Dental pulp is a cellular organic network of blood and lymphatic vessels interspersed with nerves. This neurovascular organic network is housed in a spacious tooth-chamber and the relatively narrowed root-canal spaces of a tooth. The vascularized and innervated pulp provides nutrients to cells within tissues of teeth. The pulp is compartmentalized into several zones with varying densities of cells, blood vessels, and nerves (1). This multi-zonal organ from a bioengineering perspective exhibits a relatively higher hydrostatic pressure compared to other organs, contains regenerative cells, and is encapsulated within the harder yet permeable walls of the predominantly inorganic dentin. The natural “control systems” that maintain the interstitial fluid pressure (IFP) of the pulp include lymphatic vessels and capillaries (2–4). These control systems regulate interstitial fluid flow from the pulp through the dentin tubules to the enamel coronally, and to cementum apically (1). They maintain local concentrations of plasma proteins within the zones, and the overall osmotic pressure within pulp. The higher IFP prevents bacterial invasion and maintains overall functional homeostasis not limited to pulp, but also the dentin-pulp complex (DPC) (2, 5–7). Mineralization of this functionally influential pulpal organ has been phrased as pulp mineralization (1) with minimal to no information on its causation. Clinically, it commonly manifests as pulp stone, and dentin-bridge formation (8–10).

Biominerals within pulp to date have undergone rudimentary investigations, despite being identified and categorized as free or embedded in pulp, and/or attached to dentin (11). Mineralization of the innervated and vascularized pulp can result from sterile or non-sterile inflammation (12), and from pathways similar to those reported in neurovascular skeletal and smooth muscles (13). The primary effectors that inextricably shift the IFP of the vascularized and innervated pressurized pulp include molecular- and tissue-level factors that occur locally at site-specific anatomical locations. These factors include: (1) changes in permeabilities and mineral densities of dentin and cementum that affect fluid transport between pulp and the periodontal complex (5, 14, 15), and (2) inflammation-induced vasodilation and swelling (2). Secondary effectors that can potentiate the aforementioned primary effectors at the level of a DPC include: (3) anatomy-specific tooth architecture, in particular, the apical orifice “strangulation” of an already inflamed pulp (4); and consequently (4) ischemia related altered oxygen supply to the local regions of the pulp (4).

The physiologically beneficial characteristic of higher IFP, ironically can increase beyond normal limit when the tooth is insulted (orthodontic forces, pulpitis, trauma, auto-transplantation) (16). This net increase in IFP invariably “tips” the homeostatic balance and triggers acute or chronic inflammatory pathway(s). The impact of the inflammatory pathways on the pulp also depends on the magnitude, frequency and duration of the insult, and masticatory function (1, 5, 12, 14). Conceivably, this naturally engineered pressure chamber is mechanically stressed and strained, and contains pressure and chemical graidents. These gradients are encouraged by balanced concentrations of biomolecules and ions across the many zones and tissue interfaces including the dentin-pulp and -cementum interfaces.

Interfaces across the many disparate tissues of a tooth are a part and parcel of the intricate interconnected network that can affect fluid flow between pulp and the periodontal complex. The impaired cementum, dentin or pulp, as such, can alter the homeostasis of pulp at multiple length scales, which will be the topic of discussion, but within the framework of pulp biomineralization. The hypothesis of this study is that pulp mineralization is associated with pulpal tissue adaptation, and increased mineral densities and decreased permeabilities of tubular dentin and cementum. Study outcomes will confirm that the mineralized pulp in non-carious human permanent teeth is associated with altered constituents of the pulp, and compromised physicochemical characteristics of tubular dentin and cementum. Methods will include correlative spatial mapping of physicochemical and biochemical characteristics of pulp, and contextualize these properties within the DPC to reveal the inherent vunerabilities of pulp. This multiscale correlative approach will provide insights into the plausible predisposed nature of tissues (17–21), and in this case pulp, that can form biominerals as a response to significant shifts in the IFP. From a practical standpoint, it will reveal the need to question the effect of clinical interventions such as local anesthesia (22), dental restorations (23), periodontal treatment (24), and orthodontics (16), all of which can significantly affect functional homeostasis of the pulp.

