N2 and Sargenti

N2 is a zinc oxide root canal cement with an amount of formaldeyde of about 3% of the freshly mixed product. It is producted by Ghimas in Italy and distributed by Hager& Werken.
The powder contains:
– zinc oxide: 64.50%
– zinc stearate: 0,074%
– paraformaldehyde: 5,710%
– titanium oxide: 4,670%
– bismuth carbonate: 9,930%
– bismuth nitrate: 14,90%
– ferric oxide: 0,040%

The liquid contains:
– eugenol: 77.0%
– rose oil: 1.8%
– lavender oil: 1.2%
– peanut oil: 20.0%
Metal salts, such titanium oxide, bismuth nitrate and bismuth carbonate, have the role to increase adhesion to dentinal walls and to increase radiopacity.
Who did introduce N2?
Photo of Angelo Sargenti

N2 cement and the relative endodontic technique were introduced into the dental comunity by a swiss dentist, Angelo Sargenti, in 1954. Angelo Sargenti, on the basis of the results obtained by Balint Orban, magyar,  professor at Loyola University in Chicago. Balint Otban had been studying the reparative effect of formaldehyde upon the dental pulp tissue (in concentration ranging from 4 to 10% formaldheyde is able to induce pulp cells to produce dentin). By adding some paraformaldehyde to temporary filling materials, Orban ascertained reduced dentin sensitivity, and in the course of time a secondary dentin formation after a few days of retention, if the insert with a concentration of 5 – 20 % remained in place for just a few days. Secondary dentin formation, which was localized at lower dosage and diffused within the entire coronal part of the pulp, was observed in dogs after three to nine month while using paraformaldehyde Aquadont inserts with a concentration of 1 – 5 %. A 20 – 50 % formaldehyde Aquadont concentration led to bleeding and inflammations with every aspect of defense reactions of the tissue. Thus Orban came to the conclusion that the positive effect of formalin solely depends on concentration and dosage, which should be at the range of 5 % to heal sufficiently without toxic effect.
Based on this limited dosage, Sargenti and Richter introduced the N2 root canal filling material in 1954.
At the beginning of '50, the endodontic was burdened with high failure rate, especially in general dental practice. The use of this cement, along with the technique codified by Angelo SArgenti allowed to obtain a success rate comparable and perhaps higher than actual ones.
Angelo Sargenti was also a pioneer in the field of mechanical endodontic with the first electric device able to give a reciprocal rotative motion (Giromatic, Micromega).
Besides, he has been the first to introduce single-appointment endodontic.
But the greatest merit to deserve to Angelo Sargenti is for having saved millions of teeth from the forceps. In that period, the most common therapy for teeth with pulpal and periapical affections was the extraction. His technique allowed general dentists to have a reproducibile, reliable and predictable technique with high success rate unsurpassed till today.

What is formaldeyde?

Formaldehyde is a colorless and pungent smelling gas, germicidal and well soluble in water.
It can exist in three different states:
powder: as paraformaldehyde, a polymer of formaldheyde. This is the state in wich formaldehyde is used in N2.
liquid: A 35 % aqueous solution is referred to as formalin. Formaldheyde is used as formalin in Buckely solution and in Resorcinon-Formalin (RF, also called Red Russian)

Formaldehyde owns the general properties of all aldehydes. It is reductive and easily oxidizable. It reacts with ammonia and ammonia derivates and also with protein, which contains the amino group (- NH2). Together with protein, formaldehyde forms solid, hard condensation products, whereby the protein gets tanned and microorganisms are destroyed. The hardening of the cell wall prevents further cell divisions and thus their reproduction. Formaldehyde is also used to fix and harden microscopic preparations

