (present as perivascular cells ) within the pulp facilitates the recruitment of newly differentiating cells to replace others when they are lost - specifically odontoblasts .

Odontoblasts (examined in greater detail in the module on dentin) comprise the outermost region/layer of the pulp, immediately adjacent to the dentin component of the tooth. These cells are responsible for the secretion of dentin and the formation of dentinal tubules in the crown and root.

  • The peripheral aspect of dental pulp, referred to as the odontogenic zone ( 1 ), differentiates into a layer of dentin-forming odontoblasts ( A ).
  • Immediately subjacent to the odontoblast layer is the cell-free zone (of Weil). This region ( 2 ) contains numerous bundles of reticular (Korff's) fibers ( B ). These fibers pass from the central pulp region, across the cell-free zone and between the odontoblasts, their distal ends incorporated into the matrix of the dentin layer. Numerous capillaries ( C ) and nerves ( D ) are also found in this zone.
  • Just under the cell-free zone is the cell-rich zone ( 3 ) containing numerous fibroblasts ( E ) - the predominant cell type of pulp. Fibroblasts of the pulp have demonstrated the ability to degrade collagen as well as form it. Perivascular cells (undifferentiated mesenchymal cells) are present in the pulp and can give rise to odontoblasts, fibroblasts or macrophages.

Since odontoblasts themselves are incapable of cell division, any dental procedure that relies on the formation of new dentin ( F ) after destruction of odontoblasts, depends on the differentiation of new odontoblasts from these multipotential cells of the pulp. Lymphocytes, plasma cells and eosinophils are other cell types also common in dental pulp.

indicates the 4 zones or regions of dental pulp ( Lab Image 4 ) :

Vascular Supply to the Pulp


Illustrates the extensive vascular pattern of dental pulp - emphasizing its primary function - support and maintenance of the peripheral odontoblast layer. The odontoblasts in turn maintain the overlying layer of dentin.

The walls of pulpal vessels are very thin as the pulp is protected by a hard unyielding sheath of dentin. One or more small arterioles enter the pulp via the apical foramen and ascend through the radicular pulp of the root canal. Once they reach the pulp chamber in the crown they branch out peripherally ( Lab Image 4 ) to form a dense capillary network immediately under - and sometimes extending up into - the odontoblast layer. The capillaries exhibit numerous pores, reflecting the metabolic activity of the odontoblast layer. Small venules drain the capillary bed and eventually leave as veins via the apical foramen.

Blood flow is more rapid in the pulp than in most areas of the body and the blood pressure is quite high. Arteriovenous anastomoses of arteriolar size are frequent in the pulp. For many years, investigators found it very difficult to establish the presence of lymphatics in the pulp. Most believed there was no lymphatic drainage of the teeth. Tissue fluid was speculated to have drained back into the capillary or postcapillary sites of the blood vascular system. In recent years a number of studies have demonstrated the presence of thin-walled, irregularly shaped lymphatic vessels. They are larger than capillaries and have an incomplete basal lamina facilitating the resorption of tissue fluid and large macromolecules of the pulp matrix.

The continued formation of cementum at the apical foramen can lead to occlusion of the opening. The walls of pulpal veins are first affected by the cemental constriction. Vascular congestion may occur. This ultimately leads to necrosis of the pulp.

Several large nerves enter the apical foramen of each molar and premolar with single ones entering the anterior teeth. A young premolar may have as many as 700 myelinated and 2,000 unmyelinated axons entering the apex. These nerves have two primary modalities:

1. Autonomic Nerve Fibers. Only sympathetic autonomics fibers are found in the pulp. These fibers extend from the neurons whose cell bodies are found in the superior cervical ganglion at the base of the skull. They are unmyelinated fibers and travel with the blood vessels. They innervate the smooth muscle cells of the arterioles and therefore function in regulation of blood flow in the capillary network .

2. Afferent (Sensory) Fibers . These arise from the maxillary and mandibular branches of the fifth cranial nerve (trigeminal). They are predominantly myelinated fibers and may terminate in the central pulp. From this region some will send out small individual fibers that form the subodontoblastic plexus (of Raschkow) ( Lab Image 5 ) just under the odontoblast layer. From the plexus the fibers extend in an unmyelinated form toward the odontoblasts where they then loose their Schwann cell sheath. The fibers terminate as "free nerve endings" near the odontoblasts, extend up between them or may even extend further up for short distances into the dentinal tubule. They function in transmitting pain stimuli from heat, cold or pressure. The subodontoblastic plexus is primarily located in the roof and lateral walls of the coronal pulp. It is less developed in the root canals. Few nerve endings are found among the odontoblasts of the root.

illustrates the free nerve endings ( F ) arising from the subodontoblastic plexus ( E ) and passing up between odontoblasts ( A ) to enter the dentinal tubule where they terminate ( G ) on the odontoblast process ( D ). B = predentin, C = dentin

The origin and concepts involved in pain in the pulp-dentin complex will be examined in the module on dentin.