As mentioned, a multiscale correlative microspectroscopic approach (17–21) was taken to gather insights into pulp biomineralization. Methods include a length-scale mapping of physical, chemical, and biochemical characteristics of pulp, dentin, and cementum. Post analyses of the acquired data within the context of the DPC will reveal insights into plausible biomineralization pathways of pulp in non-carious human teeth.

Results will illustrate that the structural and mineral density changes in tubular dentin and cementum within human molars are associated with mineralizing neurovascular organic network. At a microscale tissue-level, data will illustrate that the mineralizing pulp is contained within tubular dentin occluded with various Zn and Mg containing mineralized nanoparticles, and microscale aggregates of the same spread into larger volumes of the organic network. Furthermore, at a macroscale tooth-level, data will illustrate that pulp mineralization is associated with decreased cementum permeability and increased accessory (lateral) channel occlusion.

The sequelae that can help explain pathophysiology of tissues are often amiss. Pulp investigated in this study was from non-carious teeth that were extracted for orthodontic or prosthodontic reasons, with no prior information. The tissues of teeth are routinely subjected to insults in multiple ways. Acute and chronic tissue responses to insults (mechanical, chemical, and/or microbial loads) (1, 5, 12, 14) are often registered as spatial (macroscale DPC, microscale tissue/matrix/cell, and nanoscale particulate/biomoelcular levels) and temporal (short and long-term that can be connected to acute and chronic inflammatory cascades) adaptations. These adaptations are often mapped as changes in physicochemical, and biochemical characteristics using multiple high resolution microscopy and spectroscopy techniques. Higher mineral densities but lower fracture resistance of dentin with age (27–30), decreased dentin permeability resulting from microbial attack (14, 31), and increased loss in tooth-sensitivity with increased cuspal attrition (5, 14) all are adapted characteristics of dentin. The white-reflecting and dark-opaque features in dentin encircling root canals in Figure 1 indicated that, dentin indeed has adapted to conditions other than normal (1, 5, 12, 14, 31–35). Although the spatial distribution of nerves in mammals has been documented to be tooth-function specific (36), consistent non-specificity of mineralized pulp to tooth type suggested that the biomineralization pathway need not be specific to tooth type (incisors to molars) and location (anterior to posterior) in the jaw. Despite, increased volumes of mineralized pulp were mostly observed in the apical portion (37) revealing a plausible macroscale effect of tooth architecture (37) on pulp mineralization.

Much of pulp mineralization at a tooth-level or a DPC-level (macroscale) can be guided by fluid flow which is governed by the general laws of physics (Poiseuille's and Darcy's laws) (38, 39) and chemistry (van't Hoff's osmotic pressure and Fick's diffusion) (40, 41). These fundamental laws illustrate that for small changes in physical features such as root canal (R) radius, dentin tubule radius and length (r, L), interstitial fluid viscosity (μ), and concentrations of chemical constituents (C₁, C₂, C_n), significant shifts in pressure and chemical gradients can occur. These shifts in pressure and chemical gradients can alter the rates of volume flow across a unit area of a tissue. Based on the structural interconnectedness between dentin and pulp, and dentin and cementum (15), the observed changes in dentin (particulate growth and/or increased pertubular dentin production) and cementum (decreased permeabilities) could result in changes in interstitial fluid pressure (4, 42, 43) of the softer pulp. These laws also explain that changes in (1) pressure and chemical gradients at a tooth-level and (2) dentin and cementum permeabilities at a tissue-level can “drive” the rates of heterogenous and/or homogenous nucleation and subsequent mineralization of an organic matrix even in the absence of cells (44, 45) (Figure 6). Fundamental physical chemistry experiments have illustrated that osteogenic molecules such as OCN and BSP in the absence of cells can act as nucleators and in association with collagen can facilitate heterogeneous nucleation, and apatite formation (44, 45). Collectively, these changes in physicochemical, and biochemical chactersitics revealed the interconnected vulnerabilities between the seemingly disparate hard and soft tissues even in the absence of cells.