Formaldehyde is the simplest of aldehydes. Our cells metabolize formaldehyde transforming it firstly in formic acid and finally in water and carbonic anhydride.
Formaldheyde is a gas with three important properties:
microbicidal activity:
formaldehyde is a high-level disinfectant. Formaldehyde inactivates microorganisms by alkylating the amino and sulfhydral groups of proteins and ring nitrogen atoms of purine bases.  it is useful as a disinfectant as it kills most bacteria and fungi (including their spores). Formaldehyde is used to inactivate bacterial products for toxoid vaccines (vaccines that use an inactive bacterial toxin to produce immunity). It is also used to kill
unwanted viruses and bacteria that might contaminate the vaccine during production. Urinary tract infections are also often treated using a derivative of formaldehyde (methenamine), a method often chosen because it prevents overuse of antibiotics and the resultant development of bacterial resistance to them. In an acid environment methenamine is converted in the kidneys to formaldehyde, which then has an antibacterial effect in the urinary tract. Some topical creams, cosmetics and personal hygiene products also contain derivatives of formaldehyde as the active ingredients that prevent the growth of potentially harmful bacteria
Firstly Grosmann and then Orstavik showed N2 owning the best antibacterial properties in any test series. Lai e coll. (Clin Oral Investig 2001 Dec;5(4):236-9
) evaluated the antimicrobial properties of commonly used endodontic sealer against four facultative anaerobic species (Streptococcus mutans, Streptococcus sanguis, Escherichia coli, and Staphylococcus aureus) and four obligate anaerobic species (Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum, and Prevotella intermedia), finding N2 proved to be the most effective against the microorganisms. Being formaldehyde a gas, its action works not only in contact, but also at distance from the point of application ("teleactivity", as called by Sargenti). Broisman e coll. (Antimicrobial effects of N2 in vitro. Oral Surg Oral Med Oral Pathol 1978; 45: 116-22) described this effect as "vapor effect"
  Fixation of non vital  tissue. Formaldheyde, as formalin, is used  in histology in order to fixate the tissue surgically removed, avoiding the decay of tissue (autolysis and putrefaction). Formaldheyde destroyes bacteria (one of the major causes of tissue decay) and make the tissue less palatable to microrganisms. This property of formaldehyde is useful in endodontic, due to the impossibility with current techniques to obtain the complete elimination of organic material (pabulum for microorganisms) and microbiota (Nair 2005; Ricucci 2009)
inducing reparative activity upon vital pulp tissue: according to the studies of Balint Orban (1934), formaldehyde at 5% concentration induces pulp cells to produce dentin.

However, clinical follow-up studies showed that the success rate of  pulpectomy with aid of formaldheyde as judged by clinical and radiographic criteria could be as high or even higher than that of vital pulpectomy (Strindberg LZ. The dependence of the results of pulp therapy on certain factors. Acta Odontol Scand 1956: 14(Suppl 21)(Grahne´n H, Hansson L. The prognosis of pulp and root canal therapy. A clinical and radiographic follow-up examination. Odontol Revy 1961: 12: 146–165) Castagnola L. Die Mortalamputation im Liechte klinischer, rontgenologischer und bakteriologischer Untersuchungen an einem ausgedehnten statistischenMaterial. Schweiz Monatsschr Zahnheilk 1950: 60: 332–357. 13. Kotilainen R, Jokinen MA, Korte I. A roentgenological study on vital pulp extirpations. Suom Hammaslaak Toim 1970: 66: 36–48)
Exhaustive on the subject is the review of Lambjerg-Hansen (Lambjerg-Hansen H. Vital and mortal pulpectomy on permanent human teeth. An experimental comparative histologic investigation. Scand J Dent Res 1974: 82: 243–332).
Interestingly, Eriksen noted in a survey of reports on prevalences of apical periodontitis that success rates of endodontic treatment in two European countries, where formaldehyde-containing dressings are widely used, were higher than that in countries where "biocompatible" approaches to endodontic therapy are advocated.. (Eriksen HM, Kirkevang L-L, Petersson K. Endodontic epidemiology and treatment outcome: general considerations. Endod Topics 2002: 2: 1–9 ).