Figure 5 illustrates the regions where the two types of dental pulp are located:

1. Coronal pulp ( A ) ( Lab Image 3 ) occupies the crown of the tooth and has six surfaces occlusal, mesial, distal, buccal, lingual and the floor.

Pulp horns ( B ) are protrusions of the pulp that extend up into the cusps of the tooth. With age, pulp horns diminish and the coronal pulp decreases in volume due to continued (secondary) dentin formation - often the result of continued masticatory trauma. At the cervix of the tooth the coronal pulp joins the second type.

2. Radicular pulp ( C ) ( Lab Image 2 ) extends from the cervix down to the apex of the tooth. Molars and premolars exhibit multiple radicular pulps. This pulp is tapered and conical. In a fashion similar to coronal pulp, it also decreases in volume with age due to continued dentinogenesis. Pulp passing through the apical foramen may be reduced by continued cementum formation.

Age-Related and Pathologic Changes in the Pulp

Specific changes occur in dental pulp with age. Cell death results in a decreased number of cells. The surviving fibroblasts respond by producing more fibrous matrix (increased type I over type II collagen) but less ground substance that contains less water. So with age the pulp becomes:

a) less cellular

b) more fibrous

c) overall reduction in volume due to the continued deposition of dentin (secondary/reactive)

illustrates the normal appearance of the pulp cavity (P) at a young stage.

illustrates some attrition of the pulp as the result of normal aging as well as trauma from wearing of the enamel at the cusp (A). Note the pulp horn (B) is not as well defined due to responsive ingrowth of secondary dentin below the worn cusp. Cementum has begun to thicken on the root (C).

Figure 8
shows the changes in pulp cavity size by middle age. The pulp horn continues to be reduced in response to increased wearing of the overlying enamel. Anoverall reduction in pulp cavity dimensions through the continued deposition of normal secondary dentin has occurred. Histology of the pulp reveals a decreased cellularity coupled with increased fibrosis. Cementum (C) deposition continues and the apical foramen subsequently has undergone a reduction in diameter (D).

Figure 9
is illustrative of the pulp cavity in old age. Continued wearing of the enamel on the cusp has resulted in the formation of dead tracts of dentin (E). It has also stimulated the formation of reactive secondary dentin (F) that has obliterated the pulp horn and now grows into the coronal pulp cavity. The pulp cavity, coronal and radicular regions, has been markedly reduced from that in the young stages. Cementum (C) continues to be deposited and the apical foramen (D) isnow considerably narrower.

Aging decreases the ability of dental pulp to respond to injury and repair itself. The fact that the pulp is surrounded by mineralized dentin makes relatively minor pathologic events like inflammation, that cause swelling elsewhere, lead to a compression of the pulp leading to intense pain. This generally results in the death of the pulp.

Calcified Bodies in the Pulp (Pulp Stones) ( Lab Image 6 , Lab Image 7 )

Small calcified bodies are present in up to 50% of the pulp of newly erupted teeth and in over 90% of older teeth. These calcified bodies are generally found loose within the pulp but may eventually grow large enough to encroach on adjacent dentin and become attached. These bodies are classified by either their development or histology:

Epithelio-Mesenchymal Interactions . Small groups of epithelial cells become isolated from the epithelial root sheath during development and end up in the dental papilla. Here they interact with mesenchymal cells resulting in their differentiation into odontoblasts. They form small dentinal structures within the pulp.

Calcific Degenerations . Spontaneous calcification of pulp components (collagen fibers, ground substance, cell debris, etc.) may expand or induce pulpal cells into osteoblasts. These cells then produce concentric layers of calcifying matrix on the surface of the mass - but no cells become entrapped.

Diffuse Calcification. A variation of the above whereby seriously degenerated pulp undergoes calcification in a number of locations. These bodies resemble calcific degenerations except for their smaller size and increased number.

Calcified bodies in the pulp may be composed of dentin, irregularly calcified tissue , or both . A calcified body containing tubular dentin is referred to as a "true" pulp stone or denticle ( Lab Image 7 ). True pulp stones exhibit radiating striations reminiscent of dentinal tubules. Usually those bodies formed by an epithelio- mesenchymal interaction, are true pulp stones.