In the presence of cells, the observed correlations between physical and chemical features (Figure 2) of dentin, cementum, and pulp revealed macroscale culprits that could potentiate microanatomical self-governing regions (Figures 1, 2) in the insulted pulp. The narrowed orifice at the root-tip also can potentiate the already inflammed pulp via capillary stragulation (4). However, in a partially mineralized pulp at locations farther away from the sphere of influence of insults (top, middle, bottom sections, Figure 4), the built-in control system can “erase” the initial inflammatory effects of the insults and reverse the local interstitial fluid pressures to homeostasis. On the other hand, chronic response to sustained insults can “tip” this balance toward a sustained inflammatory mode, and consequently an irreversible state. The subsequent changes in pressure gradients at the tissue and organ levels can contribute to various mechano- and chemo-biological events, and plausibly, different biomineralization pathways. Specifically, the most noted mechano-chemo-sensing biomineralization pathways involve the production of osteogenic proteins as nucleators (46–49) through (1) increased vasodilation by “mechanically strained” endothelial cells (4, 50, 51); (2) compromised capillary pressures and pericyte stimulation (50, 52) resulting in a sustained increase in interstitial fluid pressure within the pulp; and (3) capillary strangulation resulting in hypoxic conditions and reactive oxygen species (ROS) (4, 53).

Studies have already shown an obvious reduction of pulpal blood flow and marked histologic changes in the pulp as a result of intrusive orthodontic forces (16); pulp canal obliteration appears to be a sequel to revascularization and/or reinnervation of a damaged pulp after traumatic injury (54). Inflammatory biomarkers including matrix metalloproteases and neuropeptides are released by traumatic, orthodontic, thermal, and chemical insults. The function of MMPs and neuropeptides are dependent on the catalytic or co-factor functions of various heavy metals including Zn²⁺ (55–59). Increased expressions of neuropeptides including calcitonin gene-related peptide (CGRP) can alter vascular function and interstitial fluid pressure, but do so locally within the tissues of the pulp (60). The finding of silver nitrate (von Kossa) positive minerals around the α-SMA positive blood vessels and PGP 9.5 positive nerves (Figure 2B) is suggestive of neurovascular involvement, a potential culprit in pathophysiology of pulp tissue mineralization.

Localization of SIBLING proteins, OPN and BSP, and OCN around α-SMA vasculature and PGP 9.5 positive nerves, furthermore provided insights into the plausible roles of neurovasculature and osteogenic proteins (61, 62) on controlled mineralization of collagen albeit pathologic in nature. SIBLINGs express themselves in regions that are mechanically challenged, not necessarily as mineral forming, but also as mineral inhibiting proteins. OPN is one such plasma protein whose intrinsic function as an inhibitor of mineralization also is central to the biomineralization pathway; its involvement could be to control mineral growth albeit pathologic as discussed in various studies in vitro and in vivo (44, 45, 63, 64). Electron dense particles (Figure 3) and diffused nanometer sized plate-like minerals located within the macroscale mineralizing collagenous constructs, and as intrafibrillar mineral (Figure 3A) confirmed heterogeneous nucleation within pulp. OCN is expressed in fibroblast-like cells of the pulp (65), and has been shown to influence specifically intrafibrillar mineralization of collagen (66). OSX is a Zn finger transcription factor and plays an important role in pre-differentiated osteoblasts (67). As an upstream marker in the osteogenic signaling pathway, OSX also could modulate the expressions of OPN, OCN, and the more downstream marker BSP (67, 68). However, we contend that its presence is more central to the nerve, as data alludes to pulp mineralization as ectopic mineralization, and not necessarily heterotopic ossification (no osteocytes were observed).