What is the amount of formaldeyde in a root canal filled with N2 cement?
In the N2 cement formulation the formaldheyde is contained in the powder, as paraformaldheyde. The paraformaldehyde has a concentration of formaldeyde of 100%, differently from the formalin in wich the concentration of formaldeyde is commonly 35%, being in acqueous solution. 
The density of paraformaldeyde is 1,42 g/cubic cm, id est  1,42 mg/cubic mm.
A root canal has an average volume of 5-10 cubic mm (Hannig C et al. 
Volumetry of human molars with flat panel-based volume CT in vitro. Clinical Oral Investigations 2006, 10(3):253-257).
If we fill up a root canal with a powder of paraformaldehyde, we shall have a root canal with 7-14 mg of paraformaldehyde inside.
Considering that paraformaldheyde, contained in the N2 powder, has a concentration of 5,75 % and considering that N2 powder needs to be mixed with N2 liquid (eugenol) in 1:1 ratio, we should calculate the 5,75% of paraformaldheyde and then divide per two. Thus, we shall obtain a quantity of formaldheyde of 0,4-0,8 mg per canal. In a molar tooth having 3 canals, we should expect a quantity of formaldehyde at maximum of 2mg.
If we use guttapercha, the volume of cement will be furtherly reduced at least of 10 times, and consequently the quantity of formaldehyde. In a canal filled with N2 and guttapercha, we should expect a quantity of formaldehyde of 0,04.0,08 mg (id est  40-80 micrograms of formaldehyde).
What written above is in accord to the work of Kock, who tested N2 samples in order to measure the released quantity of formaldheyde (Kock M.J. International Endodontic Journal 32:10-16; 1999).
Is N2 toxic? 
The WHO published the following numbers on the topic of environment and formaldehyde in 1989:
Formaldehyde content in comestibles in mg/kg:
– 60.0 in pears
– 17.3 in apples
– 6.7 in carrots
– 5.7 in tomatoes
– 20.0 in pork
– 8.0 in sheep meat
– 20.0 in sea fish (smoked)
– 20.0 in cod
– up to 3.3 in cow's and goat's milk
– up to 3.3 in cheese products
Formaldehyde content from the environment in mg/day:
– 0.02 from ambient air
– 0.5 – 2.0 from indoor air
– 1.5 – 14 from food (adults)
– 0.1 from drinking water
– 1.0 from smoking (20 cigarettes)
As we can see from the above data, the amount of paraformaldheyde contained in N2 cement is 1000 times less than amount of paraformaldehyde each day we eat and breath.
We have also to consider that this minuscule amount of formaldheyde is to be realeasing in 10 days. Thus we should expect a daily release of formaldheyde form N2 a thousand lower than daily dietary income. After 1 week the N2 cement become inert and does not release formaldheyde anymore. 
The president of the '"American Society of Toxicology", Jeffrey Brent, testified as sworn expert on the occasion of a court hearing on January 8th, 1997 in Wichita Falls, Texas 10 (according to Hyatt Court Reporting, File No. 7-95-CV-057-X dated January 8th, 1997): "N2 is no more toxic than any other root canal filling material. N2 possesses no mutagenic or carcinogenic potential. A study, during which formaldehyde was charged with radioactive C14 prior to its application, reportedly proved that C14, through intense C14 activity, was still detectable in other body parts, from which it was erroneously concluded then, that paraformaldehyde was transported into these body parts accordingly. But what was ransferred in reality was just C14".

In toxicology, for determining the toxicity of substances, the scientists use to determine the lethal dose = LD50, which describes the amount of substance killing 50% of a number of laboratory animals. This value represents the extent of toxicity of chemical substances, which is required for approval of such substances according to the Chemicals Act. The higher the LD50 dose, the better it is for the pertaining substance and its non-toxicity. The relevance of LD50 values is, however, strongly limited, as most of these orally administered, hardened substances show LD50 values over 2 g/ kg of body weight and thus are not considered toxic
The LD50 dose for N2 is 5900 mg/kg, and for some other substances as follows:
– N2 LD50 = 5900 mg/kg
– Eugenol LD50 = 2680 mg/kg
– Salt LD50 = 3000 mg/kg
– Aspirin LD50 = 815 mg/kg
– Caffeine LD50 = 127 mg/kg
– Nicotine LD50 = 24 mg/kg
Provided that a molar tooth holds an average of 50 mg of filling material, as measurements have shown, the LD50 dose for an 80 kg human would only be reached if 10,720 molars were filled with 50 mg of N2 each. Systemic toxicity is thus impossible for N2.

Several authors have opposed to N2 advocating an alleged "carcinogenity" of this material. I want to premise that a material for being carcinogenic, has to be firstly mutagenic, id est able to induce mutations in DNA. The formalin (formaldheyde at 37%) is mutagen agent, but not carcinogenic. N2, with a concentration of formaldheyde of 2,7%, has not mutagenic properties and thus it cannot be carcinogenic, as tested by Bojar W e coll (Adv Clin Exp MEd 2009, 18, 6, 615-621). They checked N2 with Ames test according to  PN-EN ISO 10993-3: 2008. In fact, European Community have authorized the production and distribution of N2 cement, considering it a material clinically efficacious and safe.