Irregularly calcified tissue generally does not bear much resemblance to any known tissue and as such is referred to as a "false" pulp stone or denticle ( Lab Image 6 ). False pulp stones generally exhibit either a hyaline-like homogeneous morphology or appear to be composed of concentric lamellae.

shows both types of stones: A and B are false pulp stones, C is a true pulp stone. A is an "attached" stone (which may become embedded as secondary dentin deposition continues. B and C are "free" stones found within the pulp cavity.

The primary function of dental pulp is providing vitality to the tooth . Loss of the pulp following a root canal) does not mean the tooth will be lost. The tooth then functions without pain but, it has lost the protective mechanism that pulp provides.


Senescence of Dental Pulp

Dental pulp is the soft connective tissue that includes odontoblasts, fibroblasts, mesenchymal stem cells, nerve fibers, and vessels. This tissue is derived from dental papilla, an ectomesenchymal cell condensation in the developing tooth (Figure 1A). The dental papilla cells underlying the enamel epithelium can differentiate into odontoblasts this process is controlled by epithelial–mesenchymal interactions (Thesleff et al., 2001). Under such epithelial–mesenchymal interactions and the effects of various growth factors (e.g., bone morphogenetic proteins, fibroblast growth factors, and WNT), tooth development proceeds with this tissue (Thesleff, 2003).

The structural relationship between dentin and dental pulp is known as the �ntin–pulp complex.” As mentioned above, the pulp volume decreases with age because of secondary dentin deposition throughout life (Murray et al., 2002). This continuous deposition of dentin and dystrophic calcification in pulpal arteries interrupts blood circulation in the dental pulp of older individuals (Bernick, 1967a). When comparing the cell density of pulp in 70-year-old individuals with that in 20-year-old individuals, the cell number in older individuals was nearly half that of the younger individuals (Nanci, 2018), indicating a reduction in pulp restoration activity (Murray et al., 2002). Additionally, Hillmann and Geurtsen (1997) reported that the collagen fiber bundle aggregation and calcification in dental pulp increased with advancing age. Dystrophic calcification in the central pulp of the coronal region and root canal is evident in older individuals, which might be due to their reduced pulpal blood flow (Ersahan and Sabuncuoglu, 2018 Iezzi et al., 2019). Notably, Li et al. (2011) reported that human dental pulp cells (HDPCs) cultured under hypoxic conditions exhibited increased mineralization. Furthermore, a previous study examined the morphological alteration of the dentinal pulp wall during the aging process (Tsurumachi et al., 2008) the shape and modality of calcospherites on the pulp wall became diverse during aging. Such deposition may contribute to the growth of secondary dentin.

Tertiary dentin also deposits on secondary dentin under pathological stimuli such as dentin caries, tooth cutting, and trauma. This dentin includes two types, reactionary and reparative, which differ depending on the degree of stimuli (Smith et al., 1995). These dentinogenic activities are also reduced in older individuals (Murray et al., 2002).

With age, difficulties in endodontic treatment occur due to the constriction of pulp chamber space by hyperplasia of secondary and tertiary dentin, as well as pulp stones (i.e., ectopic calcified particles in the coronal region) and diffuse calcification in radicular pulp (Krasner and Rankow, 2004). A recent study reported the efficiency of cone-beam computed tomography in endodontic diagnosis and treatment planning (Sue et al., 2018), demonstrating its usefulness in current treatment. Additionally, guided endodontic access using cone-beam computed tomography in patients with calcified root canals has been described (Lara-Mendes et al., 2018).

Nerve fibers are widely distributed in dental pulp. In the growing dental papilla of human fetal teeth, expression of nerve growth factor and its low and high affinity receptors (p75NTR and TrkA, respectively) precede the initiation of tooth innervation (Mitsiadis and Pagella, 2016). In particular, p75NTR is thought to condense mesenchymal cells from neural crest cells during the construction of dental papilla. Therefore, these molecules are presumably involved in both tooth development and nerve growth in dental pulp. However, the distribution of nerve fibers in dental pulp decreases, probably because of degeneration with increasing age (Bernick, 1967b Figure 1B). Notably, Couve and Schmachtenberg (2018) characterized two types of Schwann cells that reside in dental pulp: non-myelinating and myelinating. They also found a reduction in the network of these cells at the dentin–pulp interface, along with decreased innervation in old dental pulp these findings suggested the progression of less symptomatic caries in older individuals because of a reduced response to environmental injuries and pathogens (Couve et al., 2018). Thus, during aging, the pulp cavity constricts and dental pulp cells subsequently reduce their functions and activities (Figure 1B).