It should not come too much as a surprise that Zn was observed, as its localization is central to most sterile and non-sterile inflammatory pathways. Inflammation encourages vesicular transport of Zn and Mg or as bions in biological fluids (48, 69, 70). These spherulitic structures also could be associated with local tissue mineralization (Figure 5). Additionally, extracellular spherulitic bodies including exosomal and apoptotic bodies with sizes ranging from <50 nm to greater than microns continue to be observed in vascular plaques in multiple anatomical locations in humans (heart, kidney, brain) (71–75). However, studies identifying mechanisms of interaction between extracellular vesicles and collagen driving the formation of micro/macro mineralization in tissue constructs are warranted.

Zn also is involved in downstream biomineralization events especially related to hypoxia induced by ischemia (i.e., HIF, NF-κB) (53, 76–79); all of which condition the tissue biochemistry and facilitate passive or active recruitment of Ca²⁺ and PO43- ions. Changes in interstitial fluid pressure can alter hemodynamic and vascular mechanotransduction, induce osteogenic protein production, shift oxygen tension, and generate ROS. Further research on hemodynamic force-induced inflammation in association with ROS facilitated by Zn will be necessary to elucidate yet another mechanism of pathologic mineralization, but under ischemic conditions [oxygen tension resulting from insufficient blow flow, specifically capillary strangulation (2, 4) at the root apices]. Given all the many discussed pathways that would result in pathologic biominerals in pulp, confirmation of highly probable pathways at local tissue- and tooth-levels through niche-specific simulation studies are highly warranted.

Spatial colocalization of physicochemical and biomolecular compositions of biominerals in an already existing neurovascular pulpal matrix, suggest primary and secondary biomineralization pathways. Primary pathways are specific to the tissues of the pulp and dentin, and secondary pathways are defined by the aggregate effects of the primary but at an organ level. Primary pathways include shifts in hemodynamic and interstitial fluid pressures also by sterile stimuli (i.e., mechanical, chemical, thermal) that illicit neurogenic inflammation, a process that could alter normal neural and vascular functions. These shifts in tissue compliance resulting from interstitial fluid pressure change also can differentiate various cells including pericytes, endothelial, and other resident stem cells into an osteogenic lineage and consequently promote biomineralization locally within the pulp. Reduction in permeability and oxygen tension resulting from occluded dentin tubules and lateral canals, and altered tooth architecture can further stimulate ischemic events and ROS. The presence of Zn expressions in mineralized pulp is further suggestive of Zn-related biochemical pathways being central to mineralization of the DPC. Regardless, mapping niche-specific upstream and downstream biomarkers are critical toward formulating functional assays that would guide translational approaches to mitigate primary and secondary pathologic biomineralization pathways. Furthermore, from a clinical viewpoint, these observations in pulp underpin the short and long-term prognosis of a tooth receiving anesthetic, restorative, periodontal, and/or orthodontic treatment, and can inform clinicians the subtleties related to pulpitis, dentin-bridge, and pulp stone formation.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Human Research Protection Program Institutional Review Board of UCSF (Study Number: 16-19606). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

RH contributed to study design, data acquisition, in particular, immunohistochemistry, electron microscopy, and correlative light and electron microscopy. CC contributed to clinical input, editing, and preparing Figure 6. MS contributed to editing. MK contributed to writing, data acquisition, analysis, interpretation of data specific to micro CT related scans, energy dispersive X-ray analysis, and correlative microscopy. SH contributed to study design, data interpretation, prepared Figure 6, wrote, edited, revised, and critically reviewed the manuscript with all authors. All authors gave final approval and agree to be accountable for all aspects of the work.

This research was funded by the Program in Biomineralization Studies (PiBiomS), Departments of Preventive and Restorative Dental Sciences, and Urology, Schools of Dentistry and Medicine, UCSF. PiBiomS is a partnership between the Schools of Dentistry and Medicine at UCSF to investigate the cause of various pathologic biominerals in humans.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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