Other authors have opposed to N2 advocating an alleged topic toxicity. Many studies proved in vitro  the citotoxicity in vitro of N2 cement. But this toxicity is common and often lower than that of many other substances used in dentistry and endodontics (eugenol, sodium ipoclorite, glass ionomer cements, resins, ecc.) But such in vitro toxicity, never had a clinical significance, as for many other materials. Nay, after millions of teeth treated with N2 cement, we have to exclude any form of toxicity of this material in vivo. Besides, N2 cement is well tolerated in vivo, also when extruded in the periapical tissue and it reasorbs in a few months.
We need to add that N2 cement, as other root canal  sealer, is applied inside the root canal and therefore not in contact with any vital tissue. We dot not have to forget that in the root canal we use sodium ipolorite 5,25%, system B points with 250 degree Celsius (more citotoxic than N2!). As we saw before, the amount of formaldheyde inside the canal should be no more than 0,04-0,08 mg. If we consider that, in case of extrusion in the periapex, only a maximum of 10% of N2 shall be at contact with periapical tissue, then only 0,004-0,008 mg (id est 4-8 micrograms) shall be in contact with vital tissue!!
The radiologic studies performed by Overdiek (1960, 1968) prove the absence of toxicity in vivo. Zerosi, Amici and Baratieri (Ricerche istologiche sul comportamento dei tessuti periapicali dopo l'otturazione con N2. Rassegna Trimestrale di Odontoiatria, m.4, 1959, Pavia) examined apical regions by means of biopsies of teeth treated with N2 and did not find any pathologic change suggestive of toxicity. Snyder, Seltzer and Moodnik (Effects of N2 in Experimental Endodontic Therapy. Oral Surg Oral Med Oral Pathol, 5, 635, 1966) got the same results through experiments in animals, in wich they extruded intentionally N2 beyond the apical foramen: N2 was well tolerate by periapical tissues. 