All About Salivary Stones, Star of That Terrifying New Viral Video

After we recovered from the NSFW clip in which a salivary stone pops out of a man's mouth, we asked a dentist to explain what exactly it was&mdashand whether it could happen to us.

Today in viral videos, the Internet is having a collective freak-out over footage of a worm-like shape emerging from beneath a man’s tongue. The cell-phone video (below—watch at your own risk!) looks a bit like something out of an Alien movie. But it’s actually depicting a somewhat common medical phenomenon, albeit a pretty extreme case.

This phenomenon, as YouTube user Brandon Douglas wrote, is known as a salivary stone. According to Douglas’ comments on Reddit, he had been experiencing pain for about five days. A doctor told him there was a blockage, and possibly a stone, in his sublingual (under-the-tongue) salivary gland.

When Douglas, an American stationed at a Navy base in Bahrain, felt the blockage pushing its way out, he grabbed his phone and started recording. “I woke up in the morning and it felt very hard,” he wrote. “I went to the bathroom and flexed my tongue and a stone came out!”

That stone was several centimeters long, and left a noticeable hole in Douglas’ mouth after he pulled it out entirely. But what the heck was it, and could it happen to any of us? Here’s what we know.

Intro to salivary stones

Salivary stones, also known as salivary duct stones or sialolithiasis, are made up of calcium and other minerals naturally found in saliva. When saliva ducts—tiny openings in your mouth that produce saliva�ome blocked, those minerals can build up and harden beneath the surface of the skin.

This can cause pain and swelling, especially when saliva flow is stimulated. On Reddit, Douglas wrote that the stone was 𠇎xtremely irritating” for several days. “Whenever I would eat there would be nowhere for saliva on that side to go so the gland would just swell up and be really sore.”

But it’s not always painful or obvious, says Mary Gadbois, DDS, a dentist in private practice in Columbus, Missouri. 𠇊 lot of times people aren’t aware of it until it starts to displace their tongue,” she says. “We had one patient who came in not because his mouth hurt, but because his dentures didn’t fit anymore.”

Salivary stones can form when a duct is damaged due to some type of trauma, she says, or when saliva flow is slowed. People with dry mouth�use of certain medications, dehydration, or other medical conditions𠅊re much more prone to them.

What should you do if you think you have one?

Most salivary stones are small, and they often can be coaxed out out on their own by stimulating salivary flow. Dr. Gadbois recommends drinking lots of water and sucking on tart lemon candies throughout the day.

“That can create this river behind the stone and push it out,” she says, “kind of like how you can sometimes flush out kidney stones by drinking a lot of water.”

Your doctor or dentist may encourage you to try this for a few days, Dr. Gadbois adds. If it doesn’t come out on its own—or if you’re in a lot of pain—you may need surgery.

“We’ll get them numb and make a small incision and cut it out,” she says. “Normally we don’t even have to put a stitch in and it heals fine on its own.”

Doctors often put patients to sleep to do this surgery, Dr. Gadbois says, while dentists, who are more accustomed to doing oral surgery while patients are awake, tend to use local anesthesia.

Should you try to pull it out?

Dr. Gadbois doesn’t recommend using tweezers or anything sharp to remove a stone, but says that gentle massaging around the duct can sometimes help jumpstart the removal process.

If it doesn’t come out easily, however, see a professional. “If you can get to a dentist, it’s not expensive or complicated to have them numb you and extract it quickly,” she says. “That’s probably a lot nicer for the patient, versus trying to do it at home.”

Once it’s out, says Dr. Gadbois, talk to your doctor or dentist about what you can do to prevent other salivary stones in the future. That may involve staying well hydrated, or taking a closer look at your medications to see if any may be cutting off your saliva supply.

Except in cases of infection or extensive surgeries, people usually recover from salivary stones quickly and fully. When asked on Reddit what’s next for him, Douglas responded with relief: “Just ate an entire pizza without any pain. I regret nothing.”


5. Conclusion

Since the introduction of bisphosphonates they have been used to treat multiple bone disorders and cancers. In routine dental practice clinicians come across many patients who are receiving bisphosphonates as part of their therapy. Most commonly postmenopausal female patient who are receiving bisphosphonates as a treatment for osteoporosis which is very common for their age group, are encountered. These patients are at increased risk of developing ONJ when any dental treatment is done or patient is suffering from dental disease. So it becomes important to identify such patients and follow a suggested protocol to avoid complications. It is also important to identify various risk factors for the patient who might develop bisphosphonate induced ONJ prior to any dental procedure.