In light of such chemical, histological and clinical evidences,  is it yet plausible to think that a so minuscle amount of formaldheyde has any detrimental effect?
The answer is absolutely NO.
What is the goal of endodontic therapy? What are the causes of success and failure?
The success in endodontics is conditioned on numerous variables, not yet completely understood. Nevertheless, an overwhelming evidence correlates the failure with microbial presence inside the root canal system (Nair PNR. Pathogenesis of apical periodontitis and the causes of endodontic failures. Critical Reviews in Oral Biology and Medicine. 2004).
In 1965 Kakehashi et al. showed that no apical periodontitis developed in germ-free rats when their molar-pulps were kept exposed to the oral cavity, as compared with control rats with a conventional oral microflora in which massive periapical radiolucencies occurred.
Apical periodontitis is a sequel to endodontic infection and manifests itself as the host defense response to microbial challenge emanating from the root canal system. It is viewed as a dynamic encounter between microbial factors and host defenses at the interface between infected radicular pulp and periodontal ligament that results in local inflammation, resorption of hard tissues, destruction of other periapical tissues, and eventual formation of various histopathological categories of apical periodontitis, commonly referred to as periapical lesions. The treatment of apical periodontitis, as a disease of root canal infection, consists of eradicating microbes or substantially reducing the microbial load from the root canal and preventing re-infection by orthograde root filling. Apical periodontitis is inflammation and destruction of periradicular tissues caused by etiological agents of endodontic origin. It is generally a sequel to endodontic infection. Initially, the tooth pulp becomes infected and necrotic by an autogenous oral microflora. The endodontic environment provides a selective habitat for the establishment of a mixed, predominantly anaerobic, flora. Collectively, this habitat-adapted polymicrobial community residing in the root canal has several biological and pathogenic properties, such as antigenicity, mitogenic activity, chemotaxis, enzymatic histolysis, and activation of host cells. The microbial invaders in the root canal can advance, or their products can egress, into the periapex. In response, the host mounts an array of defenses consisting of several classes of cells, intercellular messengers, antibodies, and effector molecules. The microbial factors and host defense forces encounter, clash with, and destroy much of the periapical tissue, resulting in the formation of various categories of apical periodontitis lesions. In spite of the formidable defense, the body is unable to destroy the microbes well-entrenched in the sanctuary of the necrotic root canal, which is beyond the reaches of body defenses. Therefore, apical periodontitis is not self-healing. The treatment of apical periodontitis consists of eliminating infection from the root canal and preventing re-infection by a the seal of the root canal space. Nevertheless, endodontic treatment can fail for various reasons: first of the impossibility to eliminare compeletly the microorganism and the impossibility to obtain an ermetic  root canal filling.
Having the root canal system a complex geometry (Hess studies docet) not approachable by means of current instruments and techniques, we shall get at the end of the cleaning and shaping procedures a root canal system yet scattered with microorganisms and pulp remains (Nair PNR. Microbial status of apical root canal system of human mandibular first molars qith primary apical periodontitis after "one-visit" endodonic treatment. Oral Sur Oral Med ORal Pathol Oral Radiol Endodod 2005 Feb; 99(2):231-52)
Ricucci et al. (J Endod 2009;35:493–502) evaluated histologic findings from 24 patients affected by apical periodontitis. In all cases bacterial colonies were present within apical ramifications, which could not be reached by instruments, and where irrigants would have restricted access. These findings are in agreement with Nair et al (2005). The fact that virtually all failed cases harbored an intraradicular infection underlines the difficulty of adequately disinfecting root canals with current techniques and substances before obturation. These findings also demonstrated that root canal obturation fails to entomb residual bacteria in the root canal system and then prevent their access to the periradicular tissues to induce or maintain disease.
The above figure is from Ricucci. Asymptomatic case. (A) Tooth 10 in a 38-year-old woman had necrotic pulp and an apical periodontitis lesion. She reported no symptoms, and both percussion and palpation tests were negative. After working length determination, the canal was instrumented manually in the apical third up to a K-file size 60, and with Gates-Glidden burs in the coronal two thirds. Abundant irrigation was performed with 1% NaOCl after each instrument. The tooth was treated during 2 visits and restored with a ceramo-metallic crown after post and core build-up. (B) Follow-up radiograph taken after 4 years showing that the lesion had reduced in size but was still present. Tooth was asymptomatic. (C) Residual radiolucency was still evident after 19 years. Tooth had remained asymptomatic for the whole period. Percussion and palpation always gave negative responses. Clinical and radiographic signs of recurrent distal caries were now present, and a new restoration had become indicated. Apical surgery was scheduled. The root tip was removed with the pathologic periradicular tissue attached, which was located mostly on the palatal side. A root-end cavity was prepared, and a filling was inserted (not shown). (D) Section passing approximately at the center of the root canal. Resorption in the foraminal area is evident. Although on the radiographs the root canal filling seemed confined within root canal limits, it actually extended beyond. There is severe chronic inflammation at the material/tissue contact area (hematoxylin–eosin stain; original magnification25, inset400). (E) Section passing in the same area as that shown in (D) (Taylor’s modified Brown & Brenn, original magnification 25). (F) Magnification of the area indicated by the arrow in (E). Small bacterial colonies are visualized between the filling material and the root canal wall (Taylor’s modified Brown & Brenn, original magnification 400). (G) Another section
where small bacterial colonies are apparently ‘‘entombed’’ between the material and the wall (Taylor’s modified Brown & Brenn, original magnification1000). (H)Bacterial colonization deep within dentin tubules (Taylor’s modified Brown & Brenn, original magnification 100, inset 1000).
The present results indicate that bacterial persistence in the root canal in areas not affected by treatment is the major cause of post-treatment apical periodontitis. Nevertheless, it must be pointed out that bacteria persisting in the root canals after instrumentation or intracanal medication will not always cause failures, because some lesions can heal even when bacteria are found in the canal at the time of filling (Sjogren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997;30:297–306.  Fabricius L, Dahle´n G, Sundqvist G, Happonen RP, Mo ller AJR. Influence of residual bacteria on periapical tissue healing after chemomechanical treatmen and root filling of experimentally infected monkey teeth. Eur J Oral Sci 2006:114:278–85.).
Explanations for this might be that residual bacteria:
1) die after filling because of the antibacterial activity of filling materials, access denied to nutrients, or disruption of bacterial interactions
2) are present in subcritical counts to maintain inflammation
3) are located in areas with no access to the periradicular tissues (Siqueira JF Jr, Roˆ c¸as IN. Clinical implications and microbiology of bacterial persistence after treatment procedures. J Endod 2008;34:1291–301. e3)

Actually, bacteria that endure endodontic procedures can influence the treatment outcome provided they
1) withstand periods of famine
2) resist to disturbances in the community ecology
3) reach sufficient numbers to wreak havoc on the host
4) have frank access to the periradicular tissues
5) produce and release virulence factors at enough concentrations to harm the periradicular tissues

In the folllowing images you'll be able to appreciate the root canal anatomy, tough only macroscopical.


Tavole di Hess





One of the studies of Nair  is extremely enlightening. (Nair et al.  Microbial status of root canal system of human mandibular first molars with primary apical periodontitis after "one visit" endodontic treatment. Oral Surgery Oral Medicine Oral Pathology , Oral Radiology and Oral Endodontology 2005). Sixteen diseased mesial roots of mandibular first molars were treated endodontically, each in one visit. Mesio-buccal canals were instrumented using stainless steel hand files and mesio-lingual canals with a nickel-titanium rotary system. The canals were irrigated with 5.25% sodium hypochlorite (NaOCl) during the instrumentation procedures, rinsed with 10 mL of 17% ethylenediamine tetraacetic acid (EDTA), and obturated with gutta-percha and zinc oxide eugenol cement. Thereafter, the apical portion of the root of each tooth was removed by flap-surgery. The specimens were fixed, decalcified, subdivided in horizontal plane, embedded in plastic, processed, and evaluated by correlative light and transmission electron microscopy. Fourteen (88%) of the 16 endodontically treated teeth revealed residual intracanal infection after instrumentation, antimicrobial irrigation, and obturation. The microbes, pulpal remains and dentinal chips were located in inaccessible recesses and diverticula of instrumented main canals, the intercanal isthmus, and accessory canals, mostly as biofilms. 

Thus Nair in his work concluded that, in the light of the anatomical complexity of the root canal system and of the organization of the flora as biofilms in inaccessible areas of the canal system, it is not feasible to eliminate all the organic content and microorganisms by means of current instruments and techniques

The following images are from Nair


Fig 2. Photomicrograph of a transverse section (A) through the apical portion of the mesial root of the tooth removed by surgery from the radiolucent area in Fig 1, D. The rectangular demarcated are in A is magnified in B. The mesio-lingual (ML) and mesiobuccal (MB) canals (magnified in C) communicate and are root-filled (GP). The rectangular demarcated area in B is magnified in E. The main canals show recesses and diverticulations; those in the rectangular demarcated area in C are magnified in D. One noninstrumented accessory canal (AC in E) is enlarged in F. Note the diverticulation of ML in D and the larger accessory canal (E, F) clogged with bacteria (BA); the transmission electron microscopic view of the latter is shown in Fig 3. Black arrowheads in F show cross-sectioned profiles of anastomoses of the root canal system. Original magnifications: A, 316; B, 340; C and E, 3100; D, 3400; F, 3260.
Fig 4. Light microscopic view of a transverse section through the apical portion of the mesial root of a right mandibular first molar (MX-11, Table I). The surgical artifact (AT) into the root dentine did not reach or damage the instrumented mesio-lingual and mesio-buccal canals, which were incompletely obturated with gutta-percha cones. The isthmus (IS) connecting the canals is magnified in B; the area indicated with the black arrowhead is further magnified in stages in C and D, respectively. Note the uninstrumented isthmus with arcading profiles of Howship’s lacune (HL) clogged with blue stained bacterial mass (BA). A transmission electron microscopic view is shown in Fig 5. Original magnifications: A, 316; B, 344; C, 3240; D, 3400.






Fig 6. Light photomicrographs of apical root discs of the mesial root of a right mandibular first molar (MX-08, Table I). The identification notch is on the buccal aspect in A. The mesio-lingual (ML) and mesio-buccal (MB) canals are wide apart in the more cervical section (A) but only the mesio-buccal canal is still present in the more apical root segment (B). The rectangular demarcated area in B is magnified in C. The tangentionally cut segment of a very narrow isthmus (IS) is further magnified in stages in D and E. The contents of the isthmus cannot be resolved at the respective magnifications but are distinctly clear in the electron micrograph of
the area presented in Fig 7. Original magnifications: A and B, 315; C, 3100; D, 3180; E, 3300.
What is the rationale for the use of formaldheye in the endodontic cements?
In the light of what showed and said, the role of formaldehyde in endodontic cement can be summarized in the following points:
– formaldehyde is a gas and can reach an area distant from the point of application, otherwise not reachable with instrumentations and irrigants (teleactive action of formaldeyde, called also vapor effect). Thus formaldehyde can kill bacteria in inaccessible area, as diverticula, ishtmuses, recessess, lateral canals, ecc. This property explains wherefore it is frequent to observe periapical healing also when the  canal is not negotiable along the entire lenght. 
– formaldehyde has the property of fixation of non vital tissue, as necrotic pulp, thus avoiding that this tissue can become a pabulum for microrgamisms. 
– formadehyde at the concentration of N2  has a reparative action upon residual vital pulp. This is an unsurpassable advantage, beign the pulp the first barrier against the passage of bacteria into the bone, once the bacteria have crossed the enamel and dentin. The pulp has a reparative mechanism that pariapex does not have: dentin formation! Dentin formation is the best barrier against  microrganisms, because reduce leakage.
in vital teeth, allowing to instrument short to the anatomic apex, formaldehyde consents to realize a more conservative access cavity and canal shape with obviuosly minor dentin asportation. This is undeniable advantage for long term prognosis of tooth (Clark D, Khademi JA. Case studies in modern molar endodontic access and directed dentin conservation. Dent Clin North Am. 2010;54:275-289. Clark D, Khademi JA. Modern molar endodontic access and directed dentin conservation. Dent Clin North Am. 2010;54:249-273)We know that mechanical failure (fracture) of endodontically treated teeth is the major cause of failures. Dentin is the ultimate result of natural selection. Its thickness, its morphology, its ultrastructure are the ultimate products over millions of years of Nature Teleology. Nature has selected these properties in order to endure mastication loads for the entire life of humans. Besides, the nature have selected the pulp in order to produce dentin (primary dentin) and repair it (secondary and terziary dentin). An endodontically treated tooth have lost its ability to repair dentin, having lost pulp cells. Dentin reparation has two fundamental roles: reinforce the tooth and diminish leakage. Thus, in a tooth already mutilated due to the caries and due to the loss of pulp, we need to be careful to sacrifice further dental structure! A tooth with bacteria in the root canals can rely on periapical tissue for healing. Instead a tooth with scarce dentin cannot rely on any reparative possibility.
Therefore N2 allows clinician to be more conservative respect to dental structure.
What does Sargenti suggest in endodontic therapy of vital pulp? What is the rational of his suggetions?
The ideal goal of endodontic therapy is to mantain or recover the health of pulp and/or of the periapical tissue Sargenti had been always insisting do not violate the apical third in a vital pulp, defining this area "apical sanctuary"
–  in a vital tooth (sound pulp or pulpitis) the pulp apical third is generally vital and not infected. Besides, the apical thid of the puilp has a great capcity od reparation due to the minimal amount of tissue, a good blood supply (for the proximity to the foramen) and the absence of bacteria.  
– leaving a stump of vital pulp in the apical third can act as a first barrier against  the bacterial invasion. According to the review of  Gesi and Bergenholts (Pulpectomies: studies on outcome Endodontic Topics 2003, 5, 57–70), as far as the length of instrumentation is concerned, the results of numerous studies do indicate that it is more favorable to stop short of the anatomic apex than long. (Strindberg LZ. The dependence of the results of pulp therapy on certain factors. Acta Odontol Scand 1956: 14(Suppl 21).Grahne´n H, Hansson L. The prognosis of pulp and root canal therapy. A clinical and radiographic follow-up examination. Odontol Revy 1961: 12: 146–165. Ketterl W. Histologische Untersuchungen an vitalexstirpierten Za¨hnen. Stoma 1963: 16: 85–97. Ketterl W. Kriterien fu¨r den Erfolg der Vitalextirpation. Dtsch Zahna¨rztl Z 1965: 20: 407–416).
Therefore, , it is hard to rationalize, as sometimes advocated (Buchanan LS. One visit endodontics: a new model of reality. Dent Today 1996: 15:36-43) , that the apical foramen should be pierced and root canals overfilled with so called puffs. It seems that overfills of this nature only demonstrate the inexactness of the filling technique being used. If such an approach regularly is sought, a fair number of root canals will become unnecessarily overinstrumented and overfilled.
N2 induces the formation of a so called "sclerotic zone" on the pulp at contact with N2.  (Rowe A.H.R. Treatment with N2 Root Canal Sealer. Histological Report, British Dental Journal, Nr. 1, 1964. Branchini C. Ricerche istologiche sulla guarigione del resto pulpare e del periodonzio apicale dopo riempimento canalare con N2. Rivista Italiana Stomatologia, n.10, 1964).
Such a sclerotic zone insulates the remaining pulp, wich conserves vitality. 

Nel trattamento dei denti vitali, la zona sclerotica che separa l'N2 dai resti della polpa vitale, ha una importanza essenziale per il successo nei denti con polpa vitale. Esperienze in microscopia elettronica da parte di Novak, Merker and Kvapilova (1971) hanno confermato la presenza della zona sclerotica al di sotto del cemento canalare contenente formaldeide. Polpe di incisivi di cani sono state osservate al microscopio elettronico dopo applicazione di preparati con formaldeide. I risultalti confermano quelli ottenuti con la microscopia ottica "la formazione di una barriera è indotta dall'effetto della paraformaldeide"
Al di sopra della barriera il tessuto appare necrotico, sotto di essa nessun cambiamento può essere riscontrato anche con un microscopio elettronico. La barriera è formata da cellule densamente impacchettate, la superficie delle quali è estremamente allargata da un gran numero di stretti processi. Le cellule del sangue giocano un ruolo evidente nella costruzione della barriera cellulare.
Angelo Sargenti ha pertanto sempre insistito sulla importanza di lasciare la zona apicale intatta nei denti con polpa vitale, zona che lui chiamava "santuario apicale". La polpa apicale a contatto con l'N2 rimaneva sterile e vitale. 
La tecnica proposta da Sargenti, a dispetto di quanto sostenevano molti suoi detrattori, prevedeva nei vitali la rimozione della quasi totalità del contenuto pulpare dal canale radicolare, cercando di lasciare intatta la regione apicale. Solo nei casi con geometrie canalari complesse e con polpa vitale (siamo negli anni 50, senza microscopio, senza strumenti al Ni-Ti, senza localizzatori elettronici d'apice, etc.), Sargenti consigliava di arrestarsi con la preparazione e l'otturazione prima della curva nei molari con radici che non permettono di strumentare fino al terzo apicale. Infatti aveva osservato che l'N2 anche in questi casi permetteva un elevata percentuale di successi.
Nei canali necrotici Angelo Sargenti consigliava sempre di strumentare fino al forame apicale. 
Inoltre, i ritrovamenti istologici di cambiamenti regressivi della polpa (riduzione del numero di cellule, fibrosi, atrofia reticolare, calcificazioni distrofiche, ecc.) che spesso si ritrovano in preparati istologici con N2, non sono da ascriversi all'azione della formaldeide , in quanto sono reperti normali in polpe fisiologiche ed in polpe sottoposte a pulpotomia senza N2. 
What are the evidences of clinical efficacy of N2?
I'm going to present some clinical cases of necrotitc teeth with large area of radiolucency.
       pre-op (2002)                       post-op              6 month follow up    12 months follow-up  11 years follow-up (2013)
                       Post-op (2003)                                                    10 years follow-up (2013)
         pre-op (2003)                  3 month follow up            6 month follow up           9 years follow up (2012)
                     pre-op (2009)                                     post-op                                      3 years follow-up (2012)
Teleactive effect (vapor effect) of N2 on first lower premolar, 3.4.  The patient came to my office with pain and swelling owing to the necrosis of 3.4. In the pre-op a large periapical radiolucency is visible.  The canal was not negotiable till the apex. Notwithstanding after 4 year follow-up no radiolucency is visible.
                           pre-op  (2008)                                            4 year follow-up (2012)